Rosine Saleh

Ghaith Wedeh

Harald Herrmann

Siham Bibi

Sabine Cerny-Reiterer

Irina Sadovnik

Katharina Blatt

Emir Hadzijusufovic

Sylvie Jeanningros

Catherine Blanc

Magali Legarff-Tavernier

Elise Chapiro

Florence Nguyen-Khac

Fr Éd Éric Subra

Patrick Bonnemye

Patrice Dubreuil

Vanessa Desplat

Hél Ène

Merle-B Éral

Michael Willmann

Thomas R Ülicke

Dr Peter Valent
email: peter.valent@meduniwien.ac.at

Michel Arock

G Eisenwort

L Müllauer

J Schwaab

T Hoffmann

J E Bradner

D Radia

W R Sperr

G Hoermann

A Reiter

H-P Horny

J Zuber

Abdellah Akil

Mohammad Zahid Mustafa

Ama Gassama-Diagne

TGF), OSM, fibroblast growth factor (FGF) Eosinophilia Interleukins

Keywords: cytokines, chemokines Anticoagulation, bleeding Heparin, tryptase Fibrinolysis, bleeding Histamine, LTs, VEGF, cytokines Bronchoconstriction Histamine, PAF, LTs, PGD2, endothelin Pulmonary edema Histamine, PAF, LTs Gastric hypersecretion Histamine, LTs Cramping, abdominal pain Histamine, PAF, LTs Diarrhea Histamine Bone remodeling IL-6, tryptase, FGF, receptor activator HCV, SUMO1, Lipid droplets

A adenine 17-AAG 17-demethoxygeldanamycin ABL Abelson murine leukemia viral oncogene homolog AEC sodium acetate Ag antigen ALL acute lymphoblastic leukemia Allergin1 allergy inhibitor receptor 1 allo-HSCT allogeneic hematopoietic stem cell transplantation AML acute myeloid leukemia APC antigen-presenting cell APC allophycocyanin APRIL a proliferation-inducing ligand ATRA all-trans-retinoic acid ASDs autism spectrum disorders ASM aggressive SM ASXL1 additional sex combs-like 1 ATP adenosine triphosphate Bas basophils BAZ1B bromodomain adjacent to zinc finger domain protein 1B BCL-2 B-cell lymphoma-2 BCL10 B cell lymphoma 10 BD Becton Dickinson BD1/BD2 bromodomain tandems BET bromodomain and extra C-terminal domain BIM Bcl-2 (B-cell lymphoma-2) interacting modulator of cell death BLYS B-lymphocyte stimulator factor BM bone marrow BMMCs bone marrow derived mast cells BP blocking peptide BRD bromodomain BRD4 bromodomain containing protein 4 BRDT BRD testis-specific form BRD4S bromodomain containing protein 4 short form BRD4L bromodomain containing protein 4 long form BSA bovine serum albumin BTK Bruton's tyrosine kinase C cytosine CAD coronary artery disease C3aR receptor for complement component 3a C5aR receptor for complement component 5a C3b complement component 3b C3biR receptor for complement component 3bi CB cord blood CCR CC-chemokine receptor CD cluster of differentiation 2-CdA cladribine (2-chlorodeoxyadenosine) CEREMAST French Reference Center for Mastocytosis CHD4 chromodomain helicase DNA-binding protein 4 CHO chinese hamster ovary CI combination index CMML-eo CMML with eosinophilia CNS central nervous system CPA carboxypeptidase A CTMC connective tissue mast cell (in mice) CtR complement receptor CR complete remission CRAC calcium release-activated calcium CRH corticotropin-releasing hormone CrkL Crk (CT10 regulation of kinase) -like protein CSF-1 colony-stimulating factor-1 CTM c-terminal motif CXCR CXC-chemokine receptor CXCL8 CXC-chemokine ligand 8 cys-LT cysteinyl (cys) leukotrienes (LTs)

Figure 1: Metachromatic granules of peripheral blood (PB)-derived human MCs visualized after toluidine blue staining (Figure from Metcalfe et al. 2008).

Table IX: MC mediators and the clinical mediator-related symptoms

Mast cells (MCs) are long-lived, multifunctional, tissue dwelling granulated immune cells that had been first described by Paul Ehrlich (1854-1915) in 1878. Ehrlich reported MCs that he found in connective tissues as cells containing basophilic granules that can be metachromatically stained in red with aniline dye. He coined for these aniline-reactive granular cells the term "mastzellen" (in German), that means well-fed cells, in the belief that the large amount of granules they contain might develop as a result of hypernutrition [START_REF] Ehrlich | Beiträge zur Theorie und Praxis der histologischen Färbung[END_REF]). The word "mast" may be derived from German "mästen" (from masticate), or from Greek, which means breast. Later, Ehrlich with Westphal (one of his pupils) reported the solubility the MC granulations in water [START_REF] Westphal | U¨ber Mastzellen. In:Farbenanalytische Untersuchungen[END_REF]. Ehrlich also got Nobel Prize in 1908 for his observations about MCs and their localization in contact with nerve fibers, blood vessels and gland canals.

Paul Ehrlich´s observations are considered as a milestone in the study of MC biology and MCs have later become the subject of numerous investigations and receive today increasing attention. At

Ehrlich´s time, the origin of MCs was obscure as well as their heterogeneity, their biological roles or the structure and the composition of their metachromatic granulations. MCs are identified by the presence of metachromatic granules in their cytoplasm, when stained with toluidine blue (Figure 1).

The prominent cytoplasmic granules of MCs are rich in sulfated glycosaminoglycans (GAGs) that are responsible for their metachromatic staining by basic dyes such as toluidine blue and aniline dyes.

These acidic amino sugars consist predominantly of chondroitin sulfates.

MCs are found at the interface between the host and the external environment such as skin, gastrointestinal (GI) tract and respiratory mucosa [START_REF] Ehrlich | Beiträge zur Theorie und Praxis der histologischen Färbung[END_REF][START_REF] Metcalfe | Mast cells[END_REF][START_REF] Jamur | Identification and characterization of undifferentiated mast cells in mouse bone marrow[END_REF]Galli et al. 2005;[START_REF] Metcalfe | Mast cell biology in evolution[END_REF]. In 1937, almost 60 years after Ehrlich's description of MCs, Holmgren and Wilander reported that tissues containing large number of MCs are particularly rich in heparin [START_REF] Holmgren | Chemistry and function of mast cells[END_REF]. Ten years later, it was shown that both histamine and Intoduction 2 heparin are released from dog liver during anaphylactic shock [START_REF] Rocha | Liberation of histamine and heparin by peptone from the isolated dog's liver[END_REF]. Jorpes reported for the first time that heparin is produced in all species by tissue MCs and that for certain tissues there is a good correlation between the histamine content and the heparin content [START_REF] Jorpes | Anticoagulant therapy in thrombosis[END_REF]. A few years later, Riley and West reported that MCs have an important content of histamine, and play a possible role in histamine release in tissues [START_REF] Riley | The presence of histamine in tissue mast cells[END_REF].

The heterogeneity of MCs was first reported by Enerbäck in the 60s [START_REF] Enerbäck | Mast cells in rat gastrointestinal mucosa. I. Effects of fixation[END_REF]. He showed that rat MCs in intestinal mucosa could not be identified on histological sections because of their sensitivity to routine formalin fixation in contrast to skin MCs. These mucosal MCs stained blue after suitable fixation and staining with Alcian blue and safranin, whereas the connective tissue MCs stained red with safranin [START_REF] Enerbäck | Mast cells in rat gastrointestinal mucosa. I. Effects of fixation[END_REF][START_REF] Enerbäck | Mast cell heterogeneity: the evolution of the concept of a specific mucosal mast cell. In:Mast Cell Differentiation and Heterogeneity[END_REF]. William Bate Hardy also contributed to the shaping of the concept of "heterogeneity of MCs", since he focused on the 'wandering cells' in different mammalian species and distinguished two types of granular cells, corresponding today to the connective tissue-type MCs and mucosal-type MCs. He also described the degranulation of rat MCs [START_REF] Kanthack | The morphology and distribution of the wandering cells of mammalia[END_REF][START_REF] Hardy | The wandering cells of the alimentary canal[END_REF]. In humans, MCs are regionally heterogeneous in terms of content in proteases: lung and intestinal mucosa MCs disgorge the neutral protease tryptase but not chymase, whereas skin and intestinal submucosal MCs release tryptase and chymase.

MCs have long regarded as key effector cells in the pathogenesis of allergic diseases. However, their ability to generate and release various cytokines points to their role in diverse pathophysiological processes such as wound healing, inflammatory reactions, angiogenesis, fibrosis and tumours [START_REF] Gounaris | Mast cells are an essential hematopoietic component for polyp development[END_REF][START_REF] Ribatti | Mast cells as therapeutic target in cancer[END_REF].

Nowadays, it is well known that MC granules contain preformed mediators that can be released after stimulation [START_REF] Metcalfe | Mast cells[END_REF]) and that MCs are important effector cells involved in host defense mechanisms and allergic processes. In fact, these cells play a key role in innate and acquired immunity where they contribute to inflammatory reactions through the release of different proinflammatory mediators such as histamine, heparin, lipid mediators or cytokines. During a type-I allergic reaction, immunoglobulin E (IgE) produced by B-lymphocytes binds to the membrane IgE receptor (the high affinity receptor for IgE, also referred to as FcεRI) on MC surface. When multivalent antigen crosslinks the antigen-specific IgE bound to FcεRI receptors on MCs, preformed MC granuleassociated vasoactive/proinflammatory mediators, including histamine, proteases and heparin are released immediately, while lipid mediators or cytokines are synthesized in a delayed manner. These mediators, and particularly histamine and lipid derivatives account for the successive adverse events associated with allergy such as increased vascular permeability, mucus secretion, and smooth muscle contraction occur [START_REF] Metzger | The receptor with high affinity for IgE[END_REF][START_REF] Kinet | The high-affinity IgE receptor (FcεRI): from physiology to pathology[END_REF][START_REF] Siraganian | Mast cell signal transduction from the high-affinity IgE receptor[END_REF].

Development of mast cells

The origin of MCs was demonstrated for the first time in 1978 by Kitamura et al. who performed in vivo experiments using genetically MC-deficient mutant mice and suggested that MCs derive from bone marrow (BM) precursor cells [START_REF] Kitamura | Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation[END_REF]. The hematopoietic origin of MCs in humans was later confirmed after allogeneic BM transplantation in a leukemic patient, where MCs isolated from the recipient's BM displayed the donor's genotype after 7 months of transplantation [START_REF] Födinger | Origin of human mast cells: development from transplanted hematopoietic stem cells after allogeneic bone marrow transplantation[END_REF]. MCs develop from uncommitted hematopoietic stem cells that in turn give rise to committed CD34+ KIT+ CD13+ progenitor cells in the BM [START_REF] Kirshenbaum | Demonstration that human mast cells arise from a progenitor cell population that is CD34(+), c-kit(+), and expresses aminopeptidase N (CD13)[END_REF]). These progenitors are detectable in the peripheral blood (PB), BM, and umbilical cord blood (CB) [START_REF] Ogawa | Suspension culture of human mast cells/basophils from umbilical cord blood mononuclear cells[END_REF][START_REF] Valent | The growth and differentiation of mast cells[END_REF]Valent et al. 1992;[START_REF] Mitsui | Development of human mast cells from umbilical cord blood cells by recombinant human and murine c-kit ligand[END_REF][START_REF] Arock | Differentiation of References human mast cells from bone-marrow and cord-blood progenitor cells by factors produced by a mouse stromal cell line[END_REF]Valent 1995; [START_REF] Metcalfe | Mast cells[END_REF]) and express the receptor for stem cell factor (SCF) (SCF-receptor: CD117, or KIT) which binds to SCF, also termed KIT ligand (KL, also formerly termed steel factor (SF) and MC growth factor (MGF)). MC progenitors emigrate via the bloodstream into different tissues where they differentiate under the influence of various growth factors, including SCF (Abraham & Arock 1998;[START_REF] Metcalfe | Mast cells[END_REF][START_REF] Valent | The growth and differentiation of mast cells[END_REF]Valent 1995;[START_REF] Ogawa | Suspension culture of human mast cells/basophils from umbilical cord blood mononuclear cells[END_REF]Valent et al. 1992;[START_REF] Mitsui | Development of human mast cells from umbilical cord blood cells by recombinant human and murine c-kit ligand[END_REF][START_REF] Saito | Selective growth of human mast cells unduced by Steel factor, IL-6 and prostaglandin E2 from cord blood mononuclear cells[END_REF]Galli et al. 1994;Arock et al. 1993;Wedemeyer et al. 2000). More specifically, a study showed that MCs are closely related to monocytic lineage since CD34+ KIT+ CD13+ cell population can give rise to both mature monocytes and MCs, hence in vitro promastocytes are preferentially described as CD34+ KIT+ CD13+ and negative for CD14, which is a lipopolysaccharide receptor specific for monocyte lineage [START_REF] Kirshenbaum | Demonstration that human mast cells arise from a progenitor cell population that is CD34(+), c-kit(+), and expresses aminopeptidase N (CD13)[END_REF]).

Stem cell factor and its effects on mast cell development

Stem cell factor (SCF) is produced mainly by stromal cells such as fibroblasts and endothelial cells.

The mature protein SCF is either expressed on cell surface or released as a soluble growth factor.

Fibroblasts and endothelial cells display the transmembrane form of SCF and also release the soluble form [START_REF] Metcalfe | Mast cells[END_REF][START_REF] Broudy | Stem cell factor and hematopoiesis[END_REF][START_REF] Prussin | IgE, mast cells, basophils, and eosinophils[END_REF]. MCs are the only hematopoietic cells displaying a very high KIT expression during all stages of their development. Stem cell factor (SCF) has multiple biological effects on MCs, including modulating differentiation and homing, viability, MC hyperplasia and enhancement of mediator formation (Galli et al. 1995;[START_REF] Broudy | Stem cell factor and hematopoiesis[END_REF][START_REF] Ashman | The biology of stem cell factor and its receptor C-kit[END_REF]). Stem cell factor (SCF) can promote the survival of hematopoietic stem cells in vitro by increasing their entry into cell cycle [START_REF] Broudy | Stem cell factor and hematopoiesis[END_REF]. Moreover, MC progenitor stimulation by SCF leads to formation of granules and maturation of MCs. Recombinant human SCF (rhSCF) was reported to induce in vitro differentiation of human MCs from their BM and PB precursor cells in long-term culture and to upregulate MC releasability (Valent et al. 1992;[START_REF] Kirshenbaum | Effect of IL-3 and stem cell factor on the appearance of human basophils and mast cells from CD34+ pluripotent progenitor cells[END_REF]). In addition, SCF has an important role in cell-matrix adhesion; it stimulates MCs to bind to fibronectin and, to a lesser extent, to vitronectin. In addition, the stimulation of integrin adhesiveness requires activation of the KIT protein tyrosine kinase (TK) [START_REF] Kinashi | Steel factor and c-kit regulate cell-matrix adhesion[END_REF].

Effects of other cytokines and biological molecules on mast cell development in various species

In vitro generation of MCs requires absolutely SCF only for human promastocytes, but not for murine MC progenitors. Murine promastocytes develop into functional mucosal type MCs (MMCs) in culture in the presence of interleukin-3 (IL-3), and require SCF in addition to IL-3 only for the generation of connective type MCs (CTMCs) [START_REF] Schrader | In in vitro production and cloning of the P cell, a bone marrow-derived null cell that expresses H-2 and Ia-antigens, has mast cell-like granules, and is regulated by a factor released by activated T cells[END_REF][START_REF] Ihle | Biologic properties of homogeneous interleukin 3. I. Demonstration of WEHI-3 growth factor activity, mast cell growth factor activity, p cell-stimulating factor activity, colony-stimulating factor activity, and histamineproducing cell-stimulating factor activity[END_REF]. Apart from SCF and IL-3, several growth factors such as interleukin-4 (IL-4), interleukin-9 (IL-9), interleukin-10 (IL-10), and nerve growth factor (NGF) were found to regulate the development and differentiation of murine MCs [START_REF] Matsuda | Nerve growth factor induces development of connective tissue-type mast cells in vitro from murine bone marrow cells[END_REF]Valent et al. 1992;[START_REF] Rennick | Cofactors are essential for stem cell factor-dependent growth and maturation of mast cell progenitors: comparative effects of interleukin-3 (IL-3), IL-4, IL-10, and fibroblasts[END_REF]Valent 1995;[START_REF] Metcalfe | Mast cells[END_REF][START_REF] Bischoff | Il-4 enhances proliferation and mediator release in mature human mast cells[END_REF]. In addition, IL-4 and IL-6 are known to promote the differentiation and/or maturation of human MCs, whereas IL-9 and IL-10 has no such effect on the same cells (Valent et al. 1992;Galli et al. 1994;Valent 1995;[START_REF] Saito | Selective growth of human mast cells unduced by Steel factor, IL-6 and prostaglandin E2 from cord blood mononuclear cells[END_REF][START_REF] Bischoff | Il-4 enhances proliferation and mediator release in mature human mast cells[END_REF][START_REF] Nakahata | Cytokines regulate development of human mast cells from hematopoietic progenitors[END_REF].

Moreover, IL-10 supports MC differentiation [START_REF] Rennick | Cofactors are essential for stem cell factor-dependent growth and maturation of mast cell progenitors: comparative effects of interleukin-3 (IL-3), IL-4, IL-10, and fibroblasts[END_REF] and may have anti-inflammatory effects on IgE/anti-IgE-challenged human MCs by inhibiting their release of IL-8, TNF-α and histamine (Royer et al. 2001;Kennedy Norton et al. 2008).

In addition, thrombopoietin (TPO), a hepatocyte-derived growth factor ligand for TPO receptor (CD110), was reported to enhance the development of a subpopulation of CD117 low /CD110+ human MCs derived from CD34+ pluripotent and CD34+/CD13+/CD117+ progenitor cells when used at specific concentration alone or in combination with SCF [START_REF] Kirshenbaum | Thrombopoietin alone or in the presence of stem cell factor supports the growth of KIT (CD117)low/ MPL(CD110)+ human mast cells from hematopoietic progenitor cells[END_REF], Metcalfe 2008).

Of note, lysophosphatidic acid (LPA) which is a phospholipid mediator produced and released by platelets, fibroblasts, and adipocytes upon phospholipase D-mediated reduction of lysophophatidylcholine, was found to accelerate the development of human MCs in SCF-containing serum-free culture systems. LPA is comitogenic with SCF although it did not prolong MC survival [START_REF] Bagga | Lysophosphatidic acid accelerates the development of human mast cells[END_REF], Metcalfe 2008).

Finally, leukotriene D4 (LTD4), which is one of the cysteinyl (cys) leukotrienes (LTs) (cys-LT) and has the highest affinity for type 1 cys-LT G-protein-coupled receptor (CysLT1), was reported to increase the proliferation of human MCs derived from CB through mitogen-activated protein kinase/extracellular signal regulated kinase (MEK/ERK) and transactivation of the KIT receptor [START_REF] Jiang | Cutting edge: Interleukin 4dependent mast cell proliferation requires autocrine/intracrine cysteinyl leukotriene-induced signaling[END_REF], Metcalfe 2008).

Besides, MC development is also negatively regulated by other cytokines that inhibit their SCFdependent differentiation from CD34+ progenitors. These negative regulators include interferongamma (IFN-γ) for human MCs [START_REF] Kirshenbaum | Inhibition of human mast cell growth and differentiation by Interferon gamma-1β[END_REF][START_REF] Kulka | High-resolution tracking of cell division demonstrates differential effects of TH1 and TH2 cytokines on SCF-dependent human mast cell production in vitro: correlation with apoptosis and Kit expression[END_REF]Metcalfe 2008), and transforming growth factor beta (TGF-β) for murine MCs (Kashyap et al. 1998). IFN-γ has an effect on early stages of MC development, while IL-5 inhibits MC development in later stages [START_REF] Kulka | High-resolution tracking of cell division demonstrates differential effects of TH1 and TH2 cytokines on SCF-dependent human mast cell production in vitro: correlation with apoptosis and Kit expression[END_REF]Metcalfe 2008).

Although IL-3 and IL-4 enhance human MC maturation, they inhibit MC development from their circulating progenitor cells in long-term culture [START_REF] Sillaber | Inhibition of stem cell factor dependent formation of human mast cells by interleukin-3 and interleukin-4[END_REF][START_REF] Xia | Effect of recombinant human IL-4 on tryptase, chymase, and Fc epsilon receptor type I expression in recombinant human stem cell factor-dependent fetal liver-derived human mast cells[END_REF][START_REF] Oskeritzian | Recombinant human (rh)IL-4-mediated apoptosis and recombinant human IL-6-mediated protection of recombinant human stem cell factor-dependent human mast cells derived from cord blood mononuclear cell progenitors[END_REF]. 1999; [START_REF] Kulka | High-resolution tracking of cell division demonstrates differential effects of TH1 and TH2 cytokines on SCF-dependent human mast cell production in vitro: correlation with apoptosis and Kit expression[END_REF]. Interleukin-4 decreases the number of MCs developing from fetal liver cells in SCF-containing medium and inhibits the expression of KIT and tryptase during SCFdependent development of human MCs (Nilsson et al. 1994;[START_REF] Xia | Effect of recombinant human IL-4 on tryptase, chymase, and Fc epsilon receptor type I expression in recombinant human stem cell factor-dependent fetal liver-derived human mast cells[END_REF]. is only active on the primitive MC-committed progenitors, where it promotes proliferation. However, IL-3 receptor alpha also known as CD123 is not any more expressed at later stages of MC development [START_REF] Valent | Cell surface structures on human basophils and mast cells: biochemical and functional characterization[END_REF][START_REF] Agis | Mast cell-lineage versus basophil lineage involvement in myeloproliferative and myelodysplastic syndromes: diagnostic role of cell-immunophenotyping[END_REF] and this interleukin is considered to recruit committed progenitors into non-MC lineage, resulting in a decreased MC development even in the presence of SCF in culture [START_REF] Sillaber | Inhibition of stem cell factor dependent formation of human mast cells by interleukin-3 and interleukin-4[END_REF].

Regarding IL-4, its effects on human MC proliferation depend on the stage of maturation of MCs.

Interleukin-4 may decrease the proliferation of MC-committed progenitors induced by SCF. That can be explained by the fact that IL-4 down-regulates KIT expression and enhances FcεRI expression [START_REF] Sillaber | Inhibition of stem cell factor dependent formation of human mast cells by interleukin-3 and interleukin-4[END_REF][START_REF] Toru | Induction of the high-affinity IgE receptor (Fc epsilon RI) on human mast cells by IL-4[END_REF][START_REF] Oskeritzian | Recombinant human (rh)IL-4-mediated apoptosis and recombinant human IL-6-mediated protection of recombinant human stem cell factor-dependent human mast cells derived from cord blood mononuclear cell progenitors[END_REF][START_REF] Kulka | High-resolution tracking of cell division demonstrates differential effects of TH1 and TH2 cytokines on SCF-dependent human mast cell production in vitro: correlation with apoptosis and Kit expression[END_REF]. However, several reports suggest that IL-4 may promote the proliferation of mature tissue MCs [START_REF] Sillaber | Inhibition of stem cell factor dependent formation of human mast cells by interleukin-3 and interleukin-4[END_REF][START_REF] Oskeritzian | Recombinant human (rh)IL-4-mediated apoptosis and recombinant human IL-6-mediated protection of recombinant human stem cell factor-dependent human mast cells derived from cord blood mononuclear cell progenitors[END_REF][START_REF] Bischoff | Il-4 enhances proliferation and mediator release in mature human mast cells[END_REF][START_REF] Nakahata | Cytokines regulate development of human mast cells from hematopoietic progenitors[END_REF][START_REF] Kulka | High-resolution tracking of cell division demonstrates differential effects of TH1 and TH2 cytokines on SCF-dependent human mast cell production in vitro: correlation with apoptosis and Kit expression[END_REF]Metcalfe 2008).

Mast cell morphology, phenotype and heterogeneity

During the development and differentiation of human MCs, four morphologically distinct stages of maturation have been defined [START_REF] Agis | Mast cell-lineage versus basophil lineage involvement in myeloproliferative and myelodysplastic syndromes: diagnostic role of cell-immunophenotyping[END_REF]Sperr et al. 2001;Horny et al. 2007). The most immature form of MCs is the non-granulated blast (Figure 2a-b), followed by metachromatically granulated blast. Mature MCs are well-granulated, ovoid or elongated irregularly cells with a round nucleus. Between non-granulated blasts and mature MCs, there is a promastocyte stage distinguished by its bi-or multi-lobed nuclei and generally hypogranulated cytoplasm. This cell is also termed atypical MC type II (Figure 2c) [START_REF] Agis | Mast cell-lineage versus basophil lineage involvement in myeloproliferative and myelodysplastic syndromes: diagnostic role of cell-immunophenotyping[END_REF]Horny et al. 2007;Sperr et al. 2001). In all granulated stages, MCs are tryptase-positive.

In fact, human MCs express two main soluble tryptases that are secreted as tetramers during MC degranulation in response to different stimuli: α-and β-tryptases (βI, II, and III). Of these tryptases, β- tryptase is the most biologically important as it does not have a catalytic domain defect which is responsible for the reduction of activity [START_REF] Caughey | Mast cell tryptases and chymases in inflammation and host defense[END_REF][START_REF] Pejler | Mast cell proteases[END_REF]Metcalfe 2008;[START_REF] Pejler | Mast cell proteases: multifaceted regulators of inflammatory disease[END_REF]. The murine tryptases (mouse mast cell protease mMCP-6 and mMCP-7) are also tetrameric and most probably constitute the counterparts to human α-and β-tryptase. In addition, both human and mouse MCs express a monomeric, membrane-bound tryptase as well as a number of other trypsin-like serine proteases [START_REF] Pejler | Mast cell proteases[END_REF][START_REF] Caughey | Mast cell tryptases and chymases in inflammation and host defense[END_REF][START_REF] Stevens | Protease-proteoglycan complexes of mouse and human mast cells and importance of their beta-tryptase-heparin complexes in inflammation and innate immunity[END_REF][START_REF] Pejler | Mast cell proteases: multifaceted regulators of inflammatory disease[END_REF].

In humans, tryptase is used as a specific immunohistochemical marker in order to detect and enumerate tissue MCs as this protease is present in all MC subtypes. Moreover, tryptase is of diagnostic importance because it can be measured in the serum and serves as an indicator of the total number of MCs. Baseline levels in healthy individuals is comprised between 0-15 ng/ml in healthy adult individuals (Horny et al. 2007).

The number of MC granules was reported to vary substantially between species and even in the same specie, and these organelles often display sub-structural patterns distinctive to the species (Dvorak 2005). The serine proteases including tryptases and chymases are the major protein components of MC secretory granules. The properties and the type of these proteases vary not only among species, but also among different MC subtypes [START_REF] Pejler | Mast cell proteases[END_REF][START_REF] Caughey | Mast cell tryptases and chymases in inflammation and host defense[END_REF][START_REF] Stevens | Protease-proteoglycan complexes of mouse and human mast cells and importance of their beta-tryptase-heparin complexes in inflammation and innate immunity[END_REF]Metcalfe 2008;[START_REF] Pejler | Mast cell proteases: multifaceted regulators of inflammatory disease[END_REF]. Table I represents the distribution of human and murine MC proteases among different MC subtypes and the predominant localization of these subtypes. Two subtypes of MCs have been described in human tissues based on the proteases content of their granules and on their localization. MCs containing only tryptase (α and β), named MCT, are most abundant in mucosal tissues, whereas those containing tryptase together with chymase, carboxypeptidase and cathepsin G, called MCTC are mainly found in connective tissues (Irani et al. 1986;[START_REF] Schechter | Identification of cathepsin G-like protease in the MCTC type of human mast cell[END_REF][START_REF] Irani | Human mast cell heterogeneity[END_REF]Vliagoftis & Befus 2005;Metcalfe 2008). These two subtypes are also heterogeneous with respect to their cytokine content, suggesting that they have different biologic functions as a result of their capacity to generate and release different cytokines. For instance, IL-5 and IL-6 seem to be expressed almost exclusively by MCT both in lung and in skin, whereas IL-4 is expressed primarily by MCTC [START_REF] Bradding | Heterogeneity of human mast cells based on cytokine content[END_REF]Vliagoftis & Befus 2005). A third, but minor, population of MCs which is found in lung and bowel and expresses chymase and cathepsin G, but not tryptase, and is thus termed MCC, has been reported and less studied [START_REF] Weidner | Ultrastructural and immunohistochemical characterization of normal mast cells at multiple body sites[END_REF]Metcalfe 2008). MCT subtype can be found mainly in the lung, the mucosa of the gut and the nose, whereas the MCTC subtype resides in the skin, tonsils and the submucosa of the gut (Irani et al. 1986;[START_REF] Schwartz | Quantitation of histamine, tryptase, and chymase in dispersed human T and TC mast cells[END_REF][START_REF] Irani | Human mast cell heterogeneity[END_REF][START_REF] Metcalfe | Mast cells[END_REF]Vliagoftis & Befus 2005;[START_REF] Prussin | IgE, mast cells, basophils, and eosinophils[END_REF]. The microenvironment of the tissue in which MCs reside determines their sensitivity towards activating stimuli independently of their subtype. As an instance, skin MCs can be activated by poly-L-lysine, compound 48/80, the neuropeptide substance P, or morphine, whereas MCs of human lung, adenoids, tonsils, and colon are not responsive to these nonimunologoc stimuli [START_REF] Lowman | Human mast cell heterogeneity: histamine release from mast cells dispersed from skin, lung, adenoids, tonsils, and colon in response to IgE dependent and nonimmunologic stimuli[END_REF]). In the mouse, MCs are divided into the connective tissue MC (CTMC) and mucosal MC (MMC) subtypes. CTMCs express predominantly two different chymases (the β-chymase mouse mast cell protease 4 (mMCP-4) and the α-chymase (mMCP-5),

whereas MMCs express predominantly two types of β-chymases (mMCP-1 and mMCP-2). There are many distinguishing characteristics between these two MC populations. The granule core proteoglycan in CTMCs is primarily heparin (highly sulfated proteoglycan), whereas in MMCs, it is primarily chondroitin sulfate (lower level of sulfatation). There are also significant differences in morphology, histochemical characteristics, content of mediators, responsiveness to growth factors, sensitivity to drugs and stimuli, and biological functions [START_REF] Arock | Culture of murine mastocytes: heterogeneity of the ultrastructural aspects[END_REF][START_REF] Schwartz | Quantitation of histamine, tryptase, and chymase in dispersed human T and TC mast cells[END_REF]Welle 1997;Vliagoftis & Befus 2005;[START_REF] Rönnberg | Mast cell proteoglycans[END_REF]. Culture of normal mouse hematopoietic cells for few weeks in presence of murine IL-3 develops into a heterogeneous MC population that resembles to mucosal MCs and clearly differ from serosal MCs [START_REF] Arock | Culture of murine mastocytes: heterogeneity of the ultrastructural aspects[END_REF].

MCs express a myriad of receptors on their surfaces. Among the most important receptors on MCs, two of them are KIT (CD117) and FcεRI [START_REF] Ishizaka | Induction of calcium flux across the rat mast cell membrane by bridging IgE receptors[END_REF][START_REF] Metzger | The receptor with high affinity for IgE[END_REF][START_REF] Valent | Cell surface structures on human basophils and mast cells: biochemical and functional characterization[END_REF]Valent 1994;[START_REF] Okayama | Development, migration, and survival of mast cells[END_REF][START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF][START_REF] Kim | The multiple roles of phosphoinositide 3-kinase in mast cell biology[END_REF]. FcεRI is only expressed on mature MCs, but not on immature MC progenitors, whereas KIT is always expressed on MCs regardless their maturation stage [START_REF] Galli | Mast cell and basophil development[END_REF][START_REF] Welker | Nerve growth factor-beta induces mast-cell marker expression during in vitro culture of human umbilical cord blood cells[END_REF][START_REF] Schernthaner | Detection of differentiation-and activation-linked cell surface antigens on cultured mast cell progenitors[END_REF]Valent et al. 2010). Furthermore, FcεRI is essential for the initiation of inflammatory-related signaling, and KIT, the SCF receptor, plays an important role in MC development and homestasis [START_REF] Galli | The c-kit receptor, stem cell factor, and mast cells. What each is teaching us about the others[END_REF][START_REF] Galli | Mast cells and basophils[END_REF][START_REF] Okayama | Development, migration, and survival of mast cells[END_REF][START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF][START_REF] Kim | The multiple roles of phosphoinositide 3-kinase in mast cell biology[END_REF].

KIT, the tyrosine kinase receptor for stem cell factor (SCF)

As mentioned above, MCs and MC precursors express the receptor for SCF, namely KIT which is encoded by the KIT proto-oncogene (Valent 1994). The KIT proto-oncogene is identified as the cellular and untruncated counterpart of the gene in the Hardy-Zuckerman4 (HZ4) feline sarcoma virus genome (v-Kit) [START_REF] Besmer | A new acute transforming feline retrovirus and relationship of its oncogene v-kit with the protein kinase gene family[END_REF][START_REF] Yarden | Human protooncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand[END_REF]) and contains 21 exons transcribed/translated into a transmembrane tyrosine kinase receptor (TKR) that is structurally similar to the receptors for colony-stimulating factor-1 (CSF-1) and platelet derived growth factor (PDGF) [START_REF] Chabot | The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus[END_REF].

The KIT TKR is encoded at the white spotting (W) on chromosome 4q12 in human [START_REF] Giebel | Organization and nucleotide sequence of the human KIT (mast/stem cell growth factor receptor) proto-oncogene[END_REF]) and on chromosome 5 in mouse [START_REF] Yarden | Human protooncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand[END_REF][START_REF] Qiu | Primary structure of c-kit: relationship with the CSF-1/PDGF receptor kinase family-oncogenic activation of v-kit involves deletion of extracellular domain and C terminus[END_REF]. A number of mutations at the W locus have been documented [START_REF] Russell | Hereditary anemias of the mouse: a review for geneticists[END_REF]) and produce phenotypes ranging from embryonic lethality to severe macrocytic anemia, depigmentation, and sterility.

The protein KIT (CD117) has a molecular mass of 145 kDa with 976 amino acids in length [START_REF] Giebel | Organization and nucleotide sequence of the human KIT (mast/stem cell growth factor receptor) proto-oncogene[END_REF], and belongs to a family of transmembrane growth factor receptors with intrinsic TK activity [START_REF] Qiu | Primary structure of c-kit: relationship with the CSF-1/PDGF receptor kinase family-oncogenic activation of v-kit involves deletion of extracellular domain and C terminus[END_REF][START_REF] Chabot | The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus[END_REF]. KIT is expressed at both RNA and protein levels in normal cell types including hematopoietic stem cells and progenitors [START_REF] Andre | c-kit mRNA expression in human and murine hematopoietic cell lines[END_REF][START_REF] Simmons | c-kit is expressed by primitive human hematopoietic cells that give rise to colony-forming cells in stroma-dependent or cytokine-supplemented culture[END_REF], melanocytes [START_REF] Halaban | Pigmentation and proliferation of human melanocytes and theeffects of melanocyte-stimulating hormone and ultraviolet B light[END_REF], germ cells [START_REF] Manova | Gonadal expression of c-kit encoded at the W locus of the mouse[END_REF][START_REF] Orr-Urtreger | Developmental expression of c-kit, a proto-oncogene encoded by the W locus[END_REF][START_REF] Strohmeyer | Expression of the ckit proto-oncogene and its ligand stem cell factor (SCF) in normal and malignant human testicular tissue[END_REF], interstitial cells of Cajal [START_REF] Huizinga | W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity[END_REF], glandular epithelial cells of breast [START_REF] Lammie | Expression of c-kit and kit ligand proteins in normal human tissues[END_REF][START_REF] Tsuura | Preferential localization of c-kit product in tissue mast cells, basal cells of skin, epithelial cells of breast, small cell lung carcinoma and seminoma/dysgerminoma in human: Immunohistochemicalstudy on formalin-fixed, paraffin-embedded tissues[END_REF], normal MCs [START_REF] Metcalfe | Regulation of normal and neoplastic human mast cell development in mastocytosis[END_REF], and melanocytes [START_REF] Natali | Expression of c-kit receptor in normal and transformed human nonlymphoid tissues[END_REF]. The KIT protein is expressed not only in normal cells but also in neoplastic cells from gastrointestinal stromal tumours (GISTs) [START_REF] Sarlomo-Rikala | CD117: a sensitive marker for gastrointestinal stromal tumors that is more specific than CD34[END_REF][START_REF] Arber | Paraffin section detection of the c-kit gene product CD117) in human tissues: Value in the diagnosis of mast cell disorders[END_REF][START_REF] Andersson | The complexity of KIT gene mutations and chromosome rearrangements and their clinical correlation in gastrointestinal stromal (pacemaker cell) tumors[END_REF][START_REF] Went | Prevalence of KIT expression in human tumors[END_REF], small cell lung cancer [START_REF] Sekido | Preferential expression of c-kit protooncogene transcripts in small cell lung cancer[END_REF][START_REF] Matsuda | Expression of the c-kit protein in human solid tumors and in corresponding fetal and adult normal tissues[END_REF], colon cancer [START_REF] Toyota | Expression of c-kit and kit ligand in human colon carcinoma cells[END_REF], neuroblastoma [START_REF] Beck | Expression of stem cell factor and its receptor by human neuroblastoma cells and tumors[END_REF], seminomas [START_REF] Strohmeyer | Expression of the ckit proto-oncogene and its ligand stem cell factor (SCF) in normal and malignant human testicular tissue[END_REF], breast cancer [START_REF] Hines | Coexpression of the c-kit and stem cell factor genes in breast carcinomas[END_REF], MC disorders [START_REF] Arber | Paraffin section detection of the c-kit gene product CD117) in human tissues: Value in the diagnosis of mast cell disorders[END_REF]) and mastocytosis [START_REF] Pardanani | Pathogenesis, clinical features, and treatment advances in mastocytosis[END_REF]Arock et al. 2015), acute myeloid leukemia (AML) [START_REF] Bene | The reliability and specificity of c-kit for the diagnosis of acute myeloid leukemias and undifferentiated leukemias. The European Group for the Immunological Classification of Leukemias (EGIL)[END_REF], multiple Phosphorylation of cytoplasmic tyrosine residues occurs via SCF binding. The human (mouse) numbering system is displayed. Two isoforms of Kit in the mouse and four in humans have been identified and result from alternate mRNA splicing [START_REF] Ashman | The biology of stem cell factor and its receptor C-kit[END_REF]. The four isoforms of human KIT which differ by the presence or absence of a tetrapeptide sequence, GNNK, in the extracellular region, or of an S residue in the interkinase region, as indicated. G refers to glycine; N, asparagine; K, lysine S; serine. An additional splice variant resulting in the presence or absence of a single serine residue in the interkinase region of the cytoplasmic domain occurs in human KIT (Crosier et al. 1993) (Figure adapted from [START_REF] Ashman | The biology of stem cell factor and its receptor C-kit[END_REF], Roskoski 2005[START_REF] Orfao | Recent advances in the understanding of mastocytosis: the role of KIT mutations[END_REF] and Bibi et al. 2014).

myeloma (MM) (Escribano et al. 1998), myelodysplastic syndromes (MDS) [START_REF] Orfao | Immunophenotyping of acute leukemias and myelodysplastic syndromes[END_REF], myeloproliferative disorders (MPDs) [START_REF] Nakata | c-kit point mutation of extracellular domain in patients with myeloproliferative disorders[END_REF], T-cell acute lymphoblastic leukemia (ALL) [START_REF] Bene | The reliability and specificity of c-kit for the diagnosis of acute myeloid leukemias and undifferentiated leukemias. The European Group for the Immunological Classification of Leukemias (EGIL)[END_REF]), B-cell non-Hodgkin lymphoma [START_REF] Bravo | Expression of high amounts of the CD117 molecule in a case of B-cell non-Hodgkin's lymphoma carrying the t(14:18) translocation[END_REF], and B-cell precursor ALL [START_REF] Bene | The reliability and specificity of c-kit for the diagnosis of acute myeloid leukemias and undifferentiated leukemias. The European Group for the Immunological Classification of Leukemias (EGIL)[END_REF]. In normal cells, KIT has been reported to play a major role in gametogenesis [START_REF] Kissel | Point mutation in kit receptor tyrosine kinase reveals essential roles for kit signaling in spermatogenesis and oogenesis without affecting other kit responses[END_REF], melanogenesis [START_REF] Nocka | Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant mice -evidence for an impaired c-kit kinase in mutant mice[END_REF][START_REF] Halaban | Pigmentation and proliferation of human melanocytes and theeffects of melanocyte-stimulating hormone and ultraviolet B light[END_REF], MC development and function (Valent et al. 2005;[START_REF] Metcalfe | Regulation of normal and neoplastic human mast cell development in mastocytosis[END_REF]), gastrointestinal function [START_REF] Miettinen | KIT (CD117): a review on expression in normal and neoplastic tissues, and mutations and their clinicopathologic correlation[END_REF] and hematopoiesis in the differentiation of erythroid, lymphoid, megakaryocytic and myeloid precursors [START_REF] Nocka | Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant mice -evidence for an impaired c-kit kinase in mutant mice[END_REF].

The specific ligand of KIT, SCF, is primarily, but not exclusively, produced by stromal cells, such as fibroblasts, in two major forms; a soluble form and a membrane-bound form, as a result of differential splicing and proteolytic cleavage [START_REF] Flanagan | The kit ligand: a cell surface molecule altered in steel mutant fibroblasts[END_REF][START_REF] Toksoz | Support of human hematopoiesis in long-term bone marrow cultures by murine stromal cells selectively expressing the membrane-bound and secreted forms of the human homolog of the steel gene product, stem cell factor[END_REF][START_REF] Broudy | Stem cell factor and hematopoiesis[END_REF][START_REF] Ashman | The biology of stem cell factor and its receptor C-kit[END_REF][START_REF] Reber | Stem cell factor and its receptor c-Kit as targets for inflammatory diseases[END_REF]. Both soluble and membrane-bound forms activate KIT. However, membrane-bound form induces more persistent TK activation and longer life span of KIT geneencoded protein than the soluble form [START_REF] Miyazawa | Membranebound Steel factor induces more persistent tyrosine kinase activation and longer life span of c-kit gene-encoded protein than its soluble form[END_REF][START_REF] Ashman | The biology of stem cell factor and its receptor C-kit[END_REF].

Of note, KIT exists in active and inactive conformations as determined by X-ray crystallography and belongs to the type III receptor tyrosine kinase (RTK) subfamily, whose members include plateletderived growth factor (PDGF) receptors α and β, macrophage colony-stimulating factor (M-CSF)

receptor, and the Fms-like Tyrosine Kinase-3 (FLT3) receptor [START_REF] Blume-Jensen | Oncogenic kinase signalling[END_REF]. Its structure (Figure 3) consists of an extracellular domain (ECD), which contains the SCF-binding domain, a single transmembrane domain (TMD) encoded by exon 10, an autoinhibitory juxtamembrane domain (JMD) and a cytoplasmic kinase domain (KD) arranged in a proximal (N-)

and a distal (C-) lobe linked by a hinge region and interrupted by a large kinase insert (kinase insertion domain (KID)) [START_REF] Zhang | Crystal structure of human stem cell factor: implication for stem cell factor receptor dimerization and activation[END_REF]Roskoski 2005;Bibi et al. 2014). The ECD is encoded by the exons 1 to 9 and is characterized by its structure containing five immunoglobulin (Ig)-like domains (D1-5). SCF binds to the second and third immunoglobulin-like domains (D2 and D3) while the fourth domain (D4) plays a role in receptor dimerization [START_REF] Chabot | The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus[END_REF][START_REF] Qiu | Primary structure of c-kit: relationship with the CSF-1/PDGF receptor kinase family-oncogenic activation of v-kit involves deletion of extracellular domain and C terminus[END_REF][START_REF] Zhang | Crystal structure of human stem cell factor: implication for stem cell factor receptor dimerization and activation[END_REF]Roskoski 2005). Activated PI3-K generates PI (3,4,5)P3 through which PH domain-containing proteins such as Bruton's tyrosine kinase (BTK) are recruited to the plasma membrane and further propagate the signal. Subsequent activation of these signaling enzymes, as well as the JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathway and the RAS-RAF-mitogen-activated protein kinase (MAPK) pathway leads to MC growth, differentiation, survival, chemotaxis and cytokine production. An increased activity of PLC-γ leads to production of DAG and IP3, responsible for enhanced levels of free cytoplasmic Ca 2+ . This is followed by actin rearrangements and chemotactic response. KIT also phosphorylates NTAL that can bind other adapter proteins, including Grb2. Activated GRB2 orchestrates activation of RAS-RAF pathway by recruiting GEFs, the guanine nucleotide exchange factor Sos (son of sevenless homologue) and the guanine nucleotide exchange factor Vav, which subsequently leads to activation of MAP kinases ERK, JNK and p38. Some other signaling proteins such as JAK, STATs, AKT, PKC and Rac are involved in gene transcription, survival, actin rearrangement, and/or in chemotaxis (Figure adapted from [START_REF] Draber | Signal transduction and chemotaxis in mast cells[END_REF].

KIT dimerization by ligation of SCF is followed by conformational changes that enable lateral interactions between membrane proximal Ig-like domains D4 and D5 of two KIT molecules [START_REF] Yuzawa | Structural basis for activation of the receptor tyrosine kinase KIT by stem cell factor[END_REF]. The proteolytic cleavage of the fifth immunoglobulin-like domain D5 of KIT from the cell surface converts the transmembrane protein into a soluble form [START_REF] Broudy | The fifth immunoglobulin-like domain of the Kit receptor is required for proteolytic cleavage from the cell surface[END_REF]. The TMD forms a helix and is encoded by exon 10. The JMD encoded by exon 11 inhibits both KIT activity and receptor dimerization by its inserting into the kinase-active site between N-and C-lobes and stabilizing the inactivated conformation [START_REF] Ma | Inhibition of spontaneous receptor phosphorylation by residues in a putative alpha-helix in the KIT intracellular juxtamembrane region[END_REF][START_REF] Mol | Structure of a c-kit product complex reveals the basis for kinase transactivation[END_REF]Hubbard 2004;[START_REF] Mol | Structural basis for the autoinhibition and STI-571 inhibition of c-Kit tyrosine kinase[END_REF]Roskoski 2005;[START_REF] Zou | Detailed conformational dynamics of juxtamembrane region and activation loop in c-Kit kinase activation process[END_REF]Lemmon & Schlessinger 2010;[START_REF] Laine | Allosteric communication across the native and mutated KIT receptor tyrosine kinase[END_REF].

Binding of the protein-tyrosine phosphatase named SHP-1 (Src homology region 2 domain-containing phosphatase-1) to the phosphorylated KIT JMD results in dephosphorylation of the tyrosine residues and termination of the intracellular signal [START_REF] Kozlowski | SHP-1 binds and negatively modulates the c-Kit receptor by interaction with tyrosine 569 in the c-Kit juxtamembrane domain[END_REF]. The structure of the class III receptors differs from that of other TKRs by the insertion of 70-100 amino acids near the middle of the kinase domain in the C-lobe. In human KIT, the kinase insert domain (KID), which is about 80 residues in length, undergoes phosphorylation and serves as a docking site for a few pivotal signal transduction proteins (Roskoski 2005).

KIT signaling pathways

KIT signaling occurs through several pathways including Src Family kinases pathway, PI3-kinase (PI3K)/AKT pathway, Janus Kinase (JAK)-signal transducer and activator of transcription (STAT) (JAK/STAT) pathway, phospholipase C gamma (PLC-) pathway, and Ras/Raf/mitogen-activated protein kinases MAPKs pathway, leading to MC growth, differentiation, survival, adhesion cytokine production and chemotaxis (Roskoski 2005;[START_REF] Lennartsson | Normal and oncogenic forms of the receptor tyrosine kinase kit[END_REF][START_REF] Gilfillan | Integrated signalling pathways for mast-cell activation[END_REF]Bibi et al. 2014;[START_REF] Cruse | Functional deregulation of KIT: link to mast cell proliferative diseases and other neoplasms[END_REF][START_REF] Draber | Signal transduction and chemotaxis in mast cells[END_REF]) (Figure 4). In fact, the activation of KIT by SCF induces KIT dimerization and autophosphorylation by its intrinsic kinase activity of at least 10 tyrosine residues (Y568, Y570, Y703, Y721, Y730, Y747, Y823, Y900, Y917 and Y936, see Figure 3) that serve as docking sites for signal transduction molecules including cytosolic adapters Shc (Src homology 2 (SH2)-domain-containing transforming protein C), Gab2 (Grb2-associated binding protein 2) and Grb2 (growth factor receptor bound protein 2), Src family PTKs (Lyn and Fyn), PLC-γ, and phosphatidylinositol 3-kinase (PI3-K) [START_REF] Linnekin | Lyn associates with the juxtamembrane region of c-Kit and is activated by stem cell factor in hematopoietic cell lines and normal progenitor cells[END_REF][START_REF] Abram | Src family tyrosine kinases and growth factor signaling[END_REF]Roskoski 2005;Lennartson et al. 2005). The early signaling event following the activation of KIT by SCF is the recruitment of Src family PTKs (Fyn and in particular Lyn) to phosphorylated tyrosine residues (Y568, Y570) in the JMD of KIT [START_REF] Linnekin | Lyn associates with the juxtamembrane region of c-Kit and is activated by stem cell factor in hematopoietic cell lines and normal progenitor cells[END_REF][START_REF] Abram | Src family tyrosine kinases and growth factor signaling[END_REF]. Src

family PTKs contain N-terminal myristoylation sites, and in some cases a palmitoylation domain, which are critical for anchoring the kinases to the plasma membrane and to the membrane microdomains (lipid rafts) (Kovarova et al. 2001). These events, which occur in the lipid rafts, are important for the signal transduction through PI3-K pathway because disruption of these lipid rafts specifically reduces the phosphorylation of PI3-K and the serine-threonine kinase AKT (also known as protein kinase B (PKB)) in response to SCF [START_REF] Arcaro | Critical role for lipid raft-associated Src kinases in activation of PI3K-Akt signalling[END_REF][START_REF] Jahn | Lipid rafts are required for Kit survival and proliferation signals[END_REF]). The PI3-K phosphorylates the plasma membrane-associated phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) to form phosphatidylinositol-3,4,5,-triphosphate (PI (3,4,5)P3) (PIP3), which in turn recruits pleckstrin homology (PH) domain-containing signaling proteins to the plasma membrane [START_REF] Foster | The phosphoinositide (PI) 3-kinase family[END_REF].

The classic class I PI3-K complex consists of a regulatory p85 subunit and a catalytic subunit p110 [START_REF] Munugalavadla | Requirement for p85alpha regulatory subunit of class IA PI3K in myeloproliferative disease driven by an activation loop mutant of KIT[END_REF]. Activated KIT receptor can also directly bind the regulatory subunit of PI3-K (p85α) and thus contributes to subsequent generation of membrane-associated PIP3 (Roskoski 2005;Gilfillan & Rivera 2009). Phosphatidylinositol-3,4,5,-triphosphate (PIP3) et al. 2005). Phosphorylation of PI3-K activated by SCF contributes also to MC proliferation via the small GTPase Rac and c-Jun NH2-terminal kinase (JNK), which activates the transcription factor c-Jun [START_REF] Lennartsson | Normal and oncogenic forms of the receptor tyrosine kinase kit[END_REF].

The dimerization of KIT by SCF also triggers the activation of the MAPKs, extracellular signal regulated kinase 1 and 2 (ERK1 and ERK2), JNK, and p38. Phosphorylated tyrosine residues in activated KIT are recognized by the SH2 domain of the adaptor protein growth factor receptor-bound protein 2 (Grb2), which then forms a complex with the guanine exchange factor (GEF) son-of sevenless (Sos). Sos activates the G protein Ras by promoting the exchange of GDP by GTP [START_REF] Chardin | Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2[END_REF]. GTP-bound, active Ras initiates a cascade of serine/threonine kinases (Raf, MEK) that leads to the activation of the ERK1 and ERK2 (Roskoski 2012). The pathway leading to p38 and JUN amino-terminal kinase (JNK) activation by KIT in MCs is less well defined [START_REF] Gilfillan | Integrated signalling pathways for mast-cell activation[END_REF]. The Ras-Raf-MEK-ERK pathway (Ras/Raf/mitogen-activated protein kinases MAPKs pathway) regulates many cellular processes, particularly survival, proliferation, and cytokine production in MCs. Indeed, activated Ras can interact with and initiate the activation of the serine/threonine kinase Raf-1. Active Raf-1 can then phosphorylate and activate the dual-specificity kinases Mek1 and 2. The mitogen-activated protein kinases Erk1 and Erk2 are serine/threonine kinases that are activated through phosphorylation by Mek1/2. Targets for Erks in the cytoplasm include the serine/threonine kinases RSK and Mnk1/2, as well as phospholipase A2 (PLA2). A fraction of the activated Erk and ribosomal S6 kinase (RSK) proteins translocate to the nucleus, where they phosphorylate various transcription factors, thereby influencing gene transcription [START_REF] Lennartsson | Normal and oncogenic forms of the receptor tyrosine kinase kit[END_REF]. Therefore, targeting this pathway is attractive for treatment of cancer [START_REF] Montagut | Targeting the RAF-MEK-ERK pathway in cancer therapy[END_REF] and potentially of mastocytosis. The Ras-ERK pathway is activated by the interaction of the adaptor protein Grb2 with tyrosine residues 703 and 936 (Y703, Y936) of human KIT or indirectly via proteins that associate with KIT, such as Shc or Gab2 through SH2-domain-containing protein tyrosine phosphatase (SHP2).

The Janus kinases (JAKs) constitutively associate with KIT and become transiently activated in response to SCF, thereafter activated JAKs as well as activated KIT phosphorylate cytoplasmic STATs (STAT 1, 3, and 5) which then undergo a SH2 domain-dependent dimerization [START_REF] Weiler | JAK2 is associated with the c-kit proto-oncogene product and is phosphorylated in response to stem cell factor[END_REF][START_REF] Deberry | Stat1 associates with c-kit and is activated in response to stem cell factor[END_REF][START_REF] Brizzi | STAT protein recruitment and activation in c-Kit deletion mutants[END_REF] and subsequent translocation to the nucleus. After SCF binding, STAT1, STAT5A, and STAT5B associate with KIT and become tyrosine phosphorylated [START_REF] Deberry | Stat1 associates with c-kit and is activated in response to stem cell factor[END_REF][START_REF] Brizzi | STAT protein recruitment and activation in c-Kit deletion mutants[END_REF]. SCF-induced STAT tyrosine phosphorylation has been associated with increased DNA binding [START_REF] Lennartsson | Normal and oncogenic forms of the receptor tyrosine kinase kit[END_REF]. Nuclear STATs exert their functions by binding to their promotors and by regulating expression of responsive genes including c-MYC, CIS, PIM-1 and c-FOS which play an important role in cell proliferation [START_REF] Mui | Suppression of interleukin-3induced gene expression by a C-terminal truncated Stat5: role of Stat5 in proliferation[END_REF].

STATs proteins (at least 1, 3, and 5) are also serine phosphorylated at the C-terminus, which regulates their transcriptional activity [START_REF] Gotoh | Steel factor induces serine phosphorylation of Stat3 in human growth factor-dependent myeloid cell lines[END_REF].

Interestingly, SCF, similarly to IgE-antigen complexes, can be an activator/co-activator of MCs [START_REF] Gilfillan | Integrated signalling pathways for mast-cell activation[END_REF]. Indeed, when activated by antigen, SCF markedly increases MC degranulation and potentiates and prolongs calcium signals. It also influences MC chemotaxis and adhesion [START_REF] Vosseller | c-kit receptor signaling through its References phosphatidylinositide-3'-kinase-binding site and protein kinase C: role in mast cell enhancement of degranulation, adhesion, and membrane ruffling[END_REF]. The activation of KIT by SCF phosphorylates the adaptor protein non-T cell activation linker (NTAL) which in turn recruits and phosphorylates the phospholipase C-gamma1 (PLC-1). This PLC-1 is expressed ubiquitously whereas PLC-γ2 is mainly expressed in hematopoietic cells [START_REF] Wilde | Regulation of phospholipase C gamma isoforms in haematopoietic cells: why one, not the other?[END_REF]. PLC-γ hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). DAG binds and activates the serine-threonine protein kinases PKCs, whereas IP3 induces release of Ca 2+ from intracellular stores leading to cytokine production (Roskoski 2005). The PLC-γ association with KIT is dependent on tyrosine residues 730 and 936 of the human receptor [START_REF] Herbst | Formation of signal transfer complexes between stem cell and platelet-derived growth factor receptors and SH2 domain proteins in vitro[END_REF][START_REF] Gommerman | Differential stimulation of c-Kit mutants by membrane-bound and soluble Steel Factor correlates with leukemic potential[END_REF].

However, soluble SCF has been reported to fail to increase the activity or phosphorylation of PLC-γ [START_REF] Koike | Stem cell factor-induced signal transduction in rat mast cells. Activation of phospholipase D but not phosphoinositide-specific phospholipase C in c-kit receptor stimulation[END_REF][START_REF] Kozawa | Involvement of phosphatidylinositol 3'-kinase in stem-cell-factor-induced phospholipase D activation and arachidonic acid release[END_REF] suggesting activation of phospholipase D after SCF stimulation. In contrast, the membrane-bound form of SCF phosphorylates PLC-γ, suggesting its role in SCF-induced proliferation and survival [START_REF] Gommerman | Differential stimulation of c-Kit mutants by membrane-bound and soluble Steel Factor correlates with leukemic potential[END_REF][START_REF] Trieselmann | Mast cells stimulated by membrane-bound, but not soluble, steel factor are dependent on phospholipase C activation[END_REF]. The differential effects of soluble and membrane-bound SCF on signal transduction may be best explained by the fact that the membrane-bound SCF results in slower internalization of active KIT, whereas soluble SCF induces rapid internalization [START_REF] Yee | Mechanism of down-regulation of c-kit receptor. Roles of receptor tyrosine kinase, phosphatidylinositol 3'-kinase, and protein kinase C[END_REF][START_REF] Miyazawa | Ligand-dependent polyubiquitination of c-kit gene product: a possible mechanism of receptor down modulation in M07e cells[END_REF][START_REF] Miyazawa | Membranebound Steel factor induces more persistent tyrosine kinase activation and longer life span of c-kit gene-encoded protein than its soluble form[END_REF]. Although it is well established that SCF protects KIT-expressing cells against apoptosis [START_REF] Zsebo | Radioprotection of mice by recombinant rat stem cell factor[END_REF][START_REF] Neta | Inhibition of c-kit ligand/steel factor by antibodies reduces survival of lethally irradiated mice[END_REF][START_REF] Liebmann | Protection from lethal irradiation by the combination of stem cell factor and tempol[END_REF][START_REF] Blume-Jensen | The kit receptor promotes cell survival via activation of PI 3-kinase and subsequent Akt-mediated phosphorylation of Bad on Ser136[END_REF], the biological effects of PLC-γ are less known. However, PLC-γ1 has a critical role in SCF-induced protection against apoptosis evoked by irradiation [START_REF] Maddens | Kit signaling inhib-its the sphingomyelin-ceramide pathway through PLC gamma 1: implication in stem cell factor radioprotective effect[END_REF] as well as by the cytotoxic agent daunorubicin (Plo et al. 2007).

Regarding KIT-mediated chemotaxis, SCF is known as a major chemotactic attractant for MCs and their precursors [START_REF] Chabot | The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus[END_REF][START_REF] Meininger | The c-kit receptor ligand functions as a mast cell chemoattractant[END_REF]Nilsson et al. 1994). Once bound to KIT, SCF causes KIT tyrosine phosphorylation and formation of docking sites for SH2 domain-containing molecules, such as Src family kinases Lyn and Fyn. This results in activation of the kinases and further propagation of the signal. Activation of Fyn leads to phosphorylation of Gab2 and subsequent activation of the small GTPase Rac that is responsible for the cytoskeletal reorganization and MC migration [START_REF] Linnekin | Lyn associates with the juxtamembrane region of c-Kit and is activated by stem cell factor in hematopoietic cell lines and normal progenitor cells[END_REF][START_REF] Timokhina | Kit signaling through the PI 3-kinase and Src kinase pathways: an essential role for Rac1 and JNK activation in mast cell proliferation[END_REF][START_REF] Ueda | Critical roles of c-Kit tyrosine residues 567 and 719 in stem cell factor-induced chemotaxis: contribution of src family kinase and PI3-kinase on calcium mobilization and cell migration[END_REF][START_REF] Samayawardhena | Involvement of Fyn kinase in Kit and integrinmediated Rac activation, cytoskeletal reorganization, and chemotaxis of mast cells[END_REF].

Phosphorylation of the tyrosine residue Y719 of KIT is crucial for recruitment of PI3-K, which plays an important role in MC chemotaxis towards different chemoattractants [START_REF] Ueda | Critical roles of c-Kit tyrosine residues 567 and 719 in stem cell factor-induced chemotaxis: contribution of src family kinase and PI3-kinase on calcium mobilization and cell migration[END_REF]. The protein tyrosine phosphatase PTPα is required for proper functioning of the Gab2/SHP2/Vav/RAC/JNK signaling axis, and reduced migration towards SCF has been reported in PTPα knockout mice [START_REF] Samayawardhena | Protein-tyrosine phosphatase alpha regulates stem cell factor-dependent c-Kit activation and migration of mast cells[END_REF]. Chemotaxis towards SCF requires the phosphatase SHP2 which directly activates Lyn kinase by dephosphorylation of its inhibitory tyrosine, resulting in enhanced phosphorylation of Vav1, Rac activation and actin polymerization [START_REF] Draber | Signal transduction and chemotaxis in mast cells[END_REF]. Of note, BM derived MCs (BMMCs) from conditional SHP2 knockout mice exhibit decreased chemotaxis, and chemotaxis is also reduced in cells treated with a SHP2 inhibitor [START_REF] Sharma | SHP2 phosphatase promotes mast cell chemotaxis toward stem cell factor via enhancing activation of the Lyn/Vav/Rac signaling axis[END_REF].

Regulators of KIT kinase activity and KIT signaling

Since KIT activates multiple signaling pathways, its negative regulation also occurs through various mechanisms.

Receptor internalization, degradation and ubiquitination attenuate the strength and the duration of KIT signaling and thus have been considered as negative regulators of receptor signal transduction [START_REF] Cruse | Functional deregulation of KIT: link to mast cell proliferative diseases and other neoplasms[END_REF]. It is well know that ubiquitin molecules are covalently attached to lysine residues on target proteins, and this labels them for degradation via the 26S proteosome complex [START_REF] Lennartsson | Normal and oncogenic forms of the receptor tyrosine kinase kit[END_REF]. Activation of KIT leads to its poly-ubiquitination [START_REF] Miyazawa | Ligand-dependent polyubiquitination of c-kit gene product: a possible mechanism of receptor down modulation in M07e cells[END_REF]. The ubiquitin ligase Cbl has an important role in KIT ubiquitination and subsequent internalization, then degradation by the proteasome [START_REF] Miyazawa | Ligand-dependent polyubiquitination of c-kit gene product: a possible mechanism of receptor down modulation in M07e cells[END_REF] because it becomes phosphorylated in response to SCF through Src family kinases pathway and binds directly via its SH2 domain to Y568 and Y936 of activated KIT or through the adaptor protein Grb2 or tyrosine phosphorylated proteins CrkL, or the adapter proteins APS [START_REF] Wisniewski | c-kit ligand stimulates tyrosine phosphorylation of the c-Cbl protein in human hematopoietic cells[END_REF][START_REF] Sattler | Steel factor induces tyrosine phosphorylation of CRKL and bind-ing of CRKL to a complex containing c-kit, phosphatidylinositol 3-kinase, and p120(CBL)[END_REF][START_REF] Joazeiro | The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase[END_REF][START_REF] Wollberg | The adapter protein APS associates to the multifunctional docking sites Tyr568 and Tyr936 in c-Kit: possible role in v-Kit transformation[END_REF][START_REF] Sun | Grb2 mediates negative regulation of stem cell factor receptor/c-Kit signaling by recruitment of Cbl[END_REF]). and that this internalization depends on the recruitment of Src but not of PI3-K upon SCF stimulation using the induction of vesicular structures as a sign of efficient internalization of the receptor [START_REF] Jahn | Analysing c-kit internalization using a functional c-kit-EGFP chimera containing the fluorochrome within the extracellular domain[END_REF]. In fact, KIT can be internalized via the clathrin-coated pit mechanism and signal transduction by Src family kinases is not required for entry of KIT into clathrin-coated pits. In addition, KIT internalization is impaired after treatment with Src kinase inhibitor and also in Lyndeficient cells, suggesting that Src family kinases, in particular Lyn, are involved in this process [START_REF] Broudy | Signaling via Src family kinases is required for normal internalization of the receptor c-Kit[END_REF]. The PI3-K activity and Ca 2+ influx also influence the late stages of KIT endocytosis and inhibition of these two signals prevents SCF-mediated internalization, leading to KIT degradation [START_REF] Gommerman | Phosphatidylinositol 3-kinase and Ca2+ influx dependence for ligand-stimulated internalization of the c-Kit receptor[END_REF].

The protein kinases C (PKCs), which are serine-threonine kinases, can be activated through PI3-K and PLC pathways which generate DAG and intracellular Ca 2+ , the triggers of PKC activation. PKCs downregulate KIT in two ways. Indeed, PKC phosphorylates serine KIT residues 741 and 746 in the KID, resulting in the inhibition of KIT kinase activity [START_REF] Blume-Jensen | Modulation of Kit/stem cell factor receptor-induced signaling by protein kinase C[END_REF][START_REF] Blume-Jensen | Identification of the major phosphorylation sites for protein kinase C in kit/stem cell factor receptor in vitro and in intact cells[END_REF]. In addition, activated PKC also induces proteolytic release of the ECD of KIT (about 100 KDa) [START_REF] Yee | Mechanism of kit ligand, phorbol ester, and calciuminduced down-regulation of c-kit receptors in mast cells[END_REF][START_REF] Yee | Mechanism of down-regulation of c-kit receptor. Roles of receptor tyrosine kinase, phosphatidylinositol 3'-kinase, and protein kinase C[END_REF].

SHIP is a 5'-inositol phosphatase which hydrolyze phosphatidylinositol-3,4,5-trisphosphate (PIP3) into PIP2. Interestingly, SHIP becomes phosphorylated upon SCF stimulation of MCs [START_REF] Liu | The Src homology 2 (SH2) domain of SH2-containing inositol phosphatase (SHIP) is essential for tyrosine phosphorylation of SHIP, its association with Shc, and its induction of apoptosis[END_REF].

Disruption of the SHIP gene has revealed that it is a negative regulator of SCF-induced MC degranulation [START_REF] Huber | The src homology 2containing inositol phosphatase (SHIP) is the gatekeeper of mast cell degranulation[END_REF]. Furthermore, SHIP -/-mice have granulocytes and macrophages with increased responsiveness to IL-3, GM-CSF, and SCF compared to wild type (WT) cells, supporting a role for SHIP as a negative regulator of cytokine signaling [START_REF] Helgason | Targeted disruption of SHIP leads to hemopoietic perturbations, lung pathology, and a shortened life span[END_REF]). This tyrosine phosphatase SHIP negatively regulates KIT activity by interacting with its JMD [START_REF] Tauchi | The ubiquitously expressed Syp phosphatase interacts with c-kit and Grb2 in hematopoietic cells[END_REF][START_REF] Kozlowski | SHP-1 binds and negatively modulates the c-Kit receptor by interaction with tyrosine 569 in the c-Kit juxtamembrane domain[END_REF].

SHP1 (SH2 (Src homology-2) domain containing phosphatase 1) plays also a negative role when it is recruited to the tyrosine residue Y568 in the JMD of KIT and therefore dephophorylates KIT directly or indirectly via other proteins associated with KIT [START_REF] Kozlowski | SHP-1 binds and negatively modulates the c-Kit receptor by interaction with tyrosine 569 in the c-Kit juxtamembrane domain[END_REF].

The suppressor of cytokine signaling SOCS-1 binds to KIT [START_REF] Bayle | Suppressor of cytokine signaling 6 associates with KIT and regulates KIT receptor signaling[END_REF]) as well as to several downstream signal transduction proteins of KIT, including Grb2 and the guanine nucleotide exchange factor Vav and thereby inhibits JAK2 and SCF-mediated proliferation [START_REF] De Sepulveda | Socs1 binds to multiple signalling proteins and suppresses steel factor-dependent proliferation[END_REF].

SOCS-1 binds to Y567 in the JMD of KIT, ablating the activation of SCF-mediated MAPK pathway.

SOCS-1 protein levels increase after SCF stimulation of MCs, counteracting the KIT-related induction of proliferation [START_REF] De Sepulveda | Socs1 binds to multiple signalling proteins and suppresses steel factor-dependent proliferation[END_REF]. BMMCs from SOCS-1 deficient mice have reduced proliferative response to both IL-3 and SCF, as compared with WT cells, due to accumulation of proteases in the absence of SOCS-1, resulting in degradation of essential signal transduction molecules. This data indicates that SOCS1 is required for the expression and/or stability of an endogenous protease inhibitor, which protects MCs from their own proteolytic enzymes [START_REF] Ilangumaran | Suppressor of cytokine signaling 1 regulates an endogenous inhibitor of a mast cell protease[END_REF]. SOCS-6 also negatively regulates KIT signaling [START_REF] Bayle | Suppressor of cytokine signaling 6 associates with KIT and regulates KIT receptor signaling[END_REF]).

Another potential negative regulator of KIT is the adapter protein containing PH and SH2 domains APS. APS exists as a dimer and interacts with the JMD of KIT. This interaction facilitates the recruitment of Cbl, a negative regulator of TKs, into the complex [START_REF] Yokouchi | APS, an adap-tor protein containing PH and SH2 domains, is associated with the PDGF receptor and c-Cbl and inhibits PDGF-induced mitogenesis[END_REF][START_REF] Wollberg | The adapter protein APS associates to the multifunctional docking sites Tyr568 and Tyr936 in c-Kit: possible role in v-Kit transformation[END_REF]. Both KIT Y936 and Y570 seem to regulate the binding of APS and the recruitment of Cbl [START_REF] Cruse | Functional deregulation of KIT: link to mast cell proliferative diseases and other neoplasms[END_REF]. Lnk, a homologue of APS, is also tyrosine phosphorylated and regulates negatively KIT-mediated proliferation [START_REF] Takaki | Enhanced hematopoiesis by hematopoietic progenitor cells lacking intracellular adaptor protein, Lnk[END_REF]. Dok-1 (downstream of tyrosine kinase 1) is another potential negative regulator of KIT and may interact indirectly with the JMD [START_REF] Liang | Phosphatidylinositol 3kinase and Src family kinases are required for phosphorylation and membrane recruitment of Dok-1 in c-Kit signaling[END_REF].

The GTPase activating protein neurofibromin-1 (NF-1), which is a tumor suppressor commonly lost in juvenile myelomonocytic leukemia (JMML) [START_REF] Aricò | Juvenile myelomonocytic leukemia[END_REF], has been suggested to be a negative regulator of KIT activity [START_REF] Zhang | Nf1 regulates hematopoietic progenitor cell growth and ras signaling in response to multiple cytokines[END_REF]. In NF-1 -/-progenitor cells, SCF increases colony formation and induces a stronger ERK activation as compared with normal cells [START_REF] Lennartsson | Normal and oncogenic forms of the receptor tyrosine kinase kit[END_REF]).

As described above, exon 11 of the cytosolic region of the JMD plays a critical role in the negative regulation of KIT dimerization. Indeed, residues 553-663 of exon 11 form a putative alpha helix that inhibits activation of ligand-unoccupied receptor [START_REF] Ma | Inhibition of spontaneous receptor phosphorylation by residues in a putative alpha-helix in the KIT intracellular juxtamembrane region[END_REF][START_REF] Mol | Structure of a c-kit product complex reveals the basis for kinase transactivation[END_REF][START_REF] Mol | Structural basis for the autoinhibition and STI-571 inhibition of c-Kit tyrosine kinase[END_REF].

Mutations in the JMD of KIT play a critical role in regulating kinase activity by modulating the dimerization and potentially altering interaction with negative regulatory molecules including APS, Dok-1 and SHIP. Mutations in the JMD of KIT could alter the interaction with one or more of these negative regulators and thus result in cellular transformation [START_REF] Lennartsson | Normal and oncogenic forms of the receptor tyrosine kinase kit[END_REF].

Mutations in the KD (PTD phosphotransferase domain) at codon 816 of KIT alter the conformation of the mobile activation loop (A-loop), weaken the binding of the JMD to the KD and suppress the negative autoinhibitory activity of the JMD on the KIT activity [START_REF] Heinrich | Inhibition of KIT tyrosine kinase activity: a novel molecular approach to the treatment of KIT-positive malignancies[END_REF][START_REF] Laine | Mutation D816V alters the internal structure and dynamics of c-KIT receptor cytoplasmic region: implications for dimerization and activation mechanisms[END_REF] as the tyrosine residues Y568 and Y570 in JMD are phosphorylated and thus their inhibition of KIT kinase is further disrupted [START_REF] Blume-Jensen | Oncogenic kinase signalling[END_REF][START_REF] Laine | Mutation D816V alters the internal structure and dynamics of c-KIT receptor cytoplasmic region: implications for dimerization and activation mechanisms[END_REF]Lennartsson & Ronnstrand 2012). Structural studies of the active form of KIT WT suggest that Y823 of the A-loop is one of the last residues to be autophosphorylated [START_REF] Mol | Structure of a c-kit product complex reveals the basis for kinase transactivation[END_REF]. In addition, the recombinant KD with the D816Y mutation but not the WT KD was found to form dimers in solution [START_REF] Kim | Mechanism of activation of human c-KIT kinase by internal tandem duplications of the juxtamembrane domain and point mutations at aspartic acid 816[END_REF], supporting the idea that extended JMD could make contact with another KIT receptor, thereby promoting dimerization in the absence of ligand. This further data led to the conclusion that the monomeric KIT KD is close to the active conformation but positioned in a way that prevents transactivation. Dimerization alters the conformation and orientation of the kinase domains allowing efficient autophosphorylation. This may explain the differences in substrate specificities of the JMD mutants, which can dimerize like wild-type KIT, and of the kinase domain mutant that may dimerize through a different mechanism. Furthermore, this could also explain the difference in the sensitivity of these two mutants toward imatinib (STI571) [START_REF] Lennartsson | Normal and oncogenic forms of the receptor tyrosine kinase kit[END_REF].

Gain-of-function mutations in KIT and mechanisms for constitutive activation

The oncogenic KIT mutations associated with human neoplasms are gain-of-function mutations that occur most frequently in the exon 11 encoding for the JMD or in the exon 17 encoding for KD. These gain-of-function mutations lead to SCF-independent TK activity and therefore result in ligandindependent proliferation, survival, and differentiation [START_REF] Kitayama | Neoplastic transformation of normal hematopoietic cells by constitutively activating mutations of c-kit receptor tyrosine kinase[END_REF]Piao & Bernstein 1996;[START_REF] Ashman | Effects of mutant c-Kit in early myeloid cells[END_REF]Lennartsson & Ronnstrand 2012). Two gain-of-function mutations V560G and D816V were identified in the JMD and the second part of KD of KIT, respectively in the human MC line HMC-1 (Furistu et al. 1993). Since then, a variety of activating mutations in the JMD have been identified, including deletions, point mutations, tandem duplications, and combinations of deletions and point mutations. These mutations are frequently associated with GISTs [START_REF] Duensing | Biology of gastrointestinal stromal tumors: KIT mutations and beyond[END_REF].

Activating mutations in the second part of the KD have been identified as point mutations at codon 816 of human KIT, which corresponds to codon 814 in murine Kit. Aspartic acid 816/814 is located in the A-loop and can be mutated to tyrosine, histidine, asparagine, or valine with similar consequences (Furistu et al. 1993;[START_REF] Moriyama | Role of aspartic acid 814 in the function and expression of c-kit receptor tyrosine kinase[END_REF]Longely et al. 1996). Activating mutations in the KD of KIT are associated with human malignancies including mastocytosis as well as MC leukemia (MCL) (Nagata et al. 1995;Longely et al. 1996;[START_REF] Boissan | c-Kit and c-kit mutations in mastocytosis and other hematological diseases[END_REF][START_REF] Garcia-Montero | KIT mutation in mast cells and other bone marrow hematopoietic cell lineages in systemic mast cell disorders: a prospective study of the Spanish Network on Mastocytosis (REMA) in a series of 113 patients[END_REF]Brockow & Metcalfe 2010;Bodemer et al. 2010;Verstoovsek 2013), germ cell tumors (D816H) [START_REF] Tian | Activating c-kit gene mutations in human germ cell tumors[END_REF], and less commonly melanoma [START_REF] Tran | A potential role for nilotinib in KIT-mutated melanoma[END_REF] and acute myeloid leukemia (AML) [START_REF] Ashman | Effects of mutant c-Kit in early myeloid cells[END_REF][START_REF] Malaise | Clinical implications of c-Kit mutations in acute myelogenous leukemia[END_REF][START_REF] Ashman | Therapeutic targeting of c-KIT in cancer[END_REF]. Other activating KIT mutations in the exon 8, 9 coding for ECD and exon 11 coding for TMD have been described in GISTs, childhood mastocytosis, and AML [START_REF] Hirota | Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors[END_REF][START_REF] Kohl | KIT exon 8 mutations associated with core-binding factor (CBF)-acute myeloid leukemia (AML) cause hyperactivation of the receptor in response to stem cell factor[END_REF]Bodemer et al. 2010;Lennartsson & Ronnstrand 2012), but their incidence is much lower than that of mutations in exon 17 and 11 (Lennartsson & Ronnstrand 2012). These mutations in the ECD and TM of KIT increase its TK activity by stabilizing the receptor dimers and facilitating its activation [START_REF] Kohl | KIT exon 8 mutations associated with core-binding factor (CBF)-acute myeloid leukemia (AML) cause hyperactivation of the receptor in response to stem cell factor[END_REF][START_REF] Yuzawa | Structural basis for activation of the receptor tyrosine kinase KIT by stem cell factor[END_REF].

The activating mutations of KIT can be classified to either regulatory or enzymatic. The JMD mutations are considered regulatory since they likely change the conformation of KIT and lead to constitutive dimerization and activation by suppressing the auto-inhibitory effect of JMD on KIT activity, whereas mutations of the KD are catalytic and considered of enzymatic type [START_REF] Kitayama | Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines[END_REF][START_REF] Longley | Classes of c-KIT activating mutations: proposed mechanisms of action and implications for disease classification and therapy[END_REF][START_REF] Laine | Mutation D816V alters the internal structure and dynamics of c-KIT receptor cytoplasmic region: implications for dimerization and activation mechanisms[END_REF].

Although both JMD and KD mutations of KIT result in its ligand-independent constitutive phosphorylation, the magnitude of phosphorylation generated by the KD mutants is generally greater than that of the JMD mutants [START_REF] Hirota | Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors[END_REF]. The KD mutant KIT D816V in 293T cells has enhanced enzymatic activity when compared with WT KIT or with the JMD mutant KIT V559G (Furitsu et al. 1993;[START_REF] Lam | A transforming mutation enhances the activity of the c-Kit soluble tyrosine kinase domain[END_REF][START_REF] Chan | Autoinhibition of the kit receptor tyrosine kinase by the cytosolic juxtamembrane region[END_REF]. Moreover, the KD mutant (Kit D814V) exhibits an enhanced affinity for ATP, whereas the JMD deletion mutant and WT mutant bound ATP with similar affinity [START_REF] Lam | A transforming mutation enhances the activity of the c-Kit soluble tyrosine kinase domain[END_REF][START_REF] Chan | Autoinhibition of the kit receptor tyrosine kinase by the cytosolic juxtamembrane region[END_REF]. Interestingly, these types of mutations differ in their sensitivity to inhibitors of KIT WT, including imatinib (STI571) and SU9529. Indeed, JMD mutants are sensitive to these inhibitors, in contrast to the KD mutants [START_REF] Ma | The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory-type mutations[END_REF][START_REF] Frost | Juxtamembrane mutant V560GKit is more sensitive to Imatinib (STI571) compared with wild-type c-kit whereas the kinase domain mutant D816VKit is resistant[END_REF]. However, even some JMD KIT mutants, such as V559I, do not respond to imatinib [START_REF] Ashman | Therapeutic targeting of c-KIT in cancer[END_REF].

In addition, a shift in substrate specificity has been observed with oncogenic KIT mutants. This phenomenon could contribute to differential activation of transforming pathways by mutant forms of KIT. KIT D816V was reported to exhibit a substrate specificity that resembled that of Src and Abelson murine leukemia viral oncogene homolog (ABL) tyrosine kinases. Therefore, this mutant KIT could activate ERK1/2 independently of Src whereas Src activity is essential for ERK1/2 in KIT WT [START_REF] Sun | The D816V mutation of c-Kit circumvents a requirement for Src family kinases in c-Kit signal transduction[END_REF]. In vitro kinase assays using optimal peptide substrates for EGF receptor and ABL kinase showed a shift in substrate specificity with the D814Y mutation. Both WT and KIT D814Y phosphorylated an EGFR peptide, but KIT D814Y phosphorylated an ABL peptide much more efficiently (Piao et al. 1996). A similar shift in specificity was reported with a JMD deletion mutation designated K27 at codons 547-555 compared with wild-type KIT (Casteran et al. 2003).

Furthermore, feline sarcoma oncogene (FES) TK is activated by the D816V mutant and regulates negatively STAT pathway but induces mTOR phosphorylation [START_REF] Voisset | The tyrosine kinase FES is an essential effector of KITD816V proliferation signal[END_REF]. Of note, the mTOR complex (mTORC1 and mTORC2) is upregulated in neoplastic immature MCs [START_REF] Smrz | mTORC1 and mTORC2 differentially regulate homeostasis of neoplastic and non-neoplastic human mast cells[END_REF]. The mTORC1 may contribute to MC survival, whereas mTORC2 plays a crucial role only for homeostasis of neoplastic and dividing immature MCs, thus mTORC2 inhibition via a targeted approach may reduce the proliferation in MC disorders without affecting the normally differentiated MCs [START_REF] Smrz | mTORC1 and mTORC2 differentially regulate homeostasis of neoplastic and non-neoplastic human mast cells[END_REF]). Interestingly, rapamycin, which inhibits mTOR, has been reported to decrease the growth and survival of KIT D816V positive MCs [START_REF] Gabillot-Carré | Rapamycin inhibits growth and survival of D816V-mutated c-kit mast cells[END_REF].

As previously underlined, the signal transduction induced by oncogenic mutants of KIT quantitatively and qualitatively differs from that induced by KIT WT. As an example, p38-MAPK pathways are preferentially activated over ERK-MAPK pathways in JM deletion mutants (Casteran et al. 2003).

Similarly, the mitogenic pathways, such as AKT and STAT pathways are also constitutively activated in JM and D816V mutants [START_REF] Chian | Phosphatidylinositol 3 kinase contributes to the transformation of hematopoietic cells by the D816V c-Kit mutant[END_REF]Ning et al. 2001;Casteran et al. 2003). Mutations in the ECD of KIT such as those found in some pediatric mastocytosis patients result in preferential AKT activation as compared to D816V mutations when introduced into rodent cells (Yang et al. 2010).

We have already described that KIT WT dimerizes and autophosphorylates after stimulation with SCF. JMD gain-of-function mutants form stable dimers in the absence of SCF. Similar to KIT WT, JMD mutants dimerize through the extracellular domain. However, it is unknown whether KIT D816V dimerizes spontaneously with KIT WT, or with itself, or is capable of transmitting oncogenic signals as a single molecule (Bibi et al. 2014). Nevertheless, it is well-known that the KIT D816V oncogenic mutation evokes aberrant signaling pathways of the receptor (Figure 5). Particularly, STAT5 has been reported to be involved in KIT D816V downstream signaling and thus in growth and survival of in neoplastic MCs. Indeed, neoplastic MCs express both cytoplasmic and nuclear phospho-STAT5 and knockdown of STAT5 in KIT mutant cells results in growth inhibition of neoplastic MCs (Harir et al. 2008;Baumgartner et al. 2009). AKT activation is also involved in KIT-dependent differentiation and growth of neoplastic MCs harboring oncogenic KIT mutants (Harir et al. 2008). Indeed, AKT was found phosphorylated in neoplastic MCs from patients with systemic mastocytosis (SM) harboring the KIT D816V mutant [START_REF] Gabillot-Carré | Rapamycin inhibits growth and survival of D816V-mutated c-kit mast cells[END_REF].

Furthermore, the Feline Sarcoma oncogene (FES) TK has been documented to be crucial for negative regulation of STAT pathway and for positive regulation of mTOR phosphorylation downstream of KIT D816V [START_REF] Voisset | The tyrosine kinase FES is an essential effector of KITD816V proliferation signal[END_REF]. By contrast, this TK becomes activated following KIT WT stimulation via SCF but its phosphorylation is transient and results in a different final outcome:

proliferation and survival are not dependent on FES while SCF-induced chemotaxis is [START_REF] Voisset | FES kinase participates in KITligand induced chemotaxis[END_REF]. Interestingly, FES is implicated in regulating the cross-talk between KIT and β1-integrins to promote cytoskeletal reorganization and motility of murine MCs. In addition, SCF-induced chemotaxis of BMMCs was found defective in FES-deficient BMMCs, and restored in FES-rescued BMMCs [START_REF] Smith | Fps/Fes protein-tyrosine kinase regulates mast cell adhesion and migration downstream of Kit and beta1 integrin receptors[END_REF].

1.4. The High affinity receptor for IgE (FcεRI)

Structure and expression of the FcεRI

The high affinity receptor for immunoglobulin E, designated Fc epsilon RI (FcεRI), is a member of multichain immune recognition receptor (MIRR) family responsible for linking pathogen-or allergenspecific IgE with cellular immunologic effector functions. This receptor binds the Fc fragment of IgE.

The domain 3 of the constant region of heavy-chain of IgE is essential for the binding of the ligand to the receptor. In rodent, FcεRI is a tetramer (αβγ2), whereas it exists in trimeric (αγ2) as well as in tetrameric form (αβγ2) in human [START_REF] Kinet | The high-affinity IgE receptor (FcεRI): from physiology to pathology[END_REF]Borkowski et al. 2001;[START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF][START_REF] Blank | The high-affinity immunoglobulin E receptor as pharmacological target[END_REF]. The structures and functions of both tetrameric and trimeric forms of FcεRI are presented in Figure 6. The cellular distribution of human and rodent FcεRI is also different. Rodent FcεRI is only expressed on MCs and basophils (Bas) (and non-B, non-T cells) [START_REF] Kinet | The high-affinity IgE receptor (FcεRI): from physiology to pathology[END_REF], whereas in human the trimeric αγ 2 FcεRI construct lacking the β-chain is expressed on Langerhans cells, eosinophils, platelets as well as on antigen presenting cells such as monocytes, macrophages, and dendritic cells (DCs) [START_REF] Gounni | High-affinity IgE receptor on eosinophils is involved in defence against parasites[END_REF][START_REF] Maurer | Expression of References functional high affinity immunoglobulin E receptors (Fc epsilon RI) on monocytes of atopic individuals[END_REF][START_REF] Maurer | Peripheral blood dendritic cells express Fc epsilon RI as a complex composed of Fc epsilon RI alpha-and Fc epsilon RI gammachains and can use this receptor for IgE-mediated allergen presentation[END_REF][START_REF] Joseph | Expression and functions of the high-affinity IgE receptor on human platelets and megakaryocyte precursors[END_REF][START_REF] Ying | High-affinity immunoglobulin E receptor (Fc epsilon RI)-bearing eosinophils, mast cells, macrophages and Langerhans' cells in allergen-induced late-phase cutaneous reactions in atopic subjects[END_REF][START_REF] Kinet | The high-affinity IgE receptor (FcεRI): from physiology to pathology[END_REF]Borkowski et al. 2001;[START_REF] Saini | How IgE upregulates the allergic response[END_REF][START_REF] Prussin | IgE, mast cells, basophils, and eosinophils[END_REF][START_REF] Knol | Requirements for effective IgE cross-linking on mast cells and basophils[END_REF][START_REF] Rivera | New insights on mast cell activation via the high affinity receptor for IgE[END_REF]. In humans, the tetrameric αβγ 2 FcεRI complex is expressed only on Basophils (Bas) and MCs [START_REF] Nadler | Signal transduction by the high-affinity immunoglobulin E receptor Fc epsilon RI: coupling form to function[END_REF]Borkowski et al. 2001;[START_REF] Blank | The ins and outs of IgE-dependent mast-cell exocytosis[END_REF][START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF][START_REF] Rivera | New insights on mast cell activation via the high affinity receptor for IgE[END_REF][START_REF] Blank | The high-affinity immunoglobulin E receptor as pharmacological target[END_REF]. Indeed, on the MC and Bas surfaces, a mixture of αβγ 2 and αγ 2 complexes is expressed, whereas only αγ 2 structure is expressed on the antigen presenting cells [START_REF] Turner | Signalling through the high-affinity IgE receptor Fc epsilonRI[END_REF]). However, it has been recently reported that under certain conditions, including viral and plasmodium infection, rodent receptors can also be expressed in dendritic cells and neutrophils in the absence of β subunit [START_REF] Grayson | Induction of high-affinity IgE receptor on lung dendritic cells during viral infection leads to mucous cell metaplasia[END_REF][START_REF] Porcherie | Critical role of the neutrophil-associated high-affinity receptor for IgE in the pathogenesis of experimental cerebral malaria[END_REF]. The tetrameric complex includes a single IgE-binding α subunit, a single β subunit, and a disulfide-linked γ dimer whereas the trimeric structure comprises of one α subunit and γ dimer. The α-subunit, which binds IgE [START_REF] Blank | Characterization of truncated alpha chain products from human, rat, and mouse high affinity receptor for immunoglobulin E[END_REF] comprises two extracellular immunoglobulin-like domains (D1 and D2), one transmembrane domain containing an aspartic residue, and a short cytoplasmic tail. The extracellular domain D2 contains two sites binding one of the two IgE heavy chains [START_REF] Garman | Structure of the Fc fragment of human IgE bound to its high-affinity receptor Fc epsilonRI alpha[END_REF]. The αchain is heavily glycosylated since it contains seven N-linked glycosylation sites. These N-linked oligosaccharides are required in the endoplasmic reticulum (ER) to mediate the proper interaction between the α-chain and the ER folding machinery [START_REF] Letourneur | Glycosylation of human truncated Fc epsilon RI alpha chain is necessary for efficient folding in the endoplasmic reticulum[END_REF][START_REF] Kinet | The high-affinity IgE receptor (FcεRI): from physiology to pathology[END_REF]. Thus, the cytoplasmic part of -chain has no role in downstream signaling while the β and γ chains contain unique cytoplasmic domains essential for downstream signaling, called immunoreceptor tyrosine based activation motifs (ITAMs). In fact, the aggregation of IgE-bound FcɛRI induced by specific multivalent antigen (Ag) induces the phosphorylation of tyrosine residues in these motifs, which initiates in turn an intracellular signal transduction cascade and, as a consequence, the release of mediators of the allergic response [START_REF] Benhamou | Tyrosine phosphorylation coupled to IgE receptor-mediated signal transduction and histamine release[END_REF][START_REF] Kinet | The high-affinity IgE receptor (FcεRI): from physiology to pathology[END_REF][START_REF] Knol | Requirements for effective IgE cross-linking on mast cells and basophils[END_REF][START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF]. The sequence of the ITAMs differs between the two chains. The γ-chain ITAM sequence is (Asp/Glu)-X-X-Tyr-X-X-Leu-(X)-Tyr-X-X-Leu/Ile), where X stands for any amino acid. The β-chain has a slightly different sequence with a third tyrosine between the canonical tyrosine residues and the short spacer region. The tyrosine residues represent phosphor-acceptor sites that are important for the course of receptor-associated protein PTKs. The ITAMs further comprise the docking sites for cytoplasmic proteins containing the Scr-homoly 2 (SH2) domain which are the linkage between receptor and the following signal transduction cascade [START_REF] Knol | Requirements for effective IgE cross-linking on mast cells and basophils[END_REF][START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF]. The 33 kDa signal-amplifying β chain consists of 243 amino acids and has four transmembrane domains, two extracellular loops and a single cytoplasmic loop, separating amino and carboxy terminal cytoplasmic tails [START_REF] Kinet | The high-affinity IgE receptor (FcεRI): from physiology to pathology[END_REF][START_REF] Knol | Requirements for effective IgE cross-linking on mast cells and basophils[END_REF]). The β subunit (FcɛRIβ) amplifies the signal strength and enhances the cell surface expression of unoccupied receptors [START_REF] Knol | Requirements for effective IgE cross-linking on mast cells and basophils[END_REF][START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF]Borkowski et al. 2001). Furthermore, the FcɛRIβ functions to amplify responses under a low-intensity stimulus, whereas at a high intensity of stimulus, it might exert a negative regulatory role [START_REF] Xiao | Positive and negative regulation of mast cell activation by Lyn via the FcepsilonRI[END_REF]. The γ-chains have a transmembrane structure consisting of five amino acid extracellular domains, a 21 amino acid transmembrane component and a cytoplasmic part of 36 amino acids. The γ-chains represent the major FcεRI signaling units of the FcεRI [START_REF] Knol | Requirements for effective IgE cross-linking on mast cells and basophils[END_REF][START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF]. Interestingly, both the β-and γ-chains are shared with other Fc receptors. For example, the high and low affinity receptor for IgG (FcγRI and FcγRIII, respectively) and the Fc receptors for IgA (FcαRI) associate with the γ-chain depending on the cellular contex [START_REF] Knol | Requirements for effective IgE cross-linking on mast cells and basophils[END_REF][START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF]).

Regulation of FcεRI expression

Cell surface expression of FcεRI is regulated by the concentration of IgE (Borkowski et al. 2001;[START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF]. A relationship between the concentration of IgE in serum and cell-bound IgE exists and was first observed in human Bas in the PB [START_REF] Malveaux | IgE receptors on human basophils. Relationship to serum IgE concentration[END_REF]Borkowski et al. 2001;[START_REF] Kawakami | Regulation of mast-cell and basophil function and survival by IgE[END_REF][START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF].

IgE monomer binds the FcεRI α-chain with high affinity and this binding strongly upregulates the expression of FcεRI [START_REF] Kawakami | Regulation of mast-cell and basophil function and survival by IgE[END_REF][START_REF] Mirkina | Inhibition of human cord blood-derived mast cell responses by anti-Fc epsilon RI mAb 15/1 versus anti-IgE Omalizumab[END_REF][START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF], by a mechanism which involves stabilization of the receptor at the cell surface by preventing receptor internalization and degradation, by recruiting a preformed pool of receptors comprising recycled and recently synthesized receptors and by stimulating continued basal level of protein synthesis (Borkowski et al. 2001). In addition, low FcεRI expression levels on MCs from IgE-deficient mice could be significantly increased by in vitro incubation with or by in vivo injection of IgE (Yamaguchi et al. 1997;Kawakamai & Galli 2002;[START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF]). Furthermore, it has been described that IgE bound to their receptors will rest permanently at the cell surface whereas empty receptors are expressed only for some hours [START_REF] Macglashan | Endocytosis, recycling, and degradation of unoccupied FcepsilonRI in human basophils[END_REF][START_REF] Macglashan | IgE receptor and signal transduction in mast cells and basophils[END_REF]. Figure 7 shows the regulation of cell surface expression of FcεRI on human MCs and Bas.

Signal transduction downstream FcεRI

Although FcεRI has no intrinsic TK activity, the interaction between the antigen (Ag) and the receptor-bound IgE induces the activation of various signaling pathways [START_REF] Kawakami | Regulation of mast-cell and basophil function and survival by IgE[END_REF][START_REF] Rivera | New insights on mast cell activation via the high affinity receptor for IgE[END_REF]). In the last few years, two pathways of the FcεRI signaling cascade were described and analyzed, a primary pathway and a complementary pathway regulating the FcεRI-mediated degranulation [START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF]. To explain the molecular mechanism of the initial steps of FcεRI signaling, including the phosphorylation of the receptor, three models have been proposed: a transphosphorylation model, a lipid raft model, and a PTK-protein tyrosine phosphatase (PTP) interaction model (Bugajev et al. 2010).

The activation of PTKs such as Src, Syk and Tec family is induced through the crosslinking of the FcεRI by specific IgE and the corresponding antigen, and caused by phosphate transfers [START_REF] Kawakami | Redundant and opposing functions of two tyrosine kinases, Btk and Lyn, in mast cell activation[END_REF][START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF][START_REF] Rivera | New insights on mast cell activation via the high affinity receptor for IgE[END_REF][START_REF] Macglashan | IgE receptor and signal transduction in mast cells and basophils[END_REF]. After the aggregation of the receptor, phosphorylation of the tyrosine residues of the ITAMs of the β and the γ chains occurs. This phosphorylation is often caused through transphosphorylation by Lyn, a nonreceptor Src family PTK which is mainly associated with the β subunit of the receptor [START_REF] Kawakami | Regulation of mast-cell and basophil function and survival by IgE[END_REF][START_REF] Knol | Requirements for effective IgE cross-linking on mast cells and basophils[END_REF][START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF][START_REF] Rivera | New insights on mast cell activation via the high affinity receptor for IgE[END_REF][START_REF] Gilfillan | The tyrosine kinase network regulating mast cell activation[END_REF]. Lyn kinase activity is the key for phosphorylation of the tyrosine residues in ITAM motifs through transphosphorylation [START_REF] Pribluda | Transphosphorylation as the mechanism by which the high-affinity receptor for IgE is phosphorylated upon aggregation[END_REF]. The phosphorylated ITAMs of the β and the γ chain are scaffolds for the binding of the cytoplasmic signaling molecules with SH2 domains like additional Lyn and Syk as well as Fyn (another src family PTK). While Lyn and Fyn are associated with the β chain, Syk is associated with the γ chain [START_REF] Kawakami | Regulation of mast-cell and basophil function and survival by IgE[END_REF][START_REF] Knol | Requirements for effective IgE cross-linking on mast cells and basophils[END_REF][START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF][START_REF] Rivera | New insights on mast cell activation via the high affinity receptor for IgE[END_REF][START_REF] Gilfillan | The tyrosine kinase network regulating mast cell activation[END_REF][START_REF] Alvarez-Errico | Adapters in the organization of mast cell signaling[END_REF]. Fyn is also known to associate with FcɛRI and to play a complementary role by activating PI3-K [START_REF] Parravicini | Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation[END_REF]. However, unlike Lyn, which can be found primarily in lipid rafts, only a small amount of Fyn seems to reside in these domains [START_REF] Kovarova | Cholesterol deficiency in a mouse model of Smith-Lemli-Opitz syndrome reveals increased mast cell responsiveness[END_REF]. Through its interaction with the phosphorylated γ chain, Syk adopts an active confirmation, leading to its phosphorylation by Lyn and subsequently transphosphorylation of other Syk molecules, which amplifies Syk activation and allows downstream phosphorylation of its targets [START_REF] Eiseman | Engagement of the high-affinity IgE receptor activates src protein-related tyrosine kinases[END_REF][START_REF] Hutchcroft | Fc epsilon RI-mediated tyrosine phosphorylation and activation of the 72-kDa protein-tyrosine kinase, PTK72, in RBL-2H3 rat tumor mast cells[END_REF][START_REF] Jouvin | Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor[END_REF][START_REF] Parravicini | Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation[END_REF][START_REF] Kawakami | Regulation of mast-cell and basophil function and survival by IgE[END_REF][START_REF] Siraganian | Mast cell signal transduction from the high-affinity IgE receptor[END_REF][START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF]. Then, active Syk phosphorylates many substrates/proteins such as the adaptor protein linkers for activation of T cells (LAT) (Saitoh et al. 2000), non-T cell activation linker (NTAL) also known as linker for activated B cell (LAB) [START_REF] Zhu | Positive and negative regulation of FcepsilonRI-mediated signaling by the adaptor protein LAB/NTAL[END_REF]), SH2-containing leukocyte-specific protein of 76 kDa (SLP76) (Hendricks-Taylor et al. 1997), Vav (Teramoto et al. 1997), phospholipase C-gamma (PLC-γ: PLC-γ1 and PLC-γ2). In MCs, PLC-γ2 is absolutely required for FcɛRI-mediated calcium mobilization, degranulation, and cytokine production, which cannot be compensated by the concurrently expressed PLC-γ1 [START_REF] Wang | Phospholipase Cgamma2 is essential in the functions of B cell and several Fc receptors[END_REF].

Whatsoever, these events further lead to the activation of several signaling cascades including PI3-K, RAS/ERK, c-Jun N-terminal kinase (JNK), and BTK [START_REF] Kawakami | Regulation of mast-cell and basophil function and survival by IgE[END_REF][START_REF] Siraganian | Mast cell signal transduction from the high-affinity IgE receptor[END_REF][START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF][START_REF] Rivera | New insights on mast cell activation via the high affinity receptor for IgE[END_REF]Kalesnikoff & Galli 2008;[START_REF] Siraganian | Mast cell signaling: the role of protein tyrosine kinase Syk, its activation and screening methods for new pathway participants[END_REF]) (Figure 8).

PLC-γ1 and PLC-γ2 belong to PLC family and catalyze the hydrolysis of plasma membrane phosphatidylinositol 4,5-biphosphate (PIP2) to generate inositol 1,4,5-triphosphate (IP3) and 1,2diacylglycerol (DAG), leading to the activation of protein kinase C (PKC) and the mobilization of intracellular Ca 2+ from the endoplasmic reticulum (ER). The release of Ca 2+ from the ER leads to stromal interaction molecule 1 (STIM1)-mediated opening of the store-operated Ca 2+ channel ORAI1, which leads to the influx of extracellular Ca 2+ . The influx of Ca 2+ is amplified by an additional mechanism which is mediated by short transient potential Ca 2+ channel 1 (TRPC1). Indeed, the depletion of ER Ca 2+ stores results in Ca 2+ influx across the plasma membrane by store-operated calcium release-activated calcium (CRAC) channels in the plasma membrane. ORAI1 is the pore subunit of the CRAC channel while STIM1 is ER Ca 2+ sensor. The depletion of calcium in the ER induces STIM1 formation of multimers in the ER membrane which move to sites of ER plasma membrane apposition and interact directly with ORAI channels which leads to the influx of extracellular Ca 2+ and a calcium release-activated current (ICRAC). Further the increase in intracellular Ca 2+ , with the participation of membrane fusion proteins called SNAREs (soluble Nethyl-maleimide-sensitive factor [NSF] attachment protein receptors), allows granular fusion and release of their content [START_REF] Blank | The ins and outs of IgE-dependent mast-cell exocytosis[END_REF]Kalesnikoff & Galli 2008;[START_REF] Siraganian | Mast cell signaling: the role of protein tyrosine kinase Syk, its activation and screening methods for new pathway participants[END_REF]. The increase in intracellular Ca 2+ levels and the activation of PKC are thus essential triggers of FcεRIregulated degranulation [START_REF] Turner | Signalling through the high-affinity IgE receptor Fc epsilonRI[END_REF][START_REF] Siraganian | Mast cell signal transduction from the high-affinity IgE receptor[END_REF][START_REF] Knol | Requirements for effective IgE cross-linking on mast cells and basophils[END_REF][START_REF] Blank | The ins and outs of IgE-dependent mast-cell exocytosis[END_REF]).

Thus, granules translocate from the cell interior towards the plasma membrane in a microtubuledependent manner. Moreover, Phosphatidylinositol-3,4,5-trisphosphate (PI (3,4,5)P3) is an important lipid mediator that regulates the activity of various enzymes, such as BTK, the serine-threonine kinase AKT, the phosphoinositide-dependent kinase PDK1, phospholipase D (PLD) [START_REF] Hitomi | Phospholipase D1 regulates high-affinity IgE receptor-induced mast cell degranulation[END_REF]) and sphingosine kinase (SK), and the formation of other lipid mediators, such as diacylglycerol (DAG) and sphingosine 1-phosphate (S1P). S1P can act intracellularly to regulate Ca 2+ influx leading to degranulation independently of PLC and IP3 or extracellularly after secretion from the cell by binding to the S1P1 or S1P2 surface receptor, thereby inducing cytoskeletal rearrangement or enhancing degranulation respectively (Kalesnikoff & Galli 2008).

An additional pathway results in the Fyn-dependent tyrosine phosphorylation of the Gab2, which further binds the p85 subunit of PI3-K. This step results in the activation of the phosphoinositidedependent kinase 1 (PDK1) and subsequently in the initiation of the protein kinase Cδ (PKCδ) which further regulates degranulation. Moreover, the activation of PKCδ can cause activation of AKT, resulting in anti-apoptotic effects as well as starting cytokine production [START_REF] Parravicini | Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation[END_REF][START_REF] Siraganian | Mast cell signal transduction from the high-affinity IgE receptor[END_REF][START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF][START_REF] Rivera | New insights on mast cell activation via the high affinity receptor for IgE[END_REF].

The increase in intracellular Ca 2+ concentration also activates the Ca 2+ /calmodulin-dependent serine inhibitor IκB. FcεRI stimulation leads to phosphorylation, thus degradation of IκB, which allows the release and nuclear translocation of the NFκB proteins. Protein kinase Cβ (PKCβ), B cell lymphoma 10 (BCL10), and mucosa-associated-lymphoid-tissue lymphoma-translocation protein 10 (MALT10) are other proteins involved in this pathway [START_REF] Ruland | Differential requirement for Malt1 in T and B cell antigen receptor signaling[END_REF]. Activation of the PI3-K and mitogenactivated protein (MAP) kinases and of the NFAT and NFκB transcription factors then promotes transcription factors for the production of several cytokines. Another substrate of PKC and the MAP kinases is cytoplasmic phospholipase A2 (PLA2) which releases arachidonic acid that is metabolized by the cycloxygenase or the lipoxygenase pathways to form the inflammatory mediators, prostaglandins and leukotrienes.

To sum up, each of the Lyn and the Fyn pathways is crucial for specific signaling events. The Lyn pathway plays an important role in the Ca 2+ signaling, whereas the Fyn pathway is essential for the degranulation and the maintenance and/or the amplification of the Ca 2+ signal. In Fyn-deficient mice, PI3-K and degranulation are affected, but only little effects can be observed on Ca 2+ signaling. By contrast, the Ca 2+ signaling is inhibited and degranulation is enhanced in Lyn deficient mice, suggesting that the Lyn pathway is a negative regulator of the Fyn pathway [START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF]). An important fact is that both the Lyn and the Fyn pathways are dependent on the Syk pathway because the Ca 2+ mobilization, as well as the degranulation, are diminished in Syk-deficient mice. This hypothesis could also be supported in Syk-deficient non-releaser Bas, where through incubation with IL-3 and the presence of Syk mRNA, Syk protein expression and the FcεRI-dependent secretion could be re-established [START_REF] Kepley | Multiple defects in FcεRI signalling in Syk-deficient nonreleaser basophils and IL-3-induced recovery of Syk expression and secretion[END_REF][START_REF] Sanderson | Syk and Lyn mediate distinct Syk phosphorylation events in FcɛRI-signal transduction: implications for regulation of IgE-mediated degranulation[END_REF].

FcεRI aggregation also activates and recruits negative regulators which limit the intensity of the positive signals activated downstream of this receptor (Figure 9). In addition to its role in initiating the activating signals as mentioned above, Lyn also negatively regulates FcεRI-induced signaling events by activating negative regulators [START_REF] Kawakami | Redundant and opposing functions of two tyrosine kinases, Btk and Lyn, in mast cell activation[END_REF][START_REF] Odom | Negative regulation of immunoglobulin E-dependent allergic responses by Lyn kinase[END_REF]Hernandez-Hansen et al. 2004;[START_REF] Xiao | Positive and negative regulation of mast cell activation by Lyn via the FcepsilonRI[END_REF]. Several other negative regulators include for c-Cbl proteins which, by promoting ubiquitination of the activated receptor subunits and associated protein tyrosine kinases, decrease their levels [START_REF] Rao | The non-receptor tyrosine kinase Syk is a target of Cbl-mediated ubiquitylation upon B-cell receptor stimulation[END_REF]Kalesnikoff & Galli 2008;[START_REF] Gasparrini | Cbl family proteins: balancing FcεRImediated mast cell and basophil activation[END_REF], the lipid Intoduction 29 phosphatases including SHIP (Src homology 2 (SH2) domain-containing inositol 5′-phosphatases, SHIP-1 and -2) [START_REF] Huber | The src homology 2containing inositol phosphatase (SHIP) is the gatekeeper of mast cell degranulation[END_REF][START_REF] Kalesnikoff | SHIP negatively regulates IgE + antigen-induced IL-6 production in mast cells by inhibiting NF-kappa B activity[END_REF][START_REF] Kalesnikoff | The role of SHIP in cytokine-induced signaling[END_REF]Hernandez-Hansen et al. 2004;[START_REF] Xiao | Positive and negative regulation of mast cell activation by Lyn via the FcepsilonRI[END_REF][START_REF] Gimborn | SHIP down-regulates FcepsilonR1-induced degranulation at supraoptimal IgE or antigen levels[END_REF][START_REF] Leung | The inositol 5'-phosphatase SHIP-2 negatively regulates IgEinduced mast cell degranulation and cytokine production[END_REF][START_REF] Molfetta | Negative signals from FcepsilonRI engagement attenuate mast cell functions[END_REF]) and PTEN (phosphatase and tensin homologue deleted on chromosome 10) [START_REF] Furumoto | Cutting Edge: Lentiviral short hairpin RNA silencing of PTEN in human mast cells reveals constitutive signals that promote cytokine secretion and cell survival[END_REF][START_REF] Molfetta | Negative signals from FcepsilonRI engagement attenuate mast cell functions[END_REF]) both of which hydrolyze PIP3. Amongst the negative regulators, the following have also been described: the tyrosine phosphatases SHP1 (SH2 domain-containing protein tyrosine phosphatase) that can dephosphorylate Syk [START_REF] Odom | Negative regulation of immunoglobulin E-dependent allergic responses by Lyn kinase[END_REF][START_REF] Molfetta | Negative signals from FcepsilonRI engagement attenuate mast cell functions[END_REF]), the linker for activation of X cells (LAX where X denotes an as-yet-unidentified cell) [START_REF] Zhu | Negative regulation of Fc epsilonRI-mediated signaling and mast cell function by the adaptor protein LAX[END_REF] which binds Grb2 and the p85 subunit of PIK3, the exchange factor RabGEF1 (Rab5 guanine nucleotide exchange factor) which controls FcεRI internalization and can bind to GTP-bound Ras and negatively regulates Ras/Raf1/ERK pathways [START_REF] Tam | RabGEF1 is a negative regulator of mast cell activation and skin inflammation[END_REF][START_REF] Molfetta | Negative signals from FcepsilonRI engagement attenuate mast cell functions[END_REF]Kalesnikoff & Galli 2008). The downstream tyrosine kinase (Dok1) which upon activation binds RasGAP; the exchange factor enhancing the intrinsic GTPase activity of Ras; and thus negatively regulates Ras/Raf1/ERK pathways and TNF- production [START_REF] Abramson | Dok protein family members are involved in signaling mediated by the type 1 Fcepsilon receptor[END_REF][START_REF] Molfetta | Negative signals from FcepsilonRI engagement attenuate mast cell functions[END_REF]Kalesnikoff & Galli 2008), and RGS13 (regulator of G protein signaling (RGS) 13 which binds to the p85α subunit of PI3K and disrupts its association with Gab2 and Grb2 [START_REF] Bansal | Suppression of immunoglobulin Emediated allergic responses by regulator of G protein signaling 13[END_REF]Kalesnikoff & Galli 2008).

Another important fact is that MCs are able to recover and to regranulate after IgE-mediated activation via FcεRI, especially in allergic responses (Dvorak 2005;[START_REF] Xiang | IgE-receptor activation induces survival and Bfl-1 expression in human mast cells but not basophils[END_REF]. Once bound to FcεRI on resting MCs, IgE can persist on the cell surface for long time periods. IgE does not only interact with the FcεRI, but also with the IgG receptors FcγRII and FcγRIII as well as with galectin-3 [START_REF] Kawakami | Regulation of mast-cell and basophil function and survival by IgE[END_REF].

Other negative signals are generated by recruitment of negative signaling molecules such as SHIP or SHP to immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptors which promote dephosphorylation reactions and mechanistically suppress IgE-mediated cell activation [START_REF] Katz | Inhibitory receptors and allergy[END_REF][START_REF] Bruhns | Regulation of allergy by Fc receptors[END_REF][START_REF] Daëron | Immunoreceptor tyrosine-based inhibition motifs: a quest in the past and future[END_REF]. These ITIM-bearing receptors include the low affinity Fc receptor for IgG (FcγRIIB also known as CD32) [START_REF] Ono | Role of the inositol phosphatase SHIP in negative regulation of the immune system by the receptor Fc(gamma)RIIB[END_REF][START_REF] Malbec | Fc epsilon receptor I-associated lyn-dependent phosphorylation of Fc gamma receptor IIB during negative regulation of mast cell activation[END_REF][START_REF] Ott | Downstream of kinase, p62(dok), is a mediator of Fc gamma IIB inhibition of Fc epsilon RI signaling[END_REF][START_REF] Katz | Inhibitory receptors and allergy[END_REF][START_REF] Siraganian | Mast cell signal transduction from the high-affinity IgE receptor[END_REF][START_REF] Tam | RabGEF1 is a negative regulator of mast cell activation and skin inflammation[END_REF]), Platelet endothelial cell adhesion molecule-1 PECAM-1 (also known as CD31) (Wong et al. 2002), mast-cell-function-associated antigen (MAFA) [START_REF] Ortega | Possible interactions between the Fc epsilon receptor and a novel mast cell function-associated antigen[END_REF]Guthmann et al. 1995;Guthmann et al. 1995;[START_REF] Jürgens | Proximity relationships between the type I receptor for Fc epsilon (Fc epsilon RI) and the mast cell function-associated antigen (MAFA) studied by donor photobleaching fluorescence resonance energy transfer microscopy[END_REF][START_REF] Xu | SH2 domain-containing inositol polyphosphate 5'phosphatase is the main mediator of the inhibitory action of the mast cell function-associated antigen[END_REF][START_REF] Abramson | An unusual inhibitory receptor--the mast cell functionassociated antigen (MAFA)[END_REF][START_REF] Abramson | Selective inhibition of the Fc epsilon RI-induced de novo synthesis of mediators by an inhibitory receptor[END_REF], the glycoprotein 49B1 (GP49B1) [START_REF] Katz | Mouse mast cell References gp49B1 contains two immunoreceptor tyrosine-based inhibition motifs and suppresses mast cell activation when coligated with the high-affinity Fc receptor for IgE[END_REF][START_REF] Lu-Kuo | gp49B1 inhibits IgE-initiated mast cell activation through both immunoreceptor tyrosine-based inhibitory motifs, recruitment of src homology 2 domain-containing phosphatase-1, and suppression of early and late calcium mobilization[END_REF][START_REF] Katz | Inhibitory receptors and allergy[END_REF], the leukocyte immunoglobulin-like receptors (LIRs) [START_REF] Bellón | Mutational analysis of immunoreceptor tyrosine-based inhibition motifs of the Ig-like transcript 2 (CD85j) leukocyte receptor[END_REF], the paired Ig-like receptor B (PIR-B) [START_REF] Maeda | Requirement of SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 for paired immunoglobulin -like receptor B (PIR-B)mediated inhibitory signal[END_REF][START_REF] Yamashita | Inhibitory and stimulatory functions of paired Ig-like receptor (PIR) family in RBL-2H3 cells[END_REF][START_REF] Uehara | Inhibition of IgE-mediated mast cell activation by the paired Ig-like receptor PIR-B[END_REF], the signal regulator protein  (SIRP) [START_REF] Liénard | Signal regulatory proteins negatively regulate immunoreceptor-dependent cell activation[END_REF], the myeloid-associated immunoglobulin-like receptor (MAIR-1), also called CD300a [START_REF] Yotsumoto | Paired activating and inhibitory immunoglobulin-like receptors, MAIR-I and MAIR-II, regulate mast cell and macrophage activation[END_REF][START_REF] Kumagai | Identification and characterization of a new pair of immunoglobulin-like receptors LMIR1 and 2 derived from murine bone marrowderived mast cells[END_REF][START_REF] Bachelet | The inhibitory receptor IRp60 (CD300a) is expressed and functional on human mast cells[END_REF], and the allergy inhibitor receptor 1 (Allergin1) [START_REF] Katz | Inhibitory receptors and allergy[END_REF][START_REF] Hitomi | An immunoglobulin-like receptor, Allergin-1, inhibits immunoglobulin E-mediated immediate hypersensitivity reactions[END_REF].

During the activation steps by Lyn, the tyrosine residues in ITIMs are phosphorylated and then recruit inhibitory effectors such as SHIP, SHP-1 or SHP-2. In some but not all cases, the negative signals are generated by Ag-induced co-aggregation of FcεRI with negative receptors. A chimeric fusion protein containing part of the Fc portion of IgG and IgE crosslinks FcεRI to FcγRIIB, and inhibits antigeninduced Syk phosphorylation and the degranulation of human Bas [START_REF] Zhu | A novel human immunoglobulin Fc gamma Fc epsilon bifunctional fusion protein inhibits Fc epsilon RI-mediated degranulation[END_REF]. Finally, ES-62 the glycoprotein secreted by filarial nematodes forms a complex with toll like receptor 4 (TLR4)

(which causes the sequestration and subsequent proteosome-independent degradation of PKCα) to block FcεRI-induced PLD-coupled, SK (sphingosine kinase)-mediated Ca 2+ mobilization and NFκB activation [START_REF] Melendez | Inhibition of Fc epsilon RI-mediated mast cell responses by ES-62, a product of parasitic filarial nematodes[END_REF]) (Figure 9).

As previously described, signaling pathways elicited by KIT share several similar features with FcεRImediated events such as involvement of Src family PTKs, PLC-γ1, PI3-K, calcium mobilization and MAPK-cascade activation (Figure 4). However, activated KIT neither phosphorylates LAT [START_REF] Tkaczyk | NTAL phosphorylation is a pivotal link between the signaling cascades leading to human mast cell degranulation following Kit activation and Fc epsilon RI aggregation[END_REF]) nor recruits or activates Syk. The ability of KIT to potentiate FcεRI-dependent degranulation seems related to the involvement of NTAL and BTK and their capability to regulate PLC-γ1-dependent calcium mobilization and PKC activation (Gilfillan & Beaven 2011). Of note, KIT can phosphorylate tyrosine residues in NTAL, which are different from those phosphorylated by Syk in FcεRI-activated cells [START_REF] Iwaki | Kit-and Fc epsilonRIinduced differential phosphorylation of the transmembrane adaptor molecule NTAL/LAB/LAT2 allows flexibility in its scaffolding function in mast cells[END_REF]. However, PLC-γ activation and calcium mobilization evoked by KIT are slower and of lower magnitude than those elicited by the FcεRI (Hundely et al.

2004

). It is also clear that these signals by themselves cannot induce degranulation. Indeed, SCF by itself is unable to induce degranulation because of its inability to induce a detectable increase in tyrosine phosphorylation of LAT [START_REF] Tkaczyk | NTAL phosphorylation is a pivotal link between the signaling cascades leading to human mast cell degranulation following Kit activation and Fc epsilon RI aggregation[END_REF]) and activation of PKC (Hundely et al. 2004).

Finally, very recent data demonstrate that the G protein-coupled receptors GPCR kinase 2 (GRK2)

modulates FcRI signaling in MCs via at least two mechanisms. One involves GRK2-RH (regulator of G protein signaling homology domain, GRK2-RH) and modulates tyrosine phosphorylation of Syk (spleen tyrosine kinase), and the other is mediated via the phosphorylation of p38 and AKT [START_REF] Subramanian | Regulation of FcεRI signaling in mast cells by G protein-coupled receptor kinase 2 and its RH domain[END_REF]).

Other mast cell receptors

Besides KIT and FcεRI, MCs express several other surface receptors that enable them to perform various functions including recognition of microbes, signaling to other immune cells, interactions with neurons and positive or negative modulation of other surface receptors [START_REF] Pundir | The role of G protein-coupled receptors in mast cell activation by antimicrobial peptides: is there a connection?[END_REF]Gilfillan & Beaven 2011;[START_REF] Sandig | TLR signaling in mast cells: common and unique features[END_REF][START_REF] Suurmond | Toll-like receptor triggering augments activation of human mast cells by anti-citrullinated protein antibodies[END_REF].

Mast cells (MCs) express Toll-like receptors (TLRs) which are capable of binding microbial particles [START_REF] Marshall | Mast-cell responses to pathogens[END_REF]). Indeed, TLRs enable MCs to respond to products of gram-negative and grampositive bacteria. Furthermore, the tissue origin of MCs and/or culture conditions can influence the expression of different TLRs [START_REF] Marshall | Mast cells in innate immunity[END_REF]. The most well-studied TLRs on MCs, TLR2 and TLR4, which have been reported to differentially activate human MCs (Varadaradjalou et al. 2003), and to act synergistically with antigen to enhance cytokine production (Gilfillan & Beaven 2011;[START_REF] Suurmond | Toll-like receptor triggering augments activation of human mast cells by anti-citrullinated protein antibodies[END_REF].

Mast cells (MCs) express also receptors for various cytokines, such as, among others IL-4, IL-9 and IL-10. They express also CD87 (urokinase receptor) and integrins receptors [START_REF] Sperr | Differential expression of cell surface integrins on human mast cells and human basophils[END_REF][START_REF] Sillaber | Molecular and functional characterization of the urokinase receptor on human mast cells[END_REF][START_REF] Agis | Mast cell-lineage versus basophil lineage involvement in myeloproliferative and myelodysplastic syndromes: diagnostic role of cell-immunophenotyping[END_REF]Valent et al. 2001;[START_REF] Ghannadan | Detection of novel CD antigens on the surface of human mast cells and basophils[END_REF]Valent et al. 2010). The expression of activation-linked surface antigen CD88 (C5aR) on MCs depends on the localization (organ) or type of disease [START_REF] Füreder | Differential expression of complement receptors on human basophils and mast cells. Evidence for mast cell heterogeneity and CD88/C5aR expression on skin mast cells[END_REF][START_REF] Agis | Mast cell-lineage versus basophil lineage involvement in myeloproliferative and myelodysplastic syndromes: diagnostic role of cell-immunophenotyping[END_REF]Valent et al. 2010). As an example, normal foreskin MCs express this antigen, whereas normal lung MCs lack it [START_REF] Füreder | Differential expression of complement receptors on human basophils and mast cells. Evidence for mast cell heterogeneity and CD88/C5aR expression on skin mast cells[END_REF][START_REF] Agis | Mast cell-lineage versus basophil lineage involvement in myeloproliferative and myelodysplastic syndromes: diagnostic role of cell-immunophenotyping[END_REF]Valent et al. 2001;[START_REF] Ghannadan | Detection of novel CD antigens on the surface of human mast cells and basophils[END_REF]Valent et al. 2010). Besides, in patients with rheumatoid arthritis, synovial MCs habor CD88, whereas CD88 is not expressed on MCs in osteoarthritis [START_REF] Kiener | Expression of the C5a receptor (CD88) on synovial mast cells in patients with rheumatoid arthritis[END_REF]Valent et al. 2001).

Normal MCs do not express CD25 (IL-2 receptor (IL-2RA)), IL-3 receptor and granulocytemacrophage colony-stimulating factor receptor (GM-CSFR) (Valent et al. 1989;[START_REF] Agis | Mast cell-lineage versus basophil lineage involvement in myeloproliferative and myelodysplastic syndromes: diagnostic role of cell-immunophenotyping[END_REF]Valent et al. 2001;[START_REF] Schernthaner | Detection of differentiation-and activation-linked cell surface antigens on cultured mast cell progenitors[END_REF]Valent et al. 2010). In addition, MCs express a number of activation-related surface antigens such as CD63 and CD203c, and also chemokine receptors, or complement receptors (Bühring et al. 1999, Valent et al. 2001;[START_REF] Ghannadan | Detection of novel CD antigens on the surface of human mast cells and basophils[END_REF][START_REF] Schernthaner | Detection of differentiation-and activation-linked cell surface antigens on cultured mast cell progenitors[END_REF]Valent et al. 2010). However, the expression of some of these receptors depends on the differentiation stage. Thus, dependent on maturation, activation state, disease or localization, MCs are heterogeneous cells (Valent et al. 2001;[START_REF] Ghannadan | Detection of novel CD antigens on the surface of human mast cells and basophils[END_REF]Valent et al. 2010), but they all invariably express the activation-linked surface antigen CD9, but also CD33 (Siglec-3), CD44 (the homing receptor Pgp-1), CD45 (leukocyte common antigen), the membrane cofactor protein CD46, the decay-accelerating factor CD55 and the membrane attack complex inhibitory factor CD59 (Valent et al. 1989;Valent et al.1992;Valent et al. 2001;[START_REF] Ghannadan | Detection of novel CD antigens on the surface of human mast cells and basophils[END_REF]Krauth et al. 2007;Valent et al. 2010;[START_REF] Teodosio | The immunophenotype of mast cells and its utility in the diagnostic work-up of systemic mastocytosis[END_REF].

Finally, apart from the inhibitory FcγRIIB, MCs express other activating high or low affinity receptors for IgG, FcγRI (CD64) and FcγRIII (CD16), which share the same γ subunit dimer with FcεRI, while their α-chains bind IgG. Their expression may vary according to the presence of cytokines, local environment, and species origin of the cells [START_REF] Kraft | New developments in FcepsilonRI regulation, function and inhibition[END_REF][START_REF] Draber | Signal transduction and chemotaxis in mast cells[END_REF].

Mast cell mediators

Activated MCs can release three classes of mediators: preformed (granule-bound) mediators, lipidderived mediators, and cytokines and chemokines. The preformed mediators are stored in the secretory granules in the cytoplasm and are released within minutes after activation [START_REF] Prussin | IgE, mast cells, basophils, and eosinophils[END_REF]Metcalfe 2008;[START_REF] Amin | The role of mast cells in allergic inflammation[END_REF][START_REF] Da Silva | Mast cell function: a new vision of an old cell[END_REF]. These mediators include histamine, the MC exclusively expressed serine proteases including tryptase, chymase, and carboxypeptidase, [START_REF] Metcalfe | Mast cells[END_REF][START_REF] Prussin | IgE, mast cells, basophils, and eosinophils[END_REF].

Especially, histamine has major effects on various cell types in physiologic and pathologic tissues [START_REF] Jutel | Histamine, histamine receptors and their role in immune pathology[END_REF]. Basophils (Bas) are the major source of histamine in the circulation, while MCs are the primary source of histamine in tissues [START_REF] Graham | Mast cells as sources of tissue histamine[END_REF][START_REF] Valentine | The relationship of the basophil to blood histamine in man[END_REF][START_REF] Crivellato | The history of the controversial relationship between mast cells and basophils[END_REF]. Histamine and other released inflammatory mediators have an influence on vascular cells, smooth muscle cells, connective tissue cells, mucous glands and various inflammatory cells.

Histamine plays a special role after local infection, where it causes an increased recruitment of diverse inflammatory cells [START_REF] Amin | The role of mast cells in allergic inflammation[END_REF]. Through its binding on histamine receptors (HRs; H1R and H2R), histamine has effects on endothelial cells, smooth muscles (contraction), nerve endings and mucous secretion [START_REF] Olafsson | Cutaneous and systemic mastocytosis in adults. A clinical, histopathological and immunological evaluation in relation to histamine metabolism[END_REF]. Histamine is metabolized into to N-methyl histamine, methylimidazole acetic acid, and imidazole acetic acid, all metabolites being excreted by the kidneys.

Histamine in secretory granules is found in anionic association with acidic residues of the glycosaminoglycan side chains of heparin and chondroitin sulphate E and dissociates in extracellular fluids by exchanging with sodium ions. The release of histamine stored in the cytoplasmic granules of MCs and Bas is caused by a vesicular transport through cell degranulation induced by various stimuli such as chemical substances, endogenous mediators as well as through immune mechanisms [START_REF] Dvorak | Vesicular transport of histamine in stimulated human basophils[END_REF][START_REF] Jutel | Histamine, histamine receptors and their role in immune pathology[END_REF]Crivellato et al. 2010;[START_REF] Amin | The role of mast cells in allergic inflammation[END_REF].

Through the binding of the receptor FcεRI, MCs undergo a conformational change. First, intracellular degranulation chambers are shaped through granule membrane fusions as well as the solubilisation of the granules before they are secreted. Second, formed granule vesicles travel from the storage granule compartment to the plasma membrane through the cytoplasm, where the granule vesicles fuse with the membrane, leading to the release of the preformed mediators [START_REF] Dvorak | Vesicular transport of histamine in stimulated human basophils[END_REF]Crivellato et al. 2010;[START_REF] Amin | The role of mast cells in allergic inflammation[END_REF]).

Mast cell activators

MCs undergo activation by direct and indirect mechanisms upon the exposure to pathogens (Figure 10). Direct mechanisms include TLR-mediated interactions between MCs and pathogen which frequently do not result in MCs degranulation but lead to cytokine, chemokine and lipid mediator production (Figure 10a). Indirect activation of MCs includes Fc receptor-mediated activation (Figure 10b), and complement receptor-mediated activation (Figure 10c). Fc receptor-mediated activation (activation via crosslinking of the FcεRI) leads to MC degranulation and production of newly generated mediators. Complement receptor-mediated activation of MCs through the receptor for C5a leads to MC degranulation. Complement receptor mediated activation can occur through various receptors for complement components (such as CR3, the receptor for complement component 3b, C3b), and receptors for C3a (C3aR) [START_REF] Marshall | Mast-cell responses to pathogens[END_REF]).

In addition, it is well known that the pattern and the extent of mediators released upon MC degranulation and activation depend on the subtype and localization of the MCs (cytokine milieu) and the type and intensity of the stimulus and triggers [START_REF] Gilfillan | Integrated signalling pathways for mast-cell activation[END_REF]. For example, the FcεRI-induced mediator secretion can be augmented by activation of MCs via the specific SCF receptor and additional activation of Bas through the IL-3 receptor [START_REF] Sperr | Specific activation of human mast cells by the ligand for c-kit: comparison between lung, uterus and heart mast cells[END_REF]; Okayama et al. 1993;Valent 1994;[START_REF] Gebhardt | Cultured human intestinal mast cells express functional IL-3 receptors and respond to IL-3 by enhancing growth and IgE receptor-dependent mediator release[END_REF]Valent et al. 2002). In addition to aggregation of FcεRI, MCs are also activated by C3a and C5a via the C3a receptor and the C5a receptor (CD88) [START_REF] Füreder | Differential expression of complement receptors on human basophils and mast cells. Evidence for mast cell heterogeneity and CD88/C5aR expression on skin mast cells[END_REF]Valent et al. 2001;[START_REF] Prussin | IgE, mast cells, basophils, and eosinophils[END_REF], and by IgG via FcRI [START_REF] Tkaczyk | Activation of human mast cells through the high affinity IgG receptor[END_REF].

MCs might also be activated via TLRs. For instance, TLR3-mediated activation of human MCs induces them to produce type 1 interferons [START_REF] Kulka | Activation of mast cells by doublestranded RNA: evidence for activation through Toll-like receptor 3[END_REF].

Mast cell triggers include IgG, IgE+antigen, corticotropin-releasing hormone (CRH), anaphylatoxins, IL-1, immunoglobulin-free light chains, lipopolysaccharide (LPS), NGF, neurotensin (NT), SCF, substance P (SP), superantigens, urocortin (Ucn), vasoactive intestinal peptide (VIP), viral DNA sequences [START_REF] Amin | The role of mast cells in allergic inflammation[END_REF]. Additionally, MC degranulation occurs after physical stimuli such as heat and laser [START_REF] Zhang | Mast-cell degranulation induced by physical stimuli involves the activation of transient-receptor-potential channel TRPV2[END_REF][START_REF] Hügle | Beyond allergy: the role of mast cells in fibrosis[END_REF]).

Mast cell functions in physiology and in pathological situations

Mast cell function in host defense, innate and acquired immunity

Mast cells play a crucial role in host defense by modulating innate and adaptive immune responses because of their intrinsic capacity to release a wide range of proinflammatory mediators [START_REF] Abraham | Mast cells in infection and immunity[END_REF]Abraham & Arock 1998;Galli et al. 1999;Wedemeyer et al. 2000;[START_REF] Galli | Mast cells and basophils[END_REF][START_REF] Henz | Mast cells as initiators of immunity and host defense[END_REF]Galli et al. 2005). In addition, these cells express various receptors including CD43 (leukosialin), CD80, CD86, and intercellular adhesion molecule receptors (ICAM-1 and ICAM-3) enabling them to interact with B and T lymphocytes. Table III shows the major receptors of MC involved in host defense.

Since MCs are present at strategic locations enabling them to encounter pathogens, they have been well established to play a role in the defence against bacteria, certain parasites (mainly intestinal helminth parasites), and possibly against fungal and viral infections [START_REF] Malaviya | Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha[END_REF][START_REF] Supajatura | Protective roles of mast cells against enterobacterial infection are mediated by Toll-like receptor 4[END_REF][START_REF] Féger | The role of mast cells in host defense and their subversion by bacterial pathogens[END_REF][START_REF] Marshall | Mast-cell responses to pathogens[END_REF][START_REF] Sutherland | Mast cell IL-6 improves survival from Klebsiella pneumonia and sepsis by enhancing neutrophil killing[END_REF][START_REF] Shelburne | Mast cells augment adaptive immunity by orchestrating dendritic cell trafficking through infected tissues[END_REF].

Recognition of pathogens via Toll-like receptors (TLR) has been shown to contribute to MC responses to pathogens [START_REF] Supajatura | Protective roles of mast cells against enterobacterial infection are mediated by Toll-like receptor 4[END_REF].

Binding of microbial compounds to TLRs on MCs leads to release of TNF, IL-1, IL-6, and IL-8 and enhances the migration of effector cells to the infected tissues [START_REF] Marshall | Mast-cell responses to pathogens[END_REF]). Moreover, murine and human MCs have been reported to produce IFNs type I after exposure to double-stranded RNA and/or virus, the former via specific interactions with TLR3. These data suggest that MCs contribute to innate immune responses to viral infection via the production of IFNs type I, which involves NF-κB, p38, and C-Jun NH2-terminal kinase and MAP kinase [START_REF] Kulka | Activation of mast cells by doublestranded RNA: evidence for activation through Toll-like receptor 3[END_REF].

It has been shown that TNF-α released by MCs can control bacterial infection in a murine model of acute septic peritonitis [START_REF] Echtenacher | Critical protective role of mast cells in a model of acute septic peritonitis[END_REF], while nematode infections in mice lead to accumulation of mucosal MCs in the gut with release of MC protease 1 (the protease that is unique to mucosal MCs of mice) [START_REF] Knight | Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1[END_REF]. Beside the effect of MC released TNF-α, CB-derived human MCs can kill and phagocyte various gram-negative and gram-positive bacteria [START_REF] Arock | Phagocytic and tumor necrosis factor alpha response of human mast cells following exposure to gram-negative and gram-positive bacteria[END_REF]).

Thrasher et al. have recently documented the role of mucosal rat MCs in parasite expulsion during the infection with Trichinella spiralis. Rat BM-derived MCs and rat basophilic leukemia cell line (RBL-2H3) were used in that study as models for mucosal MCs and found to be activated without degranulation in response to parasite glycoproteins, demonstrating these models in the contexts of innate and adaptive responses to T. spiralis [START_REF] Thrasher | In vitro modelling of rat mucosal mast cell function in Trichinella spiralis infection[END_REF].

Furthermore, parasite infections are often associated with increased levels of circulating Bas and eosinophils, increased serum levels of IgE (which enhanced expression of FcεRI on Bas and MCs) and increased numbers of MCs and/or Bas in the affected tissues [START_REF] Metcalfe | Mast cells[END_REF]Galli et al. 1999;[START_REF] Galli | Mast cells and basophils[END_REF]Wedemeyer et al. 2000;Williams & Galli 2000).

Regarding the role of MCs in acute viral infections, to date, little is known [START_REF] Abraham | Mast cell-orchestrated immunity to pathogens[END_REF][START_REF] St John | Innate immunity and its regulation by mast cells[END_REF]. However, evidence has recently emerged that MCs significantly influence immunity and pathogenesis during dengue virus (DENV) infection (St John 2013). MCs degranulate within minutes after exposure to DENV, followed by de novo cytokine production in the subsequent hours. Detection of chymase in the serum of DENV patients with acute infection also demonstrates that MC degranulation occurs in vivo during the course of clinically significant infections (St John et al. 2011).The degranulation is though to have a protective role and to help viral clearance (St John 2013). However, MC-induced vascular leakage at a systemic level can contribute to DENV pathogenesis, both during primary and secondary infection (St John 2013).

Mast cell function in allergy, asthma, tissue remodeling and fibrosis

Immediate hypersensitivity occurs within minutes after exposure to allergen, whereas late phase occurs within hours, and in chronic phase symptoms relapse and remit overtime as in asthma (Galli et al. 1999). In the context of allergy, it is well known that MCs are involved in immediate hypersensitivity, late-phase allergic inflammation and chronic inflammation associated with diseases such as asthma.

As mentioned above, during an allergic event, B cell-released IgE binds to the FcεRI on MC surface via their Fc segment. This lets their Fab, antigen-binding segment, free to bind antigens [START_REF] Klein | Degranulation of human mast cells induces an endothelial antigen central to leukocyte adhesion[END_REF][START_REF] Metcalfe | Mast cells[END_REF][START_REF] Church | The human mast cell[END_REF][START_REF] Nakanishi | Basophils are potent antigen-presenting cells that selectively induce Th2 cells[END_REF][START_REF] Amin | The role of mast cells in allergic inflammation[END_REF]. A subsequent exposure to the same allergen crosslinks the MC-bound IgE and thus triggers the release of preformed cytokines, histamine and prostaglandins (Figure 10) (Amin 2005;[START_REF] Nakanishi | Basophils are potent antigen-presenting cells that selectively induce Th2 cells[END_REF][START_REF] Amin | The role of mast cells in allergic inflammation[END_REF].

Since MCs are positioned at the interface of the external environment in the skin and at mucosal surface, they are the central effector cells in the early allergic inflammatory responses. These cells encounter antigens that elicit allergic events [START_REF] Galli | The two faces of the mast cell[END_REF]. Figure 11 shows the interaction between FcεRI associated with IgE and the multivalent allergen, leading to the immediate release of contents of MC secretory granules, including preformed mediators mentioned above such as histamine, tryptase, chymase and proteoglycan which directly influence the surrounding tissue.

Chymase, cathepsine G and carboxypeptidase activate a rebuilding of the connective tissue matrix by metalloproteases. Histamine and heparin increase vascular permeability and trigger smooth muscle contractions. Newly synthesized mediators including prostaglandin D2 (PGD2) (the products of endogenous arachidonic acid metabolism) and leukotrienes such as LTB4, LTC4 and LTE4 are also released and are responsible for mucus hypersecretion and maintenance of inflammatory reactions. Of

Rheumatoid arthritis

Joint inflammation, cartilage erosion particular interest in humans is the production of TNFα, IL-4, IL-5, IL-6, IL-1β and IL-13 [START_REF] Abraham | Mast cells in infection and immunity[END_REF][START_REF] Arock | Phagocytic and tumor necrosis factor alpha response of human mast cells following exposure to gram-negative and gram-positive bacteria[END_REF][START_REF] Holgate | The epithelium takes centre stage in asthma and atopic dermatitis[END_REF][START_REF] Nakanishi | Basophils are potent antigen-presenting cells that selectively induce Th2 cells[END_REF][START_REF] Amin | The role of mast cells in allergic inflammation[END_REF].

The late onset reaction again is initiated by preformed and newly synthesized MC mediators. By secreting cytokines like TNFα, LTB4, IL-4 or histamine, endothelial cells are activated and upregulate expression of adhesion molecules such as P-selectin, E-selectin, vascular cell adhesion molecule (VCAM1) and ICAM-1 on their surface. Leucocytes such as neutrophils, monocytes and CD4+ T cells can bind to these molecules and migrate into the tissues by transmigration [START_REF] Koh | Neutrophils recruited to the lungs of humans by segmental antigen challenge display a reduced chemotactic response to leukotriene B4[END_REF][START_REF] Robinson | Activation of CD4+ T cells, increased TH2-type cytokine mRNA expression, and eosinophil recruitment in bronchoalveolar lavage after allergen inhalation challenge in patients with atopic asthma[END_REF][START_REF] Montefort | Bronchial biopsy evidence for leukocyte infiltration and upregulation of leukocyte-endothelial cell adhesion molecules 6 hours after local allergen challenge of sensitized asthmatic airways[END_REF][START_REF] Amin | The role of mast cells in allergic inflammation[END_REF]). MCs are also involved in late phase hypersensitive responses. In MC deficient mouse model, late and immediate phases of IgE-mediated cutaneous inflammation appear dependent on MCs [START_REF] Galli | Analyzing mast cell development and function using mice carrying mutations at W/c-kit or Sl/MGF (SCF) loci[END_REF]).

In addition, MCs seem to play a crucial role in airway remodeling by releasing tryptase onto smooth muscle and epithelium, and may play a role in skin tissue remodeling by releasing chymase in an IgEdependent manner in allergic diseases (Amin 2005) (Figure 11). Furthermore, Amin showed that MCs are specifically localized within or close to airway smooth muscle bundles in patients with allergic asthma whereas little or no MCs are found in the airways of healthy controls [START_REF] Amin | The role of mast cells in allergic inflammation[END_REF]. These cells also have an effect on epithelial damage, and on basement membrane thickening in patients with allergic asthma (Amin et al. 2000;[START_REF] Oh | Mast cell mediators in airway remodeling[END_REF]; Amin 2012), allergic rhinitis [START_REF] Amin | Inflammatory cell and epithelial characteristics of perennial allergic and nonallergic rhinitis with a symptom history of 1 to 3 years' duration[END_REF]) and middle ear infection in allergy patients (Hurst 1999). Figure 12 depicts the biological effects (labeled in red) of major MC-derived mediators including histamine, heparin, proteases, chymase, interleukins (IL-4, IL-5, IL-8, IL-9, IL-13), LTC4, FGF, PGD2, TGF-β, and TNF-α (labeled in brown) on target cells and tissues (labeled in black) (Arock et al. 2015).

Besides, increasing evidence indicate that MCs are critical for the development of inflammatory diseases, especially in the pathogenesis of arthritis, asthma, chronic dermatitis, and coronary artery disease (CAD). Table IV represents the inflammatory diseases which involve MCs (Theoharides & Cochrane 2004;[START_REF] Theoharides | The critical role of mast cells in allergy and inflammation[END_REF]. However, unlike allergic reactions, MCs rarely degranulate during autoimmune [START_REF] Benoist | Mast cells in autoimmune disease[END_REF] or inflammatory [START_REF] Woolley | The mast cell in inflammatory arthritis[END_REF] processes.

The only explanation for MC involvement in non allergic processes may be through "differential" or "selective" secretion of mediators without degranulation [START_REF] Theoharides | Differential release of serotonin and histamine from mast cells[END_REF][START_REF] Theoharides | Differential release of mast cell mediators and the pathogenesis of inflammation[END_REF].

Finally, Suurmond et al. have more recently documented that the combination of IgE-and TLRmediated activation greatly enhances cytokine production by MCs, but not their degranulation [START_REF] Suurmond | Toll-like receptor triggering augments activation of human mast cells by anti-citrullinated protein antibodies[END_REF]. Whereas MC degranulation is important for acute-phase allergic responses, cytokine production is considered to be important for the more inflammation related, late-phase responses (Naclerio 1997;[START_REF] Larché | Immunological mechanisms of allergen-specific immunotherapy[END_REF]) Therefore, the presence of TLR ligands may specifically enhance the late-phase responses in allergy [START_REF] Suurmond | Toll-like receptor triggering augments activation of human mast cells by anti-citrullinated protein antibodies[END_REF].

Of note, as described above, MCs are involved in the parasitic immune response and intrahepatic schistosomiasis is a well-known cause of liver fibrosis [START_REF] Andrade | Schistosomiasis and liver fibrosis[END_REF]. MCs are necessary for the development of eosinophils, IL-5 expression and liver fibrosis in schistosomiasis-infected animals [START_REF] Sabin | Schistosoma mansoni egg-induced early IL-4 production is dependent upon IL-5 and eosinophils[END_REF]. MCs have been implicated in the pathogenesis of fibrosis in various organs. For example, MCs drive cardiac fibrosis after ischaemic cardiopathy or pressure overloaded heart model [START_REF] Kong | The pathogenesis of cardiac fibrosis[END_REF]. Conversely, MC deficient mice are protected from cardiac fibrosis during heart failure [START_REF] Hara | Evidence for a role of mast cells in the evolution to congestive heart failure[END_REF].

Besides, in renal fibrosis due to IgA nephropathy, increased numbers of MCs are found in the kidney and correlate with fibrosis, whereas under normal circumstances MCs are not found in the kidney [START_REF] Roberts | Mast cells: the forgotten cells of renal fibrosis[END_REF]. The role of MCs, and SCF in tubulointerstitial lesions in IgA nephropathy has been recently reported [START_REF] Liu | Role of mast cells, stem cell factor and proteaseactivated receptor-2 in tubulointerstitial lesions in IgA nephropathy[END_REF][START_REF] Li | Mast cell infiltration is involved in renal interstitial fibrosis in a rat model of protein-overload nephropathy[END_REF]. MCs are also increased in the lung tissue of patients with idiopathic pulmonary fibrosis [START_REF] Tuder | A pathologist's approach to interstitial lung disease[END_REF].

Interestingly, MCs contain profibrotic cytokines such as transforming growth factor-beta (TGF-β), platelet derived growth factor (PDGF) and GM-CSF in their vesicles. MC Chymase cleaves the latent form of transforming growth factor-beta (TGF-β) from cell membrane [START_REF] Taipale | Human mast cell chymase and leukocyte elastase release latent transforming growth factor-beta 1 from the extracellular matrix of cultured human epithelial and endothelial cells[END_REF]. TGF-β is considered as a main driver for collagen production of fibroblasts and their differentiation into myofibroblasts. Expression of MC chymase in human patients with idiopathic pulmonary fibrosis is increased [START_REF] Hirata | Enhanced mast cell chymase expression in human idiopathic interstitial pneumonia[END_REF], the secretion of histamine or the activation of the renin-angiotensin system stimulates the production of collagen by fibroblasts [START_REF] Veerappan | Mast cells: a pivotal role in pulmonary fibrosis[END_REF].

In addition, it has been shown that the number of skin MCs is increased in patients with systemic sclerosis (SSc), which is considered as the prototype of all fibrotic diseases (Hawkin et al. 1985).

However tryptase serum is not elevated in these patients or in other forms of fibrosis. MCs are the main source of TGF-β in the skin of SSc patients. Hügle´s group showed the transgranulation cell-cell contact as a mechanism of fibrosis in SSc patients. In this case, TGF-β present in the MC vesicles is being transferred from MCs to fibroblasts [START_REF] Hügle | Cell-to-cell contact of activated mast cells with fibroblasts and lymphocytes in systemic sclerosis[END_REF].

Finally, IL-4 and GM-CSF stored in MCs participate in the differentiation of PB derived monocytes into functionally active and contractible myofibroblasts [START_REF] Binai | Differentiation potential of CD14+ monocytes into myofibroblasts in patients with systemic sclerosis[END_REF].

Mast cell function in vascular diseases

MCs are involved in vascular injury and atherosclerosis (Kelly et al. 2000). The content of MC granules released upon activation have anticoagulant as well as thrombogenic functions [START_REF] Szczeklik | Serum immunoglobulin E response to myocardial infarction[END_REF][START_REF] Szczeklik | Atopy and sudden cardiac death[END_REF]. Their number increased in the shoulders of atherosclerotic plaques where MCs are associated with plaque rupture culminating in luminal thrombosis [START_REF] Jeziorska | Mast cell distribution, activation, and phenotype in atherosclerotic lesions of human carotid arteries[END_REF].

Increased MC number was also reported by Kovanen et al. in the atheromatous plaque rupture in patients died from acute myocardial infarction [START_REF] Kovanen | Infiltrates of activated mast cells at the site of coronary atheromatous erosion or rupture in myocardial infarction[END_REF]. In addition, MCs have been reported to produce tissue plasminogen activator [START_REF] Sillaber | The mast cell as site of tissue-type plasminogen activator expression and fibrinolysis[END_REF][START_REF] Oh | Mast cell mediators in airway remodeling[END_REF]) and plasminogen activator inhibitor-1 [START_REF] Cho | Production of plasminogen References activator inhibitor-1 by human mast cells and its possible role in asthma[END_REF].

More recently, Bot et al have reported that MCs are effectors in atherosclerosis through the release of various mediators, such as the MC specific proteases chymase and tryptase, growth factors, histamine, and chemokines, which have detrimental effects on immediate surroundings in the vessel wall. This leads to matrix degradation, apoptosis, and enhanced recruitment of inflammatory cells, thereby actively contributing to cardiovascular diseases [START_REF] Bot | Mast cells as effectors in atherosclerosis[END_REF]).

Mast cell involvement in autoimmunity and other diseases

Several investigations have underlined the key role of MCs in coordinating the early phases of autoimmune diseases, especially those involving autoantibodies. This role of MCs was prompted by studies on multiple sclerosis which is an autoimmune demyelinating disease of central nervous system (CNS) mediated by T helper cells (Th1) and/or (Th17), which cross the blood-brain barrier, and on its animal model, experimental allergic encephalomyelitis (EAE) [START_REF] Steinman | Multiple sclerosis: a two-stage disease[END_REF]. Of note, mice lacking MCs were found to develop EAE later and less severely than do the control mice in response to injection of myelin oligodendrocyte glycoprotein. Reconstitution of the MC population in MC lacking mice restored induction of early and severe disease to wild-type levels [START_REF] Secor | Mast cells are essential for early onset and severe disease in a murine model of multiple sclerosis[END_REF][START_REF] Costanza | Mast cells in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis[END_REF].

Furthermore, various studies have reported a correlation between the number/distribution of MCs in the central nervous system (CNS) and the development of multiple sclerosis and EAE [START_REF] Olsson | Mast cells in plaques of multiple sclerosis[END_REF][START_REF] Secor | Mast cells are essential for early onset and severe disease in a murine model of multiple sclerosis[END_REF][START_REF] Brown | Mechanisms underlying mast cell influence on EAE disease course[END_REF][START_REF] Theoharides | Human mast cells stimulate activated T cells: implications for multiple sclerosis[END_REF]). In addition, increased tryptase level was observed in the cerebrospinal fluid of patient with multiple sclerosis [START_REF] Rozniecki | Elevated mast cell tryptase in cerebrospinal fluid of multiple sclerosis patients[END_REF] and the existence of MC related transcripts encoding histamine, proteases, FcεRI and other inflammatory mediators was reported in the same biological fluid [START_REF] Chabas | The influence of the proinflammatory cytokine, osteopontin, on autoimmune demyelinating disease[END_REF][START_REF] Benoist | Mast cells in autoimmune disease[END_REF][START_REF] Lock | Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis[END_REF].

Also, always in multiple sclerosis, brain MCs, which are perivascularly located, are activated by neural factors such as substance P and myelin basic protein and can secrete numerous proinflammatory and vasoactive molecules that may increase the blood-brain barrier permeability, thus facilitating the entry of inflammatory cells to the CNS [START_REF] Theoharides | Human mast cells stimulate activated T cells: implications for multiple sclerosis[END_REF][START_REF] Frenzel | Mast cells and inflammation[END_REF].

Moreover, brain MCs can stimulate activated T cells coming in contact with them, including Th17 at the blood-brain barrier. Astrocytes are also activated after the interaction with MCs via CD40/CD40 ligand complex. The activated astrocytes contribute to MC degranulation through releasing IL-33 and IL-1β [START_REF] Theoharides | Human mast cells stimulate activated T cells: implications for multiple sclerosis[END_REF][START_REF] Carlson | The Th17-ELR+ CXC chemokine pathway is essential for the development of central nervous system autoimmune disease[END_REF][START_REF] Frenzel | Mast cells and inflammation[END_REF].

Besides, MCs are effector cells in rheumatoid arthritis [START_REF] Benoist | Mast cells in autoimmune disease[END_REF][START_REF] Hueber | Mast cells express IL-17A in rheumatoid arthritis synovium[END_REF][START_REF] Merluzzi | Mast cells, basophils and B cell connection network[END_REF][START_REF] Hügle | Beyond allergy: the role of mast cells in fibrosis[END_REF]. Activating IgG receptors on MCs are engaged by IgG autoantibodies [START_REF] Merluzzi | Mast cells, basophils and B cell connection network[END_REF], which may explain the activation of MCs in this autoimmune disease. The proinflammatory cytokine IL-17A stored in MC vesicles as well as preformed MC TNF-α

Primary cutaneous Lymphoma

Poor

High MCs number at tumor periphery is associated with progressive disease

Diffuse large B-cell lymphoma

Good

High MCs count is associated with a favorable disease outcome.

Hodgkin's lymphoma

Poor High number of infiltrating MCs is associated with worse relapse-free survival.

Splenic marginal zone Lymphoma

Poor

MCs interaction with stromal cells impact on disease progression.

Angioimmunoblastic Tcell lymphoma

Poor

Accumulation of MCs helps neoangiogenesis and characterizes angio-immunoblastic T-cell lymphomas.

play a crucial role in rheumatoid arthritis [START_REF] Hueber | Mast cells express IL-17A in rheumatoid arthritis synovium[END_REF][START_REF] Hügle | Beyond allergy: the role of mast cells in fibrosis[END_REF].

MCs are also implicated in Waldenström macroglobulinemia (WM), a distinct B cell lymphoproliferative disorder which is characterized by BM infiltration with lymphoplasmacytic cells (LPCs), along with the presence of an IgM monoclonal gammopathy [START_REF] Ho | CD27-CD70 interactions in the pathogenesis of Waldenstrom macroglobulinemia[END_REF]. Bone marrow MCs provide important growth and survival signals to WM LPCs through multiple TNF-family ligands, including CD40L (CD154), a proliferation-inducing ligand (APRIL), and a B-lymphocyte stimulator factor (BLYS) [START_REF] Tournilhac | Mast cells in Waldenstrom's macroglobulinemia support lymphoplasmacytic cell growth through CD154/CD40 signaling[END_REF].

Additionaly, The role of MCs and MC-derived mediators has been evaluated in autoimmune glomerulonephritis [START_REF] Monteiro | The function of References mast cells in autoimmune glomerulonephritis[END_REF]. mMCP-4 chymase, contrary to the global antiinflammatory action of MCs, has been reported to aggravate the glomerulonephritis by promoting kidney inflammation [START_REF] Scandiuzzi | Mouse mast cell protease-4 deteriorates renal function by contributing to inflammation and fibrosis in immune complex-mediated glomerulonephritis[END_REF].

Mast cell involvement in cancer

MCs are also linked to tumor invasion [START_REF] Gounaris | Mast cells are an essential hematopoietic component for polyp development[END_REF]. Indeed, MC infiltration has been reported in several types of human tumors and in animal cancer models. Furthermore, the wide diversity in type, grade and stage of human tumors, as well as the poorly characterized distribution of MCs in tumors, present a paradigmatic situation and do not allow the unambiguous classification of MC function as pro-or anti-tumorigenic elements. Table V represents through cell-cell contact (OX40/OX40ligand croostalk between MCs and Treg cells) or by the production of soluble factors [START_REF] Gri | CD4+CD25+ regulatory T cells suppress mast cell degranulation and allergic responses through OX40-OX40L interaction[END_REF][START_REF] Piconese | Mast cells counteract regulatory T-cell suppression through interleukin-6 and OX40/OX40L axis toward Th17-cell differentiation[END_REF][START_REF] Gri | Mast cell: an emerging partner in immune interaction[END_REF]).

In addition, MCs were recently found to boost MDSC activity and contribute to the development of tumor-favoring microenvironment (Danelli et al. 2015). Figure 13 summarizes the complex crosstalk between MCs and other immune cells in tumor microenvironment.

Human mast cell lines

Isolation of tissue MCs is a costly and difficult procedure, with a poor yield. To circumvent such drawbacks, MC lines are therefore used. However, there are only limited numbers of human MC lines facilitating the in vitro study of human MC biology. Indeed, before the isolation of the ROSA cell lines in our laboratory, only 4 human MC lines were described, namely HMC-1, LAD, LUVA, and MCPV-1 cell lines.

The human mast cell line HMC-1 and its two subclones (HMC-1.1 and HMC-1.2)

The human MC line (HMC-1) was derived from the PB of a patient with a MCL (Butterfield et al. 1988). HMC-1 cells are immature cells.which stain metachromatically by toluidine blue, lack T and B lymphocyte as well as myeloid cell markers, and do not express IgE receptors. Figure 14 shows May-Grünwald Giemsa staining (MGG) of HMC-1 cells. In addition, they contain low levels of histamine, and chloroacetate esterase, aminocaproate esterase and tryptase activities (Butterfield et al. 1988).

In HMC-1 cells, KIT receptor was found to be constitutively phosphorylated on tyrosine residues, activated, and associated with PI3-K in the absence of SCF. Sequencing of whole coding region of KIT cDNA revealed that KIT genes of HMC-1 cells are composed of a normal WT allele and a mutant allele with two activating point mutations resulting in intracellular amino acid substitutions of G560 for V and V816 for D (Furitsu 1993;[START_REF] Kanakura | Activating mutations of the c-kit proto-oncogene in a human mast cell leukemia cell line[END_REF]. These mutations are responsible of SCFindependent constitutive phosphorylation of KIT (Furitsu 1993). Although the HMC-1 cell line had been widely employed for in vitro studies of human MC biology (Butterfield et al. 1988;Akin et al. 2003), being growth factor independent, the cells lack surface FcεRI expression (Nilsson et al. 1994;[START_REF] Guhl | Mast cell lines HMC-1 and LAD2 in comparison with mature human skin mast cells--drastically reduced levels of tryptase and chymase in mast cell lines[END_REF], which limits their utility, especially in examining allergic mechanisms.

Since HMC-1 cells show immature characteristics of MCs and their relevance to normal MC function is questionable, Weber et al. have described and generated a subclone (5C6) of HMC-1 cells, by limiting dilution method [START_REF] Weber | A subclone (5C6) of the human mast cell line HMC-1 represents a more differentiated phenotype than the original cell line[END_REF]. This clone has been published to represent a more mature MC than the original HMC-1 cell line, expressing MC markers more strongly than the original cells, as well as FcεRI. However, this subclone shows several limitations, and at least in our hands, is unable to degranulate after cross-linking of FcεRI.

More recently, two subclones of HMC-1, namely HMC-1(560) or HMC-1.1 and HMC-1(560,816) or HMC-1.2, were documented with different phenotypes and designated by the locations of specific mutations in the KIT protooncogene. Both sublines have a heterozygous T to G mutation at codon 560 in the JMD of the KIT gene causing an amino acid substitution of G560 for V. In contrast, only HMC-1.2 cells have the KIT D816V mutation causing a substitution of V for D in the intracellular KD [START_REF] Sundström | Functional and phenotypic studies of two variants of a human mast cell line with a distinct set of mutations in the c-kit proto-oncogene[END_REF]. HMC-1.1 cells are heterogeneous in size and exhibited some homotypic aggregation. They are also more adherent than HMC-1.2 cells, which grew in single cell suspension.

HMC-1.2 cells are smaller in size and tend to be more homogeneous. Finally, the presence of KIT D816V mutation seems to confer an advantage to the cells in terms of proliferation rate [START_REF] Sundström | Functional and phenotypic studies of two variants of a human mast cell line with a distinct set of mutations in the c-kit proto-oncogene[END_REF].

HMC-1.1 cells are sensitive to imatinib, while HMC-1.2 cells are resistant to imatinib but respond to dastatinib (Akin et al. 2003;[START_REF] Zermati | Effect of tyrosine kinase inhibitor STI571 on the kinase activity of wild-type and various mutated c-kit receptors found in mast cell neoplasms[END_REF][START_REF] Schittenhelm | Dasatinib (BMS-354825), a dual SRC/ABL kinase inhibitor, inhibits the kinase activity of wild-type, juxtamembrane, and activation loop mutant KIT isoforms associated with human malignancies[END_REF]. Besides, inhibition of kinase activity in KIT KD mutations was found to restore cell surface expression of the mutant receptor. As an instance, the KIT D816V in HMC-1.2 was found to relocalize to cell surface after dasatinib treatment [START_REF] Bougherara | Relocalization of KIT D816V to cell surface after dasatinib treatment: potential clinical implications[END_REF].

LYN and BTK are phosphorylated and activated in both HMC-1.1 and HMC-1.2 cell lines in a KITindependent manner as well as in neoplastic MCs in advanced SM. SiRNAs against LYN and BTK block the survival of HMC-1.1 and HMC-1.2 cell lines. Dasatinib was found to block also LYN and BTK activation in neoplastic MCs while bosutinib inhibits LYN and BTK activation without decreasing KIT kinase activity (Gleixner et al. 2011).

Moreover, the activation of PKC was found to have different effects in HMC-1.1 and HMC-1.2 cell lines as it translocates to nucleus only in HMC-1.2 cells upon stimulation with phorbol myristate acetate (PMA) and this may be related to the presence of the KIT D816V activating mutation in this cell line [START_REF] Tobío | C-kit mutations and PKC crosstalks: PKC translocates to nucleous only in cells HMC 560.816[END_REF].

STAT3 and STAT5 have been shown to be critical for cell proliferation elicited by KIT D816V in cell lines as well as in MCs from patients with systemic mastocytosis (SM) (Ning et al. 2001;Harir et al. 2008;Baumgartner et al. 2009). Chaix et al. have recently clarified the pathway linking KIT receptor to STAT activation and reported that STAT1, STAT3 and STAT5 are activated downstream molecules of KIT D816V mutant. Indeed, KIT D816V mutant can directly phosphorylate STAT1, 3 and 5 in vitro at tyrosine residues Y701, Y704, and Y694 respectively. Interestingly, STAT5 is transcriptionally active in the HMC-1 cell line, whereas the transcriptional activity of both STAT1 and STAT3 is absent. In addition, JAK3, which is activated downstream of KIT, has been involved in the phosphorylation of STAT proteins in HMC-1 cells (Chaix et al. 2011).

The lack of FcεRI in HMC-1 cells has led many researchers to use less physiological stimuli, such as calcium ionophores and phorbol esters to examine MC activation using these cell lines. Results of such studies are undoubtedly useful but provide limited insights into MC activation in allergic inflammation. For that, Xia et al. have recently generated and characterized a HMC-1 cell line which has been stably transfected with the human FcεRIα subunit, HMCα [START_REF] Xia | Human mast cell line-1 (HMC-1) cells transfected with FcεRIα are sensitive to IgE/antigen-mediated stimulation demonstrating selectivity towards cytokine production[END_REF]. HMCα cells express FcεRI that is functional and capable of binding human IgE. Incubation of these cells with human IgE over a period of 2-3 days leads to a stabilization of FcεRI on the cell surface [START_REF] Xia | Human mast cell line-1 (HMC-1) cells transfected with FcεRIα are sensitive to IgE/antigen-mediated stimulation demonstrating selectivity towards cytokine production[END_REF]).

LAD cell lines

LAD cell lines were developed in 2003 from a 44-year-old male with MC sarcoma/leukemia. Indeed, the BM-derived MNCs from this patient were cultured in serum free media (SFM) with SCF, with or without IL-6 and IL-3 (during the first week), resulting in generation of five cell lines (LAD1-5) that were maintained in SFM and SCF. LAD cells (both LAD-1 and LAD-2) present with many well- known MC markers and content histamine, tryptase and chymase. Indeed, 98% of LAD 2 cells are positive for tryptase and approximately 37% are positive for both tryptase and chymase. LAD 1 and 2 cells express surface FcεRI, KIT, and other markers including CD4, CD9, CD13, CD22, CD45, CD64, CD71, CD103, CD132, CD184 and CD195, and to a lesser degree, CD14, CD31 and CD32, whereas BM-derived MCs from the patient stained positive for CD13, KIT, but were negative for CD2, CD25, CD34 and CD35 (Kirshenbaum et al. 2003).

Compared to HMC-1 cells and its sublines, LAD 1 and 2 cells exhibit a more mature phenotype and closely resemble primary cultures of CD34+-derived human MCs, respond to rhSCF, and have functional FcεRI and FcγRI receptors (Kirshenbaum et al. 2003). Figure 15 shows toluidine blue staining of LAD2 cells. They can be stimulated to degranulate in an IgE-dependent manner [START_REF] Gage | Proteomic analysis of IgEmediated secretion by LAD2 mast cells[END_REF]). However, one major drawback of this cell line is its doubling time. Indeed, these cells double in numbers in approximately 3 weeks in the presence of 100 ng/ml of SCF. In addition, the cell culture only growths at high density. Thus, cell density should be maintained between 0.5×10 6 and 1×10 6 cells/ml and the cells should be splitted every 2-3 weeks. The addition of rhIL-3, rhIL-5, or rhIL-6 did not influence LAD cell numbers, while rhIL-4 reduced cell numbers. While HMC-1.2 cells harbor the KIT D816V activating mutation, LAD-1 and -2 cells do not exhibit KIT activating mutations, which may explain the requirement for SCF for the proliferation of these cells (Kirshenbaum et al. 2003).

LAD-2 cells may thus facilitate research in human MC survival and proliferation studies, as well as in MC activation through FcεRI and FcγRI, and have been distributed worldwide [START_REF] Kirshenbaum | A ten-year retrospective analysis of the distribution, use and phenotypic characteristics of the LAD2 human mast cell line[END_REF]). However, their slow doubling time, unstable phenotype in culture, and dependency on exogenous SCF for survival and proliferation, in addition to the difficulty to freeze and recover these cells, still limit their usefulness for particular applications such as high throughput screening of drugs.

LUVA cell line

The LUVA cell line is an immortalized human MC line grown from CD34+ enriched mononuclear cells (MNCs) derived from the PB of a donor with aspirin exacerbated respiratory disease, but with no clinical symptoms of mastocytosis, MC disorder or leukemia (Laidlaw et al. 2011). These cells proliferate without further addition of any cytokines or growth factors and they have no mutation in the KIT gene. They do not exhibit autophosphorylation of KIT and respond to rhSCF. In LUVA cells, KIT signaling pathway is functional, but not altered in terms of activity when compared to other MC lines (Laidlaw et al. 2011).

The LUVA cells have been published to resemble mature human MCs functionally and morphologically, to possess metachromatic cytoplasmic granules that are immunoreactive for tryptase, cathepsin G, and carboxypeptidase A3, to express surface FcεRI and to respond to IgE receptor crosslinkage leading to the release of β-hexosaminidase, PGD2, thromboxane A 2 and macrophage inflammatory protein 1-β (Laidlaw et al. 2011). However, it has to be noticed that LUVA cells are behaving differently in long-term culture as compared to their description in the original publication.

Indeed, in this publication, LUVA cells were presented as having high levels of expression of FcεRI, however, we and others have observed that these cells do not express surface FcεRI despite the very high mRNA expression for FcεRI. The reason for this discrepancy is unclear, but it is possible that this cell line tends to lose the expression of FcεRI upon long-term culture Moreover, LUVA cells in our hands seem to be immature cells in term of granulation as no granules can be seen after MGG staining (Figure 16).

MCPV-1 cell lines

The human MCPV-1 (mast cell progenitor Vienna (1-4) cell lines were obtained in an attempt by Hoermann et al. to generate a cell line reflecting the biology of RAS-mutated advanced SM [START_REF] Hoermann | CD52 is a molecular target in advanced systemic mastocytosis[END_REF]. Cord blood-derived MC progenitors were cultured in RPMI 1640 medium supplemented with 10% FCS, human SCF (100 ng/mL), and human IL-6 (100 ng/mL) for 8 weeks prior to lentiviral transduction with HRAS G12V, SV40 TAg, and TERT [START_REF] Hahn | Creation of human tumour cells with defined genetic elements[END_REF]. The human MCPV-1 was harvested and cloned by limiting dilution, generating four different clones, designated MCPV-1 (1)(2)(3)(4). Light microscopy of Wright-Giemsa-staining of MCPV-1 cells showed large, immature cells with bi-, tri-, or multilobated (often cloverleaf-like shaped) nuclei characteristic of MC precursors (Figure 17). The cells have abundant basophilic cytoplasm, cytoplasmic protrusions, and, in part, sparse granulations. Moreover, MCPV-1 cells exhibit an immunophenotype consistent with MC progenitors; they are positive for CD31, CD32, CD44, CD48, CD50, CD54, CD58, CD59, CD63, CD69, CD71, CD87, CD117, CD166 and CD203c. These cells are also positive for tryptase, and histidine decarboxylase (HDC) [START_REF] Hoermann | CD52 is a molecular target in advanced systemic mastocytosis[END_REF].

Interestingly, MCPV-1 cell line expresses the surface marker CD52, which has been recently identified as a molecular target in advanced forms of SM. This expression of CD52 in MCPV-1 cells appears to be driven through the activation of RAS and introduction of RAS mutant in the HMC-1 cells originally negative for CD52 leads to expression of CD52 on the membrane.

In addition, MAP kinase signaling is required for RAS-dependent expression of CD52 and MEK inhibitors were found to decrease such expression [START_REF] Hoermann | CD52 is a molecular target in advanced systemic mastocytosis[END_REF]. Of note, in contrast to normal MCs, neoplastic MCs in patients with SM appear to consistently express the CD52 protein, as determined by immunohistochemistry (IHC). However, substantial surface expression of CD52 was detected exclusively in patients with advanced SM [START_REF] Hoermann | CD52 is a molecular target in advanced systemic mastocytosis[END_REF]. A significant difference in CD52 mRNA and protein levels has also been recently documented between neoplastic MCs purified from patients with ISM and ASM [START_REF] Teodosio | Gene expression profile of highly purified bone marrow mast cells in systemic mastocytosis[END_REF]. Moreover, alemtuzumab efficiently mediates lysis of CD52-positive neoplastic MCs in vitro (MCPV-1 cells) and in vivo in NSG mice injected intravenously (IV) with MCPV-1 cells [START_REF] Hoermann | CD52 is a molecular target in advanced systemic mastocytosis[END_REF]).

Mastocytosis

Definition, epidemiology and diagnosis

Mastocytosis is a term used to denote a heterogeneous group of disorders characterized by the pathogenic and uncontrolled accumulation of MCs in one or more different tissues and organs including BM, skin, liver, spleen, lymph nodes and gastrointestinal (GI) tract [START_REF] Brockow | Mastocytosis[END_REF]Escribano et al. 2002;Valent et al. 2003;Horny et al. 2007;Metcalfe 2008;Amon et al. 2010;Sokol et al. 2013). Mastocytosis is considered to be a rare disease amongst BM-derived hematologic neoplasms with an estimated prevalence of about is 0.005-0.01% in Middle Europe or 1 per 10000 inhabitants [START_REF] Van Doormaal | Prevalence of indolent systemic mastocytosis in a Dutch region[END_REF][START_REF] Valent | Mastocytosis: a paradigmatic example of a rare disease with complex biology and pathology[END_REF][START_REF] Brockow | Epidemiology, prognosis, and risk factors in mastocytosis[END_REF]. No apparent gender predominance has been reported and very few familial cases have been described. The clinical presentations of mastocytosis is heterogeneous, ranging from skin-limited disease (mainly Urticaria pigmentosa; UP) which occurs mainly in childhood and spontaneously regresses (Kettelhut & Metcalfe 1997;Brilley & Phillips 2008) to more aggressive subtype with extracutaneous manifestations which is generally seen in adults and may be associated with multiorgan failure and shortened survival (Lim et al. 2009;[START_REF] Sperr | Diagnosis, progression patterns and prognostication in mastocytosis[END_REF]Sokol et al. 2013). Indeed, in childhood, the disease remains confined to the skin [START_REF] Metcalfe | The treatment of mastocytosis: an overview[END_REF] and often occurs before the age of 2 years [START_REF] Méni | Paediatric mastocytosis: a systematic review of 1747 cases[END_REF], whereas in adulthood, almost all patients with UP-like skin lesions have systemic mastocytosis (SM), usually indolent SM (ISM) [START_REF] Berezowska | Adult-onset mastocytosis in the skin is highly suggestive of systemic mastocytosis[END_REF].

Patients with SM often show an elevated serum tryptase level (>20 ng/ml). The BM is almost always involved (Escribano et al. 1998;Honry et al. 2001;Valent et al. 2003;Akin & Metcalfe 2004;Honry et al. 2007;Arock & Valent 2010). However, the skin is often affected in SM [START_REF] Pardanani | Systemic mastocytosis: disease overview, pathogenesis, and treatment[END_REF].

Neoplastic MCs in SM typically carry activating point mutations in KIT, usually at codon 816 (Nagata et al. 1995;Longley et al. 1996;[START_REF] Longley | Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis[END_REF]Fritsche-polanz et al. 2001;[START_REF] Féger | The role of mast cells in host defense and their subversion by bacterial pathogens[END_REF],

have an abnormal morphology with spindle-shaped appearance and are hypogranulated, show an aberrant expression of CD2 and/or CD25 and form compact infiltrates in extracutaneous tissues. By contrast, in cutaneous disease found in children, a mutation of codon 816 (exon 17) is found in 42% of cases, and mutations outside exon 17 are observed in 44% of the cases, half of these mutations being located in the fifth Ig loop of KIT's extracellular domain encoded by exons 8 and 9 (Bodemer et al. 2010). Interestingly, in adults, SM is sometimes (5 to 20% of the cases, depending on the studies) associated with another hematological non MC diseases (SM-AHNMD) (Arock et al. 2015).

The incidence of the prognostically unfavorable subtypes of SM including the aggressive SM (ASM)

and mast cell leukemia (MCL) is low compared to that of indolent forms of SM (ISM). ASM and MCL usually present without skin lesions but often mimic more common hematologic neoplasms like (Valent et al. 2001;Valent et al. 2001;Horny et al. 2008).

The WHO criteria to diagnose SM

Major Multifocal dense infiltrates of MCs (≥15 MCs in aggregates) in BM and/or other extracutaneous organs, confirmed by special stains such as MC tryptase.

Minor

In (Valent et al. 2001;Valent et al. 2003;Arock et al. 2015).

"B" findings (borderline-benign)

1. BM biopsy showing >30% of MCs infiltration and/or serum tryptase level exceeds 200 ng/mL 2. Signs of dysplasia or myeloproliferation, in non-MC lineage(s), but insufficient criteria for definitive diagnosis of a hematopoietic neoplasm (AHNMD), with normal or slightly abnormal blood counts.

3. Hepatomegaly without impairment of liver function, and/or palpable splenomegaly without hypersplenism, and/or lymphadenopathy on palpation or imaging.

"C" findings (consider cytoreduction)

1. BM dysfunction manifested by one or more cytopenia(s) (absolute neutrophil count ANC<10 9 /L, hemoglobin Hgb<10 g/dL, or platelets<100 x 10 9 /dL), but no obvious non-MC hematopoietic The diagnosis of mastocytosis may be challenging to establish, especially for those patients who present with features of myeloid or lymphatic tumors and do not have cutaneous involvement [START_REF] Parker | Hematologic aspects of mastocytosis: II: Management of hematologic disorders in association with systemic mast cell disease[END_REF]Valent et al. 2007). For each patient with suspected mastocytosis, a clinical workup should be performed including information about allergic reactions and skin rashes, and serum typtase level. In patients with a rash and elevated serum typtase level, histologic investigation of the BM must be performed [START_REF] Valent | Mastocytosis: a paradigmatic example of a rare disease with complex biology and pathology[END_REF]. Precise defining criteria to diagnose SM have been published by the World Health Organization (WHO) (Valent et al. 2001;Horny et al. 2008). These WHO criteria to diagnose SM are presented in table VI (Valent et al. 2001;Valent et al. 2001;Horny et al. 2008). The diagnosis of SM is established when one major criterion and one minor criterion or at least three minor criteria are present. Once the diagnosis is established, patients with SM are categorized further according to the presence of B "Borderline Benign" findings reflecting the disease burden and C "Consider Cytoreduction" findings reflecting the disease aggressiveness (Table VII) (Valent et al. 2001;Valent et al. 2003;Arock et al. 2015). Patients who have SM without B-or C-findings and without signs of an AHNMD are diagnosed as indolent SM (ISM). Patients with two or more Bfindings, but no C-findings are diagnosed with smoldering SM (SSM). Finally, patients who have one or more C-findings are candidates for therapeutic cytoreduction and are considered as aggressive SM (ASM) or MC leukemia (MCL) [START_REF] Valent | Mastocytosis: a paradigmatic example of a rare disease with complex biology and pathology[END_REF]Arock et al. 2015). MCL is excluded in patients with ASM when the MCs in BM smears represent less than 20% of total nucleated cells, whereas patients with at least 20% of MCs in BM are diagnosed with MCL [START_REF] Valent | Mastocytosis: a paradigmatic example of a rare disease with complex biology and pathology[END_REF].

KIT mutations in exon 17 at codon 816 can be detected in PB, BM aspirate, or other lesional tissue samples, and the presence of KIT D816V is one of minor criteria for the diagnosis of SM. This most common mutation arising from substitution of valine for aspartate is detected in neoplastic MCs of more than 95% of adult patients with SM and in 42% of pediatric patients with cutaneous mastocytosis (CM) (Longley et al. 1996;[START_REF] Longley | Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis[END_REF][START_REF] Orfao | Recent advances in the understanding of mastocytosis: the role of KIT mutations[END_REF]Bodemer et al. 2010;[START_REF] Cardet | Mastocytosis: update on pharmacotherapy and future directions[END_REF]. Because this mutation results in a conformational change in the KD of KIT that renders it resistant to imatinib, the TK inhibitor usually effective in GI stromal tumors (GISTs) and chronic

Variant (Abbreviation

Extracutaneous mastocytoma (ECM)

Rare benign tumor Normal mature cells Good myeloid leukemia (CML), most patients with SM do not respond to imatinib therapy [START_REF] Ma | The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory-type mutations[END_REF][START_REF] Zermati | Effect of tyrosine kinase inhibitor STI571 on the kinase activity of wild-type and various mutated c-kit receptors found in mast cell neoplasms[END_REF]Akin et al. 2003).

Classification of mastocytosis

The pathological accumulation of MCs in mastocytosis and the release of their mediators are responsible for the clinical presentations and symptoms including pruritus, flushing, nausea, diarrhea and vascular instability. In addition, inSM severe symptoms like osteoporosis, fractures, and even organ failure can occur. This complex pattern of symptoms and their severity lead to a classification listed in 2008 by the World Health Organization (WHO) and subsequent prognosis for each case of mastocytosis (Horny et al. 2007;Horny et al. 2008;Arock & Valent 2010) (Table VIII).

Cutaneous Mastocytosis (CM)

MC infiltrates in CM are restricted to the skin and variants of CM are defined by their clinical presentation. Patients with CM show a wide variety of skin alterations (Rueff et al. 2006). Even though all skin lesions in CM are due to an abnormally elevated MC number in the dermis, the number and morphology of efflorescences may vary from patients to patient [START_REF] Longley | The mast cell and mast cell disease[END_REF].

In addition, while there is no systemic infiltration and the disease is limited to the skin, it can be associated with systemic symptoms due to the release of MC mediators (Bodemer et al. 2010).

The most common subvariant of CM is termed UP and presents as disseminated macular or maculopapular rash. UP is characterized by symmetrically distributed 0.5 cm red-brown macules and papules, which induce erythema, wheal and pruritus, the so-called Darrier's sign, when rubbed or scratched [START_REF] Wolff | Clinical and histopathological aspects of cutaneous mastocytosis[END_REF][START_REF] Hartmann | Mastocytosis: recent advances in defining the disease[END_REF]. Patient with CM often exhibit the brownishred macupapular lesions of UP. In UP, MCs are either loosely scattered, with a marked tendency to aggregate around blood vessels and adnexae in the upper dermis, or form small compact infiltrates.

Larger compact transdermal infiltrates of MCs are seen in nodular variant of UP and solitary mastocytoma of the skin. Diffuse CM is less frequently diagnosed. The solitary localized mastocytoma (of the skin) is also rare [START_REF] Kanwar | Diffuse cutaneous mastocytosis: a rare entity[END_REF], and has a benign clinical course. Thus, most MC tumors of the skin appear to be benign. Intradermal MCs always coexpress tryptase and CD117, while only few cells in some cases express CD25 [START_REF] Berezowska | Adult-onset mastocytosis in the skin is highly suggestive of systemic mastocytosis[END_REF]).

Indolent systemic mastocytosis (ISM)

Indolent systemic mastocytosis (ISM) is the most common variant of SM and involves the BM, usually with limited increase of MC burden. Most of the patients with ISM have skin involvement, usually UP, and some patients may present with other organ involvement such as the GI tract, lymph node, spleen or liver, but without organ dysfuction (Valent et al. 2001). In general, the MC burden in typical ISM does not exceed 30% in BM biopsy and a few percent in BM smears. The shift into an aggressive variant or development of an associated malignancy is possible but not frequent in these patients. It is important to know that almost all patients (>95%) with adult-onset UP-like skin lesions might have ISM, hence the BM biopsy is recommended for such patients [START_REF] Ridell | The bone marrow in urticaria pigmentosa and systemic mastocytosis. Cell composition and mast cell density in relation to urinary excretion of tele-methylimidazoleacetic acid[END_REF][START_REF] Czarnetzki | Bone marrow findings in adult patients with urticaria pigmentosa[END_REF][START_REF] Valent | Mastocytosis: a paradigmatic example of a rare disease with complex biology and pathology[END_REF]. ISM shows a prolonged clinical course in almost all patients with excellent prognosis, nearly normal life expectancy, and survival of two decades and more (Pardanani et al. 2009;[START_REF] Escribano | Prognosis in adult indolent systemic mastocytosis: a long-term study of the Spanish Network on Mastocytosis in a series of 145 patients[END_REF][START_REF] Pardanani | WHO subvariants of indolent mastocytosis: clinical details and prognostic evaluation in 159 consecutive adults[END_REF]. Serum tryptase level is raised in comparison to CM with levels ranging from 20 to 200 ng/ml. A rare subvariant of ISM is the so-called smoldering systemic mastocytosis (SSM) (Valent et al. 2002) with pronounced MC infiltration (>30% in BM biopsy), organomegaly and tryptase levels above 200 ng/ml. Smoldering systemic mastocytosis (SSM) is defined by the presence of 2 or more "B-findings" (Table VII) [START_REF] Pardanani | WHO subvariants of indolent mastocytosis: clinical details and prognostic evaluation in 159 consecutive adults[END_REF]. The life expectancy of patient with SSM is variable (Valent et al. 2002). Whatsoever, KIT D816V is the most frequent abnormality found in >80% of all ISM patients and can be detected by sensitive quantitative polymerase chain reaction techniques [START_REF] Garcia-Montero | KIT mutation in mast cells and other bone marrow hematopoietic cell lineages in systemic mast cell disorders: a prospective study of the Spanish Network on Mastocytosis (REMA) in a series of 113 patients[END_REF]Kristensen et al. 2011). More recently, circulating non-MCs elements harboring the KIT D816V mutation have been detected in PB and found to be characteristic of ISM [START_REF] Garcia-Montero | KIT mutation in mast cells and other bone marrow hematopoietic cell lineages in systemic mast cell disorders: a prospective study of the Spanish Network on Mastocytosis (REMA) in a series of 113 patients[END_REF]. In addition, it has been shown that allele burden of the KIT D816V mutation in PB predicts for the risk of progression from ISM to ASM [START_REF] Valent | Mastocytosis: a paradigmatic example of a rare disease with complex biology and pathology[END_REF]Kirstensen et al. 2014). Besides, another subvariant, termed "well- Systemic mastocytosis (SM) is found in 5-20% of cases associated with another hematologic malignancy, and is thus termed SM with an associated hematologic non-MC lineage disease (SM-AHNMD). The AHNMD can be a myelodysplastic /myeloproliferative syndrome, a myeloid leukemia or a myelodysplastic syndrome (Table VIII) (Horny et al. 2008;Stoecker & Wang 2012), while lymphoproliferative neoplasms are much less commonly implicated (Sperr et al. 2000;Pardanani et al. 2009;[START_REF] Sotlar | Variable presence of KITD816V in clonal haematological non-mast cell lineage diseases associated with systemic References mastocytosis (SM-AHNMD)[END_REF].

The KIT D816V mutation is identified in >70% of the patients with SM-AHNMD [START_REF] Sotlar | Variable presence of KITD816V in clonal haematological non-mast cell lineage diseases associated with systemic References mastocytosis (SM-AHNMD)[END_REF].

However, in a subset of patients with SM-CEL (SM with chronic eosinophilic leukemia), FIP1L1/PDGFRA mutant, but no KIT D816V mutation, is found [START_REF] Valent | Mastocytosis: a paradigmatic example of a rare disease with complex biology and pathology[END_REF]). In addition, none of the rare SM patients with lymphoproliferative AHNMDs presented the KIT D816V mutation [START_REF] Sotlar | Variable presence of KITD816V in clonal haematological non-mast cell lineage diseases associated with systemic References mastocytosis (SM-AHNMD)[END_REF].

The prognosis of patients with SM-AHNMD is dictated by the course and progression of AHNMD (Pardanani et al. 2009;Arock & Valent 2010). Since the AHNMD may obscure the SM compartment, SM-AHNMD is a primarily morphological diagnosis based on a thorough investigation of BM trephine specimens including tryptase and CD25 immunohistochemistry [START_REF] Horny | Systemic mastocytosis with associated clonal haematological non-mast cell lineage diseases: a histopathological challenge[END_REF]).

Aggressive systemic mastocytosis (ASM)

As mentioned above, ISM can develop into an aggressive form with progressive infiltration of immature and atypical MCs and subsequent organ destruction and failure ('C findings') [START_REF] Valent | Biology, classification and treatment of human mastocytosis[END_REF][START_REF] Garcia-Montero | KIT mutation in mast cells and other bone marrow hematopoietic cell lineages in systemic mast cell disorders: a prospective study of the Spanish Network on Mastocytosis (REMA) in a series of 113 patients[END_REF]. ASM is much less common than ISM, representing only about 5% of patients with SM, and follows an accelerated course (Valent et al. 2000;Valent et al. 2003). However, in a subgroup of patients, a slower progression is seen (Valent et al. 2001). The life expectancy of patients with ASM is poor. Patients with ASM share common clinical features with SM-AHNMD patients and MCL patients in that they are more likely to be older, present with end-organ damage (organopathy), resulting from massive MC infiltration and usually without UP-like skin lesions, invariably elevated serum tryptase levels reflecting a significant MC burden, and activating KIT mutations.

In ASM, the criteria to diagnose SM are fulfilled and MCL and AHNMD are excluded after BM and PB examinations. The histology of the BM in ASM shows a variable degree of infiltration. MCs usually comprise less than 20% of nucleated cells in BM smears, are tryptase+ and KIT (CD117) + and in a majority of cases express CD2 and/or CD25. In addition, they do express aberrantly CD30 (Valent et al. 2011). In ASM, malignant MCs are hypogranulated and show atypical and high-grade morphology with bi-or multilobed nuclei (Valent et al. 2001). Moreover, if most patients with ASM have KIT D816V mutation, other KIT mutations such as V559I and D820G have been also found [START_REF] Pignon | A new c-kit mutation in a case of aggressive mast cell disease[END_REF][START_REF] Nakagomi | Juxtamembrane-type c-kit gene mutation found in aggressive systemic mastocytosis induces imatinib-resistant constitutive KIT activation[END_REF].

Contrasting to ISM and SSM, C findings are detectable in ASM as a sign of organopathy due to infiltration by neoplastic MCs. In particular, patients show abnormal myelopoiesis with significant blood count abnormalities, hepatomegaly with impairment of liver function due to MC infiltration (often with ascites), large osteolyses (sometimes with pathologic fractures), malabsorption with weight loss due to GI-tract infiltration, splenomegaly with hypersplenism, or life-threatening impairment of organ function in other organ systems. The most commonly affected organs are liver, BM, spleen, and the GI tract (Horny et al. 2007;Valent et al. 2011).

Abnormalities in blood counts may be cytopenia(s), leukocytosis, monocytosis, eosinophilia, basophilia, or thrombocytosis. However, no circulating MCs are detectable (Valent et al. 2001). A distinct and rare subvariant of ASM with prominent eosinophilia in blood and tissues and generalized lymphadenopathy (clinically mimicking malignant lymphoma) has been described as lymphadenopathic mastocytosis with eosinophilia [START_REF] Hauswirth | A case of smouldering mastocytosis with peripheral blood eosinophilia and lymphadenopathy[END_REF].

Mast cell leukemia (MCL)

Mast cell leukemia (MCL) is a rare and highly aggressive subvariant of mastocytosis involving less than 0.5% of all patients with mastocytosis in the database of the French Reference Center for Mastocytosis (CEREMAST) (Georgin-Lavialle et al. 2013). MCL is characterized by leukemic infiltration of various organs by highly atypical MCs (Figure 18), including the BM (Valent et al. 2003), by progressive organopathy including BM failure from MC infiltration, and by the presence of a considerable number of atypical MCs on the BM smear (MC numbers in BM smears must exceed 20% of all nucleated cells in smears). Circulating MCs are found in most cases with MCL (more than 10% of blood cells in typical MCL), while aleukemic variants of MCL are rare in which no circulating MCs are seen [START_REF] Horny | Aleukemic mast cell leukemia (formerly: malignant mastocytosis): an extremely rare form of leukemia. A case report and simultaneously a contribution to revised classification of mastocytosis[END_REF].

MCL shares several clinicobiologic aspects with ASM (Valentini et al. 2008). Hepatosplenomegaly, anemia, and thrombocytopenia are usually present and serum tryptase level is usually high. MCs in MCL are usually CD2 and/or CD25 positive. However, the positive coexpression of CD2 and CD25 was found to be very frequent among MCL patients who are KIT D816V positive (66%) but not in those who are KIT D816V negative (25%) (Georgin-Lavialle et al. 2013). However, one-third of MCL cases reported in the literature have a double-negative CD2/CD25 immunophenotype [START_REF] Valent | Mast cell leukemia--a rare form of myeloid leukemia[END_REF]Horny et al. 2007;[START_REF] Arredondo | Myelomastocytic leukemia versus mast cell leukemia versus systemic mastocytosis associated with acute myeloid leukemia: a diagnostic challenge[END_REF].

Although the KIT D816V mutation has been described in the literature as the most frequently encountered KIT mutation in patients with SM, this is not the case in MCL patients [START_REF] Joris | Mast Cell Leukemia: c-KIT Mutations Are Not Always Positive[END_REF].

Indeed, KIT WT or exon 9 to 13 KIT mutations are more commonly found in MCL and thus complete KIT gene sequencing is needed (Akin et al. 2004 (Valent et al. 2014).

MCL is rarely associated with clonal hematologic non-MC disease (Valent et al. 2010), but the prognosis of these few patients is always poor. Only 4 cases were reported in the literature: three cases of AHNMD were myelodysplastic syndromes and one case was chronic myelomonocytic leukemia.

Of note, AHNMD was only found in the MCL patients with KIT D816V mutation.

All in all, the prognosis of patients with MCL is very poor (Valent et al. 2001;[START_REF] Sperr | Diagnosis, progression patterns and prognostication in mastocytosis[END_REF].

Indeed, therapy usually fails and the mean survival time is only of 6.6 months (range 2-14 months) [START_REF] Noack | Aleukemic mast cell leukemia with abnormal immunophenotype and c-kit mutation D816V[END_REF]).

Mast cell sarcoma (MCS)

Mast cell sarcoma (MCS) is an exceedingly rare clinicopathologic variant of mastocytosis characterized by a unifocal accumulation of highly atypical neoplastic MCs that grow in a locally destructive manner. MCS has been documented in at least 6 human cases in the literature [START_REF] Horny | Mast cell sarcoma of the larynx[END_REF][START_REF] Kojima | Mast cell sarcoma with tissue eosinophilia arising in the ascending colon[END_REF][START_REF] Guenther | Temporary response of localized intracranial mast cell sarcoma to combination chemotherapy[END_REF]Valent et al. 2001;[START_REF] Bautista-Quach | Mast cell sarcoma in an infant: a case report and review of the literature[END_REF][START_REF] Ryan | Mast cell sarcoma: a rare and potentially under-recognized diagnostic entity with specific therapeutic implications[END_REF].

It is noteworthy that most reported MCSs occur in tissues not commonly involved by SM and the organs involved are different from BM or skin (larynx, colon, meningeal site, intracranial site) [START_REF] Horny | Mast cell sarcoma of the larynx[END_REF][START_REF] Kojima | Mast cell sarcoma with tissue eosinophilia arising in the ascending colon[END_REF]Valent et al. 2001;Horny et al. 2007). Furthermore, the cell atypia in MCS may be similar to that seen during MCL. In fact, MCs in MCS display a high-grade cytology and exhibit bi-or polylobed nuclei. They are rather immature (often blast like) with a hypogranulated cytoplasm, and a high nucleus/cytoplasm ratio. Since cytomorphological atypia of this rare entity Intoduction 57 (MCS) is usually very high (according to a grade 3 sarcoma), it is impossible to achieve the correct diagnosis without appropriate immunohistochemical stainings, particularly in differentiating this lesion from other neoplasms that are more likely to occur in infancy. Of note, neoplastic MCs in MCS are tryptase+ and KIT+ (Valent et al. 2001).

Finally, the prognosis of patients with MCS is poor. However, patients receiving chemotherapy and radiation may survive a few years, although MCS can progress to secondary MCL (Valent et al. 2001;Pardanani 2013).

Extracutaneous mastocytoma (ECM)

Extracutaneous mastocytoma (ECM) is a localized benign tumor characterized by a unifocal MC tumor without no aggressive or destructive growth pattern and no evidence of SM (Valent et al. 2001;Castells 2006;Horny et al. 2007;Arock et al. 2015). ECM is extremely rare and has been found almost exclusively in the lung (Scherwin et al. 1965;[START_REF] Charrette | Solitary mast cell "tumor" of lung. Its place in the spectrum of mast cell disease[END_REF][START_REF] Kudo | Solitary mast cell tumor of the lung[END_REF][START_REF] Mylanus | A solitary manifestation of mastocytosis in the head and neck[END_REF]Valent et al. 2001). In pulmonary ECM, the clinical features are those of an intrathoracic tumor with no specific macroscopic or clinical signs. In contrast to MCS, MCs in ECM show a lowgrade cytology and exhibit relatively monomorphic strongly metachromatic round mature phenotype.

The prognosis for patients with ECM is good (Scherwin et al. 1965;[START_REF] Charrette | Solitary mast cell "tumor" of lung. Its place in the spectrum of mast cell disease[END_REF][START_REF] Kudo | Solitary mast cell tumor of the lung[END_REF][START_REF] Mylanus | A solitary manifestation of mastocytosis in the head and neck[END_REF]Valent et al. 2001) and no progression to ASM or MCL occurs (Valent et al. 2001).

Clinical features of mastocytosis

Mastocytosis presents with a wide variety of clinical manifestations ranging from asymptomatic disease to a highly aggressive course with multi-system involvement. The clinical symptoms are related to the excessive release of MC mediators and/or to MC burden and can be observed in all categories of mastocytosis. Typical skin symptoms include pruritus, UP and flushing [START_REF] Sperr | Diagnosis, progression patterns and prognostication in mastocytosis[END_REF]). In patients with ISM, symptoms are related to local or remote effects of excess mediator release from MCs, either spontaneously or in response to triggering stimuli [START_REF] Castells | Mastocytosis: mediator-related signs and symptoms[END_REF]Castells 2006) [START_REF] Barete | Systemic mastocytosis and bone involvement in a cohort of 75 patients[END_REF][START_REF] Rossini | Bone mineral density, bone turnover markers and fractures in patients with indolent systemic mastocytosis[END_REF][START_REF] Rossini | Bone involvement and osteoporosis in mastocytosis[END_REF]. Less commonly, osteosclerosis is also seen, and in some patients both osteoporosis and osteosclerosis may be observed in different sites [START_REF] Johansson | Bone density, bone markers and bone radiological features in mastocytosis[END_REF].

In addition, in adult mastocytosis, neurologic symptoms including lack of concentration, irritability, anxiety and/or depression are frequent and the pathophysiology of these symptoms in a majority of cases is not known but could be linked to tissue MC infiltration, MC mediator release, or both [START_REF] Moura | Neuropsychological features of adult mastocytosis[END_REF]. Patients with systemic mastocytosis (SM, discussed later) also complain from "brain fog symptoms" which include the loss of attention, focus, short term memory and ability to multitask [START_REF] Moura | Evidence for cognitive impairment in mastocytosis: prevalence, features and correlations to depression[END_REF][START_REF] Jennings | The Mastocytosis Society survey on mast cell disorders: patient experiences and perceptions[END_REF], headaches, and autism spectrum disorders (ASDs) [START_REF] Theoharides | Brain "fog," inflammation and obesity: key aspects of neuropsychiatric disorders improved by luteolin[END_REF].

By contrast to ISM, it may be difficult to attribute symptoms to mastocytosis in patients with SM-AHNMD, as the patients may have signs and symptoms related to the AHNMD. Moreover, in advanced SM such as in ASM and MCL, destructive infiltration of neoplastic MCs is responsible for the symptoms related to end-organ damage including malabsorption and weight loss, osteolyses and pathologic bone fractures, hypersplenism, hepatomegaly with impairment of liver function often with ascites, significant cytopenia(s) (Valent et al. 2001;Valent et al. 2003;Valent et al. 2005;[START_REF] Pardanani | Pathogenesis, clinical features, and treatment advances in mastocytosis[END_REF][START_REF] Metcalfe | Systemic mastocytosis[END_REF]Lim et al. 2009;Valent et al. 2010). These symptoms represent socalled "C findings" and found in patients with ASM and patients with MCL (Valent et al. 2001;Valent et al. 2003;Valent et al. 2014).

MCs are also involved in MC activation syndrome (MCAS) which distinguishes a group of patients clinically presenting with recurrent episodes of anaphylaxis, no skin lesions, and only one or two minor diagnostic criteria for systemic mastocytosis (SM) but lacking the major criterion, i.e. compact infiltrates of MCs in BM. In MCAS, MCs may display cytomorphological atypia, aberrant expression of CD25 or the presence of KIT D816V mutant, but all three features (which would be sufficient for the diagnosis of SM) are not detectable (Horny et al. 2007;[START_REF] Valent | Definitions, criteria and global classification of mast cell disorders with special reference to mast cell activation syndromes: a consensus proposal[END_REF]. MCA can occur in any variant of mastocytosis. In such cases, the presence of MCA(S) should be recorded as such or by the subscript 'SY', for 'symptoms' (e.g. SM SY ) (Valent et al. 2001;Valent et al. 2007;[START_REF] Valent | Definitions, criteria and global classification of mast cell disorders with special reference to mast cell activation syndromes: a consensus proposal[END_REF]).

Pathophysiology of mastocytosis

During the past years, the scientific knowledge about the pathophysiology of mastocytosis has been rapidly developed. Since SCF was identified as the major cytokine responsible for MC proliferation, increased secretion of SCF was hypothesized to be involved in the pathophysiology of mastocytosis, but no data confirmed this hypothesis. This lack of SCF deregulation led the scientists to search for abnormalities in the SCF receptor, KIT. The presence of such abnormalities (point mutations) in KIT was firstly found in the HMC-1 cell line (Furistu et al. 1993), and then confirmed in patient's pathological samples. Thus, it is well known today that most cases of mastocytosis are associated with an activating KIT defect. Interestingly, all the various KIT mutations found in different variants of As mentioned previously, the finding of activating point mutation arising from substitution of valine for aspartate at codon 816 of KIT is the molecular hallmark of mastocytosis. In 1995, Nagata et al.

identified KIT D816V mutation in the PB of four patients with mastocytosis (Nagata et al. 1995). This mutation is located in the phosphotransferase domain (PTD) of the receptor and detected in neoplastic MCs of majority of adult patients with SM regardless the variant of SM and in approximately 40% of skin biopsies from pediatric patients with CM (Longley et al. 1996;[START_REF] Longley | Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis[END_REF]Orafo et al. 2007;Bodemer et al. 2010;[START_REF] Cardet | Mastocytosis: update on pharmacotherapy and future directions[END_REF]Arock et al. 2015). Zappulla et al. showed that in a model of transgenic mouse for human KIT D816V, MC hyperplasia was observed along with the expression of the transgene in these cells, indicating the involvement of KIT defects in the pathophysiology of SM (Zappulla et al. 2005). Whereas the KIT D816V mutant is thought to play an important role in ISM that presents with MC mediator-related symptoms and have no signs of a substantial MC proliferation, in contrast the KIT D816V mutation alone is not believed to be responsible for the major manifestations and aggressiveness of SM-AHNMD, ASM and MCL [START_REF] Valent | Recent advances in mastocytosis research. Summary of the Vienna Mastocytosis Meeting 1998[END_REF]Tefferi & Pardanani 2004).

Indeed, in these patients, additional genetic defects have been identified including other KIT mutations apart from D816V [START_REF] Pignon | A new c-kit mutation in a case of aggressive mast cell disease[END_REF][START_REF] Pullarkat | Mast cell disease associated with acute myeloid leukemia: detection of a new c-kit mutation Asp816His[END_REF]Akin et al. 2004 [START_REF] Spector | Mast-cell leukemia exome sequencing reveals a mutation in the IgE mast-cell receptor β chain and KIT V654A[END_REF]). These defects might decisively contribute to the clinical heterogeneity and the aggressiveness of SM and may represent new therapeutic targets (Arock et al. 2015). Interestingly, overall survival was found significantly shorter in patients with additional aberrations (Schwaab et al. 2013). Table X summarizes the molecular somatic lesions and abnormalities found in patients with mastocytosis and their estimated frequency in patients with SM [START_REF] Valent | Mastocytosis: a paradigmatic example of a rare disease with complex biology and pathology[END_REF]. Besides the "canonical" KIT D816V mutant, other rarest KIT mutations have been identified in a few patients and may also play a role in the etiology of mastocytosis. These mutations include V560G within the JMD of KIT; D816Y, D816F, and D816H in the PTD; the E839K dominant inactivating mutation in several cases of pediatric mastocytosis; and the rare germline mutation F522C in the TMD, which may be associated with WDSM. Exceedingly rare KIT mutations that are reported to be present in less than 1% of patients with SM include R815K, insV815_I816, I817V, D820G (all in the A-loop), del419 and K509I (in the ECD), and A533D and V559I in the JMD (Orafo et al. 2007).

Most cases of KIT mutations in the ECD were found in sporadic case in children (see above Most of these mutations are of germline nature, activating and respond to imatinib treatment at least in vitro (Yang et al. 2010). Figure 19 shows the major KIT mutations found in patents with different categories of mastocytosis (Arock et al. 2015).

However, the situation in MCL contrasts to the one encountered in ISM. Indeed, KIT D816V is detected in only 46% of patients with MCL, the other patients having KIT WT and other KIT mutations in exon 9, exon 11 or exon 13 were found [START_REF] Mital | A case of mast cell leukaemia with exon 9 KIT mutation and good response to imatinib[END_REF]Georgin-Lavialle et al. 2013).

Only one case of MCL with KIT mutant in the ECD has been reported and found sensitive to imatinib [START_REF] Mital | A case of mast cell leukaemia with exon 9 KIT mutation and good response to imatinib[END_REF]

Treatment of mastocytosis

Since mastocytosis is a heterogeneous disease presenting with a wide variety of clinical manifestations as mentioned above, its treatment is variable and based on the severity of clinical symptoms, patient complaints, and on prognosis (Valent et al. 2010;Arock et al. 2015;[START_REF] Pardanani | Systemic mastocytosis in adults: 2015 update on diagnosis, risk stratification, and management[END_REF]. Despite growing scientific knowledge, no curative therapy for mastocytosis exists (Escribano et al. 2002; Brockow 2004), and the most important overall aim of treatment is the control of symptoms.

Treatment recommendations for SM are based mostly on expert opinion rather than evidence obtained from controlled clinical trials and the condition is primarily managed by a hematologist (Wilson et al. 2006;[START_REF] Siebenhaar | Treatment References strategies in mastocytosis[END_REF]. Of note, in patients with SM-AHNMD, the SM component is treated as if no AHNMD was diagnosed, and AHNMD is treated as if no coexisting SM was found (Valent et al. 2010).

Symptomatic treatment of mastocytosis

In a substantial number of patients with CM or SM, diagnosed as CM SY or SM SY , mediator-related symptoms are recorded and cytoreductive therapy is not required. In these cases, the symptomatic treatment is required to control the mediator-related symptoms and is based on agents blocking the effects of MC-derived mediators, such as histamine receptor (H1R and H2R) antagonists, MCstabilizing agents, antileukotrienes, glucocorticosteroids (GCs), and on bisphosphonates (Arock et al. 2015). Figure 20 depicts the proposed algorithm for antimediator-treatment in mastocytosis [START_REF] Siebenhaar | Treatment References strategies in mastocytosis[END_REF].

GI symptoms and ulcer can be treated by H2R antagonists including ranitidine and famotidine, proton pump inhibitors, antacids and oral sodium cromolyn, which has been shown to be effective in the control of diarrhea and abdominal cramping in many patients (Escribano et al. 2006).

Systemic administered corticosteroids can be effective in patients with frequent anaphylactic reactions or severe systemic conditions like ascites, diarrhea or malapsorption [START_REF] Metcalfe | The treatment of mastocytosis: an overview[END_REF].

In case of severe osteopenia, or frank osteoporosis, therapy with a bisphosphonate is recommended [START_REF] Laroche | Clinical and densitometric efficacy of the association of interferon alpha and pamidronate in the treatment of osteoporosis in patients with References systemic mastocytosis[END_REF]Arock & Valent 2010;[START_REF] Laroche | Interferon alpha and pamidronate in osteoporosis with fracture secondary to mastocytosis[END_REF]. Prophylactic bisphosphonates are also recommended for SM patients who receive long-term GCs or have other (additional) risk factors (Arock & Valent 2010). Bisphosphonate also resolves pain in many SM patients suffering from unexplained pain accompanied by osteopenia [START_REF] Lim | Systemic mastocytosis: a rare cause of osteoporosis and its response to bisphosphonate treatment[END_REF].

In general, antimediator-type drugs are applied in a step-wise fashion and on the basis of the organ(s) affected (Valent et al. 2007). It is standard to instruct all patients to avoid agents, drugs, situations and other symptom-trigger(s) for MC degranulation, such as NSAIDs, opioids, muscle relaxants, iodinated contrast agents, excessive heat or cold, and stress. Patients at risk for severe hypotension and vascular collapse should carry two or more epinephrine-self-injectors (adrenaline auto-injectors) and are taught to use them under prescribed conditions. In patients with IgE-dependent anaphylaxis, specific immunotherapy should be considered with recognition of potential risks (Valent et al. 2007). Recent reports have promised potential beneficial effects using omalizumab, a humanized anti-IgE antibody, in the treatment of mastocytosis [START_REF] Siebenhaar | Successful treatment of cutaneous mastocytosis and Ménière disease with anti-IgE therapy[END_REF][START_REF] Douglass | Omalizumab is effective in treating systemic mastocytosis in a nonatopic patient[END_REF][START_REF] Matito | Short-term omalizumab treatment in an adolescent with cutaneous mastocytosis[END_REF]). In the absence of specific IgE, however, immunotherapy is not recommended. Also, cytoreductive agents are not recommended for treatment of mediator-related events (Valent et al. 2007;[START_REF] Pardanani | Systemic mastocytosis in adults: 2015 update on diagnosis, risk stratification, and management[END_REF].

Oral psoralen plus UV-A (PUVA) therapy results in general and cosmetic benefits in the treatment of CM (Escribano et al. 2002;[START_REF] Worobec | Mastocytosis: current treatment concepts[END_REF][START_REF] Kinsler | Diffuse cutaneous mastocytosis treated with psoralen photochemotherapy: case report and review of the literature[END_REF], however adverse effects are possible, such as skin cancer if more than 200 treatments are required. The manifestations of the disease usually recur several months after discontinuation of PUVA, but recurrences respond as well as the original lesions. Currently, PUVA therapy is reserved for severe, unresponsive cases in adults.

Moreover, medium-dose UV-A1 therapy has been shown to be as effective as high-dose UV-A1 in reducing symptoms and the number of MCs in affected skin [START_REF] Gobello | Medium-versus high-dose ultraviolet A1 therapy for urticaria pigmentosa: a pilot study[END_REF]. Also, treatment of cutaneous MC lesions with the 585-nm flashlamp-pumped dye laser and the 532-nm Nd:YAG laser has been reported to provide cosmetic improvement [START_REF] Ellis | Treatment of telangiectasia macularis eruptiva perstans with the 585-nm flashlamp-pumped dye laser[END_REF][START_REF] Resh | The cosmetic treatment of urticaria pigmentosa with Nd:YAG References laser at 532 nanometers[END_REF]).

Non specific cytoreductive therapy

In patients with ASM, MCL and MCS, cytoreductive therapy is usually required since C findings are detected. These patients may benefit from non-targeted cytoreductive drugs, such as IFN-α (Kluin- et al. 1992;[START_REF] Hauswirth | Response to therapy with interferon alpha-2b and prednisolone in aggressive systemic mastocytosis: report of five cases and review of the literature[END_REF][START_REF] Tefferi | How we diagnose and treat WHO-defined systemic mastocytosis in adults[END_REF][START_REF] Pardanani | Systemic mastocytosis in adults: 2015 update on diagnosis, risk stratification, and management[END_REF], 2-Chlorodeoxyadenosine (cladribine or 2-CdA) [START_REF] Tefferi | Treatment of systemic mast-cell disease with cladribine[END_REF][START_REF] Kluin-Nelemans | Cladribine therapy for systemic mastocytosis[END_REF]Tefferi, & Pardanani 2004;[START_REF] Böhm | In vitro and in vivo growth-inhibitory effects of cladribine on neoplastic mast cells exhibiting the imatinib-resistant KIT mutation D816V[END_REF]Arock & Valent 2010), or hydoxyurea (HU) (Valent et al. 2005).

Nelemans

Interferon (IFN)-α

Interferon (IFN)-α (IFN-alpha-2b (IFN-α2b) in most cases) has formerly been considered first-line cytoreductive therapy in patients with ASM. It has been shown to improve symptoms of MC degranulation, UP and skin findings, symptomatic organomegaly and ascites, cytopenias, and osteoporosis, as well as to decrease BM MC infiltrations [START_REF] Kluin-Nelemans | Response to interferon alfa-2b in a patient with systemic mastocytosis[END_REF][START_REF] Worobec | Treatment of three patients with systemic mastocytosis with interferon alpha-2b[END_REF][START_REF] Lehmann | Severe osteoporosis due to systemic mast cell disease: successful treatment with interferon alpha-2B[END_REF][START_REF] Lippert | Long-term effect of interferon alpha treatment in mastocytosis[END_REF][START_REF] Takasaki | Systemic mastocytosis with extensive polypoid lesions in the intestines; successful treatment with interferon-References alpha[END_REF][START_REF] Lehmann | IFNalpha treatment in systemic mastocytosis[END_REF][START_REF] Hauswirth | Response to therapy with interferon alpha-2b and prednisolone in aggressive systemic mastocytosis: report of five cases and review of the literature[END_REF][START_REF] Butterfield | Successful treatment of systemic mastocytosis with high-dose interferon-alfa: long-term follow-up of a case[END_REF][START_REF] Tefferi | How we diagnose and treat WHO-defined systemic mastocytosis in adults[END_REF]). However, IFN-α treatment is not uniformly effective, and the frequency of major response (MR) such as complete resolution of one or more C finding) is low (Valent et al. 2010;Pardanini 2015). Also, the optimal dose and duration of IFN-α treatment for patients with SM remain unclear. In addition, a significant proportion of patients will relapse within a short period of IFN-α treatment being discontinued, illustrating the cytostatic rather than cytolytic effects of the drug [START_REF] Simon | Interest of interferon alpha in systemic mastocytosis". The French experience and review of the literature[END_REF]). Moreover, IFN-α treatment is frequently complicated by toxicities and adverse events including flu-like symptoms, bone pain, fever, cytopenias, depression, and hypothyroidism [START_REF] Lippert | Long-term effect of interferon alpha treatment in mastocytosis[END_REF][START_REF] Casassus | Treatment of adult systemic mastocytosis with interferon-alpha: results of a multicentre phase II trial on 20 patients[END_REF]Lim et

Hydoxyurea (HU)

Hydoxyurea (HU) is recommended as a reliable palliative drug in patients who are neither eligible for chemotherapy nor for HSCT (Valent et al. 2005). The HU has myelosuppressive activity, but does not exhibit any substantial anti-MC effect in SM-AHMD. It has been used as first-line therapy in patients with SM-AHNMD in a Mayo Clinic study. Median duration of response was 31.5 months (range 5-50 months) and the major toxicity was myelosuppression (Lim et al. 2009).

Novel approaches in the treatment of mastocytosis: targeted therapies

During the last few years, a number of different targeted drugs targeting tyrosine kinase (TK) activity have been used in patients with advanced MC disorders and several inhibitors against the mutated form of KIT have been developed.

Imatinib

The effective inhibitor of the WT KIT TK as well as of the BCR-ABL and PDGFR TKs (Pardanani & Tefferi 2004;[START_REF] Hoffmann | Successful treatment of progressive cutaneous mastocytosis with imatinib in a 2-year-old boy carrying a somatic KIT mutation[END_REF]Pardanani 2013). Imatinib has no effectiveness in mastocytosis patients with KIT D816V mutation [START_REF] Frost | Juxtamembrane mutant V560GKit is more sensitive to Imatinib (STI571) compared with wild-type c-kit whereas the kinase domain mutant D816VKit is resistant[END_REF]Akin et al. 2003;[START_REF] Heinrich | Phase II, open-label study evaluating the activity of imatinib in treating life-threatening malignancies known to be associated with imatinib-sensitive tyrosine kinases[END_REF]Lim et al. 2009) since this mutation leads to a steric alteration in the receptor preventing imatinib from binding to the ATP binding domain of KIT, as outlined above [START_REF] Ma | The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory-type mutations[END_REF]Vendome et al. 2005).

However, patients carrying uncommon KIT mutations in exons 9 or 10 such as F522C, K509I, del419 mutations, as well as the JMD mutations V560G and V559G, but not V559I respond to imatinib which has been found to reduce MC burden, to lower tryptase levels and to improve the clinical symptoms (Akin et al. 2004

Masitinib

Masitinib (AB1010) is another KIT inhibitor that was first registered to treat canine MC tumors (Hahn et al. 2008). It has in vitro activity against KIT WT in human MCs greater than that of imatinib, and it also inhibits PDGFR and Lyn [START_REF] Dubreuil | Masitinib (AB1010), a potent and selective tyrosine kinase inhibitor targeting KIT[END_REF]. In contrast, masitinib demonstrates weak inhibition of ABL and c-Fms and does not block the KIT D816V mutant. However, the highly selective nature of masitinib suggests that it will exhibit a better safety profile than other TK inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies [START_REF] Dubreuil | Masitinib (AB1010), a potent and selective tyrosine kinase inhibitor targeting KIT[END_REF]. Masitinib inhibits more strongly degranulation, cytokine production, and BM-derived MC migration than imatinib. Furthermore, it potently inhibits human and murine KIT with JMD activating mutations [START_REF] Dubreuil | Masitinib (AB1010), a potent and selective tyrosine kinase inhibitor targeting KIT[END_REF]

Dasatinib (BMS354825)

Dasatinib is an orally available short-acting dual ABL/Src TK inhibitor [START_REF] Lindauer | Dasatinib[END_REF] that is more potent than imatinib on a molar basis against the BCR-ABL kinase [START_REF] Olivieri | Dasatinib: a new step in molecular target therapy[END_REF]. Dasatinib is also highly active against PDGFRA and has shown efficacy in vitro against various KIT mutants including D816V [START_REF] Schittenhelm | Dasatinib (BMS-354825), a dual SRC/ABL kinase inhibitor, inhibits the kinase activity of wild-type, juxtamembrane, and activation loop mutant KIT isoforms associated with human malignancies[END_REF] In addition, dasatinib induces several adverse effects, particularly pleural effusions, in a substantial number of patients [START_REF] Lindauer | Dasatinib[END_REF]. Notably, responders in the phase II study of dasatinib were found to experience an initial exacerbation of MC mediator-release symptoms (MCMRS) and rash lasting several days before the benefit benefits of dasatinib therapy became evident [START_REF] Verstovsek | Phase II study of dasatinib in Philadelphia chromosome-negative acute and chronic myeloid diseases, including systemic mastocytosis[END_REF]. Thus, dasatinib appears to date to have modest activity in KITD816V+ SM (Pardanani 2015).

Midostaurin (PKC412)

Midostaurin (PKC412) is a potent multikinase inhibitor that demonstrates in vitro activity against KD KIT mutants (D816Y and D816V) (Growney et al. 2005;Gleixner et al. 2006). It is also active against PKC, fms related tyrosine kinase 3 (FLT3), PDGFRA, and vascular endothelial growth factor receptor Midostaurin abrogates the phosphorylation of KIT in HMC-1.1 and HMC-1.2 cells in a timedependent and dose-dependent manner and suppresses the growth of primary neoplastic human MCs harboring either KIT D816V or WT KIT [START_REF] Gleixner | Synergistic growth-inhibitory effects of ponatinib and midostaurin (PKC412) on neoplastic mast cells carrying KIT D816V[END_REF]. In addition to its growth-inhibitory effects, midostaurin has been described to inhibit histamine secretion from activated normal and neoplastic MCs. Thus, it may be a beneficial drug in ASM suffering from mediator-related symptoms (Krauth et al. 2009). Of note, midostaurin can act synergistically with nilotinib and dasatinib to inhibit the growth of neoplastic MCs with the KIT D816V mutation (Gleixner et al. 2006;[START_REF] Gleixner | Synergistic growth-inhibitory effects of ponatinib and midostaurin (PKC412) on neoplastic mast cells carrying KIT D816V[END_REF].

Interestingly, midostaurin induced remission in a patient with MCL carrying the activating KIT D816V mutation (Gotlib et al. 2005) and several clinical trials are ongoing to determine the efficacy and tolerability of midostaurin in patients with SM [START_REF] Knapper | Midostaurin a Multi-Targeted Oral Kinase Inhibitor in Systemic Mastocytosis: Report of An Open-Label Compassionate Use Program in the United Kingdom[END_REF][START_REF] Gotlib | Midostaurin (PKC412) Demonstrates a High Rate of Durable Responses in Patients with Advanced Systemic Mastocytosis: Results from the Fully Accrued Global Phase 2 CPKC412D2201 Trial[END_REF]. Initial results from a phase II study of single-agent midostaurin showed promising activity in 26 patients with advanced SM, with a 69% response rate: 38% major response, 19% good partial response, and 12% minor partial response. Importantly, all patients who achieved a major response had the KIT D816V mutation, and the response was durable for a median of 1.5 year as well as Midostaurin was relatively well tolerated (Gotlib et al. 2010). A larger international phase II clinical trial in patients with ASM or MCL (National Clinical Trial NCT00782067) presented at the 2012 American Society of Hematology Annual Meeting, demonstrated that midostaurin was associated with durable responses in patients with advanced SM, particularly in MCL [START_REF] Gotlib | KIT Inhibitor Midostaurin in Patients with Advanced Systemic Mastocytosis: Results of a Planned Interim Analysis of the Global CPKC412D2201 Trial[END_REF]. In addition, another study has recently demonstrated that, despite the absence of complete remission, PKC412 gives survival advantage for patients with advanced SM over patients who did not receive the drug [START_REF] Chandesris | Treatment of Advanced Systemic Mastocytosis with PKC412: The French Compassionate Use Programme Experience and Historical Comparison[END_REF].

However, the long-term benefit of PKC412 has not yet been determined. Therefore, development of novel effective drugs to treat ASM and MCL is still needed.

Nilotinib (AMN107)

Nilotinib is a TK inhibitor that also inhibits It has been reported that ponatinib is active against BaF3 cells stably expressing KIT Y823D in vitro [START_REF] Lierman | Ponatinib is active against imatinib-resistant mutants of FIP1L1-PDGFRA and KIT, and against FGFR1-derived fusion kinases[END_REF], inhibits the growth and survival of KIT D816V positive neoplastic MCs and can produce synergistic growth-inhibitory effects when combined with PKC412 [START_REF] Gleixner | Synergistic growth-inhibitory effects of ponatinib and midostaurin (PKC412) on neoplastic mast cells carrying KIT D816V[END_REF].

Furthermore, ponatinib inhibits the growth of primary neoplastic MCs, and abrogates the phosphorylation of KIT harboring the point mutation V560G (but less effectively the D816V mutation) and the downstream signaling transduction [START_REF] Gleixner | Synergistic growth-inhibitory effects of ponatinib and midostaurin (PKC412) on neoplastic mast cells carrying KIT D816V[END_REF]Jin et al. 2014).

Interestingly, ponatinib was also found to inhibit the growth of xenografted HMC-1.2 cells in nude mice and significantly prolong the survival of mice with ASM or MCL (Jin et al. 2014).

However, whether ponatinib, alone or in combination, is able in clinical trials to suppress the in vivo growth of neoplastic MCs in patients with advanced SM and whether potential benefits could exceed the risk of its adverse effects such as thrombotic vascular events remain to be examined.

Novel TKIs

Tandutinib (MLN518) was found to inhibit the proliferation of KIT D816V positive Ba/F3 cells and P815 cells [START_REF] Corbin | Sensitivity of oncogenic KIT mutants to the kinase inhibitors MLN518 and PD180970[END_REF]. Semaxinib (SU5416) induces apoptosis in KIT D816V positive erythroleukemic cells and inhibits KIT autophosphorylation as well as activation of AKT, ERK1/2 and Stat3 downstream signaling pathways [START_REF] Kosmider | Semaxinib (SU5416) as a therapeutic agent targeting oncogenic Kit mutants resistant to imatinib mesylate[END_REF]). EXEL-0862 was also found to induce apoptosis in vitro and ex vivo and to decrease phosphorylation of STAT3 and STAT5 in HMC-1.2 cell line [START_REF] Pan | EXEL-0862, a novel tyrosine kinase inhibitor, induces apoptosis in vitro and ex vivo in human mast cells expressing the KIT D816V mutation[END_REF]).

The dual Bcr-Abl and Lyn-kinase inhibitor bafetinib (INNO-406) was found to inhibit proliferation and block KIT phoshoprylation in HMC-1.1 cells, but not in HMC-1.2 cells or primary neoplastic cells in patients with KIT D816V-positive SM but it block LYN phosphorylation in both cell lines [START_REF] Peter | KIT polymorphisms and mutations determine responses of neoplastic mast cells to bafetinib (INNO-406)[END_REF].

Besides, the adenosine triphosphate (ATP)-based kinase inhibitors (AP23464, AP23848) showed in vitro and in vivo activity against activation-loop mutants of KIT [START_REF] Corbin | In vitro and in vivo activity of ATP-based kinase inhibitors AP23464 and AP23848 against activation-loop mutants of Kit[END_REF].

In addition, the TK inhibitor APcK110 induces apoptosis and inhibits proliferation of HMC-1.2 cells [START_REF] Faderl | Kit inhibitor APcK110 induces apoptosis and inhibits proliferation of acute myeloid leukemia cells[END_REF]. Moreover, both MLN518 and PD180970 compounds inhibit the growth of HMC-1.1cell line. MLN518 additionally targets active site mutant cell lines, inhibits the proliferation of KIT D816V positive Ba/F3 cells and P815 cells, abolishes KIT and STAT3 phosphorylation and induces apoptosis. This drug was found well tolerated in phase 1 clinical trials in AML [START_REF] Corbin | Sensitivity of oncogenic KIT mutants to the kinase inhibitors MLN518 and PD180970[END_REF].

The small molecule TK inhibitor, bosutinib, is currently approved for CML treatment [START_REF] Ai | Practical management of patients with chronic myeloid leukemia who develop tyrosine kinase inhibitor-resistant BCR-ABL1 mutations[END_REF]. It deactivates Lyn and Btk without blocking KIT activity and can inhibit the growth of neoplastic MCs in vitro (Gleixner et al. 2009;Gleixner et al. 2011). Moreover, bosutinib cooperates with PKC412 to block the growth of KIT-mutated neoplastic MCs (Gleixner et al. 2011).

Interestingly, the thiazole amine class TK inhibitor 126332 has been described to potently inhibit the proliferation of KIT D816V-expressing cells as well as the phosphorylation of KIT and of its downstream signaling molecules STAT3 and STAT5. Compound 126332 induced apoptosis and downregulated levels of Mcl-1 and survivin. Furthermore, this molecule exhibited also in vivo antineoplastic activity against cells harboring the KIT D816V mutation (Jin et al. 2014).

However, all TKIs cited above still need to be evaluated in clinical trials to explore their real antineoplastic potential in ASM and MCL.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT)

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is considered as a viable and potentially curative therapeutic option for aggressive hematologic malignancies [START_REF] Holtick | Comparison of bone marrow versus peripheral blood allogeneic hematopoietic stem cell transplantation for hematological malignancies in adults -a systematic review and meta-analysis[END_REF].

In OS and PFS were significantly affected by the type of advanced SM: 74% and 63% for SM-AHNMD, 43% and 43% for ASM, and 17% and 17% for MCL. Risk factors for poor survival included MCL (the most important factor), lower Karnofsky performance status, and patients receiving reducedintensity conditioning regimens. Acute graft-versus-host disease (GVHD) grades II-IV and chronic extensive GVHD occurred in 40% and 23% of patients, respectively. Treatment-related mortality (TRM) at 1 year was 20% (MCL patients had the highest rate: 33%). Thus, allo-HSCT should be considered in all patients with drug-resistant advanced SM who are young and fit and have a suitable donor. Although the data presented are very encouraging, future prospective studies are required to confirm the value and efficacy of this treatment approach in such categories of SM.

Potential alternative therapeutic targets

Given the fact that the neoplastic MCs can rely on KIT-independent signaling pathways for survival, and that these pathways possibly contribute to disease progression (Gleixner et al. 2007), many patients treated with TKI relapse and thus current studies focus on drug combinations. Combinations of targeted drugs may represent an interesting pharmacologic approach for the treatment of aggressive SM or MCL. However, most potent TKIs and drug combinations should be tested in clinical trials.

Moreover, another interesting approach may be to attack neoplastic MC and their progenitor cells (SM stem cells) by targeting antibodies directed against various neoplastic MC-restricted surface antigens which constitute a viable option for the treatment of SM and warrant further investigations.

Indeed, many alternative targets of interest are overexpressed either on or in the neoplastic MCs cells compared to normal MCs (Valent et al. 2010). Some of these targets are thought to contribute o the mechanism(s) involved in signaling pathways critical for the transforming abilities of mutant KIT, such as the PI3-kinase-AKT pathway. Besides, signal transduction molecules that play a pivotal role in the growth, survival, and transformation of neoplastic MCs, such as FES, the transcription factor STAT5, or the mTOR complex represent promising and novel targets. The potential functional and clinical role of some of these targets has been recently demonstrated in in vitro and ex vivo studies, when used alone or in combination with KIT inhibitors. In addition, for certain targets, specific drugs are already available that might be tested in the future in clinical trials if all the preclinical studies appear convincing.

Targets related to signaling or apoptosis

Since AKT has been reported to be involved in KIT-dependent differentiation and growth of neoplastic MCs harboring oncogenic KIT mutants, and to be phosphorylated in the HMC-1.2 cell line as well as in neoplastic MCs from patients suffering from KIT D816V+ SM, its activation is considered to play potentially a critical role in the pathogenesis of mastocytosis (Harir et al. 2008;Grimwarde et al. 2009;Yang et al. 2010). Of note, AKT inhibitors decrease the proliferation of KIT D816V+human MCs in vitro (Saleh, wedeh et al. 2014).

The heat shock protein 90 (hsp90) inhibitor-17-demethoxygeldanamycin (17-AAG) binds to hsp90 and leads to the destabilization of various hsp90-dependent kinases important in oncogenesis.

Treatment of both HMC-1.1 cell line and with HMC-1.2 cell line with 17-AAG downregulates both the level and activity of KIT and downstream signaling molecules AKT and STAT3, indicating that 17-AAG may be effective in the treatment of SM, and MCL [START_REF] Fumo | 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is effective in down-regulating mutated, constitutively activated KIT protein in human mast cells[END_REF].

It is well known today that FES TK is phosphorylated on tyrosine residues in KIT D816V+ cells and reduction of FES expression using RNA interference (RNAi) decreases also the proliferation of these cells [START_REF] Voisset | The tyrosine kinase FES is an essential effector of KITD816V proliferation signal[END_REF]). However, to date there is no specific FES inhibitor.

As mentioned above, rapamycin inhibits mTOR and thus the survival of KIT D816V+ MCs [START_REF] Gabillot-Carré | Rapamycin inhibits growth and survival of D816V-mutated c-kit mast cells[END_REF]. Inhibition of mTORC2 via a targeted approach may reduce the proliferation in MC disorders without affecting the normal differentiated MCs and therefore might be a promising therapeutic alternative in the future [START_REF] Smrz | mTORC1 and mTORC2 differentially regulate homeostasis of neoplastic and non-neoplastic human mast cells[END_REF].

Regarding STAT5, it is well admitted that its expression plays a critical role of in MC development and survival [START_REF] Shelburne | Stat5 expression is critical for mast cell development and survival[END_REF]Chaix et al. 2011). Interstingly, it has been reported that phosphorylated STAT5 (p-STAT5) is found in the cytoplasm of neoplastic MCs from patients with SM [START_REF] Harir | Constitutive activation of Stat5 promotes its cytoplasmic localization and association with PI3-kinase in myeloid leukemias[END_REF]Baumgartner et al. 2009) and that knockdown of STAT5, AKT, or both resulted in cell growth inhibition (Harir et al. 2008). Moreover, it has been reported that KIT D816V

promotes the direct activation of STAT5 and that p-STAT5 contributes to growth of neoplastic MCs as shown using knockdown experiments (Harir et al. 2008). Recently, STAT5 has been reviwed to be one major cellular effector in mastocytosis by controlling the mutant KIT-mediated aberrant growth signaling. However, the pharmacological inhibition of STAT5 remains challenging, and new STAT5 inhibitors active at pharmacological doses in both indolent and aggressive forms of SM are still requied (Bibi et al. 2014).

The BH3-only (BCL-2 homology) proteins, a group of proapoptotic factors, have been described to modulate MC survival in vitro and in vivo [START_REF] Hartmann | Expression of Bcl-2 and Bcl-xL in cutaneous and bone marrow lesions of mastocytosis[END_REF][START_REF] Ekoff | The BH3-only protein Puma plays an essential role in cytokine deprivation induced apoptosis of mast cells[END_REF]

Antigen targeting

The cell surface antigens expressed on neoplastic MCs and/or their progenitor cells such as CD13 (aminopeptidase N), CD25, CD30, CD33, CD44, CD52, CD87, and CD117 have been considered as potential targets of antibody-based therapy in advanced MC disorders [START_REF] Sotlar | CD25 indicates the neoplastic phenotype of mast cells: a novel immunohistochemical marker for the diagnosis of systemic mastocytosis (SM) in routinely processed bone marrow biopsy specimens[END_REF]Valent et al. 2010;Valent et al. 2011;Arock et al. 2015). In fact, CD30, CD33, and CD52 are considered the most promising and/or relevant antigens in the treatment of mastocytosis (Arock et al. 2015). Neoplastic

MCs in patients with ASM and MCL express CD30 and CD52 at higher levels than those of MCs from patients with ISM (Valent et al. 2011;[START_REF] Sotlar | Aberrant expression of CD30 in neoplastic mast cells in high-grade mastocytosis[END_REF][START_REF] Hoermann | CD52 is a molecular target in advanced systemic mastocytosis[END_REF] Because of its expression on the putative neoplastic stem cells in SM, CD33 is considered as an attractive target in mastocytosis [START_REF] Florian | Detection of molecular targets on the surface of CD34+/CD38--stem cells in various myeloid malignancies[END_REF]. Interestingly, neoplastic MCs and their progenitors respond in vitro to Mylotarg, a drug composed by a humanized CD33 antibody (gemtuzumab) and the cytostatic agent calicheamicin (ozogamicin) (Krauth et al. 2007). However, whether patients with advanced SM may benefit of Mylotarg is still unknown.

The cell surface antigen CD52 (CAMPATH-1) has been recently identified as a molecular target expressed abundantly on the surface of primary neoplastic MCs in patients with advanced SM [START_REF] Hoermann | CD52 is a molecular target in advanced systemic mastocytosis[END_REF]. The CD52-targeting antibody alemtuzumab has been found to induce cell death in CD52+ primary neoplastic MCs obtained from patients with SM as well as an immature human MC line (MCPV-1). Moreover, alemtuzumab improves significantly the survival of NSG xenotransplanted with MCPV-1 cells [START_REF] Hoermann | CD52 is a molecular target in advanced systemic mastocytosis[END_REF].

To conclude, current therapy for patients with SM refractory to interferon-α or cladribine is inadequate and it is recommended to involve such patients in experimental treatment protocols [START_REF] Tefferi | How we diagnose and treat WHO-defined systemic mastocytosis in adults[END_REF]. Patients with very poor prognostic ASM, MCL or SM-AHNMD (because of the AHNMD) should be considered for enrollment in prospective clinical trials. Therefore, there is still a need to identify novel druggable targets in neoplastic MCs and their progenitors in order to optimize therapy and to prolong survival of these patients. The promising novel targets should be thereafter evaluated alone or in combination with TKIs or other chemotherapy in patients with advanced SM.

Bromodomain containing protein 4 (BRD4)

Bromodomains (BRDs): readers of acetyl lysine residues

Epigenetics has been defined as heritable changes in the pattern of post-translational modifications (PTMs, also referred to as the epigenetic codes), which lead to the development of new phenotypes that are not encoded in the DNA sequences [START_REF] Holliday | The inheritance of epigenetic defects[END_REF]. Readers of PTMs are structurally diverse proteins that contain one or more effector modules that recognize and read covalent modifications of proteins and DNA [START_REF] Filippakopoulos | Targeting bromodomains: epigenetic readers of lysine References acetylation[END_REF]. Acetylation of lysine residues, the first PTMS of histone described in 1964 [START_REF] Allfrey | Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis[END_REF]) is a reversible and widespread PTM, and extensively relevant to modulation of cellular processes, including protein conformation and interaction [START_REF] Kouzarides | Acetylation: a regulatory modification to rival phosphorylation?[END_REF][START_REF] Choudhary | The growing landscape of lysine acetylation links metabolism and cell signalling[END_REF]. Moreover, lysine acetylation is abundant in large macromolecular complexes that function in chromatin remodeling, DNA damage, and cell cycle control [START_REF] Choudhary | Lysine acetylation targets protein complexes and co-regulates major cellular functions[END_REF]) and particularly in histones.

Of note, three types of proteins have been identified to regulate lysine acetylation including bromodomain (BRD) proteins (Barneda-Zahonero & Parra 2012; Filippakopoulos & Knapp 2014), histone acetyltransferases (HATs), histone deacetylases (HDACs) [START_REF] Shahbazian | Functions of site-specific histone acetylation and deacetylation[END_REF] and sirtuins (SIRTs) [START_REF] Dhalluin | Structure and ligand of a histone acetyltransferase bromodomain[END_REF][START_REF] Anand | BET bromodomains mediate transcriptional pause release in heart failure[END_REF][START_REF] Seto | Erasers of histone acetylation: the histone deacetylase enzymes[END_REF]Chen et al. 2014).

Indeed, BRD proteins act as readers of lysine acetylation state as they bind acetylated lysine residues (K Ac ). The HATs act as writers as they perform the enzymatic transfer of an acetyl group from the acetylCoA to the ε-amino group of lysine side chains of histone protein tails and display target selectivity [START_REF] Shahbazian | Functions of site-specific histone acetylation and deacetylation[END_REF], whereas HDACs and SIRTs remove acetyl groups and act as erasers [START_REF] Anand | BET bromodomains mediate transcriptional pause release in heart failure[END_REF]Fu et al. 2015).

Lysine acetylation leads to neutralization of the basic charge of the lysine residues and changes the electrostatic interaction between histones or histones and DNA and hence increases DNA accessibility to the binding of transcriptional factors, and is thus associated with activated states of chromatin by changing the nucleosome positioning and chromatin architecture [START_REF] Turner | Histone acetylation and an epigenetic code[END_REF]. More importantly, lysine acetylation provides an instructive pattern for the recruitment to chromatin of factors involved not only in transcriptional regulation, but also in DNA repair and replication [START_REF] Kouzarides | Chromatin modifications and their function[END_REF][START_REF] Shahbazian | Functions of site-specific histone acetylation and deacetylation[END_REF].

The recognition of ε-N-acetylation of lysine residues is primarily initiated by bromodomains (BRDs).

BRDs are small (of about 110 amino acids in length) evolutionarily and structurally conserved protein interaction modules that were originally identified in the early 1990s by sequence alignment as a common motif in the brahma gene from Drosophila melanogaster [START_REF] Tamkun | brahma: a regulator of Drosophila homeotic genes structurally related to the yeast transcriptional activator SNF2/SWI2[END_REF], femalesterile homeotic (fsh) and four other potential transcriptional regulators [START_REF] Haynes | The bromodomain: a conserved sequence found in human, Drosophila and yeast proteins[END_REF].

Additionally, the recognition of histone acetylation code by BRDs is selective, involved in transcription, and potentially conveys transcriptional memory across cell divisions [START_REF] Kanno | Selective recognition of acetylated histones by bromodomain proteins visualized in living cells[END_REF]). In fact, the binding interaction of BRDs with acetylated histones leads to the recruitment of additional proteins to specific sites on chromatin and, as the coassociated proteins may have enzymatic activity; this can lead to additional chromatin modification. For example, where a BRD module is part of a HAT, histone acetylation can serve as a regulator of the HAT activity [START_REF] Winston | The bromodomain: a chromatin-targeting module?[END_REF]. Further, cooperative binding to other chromatin associated proteins helps achieving a high level of targeting specificity [START_REF] Garnier | BET bromodomain inhibitors: a patent review[END_REF].

The BRDs are most commonly found as single copies, frequently in combination with other epigenetic reader domains such as PHD (plant homeodomain), PWWP (named for conserved pro-trp-trp-pro motif) and Tudor [START_REF] Chung | Small molecule bromodomain inhibitors: extending the druggable genome[END_REF]. In fact, the PHD finger, which is a zinc-finger-like motif present in nuclear proteins, is considered the most frequently associated domain with BRDs, whilst the second most common association is another BRD [START_REF] Garnier | BET bromodomain inhibitors: a patent review[END_REF]. -histone acetyltransferases (HATs) and HAT-associated proteins (such as general control of amino acid synthesis protein 5-like 2 (GCN5L2; also known as KAT2A),

-P300/CBP-associated factor (PCAF; also known as KAT2B) and bromodomain-containing protein 9 (BRD9)) [START_REF] Yang | A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A[END_REF][START_REF] Dhalluin | Structure and ligand of a histone acetyltransferase bromodomain[END_REF]

2006),

-transcriptional co-activators (such as tripartite motif-containing proteins (TRIMs) and TBP- associated factors (TAFs)) [START_REF] Venturini | TIF1gamma, a novel member of the transcriptional intermediary factor 1 family[END_REF],

-transcriptional mediators (such as TAF1, also known as TAFII250) [START_REF] Jacobson | Structure and function of a human TAFII250 double bromodomain module[END_REF],

-nuclear scaffolding proteins (such as polybromo 1 (PBRM1, also known as PB1)) [START_REF] Xue | The human SWI/SNF-B chromatinremodeling complex is related to yeast rsc and localizes at kinetochores of mitotic chromosomes[END_REF],

-the bromodomain and extra C-terminal domain (BET) family of proteins [START_REF] Wu | The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation[END_REF][START_REF] Brès | The multi-tasking P-TEFb complex[END_REF][START_REF] Muller | Bromodomains as therapeutic targets[END_REF].

BRD proteins mostly contain one or two BRDs, while some proteins, such as PB1, contain more than two BRDs [START_REF] Lovén | Selective inhibition of tumor oncogenes by disruption of super-enhancers[END_REF].

The presence of a BRD ensures appropriate targeting to specific K Ac sites, where their accompanying domains govern the mechanism by which a particular BRD-containing protein will mediate gene regulation. Bromodomains have also been involved in binding to other acetylated proteins such as

RelA (Huang et al. 2009), p53 [START_REF] Barlev | Acetylation of p53 activates transcription through recruitment of coactivators/histone acetyltransferases[END_REF]) and human immunodeficiency virus (HIV) Tat [START_REF] Dorr | Transcriptional synergy between Tat and PCAF is dependent on the binding of acetylated Tat to the PCAF bromodomain[END_REF].

Structure of bromodomains

The first BRD structure was determined by nuclear magnetic resonance (NMR) spectroscopy in 1999 [START_REF] Dhalluin | Structure and ligand of a histone acetyltransferase bromodomain[END_REF]) and since then, the solution and/or crystal structures of over 40 BRDs have been determined [START_REF] Vidler | Druggability analysis and structural classification of bromodomain acetyl-lysine binding sites[END_REF]Filippakopoulos et al. 2012).

Despite having large sequence variations, BRD modules share a conserved fold that comprises a lefthanded bundle of four α helices (named αZ, αA, αB, and αC), linked by loop regions of variable length and charge (known as ZA and BC loops), which line the K Ac binding site and determine binding specificity [START_REF] Dhalluin | Structure and ligand of a histone acetyltransferase bromodomain[END_REF]Filippakopoulos et al. 2012) (Figure 22A,B). The C-and N-termini are highly diverse and may comprise additional helices that extend the canonical BRD fold (e.g., the sixth BRD of PB1 has an additional C-terminal helix) or largely extended kinked helices that are present as C-or N-terminal extensions (for instance in TAF1L or ATAD2) (Filippakopoulos et al. 2012). Besides, cocrystal structures with peptides have demonstrated that K Ac is recognized by a central deep hydrophobic cavity (Figure 22C), where it is anchored by a hydrogen bond to an asparagine residue (N140) presents in most BRDs [START_REF] Dhalluin | Structure and ligand of a histone acetyltransferase bromodomain[END_REF][START_REF] Owen | The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase gcn5p[END_REF].

More recently, it has been demonstrated that some BRDs bind to two acetylated lysine histone marks that are simultaneously recognized by the same BRD module [START_REF] Morinière | Cooperative binding of two acetylation marks on a histone tail by a single bromodomain[END_REF]). This property is shared by all members of the BET subclass of BRDs (Filippakopoulos et al. 2012).

The most notable structural difference within the BRD core fold is a hairpin insertion located between helice αZ and the long ZA loop which is present in all the members of the family VIII (Figure 21).

Moreover, while the binding site is similar in all structures of BRDs, molecular dynamics calculations indicate that there are significant flexibility differences in the binding pocket [START_REF] Steiner | Does bromodomain flexibility influence histone recognition?[END_REF].

In contrast to the conserved fold of BRDs, their surface properties are highly diverse. The electrostatic potential of the surface area around the K Ac binding site ranges from highly positively to strongly negatively charged, suggesting that BRDs recognize largely different sequences (Filippakopoulos et al. 2012).

Moreover, differences in the acetyl-binding pockets of BRD are important not only for the characterization of the BRD proteins but also for design of selective BRD inhibitors [START_REF] Dhalluin | Structure and ligand of a histone acetyltransferase bromodomain[END_REF]Filippakopoulos et al. 2012).

The BET family of proteins

The BET protein members

The BET proteins are a novel and small class of transcriptional regulators, all localize in the nucleus, and, as mentioned earlier, belonging to the subfamily II of BRD-containing proteins (Florence & Faller 2001). This subfamily is characterized by the presence of two BRD tandems (BD1, BD2), and the extra-terminal domain as outlined in details in the next paragraph. Additionally, in mouce and human, BET family is comprised of the three ubiquitously expressed and widely spread BRD2, BRD3

and BRD4, and the testis-specific form (BRDT), which is expressed specifically in testis, and ovary (Shang et al. 2004;Paillissan et al. 2007). Drosophila melanogaster's fsh (female sterile homeotic (fsh)), and Saccharomyces cerevisiae's Bdf1 and Bdf2 are corresponding homologs in other species [START_REF] Wu | The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation[END_REF].

Furthermore, BET proteins exist in two (i.e. long and short) isoforms generated by alternatively spliced transcripts differing at their 3' ends. The predominant long isoform of human and mouse BRD4 is often the only isoform detected [START_REF] Dey | A bromodomain protein, MCAP, associates with mitotic chromosomes and affects G(2)-to-M transition[END_REF]Shang et al. 2004;[START_REF] Devaiah | Cross-talk among RNA polymerase II kinases modulates Cterminal domain phosphorylation[END_REF] and is responsible for almost all the biological functions attributed to BRD4 [START_REF] Devaiah | Cross-talk among RNA polymerase II kinases modulates Cterminal domain phosphorylation[END_REF], while the function of the short form (BRD4S, also known as HUNK1 in humans), if expressed [START_REF] French | BRD4-NUT fusion oncogene: a novel mechanism in aggressive carcinoma[END_REF] RelA, the subunit of NF-Kappa (Huang et al. 2009). In addition to their different affinity, BRDs of the BET family are known to have modest affinity for mono-acetylated lysine in a range of polypeptide contexts; however, their affinity is significantly greater when multiple acetylation sites exist within a span of one to five amino acids which occurs on histone tails and on certain transcription factors such as GATA-1 and TWIST (Dey et al. 2003;[START_REF] Morinière | Cooperative binding of two acetylation marks on a histone tail by a single bromodomain[END_REF][START_REF] Lamonica | Bromodomain protein Brd3 associates with acetylated GATA1 to promote its chromatin occupancy at erythroid target genes[END_REF]Filippakopoulos et al. 2012;Shi et al. 2014). In certain settings, this increase in affinity has been attributed to bidentate acetyl-lysine recognition that occurs within a single bromodomain pocket [START_REF] Morinière | Cooperative binding of two acetylation marks on a histone tail by a single bromodomain[END_REF][START_REF] Gamsjaeger | Structural basis and specificity of acetylated transcription factor GATA1 recognition by BET family bromodomain protein Brd3[END_REF]Filippakopoulos & Knapp 2012). BET proteins can also bind to transcription factors in a BRD-independent manner to function as transcriptional cofactors [START_REF] Wu | Phospho switch triggers Brd4 References chromatin binding and activator recruitment for gene-specific targeting[END_REF].

Interestingly, the chromosomal locations of these BRD genes are adjacent to the four Notch genes found in the human genome with BRD2 and Notch4 on chromosome 6, BRD3 and Notch1 on chromosome 9, BRD4 and Notch 3 on chromosome 19, and BRDT and Notch2 on chromosome 1 [START_REF] Kasahara | Genome dynamics of the major histocompatibility complex: insights from genome paralogy[END_REF][START_REF] Houzelstein | Growth and early postimplantation defects in mice deficient for the bromodomain-containing protein Brd4[END_REF], indicating a likely functional relationship between human BRD and Notch gene families [START_REF] Wu | The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation[END_REF].

Retention on chromosomes during mitosis is likely a distinctive feature of the BET family (Chua & Roeder 1995;[START_REF] Dey | A bromodomain protein, MCAP, associates with mitotic chromosomes and affects G(2)-to-M transition[END_REF]Dey et al. 2003;[START_REF] Kanno | Selective recognition of acetylated histones by bromodomain proteins visualized in living cells[END_REF]) because proteins of other BRD families are displaced from chromosomes during mitosis [START_REF] Martínez-Balbás | Displacement of sequencespecific transcription factors from mitotic chromatin[END_REF][START_REF] Muchardt | The hbrm and BRG-1 proteins, components of the human SNF/SWI complex, are phosphorylated and excluded from the condensed chromosomes during mitosis[END_REF][START_REF] Kruhlak | Regulation of global acetylation in mitosis through loss of histone acetyltransferases and deacetylases from chromatin[END_REF]. The presence of two BRD tandems may explain this unique feature of BET proteins as the deletion of bromodomain 1 (BD1) displaces BRD2 as well as BRD4 from mitotic chromosomes (Dey et al. 2003;[START_REF] Garcia-Gutierrez | Association of bromodomain BET proteins with chromatin requires dimerization through the conserved motif B[END_REF]. Similarly, a double BRD2 mutant with point mutations Y152K and N428A in BD1 and BD2, respectively, previously shown to affect BRD2 functionality [START_REF] Huang | Solution structure of the second bromodomain of Brd2 and its specific interaction with acetylated histone tails[END_REF][START_REF] Nakamura | Crystal structure of the human BRD2 bromodomain: insights into dimerization and recognition of acetylated histone H4[END_REF], has been reported to not associate to mitotic chromosomes [START_REF] Garcia-Gutierrez | Association of bromodomain BET proteins with chromatin requires dimerization through the conserved motif B[END_REF]. However, both BD1 and BD2 alone are not sufficient to promote association to mitotic chromosomes and other factors containing BRD tandems such as TAFII250 dissociate from mitotic chromosomes [START_REF] Kruhlak | Regulation of global acetylation in mitosis through loss of histone acetyltransferases and deacetylases from chromatin[END_REF]. Nevertheless, the small conserved B motif has been identified as a dimerization domain involved in association to mitotic chromosomes and proper recognition of acetylated chromatin by BET proteins (Garcia-Gutierrez et al.

2012).

The BET domain architecture

The domain architecture of mammalian BET proteins is depicted in Figure 23 A conserved region of 12 amino acids (KGVKRKADTTTP), found at the end of the A motif between the two BET BRD modules was recently shown to act as a nuclear localisation signal and its deletion results in mislocalisation of the mutant proteins in HEK293T cells [START_REF] Fukazawa | The conserved 12-amino acid stretch in the inter-bromodomain region of BET family proteins functions as a nuclear localization signal[END_REF].

While the function of BRDs has been well characterized, the function of other domains has been poorly investigated [START_REF] Denis | RING3 kinase transactivates promoters of cell cycle regulatory genes through E2F[END_REF][START_REF] Rahman | The Brd4 extraterminal domain confers transcription activation independent of pTEFb by recruiting multiple proteins, including NSD3[END_REF]. It is well established that BRDs of BET proteins bind to acetylated chromatin. While BRD2 seems to preferentially bind histone H4 at the mono-acetylated state (acetyl-K12), BRD4 and BRDT more efficiently recognize di-acetylated histone H4, at K5/K12 and K5/K8 positions, respectively (Dey et al. 2003;[START_REF] Kanno | Selective recognition of acetylated histones by bromodomain proteins visualized in living cells[END_REF][START_REF] Leroy | The double bromodomain proteins Brd2 and Brd3 couple histone acetylation to transcription[END_REF][START_REF] Morinière | Cooperative binding of two acetylation marks on a histone tail by a single bromodomain[END_REF][START_REF] Sasaki | Real-time imaging of histone H4 hyperacetylation in living cells[END_REF][START_REF] Ito | Real-time imaging of histone H4K12-specific acetylation determines the modes of action of histone deacetylase and bromodomain inhibitors[END_REF]. While many groups provided the evidences that the two isoforms of BRD4 (BRD4S and BRD4L) share the same N-terminal region except for the final three amino acids, have opposing effects in breast cancer growth and progression [START_REF] Crawford | Bromodomain 4 activation predicts breast cancer survival[END_REF][START_REF] Alsarraj | Deletion of the proline-rich region of the murine metastasis susceptibility gene Brd4 promotes epithelial-tomesenchymal transition-and stem cell-like conversion[END_REF][START_REF] Alsarraj | BRD4 short isoform interacts with RRP1B, SIPA1 and components of the LINC complex at the inner face of the nuclear membrane[END_REF]) and reside in physically different subnuclear compartments [START_REF] Alsarraj | BRD4 short isoform interacts with RRP1B, SIPA1 and components of the LINC complex at the inner face of the nuclear membrane[END_REF]), Floyd et al. [START_REF] Floyd | The bromodomain protein Brd4 insulates chromatin from DNA damage signalling[END_REF] have shown that BRD4

encodes A, B and C splice isoforms with two BRDs and an ET domain presenting in each isoform.

They demonstrated that the A isoform contains a CTD, which is notably absent in the B and C isoforms, and it is replaced by a divergent short 75 amino acid segment in the B isoform [START_REF] Floyd | The bromodomain protein Brd4 insulates chromatin from DNA damage signalling[END_REF]. Moreover, despite possessing identical BRDs, BRD4S has been reported to have an expanded histone binding affinity relative to BRD4L [START_REF] Alsarraj | BRD4 short isoform interacts with RRP1B, SIPA1 and components of the LINC complex at the inner face of the nuclear membrane[END_REF].

Bromodomain-mediated interactions with acetylated chromatin result in the localization of BET proteins to discrete locations along the chromosome, where they recruit other regulatory complexes to influence gene expression (Dey et al. 2003). The ET domain then serves as an interface to localize different complexes or proteins to chromatin. The identity of these associated factors may depend upon which modular domains are present. The ET domain may also interact with sequence-specific factors in order to help localize the BET protein to the active chromatin (Florence & Faller 2001).

Of BET family members, the function of BRD4 has been most thoroughly studied and will be the primary subject of the following paragraphs.

The structures of the BRD4 BD1 and BD2 have been solved and consist of 4 α-helices and 2 loops linking these α-helices [START_REF] Vollmuth | Structures of the dual bromodomains of the P-TEFb-activating protein Brd4 at atomic resolution[END_REF][START_REF] Devaiah | Cross-talk among RNA polymerase II kinases modulates Cterminal domain phosphorylation[END_REF]. The BD1 primarily binds to H3 at K14 Ac , while the BD2 domain binds to H4 at K5 Ac and K16 Ac [START_REF] Vollmuth | Structures of the dual bromodomains of the P-TEFb-activating protein Brd4 at atomic resolution[END_REF][START_REF] Devaiah | Cross-talk among RNA polymerase II kinases modulates Cterminal domain phosphorylation[END_REF]; that explains how BRD4 is retained on the chromatin during mitosis in a range of cell types. The solved ET domain structure consists of 3 α-helices and a loop [START_REF] Vollmuth | Structures of the dual bromodomains of the P-TEFb-activating protein Brd4 at atomic resolution[END_REF];

Devaiah & Singer 2012).

Biological functions of BRD4 and BET proteins

BRD4 and BET proteins are mitotic bookmarks, and cell cycle regulators

Upon its binding to acetylated chromatin, BRD4 maintains the higher-order chromatin structure (Wang et al. 2012). Unlike non-BET BRD proteins [START_REF] Dey | A bromodomain protein, MCAP, associates with mitotic chromosomes and affects G(2)-to-M transition[END_REF][START_REF] Umehara | Structural basis for acetylated histone H4 recognition by the human BRD2 bromodomain[END_REF][START_REF] Belkina | BET domain co-regulators in obesity, inflammation and cancer[END_REF]), BRD4 as well as BRD2 [START_REF] Leroy | The double bromodomain proteins Brd2 and Brd3 couple histone acetylation to transcription[END_REF]) remains bound to mitotic chromatin, therefore maintaining the epigenetic memory [START_REF] Nishiyama | Brd4 is required for recovery from antimicrotubule drug-induced mitotic arrest: preservation of acetylated chromatin[END_REF][START_REF] Yang | Brd4 recruits P-TEFb to chromosomes at late mitosis to promote G1 gene expression and cell cycle progression[END_REF][START_REF] Belkina | BET domain co-regulators in obesity, inflammation and cancer[END_REF]. Also, BRD4 is considered as an essential protein as Brd4-/embryos die shortly after implantation owing to mitotic defect [START_REF] Dey | A bromodomain protein, MCAP, associates with mitotic chromosomes and affects G(2)-to-M transition[END_REF][START_REF] Maruyama | A Mammalian bromodomain protein, brd4, interacts with replication factor C and inhibits progression to S phase[END_REF]Dey et al. 2003;[START_REF] Belkina | BET domain co-regulators in obesity, inflammation and cancer[END_REF]. Such defects may occur owing the failure of Aurora B spindle checkpoint during mitosis, as Aurora B is BRD4 dependent [START_REF] You | Regulation of aurora B expression by the bromodomain protein Brd4[END_REF][START_REF] Belkina | BET domain co-regulators in obesity, inflammation and cancer[END_REF]. Furthermore, Brd4

heterozygotes mice display severe pre-and postnatal growth defects as well as several anatomical abnormalities including head malformation, absence of subcutaneous fat, cataracts, and abnormal liver cells [START_REF] Houzelstein | Growth and early postimplantation defects in mice deficient for the bromodomain-containing protein Brd4[END_REF]. Moreover, knockdown of Brd4 leads to a significant reduction in the growth of cultured cells [START_REF] Wu | Brd4 links chromatin targeting to HPV transcriptional silencing[END_REF]) and embryonic stem cells do not grow in culture without its presence [START_REF] Nishiyama | Brd4 is required for recovery from antimicrotubule drug-induced mitotic arrest: preservation of acetylated chromatin[END_REF]. Interestingly, the inhibition of Brd4 causes a G2/M cell cycle arrest [START_REF] Dey | A bromodomain protein, MCAP, associates with mitotic chromosomes and affects G(2)-to-M transition[END_REF] and its overexpression in turn is associated with a G1/S arrest (Dey et al. 2003).

Together these data reveal the role of BRD4 as a regulator of the cell cycle and cell growth at various stages. Similar to Brd4, knockdown of Brd2 is lethal in mice [START_REF] Gyuris | The chromatin-targeting protein Brd2 is required for neural tube closure and embryogenesis[END_REF][START_REF] Shang | Double bromodomaincontaining gene Brd2 is essential for embryonic development in mouse[END_REF][START_REF] Wang | Brd2 disruption in mice causes severe obesity without Type 2 diabetes[END_REF], whereas no Brd3 knockout model system is available [START_REF] Belkina | BET domain co-regulators in obesity, inflammation and cancer[END_REF].

Furthermore, BRD4 remains associated with chromatin during mitosis, leading to rapid de-compaction of the surrounding chromatin and to post-mitotic transcription [START_REF] Devaiah | Two faces of brd4: mitotic bookmark and transcriptional lynchpin[END_REF]. BRD4 functions as a positive transcriptional elongation factor complex (PTEFb) recruiter during interphase and as a mitotic bookmark responsible for accelerating post mitotic gene activation [START_REF] Dey | Brd4 marks select genes on mitotic chromatin and directs postmitotic transcription[END_REF][START_REF] Devaiah | Two faces of brd4: mitotic bookmark and transcriptional lynchpin[END_REF]. Indeed, BRD4 accelerates the dynamics of mRNA synthesis by decompacting chromatin, facilitating transcriptional re-activation and marks the start sites of many M/G1 genes, and stimulates the expression of G1 genes by binding to multiple G1 gene promoters in a cell cycle-dependent manner and thus promotes cell cycle progression to S phase as shown using a small hairpin (sh) RNA approach [START_REF] Mochizuki | The bromodomain protein Brd4 stimulates G1 gene transcription and promotes progression to S phase[END_REF][START_REF] Zhao | Gene bookmarking accelerates the kinetics of post-mitotic transcriptional re-activation[END_REF]). The Brd4 shRNA vector stably knocked down Brd4 protein expression by about 90% in NIH3T3 cells and mouse embryonic fibroblasts and Brd4 knockdown cells were found to be growth impaired and grew more slowly than control cells [START_REF] Mochizuki | The bromodomain protein Brd4 stimulates G1 gene transcription and promotes progression to S phase[END_REF]. Also, BRD4 seems to be required for the G2 to M phase transition of the cell cycle because microinjection of BRD4-specific antibodies leads to cell cycle arrest [START_REF] Belkina | BET domain co-regulators in obesity, inflammation and cancer[END_REF].

Intoduction 86 Moreover, through binding to acetylated histone tails, BET proteins have been described as transcriptional activators with a prominent role in the control of cell cycle-associated genes. Brd4 has been involved in controlling expression of cyclin D1 and other G1-associated genes required for progression to the S phase [START_REF] Mochizuki | The bromodomain protein Brd4 stimulates G1 gene transcription and promotes progression to S phase[END_REF][START_REF] Yang | Brd4 recruits P-TEFb to chromosomes at late mitosis to promote G1 gene expression and cell cycle progression[END_REF]. Similarly, Brd2 has been shown

to play a crucial role as cell cycle regulators of normal cells as it has been implicated in controlling the expression of cyclins A2 and D1 [START_REF] Sinha | Bromodomain analysis of Brd2-dependent transcriptional activation of cyclin A[END_REF][START_REF] Denis | Identification of transcription complexes that contain the double bromodomain protein Brd2 and chromatin remodeling machines[END_REF][START_REF] Leroy | The double bromodomain proteins Brd2 and Brd3 couple histone acetylation to transcription[END_REF][START_REF] Belkina | BET domain co-regulators in obesity, inflammation and cancer[END_REF]). In fact, the key transcriptional regulators genes of S phase, E2F1 and E2F2, are associated with BRD2 multi-protein complexes [START_REF] Belkina | BET domain co-regulators in obesity, inflammation and cancer[END_REF]). However, BRD3-dependent functional relationships with the cell cycle control machinery in normal cells are poorly understood [START_REF] Belkina | BET domain co-regulators in obesity, inflammation and cancer[END_REF]Fu et al. 2015), although there is some evidence that forced expression of Brd3 in nasopharyngeal carcinoma (NPC) downregulates the RB-E2F pathway, which regulates the transition from G1 to S phase [START_REF] Zhang | microRNA-141 is involved in a nasopharyngeal carcinoma-related genes network[END_REF][START_REF] Belkina | BET domain co-regulators in obesity, inflammation and cancer[END_REF]).

BRD4 and BET proteins are protein scaffolds

In view of their role as protein scaffolds, BET family proteins were firstly detected as protein scaffolds for the recruitment of transcription factors and chromatin organizers required in transcription initiation and elongation. They recruit a variety of proteins to chromatin and transcription sites (Fu et al. 2015).

BRD4 recruits PTEFb via its BD2 to site of active transcription and may be recruited by transcriptional mediator complexes independent of PTEFb [START_REF] Wu | The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation[END_REF]. Additionally, the ET domain of BRD4 recruits independently transcription-modifying factors, including glioma tumor suppressor candidate region gene 1 (GLTSCR1); NSD3, a SET domain-containing histone methyltransferase; JMJD6, a histone arginine demethylase; and CHD4 (chromodomain helicase DNAbinding protein 4), a catalytic component of the NuRD nucleosome remodeling complex [START_REF] Rahman | The Brd4 extraterminal domain confers transcription activation independent of pTEFb by recruiting multiple proteins, including NSD3[END_REF][START_REF] Liu | Brd4 and JMJD6-associated anti-pause enhancers in regulation of transcriptional pause release[END_REF]. Different from BRD4, BRD2 binds to ε-aminoacetyl groups of nucleosomal histone lysines in a PTEFb-independent manner and, with related BRD proteins, it provides a scaffold on chromatin to recruit E2F proteins [START_REF] Denis | RING3 kinase transactivates promoters of cell cycle regulatory genes through E2F[END_REF][START_REF] Peng | Brd2 is a TBP-associated protein and recruits TBP into E2F-1 transcriptional complex in response to serum stimulation[END_REF], HDACs [START_REF] Denis | Identification of transcription complexes that contain the double bromodomain protein Brd2 and chromatin remodeling machines[END_REF],

HAT [START_REF] Sinha | Bromodomain analysis of Brd2-dependent transcriptional activation of cyclin A[END_REF] and proteins involved in chromatin remodeling including SW1/SNF subunits and elements of the mediator complex [START_REF] Houzelstein | Growth and early postimplantation defects in mice deficient for the bromodomain-containing protein Brd4[END_REF][START_REF] Belkina | BET domain co-regulators in obesity, inflammation and cancer[END_REF], thereby coupling histone acetylation to transcription. Similar with BRD2, BRD3 may regulate cell processes through E2F-RB pathway. Moreover, it can directly recognized acetylated transcription factor, GATA1, which is essential for the targeting of GATA1 to chromatin [START_REF] Vollmuth | Structures of the dual bromodomains of the P-TEFb-activating protein Brd4 at atomic resolution[END_REF]).

BRD4 and BET proteins function as transcriptional regulators

BET proteins play a key role in regulating the transcription of growth-promoting genes [START_REF] Houzelstein | Growth and early postimplantation defects in mice deficient for the bromodomain-containing protein Brd4[END_REF]. Besides binding acetylated chromatin via its BRDs, BRD4 interacts with nonhistone proteins to regulate transcription, DNA replication, cell cycle progression and other cellular activities [START_REF] Wu | The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation[END_REF]. The role of BRD4 in transcriptional regulation was first suggested by the constituents of its associated protein complex including Mediator and PTEFb [START_REF] Jiang | Mammalian mediator of transcriptional regulation and its possible role as an end-point of signal transduction pathways[END_REF][START_REF] Jang | The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase IIdependent transcription[END_REF][START_REF] Yang | Recruitment of P-TEFb for stimulation References of transcriptional elongation by the bromodomain protein Brd4[END_REF][START_REF] Leroy | The double bromodomain proteins Brd2 and Brd3 couple histone acetylation to transcription[END_REF][START_REF] Garnier | BET bromodomain inhibitors: a patent review[END_REF]. Indeed, BRD4 was first identified as an interaction partner of mouse Mediator complex, a multiprotein coactivator that links transcription factors to polymerase II activation [START_REF] Jiang | Mammalian mediator of transcriptional regulation and its possible role as an end-point of signal transduction pathways[END_REF]. It was also found to associate with Mediator in human cells [START_REF] Wu | The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation[END_REF]Dawson et al. 2011). Moreover, BRD4 and Mediator occupy similar sites across the genome and can stabilize one another's occupancy at certain regions [START_REF] Donner | CDK8 is a positive regulator of transcriptional elongation within the serum response network[END_REF][START_REF] Lovén | Selective inhibition of tumor oncogenes by disruption of super-enhancers[END_REF].

The bromodomain protein BRD4 is one major regulator of PTEFb and stimulates RNA polymerase IIdependent transcription [START_REF] Jang | The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase IIdependent transcription[END_REF][START_REF] Yang | Recruitment of P-TEFb for stimulation References of transcriptional elongation by the bromodomain protein Brd4[END_REF]Chen et al. 2014). In contrast to BRD2 and BRD3, BRD4 presents CTD which is essential for BRD4 association with PTEFb [START_REF] Bisgrove | Conserved P-TEFb-interacting domain of BRD4 inhibits HIV transcription[END_REF]). Indeed, tow regions of BRD4 bind directly to PTEFb: the CTD interacts with cyclin T1 and CDK9 while BD2 recognizes the acetylated region of cyclin T1 [START_REF] Jang | The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase IIdependent transcription[END_REF][START_REF] Bisgrove | Conserved P-TEFb-interacting domain of BRD4 inhibits HIV transcription[END_REF][START_REF] Schröder | Two-pronged binding with bromodomain-containing protein 4 liberates positive transcription elongation factor b from inactive ribonucleoprotein complexes[END_REF]). These interactions result in preventing PTEFb from association with 7SK sn RNA/HEXIM1 (7SK small nuclear RNA/hexamethylene bis-acetamide inducible protein 1), a ribonucleoprotein complex that sequesters PTEFb in an inactive state [START_REF] Jang | The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase IIdependent transcription[END_REF][START_REF] Yang | Recruitment of P-TEFb for stimulation References of transcriptional elongation by the bromodomain protein Brd4[END_REF], resulting in phosphorylation of the CTD of RNA polymerase II primarily at serine 2 (ser2) of its heptad (YSPTSPS) repeats and thereby facilitating productive mRNA transcript elongation [START_REF] Price | P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II[END_REF][START_REF] Garriga | Cellular control of gene expression by T-type cyclin/CDK9 complexes[END_REF][START_REF] Nechaev | Pol II waiting in the starting gates: Regulating the transition from transcription initiation into productive elongation[END_REF][START_REF] Devaiah | Cross-talk among RNA polymerase II kinases modulates Cterminal domain phosphorylation[END_REF]Devaiah et al. 2012;[START_REF] Shi | The mechanisms behind the therapeutic activity of BET bromodomain inhibition[END_REF]. Notably, the interaction between BRD4 and PTEFb is particularly important for rapid transcriptional induction, as occurs at mitosis exit and in response to signaldependent activation of DNA-binding transcription factors [START_REF] Yang | Brd4 recruits P-TEFb to chromosomes at late mitosis to promote G1 gene expression and cell cycle progression[END_REF][START_REF] Dey | Brd4 marks select genes on mitotic chromatin and directs postmitotic transcription[END_REF][START_REF] Zippo | Histone crosstalk between H3S10ph and H4K16ac generates a histone code that mediates transcription elongation[END_REF]. The recruitment of active PTEFb is crucial for the sustained presence of RNA polymerase II in active genes and for the transcription initiation and elongation. Indeed, PTEFb and RNA polymerase II are crucial for the expression of cell proliferation supporting genes, including c-MYC and its target genes [START_REF] Rahl | c-Myc regulates transcriptional pause release[END_REF].

Furthermore, Devaiah et al. [START_REF] Devaiah | Cross-talk among RNA polymerase II kinases modulates Cterminal domain phosphorylation[END_REF]Devaiah et al. 2012) have reported BRD4 as an atypical RNA polymerase II ser2 kinase which directly phosphorylates the heptad repeats of RNA polymerase II at ser2 and remains associated with RNA polymerase II until productive elongation, suggesting additional mechanism through which BRD4 might directly regulate this elongation.

However, the biochemical mechanism underlying that intrinsic kinase activity is to date unclear since BRD4 lacks homology to other known kinase domains [START_REF] Shi | The mechanisms behind the therapeutic activity of BET bromodomain inhibition[END_REF].

Moreover, the ET domain of BRD4 has been linked to transcriptional regulation independent of PTEFb by interacting with other chromatin regulators including the lysine methyltransferase NSD3

and the arginine demethylase JMJD6 [START_REF] Rahman | The Brd4 extraterminal domain confers transcription activation independent of pTEFb by recruiting multiple proteins, including NSD3[END_REF][START_REF] Liu | Brd4 and JMJD6-associated anti-pause enhancers in regulation of transcriptional pause release[END_REF]). The ET domain has been also found to associate with ATP-dependent nucleosome remodeling enzymes SW1/SNF and CHD4 [START_REF] Rahman | The Brd4 extraterminal domain confers transcription activation independent of pTEFb by recruiting multiple proteins, including NSD3[END_REF]. These interactions imply that BRD4 alters chromatin structure at its occupied sites [START_REF] Shi | The mechanisms behind the therapeutic activity of BET bromodomain inhibition[END_REF].

Involvement of BET proteins in cancers and impact of BET bromodomain inhibitors

As key drivers of transcriptional events, BET proteins have been reported to be deregulated in a variety of diseases, such as viral infections, inflammation, obesity, metabolic disorders, autoimmune diseases, neurological disorders and cancers (for a review on the role of BET proteins in various diseases, see [START_REF] Filippakopoulos | Targeting bromodomains: epigenetic readers of lysine References acetylation[END_REF]. Here, we will only present the present knowledge on the involvement of BET proteins, and particularly BRD4, in malignant diseases, since we have focused our work on this field.

Role of BET proteins in solid tumors

Accumulating evidences have revealed the involvement of BET family proteins in tumorigenesis [START_REF] Kadota | Identification of novel gene amplifications in breast cancer and coexistence of gene amplification with an activating mutation of PIK3CA[END_REF]Balkina & Denis 2012;[START_REF] Barbieri | Bromodomains as therapeutic targets in cancer[END_REF][START_REF] Khochbin | When are the BET factors the most sensitive to bromodomain inhibitors?[END_REF][START_REF] Zhang | BRD4 promotes tumor growth and epithelial-mesenchymal transition in hepatocellular carcinoma[END_REF].

The first clue linking BRD4 with tumor development was the finding of BRD4-NUT fusion oncogene in nuclear protein in testis (NUT) midline carcinoma (NMC), a rare and aggressive form of intrathoracic squamous carcinoma. Through the chromosomal rearrangement of NUT gene, BRD4 contributes to NMC development (Kubonichi et al. 1991;[START_REF] French | BRD4 bromodomain gene rearrangement in aggressive carcinoma with translocation t(15;19)[END_REF][START_REF] French | BRD4-NUT fusion oncogene: a novel mechanism in aggressive carcinoma[END_REF]French 2010;[START_REF] French | Pathogenesis of NUT midline carcinoma[END_REF]. Of note, in the majority of the cases of NMCs, most of the coding sequence of NUT on chromosome 15q14 is fused with BRD4 creating chimeric genes that encode BRD4-NUT fusion protein which blocks differentiation and supports a high cell proliferation rate, while in the remaining cases, NUT is fused to BRD3 (French et al. 2008;French 2010). Also, the BRD4 inhibitor JQ1 treatment leads to terminal differentiation of BRD4-NUT-positive NMC cell lines and shows antitumor properties in mouse xenograft models of NMC [START_REF] Filippakopoulos | Selective inhibition of BET bromodomains[END_REF], leading to the initiation of clinical studies with I-BET762 [START_REF] Shi | The mechanisms behind the therapeutic activity of BET bromodomain inhibition[END_REF][START_REF] Jung | Targeting BET References bromodomains for cancer treatment[END_REF]. Figure 24 shows the structure of JQ1 [START_REF] Belkina | BET domain co-regulators in obesity, inflammation and cancer[END_REF]).

In addition to genomic rearrangement, BET proteins contribute to cancer development through other mechanisms as they are known to be deregulated in cancer [START_REF] Belkina | BET domain co-regulators in obesity, inflammation and cancer[END_REF]Wang & Filippakopoulos 2014;Chen et al. 2014;[START_REF] Zhou | Therapeutic targeting of BET bromodomain protein, Brd4, delays cyst growth in ADPKD[END_REF]. In fact, BRD2, BRD3, and BRD4 proteins are upregulated in glioblastoma multiforme, the most common and malignant adult brain tumor [START_REF] Pastori | BET bromodomain proteins are required for glioblastoma cell proliferation[END_REF][START_REF] Pastori | The Bromodomain protein BRD4 controls HOTAIR, a long noncoding RNA essential for glioblastoma proliferation[END_REF]. BRD4 is also found upregulated in in Pkd1 mutant renal epithelial cells and tissues [START_REF] Zhou | Therapeutic targeting of BET bromodomain protein, Brd4, delays cyst growth in ADPKD[END_REF]. Recently, BRD2 has been shown to induce lung adenocarcinoma through its interaction with RUNX3 in a KRAS-dependent fashion (Lee et al. 2013).

Besides, the expression of BRD4 has been found deregulated in breast cancer biopsies [START_REF] Crawford | Bromodomain 4 activation predicts breast cancer survival[END_REF], and in human colon cancer cell lines and primary tumors [START_REF] Rodriguez | Aberrant epigenetic regulation of bromodomain BRD4 in human colon cancer[END_REF]). In addition, the BRD4 transcriptional signature in breast cancer was found to discriminate rate of disease progression [START_REF] Crawford | Bromodomain 4 activation predicts breast cancer survival[END_REF].

Interstingly, the functional effects of BRD4 on metastasis and stem cell transformation have been recently determined to reside in the C terminus proline rich domain of the protein since deletion of this region results in epithelial-to-mesenchymal transition (EMT) and stem cell-like conversion [START_REF] Alsarraj | Deletion of the proline-rich region of the murine metastasis susceptibility gene Brd4 promotes epithelial-tomesenchymal transition-and stem cell-like conversion[END_REF]Alsarraj & Hunter 2012). Very recently, BRD4 has been also found to promote both tumor growth and EMT in hepatocellular carcinoma [START_REF] Zhang | BRD4 promotes tumor growth and epithelial-mesenchymal transition in hepatocellular carcinoma[END_REF].

Furthermore, BRDT transcript has been identified as a cancer testis antigen in 25% of non-small cell lung cancer and 1 of 12 cases of both esophageal cancer and squamous cell carcinoma of the head and neck [START_REF] Scanlan | Expression of cancer-testis antigens in lung cancer: definition of bromodomain testis-specific gene (BRDT) as a new CT gene, CT9[END_REF].

Notably and MYC [START_REF] Shang | Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription[END_REF][START_REF] Castro-Rivera | Estrogen regulation of cyclin D1 gene expression in ZR-75 breast cancer cells involves multiple enhancer elements[END_REF]. Consistently, BRD4 is required for proliferation of ERα-positive breast and endometrial cancer cells and uterine growth in mice [START_REF] Nagarajan | Bromodomain protein BRD4 is required for estrogen receptor-dependent enhancer activation and gene transcription[END_REF]. Interestingly, BRD4 occupies transcriptional start sites and EREs upon estrogen stimulation and also regulates ERα-dependent eRNA production, which requires CDK9 [START_REF] Hah | Enhancer transcripts mark active estrogen receptor binding sites[END_REF][START_REF] Nagarajan | Bromodomain protein BRD4 is required for estrogen receptor-dependent enhancer activation and gene transcription[END_REF].

Moreover, the interaction with TWIST, a basic helix-loop-helix transcription factor controlling mesoderm formation during normal development and also (EMT) during cancer progression, implicates a role for BRD4 in the biology of epithelial cancers, which is possibly related to the therapeutic effects of BET inhibitors observed in xenograft models of breast cancer (Shi et al. 2014).

Role of BET proteins in hematopoietic malignancies

The constitutive expression of Brd2 in B cell progenitors in mouse models has been also shown to promote a B cell malignancy which has a proteomic signature similar to human diffuse large B cell lymphoma, suggesting that BRD2, in addition to BRD4, might be relevant to lymphoid neoplasms [START_REF] Greenwald | E mu-BRD2 transgenic mice develop B-cell lymphoma and leukemia[END_REF]Staudt & Dave 2005;[START_REF] Romesser | Development of a malignancy-associated proteomic signature for diffuse large B-cell lymphoma[END_REF][START_REF] Delmore | BET bromodomain inhibition as a therapeutic strategy to target c-Myc[END_REF]Tolani et al. 2013).

In addition, using small hairpin RNA (shRNA) library targeting 243 known chromatin regulators, BRD4 has been identified as a required factor for the maintenance of acute myeloid leukemia (AML).

Indeed, knockdown of BRD4 in vitro and in vivo results in a robust antileukemic activity against AML (Zuber et al. 2011;[START_REF] Blobel | Short hairpin RNA screen reveals bromodomain proteins as novel targets in acute myeloid leukemia[END_REF].

Importantly, the highly selective and recently reported BET bromodomain inhibitors including JQ1

Role of BET inhibitors in solid tumors and hematopoietic malignancies

BET inhibitiors exhibit remarkable preclinical effects in various somatic cancers, such as melanoma [START_REF] Segura | BRD4 sustains melanoma proliferation and represents a new target for epigenetic therapy[END_REF] [START_REF] Bihani | Resistance to everolimus driven by epigenetic regulation of MYC in ER+ breast cancers[END_REF].

The MYC isoform NMYC has also found to be downregulated in neuroblastoma by BET inhibitors [START_REF] Puissant | Targeting MYCN in neuroblastoma by BET bromodomain inhibition[END_REF]Wyce et al. 2013;[START_REF] Shahbazi | Histone deacetylase 2 and N-Myc reduce p53 protein phosphorylation at serine 46 by repressing gene transcription of tumor protein 53-induced nuclear protein 1[END_REF]. However, MYC is not always the main transcriptional target of BET proteins [START_REF] Lockwood | Sensitivity of human lung adenocarcinoma cell lines to targeted inhibition of BET epigenetic signaling proteins[END_REF][START_REF] Cheng | Inhibition of BET bromodomain targets genetically diverse glioblastoma[END_REF][START_REF] Lenhart | Sensitivity of Small Cell Lung Cancer to BET Inhibition Is Mediated by Regulation of ASCL1 Gene Expression[END_REF],

suggesting the existence of additional, downstream targets of BET proteins and that MYC expression is driven by unique, tumor type-specific regulatory mechanisms (Wyspiańska et al. 2013;[START_REF] Kumar | GLI2-dependent c-MYC upregulation mediates resistance of pancreatic cancer cells to the BET bromodomain inhibitor JQ1[END_REF]. For instance, JAK2V617F-driven AML line HEL (human erythroleukemic) displays sensitivity to JQ1 without a pronounced effect on MYC expression (Wyspiańska et al. 2013). Transcriptional profiling of these cells following BET inhibition shows prominent downregulation of LMO, a leukemogenic transcription coregulatory (Wyspiańska et al. 2013).

Furthermore, the anticancer effects of BET inhibitors may be mediated in MYC-independent manner through suppression of the NF κ B pathway. Beside its role in in the immune system, NF κ B is well known as a heterodimeric transcription factor that enhances cancer cell proliferation and plays an important role in the pathogenesis of cancer [START_REF] Hayden | NF-κB, the first quarter-century: remarkable progress and outstanding questions[END_REF]. Treating cells with JQ1 disrupts the RelA/Brd4 interaction (Huang et al. 2009) and leads to proteasome-mediated RelA degradation, which may further suppress the NF k B pathway [START_REF] Zou | Brd4 maintains constitutively active NF-κB in cancer cells by binding to acetylated RelA[END_REF].

Interestingly, targeting viral replication by BET inhibition could have therapeutic benefits in many virus-induced cancers. Through its binding to the HPV proteins indicated earlier [START_REF] You | Interaction of the bovine papillomavirus E2 protein with Brd4 tethers the viral DNA to host mitotic chromosomes[END_REF]Schweiger et al. 2006), BRD4 has been reported to play a role in the initiation of viral DNA replication but not for continuing replication. Indeed, human papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers (in over than 90% of the cases,

Parkin & Bray 2006) and BRD4 is implicated in the regulation of DNA replication by the two viral proteins, E1 and E2 (HPV19 E1-E2) which can be effectively disrupted after treatment with JQ1 [START_REF] Gauson | Evidence supporting a role for TopBP1 and Brd4 in the initiation but not continuation of human papillomavirus 16 E1/E2mediated DNA replication[END_REF].

Similarly, in KSHV infection and Epstein-Barr-virus-associated cancers like the Burkitt's lymphomas, BRD4 is involved in the maintenance of viral infections via its interactions with viral encoded proteins [START_REF] You | Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen interacts with bromodomain protein Brd4 on host mitotic chromosomes[END_REF][START_REF] Lin | The EBNA1 protein of Epstein-Barr virus functionally interacts with Brd4[END_REF], while in primary effusion lymphoma (PEL) which is an aggressive form of non Hodgkin's B cell lymphoma associated with infection by KSHV, JQ1 and I-BET151 exert their activities through genome-wide perturbation of MYC-dependent genes (Tolani et al. 2013).

Recently, BRD4 has been additionally identified directly linked to the Merkel cell polyomavirus (MCV) protein large T antigen (LT) which is found in 80% of Merkel cell carcinoma cases, a rare and highly aggressive neuroectodermal tumor arising from mechanoreceptor Merkel cells [START_REF] Feng | Clonal integration of a polyomavirus in human Merkel cell carcinoma[END_REF]Wang et al. 2012). Merkel cell polyomavirus is the first human polyomavirus to be definitively linked to cancer. BRD4 interacts and colocalizes with MCV large T antigen (LT)/replication origin complex in the nucleus and recruits replication factor C (RFC) to the viral replication sites (Wang et al. 2012).

Given that diverse transcriptional programmes are attenuated in a tissue-specific manner by BET inhibitors [START_REF] Roe | BET Bromodomain Inhibition Suppresses the Function of Hematopoietic Transcription Factors in Acute Myeloid Leukemia[END_REF], and preclinical targeting of BET proteins has had initial success in addition to the diverse and tissue-specific roles of individual BRDs, it will be challenging to target specific diseases without severe side effects. Despite the emerging preclinical and clinical evidences for therapeutic and promising benefits of BET inhibition [START_REF] Chaidos | Inhibition of bromodomain and extra-terminal proteins (BET) as a potential therapeutic approach in haematological malignancies: emerging preclinical and clinical evidence[END_REF][START_REF] Yu | Small-molecule BET inhibitors in clinical and preclinical development and their therapeutic potential[END_REF], concerns have to be raised regarding their safety. For instance, upon targeting BET proteins, care must be taken to monitor the effects on super-enhancer-controlled stem cell specific transcription factors such as OCT4 and PRDM14, which control stem cell identity and cell fate decisions (Di Micco et al.

2014).

Moreover, BET inhibition strongly modulates the innate immune response and may increase the risk of infections when used clinically. As an instance, in mice infected with the bacterial pathogen Listeria monocytogenes, JQ1 strongly downregulated the expression of nitric oxide (NO) synthase 2, which reduced NO production and increased the susceptibility to develop colitis [START_REF] Wienerroither | Regulation of NO synthesis, local inflammation, and innate immunity to pathogens by BET family proteins[END_REF].

More interestingly, recent findings have revealed a transcription-independent role of BRD4 in DNA damage response (DDR). In fact, BRD4 isoform B, the isoform lacking the PTEFb-interacting region as mentioned earlier, functions as an endogenous inhibitor of DDR signaling upon ionizing radiation by recruiting the condensin II chromatin remodeling complex to acetylated histones through BRDmediated interactions. Consistent with these findings, knockdown or inhibition of BRD4 isoform B promotes cancer cell survival following ionizing radiation in association with increased H2AX phosphorylation, raising that induction of DDR signaling by BRD4 inhibitor might promote radioresistance [START_REF] Floyd | The bromodomain protein Brd4 insulates chromatin from DNA damage signalling[END_REF]. A similar effect was previously reported for a C-terminal truncation BRDT, as outlined earlier, that also stimulates chromatin compaction (Pivot-Pajot et al.

2003)

, suggesting that a regulatory role in chromatin decompaction may be attributed to the CTM found on the longer BET variants (BRD4 and BRDT) (Wang & Filippakopoulos 2015).

RESEARCH OUTLINE

It is well established in the literature that human MC models described before 2014 have many limitations. As an instance, none of them has a functional FcεRI and a short doubling time. Thus there was still a need to develop a relevant, inexpensive and reproducible in vitro model representing at the best the human MCs in terms of the main morphological and biological properties. In addition, no fully relevant models of advanced SM harboring only the KIT D816V mutant, the most commonly mutant found in SM, were still available. In addition, no curative treatment can be offered to date to patients with advanced SM, for whom prognosis remains poor to date. Thus, the two mjor aims of our work were:

1) to establish an in vitro model representing morphologically and biologically the human MCs (ROSA KIT WT cell line) and another in vitro model representing neoplastic human MCs (ROSA KIT D816V cell line) mimicking at the best the phenotype of malignant cells found in patients with advanced SM.

2) to investigate the potential of the epigenetic reader BRD4 as a new therapeutic target in advanced SM (ASM and MCL). Thereafter, the goal of our work was to demonstrate that BRD4 inhibition alone or in combination with inhibitors of other relevant signaling pathways, might be a new and effective therapeutic approach to treat patients with advanced SM.

RESULTS

RESULTS Publication 1. "A new human mast cell line expressing a functional IgE receptor converts to tumorigenic growth by KIT D816V transfection"

In this first article, we reported the establishment and characterization of a novel FcεRI-positive SCFdependent human MC line, named ROSA KIT WT cell line, from normal umbilical CB-derived CD34+ enriched mononuclear cells (MNCs). CD34+ cells were cultured in the presence of recombinant human SCF (rhSCF), IL-6 and IL-3 (only for the first week) as described (Arock et al. 2008). After 8 weeks, cells continued to grow in culture with a short doubling time (around 48 hours). The ROSA KIT WT cells are round cells with a metachromatic cytoplasm and strongly positive for tryptase and KIT, but they are negative for chymase. They can differentiate terminally (MCTC) after their co-culture with a monolayer of stromal cells (MS-5 cells). The ROSA KIT WT cells display a relatively mature MC phenotype as they express FcεRI receptor, KIT (CD117), CD33, CD4, CD9, CD203c, and CD300a, while they do not express CD2 and CD25. These cells are clonogenic and easily freezable. Moreover, the stimulation with IgE + anti-IgE leads to activation and degranulation of the cells. Thus, the ROSA KIT WT cell line seems to be the best model available today for studying the mechanisms involved in the differentiation and activation of human MCs, as well as for high throughput screening of antiallergic agents (Saleh, Wedeh et al. 2014).

We then transfected ROSA KIT WT cells with a lentivirus coding for Green Fluorescent Protein (GFP) + KIT D816V. Then, GFP+ cells were sorted and cultured without SCF, giving rise to a SCFindependent cell line. The ROSA KIT D816V cells display more mature phenotype than ROSA KIT WT cells, with a significant proportion of spindle-shaped cells, which is a hallmark of neoplastic MCs in BM of patients with SM. Interestingly, we found that the responsiveness of ROSA KIT D816V cells to IgEdependent stimuli was weak compared with that of ROSA KIT WT cells. Interestingly, we have also reported that the KIT D816V+ MCs obtained from patients with SM do not show increased FcεRIdependent histamine release or CD63 upregulation. Moreover, ROSA KIT D816V cells engraft efficiently NOD/SCID IL-2Rγ-/-(NSG) mice, giving rise to an aggressive SM (ASM)-like disease. Of note, the NSG mice bearing ROSA KIT D816V -derived tumors do not show mediator-related symptoms. Thus,

Introduction

Mast cells (MCs) are tissue-resident cells that play an important role in inflammatory and allergic reactions. 1 Two types of human MCs have been described: MC T containing only tryptase, and MC TC containing substantial amounts of tryptase and chymase. 2,3 MC T cells are mostly found in mucosa, and MC TC cells are more commonly detected in the connective tissues. 4-7 The development of tissue MCs from hematopoietic progenitors is regulated by stem cell factor (SCF), the ligand of KIT. 8-10 MCs express large amounts of vasoactive and immunomodulatory mediators as well as high-affinity receptors for immunoglobulin E (IgE; FceRI). As a result, MCs are potent effector cells of type I allergic reactions. 11-13 Moreover, MCs play a pivotal role in the initiation and regulation of immune responses against various pathogens. 14-17 The MC lineage can give rise to a distinct myeloid neoplasm, namely mastocytosis, characterized by MC expansion in one or more organ systems. 18-21 In mastocytosis patients, clinical symptoms result from MC-derived mediators and from the destructive invasion of MCs into various organs. 22 However, the exact mechanisms underlying MC activation in systemic mastocytosis (SM) remain unknown. In SM, the KIT D816V mutation in the catalytic domain of KIT is found in up to 90% of all patients. 23 This mutant initiates a number of signal transduction events and is thought to trigger abnormal MC activation in patients with SM. However, not all patients with SM suffer from these Submitted October 23, 2013;accepted March 19, 2014. Prepublished online as Blood First Edition paper, March 27, 2014;DOI 10.1182DOI 10. /blood-2013-10-534685-10-534685.

R.S. and G.W. contributed equally to this work as joint first authors.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.

© 2014 by The American Society of Hematology BLOOD, 3 JULY 2014 x VOLUME 124, NUMBER 1 symptoms, even if their neoplastic MCs display KIT D816V. Other studies have shown that such mediator-related symptoms are seen especially in SM patients who suffer from a coexisting allergy. 22,24-26 Studies of the biology of human MCs have mainly been conducted by using MC lines or MCs obtained from primary cultures of CD34 1 cells. 27 The only 3 human MC lines available so far are HMC-1, 28 LAD2, 29 and LUVA. 30 None of these cell lines coexpress KIT D816V and a functional IgE receptor. Here, we describe the establishment and characterization of a novel FceRI-positive SCFdependent human MC line, ROSA KIT WT , and of a factor-independent subclone, ROSA KIT D816V , produced by stably expressing KIT D816V in ROSA KIT WT cells. ROSA KIT D816V cells expressed the IgE receptor and engrafted NOD/SCID IL-2Rg 2/2 (NSG) mice. However, unexpectedly, the responsiveness of ROSA KIT D816V cells to IgE-dependent stimuli was weak when compared with normal MCs, ROSA KIT WT cells, or normal basophils.

Materials and methods

Establishment and characterization of a new human SCF-dependent MC line, ROSA KIT WT

A sample of normal umbilical cord blood (CB) was collected after informed consent was given by the patient in September 2009 (Clinic Ambroise Paré, Bourg-la-Reine, France). Mononuclear cells were separated by density centrifugation (Ficoll-Hypaque; GE Healthcare). CD34 1 cells were enriched by immunomagnetic sorting by using the MACS system (Miltenyi Biotec) and were cultured in the presence of recombinant human SCF (rhSCF) (80 ng/mL; R&D Systems) exactly as described. 27 After 8 weeks, cells continued to grow in culture with virtually all cells resembling MCs.

The complete coding sequences of the KIT gene expressed in ROSA cells were analyzed as previously described 31 and were found to be normal (wildtype). The resulting SCF-dependent MC line was termed ROSA KIT WT . This cell line was further transfected with KIT D816V, converting it into an SCFindependent clone, ROSA KIT D816V (see supplemental Methods available on the Blood Web site).

Morphologic and immunocytologic characterization of the ROSA cell lines

The 2 cell lines were analyzed by microscopy on cytospin preparations stained with May-Grünwald Giemsa (MGG) or toluidine blue, or on MGG-stained smears produced from a cell pellet. Transmission electronic microscopy (TEM) was performed on ROSA KIT WT cells by using standard techniques. 32 Immunocytochemical staining was carried out on cytospin preparations of ROSA cells by using antibodies (Abs) against tryptase, chymase, and KIT (CD117) as described. 33

Analysis of the effects of various cytokines on ROSA cells

In the first set of experiments, ROSA KIT WT or ROSA KIT D816V cells (3 3 10 5 per milliliter in 96-well plates; 100 mL per well) were incubated in medium with or without rhSCF (80 ng/mL) at 37°C and 5% CO 2 for 4 days. On days 1, 2, 3, and 4, 10 mL of WST-1 (Water Soluble Tetrazolium salt-1; Roche Applied Science) were then added to the corresponding wells, and the cells were incubated for 3 additional hours at 37°C. The numbers of viable cells were measured by reading the absorbance at 570 nm using a Multiskan-MS plate reader (Thermo Labsystems). Because rhSCF has no significant (increasing) effects on the growth of ROSA KIT D816V cells, we treated ROSA KIT WT (cells with or without SCF) or ROSA KIT D816V (cells without SCF) with various cytokines, each at 100 ng/mL: interleukin-3 (IL-3), IL-4, IL-6, IL-9, interferon gamma (IFN-g), granulocyte macrophage colony-stimulating factor (GM-CSF), and nerve growth factor (NGF) (all from R&D Systems) at 37°C and 5% CO 2 for 3 days. Then, 10 mL of WST-1 was added, and the cells were incubated for 3 additional hours at 37°C before the numbers of viable cells were measured.

Assays of cell activation and mediator release in ROSA cells

ROSA KIT WT or ROSA KIT D816V cells were first primed with recombinant human IL-4 (20 ng/mL; BioLegend) and human monoclonal IgE (2 mg/mL; Calbiochem) for 5 days to enhance the expression of FceRI before stimulation. 27 To determine the expression of CD203c and b-hexosaminidase release, cells were incubated in control medium or medium containing antihuman IgE (5 mg/mL) (BioValley) or 1 mM of calcium ionophore (Cai) A23187 (Sigma) for 1 hour. Then, cells were stained by an allophycocyanin-conjugated anti-human CD203c Ab. The fluorescence intensity was measured by using a FACSCalibur flow cytometer (BD Biosciences). For the b-hexosaminidase release assay, stimulated cells were centrifuged, and b-hexosaminidase activity was measured by spectrophotometry in the supernatants and in cell lysates. 34 The percentage of b-hexosaminidase release was determined as described. 35 To determine histamine release, cells were incubated in control buffer or buffer containing mouse anti-human IgE (clone Depsilon2; Immunotech) (0.1 to 10 mg/mL) for 30 minutes at 37°C. Thereafter, cells were centrifuged, and the histamine content of the cell-free supernatants and the cell pellets was analyzed by radioimmunoassay (Immunotech). The percentage of histamine release was calculated as described. 36 To determine prostaglandin D2 (PGD2) and tumor necrosis factor a (TNF-a) release, cells were incubated in control medium, anti-IgE Ab (5 or 10 mg/mL), or Cai (1 mM) at 37°C for 1 hour (for PGD2 release) or 6 hours (for TNF-a release) and then centrifuged. PGD2 and TNF-a levels were determined in the supernatants by using a specific enzyme immunoassay for PGD2 (Cayman Chemical) and a specific enzyme-linked immunosorbent assay for TNF-a (R&D systems) according to the manufacturers' instructions.

Western blotting

HMC-1.2 cells exhibiting 2 KIT point mutations, V560G and D816V (kindly provided by Dr J. H. Butterfield, Mayo Clinic, Rochester, MN), ROSA KIT WT cells, and ROSA KIT D816V cells were incubated in control medium or with rhSCF (80 ng/mL) for 5 minutes. Western blots were performed with Abs against total or phosphorylated STAT5 (all from Santa Cruz Biotechnology), total KIT, phosphorylated KIT, and total or phosphorylated AKT (all from Cell Signaling Technology). A mouse monoclonal IgG1 anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Ab (Santa Cruz Biotechnology) was used as loading control.

Assessment of the antiproliferative effects of tyrosine kinase inhibitors on ROSA cells

Cells (3 3 10 5 per milliliter; 100 mL per well) were incubated in 96-well plates with 1 mM of imatinib, 1 mM dasatinib (both supplied by Sequoia Research) or 1 mM midostaurin (Sigma) diluted in dimethylsulfoxide (DMSO; 0.1% final) or with DMSO alone (0.1%) in serum-free medium (with rhSCF at 80 ng/mL for ROSA KIT WT ) at 37°C (5% CO 2 ) for 48 hours. The number of viable cells was then determined in each condition as described in supplemental Methods.

Xenotransplantation of ROSA cells in NSG mice

ROSA KIT WT or ROSA KIT D816V cells (0.4 to 5 3 10 6 ) were either injected into the tail vein (intravenously) or transplanted under the skin (subcutaneously) bilaterally dorsocaudal of the scapula (3 to 5 mice per group) of adult NSG mice (The Jackson Laboratory). NSG mice were irradiated (2.4 Gy) 24 hours prior to intravenous injection. After injection, mice were inspected daily and euthanized as soon as disease-related symptoms appeared (intravenous group), the tumor grew to a maximum of 1 cm in diameter (subcutaneous group), or after a maximum observation period of 6 months. Bone marrow (BM) cells were obtained from flushed femurs, tibias, and humeri. Subcutaneous tumors were collected and dispersed by collagenase digestion. In addition, BM, lung, liver, spleen, and tumor samples were prepared for histologic and immunohistochemical analyses (supplemental Methods).

Flow cytometry analyses of ROSA cells

ROSA KIT WT cells and ROSA KIT D816V cells were analyzed by flow cytometry using a panel of monoclonal Abs directed against various leukocyte differentiation antigens (supplemental Table 1). In select experiments, xenotransplanted ROSA cells were quantified in BM samples and tumor dispersates obtained from NSG mice by flow cytometry as described 37 by using phycoerythrin-labeled monoclonal Abs directed against human CD117 (BioLegend) and CD45 (BD Biosciences). Engrafted ROSA cells were defined as CD45 1 /CD117 1 cells (at least 0.1% of all nucleated cells) detectable in mouse BM cell suspensions.

Statistics

Unless otherwise stated, data represent the mean values 6 standard deviation of at least 3 independent experiments. When appropriate, data were analyzed by using the Student t test. Differences were considered as statistically significant if P , .05.

Animal studies

Animal studies were approved by the ethics committee of the Medical University of Vienna and were carried out in accordance with guidelines for animal care and protection and protocols approved by Austrian law (GZ 66.009/0040-II/10b/2009). This study was conducted in accordance with the Declaration of Helsinki.

Results

Establishment and growth characteristics of ROSA KIT WT , a new SCF-dependent human MC line, and of its SCF-independent subclone ROSA KIT D816V

ROSA KIT WT cells have a doubling time of 24 hours, and are split every 3 to 4 days. Optimal growth is obtained at 2 3 10 5 cells per milliliter in rhSCF (80 ng/mL). Because the KIT D816V mutant is commonly found in neoplastic MCs in patients with SM, 38 we transfected ROSA KIT WT cells with a lentiviral vector encoding KIT D816V. The resulting SCF-independent line, named ROSA KIT D816V , exhibited the same doubling time as ROSA KIT WT cells cultured with rhSCF (80 ng/mL). The two ROSA cell lines have been deposited to the Collection National de Cultures de Microorganismes (CNCM, Institut Pasteur, Paris), under the number CNCM I-4551 for the KIT WT cell line and CNCM I4552 for the KIT D816V 1 cell line. In addition, the two ROSA cell lines have been patented (Patent WO/2013/064639). The ultrastructure of ROSA KIT WT cells was analyzed by transmission electron microscopy. In addition, ROSA KIT WT cells were treated with an antibody against (E) tryptase or (F) KIT (CD117). Positivity was revealed by indirect immunocytochemistry as described in "Material and methods." (G) Cytospin preparations or (H-I) glass smears of ROSA KIT D816V cells were stained by MGG. (G) Note the presence of many granules in ROSA KIT D816V cells and of apoptotic cells, a phenomenon likely to be related to terminal maturation induced by the introduction of the KIT D816V mutant. (H) On glass smear, a significant proportion of the ROSA KIT D816V cells presented with spindle-shaped appearance, which was not noticed for (I) ROSA KIT WT cells. Magnification: A and C, 3200; B, E, G, H, and I 3500; D, 32000; F, 31000.

ROSA KIT WT and KIT D816V cells are MCs by morphology, TEM, and phenotype MGG-stained ROSA KIT WT cells are round cells with a relatively high nuclear-to-cytoplasm ratio (Figure 1A-B). The slightly basophilic cytoplasm contained purple granules, their number and size being variable from cell to cell. Metachromasia was documented by staining with toluidine blue (Figure 1C). TEM studies showed that ROSA KIT WT cells have a large nucleus with loose chromatin and 1 to 2 nucleoli (Figure 1D). The cytoplasm contained ribosomes, a welldeveloped Golgi apparatus, abundant mitochondria, and secretory granules with spherical electron-dense cores surrounded by electronlucent space similar to those described in human CB-derived MCs (CBMCs). 39 In addition, almost all cells stained strongly positive for tryptase (Figure 1E) and KIT (Figure 1F) but were negative for chymase (data not shown).

Morphologic analysis of ROSA KIT D816V cells showed significant differences when compared with ROSA KIT WT cells. Indeed, ROSA KIT D816V cells appear to be more granulated (Figure 1G). Interestingly, a significant percentage of ROSA KIT D816V cells exhibited a spindle-shaped morphology on smears (Figure 1H), which was not observed with ROSA KIT WT cells (Figure 1I). However, despite signs of maturation, ROSA KIT D816V cells retained an MC T phenotype because they expressed only tryptase but not chymase (data not shown).

Conventional cytogenetics and fluorescence in situ hybridization analyses of the ROSA cell lines

In initial cultures as well as after 12 months in culture, ROSA KIT WT cells exhibited a complex karyotype, with a derivative chromosome 1 (der( 1)inv(1)(p31q21)del(1)(q24q32)) and 2 subclones: one with a complete trisomy 5, and the other was predominant with a partial trisomy 5 (1del(5)(q14q34)) (supplemental Figure 1A). Results from fluorescence in situ hybridization analysis confirmed the expression of aberrations observed by karyotyping in ROSA cells (supplemental Figure 1B-C). Interestingly, ROSA KIT D816V cells exhibited the same karyotype after lentiviral infection (not shown).

Analysis of cell surface antigens in ROSA cells and responses to IL-4

Both ROSA cell lines have a comparable phenotype with a strong positivity for FceRIa and KIT (CD117) (supplemental Table 2). The level of KIT (CD117) was higher in ROSA KIT D816V cells compared with ROSA KIT WT cells. A number of other antigens known to be expressed by human cultured MCs were also detected on ROSA cells, such as CD32, CD44, CD50, CD66a, CD166, CD203c, and CD300a (supplemental Table 2). 40 In addition, IL-4 induced a significant decrease in the expression of KIT as well as an increase in expression of FceRI in both ROSA cell lines (supplemental Figure 2). These data strongly suggest that ROSA cells are MCs.

Response of ROSA KIT WT and ROSA KIT D816V cells to various cytokines

When comparing the effect of rhSCF (80 ng/mL) on the proliferation of ROSA KIT WT cells and of ROSA KIT D816V cells during a 4-day period, we observed that ROSA KIT WT cells deprived of SCF were not able to grow, whereas ROSA KIT WT cells cultured in the presence of SCF, as well as ROSA KIT D816V cells cultured in the absence of SCF or presence of rhSCF, proliferated at an apparently similar rate (Figure 2A), suggesting that constitutive activation of the mutant KIT D816V is sufficient to induce optimal growth of ROSA cells.

We then investigated the potential effects of IL-3, IL-4, IL-6, IL-9, IFN-g, GM-CSF, or NGF (all at 100 ng/mL) on spontaneous or (A) ROSA KIT WT or ROSA KIT D816V cells were treated or not treated with rhSCF (80 ng/mL) for 4 days at 37°C. At each time point, 10 mL of 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added in corresponding wells, and the cells were incubated for 3 additional hours in an incubator at 37°C. The number of living cells was then measured for each condition by reading the absorbance at 570 nm. (B) ROSA KIT WT (with or without SCF) or ROSA KIT D816V cells (without SCF) were treated with various cytokines (all at 100 ng/mL) for 3 days. At that time, 10 mL of MTT was added in corresponding wells, and the cells were incubated for 3 additional hours in an incubator at 37°C. The number of living cells was then measured for each condition by reading the absorbance at 570 nm. Data presented are the mean 6 standard deviation (SD) from 3 independent experiments. OD, optical density.

SCF-driven proliferation of ROSA KIT WT cells in the absence or presence of SCF (80 ng/mL) for 3 days. In addition, the same cytokines were added to ROSA KIT D816V cells cultured without SCF. As shown in Figure 2B, NGF, and to a lesser extent IL-3 and GM-CSF, were able to partly rescue ROSA KIT WT cells from cell death induced by SCF withdrawal. By contrast, none of the cytokines tested had any effect on increasing proliferation of ROSA KIT WT cells in the presence of SCF or on the proliferation of ROSA KIT D816V cells in the absence of SCF, except IL-4 and IFN-g, which decreased the SCF-induced proliferation of ROSA KIT WT cells and the autonomous proliferation of ROSA KIT D816V cells (Figure 2B).

Fc«RI cross-linked ROSA KIT WT cells release MC mediators

Anti-IgE-induced activation of ROSA KIT WT cells resulted in a significant upregulation of CD203c (Figure 3A), which was not the case neither for ROSA KIT D816V cells, nor for purified neoplastic BM MCs from SM patients (supplemental Results and Figures 5 and7). Corresponding results were obtained in a b-hexosaminidase-release assay. In fact, anti-IgE-activated ROSA KIT WT cells released up to 38% of the enzyme after 1 hour (Figure 3B). As expected, calcium ionophore A23187 induced a strong increase in the expression of CD203c on ROSA KIT WT cells (Figure 3A) as well as a release of up to 80% of b-hexosaminidase (Figure 3B), which is consistent with Figure 3. Biologic effects of the cross-linking of Fc«RI on ROSA KIT WT cells. ROSA KIT WT cells were primed first with recombinant human IL-4 (recombinant human IL-4; 20 ng/mL) and human monoclonal IgE (2 mg/mL) for 5 days to increase the expression of FceRI. (A) To determine CD203c upregulation, ROSA KIT WT primed cells were stimulated by anti-human IgE (5 mg/mL) or by Ca-ionophore (Cai) (1 mM) for 1 hour. After labeling with an anti-human CD203c Ab coupled with allophycocyanin (APC), the fluorescence intensity was measured by using a FACSCalibur flow cytometer. (B) To determine b-hexosaminidase release, ROSA KIT WT primed cells were stimulated by antihuman IgE or by Cai for 1 hour. b-hexosaminidase enzymatic activity was measured in the cell-free supernatants and after cell lysis as described in Materials and methods. (C) To determine histamine release, primed ROSA KIT WT cells were stimulated with various concentrations of anti-human anti-IgE Ab at 37°C for 30 minutes. Thereafter, cells were centrifuged at 4°C, and the supernatants and cell pellets were analyzed for histamine content by using a specific radioimmunoassay kit. Results of histamine release are expressed as percentage of total histamine. (D) To examine synthesis of PGD2, primed cells were stimulated by anti-human IgE (5 or 10 mg/mL) or by Cai (1 mM) for 1 hour. Thereafter, cells were centrifuged, and the cell-free supernatants were analyzed for PGD2 content by using a specific enzyme immunoassay (EIA) kit. (E) To study the synthesis of TNF-a, primed ROSA KIT WT cells were stimulated by anti-human IgE (5 or 10 mg/mL) or by Cai (1 mM) for 6 hours. Thereafter, cells were centrifuged, and the cellfree supernatants were analyzed for TNF-a content by using a specific EIA kit. For each parameter, data presented are the mean 6 SD from 3 independent experiments. *Significantly different from the control (IL-4 1 IgE) at P , .05. previous data. 41 In addition, anti-IgE-induced activation of ROSA KIT WT cells resulted in histamine release (Figure 3C). PGD2 and TNF-a were also synthesized and released by ROSA KIT WT cells upon stimulation with anti-IgE or Cai A23187 (Figure 3D-E). Besides, the cellular histamine level averaged approximately 15 pg per cell in ROSA KIT WT cells and ROSA KIT D816V cells, which is more than in primary CBMCs (4.5 pg per CBMC), 42 LAD2 cells (3.1 pg per cell), or HMC-1 cells (0.9 pg per cell). 29 KIT phosphorylation and KIT-dependent signaling are constitutively activated in ROSA KIT D816V cells Western blotting was performed with anti-phospho-KIT Ab (Y703) on ROSA cells and on HMC-1.2 cells in the presence or absence of rhSCF. As shown in Figure 4A, in the absence of rhSCF, ROSA KIT D816V and HMC-1.2 cells exhibited a constitutively phosphorylated KIT (p-KIT), whereas ROSA KIT WT cells exhibited only low levels of p-KIT. Stimulation with rhSCF resulted in higher phosphorylation of KIT in ROSA KIT WT cells and increased phosphorylation of KIT in ROSA KIT D816V cells but not in HMC-1.2 cells (Figure 4A).

We also examined the phosphorylation of the serine residue 473 of AKT in the presence or absence of rhSCF in ROSA cells and in HMC-1.2 cells. In contrast to ROSA KIT WT cells, which require the presence of SCF to express phospho-AKT, ROSA KIT D816V cells exhibited a significant phosphorylation of AKT in the absence of rhSCF, which was even higher than in HMC-1.2 cells (Figure 4B).

Moreover, we demonstrated the constitutive phosphorylation of STAT5 in ROSA KIT D816V cells but not in ROSA KIT WT cells (even in the presence of rhSCF; Figure 4C). Finally, rhSCF did not alter p-STAT5 levels in ROSA KIT D816V , whereas it increased this expression in HMC-1.2 cells (Figure 4C). Anti-human glyceraldehyde-3-phosphate dehydrogenase demonstrated equal loading in all conditions (Figure 4D)

ROSA cells can be used as a major new model system for in vitro drug testing

We evaluated the inhibitory effects of three tyrosine kinase inhibitors, imatinib, dasatinib, and midostaurin (PKC412), each at 1 mM, on the proliferation of ROSA cell lines. In line with published results, 43 ROSA KIT WT cells were sensitive to imatinib, dasatinib, and midostaurin, whereas imatinib had no effects on ROSA KIT D816V cells (Figure 5). As expected, dasatinib and midostaurin were found to inhibit the growth of ROSA KIT D816V cells (Figure 5).

The 2 ROSA cell lines differentially engraft NSG mice BM samples (mice injected intravenously) or dispersed tumors (mice injected subcutaneously) were analyzed for the presence of CD117 1 / CD45 1 cells. Nearly all the mice injected intravenously with the 2 different types of ROSA cells developed a significant BM engraftment, with a mean percentage of CD117 1 /CD45 1 cells significantly higher in the mice injected with ROSA KIT D816V (52%) than in those injected with ROSA KIT WT cells (2.62%) (Table 1). Of 5 mice injected subcutaneously with ROSA KIT WT cells, 1 developed a subcutaneous tumor. This was similar to the other group, in which 1 of the 4 mice injected subcutaneously with ROSA KIT D816V cells presented a subcutaneous tumor (Table 1). Interestingly, cytocentrifuged BM-engrafted ROSA KIT WT cells stained with MGG kept a morphology comparable to the one observed in vitro, whereas BM-engrafted ROSA KIT D816V cells exhibited spindle-shaped features resembling those of MCs from patients with KIT D816V-positive SM (Figure 6). 44 In addition, there was a highly significant correlation between the level of BM engraftment by CD117 1 /CD45 1 cells and the level of tryptase-positive cells on BM biopsy (supplemental Figure 9). Finally, in line with previous data showing that the microenvironment can influence MC phenotype, 3 there was a clear tendency toward increased expression of chymase in subcutaneous tumors compared with BM-grafted cells (supplemental Table 5).

Discussion

MC research suffers from a lack of FceRI-positive human MC cell lines established from normal hematopoietic progenitors. Here we present a new tryptase-positive, chymase-negative, FceRI-positive SCF-dependent human MC line, ROSA KIT WT , derived from CD34 1 cells of a normal human CB, with a normal KIT receptor. Their morphology and phenotype closely resemble those of primary cultured CBMCs. 29,42,45 However, in contrast to CBMCs, ROSA KIT WT cells can be cultured for years without loss of clonal stability, changes in phenotype, or significant alteration in their functional properties. Moreover, ROSA KIT WT cells are easy to freeze and thaw by using conventional techniques (supplemental Methods). All these features suggest that ROSA cells represent a novel tool for MC research.

So far, only 2 FceRI-positive human cell lines have been established: LAD2 and LUVA. 29,30 These 2 cell lines have several limitations, such as a long doubling time for LAD2 or a weak expression of FceRI by LUVA cells. Thus, the establishment of a ROSA KIT WT cell line that has a short doubling time and stably expresses a functional FceRI at high levels represents a new advanced tool for investigating human MC functions and for highthroughput screening of anti-allergic therapeutics.

KIT is a key receptor regulating the development of normal and neoplastic MCs, as evidenced by the presence of activating mutations in the KIT gene. 22 In SM, the most frequent defect found, the KIT D816V point mutation, accounts for the pathophysiology of the disease. 46,47 However, in vitro models available for studying the impact of KIT D816V mutation on human MCs are limited. Indeed, the only model available is the HMC-1 cell line and its 2 subclones HMC-1.1 and HMC-1.2. 48 Both clones contain the KIT V560G mutation, whereas HMC-1.2 additionally exhibits the KIT D816V mutation. 48 Unfortunately, HMC-1 cells do not bear the KIT D816V mutation alone, and there is no HMC-1 subclone with a normal KIT gene, making it difficult to compare signaling pathways activated by normal or mutant forms of KIT in the same cell model. To overcome these limitations, we established the KIT D816V-positive SCFindependent ROSA KIT D816V MC line, which constitutes the first in vitro model of human MC with KIT D816V mutant alone and for which a KIT WT equivalent exists.

ROSA KIT D816V cells express CD117, CD203c, and tryptase but they also express FceRI at a level comparable to that of ROSA KIT WT cells. Interestingly, ROSA KIT D816V cells could not be activated by anti-IgE in the same way as ROSA KIT WT cells (data presented in supplemental Results and in Figure 5). These discrepant results led us to investigate whether malignant MCs from different subcategories of KIT D816V-positive SM were also insensitive to such activation. Interestingly, anti-IgE stimulated by BM MCs from such patients released little or no histamine and did not upregulate the activation marker CD63, and these data are presented in supplemental Results and in supplemental Figures 5 and7. So far, the mechanisms underlying this phenomenon remain unknown. A decrease in expression of IgE receptors or other molecules relevant to MC activation in KIT D186V-transformed cells was excluded in our experiments. However, Itoh et al 49 have recently shown that MCs chronically exposed to SCF display a marked attenuation of FceRImediated degranulation and cytokine production. Such desensitization may also play a role in KIT D816V-positive cells in which the KIT receptor is chronically phosphorylated. Since FceRI and KIT use common signaling pathways, cross-desensitization may occur. 50 Alternatively, KIT D816V may induce the expression of negative regulators of MC activation, such as phosphatases. All in all, these findings might explain why many patients with SM, even with high MC burden, do not suffer at all from IgE-related mediator-related symptoms. Nevertheless, a number of patients may suffer from mediator-release symptoms related to non-IgE triggers, such as drugs, infection, or alcohol. 51 Activation of KIT recruits a number of signaling pathways in MCs. Specifically, activation of the PI3-K/AKT pathway was shown to contribute to KIT-dependent proliferation, survival, maturation, adhesion, or activation. 52 Interestingly, the introduction of KIT D816V in ROSA cells induced the constitutive activation of AKT, which is in line with previously reported data. 53 In addition, KIT D816V recruits alternative signaling pathways not evoked by KIT WT, particularly STAT5. 54-56 Interestingly, ROSA KIT D816V cells constitutively expressed p-STAT5, suggesting that they use the same alternative pathways as MCs in mastocytosis patients. Moreover, using inhibitors of AKT or STAT5, we showed that ROSA KIT D816V cells are more sensitive to their antiproliferative effects than the ROSA KIT WT cells (supplemental Methods, Results, and Figure 8). This is consistent with a previous report showing that (1) oncogenic KIT controls neoplastic MC growth through a STAT5/PI3-kinase signaling complex activating AKT and (2) knockdown of AKT or STAT5 activity inhibits growth of neoplastic KIT D816V-positive MCs. 47 To the best of our knowledge, this is the first demonstration that de novo introduction of the KIT D816V mutation leads to constitutive phosphorylation of STAT5 in a model of human neoplastic MCs. Thus, ROSA KIT D816V cells represent valuable tools for exploring the mechanisms involved in mutant KITrelated signaling pathways and provide a robust model for screening potential STAT5 inhibitors. Moreover, in line with previously reported data, 57,58 ROSA KIT WT cells, but not ROSA KIT D816V cells, were sensitive to imatinib. Of note, ROSA cell lines were equally sensitive to dasatinib and midostaurin, which is also consistent with previous reports. 59,60 Thus, the 2 ROSA cell lines seem to be suitable models for in vitro screening of selective inhibitors of the different forms of KIT.

Mastocytosis research suffers from a lack of animal models mimicking human SM. Indeed in the 2 models already published, which were obtained by using mice transgenic for mutant KIT in which the transgenes were selectively expressed in MCs, 46,61 the targeted cells in both cases were murine MCs. Here, we show that human neoplastic KIT D816V-positive MCs can engraft at a high rate in NSG mice, not only in the BM, but also in spleen and lung, giving rise to a disease mimicking, at best, the situation encountered in SM patients. Indeed, as evidenced by immunohistochemistry for human tryptase on BM sections of NSG mice injected intravenously with ROSA KIT D816V cells, compact infiltrates of tryptase-positive human MCs can be seen. Together with the spindle-shaped appearance of the neoplastic MCs found on BM smears of the animals, and with their positivity for KIT D816V, our animal model exhibits several features resembling SM in humans, 62 making it the most similar model for human SM described so far.

In summary, we have established a new human FceRI-positive MC line exhibiting the major characteristics of normal MCs, with several advantages over available human MC models in vitro, which might support future studies on the biology of MCs and on their pharmacologic modulation. In addition, we have derived a unique model of human MCs that expresses the KIT D816V mutant producing a mastocytosis-like disease in NSG mice. These noteworthy features should facilitate the development of new pathogenetic concepts and the testing of new anti-neoplastic drug therapies.

Supplemental Methods

Lentiviral transfection of ROSA KIT WT cells with KIT D816V

Human KIT cDNA was cloned in pRRL lentiviral vector. 1 Production of lentivirus encoding KIT D816V and infection of ROSA KIT WT cells were performed exactly as previously described. 2 The efficiency of infection was measured by flow cytometry (detection of green fluorescence of GFP) after 4 days. The GFP+ cells were selected by cell sorting and placed immediately in SFM deprived of rhSCF. The cells were maintained in culture for more than 2 years by regular dilution (every 3 or 4 days) in SFM without rhSCF. The resulting human autonomous MC line was termed ROSA KIT D816V .

KIT-sequencing

Total RNA was extracted from 5.10 6 ROSA KIT WT cells or from the same number of ROSA KIT D816V cells, using an RNeasy Mini Kit (Qiagen S.A.). RNA was reverse transcribed into cDNA using a StrataScript first-strand synthesis system (Stratagene) and random hexamer primers in a total volume of 25 l, according to the manufacturer's instructions. Next, the KIT coding sequence was amplified from 2.5 l of cDNA by PCR, using HotStarTaq DNA polymerase (Qiagen S.A.) and the primers already published by us 3 for 40 cycles at 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 45 seconds. The PCR products were purified using a GeneClean III KIT (Qbiogene), and the entire KIT coding region was directly sequenced using a BigDye terminator v1.1 kit (Applied Biosystems), the sequencing primers already published, 3 and an ABI Prism 3100 sequencer (Applied Biosystems). By these means, no mutations in KIT were found in ROSA KIT WT cells whereas, as expected, ROSA KIT D816V cells exhibited two variants of the KIT gene at position 816: GAC (WT) and GTC (D816V) (data not shown).

Quantitative PCR (qPCR) detection of KIT WT and KIT D816V mRNA

Total RNA from 5.10 6 ROSA KIT WT and ROSA KIT D816V , or HMC1.2 as a control, was isolated using TRIzol (Life Technology) following exactly the manufacturer's protocol. RNA concentration and purity was determined using a Nanovue plus spectrophotometer (GE Healthcare) and samples were stored at -20°C until use. First strand cDNA synthesis was conducted using the iScript Select cDNA synthesis kit (Biorad) according to the manufacturer's instructions.

Real time PCR was performed using a LightCycler 1.5 Instrument (Roche) using LightCycler ® FastStart DNA Master PLUS SYBR Green I (Roche). Primer sequences used were designed as described by Kristensen et al. 4 : KIT WT F; a common forward primer 5'-AGAGACTTGFCAGCCAGAAAA-3', a reverse primer 5'-TTAACCACATAATTAGAATCATTCTTGATCA-3' for KIT D816V, a reverse primer 5'-TGCAGGACTGTCAAGCAGAG-3' for KIT WT. Reactions were compared to the levels of expression of mRNA for Hypoxanthine Phosphoribotransferase (HPRT) amplified using the following primers: HPRT F; 5'-ATGGACAGGACTGAACGTCTTGC-3'; and HPRT R; 5'-GACACAAACATGATTCAAATCCCTGA-3'. Real time PCR reactions were carried out in duplicate using the following cycle conditions: 95°C for 10 min, followed by 50 cycles at 95°C for 15 s, at 60 °C for 1 min and at 72°C for 1 min. All qPCR experiments included a no template control (water). A serial dilution of the cDNA mixture was used to produce a standard curve and the cDNA of HMC1.2 served as a calibrator for gene expression analysis. The mRNA expression of KIT WT and KIT D816V in ROSA cells was calculated relative to the expression of these genes in control HMC1.2, according to the delta-delta C t method (2 -∆∆Ct ).

Karyotyping and Fluorescence in situ Hybridization of the ROSA cell lines

Chromosomes were prepared after 24-48 hours of in vitro culture. Conventional R-banding analysis was performed with standard methods and karyotypes were defined according to the International System for Human Cytogenetic Nomenclature (ISCN 2013). Fluorescence in situ hybridization (FISH) analyses on metaphases were performed with commercially available probes, according to the manufacturer's protocols. The following probes provided by Kreatech (Amsterdam, Netherlands) were used: EGR1(5q31)/CSF1R(5q33)/hTERT(5p15) and whole chromosome painting (WCP)1. Results were recorded using Eclipse 90i fluorescence microscope (Nikon) fitted with appropriate filters, CCD camera and digital imaging software from Alphelys (Genikon).

Analysis of selected surface markers expressed on ROSA cell lines by flow cytometry

The expression of cell surface antigens on ROSA KIT WT and ROSA KIT D816V cells was analyzed by direct immunofluorescence using FACSCalibur and Aria flow cytometer (BD Biosciences) and the characteristics of the various antibodies used are provided in supplementary Table S1. Briefly, 2x105 untreated cells were incubated with monoclonal murine anti-human antibodies specific for FcεRIα, KIT (CD117), CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD25, CD34, CD54, CD203c (all from Biolegend), CD13, CD14, CD16, CD17, CD19, CD20, CD22, CD23, CD24, CD30, CD31, CD32, CD33, CD38, CD44, CD45, CD48, CD50, CD52, CD56, CD59, CD63, CD64, CD66a, CD69, CD71, CD87, CD90, CD95, CD96, CD105, CD114, CD115, CD116, CD123, CD127, CD133, CD134, CD138, CD144, CD146, CD150, CD164, CD166, CD213a, CD218a, CD300a, and CD309 (all from BD Biosciences). The expression of FcεRIα and KIT was also determined before or after incubation of the cell lines with rhIL-4 (20 ng/mL) (R&D) and human monoclonal IgE (2μg/mL) (Calbiochem) for 5 days. Isotype controls were used for each experiment, and at least 10,000 events were recorded for each analysis on a BD FACSCalibur and BD FACSAria.

Analysis of the clonogenicity of ROSA cells

Five hundred cells were plated in 35 mm Petri dishes containing 1 mL methylcellulose based medium (Methocult, Stem Cell technologies), supplemented with 20% fetal calf serum (FCS; PAA) and 0.3% bovine serum albumin (PAA) and with human rhSCF at 80 ng/mL for ROSA KIT WT but not for ROSA KIT D816V cells. Petri dishes were then placed in an incubator at 37°C and 5% of CO 2 . Colonies of more than 50 cells were enumerated by microscopy after 15 days of culture.

Cryopreservation

ROSA cell lines were frozen and stored according to conventional freezing methods by suspension of cells at 10 7 /mL in a freezing medium made by 90% FCS (PAA) and 10% of DMSO (Sigma), samples being cooled immediately at -80°C overnight then kept in liquid nitrogen until used.

Analysis of the effect of withdrawal of SCF on the apoptosis of ROSA KIT WT cells

ROSA KIT WT cells were seeded at 2x10 5 cells per mL in flaks, in the absence or presence of recombinant human SCF(80 ng/mL) and incubated at 37°C and 5% CO 2 for 7 days (d0 to d7). At d0, d2, d4 and d7, an aliquot (10 5 cells) was harvested for each condition, transferred in flow cytometry tubes, washed with cold PBS and treated by 10 μg/mL Annexin V-APC (BD Biosciences) and 5 μg/ml propidium iodide (PI, Biolegend) in 100 μL incubation buffer (PBS supplemented with 1% FCS) for 15 minutes at RT in the dark and then analyzed by flow cytometry using a FACSCalibur (BD Biosciences).

Analysis of the effect of non-IgE triggers on the activation of ROSA cell lines

ROSA KIT WT or ROSA KIT D816V cells were incubated in control medium or medium containing peptidoglycan purified from S. aureus (PGN) (1, 5 or 10 g/mL; Sigma), compound 48/80 (1, 5 or 10 g/mL; Sigma) or 1 µM of calcium ionophore A23187 (Sigma) for 1 hour. Then, cells were stained by an anti-human CD203c antibody (Ab) (Biolegend) coupled with APC. The fluorescence intensity was measured using a FACSCalibur (BD Biosciences). For β-hexosaminidase release assay, stimulated cells were centrifuged and β-hexosaminidase activity was measured by spectrophotometry in the supernatants and in cell lysates. 5 The percentage of -hexosaminidase release was determined as described. 6

Histamine release assay on cells from mastocytosis patients and from healthy volunteers

Major demographic and biological characteristics of mastocytosis patients involved in the study are detailed in Table S3. The histamine release assay was performed on dextran-enriched basophils (healthy donors, n=3), BM derived mononuclear cells from patients with SM (3 ISM, 2 ASM, 2 MCL; n=7) and PB derived mononuclear cells isolated from 2 ISM patients and 2 ASM patients (n=4) essentially as described. 7,8 Before challenge with anti-IgE, cells were preincubated or not with 200 ng/ml human recombinant stem cell factor (SCF) (PeproTek). After incubation for 90 minutes, cells ( 1x 10 6 /ml) were washed, and thereafter incubated with various concentrations of anti-IgE antibody E124.2.8 (Immunotech) (0.001-10 µg/ml) in histamine release buffer (HRB) (Immunotech) at 37°C for 30 minutes. Cells were then centrifuged at 4°C, and the cell-free supernatants and total suspensions recovered and analyzed for histamine content by radioimmunoassay (RIA, Immunotech). Histamine release was calculated as percent of total (cellular + extracellular) histamine. For primary cells derived from patients with different categories of systemic mastocytosis, only three concentrations of anti-IgE antibody E124.2.8 (0.1, 1 and 10 µM) were applied to induce histamine liberation.

Antibody staining and flow cytometry analysis of CD63 on stimulated BM MCs

To examine the effects of anti-IgE and C5a on expression of activation-linked cell surface antigens on bone marrow (BM)-derived mast cells (MCs), flow cytometry experiments were performed.

Unseparated BM cells from KIT D816V+ SM patients (ISM, n=3; ASM/MCL, n=3) (ISM patients were patients N° 10, 11 and 12 presented

Analysis of the effects of AKT and STAT5 inhibitors on cell proliferation

In order to evaluate the impact of AKT or of STAT5 on proliferation, cells were seeded at a starting concentration of 3x10 5 per mL in 96-well plates (100 L per well) and incubated at 37°C and 5% CO 2 for 48 hours in the presence of various concentrations (0.01 to 5 M) of a selective inhibitor of AKT1/AKT2 activity (compound A6730; Sigma), or of a selective inhibitor of STAT5 (pimozide: 1 to 6 M; Sigma), both diluted in DMSO 0.1% final concentration, or in the presence of DMSO alone (0.1%) in SFM (with rhSCF at 80 ng/mL for ROSA KIT WT but not for ROSA KIT D816V cells). Ten L of WST-1 (Roche Applied Science) were then added in each well and the cells were incubated for 3 hours at 37° C. The number of living cells was then measured for each condition by reading the absorbance at 570 nm using a plate reader Multiskan-MS (Thermo-Labsystems).

Immunohistochemistry (IHC) in mouse tissue sections

IHC was performed on slides prepared from paraffin-embedded, formalin-fixed BM, lung, liver, spleen and skin tumor biopsy specimens. The indirect immunoperoxidase staining technique was performed according to established protocols using the anti-chymase mAb B7 (work dilution: 1:50) and the antitryptase mAb G3 (1:100) (both from Chemicon). 9,10 Prior to staining, sections were pretreated with citrate buffer and microwave oven (tryptase stain) or proteinase and waterbath (chymase stain).

Endogenous peroxidase was blocked by methanol/H2O2. Slides were incubated with primary mAb diluted in 0.05 M Tris-buffered saline (TBS) (pH 7.5) plus 10% FCS for 20 hours at 4°C then washed in TBS. After incubation with biotinylated anti-mouse IgG (Vector) for 30 minutes, slides were washed three times in TBS, and then exposed to Vectastain ABC KIT for 30 minutes at room temperature. 3amino-9-ethylcarbazole (AEC) (Sigma) was used as chromogen. All slides were counterstained in Mayer´s Hemalun solution.

Supplemental Results

Clonogenicity of the ROSA cell lines

In semi-solid medium cultures, clonogenic capability of ROSA KIT WT cells (in the presence of rhSCF) was found to be around 7% (mean±SD: 6.8±1.7%), while ROSA KIT D816V cells (in absence of rhSCF) appeared to be less clonogenic (mean±SD: 4.08±1.7%), which is consistent with their more differentiated state.

Withdrawal of SCF on ROSA KIT WT cells induces rapid cell apoptosis

In a first set of experiments, we investigated the effect of the complete withdrawal of SCF on the survival of ROSA KIT WT cells. For this purpose, ROSA KIT WT cells were placed in their usual medium of culture, containing or not SCF (at the optimal predetermined concentration of 80 ng/mL), then incubated for 7 days. At day 0, day 2, day 4 and day 7, an aliquot of the cells placed in each condition was harvested and double labeled with Annexin-VAPC and propidium iodide (PI) in order to determine the percentage of apoptotic cells by flow cytometry. As shown in Figure S3, withdrawal of SCF induced a time-dependent increase in the percentage of apoptotic cells, which rose up to 40% at day 7, whereas the percent of apoptotic cells remained very low (around 5%) whatever the time of treatment when the cells were cultured in the continuous presence of SCF. In addition, a visual examination of the cultures at day 7 under an inverted microscope with phase contrast revealed that nearly 100% of the cells deprived of SCF appeared dead (most of the cells appeared as grey elements in this condition).

ROSA KIT D816V cells express equivalent amounts of mRNA coding for KIT WT and KIT D816V

Since ROSA KIT D816V cell line was derived from ROSA KIT WT cells (which only express KIT WT gene) by lentiviral transfection of a construct coding for the KIT D816V gene, we decided to measure the level of transcription of both genes into mRNA coding for KIT WT and for KIT D816V in this mutant cell line. We thus analyzed by quantitative RT-PCR (qRT-PCR) the amount of each type of mRNA, using for this purpose primers specific for KIT WT mRNA, as well as primers specific for KIT D816V mRNA. In addition, in the same experiments, we compared the expression of KIT WT mRNA in ROSA KIT WT cells and in ROSA KIT D816V cells. The results of the expression of each mRNA were calculated relative to the expression of these mRNA in HMC1.2 cells. As reported in Figure S4, the relative level of mRNAs coding for KIT WT was similar in ROSA KIT WT cells and in ROSA KIT D816V cells, which was expected since the mutant cell line can be considered as a daughter cell line derived from the ROSA KIT WT cell line. In addition, ROSA KIT D816V expressed only slightly more (1.2x) mRNA coding for the KIT D816V receptor than mRNA coding for the wild-type receptor (Figure S4), which is not significantly different and witnesses that both variants of KIT were quite equally expressed (at least at the mRNA level) in ROSA KIT D816V cells. Finally, also as expected, ROSA KIT WT cells did not expressed mRNA coding for the mutant KIT receptor (Figure S4).

ROSA KIT D816V cells and primary KIT D816V+ mast cells exhibit constitutively activated KIT, but show a largely reduced or absent response to anti-IgE antibody

Although expressing the FcRI at the same level than ROSA KIT WT cells, ROSA KIT D816V cells did not release histamine (Figure S5A), ß-hexosaminidase, TNF- or PGD2 after activation by anti-IgE (data not shown). At the same time, ROSA KIT D816V cells were able to up-regulate CD203c and to release up to 80% of their ß-hexosaminidase following stimulation with calcium ionophore. Interestingly, neoplastic KIT D816V+ BM MCs obtained from patients with ISM (Figure S5B), ASM or MCL (Figure S5C) released only little if any histamine when stimulated through the FcRI. This failure to respond to IgE-induced activation was not related to an intrinsic defect in FcRI, since (non-clonal) basophils obtained from the same patients could be fully activated through the FcRI (Figure S5D). Moreover, basophils derived from healthy donors were activated and released substantial amounts of histamine under the same conditions (Figure S5E).

ROSA cell lines respond differentially to activation by non-IgE triggers

Contrasting to ROSA KIT WT cells (Figure 3), we observed that ROSA KIT D816V cells were unresponsive to IgE-mediated activation (Figure S5A). For this reason, we sought to investigate whether this lack of response of the mutant cell line was limited to FcRI triggering or was a general phenomenon of hyporesponsiveness towards other, non-IgE triggers. For this purpose, we stimulated ROSA KIT WT cells and ROSA KIT D816V cells for 1 hour with different concentrations of peptidoglycan purified from S. aureus (PGN), which is known to activate human MCs through TLR2, 11 or of compound 48/80, a cationic polymer inducing degranulation of human MCs. 12 We then analyzed the level of expression by these cells of the activation marker, CD203c, and compared it to the one of unstimulated cells or of cells stimulated with 1 µM of calcium ionophore A23187. In addition, we measured the percentage of -hexosaminidase released by the two cell lines stimulated similarly. As reported in Figure S6, the level of stimulation evoked by a treatment with increasing concentrations of peptidoglycan (PGN) or compound 48/80, each used at 1, 5 or 10 g/mL appears dose-dependent in ROSA KIT WT cells for upregulation of CD203c as measured by flow cytometry (Figure S6A), as well as for -hexosaminidase release (Figure S6C). By contrast, it appears that those two triggers are not able to induce activation at the same level for ROSA KIT D816V cells. Indeed, in ROSA KIT WT cells, PGN and compound 48/80 induced respectively up to 35 and 55% of mean -hexosaminidase release at the optimal dose of 10 g/mL, whereas in ROSA KIT D816V , the same triggers, used at the same dose, induced respectively only a mean -hexosaminidase release of 20% and 22% (Figure S6B). The same differences can be noticed for up-regulation of CD203c (Figure S6D). Although there is a trend to a tendency for ROSA KIT D816V cells to increase their -hexosaminidase release and to up-regulate CD203c with increasing concentrations of triggers, the values obtained with PGN or compound 48/80 used at 10 g/mL were not found statistically different (at p<0.05) than the one obtained for control, unstimulated cells. These data suggest that the presence of the activating KIT D816V mutation seems to be also able to desensitize human MCs towards non-IgE triggers. The mechanism of this desensitization remains unknown, although one cannot exclude that, as for IgE-triggering, a mechanism of competition for a number of intracellular common substrates might exist, due to the presence of the constitutively activated mutant.

Effects of anti-IgE and C5a on expression of CD63 on mast cells obtained from patients with ISM or ASM/MCL and from normal/reactive bone marrow

We found that 10 µg/ml anti-IgE as well as 100 nM C5a upregulated the expression of CD63 on mast cells in normal bone marrow, whereas CD63 expression was not affected by anti-IgE or C5a in mast cells derived from patients with ISM or advanced SM (ASM/MCL) (Figure S7). These data corroborate those obtained in vitro with the ROSA KIT D816V cells which were found unresponsive to IgE and non-IgE triggers (Figures S5A, S6B and S6D), as well with ex vivo data on BM mononuclear cells of SM patients, which failed to release histamine upon FcRI triggering (Figure S5B andS5C).

Activated AKT and STAT5 are critical for the proliferation of ROSA KIT D816V cells

The potential role of AKT and STAT5 in the proliferation of ROSA cells was evaluated by treating both cell lines with an AKT1/2 inhibitor (compound A6730; 0.01 to 5 M) or with a STAT5 inhibitor (pimozide; 1 to 6 M). Interestingly, as shown in Figure S8A, while the IC50 of compound A6730 was found to be 3 M for ROSA KIT WT cells, the same compound inhibited 50% of the proliferation of ROSA KIT D816V cells already at 0.5 M. Regarding pimozide, IC50 for ROSA KIT D816V cells was found around 1 M, whereas it was > 6 M for ROSA KIT WT cells (Figure S8B).

Engraftment of ROSA KIT D816V cells and ROSA KIT WT cells in NSG mice as determined by tryptase staining

Since we evidenced that intravenously injected ROSA KIT WT cells and particularly ROSA KIT D816V cells were able to engraft NSG mice in the bone marrow (Table 1), we sought to investigate for possible infiltration by the same cellular elements of other organs of the grafted NSG mice. For this purpose, tryptase staining by IHC was performed on sections prepared from paraffin-embedded, formalin-fixed BM, lung, liver and spleen of 5 NSG mice injected with ROSA KIT WT cells and 5 NSG mice injected with ROSA KIT D816V cells. Engraftment levels were defined using the following score: ++, infiltration grade >50%; +, infiltration 10-50%; +/-, 2-10%; -/+, only a very few cells detected; -, no engraftment found. Data presented in Table S4 showed that in NSG mice injected i.v. with ROSA KIT D816V cells, the two main organs involved (apart BM) were spleen and lungs, with a more important involvement for the spleen (one mouse found ++, 3 found + and one found +/6) than for the lung (2 mice were found +, one mouse was found +/-and two mice were found negative). Interestingly, the liver seemed to be spared by ROSA KIT D816V infiltration since 4 mice were found negative and only one mouse was scored +/-(this latter animal having the highest score concerning spleen involvement). Regarding NSG mice injected i.v. with ROSA KIT WT cells, and in line with the lower level of BM involvement already observed for these animals, we observed a lower level of spleen involvement (score ranging from -/+ to +) and no lung or liver involvement, excepted for one mouse who presented with a low level (-/+) of involvement for both organs (Table S4). All in all, these data confirmed the highest tumorigenic potential of ROSA KIT D816V cells as compared to their KIT WT counterpart. Table S1:

Supplemental Tables

Figure S3: Effect of withdrawal of SCF on the apoptosis of ROSA KIT WT cells. ROSA KIT WT were treated or not with recombinant human SCF (80 ng/ml), then cultured for 7 days in an incubator at 37°C (5% CO2). At the beginning of the culture (d0) and at each time point (d2, d4 and d7), an aliquot of the cells (10 6 ) in both conditions was labeled with Annexin V-APC/propidium iodide (PI). The percentage of Annexin V positive/PI positive cells was then measured for each condition by flow cytometry with a FACSCalibur flow cytometer. Data presented are the mean ± SD from 3 independent experiments.

Figure S4

: Quantitative expression analysis of mRNA coding for KIT WT or for KIT D816V in ROSA KIT WT or ROSA KIT D816V cells as compared to HMC1.2 cells using qPCR with specific primers. Total RNA from 5.10 6 ROSA KIT WT and ROSA KIT D816V cells, from 5.10 6 HMC1.2 cells used as the control, was isolated then reverse transcribed into cDNA. Real time PCR was then using primers specific for KIT WT or for KIT D816V cDNA. Reactions were compared to the levels of expression of mRNA for Hypoxanthine Phosphoribotransferase (HPRT). A serial dilution of the cDNA mixture was used to produce a standard curve and the cDNA of HMC1.2 was used as a calibrator for gene expression analysis. The mRNA expression of KIT WT and KIT D816V in the two ROSA cell lines was calculated relative to the expression of these genes in control HMC1.2, according to the delta-delta C t method (2 -∆∆Ct ). Data are reported as normalized fold expression of each mRNA using the 2 -∆∆Ct cells isolated from 7 patients with mastocytosis after preincubation with human recombinant SCF. Bone marrow (BM) derived primary mononuclear cells (MNC) obtained from 3 patients with indolent systemic mastocytosis (ISM) (patients N° 2, 3 and 5 of the Table S3), from 2 patients with aggressive systemic mastocytosis (ASM) (patients N° 1 and 6 of the Table S3) and from 2 patients with mast cell leukemia (MCL) (patients N° 4 and 8 of the Table S3) were incubated with 200 ng/ml human recombinant SCF at 37°C for 90 minutes. Then, cells were incubated with three concentrations of anti-IgE for 30 minutes. Thereafter, histamine was quantified as described above in the supplemental methods section. A: mean ± SD of the percent of histamine release for the ISM patients; B: mean ± SD of the percent of histamine release for 2 ASM and 2 MCL patients. Results represent the mean ± SD of 7 independent experiments. D: percentage of histamine release from PB dextran-enriched mononuclear cells isolated from 2 patients with ISM and 2 others with ASM. E: Percentage of histamine release from PB dextran-enriched basophils isolated from 3 normal donors. PB derived primary mononuclear cells (MNC) obtained from 4 patients with systemic mastocytosis (2 ISM and 2 ASM), as well as Dextran-enriched basophils from 3 healthy donors, were preincubated in medium containing 200 ng/ml human recombinant SCF at 37°C for 90 minutes. Then, cells were incubated with three concentrations of anti-IgE for 30 minutes. Thereafter, histamine was quantified in cell lysates and supernatants by RIA. Histamine release is expressed as the percentage of total histamine and results represent the mean ± SD of four independent experiments. Data were analyzed by the Student's t-test and differences were considered to be statistically significant when p was <0.05. Asterisk (*): p <0.05, asterisk (**): p>0.05.

RESULTS Publication 2. "Identification of bromodomain-containing protein-4 as a novel marker and epigenetic target in mast cell leukemia"

In this second article, we reported a role for BRD4 as potential target in neoplastic MCs in advanced SM (Wedeh et al. 2015). Indeed, we found that BM-derived neoplastic MCs from patients with ASM and MCL expressed substantial amounts of cytoplasmic BRD4 compared to MCs in patients with ISM. In addition, we observed that the human MC lines HMC-1.1, HMC-1.2, ROSA KIT WT and ROSA KIT D816V expressed cytoplasmic and nuclear BRD4. Moreover, the shRNA-induced knock-down of BRD4 resulted in a significant growth inhibition of HMC-1 cells and ROSA cells, and induced apoptosis in these cells. Also, we found that JQ1, a specific inhibitor of BRD4, induced a cell cycle arrest in G1 in these cells. Furthermore, JQ1 down-regulated dose-dependently the expression of CD63 and CD71 in HMC-1 cells and ROSA cells. In addition, we have observed that JQ1 downregulated the expression of FcεRIα in ROSA KIT WT cells. Consistent with this latter data, we found that JQ1 blocked IgE-mediated histamine release and counteracted IgE-mediated up-regulation of CD63 and CD203c in ROSA KIT WT cells. Besides, the growth-inhibitory effects of JQ1 on neoplastic human MC lines were associated with down-regulation of the expression of BCL-2, MCL-1, BCL-xL and MYC mRNA, and up-regulation of the expression of BIM mRNA.

We next examined the mechanisms underlying the expression of BRD4 in neoplastic MCs in SM. We found that SCF upregulated BRD4 mRNA expression in ROSA KIT D816V cells but not in HMC-1 cells.

To explore underlying signalling pathways, we applied KIT-targeting drugs and a panel of signal transduction inhibitors. Interestingly, the KIT-targeting drugs PKC412 and dasatinib suppressed the expression of BRD4 mRNA in all human MC lines tested. Besides, Imatinib was found to suppress theexpression of BRD4 mRNA in HMC-1.1 cells and ROSA KIT WT cells, but was not able block BRD4 expression in HMC-1.2 or ROSA KIT D816V cells. In addition, we observed that the MEK inhibitors RDEA119 and PD0325901, the PI3K/mTOR blocker BEZ235 and all-trans retinoic acid (ATRA) were capable to downregulate the expression of BRD4 mRNA in HMC-1 cells and ROSA cells.

In the last part of our work, we screened for suitable anti-neoplastic drugs that might be applied in combination with JQ1 in patients with advanced SM. We found that JQ1 strongly synergized with PKC412 and with ATRA in suppression of the growth of HMC-1 cells and ROSA cells and in induction of apoptosis. Interestingly, we observed that the combination ´JQ1+PKC412´ was able to overcome the SCF-induced resistance in ROSA KIT D816V cells.

INTRODUCTION

Systemic mastocytosis (SM) is a hematopoietic stem cell disorder characterized by pathologic expansion and accumulation of clonal mast cells (MCs) in one or more internal organs. 1-5 Indolent and advanced types of the disease have been described. 1-5 The classification of the World Health Organization (WHO) delineates four major categories of SM, namely indolent SM (ISM), SM with an associated clonal hematopoietic non-MC-lineage disease (SM-AHNMD), aggressive SM (ASM) and MC leukemia (MCL). 6-8 The smoldering subtype of SM, initially considered a subtype of ISM, is now also regarded a separate SM category. 9 In ISM, the prognosis is quite favorable. In fact, despite mediatorrelated symptoms, cosmetic problems and the neoplastic nature of the disease, patients with ISM have a normal or near-normal life expectancy. 5-8 By contrast, in patients with advanced SM (ASM, SM-AHNMD, MCL) the prognosis is grave. 6-8,10-15 In these patients, cytoreductive therapy is administered to suppress MC expansion in various organ systems. 9,10,12-16 However, unfortunately, clonal MCs in these patients are often resistant against anticancer drugs. Therefore, research efforts focus on new potential drug targets in neoplastic MCs and the development of novel targeted drugs for advanced SM. 16-18 One potential drug target in ASM and MCL is KIT D816V, an SM-related oncoprotein. 17,18 This transforming mutant is detectable in a majority of all patients with SM, including ASM and MCL. 19-21 KIT D816V is expressed in MCs as well as in MC progenitors in these patients, and is considered to contribute substantially to differentiation and survival of neoplastic MCs. 21-23 However, so far, only a few tyrosine kinase inhibitors were found to counteract the growth and survival of KIT D816V+ neoplastic MCs in patients with advanced SM. 18,24-27 One of the most encouraging agents is the multikinase inhibitor PKC412, also known as midostaurin. However, despite impressive efficacy, responses to PKC412 in ASM and MCL are often incomplete or short lived. 27,28 Therefore, research is seeking additional drug targets expressed by neoplastic MCs.

During the past few years, chromatin-regulatory molecules and related antigens have been examined as potential therapeutic targets in diverse human malignancies. 29-31 One promising class of targets are the bromodomain-containing proteins. 30-32 To identify candidate therapeutic targets in the chromatinregulatory network in acute myeloid leukemia (AML), we have recently performed a multiplexed short hairpin RNA (shRNA) screen in a genetically engineered mouse model of AML. 32 In this screen, we identified the 'epigenetic reader' bromodomaincontaining protein-4 (BRD4) as a promising new target in AML. 32 Thus, inhibition of BRD4 by exposure to RNA interference or treatment with JQ1, a drug blocking the specific interactions between BRD4 and acetylated histones, resulted in major antileukemic effects in murine and human AML cells. 32,33 The aims of the present study were to explore whether neoplastic MCs in MCL and other types of SM express BRD4 and whether BRD4 serves as a druggable target in these incurable malignancies.

PATIENTS AND METHODS Reagents

A description of reagents is provided in the Supplementary Materials. A specification of monoclonal antibodies used is shown in Supplementary Table S1.

Patients and isolation of primary neoplastic MCs Diagnostic bone marrow (BM) samples were obtained from the iliac crest of 62 patients with SM, including 46 with ISM (including one with smoldering subtype of SM), 8 with ASM, 5 with SM-AHNMD and 3 with MCL. Diagnoses were established according to WHO criteria. 6-8 Patient characteristics are shown in Supplementary Table S2. BM aspirates were collected in syringes containing preservative-free heparin. All patients gave written informed consent before BM examination. The study was approved by the ethics committee of the Medical University of Vienna. Aspirated BM cells were layered over Ficoll to isolate mononuclear cells. Mononuclear cells were examined for drug responses, expression of BRD4 mRNA and presence of KIT mutations at codon 816 as reported. 21,34 In one patient with MCL, a KIT D816H mutation was found. In most other patients, neoplastic cells were found to carry KIT D816V (Supplementary Table S2). Control samples (normal/reactive BM) were from patients with idiopathic cytopenia (n = 2), lymphomas without BM infiltration (n = 6), autoimmune thyroiditis (n = 1) and chronic myeloid leukemia in major molecular response (n = 1).

Cell lines

The MCL cell line HMC-1 35 was kindly provided by Dr JH Butterfield (Mayo Clinic, Rochester, MN, USA). Two subclones were used, HMC-1.1 expressing KIT V560G and HMC-1.2 harboring KIT V560G and KIT D816V. 24,36 HMC-1 cells were maintained in Iscove's modified Dulbecco's medium containing 10% fetal calf serum, L-glutamine and antibiotics (at 37 °C with 5% CO 2 ). The recently established human MC lines ROSA KIT WT and ROSA KIT D816V were cultured in Iscove's modified Dulbecco's medium with 10% fetal calf serum. 37 ROSA KIT WT cells were maintained in stem cell factor (SCF), and ROSA KIT D816V cells were maintained without SCF. Chinese hamster ovary cells transfected with the murine SCF gene served as a source of SCF. 37 In select experiments, recombinant human SCF (rhSCF) was used. The identity of the HMC-1 and ROSA cell lines was confirmed by mutation analysis and phenotyping. For knockdown experiments, pRRL-SFFV-GFPmir-E was constructed based on pRRL-SGEP 38 by removing the PGK-Puro cassette. Knockdown-validated shRNAs targeting BRD4 or Renilla luciferase (Supplementary Table S3) were cloned using XhoI/EcoRI from existing miR-E constructs. Lentivirus was produced by transfection of HEK-293FT cells with shRNA constructs and third-generation lentiviral packaging vectors (pRSV-Rev; Addgene no. 12253; pMD2.G, Addgene no. 12259; Addgene, Cambridge, MA, USA; and pcDNA3.GP4xCTE, kindly provided by Dr A Schambach, Hannover Medical School, Hannover, Germany) as described previously. 38 HMC-1 and ROSA cells were transduced using spin infection (800 g for 90 min at 32 °C) in the presence of polybrene (7 μg/ml). GFP+ cells were sorted on a FACSAria (Becton Dickinson Biosciences, San Jose, CA, USA).

Immunocytochemistry and immunohistochemistry

To study the expression of BRD4 in neoplastic MCs, immunocytochemistry and immunohistochemistry were performed according to published protocols. 39,40 A description of staining techniques used in this study is provided in the Supplementary Material.

Evaluation of MC proliferation

Primary cells (mononuclear cells, 10 × 10 4 cells per well) and cell lines (1-2 × 10 4 cells per well) were cultured in 96-well microtiter plates in RPMI-1640 medium plus 10% fetal calf serum in the absence or presence of JQ1 (5-5000 nM) at 37 °C. After 48 h, 3 H-labeled thymidine (0.5 μCi per well) was applied for 16 h. Cells were then harvested in filter membranes and bound radioactivity was measured in a β-counter. In a separate set of experiments, drugs were applied in the absence or presence of either Chinese hamster ovary supernatant containing SCF or rhSCF (200 ng/ml) for 24 h. In another set of experiments, drug combinations were tested, using JQ1 and other drugs (PKC412, cladribine, 5-azacytidine, decitabine, ATRA (all-trans retinoic acid)). After an initial screen, PKC412 and ATRA were identified as potent drug partners. HMC-1 cells were incubated with suboptimal concentrations of JQ1 and ATRA or JQ1 and PKC412 at a fixed ratio of drug concentrations. Synergism was defined as supraadditive effect that was confirmed by calculating combination index (CI) values using Calcusyn software (Calcusyn Biosoft, Ferguson, MO, USA) as reported. 24,26

Assessment of apoptosis and cell cycle progression in neoplastic MCs

A detailed description of apoptosis evaluation techniques and cell cycle progression analysis is provided in the Supplementary Materials.

Evaluation of the expression of activation antigens in drugexposed cells

Recent data suggest that JQ1 induces maturation in AML cells. 32,33 To study the effects of JQ1 on MC differentiation, flow cytometry experiments were performed. HMC-1.1, HMC-1.2, ROSA KIT WT and ROSA KIT D816V cells were incubated in JQ1 (100-5000 nM) at 37 °C for 48 h. Then, viable cells were examined for the expression of CD2 (LFA-2), CD9, CD11b (C3biR), CD54 (ICAM-1), CD63 (LAMP-3), CD71 (transferrin receptor), CD95 (FAS), CD117 (KIT), CD203c (E-NPP3) and FcεR1α by flow cytometry on a FACSCalibur. In addition, cells were examined for morphologic signs of maturation by Wright-Giemsa staining.

Histamine release experiments

Histamine release experiments were performed on ROSA KIT WT cells following a standard protocol. 41,42 A description of the histamine release assay is provided in the the Supplementary Materials.

Quantitative PCR

A detailed description of quantitative PCR experiments is provided in the Supplementary Materials. Primers used in PCR assays are shown in Supplementary Table S4.

Statistical analysis

Differences in growth and apoptosis in drug-exposed cells were determined by appropriate statistical analysis, including analysis of variance and the Student's t-test. Results were considered to be significantly different when the P-value was o 0.05.

RESULTS

Cytoplasmic BRD4 is expressed abundantly in neoplastic MCs in ASM and MCL In all 26 SM patients tested, neoplastic MCs were found to exhibit nuclear BRD4 (Figure 1a and Supplementary Table S2). In ASM and MCL, neoplastic MCs also expressed substantial amounts of cytoplasmic BRD4 (Figure 1a). By contrast, in most patients with ISM, MCs expressed only little if any cytoplasmic BRD4 (Figure 1a and Supplementary Table S2). We were also able to show that MCs obtained from patients with advanced SM express BRD4 mRNA (Figure 1b). The MC lines HMC-1.1, HMC-1.2, ROSA KIT WT and ROSA KIT D816V also expressed BRD4 at the mRNA and protein levels (Figure 1b and Supplementary Figure S1). Finally, we confirmed the expression of cytoplasmic BRD4 in primary neoplastic MCs in advanced SM (Supplementary Figure S1). Preincubation of anti-BRD4 antibody with a BRD4-specific blocking peptide or antibody omission resulted in a negative stain (Supplementary Figure S1). Neither normal CD34+ progenitor cells nor SCF-dependent cultured (non-neoplastic) MCs expressed substantial amounts of BRD4 (Supplementary Figure S1).

Regulation of expression of BRD4 in neoplastic MCs SCF was found to upregulate BRD4 mRNA expression in ROSA KIT D816V but not in HMC-1 cells (Supplementary Figure S2). To explore the underlying signaling pathways, we applied KIT-targeting drugs and a panel of signal-transduction inhibitors. As shown in Supplementary Figures S2 andS3, the KIT-targeting drugs PKC412 and dasatinib dose-dependently suppressed the expression of BRD4 mRNA in all cell lines tested (Supplementary Figure S3). Imatinib was found to suppress BRD4 mRNA expression in HMC-1.1 and ROSA KIT WT cells but not in HMC-1.2 or ROSA KIT D816V cells (Supplementary Figure S2). These data are in line with the notion that KIT D816V confers resistance against imatinib. 17,18 The mitogen-activated protein kinase kinase (MEK) inhibitors RDEA119 and PD0325901, the PI3K/mTOR (phosphoinositide 3-kinase/mammalian target of rapamycin) blocker BEZ235 and ATRA were also found to downregulate BRD4 expression in HMC-1 and ROSA cells. Rapamycin induced a slight decrease in BRD4 mRNA expression in HMC-1.2 and ROSA KIT D816V cells but not in ROSA KIT WT or HMC-1.1 cells. BRD4 mRNA expression remained unchanged after exposure to the other drugs examined, including the signal transducer and activator of transcription 5 blockers piceatannol and pimozide, cladribine (2CdA), and the demethylating agents, 5-azacytidine and decitabine. INCB018424 (ruxolitinib) slightly reduced the expression of BRD4 mRNA in ROSA KIT D816V and HMC-1.2 cells (Supplementary Figure S2).

The BRD4-targeting drug JQ1 inhibits the proliferation of neoplastic MCs We have recently shown that shRNAs against BRD4 and the BRD4targeting drug JQ1 inhibit the growth of AML cells. 32,33 To demonstrate a role for BRD4 as potential target in neoplastic MCs, we applied BRD4-specific shRNAs. BRD4 knockdown was confirmed by quantitative PCR. As visible in Figure 2a and Supplementary Figures S4A andS4B, the shRNA-induced knockdown of BRD4 resulted in a significant growth inhibition of HMC-1 and ROSA cells. The knockdown of BRD4 was also found to decrease MYC mRNA levels in all cell lines (not shown). JQ1 was found to inhibit 3 H-thymidine uptake in HMC-1.1 and HMC-1.2 cells as well as in ROSA KIT WT and ROSA KIT D816V cells, with IC 50 values of 500-1000 nM (Figures 2b andc). Similar effects were seen with volasertib and BI2536, two other drugs targeting BRD4 (Supplementary Figure S4C). JQ1 also suppressed growth in primary neoplastic MCs obtained from patients with ASM or MCL (Figure 2d). Interestingly, IC 50 values obtained with JQ1 in primary cells were lower compared with IC 50 values produced with the MC lines tested. Finally, JQ1 was found to induce a G1 cell cycle arrest in all MC lines examined (Supplementary Figure S5). JQ1 induces apoptosis in neoplastic MCs JQ1 was found to induce dose-dependent apoptosis in HMC-1 and ROSA cells as evidenced by light microscopy, AnnexinV staining and active caspase-3 staining (Figures 3a andb and Supplementary Figures S6A). The apoptosis-inducing effect of JQ1 was similar in HMC-1.1 cells compared with HMC-1.2 cells (Figures 3a andb and Supplementary Figure S6A) and was also comparable in ROSA KIT WT and ROSA KIT D816V cells (Figures 3a andb and Supplementary Figure S6A). We were also able to confirm the apoptosis-inducing effects of JQ1 on HMC-1 and ROSA cells in a terminal deoxynucleotidyl transferase dUTP nick-end labeling assay (Supplementary Figure S6B). Finally, we were able to show that shRNAs against BRD4 induce apoptosis in HMC-1 and ROSA cells (not shown).

JQ1 downregulates the expression of CD63, CD71 and FcεRI in neoplastic MCs

Recent data suggest that JQ1 can induce maturation in AML cells. 32,33 In the present study, we found that JQ1 dosedependently downregulates the expression of CD63 and CD71 in HMC-1 and ROSA cells (Supplementary Figure S7A) and HMC-1.2 cells. In ROSA KIT WT and ROSA KIT D816V cells, JQ1 was also found to suppress the expression of the FcεRI (Supplementary Figure S7B). Moreover, we found that preincubation of ROSA KIT WT cells with JQ1 blocks IgE-mediated histamine release (Supplementary Figure S8) and counteracts IgE-mediated upregulation of CD63 and CD203c (Supplementary Figure S9). Although JQ1 alone failed to upregulate the expression of differentiation antigens on neoplastic MCs, the drug combination 'JQ1+ATRA' was found to upregulate the expression of CD11b (C3biR) and CD54 (ICAM-1) in HMC-1 and ROSA cells (Supplementary Figure S10A). By contrast, when used as a single agent, ATRA did not upregulate CD54 or CD11b in HMC-1 or ROSA cells (not shown). ATRA also failed to modulate the expression of CD63 and CD71, and neither JQ1 nor ATRA (as single agents or in combination) produced visible signs of maturation in HMC-1 or ROSA cells (Supplementary Figure S10B).

Effects of JQ1 on the expression of MYC and BCL-2 family members in MCs

Recent data suggest that JQ1 modulates the expression of MYC and various BCL-2 family members in leukemic cells. 43-45 We found that JQ1 downregulates the expression of BCL-2, MCL-1 and BCL-xL mRNA levels, as well as MYC mRNA expression levels, in HMC-1 and ROSA cells (Supplementary Figure S11). In addition, we found that JQ1 upregulates BIM mRNA levels in all four cell lines tested (Supplementary Figure S11). JQ1 synergizes with PKC412 and with ATRA in producing growth inhibition in HMC-1 and ROSA cells As BRD4-targeting drugs may best be applied in combination with other antineoplastic drugs in patients with advanced SM, we screened for suitable drug partners of JQ1. A number of candidates, including cladribine (2CdA), 5-azacytidine and decitabine, did not show synergistic effects with JQ1 (not shown). However, we found that JQ1 strongly synergizes with PKC412 and with ATRA in suppressing the growth of HMC-1 and ROSA cells (Supplementary Figure S12A). In addition, JQ1 was found to synergize with PKC412 and ATRA in inducing apoptosis in HMC-1 and ROSA cells (Supplementary Figures S12B andS12C). Synergism was confirmed by calculating CI values ( o1) (not shown).

KIT activation confers resistance against JQ1 in ROSA KIT D816V cells In a final step, we screened for potential resistance mechanisms that could interfere with JQ1 effects. As KIT has been implicated in MYC activation and upregulates BRD4 expression, we asked whether KIT activation could interfere with JQ1 effects. Indeed, as visible in Figures 4a andb, incubation of ROSA KIT D816V cells with SCF blocked their responsiveness against JQ1. Interestingly, however, SCF did not alter responsiveness of HMC-1.1 and HMC-1.2 cells to JQ1 (Supplementary Figure S13A). We also asked whether the combination 'JQ1+PKC412' would overcome KITmediated resistance. Indeed, this drug combination was found to produce strong synergistic apoptosis-inducing effects in the presence of SCF in ROSA KIT D816V cells (Supplementary Figure S13B).

DISCUSSION

Bromodomain-containing proteins have recently been identified as key regulators of cell cycle progression and growth in various neoplastic cells. 29-33 One of these regulators appears to be BRD4, an epigenetic reader involved in the regulation of MYC. We have recently shown that BRD4 is a druggable target in AML cells, 32,33 and similar observations have been made in other tumor models. 43-45 We here show that BRD4 is abundantly expressed in neoplastic MCs in advanced SM, and that the BRD4-targeting drug JQ1 induces growth inhibition and apoptosis in these cells.

ASMs and MCLs are incurable malignancies characterized by drug resistance and a poor survival. 1-6,10-15 In these patients, neoplastic cells are often resistant against conventional drugs and KIT-targeting tyrosine kinase inhibitors. Therefore, it is important to define new robust targets in these malignancies. We have identified BRD4 as a novel drug target in neoplastic MCs in advanced SM. To the best of our knowledge, this is the first report describing a BET-bromodomain-containing molecule as a target in neoplastic MCs.

We have recently shown that BRD4 is expressed abundantly in the cytoplasm of leukemic blasts in AML. 33 In the present study, we show that BRD4 is expressed abundantly in the cytoplasm and the nuclei of neoplastic MCs in almost all patients with advanced SM, whereas in most patients with ISM, MCs expressed only low levels or no detectable BRD4 in their cytoplasm but still expressed BRD4 in their nuclei. HMC-1 and ROSA cells also expressed cytoplasmic and nuclear BRD4. The abundant expression of cytoplasmic BRD4 in MCs in advanced SM may have several explanations. One hypothesis is that cytoplasmic expression of BRD4 is indicative of a higher proliferative potential of neoplastic cells. Alternatively, KIT-independent lesions acquired during disease progression promote cytoplasmic BRD4 expression. Indeed, apart from KIT D816V, several additional molecular lesions and pro-oncogenic signaling pathways are expressed in neoplastic MCs in advanced SM. 46,47 The exact role and biochemical function of cytoplasmic BRD4 in neoplastic MCs remain unknown. However, there is evidence that BRD4 exerts kinase activity and interacts with cytoplasmic antigens, such as SPA-1, in malignant cells. 48-50 Whether BRD4 also fulfills these functions in neoplastic MCs remains unknown.

So far, little is known about the mechanisms contributing to the expression of BRD4 in neoplastic MCs. In an attempt to define signaling pathways potentially involved in the expression of BRD4, we applied various signal-transduction inhibitors. The multitargeted tyrosine kinase inhibitors PKC412 and dasatinib, which are known to block the kinase activity of KIT D816V, were found to downregulate the expression of BRD4 in HMC-1 and ROSA cells. In addition, we found that various MEK inhibitors and the PI3K/mTOR blocker BEZ235 can downregulate the expression of BRD4 in neoplastic MCs. As MEK as well as PI3K are downstream of KIT, these data suggest that KIT D816V contributes to the expression of BRD4 in neoplastic MCs. This conclusion was supported by the observation that SCF promotes the expression of BRD4 in ROSA KIT D816V cells. However, SCF failed to upregulate the expression of BRD4 in HMC-1 cells. These observations suggest that the expression of BRD4 in MCs depends not only on KIT activation but also on other factors. An interesting observation was that BRD4 mRNA expression levels are slightly higher in HMC-1.1 compared with that in HMC-1.2 cells. The reason for this phenomenon remains unknown.

We have recently shown that the BRD4-targeting drug JQ1 exerts strong antineoplastic effects in drug-resistant AML cells. 33,34 In the present study, we were able to show that JQ1 induces growth inhibition and apoptosis in primary neoplastic cells obtained from patients with advanced SM as well as in HMC-1 and ROSA cells. In each cell line tested, the effects of JQ1 were dose-dependent, with reasonable IC 50 values, and in each case, growth inhibition was associated with induction of apoptosis.

In AML, JQ1 effects were found to be associated with signs of maturation. 32,33 We were therefore interested to learn whether exposure to JQ1 would lead to maturation in neoplastic MCs. However, we were not able to detect a morphologically visible maturation-inducing effect of JQ1 in HMC-1 or ROSA cells. Moreover, we were unable to demonstrate that JQ1 induces the upregulation of differentiation-associated antigens in MCs. Rather, JQ1 was found to even downregulate the expression of several key surface antigens in HMC-1 and/or ROSA cells, including CD63 and CD71. Furthermore, JQ1 was found to downregulate the expression of FcεRI and to suppress IgE-dependent histamine release in ROSA cells. Collectively, these data suggest that JQ1 blocks proliferation as well as activation of neoplastic MCs, which may be relevant clinically, as patients with advanced SM not only suffer from organ infiltration but also from the consequences of MC activation and mediator release. 2-6 The intriguing effects of JQ1 on growth and survival of neoplastic MCs prompted us to ask for optimal drug partners and synergistic antineoplastic effects. Of all drug partners tested, PKC412 and ATRA were found to synergize with JQ1 in inhibiting growth and survival in HMC-1 and ROSA cells.

In a final step, we asked whether resistance against JQ1 may occur in neoplastic MCs. In these experiments, we found that SCF introduces resistance against JQ1 in ROSA KIT D816V cells but not in HMC-1 cells. We also asked whether the synergistic activity of the drug combinations JQ1+ATRA and JQ1+PKC412 would overcome SCF-mediated resistance. Indeed, the combination JQ1+PKC412 was found to overcome SCF-mediated resistance in ROSA, whereas the combination JQ1+ATRA was unable to overcome resistance. This is best explained by the fact that SCF-induced resistance is mediated by the activation of KIT, the principle target of PKC412.

In summary, we have shown that BRD4 is a druggable target in neoplastic MCs. Inhibition of BRD4 by JQ1 is a potent approach to eradicate neoplastic MCs, and drug effects can be potentiated by the addition of PKC412 or ATRA. Whether BET-targeting drugs exert beneficial effects in patients with advanced SM remains to be determined.

Immunocytochemistry (ICC) and immunohistochemistry (IHC)

To study the expression of BRD4 in neoplastic mast cells (MCs), ICC and IHC were performed according to published protocols. 2,3 ICC was performed on HMC-1 cells, ROSA cells, normal human CD34+ progenitor cells, normal stem cell factor (SCF)cultured peripheral blood (PB)-derived MCs and primary neoplastic MCs obtained from patients with indolent systemic mastocytosis (ISM) (n=5), aggressive systemic mastocytosis (ASM) (n=1) or mast cell leukemia (MCL) (n=2). Cells were spun on cytospin-slides and incubated with a polyclonal rabbit anti-BRD4 antibody (Sigma-Aldrich; 1:20) at 4°C for 20 hours. Cells were then washed and incubated with biotinylated goat-anti-rabbit IgG for 30 minutes. Alkaline phosphatase complex (Biocare, Walnut Creek, CA, USA) was used as chromogen. In control experiments, the anti-BRD4 antibody was preincubated with a BRD4-specific blocking peptide (Bethyl Lab, Montgomery, TX, USA). IHC was performed on sections prepared from paraffin-embedded BM biopsy specimens (ISM, n=15; SM-AHNMD, n=4; ASM, n=5; MCL, n=2; Table 1) using the indirect immunoperoxidase staining technique as reported. 2,3 Prior to staining, sections were pretreated by microwave oven. Slides were incubated with anti-BRD4 antibody (1:20; pH 7.5) at 4°C for 20 hours. Then, slides were washed and incubated with biotinylated anti-mouse IgG (Vector, Burlingame, CA, USA) for 30 minutes, washed, and then exposed to Vectastain ABC KIT (30 minutes). AEC (Sigma Aldrich) was used as chromogen. Stained sections were analyzed by Olympus AX-1 microscope equipped with a 60x/0.9 UPlan-Apo objective lens. Images were taken on an Olympus DP21 camera (Olympus, Hamburg, Germany) and adjusted by Adobe Photoshop CS2 software Version 9.0 (Adobe Systems, San Jose, CA, USA).

Assessment of apoptosis and cell cycle progression in neoplastic MCs

HMC-1 and ROSA cells were incubated in control medium or in various concentrations of JQ1 at 37°C for 48 hours. Then, cells were subjected to flow cytometry following published protocols. 3-5 Apoptosis was measured by combined AnnexinV/propidium-iodide staining or staining for active caspase-3. 3,5 For staining of caspase-3, cells were fixed in 2% paraformaldehyde and permeabilized in methanol at -20°C (15 minutes). Rabbit anti-human mAb C92-605 against active caspase-3 was applied for 30 minutes. Then, cells were washed and analyzed on a FACSCalibur (Becton Dickinson Biosciences). Drug-induced apoptosis was also measured on Wright-Giemsa-stained cytospin slides prepared from HMC-1 and ROSA cells. In select experiments, drug-induced apoptosis (5,000 nM JQ1 for 48 hours) was confirmed by Tunel Assay as described previously. 4 Cell cycle analysis was performed with HMC-1 and ROSA cells after exposure to control medium or JQ1 (250-2,500 nM) at 37°C for 24 hours. Cell cycle progression was analyzed by flow cytometry as reported. 3 Quantitative PCR (qPCR) qPCR was performed with RNA isolated from HMC-1 cells, ROSA cells, normal donors or primary neoplastic MCs. Cell lines were incubated in control medium or JQ1 (2,500 nM) for 24 hours. In select experiments cells were treated with different targeted drugs for 12 hours. Then, RNAs were isolated using the RNeasy Mini-Kit (Qiagen, Hilden, Germany). BRD4, BCL-2, MCL-1, BCL-xL, BIM, and MYC mRNA levels were quantified on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA), using iTaq SYBR Green Supermix with ROX (Bio-Rad, Hercules, CA) following published protocols. 3,6 Primers used in PCR assays are shown in Supplementary Table S3. Results were expressed as percent of ABL mRNA levels.

Histamine release experiments

Histamine release experiments were performed on ROSA KIT WT cells following a standard protocol. 7,8 Cells were incubated with IgE (10 µg/ml) (Calbiochem, Darmstadt, Germany) and IL-4 (10 ng/ml, Peprotech) in IMDM at 37°C for 80 hours.

Then, cells were split and incubated in control medium or in medium containing JQ1 (200 nM) at 37°C overnight. Cells were then washed and exposed to 3 different concentrations of anti-IgE antibody E-124.2.8 (0.1, 1, and 10 µg/ml) in histamine release buffer (HRB, Immunotech, Marseilles, France) at 37°C for 30 minutes.

Thereafter, cells were centrifuged at 4°C and the cell-free supernatants and cell suspensions were recovered and examined for histamine content by radioimmunoassay (RIA) (Immunotech). Histamine release was calculated as percent of released (extracellular) histamine relative to total (cellular+extracellular) histamine. 7,8 Supplementary Tables Supplementary Table S1 Specification of antibodies used in immunostaining experiments --------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------

Supplementary Table S2 Patients´ characteristics and expression of BRD4 in neoplastic mast cells (MCs)

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UNPUBLISHED SUPPLEMENTAL DATA SUPPLEMENTAL MATERIALS AND METHODS

Chemical reagents and drugs

The proteasome inhibitors Bortezomib and MG132 were purchased from ChemieTek and Selleckchem (Houston, TX, USA). They were dissolved in in dimethylsulfoxide (DMSO) (Sigma-Aldrich).

Canine cell lines

The canine MCL cell line NI-1 [START_REF] Hadzijusufovic | NI-1: a novel canine mastocytoma model for studying drug resistance and IgER-dependent mast cell activation[END_REF] was maintained in RPMI medium supplemented with 10% fetal calf serum (FCS), 1% amphotericin and 1% antibiotics in 5% CO2 at 37°C. The canine mastocytoma cell line C2 (DeVinney & Gold 1990) was kindly provided by Dr. W.

Gold (University of California, San Francisco, CA, USA). C2 cells were cultured in IMDM supplemented with 10% FCS, α-thioglycerol, L-glutamine, and antibiotics in 5% CO2 at 37°C.

Methods

Evaluation of the expression of STAT proteins by flow cytometry

In selected experiments, intracellular staining for p-STAT3 and p-STAT5 was conducted after 4, 16

and 48 hours of incubation of ROSA KIT WT in control medium or in medium containing different concentrations of JQ1. After incubation, cells were washed with PBS then fixed with paraformaldehyde 2% for 15 minutes at RT. Therafter, cells were centrifuged and the cell pellet was resuspended in cool methanol at -20°C for at least 20 minutes. Cells then were washed with PBS containing 0.1% of bovine serum albuminBSA and the Alexa Fluor conjugated antibodies against p-STAT 3 (Alexa Fluor ® 647 mouse anti-STAT3 (pY705), clone 4/p-STAT3, BD Biosciences) or against p-STAT 5 (Alexa Fluor ® 647 mouse anti-STAT5 (pY694), clone 47, BD Biosciences) or the corresponding isotype control (Alexa Fluor ® 647 mouse IgG1κ or IgG2a κ) were added. Cells were then incubated in the dark for 30 minutes at RT. After one washing step with PBS 0.1% BSA, cell pellet was resuspended in about 30 µL of PBS 0.1% BSA and fluorescence was analyzed with a FACSCalibur (BD Biosciences).

Western Blotting

Prior to Western Blotting, HMC-1. 7.5). The membrane was then incubated sequentially with the primary antibody overnight at 4°C with gentle shaking and the secondary horseradish peroxidase (HRP)-coupled antibody for 1 hour at RT with gentle shaking. Both primary and secondary antibodies were diluted in 5 % BST w/v, 1X TBS, and 0.1% Tween-20 Tris-buffered saline Amersham, Piscataway, NJ) for 1 hour. HRP activity was detected by using HRP substrates (Amersham TM ECL TM Western Blotting Analysis System, GE Healthcare) and chimeluminesecence film (Amersham Hyperfilm TM ECL, GE Healthcare).

The following primary Abs were used: a polyclonal Ab against p-KIT (Tyr719, Cell Signaling For densitometrical analysis of the Western Blots for phosphorylated proteins, the blots were scanned and then the expression of proteins was calculated and normalized against GAPDH for every cell line.

Reverse transcriptase polymerase chain reaction (RT-PCR)

In selected experiments, using Trizol® (Invitrogen, Carlsbad, CA, USA), total RNA was extracted from ROSA KIT WT cells preincubatated with control medium or medium containing 200 nM JQ1 for 16 hours at 37°C. The cDNA was then synthesized from 1 µg of total RNA extract using. Bio-Rad Kit

UNPUBLISHED SUPPLEMENTAL RESULTS

During our work, we have also generated several additional ROSA subclones:

 the ROSA KIT Δ417-419 insY cell line which presents with a very immature phenotype. This particular KIT defect is frequently found in pediatric patients suffering from cutaneous mastocytosis (Bodemer et al. 2010). Interestingly, we found that ROSA KIT Δ417-419 insY cells were negative for FcεRI and do not contain any cytoplasmic granulations upon MGG staining.

The ROSA KIT Δ417-419 insY cells are SCF-independent like ROSA KIT D816V cells, but were found sensitive to imatinib in contrast to ROSA KIT D816V cells.

 the ROSA KIT K509I cell line which displays a mature phenotype with higher surface expression of CD203c compared to ROSA KIT WT cells. However, ROSA KIT K509I cells are SCF-independent.

 More recently, we were also able to transduce ROSA KIT WT cells with the following lentiviral constructs (gene-IRES-GFP) pWPI empty, pWPI HRAS WT and pWPI HRAS G12V to obtain three cell lines; ROSA pWPI empty , ROSA HRAS WT and ROSA HRAS G12V . Only ROSA HRAS G12V cells became SCF-independent and we observed that RAS was overexpressed in these cells (Figure S1).

Besides, regarding the involvement of BRD4 in the biology of neoplastic MCs, we were able to generate several additional data that have not been published yet.

For instance, we observed that BRD4 is expressed in the canine neoplastic MC lines NI-1 cells and C2 cells (Figure S2). Also, we found that BRD4-targeting drug JQ1 is able to inhibit 3 H-thymidine uptake in NI-1cells and C2 cells with IC 50 values of about 100-400 nM (Figure S3).

Besides, using RT-PCR, we observed that only the α chain of the FcεRI was significantly downregulated in ROSA KIT WT cells upon treatment with JQ1 (Figure S4). Moreover, we demonstrated that the treatment of ROSA KIT WT with the proteasome inhibitors (MG-132 or bortozemib) was not able to compensate the JQ1-mediated down regulation of FcRI expression (Figure S5). Of note, the proteasome inhibitors (MG-132 or bortozemib) were found to exert inhibitory effects on the proliferation of both ROSA KIT WT cells and ROSA KIT D816V cells with IC50 <5 nM after 48 hours of A. ROSA KIT WT cells were incubated in control medium (Co) or medium containing JQ1 (2500 nM) at 37°C for 24 hours. Then, RNA was isolated and tryptase mRNA level was quantified by qPCR (left panel) as described (Wedeh et al.2015). Results show transcript expression levels as percent of ABL mRNA expression and represent the mean±S.D of three independent experiments. Asterisk (*) indicates p<0.05 compared to control. B. ROSA KIT WT cells were incubated in medium (Co) or in medium containing indicated concentrations of JQ1 for 24 hours (right panels) and then western blotting analysis for tryptase was conducted as described. GAPDH was probed to control for protein loading. Then, cells were lysed and western blotting analyses were performed using indicated antibodies as described above. Results show a representative blot and GAPDH was used as a loading control.

A B

incubation. These data are in line with previously published reports which demonstrated that treatment of HMC-1.1 cells, HMC-1.2 cells and of the human leukemia cell line KU812 (established from the peripheral blood of a patient in blast crisis of chronic myelogenous leukemia) with different concentrations of hypomethylating agents (5-azacytidine and decitabine) had no effect on the surface expression of FcεRIα on these cells [START_REF] Aichberger | Identification of proapoptotic Bim as a tumor suppressor in neoplastic mast cells: role of KIT D816V and effects of various targeted drugs[END_REF][START_REF] Westerberg | Proteasome inhibition upregulates Bim and induces caspase-3-dependent apoptosis in human mast cells expressing the Kit D816V mutation[END_REF].

Additionally, we found that JQ1 was able to significantly decrease the expression of p-STAT-3 in ROSA KIT WT cells in a dose dependent manner after 16 hours and 48, as shown in Figure S6, whereas the level of p-STAT-5 remained unaffected (Figure S6). Also, JQ1 was found to decrease the expression of tryptase in ROSA KIT WT cells at both protein and mRNA levels (Figure S7).

Finally, as assessed by Western Blotting, we found that JQ1 decreased the phosphorylation of KIT in HMC-1.1 cells and HMC-1.2 cells as well as in ROSA KIT WT cells and ROSA KIT D816V cells (Figure S7). In addition, JQ1 was found to attenuate p-AKT, p-BTK and p-ERK1/2 in a dose-dependent manner with no substantial effects on the expression of KIT, AKT, BtK and ERK1/2 in all human MC lines, as shown in Figure S8.

DISCUSSION

Despite the growing scientific advances, research on MCs sufferered for long time from a lack of FcεRI+ human MC lines mimicking at the best the properties of normal MCs. In order to overcome this difficulty, we have recently isolated and characterized a new human MC line, derived from normal hematopoietic progenitors from a cord blood sample, that we termed ROSA KIT WT . The ROSA KIT WT cells are tryptase+ chymase-FcεRI+ SCF-dependent human MCs, with a KIT receptor which we showed to present with a normal sequence (Saleh, Wedeh et al. 2014). In addition, our cell line has a short doubling time, around 48-72h and is fully activable by a number of stimulants, including the cross-linking of their FcεRI. So far, only two FcεRI+ human MC lines have been established and published: LAD2 and LUVA cells (Kirshenbaum et al. 2003;Laidlaw et al. 2011). However, these two cell lines have several limitations, such as the long doubling time of about two weeks for LAD2

and the weak or rather negative surface expressionof FcεRIα in LUVA cells. As described above, our ROSA KIT WT cells have a short doubling time and stably express a functional FcεRI. Thus, these cells provide a novel advanced tool for MC research and enable the investigation of human MC functions as well as the high-throughput screening of anti-allergic therapeutics. Notably, the morphology and phenotype of these cells closely resemble the ones of primary cultured CBMCs, which is not the case for LUVA cells (Laidlaw et al. 2011). Interestingly, in contrast to cord blood-derived MCs (CBMCs),

ROSA KIT WT cells are easy to freeze and to thaw by conventional techniques and can be cultured for months without substantial changes in phenotype or significant alteration in their functional properties or clonal stability.

Since our laboratory is interested to work on the pathophysiology and treatment options of systemic mastocytosis, a category of disease characterized by multi-organ accumulation of neoplastic MCs harboring recurrently an activating point mutation in KIT (KIT D816V) (Valent 2008), we established the ROSA KIT D816V cell line, a KIT D816V+ SCF-independent MC line which constitutes the first in vitro model of human MCs with a KIT D816V mutant alone and for which a KIT WT equivalent exists. Indeed, in vitro models yet available to study the impact of KIT D816V mutation on human MCs proliferation, survival and activation were limited. Of note, the only cell model available before was the HMC-1 cell line and its two subclones HMC-1.1 cells and HMC-1.2 cells (Butterfield et al. 1988;[START_REF] Sundström | Functional and phenotypic studies of two variants of a human mast cell line with a distinct set of mutations in the c-kit proto-oncogene[END_REF]. Both clones contain the KIT V560G mutation, whereas HMC-1.2 additionally exhibits the KIT D816V mutation. Unfortunately, HMC-1 cells do not bear the KIT D816V alone and there is no HMC-1 subclone with a normal KIT gene, making it difficult to compare signaling recruited by normal or mutant forms of KIT in the same cell model.

Interestingly, we observed that our ROSA KIT D816V cell line, which expresses CD117, CD203c, and tryptase although not expressing CD2 and CD25, acquiered in vitro (and in vivo, see below) the morphological phenotype of neoplastic MCs found in patients suffering of SM, i.e.; a spinle-shaped appearance (Valent et al. 2007), which is not the case for any other in vitro models of human mastocytosis.

Besides, activation of KIT recruits a number of signaling pathways in MCs. Specifically, activation of the PI3-K/AKT pathway was shown to contribute to KIT-dependent proliferation, survival, maturation, adhesion, or activation (Ma et al. 2011). Interestingly, the introduction of KIT D816V in ROSA KIT WT cells induced the constitutive activation of AKT, which is in line with previously reported data (Casteran et al. 2003). In addition, KIT D816V recruits alternative signaling pathways not evoked by KIT WT, particularly STAT5 (Mayerhofer et al. 2008;Baumgartner et al. 2009;Chaix et al. 2011).

Interestingly, we observed that ROSA KIT D816V cells constitutively expressed p-STAT5, suggesting that they use the same alternative pathways as MCs in mastocytosis patients.

Surprisingly, we observed that, although expressing FcεRI at the same levels than ROSA KIT WT cells, ROSA KIT D816V cells do not respond to anti-IgE-mediated activation as neither do the BM-derived neoplastic MCs from patients with ASM and MCL (Saleh, Wedeh et al. 2014). The mechanism(s) involved in such hyporesponsiveness is still unknown. However, Ito et al have recently shown that MCs chronically exposed to SCF display a marked attenuation of FcεRI-mediated degranulation and cytokine production (Ito et al. 2012). Such desensitization may also play a role in KIT D816V-positive cells in which the KIT receptor is chronically phosphorylated. Since FcεRI and KIT use common signaling pathways, cross-desensitization may occur (Hundley et al. 2004). Alternatively, KIT D816V may induce the expression of negative regulators of MC activation, such as phosphatases. All in all, these findings might explain why many patients with SM, even with high MC burden, do not suffer at all from IgE-related mediator-related symptoms. Nevertheless, a number of patients may suffer from mediator-release symptoms related to non-IgE triggers, such as drugs, infection, or alcohol (Brockow & Metcalfe 2010).

In addition, we confirmed that ROSA KIT D816V cells were unresponsive to antiproliferative effects of imatinib, a tyrosine kinase inhibitor which targets BCR/ABL1 and wild-type KIT, but previously

shown devoid of significant effects on the TK activity of the KIT D816V mutant protein (Ustun et al. 2011). However, as expected, the ROSA KIT D816V cells remained sensitive to dasatinib and to midostaurin, two TKIs with broad activity of a variety of TKs, including KIT D816V mutant receptor (Saleh, Wedeh et al. 2014). Thus, given its ability to be grown easily in large amounts, the ROSA KIT D816V cell line can be used to perform a high-throughput screening of TKIs selectively targeting the KIT D816V mutations drugs in vitro as well as in vivo after injecting these cells to mouse models.

As of today, there has been several attempts to establish in vivo models mimicking aggressive SM using various techniques and protocols, with more or less success. For instance, the HMC1.2 cell line (Butterfield et al. 1988) has been injected in nude mice, and this gave rise to solid mast cell tumors [START_REF] Schumacher | Lectin histochemistry of human leukaemic mast cells (HMC-1) transplanted into severe combined immunodeficient (scid) mice[END_REF]. However, HMC1.2 is a human mast cell line derived from a patient with a MCL, the cells contain 80 chromosomes and present with two KIT mutations (which is unusual in SM patients). Thus this model looks far from ASM in humans for all these reasons. Besides, another model was made by injecting P815 cells injected in DBA2 mice [START_REF] Demehri | Establishment of a murine model of aggressive systemic mastocytosis/mast cell leukemia[END_REF]. P815 is a mouse mastocytoma cell line which harbors a D814Y mutation in a mouse Kit gene (corresponding to a human KIT D816Y and not D816V). When injected IV in DBA2 mice, it gives rise mostly to MCL. In this case the mice died in 7-8 days. Clearly all these elements make the model far from ASM patients.

Later, our team was involved in the development of a mouse model transgenic for the human KIT D816V gene, which was selectively expressed in MCs because the gene coding for the KIT mutant was expressed under a baboon chymase promoter (Zappulla et al. 2005). This seemed to us a good model to study the pathophysiology of mastocytosis, although we observed that a SM-like disease developed only in one to 2 years and in only one third of the transgenic animals (Zappulla et al. 2005).

In addition, while in this model the KIT mutant was of human nature, it was expressed in murine mast cells. Finally, this model appears clearly not suitable for in vivo assay of drugs given the time needed to observe appearance of the disease and the frequency of diseased animals (Zappulla et al. 2005).

A more recently published in vivo model of mastocytosis consisted of mice engeenered to express conditionally a constitutively active Kit D814V murine mutant, i.e; a Cre/loxP-based bacterial artificial chromosome transgenic mouse model [START_REF] Gerbaulet | Mast cell hyperplasia, B-cell malignancy, and intestinal inflammation in mice with conditional expression of a constitutively active kit[END_REF]. Although the model is still dealing with mouse mast cells harboring a murine Kit mutant, which is far from the reality of neoplastic MCs cells in ASM patients (a human KIT D816V mutant in human MCs), however, this model has some merits since, in certain conditions, by crossing KitD814Vflox mice to A-Mcpt5-Cre line [START_REF] Gerbaulet | Mast cell hyperplasia, B-cell malignancy, and intestinal inflammation in mice with conditional expression of a constitutively active kit[END_REF], which deletes loxP-flanked DNA, the Kit D814V mutant can be driven to be selectively expressed only in mature MCs. This gives rise to a slowly progressive SM, with bone marrow involvement and with a latency of up to 52 weeks. In addition, affected mice suffered of severe colitis.

Although potentially interesting to analyze the pathophysiology of mastocytosis, this model seems not fully suitable for drug testing for several reasons such as the latency in the development of the disease (one year, which is not fully compatible with drug testing).

Given that the in vivo models of mastocytosis yet available were not fully comparable to what is observed in the human SM patients, for the reasons exposed above, we sought to investigate whether we could create, using our ROSA KIT D816V cell line, a new and more relevant in vivo model of mastocytosis mimicking at the best the human disease, in order to be able not only to investigate the pathophysiological events involved in the development of the disease, but also to screen for drugs potentially active in vivo against the KIT mutant or the signalling pathways evoked by the abberantly activated TK. Interestingly, we were able to show that, after intravenous injection in the tail vein of NOD/SCID IL-2Rγ -/-(NSG) mice, ROSA KIT D816V cells can engraft at a high rate in these animals, not only in the BM, but also in spleen and lung, giving rise to a disease mimicking at best the situation encountered in SM patients (Saleh, Wedeh et al. 2014). Indeed, as evidenced by immunohistochemistry for human tryptase on BM sections of NSG mice injected intravenously with ROSA KIT D816V cells, compact infiltrates of tryptase-positive human MCs can be seen (Saleh, Wedeh et al. 2014). Together with the spindle-shaped appearance of the neoplastic MCs found on BM smears of the animals, and with their positivity for KIT D816V, our animal model exhibits several features resembling SM in humans, making it the most similar model for human SM described so far. Both ROSA KIT WT cells and ROSA KIT D816V cells have been patented (international approved patent WO/2013/064639) and published (Saleh, Wedeh et al. 2014).

The KIT D816V mutation is known to confer resistance against imatinib (Gotlib et al. 2006;Ustun et al. 2011). So far, no curative treatment is available for patients with advanced SM (Arock et al. 2015).

In these patients, no standard treatment exists and cytoreductive therapy is usually required to keep the disease process under control (Valent et al. 2010;Arock et al. 2015). Besides antimediator therapy, and depending on the aggressiveness of the disease, targeted or non-targeted chemotherapies, such as IFN-α, 2-CdA, or hydroxyurea (HU), or even allogeneic stem cell transplantation (allo-SCT) are sometimes required to control the end-organ damage caused by MC infiltration [START_REF] Siebenhaar | Treatment References strategies in mastocytosis[END_REF]Arock et al. 2015). Despite the encouraging data obtained in clinical trials with the TKI midostaurin (PKC412) in patients with advanced SM, neoplastic MCs may bypass and rely on KITindependent pathways for survival (Gleixner et al. 2011. Arock et al. 2015). Therefore, considerable efforts and investigations are made to develop drugs targeting the oncogenic signaling machinery downstream of KIT and/or other, KIT-independent survival pathways Moreover, given that there is also an increasing need to identify other druggable targets in neoplastic MCs from patients with advanced SM, we were interested to evaluate the potential activity of BET inhibitors in neoplastic MCs in vitro and ex vivo. In fact, bromodomain-containing proteins have recently been reported to be of interest in several hematological malignancies and in solid tumors [START_REF] Filippakopoulos | Selective inhibition of BET bromodomains[END_REF]Zuber et al. 2011;[START_REF] Delmore | BET bromodomain inhibition as a therapeutic strategy to target c-Myc[END_REF]Mertz et al. 2011;Dawson et al. 2011;[START_REF] Lockwood | Sensitivity of human lung adenocarcinoma cell lines to targeted inhibition of BET epigenetic signaling proteins[END_REF]Herrmann et al. 2012;[START_REF] Puissant | Targeting MYCN in neuroblastoma by BET bromodomain inhibition[END_REF][START_REF] Bandopadhayay | BET bromodomain inhibition of MYC-amplified medulloblastoma[END_REF]Shi et al. 2014;[START_REF] Asangani | Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer[END_REF]). These proteins have also been recently identified as key transcriptional regulators of cell cycle progression and growth in various neoplastic cells (Zuber et al. 2011;Herrmann et al. 2012;[START_REF] Dawson | Targeting epigenetic readers in cancer[END_REF]Godley & Le Beau 2012;[START_REF] Belkina | BET domain co-regulators in obesity, inflammation and cancer[END_REF].

One of these regulators appears to be BRD4, an epigenetic reader involved in the regulation of MYC.

Moreover, it has been recently established that BRD4 is a druggable epigenetic target in AML cells (Zuber et al. 2011;Herrmann et al. 2012) and similar observations have been made in other tumor models (Ott et al. 2012;Fiskus et al. 2014;Roderick et al. 2014). BRD4 inhibition by the selective BET bromodomain inhibitor and the BRD4-targeting drug JQ1 has been reported to abrogate tumor growth in vito as well as in vivo [START_REF] Filippakopoulos | Selective inhibition of BET bromodomains[END_REF]. In addition, it has been recently shown that BRD4 is expressed abundantly in the cytoplasm of leukemic blast cells in AML (Herrmann et al. 2012). Consistently, in the present study, we have showed that BRD4 protein is highly expressed in the cytoplasm of neoplastic MCs derived from patients with advanced SM, and that the BRD4targeting drug JQ1 induced growth inhibition and apoptosis in these cells (Wedeh et al. 2015). More precisely, we were able to show that BRD4 is expressed abundantly in the cytoplasm of neoplastic MCs in almost all patients with advanced SM, whereas in most patients with ISM, MCs expressed only low levels or no detectable the BRD4 protein in their cytoplasm. In addition, we were also able to demonstrate that HMC-1 cells as well as ROSA cells expressed cytoplasmic and nuclear BRD4 (Wedeh et al. 2015).

Of interest, BRD4 knock-down by RNA silencing in human neoplastic MC lines decreased the proliferation of these cells, and induced apoptosis. In addition, we observed that RNA silencing of BRD4 was associated with the downregulation of MYC mRNA. These data strongly suggest that BRD4 protein likely habors functions that are crucial for the survival of neoplastic MCs. As it is well known that ASM and MCL are incurable malignancies characterized by drug resistance and a poor survival (Travis et al. 1986;Valent et al. 2001;Horny & Valent 2001;Sperr et al. 2001;Valent et al. 2003;Akin & Metcalfe 2004;Valent et al. 2004;Valent et al. 2004;Horny et al. 2007;Metcalfe 2008;Lim et al. 2009;Arock & Valent 2010), it was critical to learn more about new drug targets expressed in neoplastic MCs and to identify more effective targeted drugs that have the potential to overcome drug resistance in ASM and MCL. In the present study, we have identified BRD4 as a novel drug target in neoplastic MCs in advanced SM (Wedeh et al. 2015). To the best of our knowledge this is the first report describing a BET bromodomain-containing molecule as a regulator of cell growth and therapeutic target in neoplastic MCs.

The expression of cytoplasmic BRD4 protein in neoplastic MCs in advanced SM may have several explanations. One hypothesis is that cytoplasmic expression of BRD4 is indicative of a higher proliferative potential of neoplastic MCs. An alternative explanation would be that additional, KITindependent, lesions acquired during disease progression from indolent to advanced SM, promote cytoplasmic expression of BRD4 in neoplastic MCs. Indeed, apart from KIT D816V, several additional molecular lesions and pro-oncogenic signaling pathways are expressed and activated in neoplastic MCs in advanced SM.

Althought the exact role and biochemical function of cytoplasmic BRD4 in neoplastic MCs remain at present unknown, it is interesting to notice that BRD4 is considered to display a kinase activity (Devaiah et al. 2012) and to interact with a number of cytoplasmic antigens, such as the signal-induced proliferation-associated protein 1 (SPA-1) in malignant cells (Gleixner et al. 2011;Wilson et al. 2011;Schwaab et al. 2013). BRD4 interacts with SPA-1 in vivo and in vitro and increases Rap GAP activity of SPA-1. In addition, BRD4 and the SPA-1 molecules have been documented to regulate each other's subcellular localization and to affect cell cycle progression from G 2 through M [START_REF] Farina | Bromodomain protein Brd4 binds to GTPase-activating SPA-1, modulating its activity and subcellular localization[END_REF].

Whether BRD4 also fulfils these functions in the cytoplasm of neoplastic MCs remains unknown.

Thus, further studies are required to understand in more details the precise role of BRD4 in the context of its cytoplasmic expression in neoplastic MCs of advanced SM.

Regarding the mechanisms contributing to the expression of BRD4 in neoplastic MCs, little was known so far. In order to investigate the signaling pathways potentially involved in the expression of BRD4, we performed experiments using various signal transduction inhibitors, including TKIs such as imatinib, dasatinib, PKC412, and ponatinib, MEK inhibitors, PI3K/mTOR inhibitor, ATRA, 2CdA, demethylating agents, JAK2 inhibitors, and STAT inhibitors piceatannol and pimozide. In these experiments, the multi-targeted TKIs PKC412 and dasatinib, that are known to block the kinase activity of KIT D816V, were found to downregulate the expression of BRD4 in HMC-1.1 cells, HMC-1.2 cells, ROSA KIT WT cells and ROSA KIT D816V cells. In addition, we found that various MEK inhibitors and the PI3K/mTOR blocker BEZ235 can downregulate expression of BRD4 in neoplastic MCs. Since MEK as well as PI3-K are downstream effectors of KIT (Hu et al. 2003;Roskoski 2005;Harir et al. 2008), these data suggest that KIT D816V but also other KIT mutants and even KIT WT may contribute to the expression of BRD4 protein in neoplastic MCs. This hypothesis is supported by the observation that SCF promoted the expression of BRD4 in ROSA KIT D816V cells. However, SCF failed to upregulate expression of BRD4 in HMC-1 cells. These observations suggest that expression of BRD4 in MCs depends not only on KIT activation, but also on other factors. Moreover, it has been recently shown that the BRD4-targeting drug JQ1 exerts strong anti-neoplastic effects in drug-resistant AML cells (Sotlar et al. 2003;Herrmann et al. 2012). In the present study, we were able to show that JQ1 induces growth inhibition and apoptosis in HMC-1 cells and ROSA cells as well as in primary neoplastic mast cells obtained from patients with advanced SM. Interestingly, in each cell line tested, the effects of JQ1 on malignant cell growth were dose-dependent, with reasonable IC 50 values, and in each case, growth-inhibition was associated with induction of apoptosis. In addition, the BRD4trageting drug JQ1 was found to block in a dose-dependent manner the cell cycle progression in all human MC lines tested in our study. Moreover, we found that BRD4 is overexpressed in the cytoplasm was found to block the proliferation of these canine cell lines in a dose-dependent manner. As well, JQ1 induced a dose-dependent apoptosis in the same cell lines. To the best of our knowledge this is also the first demonstration that BRD4 inhibition may impede dog neoplastic MC proliferation and survival. This might have interesting therapeutic consequences at the level of veterinary medicine since, by contrast to the human situation, mastocytoma is one of the most common malignant disease found in dogs and has always a poor prognosis in such animals [START_REF] Cohen | Epidemiological analysis of the most prevalent sites and types of canine neoplasia observed in a veterinary hospital[END_REF]London & Sequin 2003;[START_REF] Misdorp | Mast cells and canine mast cell tumours[END_REF][START_REF] Welle | Canine mast cell tumours: a review of the pathogenesis, clinical features, pathology and treatment[END_REF][START_REF] Hosseini | Cutaneous mast cell tumor (Mastocytoma): cyto-histopathological and haematological investigations[END_REF].

In AML, JQ1 effects were found to be associated with signs of maturation (Zuber et al. 2011;Herrmann et al. 2012). We were therefore interested to explore whether exposure to JQ1 could lead to maturation in HMC-1 cells and ROSA cells. However, we were not able to detect a morphologically visible maturation-inducing effect of JQ1 on these cells. Moreover, we were unable to demonstrate that JQ1 induced upregulation of differentiation-associated antigens in the same cells. Rather, we observed that JQ1 downregulated the expression of several key surface antigens in HMC-1 cells and/or ROSA cells, including CD63 (LAMP-3) and CD71 (transferrin receptor). Besides, JQ1 was found to downregulate the expression of FcεRI on ROSA, which may point to the fact that FcεRI expression is regulated by epigenetic mechanisms in these cells. To the best of our knowledge, this is the first report describing that FcεRI expression in human MCs is regulated by an epigenetic mechanism involving a bromodomain-containing molecule. We were also able to show that this effect of JQ1 is of functional significance. Indeed, JQ1 was found to suppress FcεRI-dependent histamine release in ROSA KIT WT cells as well as IgE-anti-IgE-induced upregulation of CD63 and CD203c in these cells, presumably because of down-regulation of FcεRI expression. Also, interestingly, we found that expression of tryptase decreased in ROSA KIT WT cells upon treatment with JQ1.

Besides, JQ1 was found not only to abrogate the expression of p-STAT3 in ROSA KIT WT cells but also to decrease the expression of p-STAT5 in ROSA KIT D816V cells, HMC-1.1 cells and HMC-1.2 cells in a dose-dependent manner. These observations are consistent with a recent report in which JQ1 was shown to inhibit p-STAT5 in myeloid malignancies (Liu et al. 2014). Collectively, these data suggest that JQ1 blocks several molecular key programs involved not only in the proliferation, but also inthe activation of neoplastic MCs, which may be relevant clinically, as patients with advanced SM not only suffer from organ infiltration but also from the consequences of MC activation and mediator release (Valent et al. 2001;Valent et al. 2004;Horny et al. 2007;Metcalfe 2008;Arock & Valent 2010). We then examined if the treatment of ROSA KIT WT cells with proteasome inhibitors might rescue JQ1induced downregulation of FcεRIα surface expression on these cells. However, no effect of proteasome inhibitors was found in this context. Instead, we found that these proteasome inhibitors exert antiproliferative effects on these cells, with an IC50 of about 5 nM. In addition, these antiproliferative effects were associated with induction of apoptosis not only in ROSA KIT WT cells but also in ROSA KIT D816V cells.

Interestingly, using RT-PCR experiments, we were able to show that JQ1 induced the downregulation of the expression of mRNA coding for FcεRIα in ROSA KIT WT cells. In addition, using qPCR, we found that JQ1 treatment decreased the level of transcription of MYC as well as of BCL-2, BCL-xL and MCL-1, whereas it increased the transcription of the proapoptotic molecule BIM in human MC lines.

These observations are consistent with the recently published reports describing that JQ1 can inhibit the binding of BRD4 and of pTEFb to the super enhancers of some oncogenes including MYC, and BCL-2, leading to the depletion of their transcript levels [START_REF] Delmore | BET bromodomain inhibition as a therapeutic strategy to target c-Myc[END_REF][START_REF] Lee | Transcriptional regulation and its misregulation in disease[END_REF][START_REF] Lovén | Selective inhibition of tumor oncogenes by disruption of super-enhancers[END_REF][START_REF] Chapuy | Discovery and characterization of super-enhancer-associated dependencies in diffuse large B cell lymphoma[END_REF]. Figure 25 shows a scheme recapitulating the effects of JQ1 in neoplastic MCs. In addition, since demethylating agents have been recently shown to lead to over-expression of FcεRI on monocytes of patients with atopic dermatitis [START_REF] Liang | Demethylation of the FCER1G promoter leads to FcεRI overexpression on monocytes of patients with atopic dermatitis[END_REF], we were thus interested to know whether treatment of HMC-1.1 cells, HMC-1.2 cells and KU812 cells (which usually lack the expression of this receptor) with these agents would induce expression on FcεRI at the cell surface.

However, we found that demethylating agents do not induce FcεRI surface expression in these cells.

Moreover, the analysis of the data obtained after immunoblot conducted on HMC-1.1, HMC-1.2, ROSA KIT WT , and ROSA KIT D816V cells upon treatment with JQ1 led us to conclude that the MEK and PI3-Kinase pathways are involved in the mechanism underlying the antineoplastic effect of the drug on these cells. Therefore, our hypothesis is that the RAS signalling pathway might be involved in this effect. Our findings are consistent with several recent reports in the context of the activity of BRD4 Based on these studies, we have recently generated ROSA HRAS WT and ROSA HRAS G12V cell lines, which will enable us to further investigate the validity of our hypothesis about the involvement of RAS in the the activity of BRD4. ROSA HRAS WT cells overexpress the RAS WT but remain SCF-dependent, whereas and ROSA HRAS G12V cells are SCF-independent. We will compare the proliferation rate and the expression of BRD4 expression in both cell lines, as well as the effects of JQ1 on cell proliferation and apoptosis. It is here tempting to speculate that overexpression of RAS or RAS mutation will confer sensitivity to JQ1 in ROSA SCF-mediated resistant cells.

Whatsoever, the intriguing effects of JQ1 on growth and survival of neoplastic MCs prompted us to search for optimal drug partners through which synergistic anti-neoplastic effects can be obtained. To address this point, we tested a number of different combination partners in HMC-1 cells and ROSA cells. Of all drug-partners tested, PKC412 and ATRA were found to synergize with JQ1 in inhibiting proliferation and survival in HMC-1 cells and ROSA cells, which may have clinical implications. Of note, PKC412 is already in clinical trials for the treatment of patients with Advanced SM and seems to present beneficial effects in such cases [START_REF] Gotlib | Midostaurin (PKC412) Demonstrates a High Rate of Durable Responses in Patients with Advanced Systemic Mastocytosis: Results from the Fully Accrued Global Phase 2 CPKC412D2201 Trial[END_REF], whereas it has been already shown that ATRA may negatively impact the development of normal and malignant MCS [START_REF] Ishida | Serum inhibitors for human mast cell growth: possible role of retinol[END_REF][START_REF] Hjertson | Retinoic acid inhibits in vitro development of mast cells but has no marked effect on mature human skin tryptase-and chymase-positive mast cells[END_REF][START_REF] Alexandrakis | Inhibitory effect of retinoic acid on proliferation, maturation and tryptase level in human leukemic mast cells References (HMC-1)[END_REF][START_REF] Ohashi | Retinoids induce growth inhibition and apoptosis in mast cell tumor cell lines[END_REF][START_REF] Pinello | In vitro chemosensitivity of canine mast cell tumors grades II and III to all-trans-retinoic acid (ATRA)[END_REF]). Thus, that JQ1 synergizes with such drugs in inhibiting the growth of human malignant MC lines was expected.

In a final step, we asked whether resistance against JQ1 may occur in neoplastic MCs. In these experiments, we found that SCF introduced resistance against JQ1 in ROSA KIT D816V cells but not in HMC-1 cells. Although the underlying mechanism of this drug resistance induced by the addition of SCF in ROSA cells remains at present unknown, we asked whether the synergistic activity of the drug combinations JQ1+ATRA and JQ1+PKC412 would overcome this SCF-mediated drug resistance.

Interestingly, we found that the combination JQ1+PKC412, but not of JQ1+ATRA, was able to overcome SCF-mediated resistance in ROSA cells. Given these data, one can hypothesize that the SCF-induced resistance to JQ1 is mediated by over-activation of the KIT recptor by its ligand, and that this mechanism of resistance can only be overcome by the addition of a strong KIT-blocking agent such as PKC412.

In summary, our data show that BRD4 is a promising druggable target in neoplastic MCs in advanced SM. Our data also show that inhibition of BRD4 by JQ1 is a potent approach to eradicate neoplastic MCs, and that these interesting drug effects could be potentiated by addition of PKC412 or of ATRA.

CONCLUSIONS AND PERSPECTIVES

In the first part of our work, we have been able to establish a new human FcεRI+ Tryptase + MC line exhibiting the major characteristics of normal MCs, termed ROSA KIT WT cells. Using this cell line, which is SCF-dependent, we were able to show its full activation capabilities after cross-linking of their FcεRI. This cell line, which has a short doubling time and a stable phenotype, represents a significant advance towards other human mast cell lines already available. In addition, using lentiviral infection of these ROSA KIT WT cells, we have been capable to generate a new stable subclone, termed ROSA KIT D816V cells, since the cells were transduced with a construct expressing the KIT D816V mutant. This new subclone shares a number of characteristics with neoplastic MCs found in aggressive SM (ASM), including growth-factor independence for its proliferation and constitutive activation of KIT and of downstream signaling pathways already known to be evoked in neoplastic MCs from SM patients, such as STAT-5 and AKT. Moreover, after intravenous injections, these ROSA KIT D816V cells were able to produce a systemic mastocytosis-like disease in NSG mice, with an aggressive phenotype.

ROSA KIT D816V cells can thus be used for testing new anti-neoplastic drug therapies in vitro and in vivo and can be helpful to fully understand the pathogenicity of the disease. Finally, this malignant cell line can be easily manipulated and used to introduce the more frequent additional genetic alterations found in ASM, such as mutations in TET2, SF3B1, etc…with the aim to better understand how these somatic mutations alter the behavior and prognosis of the disease.

Given that patients with advanced categories of SM have a poor prognosis and that the therapeutic arsenal for such patients is still limited, we were thus interested, in the second part of our work, to find new targets of potential therapeutic interest in neoplastic MCs. Starting from the literature already available showing a potential role of BRD4 in several hematological malignancies, we decided to investigate the possible role of this molecule in SM. Interestingly, we have been able to show a significant expression of cytoplasmic BRD4 protein in human and canine neoplastic MC lines. In addition, we have demonstrated that neoplastic MCs derived from patients with ASM and MCL overexpress BRD4 in their cytoplasm. Moreover, we have showed that RNA silencing of BRD4 in human MC lines inhibited the proliferation of these cells, and induced their apoptosis with downregulation of MYC transcript. However, further studies are still required to determine the specific role and the particular biochemical functions of BRD4 abberantly localized in the cytoplasm of neoplastic MCs in SM. Moreover, the interaction of cytoplasmic BRD4 with other cytoplasmic antigens, such as SPA-1, in neoplastic MCs remains to be elucidated. These studies may reveal other promising novel and druggable targets in neoplastic MCs and their progenitors, leading to an optimization of the therapeutic options with the aim of increasing the survival time of patients with advanced SM.

Whatsoever, the relevance of our data obtained on neoplastic MC lines was confirmed by further experiments performed with the use of JQ1, a first-in-class prototype of bromodomain inhibitor (a benzodiazepine-based drug developed in Bradner's laboratory). Indeed, inhibition of BRD4 using this molecule, leads in our hands to sustained inhibiton of proliferation associated with the induction of apoptosis in human neoplastic MC lines as well as in primary MCs derived from patients with ASM and MCL. In addition, JQ1 was found to block dose-dependently the cell cycle progression in human malignant MC lines and to decrease in the same way the surface expression of CD63 and CD71 on these cells.

One intringuing observation made during our experiments was the fact that JQ1 potently downregulated the expression of FcεRI in ROSA KIT WT cells and suppresses FcεRI-dependent histamine release and IgE-anti-IgE-induced upregulation of CD63 and CD203c in these cells. This finding underlines the therapeutic potential of BRD4 inhibition in neoplastic MCs derived from patients with advanced SM suffering from the consequences of MC activation and mediator release.

Indeed, it appears that JQ1 may not only inhibit the proliferation of neoplastic MCs, but might also decrease the exaggerated release of MC-derived mediators in SM patients who are prone to develop MC mediators symptoms. However, this interesting effect of JQ1 on FcεRI expression and on IgEanti-IgE-dependent histamine release deserves further evaluation in human normal MCs and basophils.

Particularly, the molecular mechanisms involved in such effects need to be further investigated and, among others, effects of JQ1 on the expression and phosphorylation state of LYN, SYK or PLC- need to be evaluated on resting and FcεRI-activated cells.

Interestingly, we observed that JQ1 treatment decreased transcriptional levels of MYC levels. This observation is consistent with published data on the effects of the same molecule in other cancer cells.

Thus, one can hypothesize that JQ1 treatment could thereby suppress transcription of MYC-dependent target genes in neoplastic MC lines. However, this needs to be confirmed in further experiments.

Although the BET bromodomain is primarily known to associate with chromatin through proteinprotein interaction with acetylated histones, our data support the fact that JQ1 inhibits the activation of STAT3 and STAT5 in human MC lines. In addition, we also demonstrated that JQ1 blocks PI3-Kinase and MEK pathways involved in the survival and proliferation of neoplastic MCs. These observations may be important to identify other druggable targets.

Moreover, we found that SCF introduced resistance against JQ1 in ROSA KIT D816V cells. The underlying mechanism(s) of this SCF-induced resistance in these cells remain(s) at present unknown and require(s) further investigations. Whatsoever, we observed that SCF promoted the expression of BRD4 at the transcriptional level in these cells, underlining the potential involvement of this cytokine in the context of proliferation and survival of neoplastic MCs. However, cytokine-induced resistance to JQ1 might be due to mutations in BRD4 developed as a mechanisme of resistance in neoplastic MCs, indicating the necessity to rule out these mutations. Genome-wide transcriptome analysis comparing cells treated with control or with JQ1 in the presence and absence of SCF could be of great interest in order to uncover alternate and possible mechanisms of resistance. Importantly, the synergistic activity of the JQ1 with PKC412 can overcome SCF-mediated JQ1 resistance in neoplastic MCs. Indeed, the combination JQ1+PKC412 was found to overcome SCF-mediated resistance in ROSA cells.

Alltogether, our data show that BRD4 is a promising druggable target in neoplastic MCs in advanced SM. BRD4 inhibition provides an opportunity to target dysregulated transcriptional programs in neoplastic MCs. Of note, JQ1 has been reported to show reasonable safety profile and tolerance in mice. However, the potency of BRD4 inhibition with JQ1 or other I-BET in vivo models such as NSG mice and in clinical trials in patients with advanced SM is still unkown. Hitherto, the antineoplastic effect observed with JQ1 or other BET-bromodomain inhibitors in vivo and in patients with advanced Conclusions and Perspectives

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SM remains also to be elucidated.

In conclusion, our data highlight the promise of BET protein antagonists for the treatment of patients with advanced SM. However, while current BET inhibitors target simultaneously BRD2, BRD3 and BRD4, it might be beneficial to design more specific and selective inhibitors for BRD4, as well as to develop chemical tools that could allow to discriminate between the disease-relevant functions of each bromodomain present (BD1 and BD2) in BRD4. Continued research efforts are also needed in order to explore disease-relevant BRD4 protein-protein interactions, genomic binding sites, and downstream transcriptional effects. Par ailleurs, il a été montré que l'inhibition de BRD4 par des siRNA ou par les inhibiteurs de BET tels que le composé JQ1 induit la mort de cellules tumorales in vitro ainsi qu'in vivo dans divers cancers.

Synthèse en

Ainsi, de nombreuses publications ont rapporté le potentiel thérapeutique prometteur de ces inhibiteurs in vitro et in vivo dans plusieurs hémopathies malignes, comme le lymphome de Burkitt, le myélome multiple ou les leucémies aiguës myéloïdes (LAM) (Zuber et al. 2011;.Dawson et al. 2011;Mertz et al. 2011;[START_REF] Delmore | BET bromodomain inhibition as a therapeutic strategy to target c-Myc[END_REF].Herrmann et al. 2013).

Objectifs de la thèse

Mon projet de recherche, au cours de ma thèse, a visé dans un premier temps à établir de nouvelles lignées mastocytaires humaines normales ou néoplasiques, à partir de cellules souches CD34+ de sang de cordon. Nous avons ainsi pu établir in vitro un modèle cellulaire qui représente morphologiquement et biologiquement les mastocytes humains normaux et un autre modèle cellulaire qui représente au mieux les mastocytes maligns retrouvés chez les patients atteints de mastocytoses avancées. Nous nous sommes ensuite interessé, dans un second temps, à étudier le potentiel thérapeutique de l'inhibition du lecteur épigénétique BRD4 chez les patients atteints de mastocytoses systémiques (SM) avancées (ASM et MCL).

Introduction de la thèse :

L'introduction en anglais de ma thèse comprend les chapitres suivants :

1. Les mastocytes Comme il a été récemment montré que le composé JQ1, inhibiteur de BRD4, exerce de puissants effets anti-néoplastiques dans des cellules de leucémies aiguës myéloïdes résistantes aux chimiothérapies (Sotlar et al. 2003;Herrmann et al. 2012), nous nous sommes intéressés aux effets de cette molécule dans nos modèles. Nous avons pu montrer, en utilisant le composé JQ1 ou un shRNA spécifique pour BRD4, que l'inhibition de BRD4 conduit à l'arrêt de la prolifération de toutes les lignées mastocytaires humaines testées. Les effets de JQ1 sur la croissance des cellules mastocytaires malignes ont été démontrés comme étant dose-dépendants. De plus, nous avons montré que le knock-down de BRD4 est associé à l'induction de l'apoptose et à la diminution d'ARN messagers codants pour MYC (Wedeh et al. 2015). Un même effet inducteur d'apoptose, dose-dépendant, a été observé avec le composé JQ1 dans toutes les lignées mastocytaires humaines testées (Wedeh et al. 2015). En outre, nous avons pu monter que le composé JQ1 inhibe la progression dans le cycle cellulaire de toutes les lignées mastocytaires humaines testées.

Fait intéressant, nous avons aussi observé que l'effet de JQ1 sur BRD4 conduit à l'inhibition de la prolifération des mastocytes médullaires primaires obtenus à partir de prélèvements médullaires de En conclusion, notre travail est le premier à démontrer que BRD4 peut constituer une cible thérapeutique intéressante dans les mastocytoses systémiques graves (MCL et ASM). Les données que nous avons obtenues montrent également que l'inhibition de BRD4 par le composé JQ1 est une approche thérapeutique novatrice potentiellement capable d'éradiquer les mastocytes néoplasiques et que, de plus, les effets de cette molécule peuvent être potentialisés par l'addition de PKC412 ou d'ATRA.

Introduction

Hepatitis C virus (HCV) has long been considered a publichealth problem, with an estimated 170 million infected individuals worldwide. It is a major human pathogen responsible for development of serious liver diseases, including steatosis, fibrosis, cirrhosis, and eventually, hepatocellular carcinoma [1]. HCV is a small, enveloped, positive-sense, single-stranded RNA virus of the family Flaviviridae [2]. The HCV genome is transported to the endoplasmic reticulum, where it is translated into a large polyprotein by the host translation machinery and processed by viral and cellular proteases into the mature structural (core, E1, E2) and non-structural proteins (p7, NS2, NS3, NS4A-B, NS5A-B) [2][3][4]. HCV replication occurs in replication complexes (RCs) in a membranous web (MW) consisting of lipid droplets (LDs) [4,5].

There is not yet a vaccine to protect against HCV infection. However, a major improvement regarding treatment of HCV infection has been recently achieved. Two direct-acting antivirals (DAAs), the protease inhibitors telaprevir and boceprevir, were approved for the treatment of HCV infection in combination with pegylated interferon alfa/ribavirin (peg-IFN/RBV) [6,7]. The addition of DAAs targeting different viral proteins was found to increase the sustained virologic response (SVR) [8], defined as the lack of detection of HCV RNA in blood several months after a complete course of treatment, and therefore to improve treatment success [9][10][11]. Unfortunately, resistance to DAAs has been reported recently, due to the high genetic variability of the HCV RNA genome [12][13][14]. Therapies that target host cell pathways may overcome or limit the occurrence of viral resistance and thus improve treatment responses. In this therapeutic context, there is an increased need to define the pathways that are hijacked by HCV for its life cycle. Moreover, HCV infection is tightly associated with alterations in lipid metabolism, and lipids have been shown to play important roles during the viral life cycle [15,16]. The HCV core induces the redistribution of LDs through the clustering of these organelles in the perinuclear area, providing a platform for virus replication, assembly and production [17].

LDs have long been considered simple reservoirs for lipid storage, but they are now considered dynamic organelles that are involved in many biological processes, and several reports have highlighted their movement by attachment to microtubules [15][16][17][18][19]. Nevertheless, the molecular details of the mechanisms driving LDs biogenesis, growth and intracellular movement remain largely unknown. Modification of proteins involved in lipogenesis, such as the sterol regulatory element-binding proteins (SREBPs) and liver receptor homolog 1 (LRH-1) by SUMO has been demonstrated [20,21]. Additionally, Talamillo and coworkers have demonstrated that silencing the drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content [23]. These recent advances suggest that SUMO might have critical roles in lipogenesis [20,21,23].

The SUMO protein was first identified in mammals, where it was found to be covalently linked to the GTPaseactivating protein RanGAP1 [24]. Consequently, SUMO is expressed by all eukaryotes but is absent from prokaryotes/ archaea. Lower eukaryotes have a single SUMO gene, whereas plants and vertebrates express several SUMO paralogues [25]. In mammals, the SUMO family consists of four different isoforms, termed SUMO1 to 4. The SUMOs are post-translationally conjugated to other proteins, thereby regulating diverse cellular processes, including transcription, DNA repair, chromosome dynamics, subcellular localization of proteins, and protein-protein interactions via a mechanism essentially analogous to that of ubiquitination [26,27].

In addition to its critical roles in a variety of cellular signalling pathways, SUMO family members play important roles in the life cycle of various viruses, such as KSHV (Kaposi's sarcoma-associated virus) [28] and influenza virus [29]. In this study, we performed different experiments using HCVcc-JFH1 and investigated the importance of SUMO1 for HCV replication.

Materials and methods

Antibodies and reagents

Anti-mouse actin and oleic acid were obtained from Sigma, and anti-mouse core and anti-rabbit SUMO1 were from Abcam. Anti-mouse Alexa Fluor 488 and 546 were obtained from Invitrogen. Nuclei were stained with TO-PRO-3 Iodide or Hoechst from Invitrogen.

Cell culture

Huh7.5 cells were cultured in Dulbecco's modified Eagle medium (DMEM, Invitrogen) containing 4.5 g of glucose per liter, supplemented with 10 % heat-inactivated fetal bovine serum, 1 % nonessential amino acids (Invitrogen) and 1 % penicillin/streptomycin (Invitrogen).

Small interfering RNA and cell transfection

Endogenous expression of SUMO1 was silenced by transfection with a specific siRNA (sc-29498, Santa Cruz). Huh7.5 cells were grown in 12-well plates or on glass coverslips and infected with HCVcc. At 24 hours post-infection, cells were transiently transfected with 100 pmol of a siRNA oligonucleotide against SUMO1 using a Lipofectamine RNAiMAX Kit (Invitrogen) according to the manufacturer's protocol and incubated for another 48 hours. Control non-targeting siRNA was used as a reference (Genecust). At 48 hours post-transfection, cells were harvested for extraction of proteins and isolation of total RNA or fixed with a 3.7 % paraformaldehyde. The samples were then analyzed via western blot, qRT-PCR and immunofluorescence.

Immunoblot

Cells were collected on ice, washed and lysed in 20 mM Tris-HCl, 100 mM NaCl, 1 % Triton X-100 and 10 mM EDTA, pH 7.4, containing protease and phosphatase inhibitor cocktail (Roche diagnostics). The proteins were separated by SDS-PAGE, blotted onto a PVDF membrane, and visualized using a chemiluminescence reagent (Amersham).

Immunofluorescence staining

Huh7.5 cells were grown on glass coverslips in 12-well plates, treated according to the experiments, fixed with 3.7 % paraformaldehyde, permeabilized, and saturated with PBS supplemented with 0.7 % fish gelatin and 0.025 % saponin. Primary antibodies were diluted with permeabilization solution and incubated with cells. The

JFH1/HCVcc production and inoculation

The JFH1/HCVcc stock was prepared as described [30]. Cells were infected with HCV at 37 °C for 16 h, and unbound viruses were removed by aspiration and by washing cells with PBS and DMEM. The infected cells were harvested for extraction of proteins and isolation of total RNA or fixed with a 3.7 % paraformaldehyde. The samples were then analyzed via western blot, qRT-PCR and immunofluorescence.

RNA extraction and qRT-PCR analysis

Total RNA was isolated using RNAble solution (Eurobio), and intracellular levels of positive-strand HCV RNA were quantified by a strand-specific qRT-PCR technique described previously [36] using a Light Cycler Fast Start DNA MasterPlus SYBR Green (Roche) on a Real-Time PCR System (Roche). The triplicate mean values were calculated according to the Ct quantification method (Pfaffl 2001) using GAPDH gene transcription as reference for normalization. The average levels of HCV RNA relative to the positive control (100 %) from triplicate experiments, are shown.

Statistical analysis

Comparison of mean values was conducted using an unpaired Student's t-test. Statistical significance was determined at *P \ 0.05, **P \ 0.001, and ***P \ 0.0001.

Results

The abundance of SUMO1 is altered in infected Huh7.5 cells

We examined the expression of SUMO1 in uninfected Huh7.5 cells (Mock) and infected Huh7.5 cells (HCV) (Fig. 1). Based on the specific bands size, immunoblot analysis indicated an increase in the amount of SUMO1 (Fig. 1B and Fig. 1C). In addition to investigating protein level of SUMO1, we examined the localization of SUMO-1 in uninfected and infected cells. SUMO-1 appeared in the nucleus and cytoplasm of uninfected and infected cells (Fig. 1A).

SUMO1 regulates HCV genomic RNA replication and core expression

We assessed the requirement for SUMO1 in HCV viral protein expression and RNA replication. First, Huh7.5 cells were infected with HCV-JFH1 for 24 hours and then treated with SUMO1 siRNA for another 48 hours and analyzed by immunoblot, immunofluorescence staining and qRT-PCR. A reduction in the SUMO1 signal was observed, and this was associated with a decrease in the core protein level (Fig. 1E). Next, the cells were analyzed by confocal microscopy. Treatment of cells with SUMO1 siRNA reduced cellular expression of the core protein, as shown by immunoblotting and immunofluorescence (Fig. 1D). After that, we tested the effect of SUMO1 on HCV replication using qRT-PCR. The intracellular level of HCV RNA was quantified, and a significant drop in the level of HCV genomic RNA was observed after depletion of SUMO1 (Fig. 1F), indicating a critical role for this protein in HCV replication. The efficiency of SUMO1 siRNA was evaluated by immunoblotting and immunofluorescence staining (Fig. 1E andD).

Depletion of SUMO1 affects colocalization of HCV core protein and LDs

Attachment of the core protein to LDs is crucial for HCV replication [15]. Recent advances suggest that SUMO might have critical roles in lipogenesis [20][21][22], silencing the drosophila SUMO in the prothoracic gland has been shown to result in reduced lipid content [23]. Taking these data into account, we analysed the behaviour of LDs in SUMO1-siRNA-treated infected Huh7.5 cells. As presented in Fig. 2A, in uninfected cells, LDs were present at low levels and dispersed in the cytoplasm, whereas in infected cells, there was an increase and redistribution of LDs as clusters that colocalized with core protein around the nucleus (Fig. 2B and SI Fig. 1A). Treatment of cells with SUMO1 siRNA strongly reduced accumulation of LDs and cluster formation (Fig. 2C). Confocal images were analysed using Image J software, and the results are presented as graph under to each panel with a cartoon representing the LD content of the cell separated in three zones from the nucleus (Z1, Z2, and Z3). The LD signal was mainly present around the nucleus (Z2), and the intensity of the LD signal strongly increased with HCV infection. However, treatment of cells with SUMO1 siRNA led to a decrease in the intensity of the LD signal and a shift from the centre to the cell periphery. Fifty cells were analysed from three experiments, and the intensity of the LD signal was evaluated using Image J software. The data are presented as histograms in Fig. 2C andD. The number of LDs and the percentage of cells expressing LDs increased in the presence of HCV infection (Fig. 2D). However, the silencing of SUMO1 expression led to a statistically significant decrease in the number of LDs (Fig. 2D) and also caused a reduction in the percentage of cells expressing LDs (Fig. 2E), indicating a role of SUMO1 in HCV induced-accumulation of LDs.

123

(Fig. 3A andB). Interestingly, treatment of cells with siRNA against SUMO1 suppressed LD formation induced by oleic acid in uninfected (Fig. 3A) and in infected cells (Fig. 3B), indicating that SUMO1 is required for formation of lipid droplets. Moreover, depletion of SUMO1 affected the expression of adipose differentiation-related protein (ADRP), an important protein associated with lipid droplets (Fig. 3C), confirming that knockdown of SUMO1 leads to reduced lipid content. The efficiency of SUMO1 siRNA was evaluated by immunoblot and immunofluorescence staining (Fig. 3C andD).

Discussion

This study provides another example of SUMO being needed for viral infection. We showed that HCV infection increases SUMO1 expression, which is necessary for its replication. This work provided significant insights into the host cellular machineries exploited by HCV for its replication, as well as new fundamental data on the functional features of SUMO1. Different studies have suggested that LDs play a crucial role in the life cycle of HCV. Interactions between HCV proteins, especially the core protein and LDs, are required for the morphogenesis and production of infectious HCV [31]. Our study revealed that SUMO1 modulated the accumulation of LDs and their association with HCV core and thus contributed to the establishment of the lipid environment required for virus replication. SUMO proteins are important regulators of cell signalling and are covalently linked to other proteins in order to alter their structure, localization or function. To date, descriptions of SUMO conjugated pathogen proteins are limited to viruses, for which protein SUMOylation facilitates viral entry, nuclear translocation, changes in gene expression, and vesicular trafficking [34]. Studies conducted by Dunphy and coworkers have shown that Ehrlichia chaffeensis exploits SUMOylation pathways to promote its intracellular survival [33]. Moreover, previous work has shown that global cellular SUMOylation increases during influenza virus infection, and silencing the key enzymes required for SUMO conjugation prevents virus replication [32]. Our own work indicated also that negative changes in SUMO1 levels disturb HCV replication. This raised the possibility that this virus may manipulate SUMO and/or the fate of SUMO-modified proteins to facilitate its replication. In this report, SUMO1 was found mainly within the nucleus in both uninfected and infected cells. However, there appeared to be an increase in SUMOylation during HCV infection. This change in SUMO1 might be an important step for determining the abundance of lipid droplets and for their recruitment, a process that is necessary for HCV replication. It is difficult to provide a simple hypothesis that attempts to explain these results, because the biological effects of SUMOylation are quite diverse, and the mechanisms and signal pathways involved in most of them remain unclear. Recent advances suggest that SUMO might have critical roles in lipogenesis [20][21][22]. Moreover, silencing of SUMO in the prothoracic gland of drosophila led to reduced lipid content [23]. More experiments will be needed to identify the factors involved in the connection of SUMO1 and lipid droplets. This study provided information about the role of SUMO1 in production and redistribution of LDs, which is timely, because there is increasing evidence in the literature that lipid droplets play a crucial role in the replication of different pathogens [35]. Our study suggested that HCV may alter the localization and abundance of SUMO1, and this, in turn, might help to recruit and organize the LDs required for HCV assembly and replication. Thus, one explanation for the implication of SUMO1 in LDs redistribution could be connected to the SUMO E3 ligase function.

To conclude, this study illuminates an important function for SUMO1 in HCV replication. These findings also represent a step forward in our understanding of the relationship between HCV and its host and may open new therapeutic directions. A future key aim may be to correlate changes to the SUMO sub-proteome with the function of lipid droplet biogenesis, thereby establishing the mechanistic role of these modifications during HCV replication. Understanding the basic mechanisms underlying SUMOylation in HCV infection will provide new insights into the fundamental biology of this important pathogen. Future studies focused on the key enzymes required for general SUMOylation could also lead to identification of key cellular pathways that can be exploited by novel therapeutic targets. , représentant un équivalent des cellules néoplasiques s'accumulant dans les SM. L'utilisation de cette lignée et de cellules des patients nous a permis d'identifier BRD4 comme une nouvelle cible thérapeutique dans les ASM et les MCL. Nous avons démontré que les MC néoplasiques de patients avec ASM expriment des quantités substantielles de BRD4. Fait intéressant, nous avons aussi démontré que les lignées cellulaires HMC-1 et ROSA KIT D816V expriment aussi BRD4, et que leur prolifération est inhibée par un shRNA BRD4-spécifique. En outre, nous avons montré que le médicament JQ1, inhibiteur de BRD4, induit une inhibition de la croissance et une apoptose dose-dépendante dans les mêmes cellules. De plus, nous avons démontré que JQ1 supprime également la prolifération des MC néoplasiques primaires de patients atteints d'ASM ou de MCL à de faibles concentrations. Enfin, nous avons observé que la midostaurine (PKC412) et l'acide rétinoïque tout-trans (ATRA) coopèrent avec JQ1 pour induire des effets inhibiteurs synergiques sur l'inhibition de la survie des mêmes cellules. En conclusion, nos résultats représentent une avancée sur ce qui était précédemment connu sur l'implication de BRD4 dans les mastocytoses et nous ont permis d'identifier cette protéine comme cible thérapeutique prometteuse dans le traitement des formes avancées de SM.

Title: Identification of BRD4 as a novel therapeutic target in aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL) Keywords: BRD4, aggressive systemic mastocytosis, mast cell leukemia, therapeutic target Abstract: Human mast cells (MCs) are hematopoietic stem cell (HSC)-derived, tissue-resident, multifaceted cells involved in a myriad of physiological and pathological processes. Researches on MCs have been hampered for a long time, due to limited access to pure populations of these cells. We have established a new human MC line, ROSA KIT WT , whose properties are similar to those of primary HSC-derived MCs, providing a novel tool for research on human MC functions, and enabling the highthroughput screening of anti-allergic therapies. Among others, MCs are involved in a group of diseases termed mastocytosis, where they accumulate pathologically in various tissues. Although most cases of systemic mastocytosis (SM) are chronic with an indolent course, patients with advanced SM (aggressive SM; ASM, and mast cell leukemia; MCL) have a reduced life expectancy and a poor prognosis, since most of the therapies already available are not curative. In order to better understand the pathophysiology of advanced SM and to. find new approaches for treatment, we took advantage of the availability of the ROSA KIT WT cells to establish a new subclone, the ROSA KIT D816V cell line, representing a paradigm of the neoplastic cells accumulating in SM.

Using these malignant cell line and patients' cells, we identified the epigenetic reader bromodomain-containing protein-4 (BRD4) as a novel drug target in ASM and MCL. Indeed, we demonstrated that neoplastic MCs from ASM patients expressed substantial amounts of BRD4. Interestingly, we then demonstrated that HMC-1 and ROSA KIT D816V cell lines express BRD4, and that their proliferation is inhibited by a BRD4-specific shRNA. Moreover, we showed that the BRD4-targeting drug JQ1 induced a dose-dependent growth inhibition and apoptosis in the same cells. In addition, we demonstrated that JQ1 suppressed also the proliferation of primary neoplastic MCs of patients with ASM or MCL at low concentrations. Finally, we reported that midostaurin (PKC412) and alltrans retinoic acid (ATRA) cooperated with JQ1 in producing synergistic inhibitory effects on the survival of HMC-1 and ROSA cells. Together, our data represent a significant advance over what was previously known on the involvement of BRD4 in mastocytosis and identify this epigenetic reader bromodomain-containing protein as a promising drug target in advanced SM.

Figure 2 :

 2 Figure 2: Morphology of cultured human MCs. CD34+ cord blood (CB) progenitor cells were cultured in SCF-containing medium and stained with Wright-Giemsa after the first two weeks (a,b) and at day 42 (c) (Figure adapted from Sperr et al. 2001).

Figure 3 :

 3 Figure 3: Schematic representation of the KIT structure and of its tyrosine phosphorylation sites. Phosphorylation of cytoplasmic tyrosine residues occurs via SCF binding. The human (mouse) numbering system is displayed. Two isoforms of Kit in the mouse and four in humans have been identified and result from alternate mRNA splicing (Ashman 1999). The four isoforms of human KIT which differ by the presence or absence of a tetrapeptide sequence, GNNK, in the extracellular region, or of an S residue in the interkinase region, as indicated. G refers to glycine; N, asparagine; K, lysine S; serine. An additional splice variant resulting in the presence or absence of a single serine residue in the interkinase region of the cytoplasmic domain occurs in human KIT (Crosier et al. 1993) (Figure adapted from Ashman 1999, Roskoski 2005, Orfao 2007 and Bibi et al. 2014).

Figure 4 :

 4 Figure 4: Schematic representation of signaling pathways downstream KIT.The KIT signaling occurs through several pathways. Stem-cell factor (SCF)-mediated dimerization of KIT leads to the receptor autophosphorylation at multiple tyrosine residues in the cytoplasmic tail, resulting in the recruitment of various molecules including cytosolic adaptor molecules, such as Shc (Src homology 2 (SH2)-domain-containing transforming protein C) and Grb2 (growth-factor-receptor-bound protein 2); Src family protein kinases (Lyn, Fyn); and signaling enzymes such as phospholipase C- (PLC-) and phosphatidylinositol 3-kinase (PI3-K). Activated PI3-K generates PI(3,4,5)P3 through which PH domain-containing proteins such as Bruton's tyrosine kinase (BTK) are recruited to the plasma membrane and further propagate the signal. Subsequent activation of these signaling enzymes, as well as the JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathway and the RAS-RAF-mitogen-activated protein kinase (MAPK) pathway leads to MC growth, differentiation, survival, chemotaxis and cytokine production. An increased activity of PLC-γ leads to production of DAG and IP3, responsible for enhanced levels of free cytoplasmic Ca 2+ . This is followed by actin rearrangements and chemotactic response. KIT also phosphorylates NTAL that can bind other adapter proteins, including Grb2. Activated GRB2 orchestrates activation of RAS-RAF pathway by recruiting GEFs, the guanine nucleotide exchange factor Sos (son of sevenless homologue) and the guanine nucleotide exchange factor Vav, which subsequently leads to activation of MAP kinases ERK, JNK and p38. Some other signaling proteins such as JAK, STATs, AKT, PKC and Rac are involved in gene transcription, survival, actin rearrangement, and/or in chemotaxis (Figure adapted from[START_REF] Draber | Signal transduction and chemotaxis in mast cells[END_REF].

  thereafter recruits and activates PH (Pleckstrin homology) domain-containing proteins such as Bruton's tyrosine kinase (BTK) and the serine-threonine kinase such as AKT (also known as protein kinase B (PKB)). BTK is recruited to the plasma membrane and further propagates the signal. Activated AKT in its turn phosphorylates Bad (Blume-Jensen et al. 1998), BIM (BCL-2 [B-cell lymphoma-2] interacting modulator of cell death) or the Forkhead transcription factor FOXO3a (forkhead box, class O3A) and downregulates the pro-apoptotic target BIM, leading to cell survival (Moller et al. 2005; Lennartsson

Figure 5 :

 5 Figure 5: Aberrant signaling pathways downstream of KIT D816V mutant receptor. FES is permanently phosphorylated and negatively regulates (as indicated in red arrows) STAT pathways and activates (as indicated in red arrows) m-TOR pathway, resulting in induction of survival (black arrows) (Figure adapted from Bibi et al. 2014).

Figure 6 :

 6 Figure 6: Composition and functions of complexes of FcεRI on mast cells (MCs) and basophils (Bas) as well as on antigen presenting cells (APCs). a. The tetrameric form of the FcεRI comprises the IgE-binding α chain with two immunoglobulin-like domains, the signal amplifying membrane-tetraspanning β chain and two γ chains. Both β and γ chains contain intracellular immunoreceptor tyrosine-based activation motifs (ITAMs) whose phosphorylation of canonical tyrosine residues is essential for the signaling cascade. The tetramer form of FcεRI is expressed on MCs and Bas. b. The trimeric FcεRI lacks the β chain and is expressed by antigenpresenting cells (APCs) and eosinophils. It has influence on the uptake by major histocompatibility complex (MHC) class-II-rich compartments (Figure adapted from Kraft & Kinet 2007).

Figure 7 :

 7 Figure 7: Regulation of surface expression of FcεRI on human mast cells (MCs) and basophils (Bas). Interleukin-4 (IL-4) promotes the production of immature core-glycosylated α-chains of FcεRI in theendoplasmic reticulum (ER). These immature chains remain in ER in the absence of β-and two γ-chains, while they are exported with β and γ chains after blocking of their retention signals from ER to the cell surface. However, this process happens after the full maturation of the FcεRI by terminal glycosylation in the Golgi compartment, and is more efficient for the tetrameric form of FcεRI which is more stable on the cell surface than the trimeric FcεRI lacking the β-chain. IgE, the FcεRI ligand, stabilizes FcεRI complexes on the cell surface and blocks their internalization. The inhibition of FcεRI internalization in turn leads to the accumulation of more FcεRI complexes on the cell surface because of the ongoing FcεRI synthesis. However, the truncated β chain variant (βT) produced by alternative splicing prevents the FcεRI complex from reaching the cell surface and results in proteasomal degradation of immature FcεRI complexes (Figure adapted from Kraft & Kinet 2007).

Figure 8 :

 8 Figure 8: Intracellular signaling and cellular events following aggregation of FεRI in MCs (Figure from Kalesnikoff & Galli 2008).

  phosphatase calcineurin. Calcineurin dephosphorylates the nuclear factor for T cell activation (NFAT) which exposes NFAT nuclear-localization signals. Once in the nucleus, NFAT regulates the transcription of several cytokine genes. Transcriptions of other proinflammatory mediators are regulated by NFκB proteins which are transcription factors retained in the cytoplasm by binding to the

Figure 9 :

 9 Figure 9: The negative regulation of FcεRI-mediated signaling events (Figure from Kalesnikoff & Galli 2008).

Figure 10 :

 10 Figure 10: The variety of MC activation pathways by pathogens ensures the effective role of sentinel of MCs. CXCL8, CXC-chemokine ligand 8; GM-CSF, granulocyte/macrophage colony-stimulating factor; IL, interleukin; LTC4, leukotriene C4 (Figure adapted from Marshall 2004).

Figure 11 :

 11 Figure 11: The role of MCs in type 1 hypersensitivity. APC, antigen presenting cells; GM-CSF, granulocyte/macrophage colony-stimulating factor; IL, interleukin; LTs, leukotrienes; MHCII, major histocompatibility complex class II; PAF, platelet activating factor; PGs, prostaglandins; TCR, T cell receptor, TNF, tumour-necrosis factor (Figure adapted from Amin 2012).

Figure 12 :

 12 Figure 12: A schematic representation of the biological effects of the major MC mediators on surrounding cells and tissues. Target cells and tissues are in black, MC-derived mediators are in brown, and biological effects are in red (Figure from Arock et al. 2015).

  the correlation between MC infiltration and tumor outcome. However, it is well known that MCs are recruited into the tumor microenvironment by chemotactic factors released by cancer cells. MC mediators may thus influence cancer cell survival and also have a role in the lymphangiogenesis, angiogenesis, invasion and metastasis through extracellular matrix (ECM) remodeling (Maltby et al. 2009; Marone et al. 2015). In addition, MCs interact with other tumor-infiltrating immune cells. For instance, MCs mobilize and modulate the activity of T cells, regulatory T (Treg) cells, antigen-presenting cells (APCs) and myeloid-derived suppressor cells (MDSCs). Particularly, MCs enhance T cell migration to the tumor microenvironment and change the phenotype of Treg cells. However, Treg cells also inhibit MC degranulation and allergic responses

Figure 13 :

 13 Figure 13: The complex network of interaction between MCs and other components of the tumor microenvironment (Figure adapted from Rigoni et al. 2014).

Figure 14 :

 14 Figure 14: Morphological aspect of HMC-1 cells after May-Grünwald Giemsa (MGG) staining. Magnification x200.

Figure 15 :

 15 Figure 15: Toluidine blue staining of LAD2 cells. Magnification x200.

Figure 16 :

 16 Figure 16: Morphological aspect of LUVA cells after May-Grünwald Giemsa (MGG) staining. Magnification x200.

Figure 17 :

 17 Figure 17: Morphological appearance of MCPV-1 cells after Wright-Giemsa staining staining. Magnification x500. (Figure adapted from Hoermann et al. 2014).

malignancy 2 .

 2 Palpable hepatomegaly with impairment of liver function, ascites, and/or portal hypertension 3. Skeletal involvement with large osteolytic lesions and/or pathological fractures 4. Palpable splenomegaly with hypersplenism 5. Malabsorption with weight loss due to gastrointestinal MC infiltrates malignant lymphoma or myeloid leukemia at first presentation.

2 . 2 . 3 .

 223 differentiated systemic mastocytosis" (WDSM) is characterized by BM multifocal infiltrates by round, Intoduction 53 mature CD25 MCs and by the absence of mutation of 816 codon of KIT or of other KIT mutations (Teodosio et al. 2010) Systemic mastocytosis with an associated clonal hematologic non mast cell lineage disease (SM-AHNMD)

Figure 18 :

 18 Figure 18: Different morphological aspects of leukemic MCs circulating in the PB of patient with MCL after May-Grünwald Giemsa (MGG) staining. (a) Metachromatic granulated blast-like MCs. (b) Atypical MCs type II also known as promastocytes. (c) More immature atypical MCs type I. (d) More mature atypical MCs type I; spindle-shaped MCs. (e) Mature round MCs. Magnification: x500.

Figure 19 :

 19 Figure 19: The different KIT mutations found in mastocytosis.In adults, the structure of KIT is found WT in <20% of all patients analyzed, whereas in children, KIT is found WT in around 25% of the patients analyzed. In adults, depending of the category of mastocytosis, the KIT D81V mutant (in red) is found in at least 80% of all patients, as mentioned in the text. Other KIT mutants in position 816 less frequently retrieved in mastocytosis are depicted here in black. In children, the ECD mutants (in blue) are found in nearly 40% of patients, whereas the KIT D816V mutant (in red) located in the phosphotransferase domain (PTD) is found in around 30% of the affected children.aa, amino acid; Del, deletion; Ins, insertion; ITD, internal tandem duplication; TK, tyrosine kinase. ±, mutation found in < 1% of the adult or pediatric patients; +, mutations found in 1-5% of pediatric patients; ++, mutation found in 5-20% of pediatric patients; +++, mutation found in >80% of all adult patients and in around 30% of pediatric patients (Figure fromArock et al. 2015).

Figure 20 :

 20 Figure 20: Proposed therapeutic algorithm for antimediator-treatment in mastocytosis (Figure from Siebenhaar & Akin 2014).

  been tested for their efficacy in pilot clinical trials, such as the raft modulator miltefosine. Miltefosine has been shown to reduce the release of histamine, TNF-MCs upon IgE-mediated and non-IgE-mediated activation. In addition, topical administration of miltefosine induced a reduction of histamine-induced wheal-and-flare-type skin reactions in vivo. In a randomized, placebo-controlled, double-blind clinical trial, topical miltefosine has been shown to inhibit MC activation in patients with CM(Weller et al. 2010;[START_REF] Hartmann | Effects of topical treatment with the raft modulator miltefosine and clobetasol in cutaneous mastocytosis: a randomized, double-blind, placebo-controlled trial[END_REF][START_REF] Magerl | Randomized, double-blind, placebo-controlled study of safety and efficacy of miltefosine in antihistamine-resistant chronic spontaneous urticaria[END_REF][START_REF] Maurer | Miltefosine: a novel treatment option for mast cell-mediated diseases[END_REF]).

  . In physiologic conditions, CD30 is infrequently expressed and thus is considered a specificand useful target in advanced SM (Van Anrooij et al. 2014). Recently, a single-arm open-label trial clinical trial has been initiated (and is still ongoing) to study the potential effects of Brentuximab Vedotin (a CD30-targeted antibody conjugated with the antimitotic agent auristatin E) in CD30-positive SM with or without an AHNMD (National Clinical Trial NCT01807598).

Figure 21 :

 21 Figure 21: Phylogenetic tree of the human BRD family based on sequence alignments of predicted BRDs. The different families are named by Roman numbers (I-VIII). Structures determined in this study, by NMR, or by other groups are indicated by blue, red, and green dots, respectively. For targets with multiple BRDs, the domains have been numbered starting from the N-terminus and the number is shown in parentheses (Figure adapted from Filippakopoulos et al. 2012 and from Prinjha et al. 2012).

Furthermore, the human

  genome encodes 61 BRDs, which are present in 46 different nuclear and cytoplasmic proteins[START_REF] Filippakopoulos | Targeting bromodomains: epigenetic readers of lysine References acetylation[END_REF][START_REF] Shi | The mechanisms behind the therapeutic activity of BET bromodomain inhibition[END_REF][START_REF] Gallenkamp | Bromodomains and their pharmacological inhibitors[END_REF], where differences in the amino acid residues around the acetyl lysine binding site impact ligand specificity(Filippakopoulos et al. 2012). Proteins containing this module are often involved in transcriptional regulation and chromatin remodeling[START_REF] Garnier | BET bromodomain inhibitors: a patent review[END_REF]. Moreover, structure-based alignments have clustered human BRD families into eight distinct families by the derived phylogram analysis in which the BRDs and extra-terminal domain (BET) subfamily of proteins discused later belongs to the subfamily II as shown in Figure 21. Indeed, BRDs have been found in:

  ,-histone methyltransferases (such as ASH1L and mixed lineage leukemia protein (MLL))[START_REF] Gregory | Mammalian ASH1L is a histone methyltransferase that occupies the transcribed region of active genes[END_REF][START_REF] Malik | Mixed lineage leukemia: histone H3 lysine 4 methyltransferases from yeast to human[END_REF], -helicases (such as SWI/SNF (switch mating type/sucrose non-fermenting)-related matrix-associated actin-dependent regulators of chromatin subfamily A (SMARCAs))[START_REF] Trotter | The HSA domain of BRG1 mediates critical interactions required for glucocorticoid receptor-dependent transcriptional activation in vivo[END_REF], -ATP-dependent chromatin remodelling complexes (such as bromodomain adjacent to zinc finger domain protein 1B (BAZ1B; also known as Williams syndrome transcription factor))(Cavellán et al.

Figure 22 :

 22 Figure 22: Structure of BRD module. A. The BRD is a highly conserved motif of 110 amino acids which is bundled into four anti-parallel α-helices that are linked by diverse loop regions (ZA and BC loops). B. Major structural elements are highlighted on the structure of the first bromodomain of bromodomain-containing protein 4 (BRD4(1)) in complex with a di-acetylated histone H4 peptide. The electrostatic potential of the module is also shown, from -10 to +10 kT/e, highlighting the charged nature of the surface lining the acetyl-lysine recognition site. C. The hydrophobic nature of the acetyl-lysine binding site is evident by the aromatic and hydrophobic residues that line the central acetyl-lysine-binding cavity, shown here on BRD4(1) (Figure adapted from Filippakopoulos et al. 2012 and Filippakopoulos & Knapp 2014).

  , remained undefined[START_REF] Wu | The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation[END_REF][START_REF] Devaiah | Cross-talk among RNA polymerase II kinases modulates Cterminal domain phosphorylation[END_REF] untill 2013 whenAlsarraj et al. demonstrated that BRD4S is a nuclear membrane associated protein which interacts with metastasis susceptibility proteins such as signal-induced proliferation associated protein 1 (SIPA1) and ribosomal RNA processing 1 homolog B (RRP1B). This complex interacts with the inner nuclear protein SU2 (Sad1 and UNC84 containing 2) at the inner face of nuclear membrane and therefore plays a role in mechanotransduction of extracellular microenvironmental signal to the nucleus[START_REF] Alsarraj | BRD4 short isoform interacts with RRP1B, SIPA1 and components of the LINC complex at the inner face of the nuclear membrane[END_REF]. The genes encoding BET proteins are now designated BRD2 (formerly referred to as RING3 (really interesting new gene 3) or Fshrg1 (female sterile homeotic related gene 1)), BRD3 (also called ORFX or Fshrg2), BRD4 (Fsrg4, MCAP/HUNK1), and BRDT (Fsrg3, BRD6).BRD2 is a nuclear serine/threonine kinase possessing chromatin binding activity with preference for K12 Ac on histone H4[START_REF] Umehara | Structural basis for acetylated histone H4 recognition by the human BRD2 bromodomain[END_REF]) and transcription activity via its association with transcriptional regulators such as E2F1[START_REF] Denis | RING3 kinase transactivates promoters of cell cycle regulatory genes through E2F[END_REF][START_REF] Kanno | Selective recognition of acetylated histones by bromodomain proteins visualized in living cells[END_REF]). BRD3 and BRDT are less well characterized although mouse Brdt has been reported to induce global chromatin reorganization in an acetylation-dependent manner[START_REF] Pivot-Pajot | Acetylation-dependent chromatin reorganization by BRDT, a testis-specific bromodomain-containing protein[END_REF]. BRD4, originally named MCAP (mitotic chromosome associated protein)[START_REF] Dey | A bromodomain protein, MCAP, associates with mitotic chromosomes and affects G(2)-to-M transition[END_REF], but also called Fshrg4 or Hunk1, is a chromatin binding factor with preference for acetylated Lys14 (K14 Ac ) on histone H3 and Lys5/12 on H4(Dey et al. 2003). BRD4 also shares strong structural similarity to the yeast BET proteins; Bdf1 and Bdf2 (Devaiaha & Singer 2013). Additionally, the BRDs of BRD4 bind to acetylated lysine 310 of

Figure 23 :

 23 Figure 23: Domain architecture of BET proteins. BD1 and BD2 signify bromodomains. A, B and ET represent motif A, B and the ET domain, respectively. Colors indicate the different parts of BET proteins as mentioned in the text. Numbers indicate the amino acid boundaries of each domain or the corresponding amino acid residues of individual proteins (Figure adapted from Wu & Chiang 2007, Chung et al. 2011, Belkina & Denis 2012, and Fukazawa & Masumi 2012).

  . These proteins have a conserved modular architecture including two N-terminal tandem BRD modules (designated BD1 and BD2; responsible for acetyl-lysine recognition and shown in blue), an extra-terminal domain (ET;shown in red) on the C-terminal side of the double bromodomains(Florence & Faller 2001;[START_REF] Zeng | Bromodomain: an acetyl-lysine binding domain[END_REF][START_REF] Mujtaba | Structure and acetyl-lysine recognition of the bromodomain[END_REF][START_REF] Wu | The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation[END_REF][START_REF] Belkina | BET domain co-regulators in obesity, inflammation and cancer[END_REF], several conserved motifs including (A, B motifs; shown in orange and light purple in Figure26, respectively) as well as a serine-glutamate-aspartate-rich region (SEED; shown in yellow), and, in the case of BRDT and the long variant of BRD4 (BRD4L) , a C-terminal motif (CTM; shown in green)[START_REF] Abbate | Structure of the papillomavirus DNA-tethering complex E2:Brd4 and a peptide that ablates HPV chromosomal association[END_REF]) that is sometimes referred to as PTEFb-interacting domain (PID) because it interacts with the positive transcription elongation factor b (PTEFb)(Wang & Filippakopoulos 2015). Although BD1, BD2, and the ET domains are characteristic of the BET family proteins, motifs B and SEED are also highly conserved among BET proteins. However, CTM[START_REF] Abbate | Structure of the papillomavirus DNA-tethering complex E2:Brd4 and a peptide that ablates HPV chromosomal association[END_REF]) and motif A(Paillisson et al. 2007) are not present in every protein.

Figure 24 :

 24 Figure 24: Structure of the small-molecule inhibitor of BRD4 protein, JQ1. The parts of the molecule that displace the ε-acetyl-lysine group of the histone are circled in red (Figure adapted from Belkina & Denis 2012).

  , recent reports have uncovered BRD4 as a central regulator of estrogen receptor α (ERα) function and potential therapeutic target in ERα-dependent cancers like breast cancer (Nagarajan et al. 2014). In fact, BRD4 regulates estrogen receptor α (ERα)-induced gene expression by affecting elongation-associated phosphorylation of RNA polymerase II and histone H2B monoubiquitination in ERα-positive breast cancers. Of note, ERα controls cell proliferation and tumorigenesis by recruiting various cofactors to estrogen response elements (EREs) to control gene transcription such as cyclin D1

(

  [START_REF] Filippakopoulos | Selective inhibition of BET bromodomains[END_REF], I-BET151[START_REF] Nicodeme | Suppression of inflammation by a synthetic histone mimic[END_REF][START_REF] Seal | Identification of a novel series of BET family bromodomain inhibitors: binding mode and profile of I-BET151 (GSK1210151A)[END_REF], and I-BET762[START_REF] Mirguet | Discovery of epigenetic regulator I-BET762: lead optimization to afford a clinical candidate inhibitor of the BET bromodomains[END_REF] competitively and selectively occupy the acetyl binding pockets of the closely related BRDs, resulting in release from active chromatin and the suppression of downstream signal transduction events to RNA Polymerase II(Filippakopoulos.et al. 2010; Dawson et al 2011;[START_REF] Delmore | BET bromodomain inhibition as a therapeutic strategy to target c-Myc[END_REF][START_REF] Puissant | Targeting MYCN in neuroblastoma by BET bromodomain inhibition[END_REF][START_REF] Lovén | Selective inhibition of tumor oncogenes by disruption of super-enhancers[END_REF]. Numerous reports have thereby revealed the promising therapeutic potential of these inhibitors in vitro and in vivo in several hematopoietic malignancies including Burkitt's lymphoma, multiple myeloma, AML, and mixed-lineage leukemia (MLL)(Zuber et al. 2011; Dawson et al. 2011; Mertz et al. 2011;[START_REF] Delmore | BET bromodomain inhibition as a therapeutic strategy to target c-Myc[END_REF] Herrmann et al. 2012).

Publication 1 .

 1 "A new human mast cell line expressing a functional IgE receptor converts to tumorigenic growth by KIT D816V transfection" R Saleh * , G Wedeh * , H Herrmann, S Bibi, S Cerny-Reiterer, I Sadovnik, K Blatt, E Hadzijusufovic, S Jeanningros, C Blanc, M Legarff-Tavernier, E Chapiro, F Nguyen-Khac, F Subra, P Bonnemye, P Dubreuil, V Desplat, H Merle-Béral, M Willmann, T Rülicke, P Valent, and M Arock. Blood.

Figure 1 .

 1 Figure 1. Phenotypic characterization of the ROSA cell lines. ROSA KIT WT cells were stained by (A-B) MGG or (C) toluidine blue. (D)The ultrastructure of ROSA KIT WT cells was analyzed by transmission electron microscopy. In addition, ROSA KIT WT cells were treated with an antibody against (E) tryptase or (F) KIT (CD117). Positivity was revealed by indirect immunocytochemistry as described in "Material and methods." (G) Cytospin preparations or (H-I) glass smears of ROSA KIT D816V cells were stained by MGG. (G) Note the presence of many granules in ROSA KIT D816V cells and of apoptotic cells, a phenomenon likely to be related to terminal maturation induced by the introduction of the KIT D816V mutant. (H) On glass smear, a significant proportion of the ROSA KIT D816V cells presented with spindle-shaped appearance, which was not noticed for (I) ROSA KIT WT cells. Magnification: A and C, 3200; B, E, G, H, and I 3500; D, 32000; F, 31000.

Figure 2 .

 2 Figure 2. Effect of SCF and of various cytokines on the proliferation of the 2 different ROSA cell lines.(A) ROSA KIT WT or ROSA KIT D816V cells were treated or not treated with rhSCF (80 ng/mL) for 4 days at 37°C. At each time point, 10 mL of 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added in corresponding wells, and the cells were incubated for 3 additional hours in an incubator at 37°C. The number of living cells was then measured for each condition by reading the absorbance at 570 nm. (B) ROSA KIT WT (with or without SCF) or ROSA KIT D816V cells (without SCF) were treated with various cytokines (all at 100 ng/mL) for 3 days. At that time, 10 mL of MTT was added in corresponding wells, and the cells were incubated for 3 additional hours in an incubator at 37°C. The number of living cells was then measured for each condition by reading the absorbance at 570 nm. Data presented are the mean 6 standard deviation (SD) from 3 independent experiments. OD, optical density.

Figure 4 .

 4 Figure 4. Detection of spontaneous phosphorylation of KIT, AKT, and STAT5 in ROSA KIT D816V cells by western blotting. Cell lysates from unstimulated ROSA cell lines and from the HMC-1.2 cell line or from the same cells stimulated for 10 minutes with rhSCF were subjected to electrophoresis in sodium dodecyl sulfate polyacrylamide gel electrophoresis and treated with Abs against (A) anti-human total KIT or p-KIT Y719, (B) anti-human total AKT or p-AKT S473, or (C) anti-human total STAT5 or p-STAT5 Y694. (D) An anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. The lanes were run on the same gel but were noncontiguous.

Figure 5 .

 5 Figure 5. Effect of three tyrosine kinase inhibitors on the proliferation of the 2 ROSA cell lines. Cells were cultured for 48 hours in the presence of imatinib, dasatinib or midostaurin (1 mM, provided in dimethylsulfoxide (DMSO) 0.1% final concentration) or in the presence of DMSO alone (0.1% final concentration) in the established culture medium (containing rhSCF at 80 ng/mL for ROSA KIT WT cells but not for ROSA KIT D816V cells). At the end of this incubation, 10 mL of MTT was added in each well and the cells were incubated for 3 additional hours at 37°C. The number of living cells was then measured for each condition by reading the absorbance at 570 nm, and the results were expressed as a percentage of living cells compared with the control (DMSO 0.1% alone). Data presented are the mean 6 SD from 3 independent experiments. ***Significantly different from control (DMSO alone) at P , .0001.

Figure 6 .

 6 Figure 6. Morphologic appearance of BM-engrafted ROSA KIT WT and ROSA KIT D816V cells in NSG mice. Whole BM cells from NSG mice engrafted intravenously with (A) ROSA KIT WT or (B) ROSA KIT D816V cells were analyzed for their content of CD117 1 /CD45 1 engrafted human MCs (A and B, top panels). The cells were also cytocentrifuged on glass slides, stained with MGG and then observed by using light microscopy (bottom panels). Note the presence of spindle-shaped MCs with an abnormal distribution of granules in the BM of mice engrafted with ROSA KIT D816V cells (B, black arrows), whereas the BM MCs from mice engrafted with ROSA KIT WT cells presented with a rounded shape, similar to the one observed in culture (A, gray arrows). Magnification is 3200. FL2-H, FL2 peak emission values; PerCP, peridinin chlorophyll.

  Merle-Béral, Michael Willmann, Thomas Rülicke, Peter Valent and Michel Arock Elise Chapiro, Florence Nguyen-Khac, Frédéric Subra, Patrick Bonnemye, Patrice Dubreuil, Vanessa Katharina Blatt, Emir Hadzijusufovic, Sylvie Jeanningros, Catherine Blanc, Magali Legarff-Tavernier, Rosine Saleh, Ghaith Wedeh, Harald Herrmann, Siham Bibi, Sabine Cerny-Reiterer, Irina Sadovnik, D816V transfection KIT converts to tumorigenic growth by A new human mast cell line expressing a functional IgE receptor http://www.bloodjournal.org/content/124/1/111.full.html Updated information and services can be found at: (532 articles) Phagocytes, Granulocytes, and Myelopoiesis (1425 articles) Myeloid Neoplasia Articles on similar topics can be found in the following Blood collections http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at: Copyright 2011 by The American Society of Hematology; all rights reserved. of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society 1 Supplemental Methods, Results, Tables and Figures R. Saleh et al. A new human mast cell line expressing a functional IgE receptor converts to factorindependence and tumorigenicity by KIT D816V-transfection

  Fullerton, CA) (10 µg/ml), recombinant human C5a (Sigma-Aldrich, St. Louis, MO) (100 nM) or control buffer at 37°C for 90 minutes. Thereafter, cells were washed and stained with FITC-labeled CD63 mAb CLB-gran12 (Beckman Coulter), PE-Cy-7-labeled CD117 mAb 104D2 (ebioscience, San Diego, CA), APC-Cy7-labeled CD45 mAb HI30, and Pacific Blue-labeled CD34 mAb 581 (Biolegend, San Diego, CA) for 15 minutes at room temperature. After erythrocyte lysis with FACS-lysis solution (BD Biosciences, San Jose, CA), expression of cell surface antigens was quantified by multi-color flow cytometry on a FACSCantoII (BD Biosciences). MCs were identified as CD45+/CD117+/CD34-cells.Anti-IgE-induced and C5a-induced upregulation of CD63 on MCs was calculated from mean fluorescence intensities (MFI) obtained with stimulated (MFI stim ) and unstimulated (MFI control ) cells and was expressed as stimulation index = SI (MFI stim /MFI control ).

Figure S8 :Figure S9 :

 S8S9 Figure S8: Effect of the specific inhibition of AKT (A) and STAT5 (B) on the proliferation of the two ROSA cell lines. ROSA KIT WT (-) or ROSA KIT D816V (---) cells were seeded for 48 hours in the in the presence of various concentrations (0.01 to 5 M) of the selective inhibitor of AKT1/AKT2 (compound A6730 diluted in DMSO 0.1% final concentration) or in the presence of various concentrations (1 to 6 M) of the specific STAT5 inhibitor (pimozide diluted in DMSO 0.1% final concentration) in SFM (with rhSCF at 80 ng/mL for ROSA KIT WT but not for ROSA KIT D816V cells). At the end of this incubation, 10 L of MTT were added in each well (3-(4,5-Dimethyltiazol-2-yl)-2,5diphenyltetrazolium bromide) and the cells were incubated for 3 additional hours in an incubator at 37°C. The number of living cells was then measured for each condition by reading the absorbance at 570 nm. Data presented are the mean±SD from 3 independent experiments and are expressed as percent of proliferation in each condition relative to the control (DMSO alone) considered as 100% proliferation.

Figure 1 .

 1 Figure 1. Expression of BRD4 in neoplastic MCs. (a) BM sections obtained from patients with ISM (upper panels), ASM (middle panels) or MCL (lower panels) were stained with antibodies against BRD4 (left panels) or tryptase (right panels) as described in the text. Tryptase was used to detect MCs. Original magnification: x100. #: Patient details are listed in Supplementary Table S2. (b) BRD4 mRNA expression in HMC-1 and ROSA cells (upper panel), in normal BM cells (n = 10 donors) and in mononuclear BM cells obtained from patients with ISM (n = 42) or advanced SM (ASM/MCL) (n = 7) (lower panel). Quantitative PCR (qPCR) was performed as described in the text. Results are expressed as the percent of ABL mRNA and represent the mean ± s.d. of five independent experiments in cell lines (upper panel) and median values (indicated by horizontal bars) for each group of donors. As assessed by analysis of variance (ANOVA) and unpaired t-test, BRD4 mRNA expression was significantly higher in the ASM/MCL group compared with normal donors or ISM (P o0.05).

Figure 2 .

 2 Figure 2. BRD4 shRNA and JQ1 inhibit the proliferation of neoplastic MCs. (a) Effect of shRNA-mediated knockdown of BRD4 on proliferation of HMC-1.1, HMC-1.2, ROSA KIT WT and ROSA KIT D816V cells. Cells were transduced with a control shRNA targeting Renilla luciferase (◇-◇) or with two different shRNA constructs targeting BRD4, #602 (♦-♦) and #1817 (♦-♦). GFP+ cells were sorted, mixed with their non-transduced control cells (ratio 1:1) and maintained in culture for 7 days. The percentage of GFP+ cells was measured daily by flow cytometry. Results show the percentage of GFP+ cells in one typical experiment. (b-d) Effects of JQ1 on proliferation of HMC-1 cells (b), ROSA cells (c) and primary neoplastic MCs obtained from patients with ASM or MCL (d). Cells were incubated in the absence (Co) or presence of various concentrations of JQ1 at 37 °C for 48 h. Then, 3 H-thymidine uptake was measured. Results are expressed as the percent of control and represent the mean ± s.d. of three independent experiments (b and c) or the mean ± s.d. of triplicates (d). *P o0.05 compared with control (Co).

Figure 3 .

 3 Figure 3. JQ1 induces apoptosis in neoplastic MCs. (a) HMC-1 and ROSA cells were incubated in the absence (Co) or presence of various concentrations of JQ1 at 37 °C for 48 h. Thereafter, the percentage of apoptotic cells was determined by light microscopy. Results show the percent of apoptotic cells and represent the mean ± s.d. of three independent experiments. (b) After incubation in control medium (Co) or JQ1, HMC-1 and ROSA cells were stained with AnnexinV/propidium iodide (PI), and the percentage of apoptotic cells was determined by flow cytometry. Results show the percent of AnnexinV/PI+ cells and represent the mean ± s.d. of three independent experiments. *Po 0.05 compared with control.

Figure 4 .

 4 Figure 4. Effects of SCF on responsiveness of neoplastic MCs to JQ1. (a and b) ROSA KIT D816V cells were incubated in the absence (◇-◇ in the left image; open bars in the right image) or presence of rhSCF (200 ng/ml) (♦-♦ in the left image; black bars in the right image) at 37 °C for 24 h. Thereafter, cells were washed and incubated in control medium (Co) or in various concentrations of JQ1 (5-5000 nM) for 48 h. Then, 3 H-labeled thymidine uptake (left panel) and the percentage of active caspase-3-positive cells (right panel) were measured. Results represent the mean ± s.d. of three independent experiments. *Po 0.05 compared with Co.

  -CD, cluster of differantiation; n.c, not clustered; LFA-2, leukocyte function antigen-2; TSPAN-29/p24, tetraspanin-29/platelet antigen p24; C3biR, complement receptor 3; Pgp1, phagocyte glycoprotein 1; ICAM-1, intercellular adhesion molecule 1; LAMP-3, lysosome-associated membrane glycoprotein 3; Tf-R, transferrin receptor; FAS, Apoptosis stimulating fragment; E-NPP3, ectonucleotide pyrophosphatase/phosphodiesterase 3; FcεR1α; IgE receptor alpha chain; BRD4, bromodomain-containing protein 4; PE, phycoerythrin; APC, allophycocyanin; FITC, fluorescein isothiocyanate; IgG, immunoglobulin G; BD, Becton Dickinson. *Manufacturers´ locations: Becton Dickinson: San Jose, CA, USA; Biolegend: San Diego, CA, USA; Immunotech: Marseille, France; Beckman Coulter: Brea, CA, USA; Chemicon, Temecula, CA, USA; Sigma-Aldrich: St. Louis, MO, USA; Bethyl Labs: Montgomery, TX, USA.

A

  : HMC-1.1 cells (left panel), HMC-1.2 cells (middle panel), and ROSA KIT D816V cells (right panel) were incubated in control medium (-), rhSCF (200 ng/ml) or CHOKL (10% Chinese hamster ovary (CHO) supernatant containing murine SCF) at 37°C for 16 (open bars), 24 hours (gray bars) or 48 hours (black bars). Thereafter, cells were subjected to RNA isolation and qPCR. Results show BRD4 mRNA expression levels as percent of ABL mRNA levels, and represent the mean±S.D. of 3 independent experiments. Asterisk (*) indicates p<0.05 compared to control. B: HMC-1.1 cells,HMC-1.2 cells, ROSA KIT WT cells and ROSA KIT D816V cells were incubated in control medium (-) or medium containing imatinib (1 µM), PKC412 (1 µM), ponatinib (1 µM), ATRA (10 µM), the demethylating agents 5-azacytidine and decitabine (10 µM each), the mTOR blocker rapamycin (1 µg/ml), BEZ235 (1 µM), the MEK1/2 inhibitors RDEA119 and PD0325901 (1 µM each), the STAT5 blockers pimozide(5 µM) and piceatannol(50 µM), the JAK2 inhibitors TG101348, INCB018424 and AZD1480 (1 µM each) and 2CdA (cladribine) (1 µg/ml) at 37°C for 12 hours. Then, RNA was isolated and BRD4 mRNA levels were quantified by qPCR. Results show BRD4 mRNA levels as percent of ABL mRNA levels and represent the mean±S.D. of 3 independent experiments. Asterisk (*) indicates p<0.05 compared to control.Supplementary Figure S3PKC412 and dasatinib dose-dependently downregulate the expression of BRD4 in HMC-1 cells and ROSA cells HMC-1.1 cells (upper left panel), HMC-1.2 cells (upper right panel), ROSA KIT WT cells (lower left panel) and ROSA KIT D816V cells (lower right panel) were incubated in control medium (Co) or in medium containing 0.5, 1 or 2 µM of PKC412 or dasatinib (as indicated) at 37°C for 12 hours. Thereafter, RNA was isolated and BRD4 mRNA levels were quantified by qPCR. Results show BRD4 mRNA levels as percent of ABL mRNA levels and represent the mean±S.D. of 3 independent experiments. Asterisk (*) indicates: p<0.05. Effects of Volasertib (BI6727) and BI2536 on the proliferation of HMC-1 cells and ROSA cells C A: HMC-1.1 cells, HMC-1.2 cells, ROSA KIT WT cells and ROSA KIT D816V cells were transduced with a control shRNA targeting Renilla luciferase (shRNA-Renilla) (black bars) or with two different shRNA constructs targeting BRD4 (shRNA-BRD4 #602 and #1817) as indicated. GFP+ cells and non-transduced control cells were sorted and maintained at 37°C for 24 hours. Then, 3 H-thymidine uptake was measured. Results are expressed as percent of control and represent the mean±S.D. of triplicates. B: HMC-1.1 cells, HMC-1.2 cells, ROSA KIT WT cells and ROSA KIT D816V cells were transduced with a control shRNA (shRNA-Renilla) or with two different shRNA constructs targeting BRD4 (shRNA-BRD4 #602 and #1817). GFP+ cells were sorted and maintained at 37°C. Cell counts were assessed on day 7. Results are expressed as percent of shRNA control and represent the mean±S.D. of triplicates. C: HMC-1.1 cells, HMC-1.2 cells, ROSA KIT WT cells and ROSA KIT D816V cells were incubated in the absence (Co) or presence of increasing concentrations of either Volasertib (left panels) or BI2536 (right panels) at 37°C for 48 hours. Then, 3 H-thymidine uptake was measured. Results are expressed as percent of control (Co) and represent the mean±S.D. of 3 independent experiments. Supplementary Figure S5 JQ1 induces cell cycle arrest in HMC-1 cells and ROSA cells HMC-1.1 cells (upper left panel), HMC-1.2 cells (upper right panel), ROSA KIT WT cells (lower left panel) and ROSA KIT D816V cells (lower right panel) were incubated in control medium (Co) (open bars) or medium containing various concentrations of JQ1 (250-2,500 nM; grey and black bars) at 37°C for 24 hours. Thereafter, cells were lysed and stained with propidium iodide (PI). Results show the percentage of cells in each cell cycle phase (S, G2, G1) and represent the mean±S.D. of 3 independent experiments. Asterisk (*) indicates p<0.05 compared to control. Effects of JQ1 and ATRA on maturation of HMC-1 cells and ROSA cells B A: HMC-1.1 cells, HMC-1.2 cells, ROSA KIT WT cells and ROSA KIT D816V cells were incubated in control medium (0) or in medium containing ATRA and JQ1 in various concentrations alone or in combination (as indicated) at 37°C for 48 hours. Then, expression of CD11b (right panels) and CD54 (left panels) was analyzed by flow cytometry. Results are expressed as staining index (SI = mean fluorescence intensity values of tested antibody relative to an isotype-control) obtained with viable cells and represent the mean±S.D. of 3 independent experiments. Asterisk (*) indicates p<0.05 compared to untreated cells. B: HMC-1.1 cells, HMC-1.2 cells, ROSA KIT WT cells and ROSA KIT D816V cells were incubated in control medium (Co) or in medium containing JQ1 (100 nM) and ATRA (500 nM) alone or in combination (JQ1 + ATRA) at 37°C for 48 hours. Thereafter, cells were washed, spun on cytospin slides and stained with Wright-Giemsa solution. Original magnification: x 60. -1.1 cells, HMC-1.2 cells, ROSA KIT WT cells and ROSA KIT D816V cells were incubated in control medium (Co) or in medium containing various concentrations of JQ1, ATRA, or PKC412 alone or in combinations (as indicated) at 37°C for 48 hours. Then, cells were analyzed for uptake of 3 H-thymidine. Results are expressed as percent of control (Co) and represent the mean±S.D. of 3 independent experiments. B, C: HMC-1 cells (upper images) and ROSA cells (lower images) were incubated in control ) or various concentrations of JQ1 (black bars), PKC412 (dark gray bars, left images) or ATRA (dark gray bars, right images) alone or in combination (open bars) for 48 hours. Then, cells were stained for AnnexinV/Propidium iodide (B) or active caspase-3 expression (C) by flow cytometry. Results represent the mean±S.D. of 3 independent experiments. Asterisk (*): p<0.05 compared to control (Co). of SCF on JQ1-mediated apoptosis in HMC-1 cells. HMC-1.1 cells (left panel) and HMC-1.2 cells (right panel) were incubated in the absence (open bars) or presence of rhSCF (200 ng/ml) (black bars) at 37°C for 24 hours. Then, cells were washed and incubated in control medium (Co) or in various concentrations of JQ1 (200-5,000 nM) at 37°C for 48 hours. Thereafter, the percentage of active caspase-3 positive cells was measured. Results represent the mean±S.D. of 3 independent experiments. Asterisk (*): p<0.05 compared to Co. B: ROSA KIT D816V cells were incubation with rhSCF (200 ng/ml) and treated with JQ1 (black bars) or PKC412 (dark gray bars) alone or in combination (light gray bars) at 37°C for 48 hours. Then, the percentage of active caspase-3 positive cells was analysed by flow cytometry. Results represent the mean±S.D. of 3 independent experiments. Asterisk (*) indicates p<0.05.

  Technology, Danvers, MA, USA), a polyclonal Ab against KIT, a polyclonal Ab against p-STAT5 (Tyr694, Cell Signaling Technology), a polyclonal Ab against STAT5 (Cell Signaling Technology), a polyclonal Ab against p-p44/42 MAP (ERK1/2) (Thr202/Tyr204, Cell Signaling Technology), a polyclonal Ab against p-Akt (Ser473, Cell Signaling Technology), a polyclonal Ab against AKT (Cell Signaling Technology), a mAb against total p44/42 MAP (ERK1/2) (Cell Signalling Technology), a mAb against p-STAT3 (Millipore, Billerica, MA, USA), a mAb against STAT3 (Millipore), a mAb against BTK (Cell Signaling Technology), a polyclonal Ab against p-BTK (Tyr223, Cell Signaling Technology), a mAb against c-MYC (Cell Signalling Technology), a mAb against Glyceraldehyde 3phosphate dehydrogenase (GAPDH) (GAPDH 6C5, Santa Cruz Biotechnology, Dallas, Texas, USA) and a polyclonal AB against MC tryptase (Santa Cruz Biotechnology).The secondary Abs used were: a polyclonal goat anti-rabbit IgG conjugated to HRP (Dako, Glostruo, Denmark), a donkey anti-goat IgG conjugated to HRP (Santa Cruz Biotechnology), or a polyclonal goat anti-mouse IgG conjugated to HRP (Rockland H & L, Rockland, MA, USA).

(

  Hercules, CA, USA) according to the manufacturer's instructions. The cDNA was used for the template of PCR with Crimson Taq DNA polymerase (New England BioLabs, Ipswich, Massachusetts). The polymerase chain reaction (PCR) conditions were: denaturation 30 s (94°C); annealing 30 s (55°C); extension, 1 min (72°C); 35 cycles. Equal loading was confirmed byTable XI : Sequences of oligonucleotide primers used for RT-PCR.

Figure S1 :

 S1 Figure S1: Western Blot (WB) analysis of ROSA KIT WT cells lentivirally transduced with pWPI.

Figure S2 :

 S2 Figure S2: Expression of BRD4 in neoplastic canine MC lines. The canine MCL cell line NI-1 (upper panels) and the canine mastocytoma cell line C2 (lower panels) were spun on cytospin slides and stained with a mAb against BRD4 (left panel) or antibody omission control (Co, right panels) by indirect immunocytochemistry.Before applied, the mAb was incubated in control buffer (left panels) or with a blocking peptide (mAb + BP) specific for BRD4 (middle panels) for 1 hour. Stained slides were analysed by microscopy, and images were taken and adjusted as described. Original magnification: x 100.

Figure S3 :

 S3 Figure S3: Effect of JQ1 on the proliferation of neoplastic canine MC lines. The canine MCL cell line NI-1 (left panel) and the canine mastocytoma cell line C2 (right panel) were incubated in the absence (Co) or the presence of various concentrations of JQ1 at 37°C for 48 hours. Then, 3 H-thymidine uptake was measured. Results are expressed as percent of control and represent the mean±S.D. of 3 independent experiments. Asterisk (*) indicates p<0.05 compared to control (Co).

Figure S4 :

 S4 Figure S4: Effects of JQ1 on the expression of FcεRI subunits in ROSA cells. ROSA KIT WT cells were incubated in control medium (Co) or in medium containing 200 nM of JQ1 at 37°C for 16 hours. Then, total RNA was extracted with trizol and cDNA was synthesized by RT-PCR as described(Wedeh et al. 2015). All PCR products were resolved by 1.5% agarose gel electrophoresis and visualized with ethidium bromide. For quantitation, gels were scanned (left panels), and the relative level of FcεRIα band was quantified using the ImageJ software and normalized to the amount of HRPT (right panel). Results are shown as the mean±S.D. of the quantification of densitometry of three independent experiments. Asterisk (*) indicates p<0.05 compared to untreated cells.

Figure S5 :Figure S6 :

 S5S6 Figure S5: Effects of proteasome inhibitors on the expression of FcεRI in ROSA KIT WT cells. ROSA KIT WT cells were treated with 10 nM or, 20 nM of proteasome inhibitors (Bortezomib or MG-132) for 1 hour then incubated in medium or in medium containing 200 nM of JQ1 for 16 hours. Then, the expression of FcεRIα was analyzed by flow cytometry. Results are expressed as staining index (SI) and represent the mean±S.D. of three independent experiments.

Figure S7 :

 S7 Figure S7: Effect of JQ1 on the expression of tryptasein human MC lines. A. ROSA KIT WT cells were incubated in control medium (Co) or medium containing JQ1 (2500 nM) at 37°C for 24 hours. Then, RNA was isolated and tryptase mRNA level was quantified by qPCR (left panel) as described(Wedeh et al.2015). Results show transcript expression levels as percent of ABL mRNA expression and represent the mean±S.D of three independent experiments. Asterisk (*) indicates p<0.05 compared to control. B. ROSA KIT WT cells were incubated in medium (Co) or in medium containing indicated concentrations of JQ1 for 24 hours (right panels) and then western blotting analysis for tryptase was conducted as described. GAPDH was probed to control for protein loading.

Figure S8 :

 S8 Figure S8: Effect of JQ1 on the expression of p-KIT, p-AKT, p-BtK, and p-ERK1/2 in human MC lines. HMC-1.1 cells, HMC-1.2 cells, ROSA KIT WT cells and ROSA KIT D816V cells were incubated in control medium (Co) or medium containing increasing concentrations of JQ1 (1000, 2500 and 5000 nM) at 37°C for 24 hours.Then, cells were lysed and western blotting analyses were performed using indicated antibodies as described above. Results show a representative blot and GAPDH was used as a loading control.

  of the canine neoplastic mast cell lines NI-1, harboring several homozygous Kit mutations, including missense mutations at nucleotides 107(CT) and 1187(AG), a 12-bp duplication (nucleotide 1263), and a 12-bp deletion (nucleotide 1550) and the C2 mastocytoma cell line harboring a Kit exon 11 mutation(DeVinney & Gold 1990;[START_REF] Hadzijusufovic | NI-1: a novel canine mastocytoma model for studying drug resistance and IgER-dependent mast cell activation[END_REF], and that inhibition of BRD4 using JQ1

Figure 25 :

 25 Figure 25: Molecular scheme demonstrating the effects of JQ1 on neoplastic MCs. The figure shows the major signaling pathways evoked by the KIT D816V mutant receptor in neoplastic MCs. In this scheme, BRD4 binds to acetylated histones and recruits pTEFb to the enhancer/promoter of target genes such as c-MYC, and BCL-2. Upon its recruitment by BRD4, pTEFb phosphorylates the CTD of RNA POL II, resulting in transcription of target genes. Treatment with BET protein inhibitors JQ1 inhibits BRD4 activity causing depletion of RNA POL II phosphorylation at Ser 2, reduced binding of BRD4 and RNA POL II at target gene promoters, and decreased transcription of target genes (MYC, BCL-2, BCL-xL, and MCL-1). In addition, treatment with JQ1 inhibits cell growth and induces apoptosis of neoplastic MCs. Treatment with JQ1 also decreases FcεRI expression, IgE-mediated histamine release, CD63, CD71, and the activation of KIT, STAT3, STAT5, AKT, BTK and ERK1/2.

1 ( 2 (

 12 Pour pouvoir étudier le rôle transformant du principal mutant de KIT retrouvé au cours des mastocytoses systémiques, nous avons transfecté ces cellules ROSA KIT WT par un vecteur lentiviral apportant une construction codant pour le KIT muté en D816V. Ceci nous a permis d'établir une nouvelle lignée mastocytaire humaine SCF indépendante, dénommée ROSA KIT D816V . Nous avons ainsi pu effectuer diverses études sur le phénotype de la lignée ROSA KIT D816V , ainsi que sur la signalisation intracellulaire liée à la mutation activatrice de KIT(Saleh, Wedeh et al. 2014). Fait intéressant, nous avons pu montrer que, après injection intraveineuse dans la veine de la queue des souris NOD/SCID IL-2R -/-(souris NSG), les cellules ROSA KIT D816V peuvent greffer à un niveau élevé ces animaux, non seulement dans la moelle osseuse, mais également dans la rate et les poumons, donnant lieu à une maladie qui reproduit au mieux la situation rencontrée chez les patients atteints de mastocytose systémique aggressive. En effet, comme en témoigne l'immunomarquage de tryptase humaine sur les biopsies de moelle osseuse de souris NSG injectées par voie intraveineuse avec des cellules ROSA KIT D816V , des infiltrats compacts de mastocytes humains tryptase positifs peuvent être observés. Par ailleurs, nous avons observé que les cellules ROSA KIT D816V présentes sur les frottis médullaires des souris greffées prennent un aspect fusiforme, comparable à ce qui est observé dans les prélèvements médullaires des patients atteints de mastocytose systémique. Ainsi, notre modèle animal présente plusieurs caractéristiques ressemblant à celles des mastocytoses systémiques chez l'homme. Par ailleurs, nous avons pu montrer qu'aussi bien les cellules ROSA KIT D816V que les cellules néoplasiques de patients atteints de MCL ou d'ASM ne répondent pas à l'activation anti-IgE-dépendante, bien qu'elles expriment de façon significative le récepteur FcRI.Les deux lignées ROSA KIT WT et ROSA KIT D816V ont fait l'objet d'un brevet international publié (brevet WO/2013/064639) et d'une publication où je suis co-premier auteur(Saleh, Wedeh et al. 2014).Par la suite, nous avons pu montrer par immunohistochimie que la protéine BRD4 est surexprimée dans le cytoplasme des mastocytes médullaires des patients atteints d'ASM ainsi que de MCL, alors que les mastocytes médullaires des patients atteints d'ISM n'expriment pas de BRD4 cytoplasmique. Par ailleurs, en utilisant une technique de PCR quantitative, nous avons aussi montré l'existence d'une surexpression significative d'ARN messagers codants pour BRD4 dans les cellules des patients atteints d'ASM ainsi que de MCL, par rapport à l'expression de ce même messager dans des mastocytes normaux ou dans des cellules néoplasiques provenant de patients atteint la forme indolente de la maladie(Wedeh et al. 2015).De plus, nous avons pu montrer que les lignées mastocytaires humaines, comme la lignée HMC-1.lignée mastocytaire leucémique présentant une mutation activatrice de KIT V560G), la lignée HMC-1.lignée mastocytaire leucémique présentant deux mutations activatrices de KIT: V560G et D816V), la lignée ROSA KIT WT et la lignée ROSA KIT D816V , expriment des quantités importantes de la protéine BRD4 dans leur cytoplasme(Wedeh et al. 2015).En outre, afin d'étudier les mécanismes contribuant à la surexpression de BRD4 et les voies de signalisation potentiellement impliquées dans cette expression anormale par les mastocytes néoplasiques, nous avons traité les lignées mastocytaires humaines avec des inhibiteurs de divers signaux de signalisation, comme des inhibiteurs de tyrosine kinase (ITK) tels que l'imatinib, le dasatinib, le PKC412 et le Ponatinib, des inhibiteurs de MEK, des inhibiteurs de la voie PI3-kinase/mTOR, mais aussi par de l'acide rétinoique tout-trans (ATRA), de la cladribine (2-CdA), des agents déméthylants, des inhibiteurs de JAK2 ou des inhibiteurs de STAT5. Nous avons ainsi pu montrer que le PKC412 et le dasatinib, qui sont connus pour bloquer l'activité tyrosine kinase du mutant KIT D816V, peuvent réguler négativement l'expression de BRD4 dans les cellules HMC-1.1, HMC-1.2, ROSA KIT WT et ROSA KIT D816V . En outre, nous avons constaté que différents inhibiteurs de MEK et la voie PI3K/mTOR, comme le composé BEZ235, peuvent réguler négativement l'expression de BRD4 dans les mêmes lignées mastocytaires humaines. Comme MEK et laPI3-kinase sont des effecteurs en aval de KIT, nos données suggèrent que KIT D816V, mais aussi d'autres mutants de KIT et même le KIT WT, pourraient contribuer à l'expression de BRD4 dans les mastocytes néoplasiques. Cette conclusion est en accord avec l'observation que le SCF favorise l'expression de BRD4 dans les cellules ROSA KIT D816V . En revanche le même effet du SCF n'a pas été observé dans les cellules HMC-1. Ces résultats suggèrent donc que l'expression de BRD4 dans les mastocytes malins dépend non seulement de l'état d'activation de KIT, mais aussi d'autres facteurs(Wedeh et al. 2015).

  patients atteints de la forme agressive des mastocytoses systémiques et ce, à une CI50 plus faible que celle observée pour les lignées mastocytaires. Ces données suggèrent que la protéine BRD4 joue un rôle important dans la prolifération et la survie des mastocytes malins in vivo. Néanmoins, les mécanismes d'action et les fonctions biochimiques de la protéine BRD4 présente dans le cytoplasme des mastocytes malins restent à l'heure actuelle inconnus.Comme le composé JQ1 a été montré comme capable d'induire une maturation des cellules blastiques au cours des leucémies aiguës myéloïdes(Zuber et al 2011; Herrmann et al. 2012), nous nous sommes donc demandés si la même molécule pourrait aussi induire la maturation des cellules HMC-1 et/ou des cellules ROSA. Nous n'avons pu détecter aucun effet de ce composé sur la maturation morphologique de ces cellules. De plus, nous avons pu démontrer que le composé JQ1 induit une diminution dose-dépendante de l'expression de certains antigènes de différenciation (CDs) à la surface des cellules HMC-1 et des cellules ROSA, y compris CD63 (LAMP-3) et CD71 (récepteur de la transferrine). En outre, nous avons pu constater que JQ1 régule négativement l'expression membranaire de FcεRI sur les cellules ROSA KIT WT . Ceci peut suggérer le fait que l'expression de FcRI est régulée par certains mécanismes épigénétiques dans ces cellules. Notre observation est la première décrivant un effet de JQ1 sur l'expression du FcεRI, cette expression étant d'ailleurs pour la première fois démontrée comme étant régulée par un mécanisme épigénétique impliquant une molécule à bromodomaine dans les mastocytes humains normaux ou néoplasiques. Nous avons également pu montrer que le composé JQ1 supprime non seulement la libération d'histamine IgE-dépendante dans les cellules ROSA KIT WT mais aussi l'augmentation de CD63 et de CD203c induite par les anti-IgE dans ces mêmes cellules, et ceci de façon dose-dépendante. Cette inhibition est probablement en rapport avec la régulation négative de l'expression membranaire du FcRI sur ces cellules. Nous nous sommes ensuite demandés si le traitement des cellules ROSA KIT WT avec des inhibiteurs de protéasome pourrait contrecarrer la régulation négative du FcεRI induite par le composé JQ1. Cependant, si aucun effet de ces inhibiteurs du protéasome n'a été trouvé dans ce cadre, nous avons observé que ces molécules exercent des effets antiprolifératifs sur ces cellules, avec des CI50 de l'ordre de 5 nM. Enfin, ces effets antiprolifératifs ont été associés à une induction d'apoptose non seulement dans les cellules ROSA KIT WT mais également dans les cellules de ROSA KIT D816V . Par ailleurs, nous avons montré que l'expression de la tryptase dans les cellules ROSA KIT WT diminue après traitement avec le composé JQ1 (Wedeh et al. 2015). En outre, JQ1 s'est avérée capable d'abroger l'expression de p-STAT3 dans les cellules ROSA KIT WT et aussi de diminuer l'expression de p-STAT5 dans les cellules ROSA KIT D816V , HMC-1.1 et HMC-1.2, et ce d'une manière dose-dépendante. Ces observations sont en cohérence avec une publication récente dans laquelle les auteurs ont rapporté que le composé JQ1 est capable d'inhiber l'expression de p-STAT5 dans les cellules d'hémopathies myéloïdes malignes (Liu et al. 2014). Prises ensemble, nos données suggèrent que JQ1 peut inhiber plusieurs voies moléculaires impliquées dans l'activation et la prolifération des mastocytes malins. Par ailleurs, l'analyse par western-blot des voies de signalisation, menée sur les cellules HMC-1.1, HMC-1.2, ROSA KIT WT et ROSA KIT D816V lors du traitement par JQ1 nous a conduit à conclure que la voie MEK et celle de la PI3-Kinase sont impliquées dans le mécanisme sous-jacent de l'effet de JQ1 dans ces cellules. De ce fait, on peut envisager l'hypothèse que la voie de signalisation RAS pourrait être une des voies responsables des effets du composé JQ1. Les effets importants de ce composé sur la croissance et la survie des mastocytes néoplasiques nous ont ainsi incités à rechercher des molécules à activité pharmacologique avec lesquels des effets synergiques antinéoplasiques pourraient être observés lorsqu'ils sont utilisés en combinaison avec JQ1. Dans ce but, nous avons testé l'effet de différentes combinaisons de molécules sur les cellules HMC-1 et ROSA. De tous les médicaments testés, seuls le PKC412 et l'ATRA se sont montrés capables d'exercer une synergie avec JQ1 et d'inhiber la prolifération et la survie des cellules HMC-1 et ROSA de manière dosedépendante en synergie avec ce dernier composé. Cette mise en évidence d'une synergie entre le PKC412 ou l'ATRA et le composé JQ1 pourrait avoir des implications en termes de thérapeutique des mastocytoses systémique agressives.Dans une dernière étape de notre travail, nous nous sommes demandés si les mastocytes néoplasiques pourraient devenir résistants au composé JQ1 du fait de la présence de stem cell factor. En effet, la résistance aux thérapeutiques liée à un effet inhibiteur des cytokines est un problème majeur du traitement des hémopathies malignes. Ainsi, nous avons pu observer que le SCF réduit l'effet antiprolifératif du composé JQ1 sur les cellules ROSA KIT D816V mais de façon un peu étonnante, pas sur les cellules HMC-1.Nous nous sommes alors demandés si l'utilisation de combinaisons JQ1 + ATRA ou JQ1 + PKC412 permettrait de surmonter cette résistance induite par le SCF dans les mastocytes néoplasiques. Ainsi, nous avons pu montrer que la combinaison JQ1 + PKC412 permet de surmonter cette résistance cytokinedépendante des cellules ROSA KIT D816V , tandis que l'utilisation de la combinaison JQ1 + ATRA n'a pas permis d'observer cet effet. Cette observation pourrait être expliquée par le fait que la résistance induite par le SCF est médiée par l'activation de KIT, et que donc ce type de résistance ne peut être surmonté que par l'ajout d'un agent inhibant de façon significative l'activité tyrosine kinase de KIT, ce qui est le cas du PKC412(Wedeh et al. 2015).

1 INSERMFaculty

 1 Electronic supplementary material The online version of this article (doi:10.1007/s00705-015-2628-3) contains supplementary material, which is available to authorized users.& Abdellah Akil abdellah.akil@u-psud.fr; abdellah.akil@gmail.com of Science, UFR Biochemistry-Immunology, Univ Mohammed V, Rabat-Agdal, Morocco

A

  .Akil et al. 123 cells were washed and stained with fluorescent secondary antibodies, phalloidin and TO-PRO 3 or Hoechst. Samples were observed with a Zeiss 510 LSM confocal microscope or a Leica TCS SP5 (Leica Microsystems). The lipophilic fluorescence dye LD 540 was used to stain lipid droplets.

Fig. 1

 1 Fig. 1 SUMO1 regulates expression of the HCV core protein and genomic RNA expression. (A) Immunofluorescence analysis of SUMO1 (green), actin (red) and nuclei (blue) in uninfected (Mock) and infected (HCV) Huh7.5 cells. Scale bars represent 10 lm). (B) Immunoblot analysis of SUMO1 and HCV core in lysates of uninfected and infected Huh7.5 cells. Actin was used as a loading control. (C) Data analysis of the immunoblot staining in B, recapitulated as a histogram. (D) Immunofluorescence analysis of SUMO1 (green), core (red) and nuclei (blue) in Huh7.5 cells infected with HCV for 24 hours and then transfected with control siRNA (siNSC) or with SUMO1 siRNA (100 pmoles/assay) at 37 °C. At 48 hours post-transfection, cells were fixed with 3.7 % paraformaldehyde and stained as described in Materials and methods. Scale bars, 10 lm. (E) Immunoblot analysis of HCV core and SUMO1 in Huh7.5

Fig. 2

 2 Fig. 2 Effect of SUMO1 on LD formation and on their association with the HCV core protein. (A) Immunofluorescence labeling of lipid droplets (red), core (green) and nuclei (blue) in uninfected Huh7.5 cells (Mock) treated with control siRNA. Scale bar, 10 lm. (B) Immunofluorescence labeling of lipid droplets (red), core (green) and nuclei (blue) in infected Huh7.5 cells treated with control siRNA as in Fig. 1D. Scale bar, 10 lm. (C) Immunofluorescence labeling of lipid

Fig. 3

 3 Fig. 3 SUMO1 alters cellular accumulation of LDs. (A) Immunofluorescence analysis of LD (green) and nuclei (blue) in uninfected cells treated with 300 lM oleic acid for 24 hours and then transfected with control siRNA or SUMO1 siRNA (100 pmoles/assay) for another 48 hours. Scale bar, 10 lm. (B) Immunofluorescence analysis of LD (green) and nuclei (blue) and core (red) in infected cells treated with 300 lM of oleic acid for 24 hours, transfected with control siRNA or

Titre:

  Identification de BRD4 comme nouvelle cible thérapeutique dans le traitement des mastocytoses systémiques agressives (ASM) et des leucémies à mastocytes (MCL) Mots clés: BRD4, mastocytoses systémiques agressives, leucémies à mastocytes, cible thérapeutique Résumé: Les mastocytes humains (MC) sont des cellules tissulaires d'origine hématopoïétique impliquées dans une série de processus physiologiques et pathologiques. Les recherches sur les MC ont été entravées pendant longtemps en raison de l'accès limité à des populations pures de ces cellules. Nous avons établi une nouvelle lignée humaine de MC, ROSA KIT WT , dont les propriétés sont similaires à celles des MC primaires, constituant un nouvel outil pour la recherche sur les fonctions des MC humains, et permettant le criblage à haut débit de thérapies anti-allergiques. Les MC sont impliqués dans les mastocytoses, où ils s'accumulent pathologiquement dans divers tissus. Bien que la plupart des cas de mastocytoses systémiques (SM) sont chroniques et indolents, les patients atteints de SM avancée (SM agressive; ASM, et leucémie à mastocytes; MCL) ont un mauvais pronostic, car la plupart des thérapies disponibles ne sont pas curatives. Afin de mieux comprendre la physiopathologie des formes avancées de SM et pour trouver de nouvelles approches pour le traitement, nous avons profité de la disponibilité des cellules ROSA KIT WT pour établir un nouveau sous-clone, la lignée cellulaire ROSA KIT D816V

Table I : Distribution of human and murine MC proteases among MC subtypes

 I . MCT, mast cell tryptae; MCTC, mast cell tryptae chymase; MMC, mucosal mast cell; CTMC, connective tissue mast cell; mMCP, mouse mast cell protease; MC-CPA, mast cell carboxypeptidase A (which is a Zn-dependent metalloprotease´ type,[START_REF] Pejler | Mast cell proteases[END_REF]. MCTs have some characteristics of rodent MMCs whereas MCTCs have characteristics of rodent CTMCs (Table adapted from Vliagoftis & Befus 2005 and Pejler et al. 2010).

	Species MC subtype Predominant localization	Tryptase	Chymase	MC-CPA
	Human
	MCT	Lung	Tryptase α
			Tryptase β (I, II, III)
	MCTC	Skin	Tryptase α	CMA1	MC-CPA
			Tryptase β (I, II, III)
	Mouse
	MMC	Mucosal tissues		mMCP-1
				mMCP-2
	CTMC	Serosal serfaces	mMCP-6	mMCP-4	MC-CPA
			mMCP-7	mMCP-5
				mMCP-9

  Masson et al. have revealed insights into the mechanisms by which Cbl negatively regulates KITmediated signaling. Indeed, Cbl mediates monoubiquitination of KIT and the receptor is subsequently

targeted for lysosomal degradation

[START_REF] Masson | Direct binding of Cbl to Tyr568 and Tyr936 of the stem cell factor receptor/c-Kit is required for ligand-induced ubiquitination, internalization and degradation[END_REF]

. Moreover, It has been shown that intact lipid rafts are required for the clathrin-dependent earliest stages of efficient internalization of KIT receptor

Table II : MC mediators. TNF

 II

	-α: tumor necrosis factor-α; INF-: Interferon-; MCP, monocyte chemotactic protein; MIF: macrophage inflammatory factor; GM-CSF: granulocyte monocyte-colony stimulating factor; b-FGF: fibroblast growth factor; NGF: nerve growth factor; RANTES, regulated on activation, normal T cell expressed and secreted; SCF: stem cell factor; VEGF: vascular endothelial growth factor (Table adapted from proteoglycan and the lysosomal enzyme β-hexosaminidase which is ubiquitous to all MC subtypes in all species and quantification of the released β-hexosaminidase activity is often used as a measure of
	MC degranulation (Lundequist & Pejler 2011; da Silva et al. 2014). Currently, several studies suggest
	that numerous cytokines and growth factors are stored in MC granules along with other preformed
	mediators (Lundequist & Pejler 2011; da Silva et al. 2014). Table II presents preformed mediators of
	the MC as well as the mediators synthesized by this cell after its activation (Theoharides &
	Kalogeromitros 2006; da Silva et al. 2014).
	Three main consequences of the activation of FcεRI are known and cause mediator release of the
	MCs, as indicated earlier. The first outcome of the cross-linking is the integration of the cytoplasmic
	granules with the plasma membrane causing within minutes the release of preformed mediators such
	as histamine, tryptase and chymase. The next step of the activation is the release of newly synthesized
	lipid mediators like leukotriene C4 (LTC 4 ) or prostaglandin D2 (PGD2) caused through immediate
	synthesis of the membrane because of Ca 2+ influx. Afterwards, a delayed release of the diverse
	cytokines, growth factors and chemokines via the IgE-or IgG-dependent stimulation or other activated
	gene expressions occurs

Table III : The major MC receptors involved in host defense (Table adapted from Marshall 2004). Receptor class Examples of receptors Examples of ligands Direct receptors for pathogen products  Toll-like receptors (TLRs)

 III

		TLR1	Lipopeptide
		TLR2	PGN, zymosan and some
			LPS
		TLR3	Double -stranded RNA
		TLR4	LPS and F protein of RSV
		TLR6	PGN and zymosan
		TLR9	Bacterial DNA and CpG-
			containing DNA
	 Manosylated receptors	CD48	FimH
	Indirect

receptors for products of immune responses to pathogens  Fc receptors

		FcεRI	IgE
		FcγRI, FcγRII and FcγRIII	IgG
	 Complement receptors	CR2, CR4 and CR5	Complement components
		C5aR and C3aR	Complement	spilt
			products
	 Protease-activated	PAR2	Mast cell proteases
	receptors
	 Cytokine receptors	IL-1 and INF-γR	Immunoregulatory
			cytokines
		IL-10R and IL-12R	Immunoregulatory
			cytokines
	 Chemokine receptors	CCR3, CCR5 and CXCR4	Mast	cell
			chemoattractants

Table IV: Inflammatory diseases involving MCs (Table adapted from Arock et al. 2015).

	Disease	Pathophysiologic effects
	Asthma	Bronchonstriction, pulmonary inflammation
	Atopic dermatitis	Skin vasodilation, T-cell recruitment, inflammation, itching
	Coronary artery disease (CAD) Coronary inflammation, myocardial ischemia
	Chronic rhinitis	Nasal inflammation
	Chronic prostatitis	Prostate inflammation
	Fibromyalgia	Muscle inflammation, pain
	Interstitial cystitis	Bladder mucosal damage, inflammation, pain
	Migraine	Meningeal vasodilation, inflammation, pain
	Multiple sclerosis	Increased blood-brain barrier permeability,
		brain inflammation, demyelination
	Neurofibromatosis	Skin nerve growth, fibrosis
	Osteoarthritis	Articular erosion, inflammation, pain

Table V : Correlation between MC infiltration and tumor outcome

 V (Table adapted from Rigoni et al. 2014).

	Tumor	Prognosis	Brief description of MC activity
	Breast cancer	Good	Stromal MC infiltration, even in small quantity, is a good
			prognostic marker in breast cancer.
			MCs are recruited by stem cell factor and produce a
		Good	immunosuppressed tumor microenvironment.
	Colorectal cancer	Poor	High micro vessels density and MC number is associated
			with metastases and shorter survival.
		Poor	High density of MCs at the invasive margin of tumors
			favors colorectal cancer progression.
		Poor	In polyps, MCs and their precursors sustain inflammation and progression to cancer.
	Esophagus squamous cell	Good	MCs sustain antitumor immunity producing IL-17 and
	carcinoma		predict a favorable prognosis.
		Poor	High MCs number at the invasive edge of tumor favors
			tumor progression and decreases survival rate post-
			surgery.
	Gastrointestinal cancer	Poor	Number of MCs expressing tryptase is correlated with
			lymph node metastatization post-surgery.
	Hepatocellular carcinoma Good	High density of MCs predicts late recurrence.
	Lung cancer	Good	MCs infiltration of non-small cell lung cancer is
			associated with increase in 5-year survival, independently
		Poor	of tumor stage.
			In lung adenocarcinoma, MC density correlates with
			angiogenesis and poor prognosis.
	Malignant melanoma	Poor	Higher MC number found in invasive melanoma
			compared with benign nevi or in situ melanoma.
		Poor	High MC number correlates with high microvessels
			density and poor prognosis.
	Neurofibromatosis	Poor	MCs sustain neurofibromas initiation and progression.
	Pancreatic cancer	Poor	Higher MCs number in pancreatic cancer than in normal
			pancreas; MCs number correlates with metastatic
		Poor	potential.
			Tumor stroma infiltration by MCs is associated with poor
		Poor	prognosis. High MCs number at the tumor edge correlates with
			invasion, metastasis and tumor staging.
	Prostate cancer	Good	Intratumoral MCs negatively regulate angiogenesis and
			tumor growth.
		Poor	Peritumoral MCs stimulate the expansion of tumor.
		Poor	MCs favor the progression to adenocarcinoma but are
			absent in neuroendocrine tumor foci.

Table VI : The WHO criteria to diagnose SM

 VI

  MC infiltrates in BM or other extracutaneous organs, >25% of MCs are spindle-shaped or have atypical morphology (cytoplasmic projections, hypogranulation); or in BM smears, >25% of MCs are spindle shaped or otherwise atypical Activating point mutation at codon 816 of KIT is detected in BM, PB, or other extracutaneous organs MCs in BM, PB, or other extracutaneous organs express CD2 and/or CD25 in addition to normal MC markers as determined by flow cytometry Serum tryptase level persistently exceeds 20 ng/mL (does not count in patients who have an associated clonal hematological non-mast-cell disease; AHNMD

Table VII : The B-"Borderline Benign" findings and C-"Consider Cytoreduction" findings useful to assess MC burden and aggressiveness of SM

 VII

Table VIII : Variants and subvariants of mastocytosis according to the 2008 WHO classification (Table adapted

 VIII

from Horny et al. 2008; Arock & Valent 2010)

.

  . Exogenous and endogenous triggers, such as temperature changes, infections, physical exercise, consumption of alcohol, nonsteroidal anti-inflammatory drugs (NSAIDs) and/or emotional stress are variable from one patient to another. In addition, patients present a variable and often changing pattern of symptoms. Other mediator-related symptoms include nausea, vomiting, diarrhea, headache, dizziness, hypotension, gastroduodenal ulcers, tissue fibrosis, breathing difficulties, abdominal pain, edema, arthralgia myalgia, and arrhythmia. MC mediators involved in the most prominent clinical symptoms of mastocytosis are listed in TableIX. Of note, not all patients experience all these symptoms, and symptoms occur as attacks that may be brief or prolonged, but the duration is usually in the range of 15 to 30 minutes. In addition, symptoms may present in some

patients as anaphylactic reaction, which might be life-threatening (anaphylactic shock)

(Akin & Metcalfe 2004;[START_REF] Butterfield | Systemic mastocytosis: clinical manifestations and differential diagnosis[END_REF]

. The prevalence of anaphylaxis has been reported to be 20% to 56% in adult patients with various forms of mastocytosis

[START_REF] Florian | Indolent systemic mastocytosis with elevated serum tryptase, absence of skin lesions, and recurrent severe anaphylactoid episodes[END_REF][START_REF] González De Olano | Prevalence of allergy and anaphylactic symptoms in 210 adult and pediatric patients with mastocytosis in Spain: a study of the Spanish network on mastocytosis (REMA)[END_REF][START_REF] Brockow | Anaphylaxis in patients with mastocytosis: a study on history, clinical features and risk factors in 120 patients[END_REF]

). Nevertheless, not all SM patients bearing KIT D816V mutation suffer from anaphylaxis. Therefore, other genetic polymorphisms or mutations in MC signaling components, apart activating KIT D816V mutation, contribute to dysregulation and predispose to anaphylaxis

[START_REF] Metcalfe | Mastocytosis: molecular mechanisms and clinical disease heterogeneity[END_REF] Brown et al. 2008)

. Furthermore, osteopenia/osteoporosis has long been recognized in SM patients. Thus, Routine measurements of bone mineral density and vertebral morphometry are warranted

Table X : Molecular somatic lesions and abnormalities found in patients with mastocytosis

 X . CM, cutaneous mastocytosis; SM, systemic mastocytosis; SM-AHNMD, SM with an associated haematologic clonal non mast cell lineage disease; CEL, chronic eosinophilic leukemia; AML, acute myeloid leukemia; PMF, primary myelofibrosis (Tableadapted from Valent 2013).

	mastocytosis result in constitutive autophosphorylation of the KIT molecule, independently from its
	ligand (SCF), thereby leading to an enhanced differentiation, survival and activation of MCs
	Molecular	Estimated	frequency	in	Reported in patients with
	abnormality	patients	with SM
	KIT D816V	>80%			all SM variants, rarely in CM
	KIT D816Y	<5%			CM, ISM, SM-AHNMD
	KIT D816F	<5%			CM
	KIT D816H	<5%			MCL, ASM, SM-AHNMD
	KIT D820G	<5%			ASM
	KIT V560G	<5%			ISM
	KIT F522C	<5%			ISM
	KIT E839K	<5%			CM
	KIT V530I	<5%			SM-AHNMD
	KIT K509I	<5%			SM (familial type)
	Other KIT	<5%			CM and/or SM variants
	mutations FIP1L1/PDGFRA	<5%			SM-CEL
	AML1/ETO	<5%			SM-AML with t(8;21)
	JAK2 V617F	<5%			SM-PMF
	RAS mutations	<5%			ASM, SM-AHNMD
	TET2 mutations	<5%			SM-AHNMD, ISM, ASM
	DNMT3A	<5%			ISM, SM-AHNMD
	mutations ASXL1 mutations	<5%			SM-AHNMD
	CBL mutations	<5%			SM-AHNMD

[START_REF] Tsujimura | Ligand-independent activation of c-kit receptor tyrosine kinase in a murine mastocytoma cell line P-815 generated by a point mutation[END_REF] Akin & Metcalfe 2004; Orafo et al. 2007; Metcalfe 2008

).

5.2.2. 2-Chlorodeoxyadenosine (cladribine or 2-CdA)

  Pardanani et al. 2004), potential toxicities of this drug include myelosuppression and lymphopenia with increased risk of opportunistic infections(Hermine et al. 2010). In addition, in a study published by[START_REF] Kluin-Nelemans | Cladribine therapy for systemic mastocytosis[END_REF], none of the patients treated with 2-CdA achieved a complete remission (CR), indicating that 2-CdA is a non curative treatment option for severe SM. Moreover, 2-CdA was found ineffective in patients with progressive ASM or MCL[START_REF] Böhm | In vitro and in vivo growth-inhibitory effects of cladribine on neoplastic mast cells exhibiting the imatinib-resistant KIT mutation D816V[END_REF] as well as in SM-AHNMD(Hermine et al. 2010).

	2004; Barete et al. 2015). Besides, 2-CdA has been combined with other chemotherapy prior to
	allogeneic hematopoietic stem cell transplantation (allo-HSCT) (Ustun et al. 2014).
	Although 2-CdA has been reported to produce a long-lasting response with a sustained decrease in

al. 2009; Arock et al. 2015)

. Nevertheless, concurrent administration of GCs may improve its efficacy (up to 40% MR rate) and tolerability

(Delaporte et al. 1995;[START_REF] Hauswirth | Response to therapy with interferon alpha-2b and prednisolone in aggressive systemic mastocytosis: report of five cases and review of the literature[END_REF] Pardanini 2015)

.

2.

Cladribine (2-CdA) has been shown to exert in vivo and in vitro inhibitory effects on the growth of neoplastic MCs

[START_REF] Böhm | In vitro and in vivo growth-inhibitory effects of cladribine on neoplastic mast cells exhibiting the imatinib-resistant KIT mutation D816V[END_REF]

) and has been used in advanced SM subtypes including MCL

(Penack et al. 2005)

, and as first-line treatment in SM patients with severe mediator-related symptoms refractory or intolerant to IFN-α

[START_REF] Tefferi | Treatment of systemic mast-cell disease with cladribine[END_REF][START_REF] Kluin-Nelemans | Cladribine therapy for systemic mastocytosis[END_REF] Pardanani et al.

serum tryptase levels in two patients with progressive SSM as well as to be well tolerated with duration of remission in responding patients ranging from 2 months to 4 years

[START_REF] Böhm | In vitro and in vivo growth-inhibitory effects of cladribine on neoplastic mast cells exhibiting the imatinib-resistant KIT mutation D816V[END_REF]

  TK inhibitor imatinib (STI571) (400 mg per day) is approved by the Food and Drug Administration (FAD) for use in ASM patients without KIT D816V mutation or with unknown KIT mutation status, but with organ dysfunction due to progressive infiltration of various organs by MCs[START_REF] Bains | Current approaches to the diagnosis and treatment of systemic mastocytosis[END_REF]. However, the role of imatinib in the treatment of ISM and CM is less well established. Aqarwala et al. have demonstrated the usefulness of imatinib in the treatment of KIT816-

unmutated ISM with extensive cutaneous involvement

(Aqarwala et al. 2013)

. Moreover, patients with WDSM respond to imatinib mesylate therapy

[START_REF] Alvarez-Twose | Complete response after imatinib mesylate therapy in a patient with well-differentiated systemic mastocytosis[END_REF]

. In fact, imatinib is an

  ;Shan et al. 2006;[START_REF] De Melo Campos | Familial systemic mastocytosis with germline KIT K509I mutation is sensitive to treatment with imatinib, dasatinib and PKC412[END_REF]. In addition, dasatinib exerts in vitro cytotoxic effects against primary neoplastic MCs derived from the BM of patients with SM[START_REF] Gleixner | Synergistic growth-inhibitory effects of ponatinib and midostaurin (PKC412) on neoplastic mast cells carrying KIT D816V[END_REF]. Furthermore, dasatinib may synergize with PKC412 and chemotherapy in this regard(Gleixner et al. 2007;[START_REF] Aichberger | Treatment responses to cladribine and dasatinib in rapidly progressing aggressive mastocytosis[END_REF][START_REF] Ustun | Chemotherapy and dasatinib induce long-term hematologic and molecular remission in systemic mastocytosis with acute myeloid leukemia with KIT D816V[END_REF]. However, based upon data of phase II study of dasatinib in Philadelphia chromosome-negative

acute and chronic myeloid diseases, including SM, dasatinib therapy may benefit to a selected group of SM patients, primarily by improving their symptoms, but it does not eliminate the disease in the patients with KIT D816V mutation

[START_REF] Verstovsek | Phase II study of dasatinib in Philadelphia chromosome-negative acute and chronic myeloid diseases, including systemic mastocytosis[END_REF]

.

Purtill et al.

have reported that dasatinib exhibits modest activity in patients with SM in vivo

[START_REF] Purtill | Dasatinib therapy for systemic mastocytosis: four cases[END_REF]

. In a patient with rapidly progressing ASM, neither dasatinib nor 2-CdA produce a long-lasting response in vivo, despite encouraging in vitro results.

  Kit D814V mutation, which corresponds to the human KIT D816V mutation[START_REF] Von Bubnoff | The systemic mastocytosis-specific activating cKit mutation D816V can be inhibited by the tyrosine kinase inhibitor AMN107[END_REF]. However, nilotinib was not sufficient to inhibit the growth of KIT D816Vpositive BM MCs obtained from patients with ASM, but showed activity in cell lines expressing KIT

	WT and the very rare JM mutation (KIT D560G) (Verstovsek et al. 2006). In a phase 2 clinical study,
	an overall response rate (ORR) of 20% was seen in patients with SM (83% KIT D816V positive)
	treated with nilotinib and included two complete responses, one partial response, five incomplete
	responses, and four minor responses (Hochhaus et al. 2006).
	2.5.3.6. Ponatinib (AP24534)

KIT, BCR-ABL, and PDGFR, (Manely et al. 2010)

. Low concentrations of nilotinib reduced the growth of and induced apoptosis in transformed murine Ba/F3 cells expressing the Ponatinib (AP24534) is a potent multitargeted kinase inhibitor that has been approved by the FDA for treatment of imatinib-resistant patients with CML, including those harboring the BCR-ABL T315I mutant

(O'Har et al. 2009;[START_REF] Cortes | Ponatinib in refractory Philadelphia chromosome-positive leukemias[END_REF]

. Ponatinib has inhibitory effects on a wide variety of kinase targets, including KIT, FLT3, fibroblast growth factor receptor 1 (FGFR1), and platelet-derived growth factor receptor α (PDGFRα)

(O'Har et al. 2009;[START_REF] Gozgit | Potent activity of ponatinib (AP24534) in models of FLT3-driven acute myeloid leukemia and other hematologic malignancies[END_REF][START_REF] Gozgit | Ponatinib (AP24534), a multitargeted pan-FGFR inhibitor with activity in multiple FGFR-amplified or mutated cancer models[END_REF][START_REF] Lierman | Ponatinib is active against imatinib-resistant mutants of FIP1L1-PDGFRA and KIT, and against FGFR1-derived fusion kinases[END_REF]

) and blocks several downstream signaling molecules, including Lyn

[START_REF] Gozgit | Potent activity of ponatinib (AP24534) in models of FLT3-driven acute myeloid leukemia and other hematologic malignancies[END_REF][START_REF] Lierman | Ponatinib is active against imatinib-resistant mutants of FIP1L1-PDGFRA and KIT, and against FGFR1-derived fusion kinases[END_REF][START_REF] Okabe | Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells[END_REF]

.

  fact, Allo-HSCT remains the only potentially curative treatment option for patients with advanced SM. The effect of allo-HSCT in patients with advanced SM (SM-AHNMD, n=38; MCL, n=12; ASM,

n=7) has been recently reported in a global multi-center study

(Ustun et al. 2014)

. The median age of patients was 46 years. Most patients received a graft from HLA (HumanLeukocyte Antigens)-identical siblings (n=34) or unrelated donors (URD) (n=17). Myeloablative (n=36) conditioning (MAC) was more commonly used. Responses (defined per decrease in serum tryptase level and in BM MC percentage) were observed in 40 patients (70%), including 16 patients (28%) with CR. Overall survival (OS) and SM progression-free survival (PFS) at 3 years were 57% and 51% for all patients.

  In deed, the proapoptotic BCL-2 family member BIM has been identified as a tumor suppressor in neoplastic MCs as it is down-regulated in neoplastic MCs by SCF stimulation and by the oncogenic KIT D816V TK. Besides, midostaurin and the proteasome inhibitor bortezomib upregulate BIM in HMC-1.1 and HMC-1.2 cells[START_REF] Aichberger | Identification of proapoptotic Bim as a tumor suppressor in neoplastic mast cells: role of KIT D816V and effects of various targeted drugs[END_REF]. Besides, the recently described inhibitory receptors, CD300a and Siglec-8, have potential downregulatory properties on MC activation and survival in vivo[START_REF] Berent-Maoz | Human mast cells express intracellular TRAIL[END_REF].

	Recently, neoplastic MC in ASM and MCL were found to express antiapoptotic MCL-1, BCL-2, and
	BCL-xL and the pan-BCL-2 family blocker obatoclax (GX015-070) was found to inhibit the
	proliferation of primary neoplastic MCs and of HMC-1 cell lines. Obatoclax was also found to induce
	apoptosis and to increase the expression of PUMA, NOXA, and BIM mRNA. Moreover, obatoclax
	synergizes with several other antineoplastic drugs, including dasatinib, midostaurin, and bortezomib,
	A selective targeting of these receptors could lead to a potential innovative treatment strategy in
	mastocytosis (Karra et al. 2009).
	Furthermore, pimecrolimus and other calcineurin inhibitors or dimethylfumarate, have been reported

;[START_REF] Aichberger | Identification of proapoptotic Bim as a tumor suppressor in neoplastic mast cells: role of KIT D816V and effects of various targeted drugs[END_REF] Karlberg et al. 2010;[START_REF] Peter | The pan-Bcl-2 blocker obatoclax promotes the expression of Puma, Noxa, and Bim mRNA and induces apoptosis in neoplastic mast cells[END_REF]

. Targeting of BCL-2 family members by drugs promoting BIM (re)-expression, or by BH3-mimetics such as obatoclax, may thus be an attractive therapy concept in SM.

in producing apoptosis and/or growth arrest in neoplastic MC

[START_REF] Peter | The pan-Bcl-2 blocker obatoclax promotes the expression of Puma, Noxa, and Bim mRNA and induces apoptosis in neoplastic mast cells[END_REF]

.

The autophosphorylated KIT receptor induces NF-κB activation and resuls in the up-regulation of cyclin D3 expression and cell cycle progression. Therefore, compounds that interfere with NF-κB signaling may present a therapeutic potential in SM. The NF-κB inhibitor IMD-0354 has been documented to restrain in vitro factor-independent proliferation of neoplastic MCs with KIT mutations but not of normal MCs

[START_REF] Tanaka | A novel NF-kappaB inhibitor, IMD-0354, suppresses neoplastic proliferation of human mast cells with constitutively activated c-kit receptors[END_REF]

.

Moreover, human MCs have recently been reported to express receptors that could allow for the induction of apoptosis and inhibition of activation, like the Tumor Necrosis Factor Related Apoptosis Inducing Ligand receptor (TRAIL) and the inhibitory receptors CD300a and Siglec-8

[START_REF] Karra | Are we ready to downregulate mast cells?[END_REF][START_REF] Karra | Down-regulation of mast cell responses through ITIM containing inhibitory receptors[END_REF] Migalovich-Sheikhet et al. 2012)

. So far, TRAIL receptor is the only death receptor known to be expressed on human MCs and, therefore, constitutes an interesting therapeutic target. to induce apoptosis in MCs and in disease models of mastocytosis may be another potential therapy for mastocytosis and other MC-associated diseases

(Ma el al. 2010;[START_REF] Ma | Mast cells as targets of pimecrolimus[END_REF][START_REF] Förster | Dimethylfumarate induces apoptosis in human mast cells[END_REF]

  The mode of action and efficacy of these inhibitors has been attributed mainly to their ability to modulate transcription elongation of essential genes involved in cell cycle and apoptosis such as c-MYC (v-myc myelocytomatosis viral oncogene homolog) and BCL-2(Dawson et al. 2011; Mertz et al. 2011;

	Delmore et al. 2011; Jung et al. 2015). Indeed, JQ1 and other BET inhibitors result in preferential loss
	of Brd4 at super enhancers and consequent transcription elongation defects that preferentially impact
	genes with super enhancers, including the oncogene MYC (Lovén et al. 2013; Valent & Zuber 2014).
	Of note, MYC is known to be the main transcriptional target of BET proteins and contributes to the
	pathogenesis of certain cancer types.
	To date, BET inhibitors have been reported to downregulate MYC expression in diverse tumors

, glioblastoma and brain tumors

(Wyce et al. 2013;[START_REF] Cheng | Inhibition of BET bromodomain targets genetically diverse glioblastoma[END_REF][START_REF] Pastori | The Bromodomain protein BRD4 controls HOTAIR, a long noncoding RNA essential for glioblastoma proliferation[END_REF]

, breast cancer

(Alsarraj & Hunter 2012;[START_REF] Stuhlmiller | Inhibition of Lapatinib-Induced Kinome Reprogramming in ERBB2-Positive Breast Cancer by Targeting BET Family Bromodomains[END_REF]

, liver cancer

[START_REF] Zhang | BRD4 promotes tumor growth and epithelial-mesenchymal transition in hepatocellular carcinoma[END_REF]

, lung cancer

[START_REF] Lockwood | Sensitivity of human lung adenocarcinoma cell lines to targeted inhibition of BET epigenetic signaling proteins[END_REF] Lee et al. 2013;[START_REF] Lenhart | Sensitivity of Small Cell Lung Cancer to BET Inhibition Is Mediated by Regulation of ASCL1 Gene Expression[END_REF][START_REF] Tsai | The MZF1/c-MYC axis mediates lung adenocarcinoma progression caused by wild-type lkb1 loss[END_REF]

, colon cancer

[START_REF] Rodriguez | Aberrant epigenetic regulation of bromodomain BRD4 in human colon cancer[END_REF]

, and.prostate cancer

(Wyce et al. 2013;[START_REF] Asangani | Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer[END_REF]

.

including myeloma

[START_REF] Delmore | BET bromodomain inhibition as a therapeutic strategy to target c-Myc[END_REF][START_REF] Chaidos | Potent antimyeloma activity of the novel bromodomain inhibitors I-BET151 and I-BET762[END_REF]

, Mantle Cell Lymphoma

[START_REF] Sun | Synergistic activity of BET protein antagonist-based combinations in mantle cell lymphoma cells sensitive or resistant to ibrutinib[END_REF]

); Burkitt's lymphoma

(Mertz et al. 2011)

, AML and MLL

(Dawson et al. 2011; Zuber et al. 2011; Herrmann et al. 2012;[START_REF] Picaud | PFI-1, a highly selective protein interaction inhibitor, targeting BET Bromodomains[END_REF] Fiskus et al. 2014)

, acute lymphoblastic leukemia (ALL), and T cell ALL

(Ott et al. 2012;[START_REF] Da Costa | BET inhibition as a single or combined therapeutic approach in primary paediatric B-precursor acute lymphoblastic leukaemia[END_REF] Roderick et al. 2014;[START_REF] Tan | Co-targeting of Akt and Myc inhibits viability of lymphoma cells from Lck-Dlx5 mice[END_REF]

, medulloblastoma

[START_REF] Henssen | BET bromodomain References protein inhibition is a therapeutic option for medulloblastoma[END_REF][START_REF] Bandopadhayay | BET bromodomain inhibition of MYC-amplified medulloblastoma[END_REF][START_REF] Venkataraman | Inhibition of BRD4 attenuates tumor cell self-renewal and suppresses stem cell signaling in MYC driven medulloblastoma[END_REF]

, glioblastoma

[START_REF] Rajagopalan | Pre-clinical analysis of changes in intra-cellular biochemistry of glioblastoma multiforme (GBM) cells due to c-Myc silencing[END_REF]

, lung adenocarcinomas

[START_REF] Lockwood | Sensitivity of human lung adenocarcinoma cell lines to targeted inhibition of BET epigenetic signaling proteins[END_REF][START_REF] Tsai | The MZF1/c-MYC axis mediates lung adenocarcinoma progression caused by wild-type lkb1 loss[END_REF]

, lung cancer and non-small cell lung cancer

[START_REF] Shimamura | Efficacy of BET bromodomain inhibition in Kras-mutant non-small cell lung cancer[END_REF]

, prostate cancer

[START_REF] Asangani | Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer[END_REF]

, ovarian cancer

[START_REF] Baratta | An intumor genetic screen reveals that the BET bromodomain protein, BRD4, is a potential therapeutic target in ovarian carcinoma[END_REF][START_REF] Qiu | JQ1 suppresses tumor growth through downregulating LDHA in ovarian cancer[END_REF]

, and skin cancers

[START_REF] Segura | BRD4 sustains melanoma proliferation and represents a new target for epigenetic therapy[END_REF][START_REF] Shao | BET protein inhibitor JQ1 attenuates Myc-amplified MCC tumor growth in vivo[END_REF]

, as well as in estrogen receptor (ER)α-positive breast cancers

Table 1 .

 1 Overview of ROSA NSG experimentsTable shows engraftment of ROSA KIT WT and of ROSA KIT D816V cells in NSG mice. Percentage of engrafted human MCs was assessed by flow cytometry using their double positivity for human CD45 and human CD117 antigens. *Number of mice showing an engraftment of ROSA cells .0.1% in bone marrow after intravenous injection of total mice injected that survived more than 12 weeks. †Number of mice showing a subcutaneous tumor formation after subcutaneous injection of total mice injected (2 sites per mouse) that survived more than 12 weeks.

				Median
			BM	engraftment of	Subcutaneous
		Cells	engraftment	intravenously	tumor
		injected	(positive/	injected	growth
		3 10 6	total)*	mice (%)	(positive/total) †
	ROSA KIT WT	5	4/5 (18 wk)	2.62	1/5
	ROSA KIT D816V	0.4	4/4 (21 wk)	52	1/4

  in TableS3; ASM/MCL patients were patients N° 1, 4 and 9 of the same table; all these 6 patients having a KIT point mutation at position 816) and from normal/reactive bone marrow (n=1) were incubated with anti-IgE mAb E-124.2.8 (Beckman Coulter,

Characteristics and origins of the various antibodies used in multicolor flow cytometry experiments.

	CD16 CD56 CD146	NKP15 NCAM16.2 128018	FITC PE PE	Mouse IgG1 Mouse IgG1 Mouse IgG1	BD Biosciences BD Biosciences R&D
	CD17 CD58 CD150	G035 AICD58 A12 (7D4)	FITC FITC PE	Mouse IgG1 Mouse IgG2a Beckman Coulter BD Biosciences Mouse IgG1 Biolegend
	CD19 CD59 CD164	4G7 p282 (H19) N6B6	FITC FITC PE	Mouse IgG1 Mouse IgG2a BD Biosciences BD Biosciences Mouse IgG2a BD Biosciences
	Antigen CD20 CD63 CD166	Clone L27 CLBGran/12 105902	Fluorochrome PE FITC PE	Isotype Mouse IgG1 Mouse IgG1 Mouse IgG1	Manufacturer BD Biosciences Beckman Coulter R&D
	Isotype Control CD22 CD64 CD203c	MOPC-21 SJ10.1H11 10,1 97A6	FITC PE FITC PE	Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG1	BD Biosciences Beckman Coulter BD Biosciences Beckman Coulter
	Isotype Control CD23 CD66a CD213a	G155-178 M-L233 283340 B-K19	FITC PE PE PE	Mouse IgG2a BD Biosciences Mouse IgG1 BD Biosciences Mouse IgG2b R&D Mouse IgG1 cell sciences
	Isotype Control CD24 CD69 CD218a	MOPC-21 ML5 FN50 H44	PE FITC FITC PE	Mouse IgG1 Mouse IgG2a BD Biosciences BD Biosciences Mouse IgG1 BD Biosciences Mouse IgG1 Biolegend
	Isotype Control CD25 CD71 CD271	NA/LE 2A3 MEM-75 C40-1457	PE PE FITC PE	Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG1	BD Biosciences BD Biosciences Biolegend BD Biosciences
	Isotype Control CD26 CD87 CD273	polyclonal M-A261 VIM5 MIH18	PE PE PE PE	Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG1	R&D BD Biosciences Biolegend Biolegend
	Isotype Control CD27 CD90 CD274	G155-178 O323 5E10 MIH1	PE PE PE PE	Mouse IgG2a BD Biosciences Mouse IgG1 Biolegend Mouse IgG1 BD Biosciences Mouse IgG1 BD Biosciences
	Isotype Control CD30 CD95 CD279	133303 BerH8 DX2 EH12.2H7	PE PE PE PE	Mouse IgG2b R&D Mouse IgG1 BD Biosciences Mouse IgG1 BD Biosciences Mouse IgG1 Biolegend
	Isotype Control CD31 CD96 CD300a	MOPC-21 AC128 NK92.39 MEM-260	APC APC PE PE	Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG1	BD Biosciences Miltenyi Biotec eBiosciences abcam
	CD2 CD32 CD105 CD309	RPA-2.10 2E1 166707 89106	PE PE PE PE	Mouse IgG1 Mouse IgG2a Beckman Coulter BD Biosciences Mouse IgG1 R&D Mouse IgG1 R&D
	CD3 CD33 CD114	UCHT1 WM53 LMM741	APC PE PE	Mouse IgG1 Mouse IgG1 Mouse IgG1	BD Biosciences BD Biosciences BD Biosciences
	CD4 CD34 CD115	RPA-T4 581 61708	FITC PE PE	Mouse IgG1 Mouse IgG1 Mouse IgG1	BD Biosciences BD Biosciences R&D
	CD5 CD38 CD116	L17F12 HIT2 31916	FITC APC PE	Mouse IgG2a BD Biosciences Mouse IgG1 BD Biosciences Mouse IgG1 R&D
	CD8 CD44 CD117	RPA-T8 515 104D2	PE PE PE	Mouse IgG1 Mouse IgG1 Mouse IgG1	BD Biosciences BD Biosciences BD Biosciences
	CD9 CD45 CD123	ALB 6 2D1 32703	FITC PerCP PE	Mouse IgG1 Mouse IgG1 Mouse IgG1	Beckman Coulter BD Biosciences R&D
	CD10 CD45 CD127	HI10a 2D1 hIL-/R-M21	PE APC PE	Mouse IgG1 Mouse IgG1 Mouse IgG1	BD Biosciences BD Biosciences BD Biosciences
	CD11b CD48 CD133	ICRF44 J4-57 AC133	PE FITC PE	Mouse IgG1 Mouse IgG1 Mouse IgG1	Biolegend Beckman Coulter Miltenyi Biotec
	CD13 CD50 CD134	WM15 ICAM3.3 (CH3.3) FITC PE ACT35 PE	Mouse IgG1 Mouse IgG1 Mouse IgG1	BD Biosciences Serotec BD Biosciences
	CD14 CD52 CD138	TÜK4 HI186 DL-101	FITC PE PE	Mouse IgG2a Dako Mouse IgG2b R&D Mouse IgG1 BD Biosciences
	CD15 CD54 CD144	W6D3 84H10 123413	PE FITC PE	Mouse IgG1 Mouse IgG1 Mouse IgG2b R&D Biolegend Beckman Coulter

Table S3 : Major demographic and biological characteristics of mastocytosis patients involved in the study.

 S3

	Pat.	m/f Age	Diagnosis	Tryptase	KIT	% MC in	% MC in	% MC in
	No			(WHO)	level	codon 816	BM sections	BM	PB
					(ng/ml)	mutation		smears	smears
	1	m	71	ASM	88.6	D816V+	15%	1%	0%
	2	f	57	ISM	30.7	D816V+	5%	<1%	0%
	3	m	64	ISM	33.5	D816V+	5%	3%	0%
	4	f	54	MCL	904	D816H+	50%	60%	75%
	5	f	68	ISM-MGUS	41	n.t.	<5%	1%	0%
	6	m	75	ASM	75.5	D816V+	10%	<1%	0%
	7	f	74	SM-CMML	67.5	D816V+	5%	1%	0%
	8	f	48	MCL	332	D816V-	60%	70%	0%
	9	m	68	ASM	152	D816V+	5-10%	2%	0%
	10	m	48	ISM	40.9	D816V+	10%	1%	0%
	11	m	48	ISM	82.5	D816V+	5%	1%	0%
	12	f	48	ISM	34.1	D816V+	5%	1%	0%

m, male; f, female; ASM, aggressive systemic mastocytosis; ISM, indolent systemic mastocytosis; MCL, mast cell leukemia; MGUS, monoclonal gammopathy of undetermined significance; SM-CMML, SM with an associated Chronic Myelomonocytic Leukemia. MC: mast cell; BM: bone marrow; PB: peripheral blood; WHO: World Health Organization. *Serum tryptase levels were determined at diagnosis by commercial fluoroimmunoassay (FIA; Pharmacia, Uppsala, Sweden). Normal value for tryptase level in serum: < 15 ng/mL.

Table S4 : Engraftment of ROSA KIT D816V cells and ROSA KIT WT cells in NSG mice as determined by tryptase staining.

 S4 Tissue sections obtained from NSG mice injected with ROSA KIT D816V cells and ROSA KIT WT cells, were stained with an antibody against mast cell tryptase. Engraftment levels were defined using the following score: ++, infiltration grade >50%; +, infiltration 10-50%; +/-, 2-10%; -/+, only a very few cells detected; -, no engraftment found. Abbreviations: WT, wild type.

	Mouse number	Bone marrow	Liver	Lung	Spleen
	186/D816V	++	+/-	+	++
	187/D816V	++	+/-	+/-	+
	190/D816V	++	-	+/-	+
	191/D816V	++	-	+	+
	171/WT	++	+/-	+/-	+
	172/WT	-/+	-	-	-/+
	173/WT	+	-	-	+
	174/WT	++	-	-	+
	175/WT	+/-	-	-	-/+

Table S5 : Level of chymase expression in ROSA KIT D816V cells engrafting in NSG mice, as determined by immunohistochemistry (IHC) on biopsies of sub-cutaneous tumors or of infiltrated bone marrow (BM) samples

 S5 . NSG mice were injected i.v. or s.c. with ROSA KIT D816V cells as described in the Material and Methods section. The diseased mice were sacrificed and BM cells were obtained from flushed femurs, tibias, and humeri, whereas tumors grown from s.c. injected cells were collected and dispersed by collagenase digestion. The percentage of CD117 positive cells was determined for each sample by flow cytometry. IHC for chymase was performed on slides prepared from paraffin-embedded, formalin-fixed BM and tumor biopsy specimens. The percentage of chymase positive cells was determined using a scoring system.

	No.	ROSA Injected	Mouse No.	Flow Cytometry	IHC
				CD117 (%)	Chymase
	P751 Tumor	D816V	30	58	+
	P752 Tumor	D816V	30	58	+/-
	P757 BM	D816V	186	66	-
	P761 BM	D816V	187	76	-

Score Result: +, most cells immunoreactive; +/-, 10-50% of cells reactive; -, no chymase+ cells found.

  inhibitors in KRAS-positive tumours in other models[START_REF] Shimamura | Efficacy of BET bromodomain inhibition in Kras-mutant non-small cell lung cancer[END_REF][START_REF] De Raedt | PRC2 loss amplifies Rasdriven transcription and confers sensitivity to BRD4-based therapies[END_REF] Roy et al. 2015. Borbely et al. 2015). For instance, changes in the epigenetic state in a KRAS-driven pancreatic cancer formation model initiate duct-derived intraductal papillary mucinous neoplasia (IPMN) lesions(Roy et al. 2015). Also, JQ1 treatment impairs the tumorigenicity of Kras G12D,

	2014). Importantly, SUZ12 inactivation also triggers an epigenetic switch that sensitizes peripheral
	nerve sheath tumours to bromodomain inhibitors (De Raedt et al. 2014). More recently, co-treatment
	with JQ1 and the HDAC inhibitor was showed to reduce breast cancer cell viability through induction
	of the expression of several members of the ubiquitin-specific protease 17 (USP17) family of
	deubiquitinating enzymes that attenuate the Ras/MAPK pathway causing decrease in cell viability

pancreatic ductal adenocarcinoma (PDA) and (IPMN)-derived murine cancer cell lines both in vitro and in vivo

(Roy et al. 2015)

. Moreover, JQ1 abrogates the survival and the viability of KRASpositive non-small cell lung cancer (NSCLC) cell lines. However, the activity of JQ1 in mutant KRAS NSCLC is abrogated by concurrent alteration or genetic knock-down of LKB1

[START_REF] Shimamura | Efficacy of BET bromodomain inhibition in Kras-mutant non-small cell lung cancer[END_REF]

. Similarly, JQ1 treatment produces significant tumor regression in mutant kras mice, whereas, tumors from mutant kras and lkb1mice did not respond to JQ1

[START_REF] Shimamura | Efficacy of BET bromodomain inhibition in Kras-mutant non-small cell lung cancer[END_REF]

. Interestingly, by amplifying Ras-driven transcription through effects on chromatin, the loss of the tumour suppressor polycomb group gene SUZ12 potentiates the effects of mutations in NF1, which encodes a Ras GTPase-activating protein (RasGAP) and whose loss drives cancer by activating Ras

(De Raedt et al. (Borbely et al. 2015)

.

Français de la thèse de Doctorat d'Université de Mme Ghaith WEDEH Ecole Doctorale de Cancérologie -Université Paris XI Titre: Identification de BRD4 comme nouvelle cible thérapeutique dans le traitement des mastocytoses systémiques agressives (ASM) et des leucémies à mastocytes (MCL) Avant propos:

  Les mastocytes sont des cellules granuleuses découvertes en 1878 par Paul Ehrlich, et retrouvées dans tous les tissus de l'organisme, bien que certains tissus conjonctifs, comme la peau, en soient particulièrement riches. Les mastocytes dérivent des cellules souches hématopoïétiques non engagées Dans les tissus, ces précurseurs subissent les étapes finales de leur maturation, et en particulier, l'acquisition de granules intracellulaires matures, toujours sous l'influence de différentes cytokines comme le SCF. L'identification des mastocytes matures repose ainsi essentiellement sur la mise en évidence d'un cytoplasme rempli de très nombreuses granulations métachromatiques après coloration par des colorants basiques tels que le bleu de toluidine, sur leur positivité pour la tryptase et sur leur expression membranaire de récepteurs de haute affinité aux IgE (FcεRI). Chez l'homme on distingue deux types de mastocytes en fonction de leur contenu en protéases. Les mastocytes n'exprimant que la tryptase (MC T ) sont essentiellement retrouvés dans les muqueuses (digestives, pulmonaires, etc…), tandis que les mastocytes exprimant la tryptase, mais aussi la chymase et la carboxypeptidase (MC TC ) sont surtout présents dans les tissus conjonctifs (peau, péritoine, etc…)(Vliagoftis & Befus 2005; Metcalfe 2008).Les mastocytes jouent un rôle important dans l'initiation des réponses immunitaires innées et adaptatives en produisant les médiateurs chimiques de l'inflammation, en particulier l'histamine, l'héparine, la sérotonine, la tryptase et les cytokines. De plus, ces cellules jouent un rôle néfaste en tant qu'initiateurs des réactions allergiques immunoglobine E (IgE)-dépendantes, dites réaction allergique de type I. En effet, durant la phase de sensibilisation à un allergène multivalent, les IgE spécifiques produites par les lymphocytes B se fixent aux récepteurs de haute affinité aux IgE (FcεRI) se trouvant à la surface des mastocytes. Lors de la réintroduction de l'allergène multivalent, celui-ci vient agréger les FcεRI à la surface des mastocytes en se fixant sur les fragments Fab des IgE fixées à la surface de ces cellules. Cette Ces pathologies rares sont des néoplasies myéloïdes caractérisées par une accumulation anormale de mastocytes dans divers organes. Les organes généralement atteints sont la moelle osseuse, la peau, le foie, et le tractus gastro-intestinal(Valent et al. Les formes graves comportent une variante agressive (ASM) et une variante leucémique, aussi appelée leucémie à mastocytes (MCL). Le pronostic des patients atteints d'ASM ou de MCL est médiocre. Un certain nombre de patients atteints de SM vont présenter en même temps une autre hémopathie maligne non mastocytaire associée, le plus souvent myéloïde (SM-AHNMD)(Horny et al. 2008; Stoecker & Wang 2012). Dans ces cas, le pronostic est à la fois fonction de celui de la mastocytose et de celui de l'hémopathie maligne associée(Pardanani et al. 2009; Arock & Valent 2010).Notre laboratoire s'intéresse depuis de nombreuses années aux agents pharmacologiques capables d'inhiber la survie des mastocytes malins, et en particulier, à l'identification de nouvelles cibles thérapeutiques. En effet, comme décrit précédemment, les traitements actuels des mastocytoses systémiques, en particulier dans les formes agressives, sont décevants et même la midostaurine (PKC412) présente un intérêt limité dans la mesure où elle ne permet pas la guérison définitive de ces patients. C'est pourquoi nous nous sommes intéressés aux protéines épigénétiques qui ont émergé récemment comme des cibles anticancéreuses nouvelles, et parmi celles-ci, plus particulièrement aux protéines BET (bromodomaine et domaine terminal) qui reconnaissent les histones lysine acétylée, et de cette façon régulent l'expression des gènes. En effet, il a été montré qu'une dérégulation de leur activité́ peut être responsable de diverses maladies, dont des pathologies tumorales. Denis 2012). Les données de la littérature tendent à leur conférer une fonction importante dans le contrôle de la prolifération au sens large: régulation du cycle cellulaire, de la transcription et participation aux évènements de divisions mitotiques et méiotiques. Elles semblent servir de «plate-forme fonctionnelle» et recruter des facteurs de transcription, voire les polymérases elles-mêmes, au niveau de régions d'euchromatine, permettant ainsi l'expression spécifique de gènes. Elles pourraient également participer à l'hérédité d'une mémoire épigénétique entre les cellules et jouer un rôle dans la maturation des cellules germinales. Ainsi, les protéines BET sont indispensables chez les différents organismes par leur rôle central potentiel d'intermédiaire entre la chromatine et les facteurs nucléaires. Contrairement aux autres facteurs d'organisation de la chromatine, en particulier les autres protéines à bromodomaine, les protéines BET sont capables de rester fixées aux chromosomes durant la division mitotique. Ceci laisse supposer qu'elles puissent jouer un rôle important au cours de la mitose et qu'elles participent au maintien d'une mémoire épigénétique à travers les divisions cellulaires.

	Les protéines BET possèdent également un second domaine caractérisé qui leur est propre, le domaine
	Extra-Terminal, permettant des interactions protéine-protéine (Florence & Faller 2001; Belkina & Denis
	Les symptômes cliniques des mastocytoses sont en rapport avec, d'une part, l'infiltration des différents 2012). Ainsi, cette famille de protéine est au carrefour de l'homéostasie cellulaire et des informations
	tissus ou organes par les mastocytes pathologiques et, d'une autre part, avec la libération exagérée de épigénétiques permettant la mise en place d'un programme génétique spécifique à la fonction d'une
	médiateurs par ces cellules néoplasiques. cellule.
	Les traitements actuels des mastocytoses sont essentiellement symptomatiques et aucune thérapeutique
	spécifique ne permet d'obtenir la guérison de ces pathologies. En effet, le traitement des mastocytoses a Fait notable, il est bien établi que des translocations chromosomiques impliquant les gènes BRD3 ou
	pour but d'inhiber la libération des médiateurs par les mastocytes et/ou neutraliser les effets délétères de BRD4 sont impliquées dans des cancers extrêmement agressifs (French et al. 2008). BRD2 est
	ces médiateurs et d'inhiber la survie des mastocytes pathologiques. Le traitement des mastocytoses surexprimée dans des lymphomes et des lignées leucémiques, tandis que BRDT est impliquée dans des
	systémiques indolentes utilisent généralement des antagonistes des récepteurs de l'histamine. Dans les cancers du poumon.
	2003; Horny et al. 2007; Metcalfe 2008; Amon et al. 2010; Sokol et al. 2013). Chez les enfants, la forme mastocytoses agressives et les leucémies à mastocytes, des traitements cytoréducteurs comme
	la plus fréquente est habituellement confinée à la peau (mastocytose cutanée ou CM) et peut régresser l'interféron-alpha ou la cladribine (2-CdA) et/ou des traitements ciblant KIT sont utilisés, comme le
	spontanément à l'adolescence. Par contre, la plupart des patients adultes présentent une forme systémique PKC412 (midostaurine). Malheureusement, le pronostic de ces patients reste médiocre, même si lorsqu'ils

présentes dans la moelle osseuse

(Welle 1997)

. Ces cellules souches donnent naissance à des progéniteurs mastocytaires de phénotype CD34+, KIT+, CD13+, sous l'influence de cytokines, en particulier le stem cell factor (SCF), dont le récepteur spécifique, dénommé KIT, est un récepteur transmembranaire à activité tyrosine kinase (TK) intrinsèque. Fait intéressant, KIT est exprimé sur les cellules souches hématopoïétiques et sur toutes les cellules de la lignée mastocytaire quelque soit leur stade de maturation

(Arock et al. 2008)

. Les progéniteurs mastocytaires circulent puis migrent vers les tissus, sous l'influence de différentes cytokines (dont le SCF) et de chimiokines (dont le SDF-1)

(Arock et al. 1993; Wedemeyer et al. 2000)

. agrégation provoque une activation, la dégranulation très rapide des mastocytes et donc une libération immédiate des médiateurs chimiques (histamine) et des protéases contenus dans les granules. De façon plus retardée, au cours de la même réaction allergique, les mastocytes sont aussi capables de synthétiser de nombreux autres médiateurs comme des dérivés des prostaglandines et des leucotriènes, mais aussi de nombreuses cytokines et chimiokines à activité proinflammatoire et/ou vasoactive.

Outre son implication dans l'immunité innée et acquise, dans les phénomènes de cicatrisation, de fibrose, voire d'auto-immunité

(Abraham & Arock 1998; Galli et al. 2005; Metcalfe 2008

), le mastocyte est impliqué directement dans les mastocytoses.

(mastocytose systémique ou SM), avec atteinte, au minimum de la moelle osseuse et, très souvent, de la peau (urticaire pigmentaire). Les mastocytoses systémiques sont le plus souvent indolentes (ISM), et plus rarement graves.

La physiopathologie des mastocytoses est dominée par la présence de mutations activatrices et acquises dans la structure du gène KIT. L'anomalie la plus fréquemment rencontrée chez l'adulte est la mutation ponctuelle KIT D816V, qui est détectable chez la plupart des patients avec mastocytose systémique. Par contre, chez l'enfant, la mutation KIT D816V n'est rencontrée que chez seulement un tiers des patients atteints de mastocytose, tandis que les autres malades présentent soit des mutations différentes de KIT (40% des patients), soit n'ont pas d'anomalies de KIT. Lorsqu'il y a une mutation de KIT, elle conduit à l'autophosphorylation constitutive du récepteur en absence de son ligand, le SCF. sont traités par de nouveaux agents ciblant KIT, ou par polychimiothérapie et/ou même par greffe de cellules souches hématopoïétiques.

Les protéines BET, qui sont multifonctionnelles, sont les membres d'une famille protéique extrêmement bien conservée au cours de l'évolution au sein de nombreux organismes. Chez les mammifères, la famille compte quatre membres, dont trois qui sont ubiquitaires et qui présentent des affinités différentes pour la chromatine, ce qui pourrait expliquer qu'elles ciblent des régions spécifiques du génome, à savoir BRD2, BRD3 et BRD4. Une autre protéine de cette famille, appelée BRDT est, elle, spécifique du tissu testiculaire

(Shang et al. 2004; paillisson et al. 2007

). La particularité de cette famille est que ses membres possèdent un double bromodomaine, leur permettant une association forte à la chromatine acétylée, sur différents résidus lysines acétylés

(Florence & Faller 2001; Filippakopoulos et al. 2012; Belkina &

Parmi les protéines BET, nous nous sommes intéressés plus particulièrement à la protéine BRD4. Cette protéine est capitale pour la machinerie transcriptionnelle, car par le biais de son domaine CTM (Cterminal motif), elle recrute, au niveau de régions de chromatine acétylée, le facteur P-TEFb, permettant alors l'activation de la phase d'élongation de la transcription

(Wang & Filippakopoulos 2015)

. Par ailleurs, Les bromodomaines de BRD4 sont capables, quant à eux, de reconnaître H3K14ac, H4K5ac et H4K12ac

(Dey et al. 2003)

. Il est également intéressant de noter que BRD4 est capable d'interagir avec la sous-unité RelA du facteur de transcription NF-κB acétylé sur la lysine 301, ce qui laisse supposer que les histones ne sont pas les seuls partenaires acétylés reconnus par les bromodomaines

(Huang et al. 2009

).

  Fonctions des mastocytes dans la physiologie et dans les situations pathologiques 1.8.1. Fonction des mastocytes dans la défense de l'hôte, l'immunité innée et acquise 1.8.2. Le rôle des mastocytes dans l'allergie, l'asthme, le remodelage tissulaire et la fibrose 1.8.3. Le rôle des mastocytes dans les maladies vasculaires 1.8.4. Le rôle des mastocytes dans l'auto-immunité et d'autres maladies 1.8.5. L'implication des mastocytes dans le cancer et d'autres situations pathologiques 1.9. Les lignées mastocytaires humainesAu cours de ma thèse, nous avons établi, à partir de cellules souches CD34+ de sang de cordon cultivées à long terme en présence de SCF, une nouvelle lignée mastocytaire humaine stable, dénommée ROSA KIT WT , exprimant à la fois le récepteur FcεRI et le récepteur KIT de structure normale. Cette lignée cellulaire, que nous avons caractérisée, reste dépendante du SCF pour sa survie et sa prolifération(Saleh, Wedeh et al. 2014). Fait intéressant, son temps de doublement est court, elle est aisément congelable et parfaitement activable par aggrégation de ses récepteurs de haute affinité aux IgE.

	1.4. Le récepteur de haute affinité pour l'IgE (FcεRI)
	1.4.1. Structure et expression de FcεRI
	1.4.2. Régulation de l'expression FcεRI
	1.4.3. Voies de signalisation de FcεRI
	1.5. D'autres récepteurs mastocytaires
	1.6. Médiateurs mastocytaires
	1.7. Activateurs de mastocytes
	1.8. Résultats obtenus
	1.1. Aperçu historique
	1.2. Le développement des mastocytes
	1.2.1. Le SCF et son effet sur le développement des mastocytes
	1.2.2. Effet d'autres cytokines et d'autres molécules biologiques sur le développement des mastocytes
	chez diverses espèces
	1.3. La morphologie le phénotype et l'hétérogénéité des mastocytes
	1.3.1. KIT, le récepteur tyrosine kinase du SCF
	1.3.2. Voies de signalisation du récepteur KIT
	1.3.3. Les régulateurs de l'activité kinase KIT et de la signalisation KIT
	1.3.4. Mutations 'gain de fonction' et mécanismes de l'activation constitutive de KIT

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Centre for Advanced Studies in Vaccinology and Biotechnology (CASVAB), University of Balochistan, Quetta, Pakistan

ACKNOWLEDGEMENTS

I would like to express my gratitude to the members of my dissertation committee which is a compliment for me.

Acknowledgments

The authors thank Tina Bernthaler, Sandra Brahimi, Caroline Ohana, Gabriele Stefanzl, Verena Suppan, and Daniela Berger for excellent technical assistance.

This work was supported in part by a grant from Fondation de France and in part by the Austrian Science Fund (FWF), SFB project F4611 and F4704-B20. Ghaith Wedeh is a fellow of the Marie Curie Eurocancer Stemcell (CSC) Training Network.

ACKNOWLEDGEMENTS

We thank Daniela Berger and Gabriele Stefanzl for excellent technical assistance, and Günther Hofbauer and Andreas Spittler (both at the Cell Sorting Core Unit of the Medical University of Vienna) for excellent technical support and advice. This study was supported by Austrian Science Fund, SFB Grant Nos. F4611, F4704-B20 and F4710-B20.

Acknowledgments A.A. was supported by a fellowship from RII Pasteur, University of Paris XI and CHB association. CG was supported by a grant from ANRS. This work was supported by a grant from Association pour la Recherche sur le Cancer (ARC/SUBV/ CKLQ6) to AGD. We thank all the members of the INSERM U785 for their helpful discussions. We thank A. Jalil, E. Perret from the imagery services at IGR (Villejuif) and Institut Pasteur (Paris), and Dr. Di Liu from University of Alabama at Birmingham.

Authorship

Contribution: M.A. and P.V. designed the experiments; R.S., G.W., H.H., S.C.-R., I.S., K.B., E.H., S.B., S.J., C.B., E.C., F.S., and P.B. performed the experiments; M.A., M.L.-T., F.N.-K., H.M.-B., M.W., T.R., and P.V. analyzed the data; P.D. and V.D. contributed essential reagents; M.A. and P.V. wrote the paper; and all authors approved this manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Michel Arock, Laboratoire de Biologie et Pharmacologie Appliquée (LBPA), Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR) 8113, Ecole Normale Supérieure de Cachan, 61 Ave du Président Wilson,94235 Cachan Cedex, France; e-mail: arock@ens-cachan.fr.

Table S2: Analysis by flow cytometry of several surface markers expressed by ROSA cell lines. The expression of selected cell surface antigens was analyzed by direct immunofluorescence using FACSCalibur and Aria flow cytometer after cell labeling by specific monoclonal anti-human antibodies directed against the specified CD. Intensity of the labeling for each antigen is expressed as Mean Fluorescence Intensity ratio (MFI test mAb/MFI control mAb) MFI ratio < 1.19:-, MFI ratio 1.2 -2.9:+/-, MFI ratio 3-9.9: +, MFI ratio 10-99.9: ++, MFI ratio >100:+++. N.t.: not tested.

ROSA KIT A: primed ROSA KIT D816V cells were stimulated with various concentrations of anti-human anti-IgE antibody at 37°C for 30 min. Thereafter, cells were centrifuged at 4°C, and the supernatants and cell pellets were analyzed for histamine content using a specific radioimmunoassay kit. Results of histamine release are expressed as percent of total histamine and represent the mean ± SD of three independent experiments. B and C: percentage of histamine release from BM derived mononuclear Supplemental S3; ASM/MCL, n=3, Patients N° 1, 4 and 9 of the Table S3) and from normal/reactive bone marrow (n=1) were incubated with anti-IgE mAb (10 µg/ml), recombinant human C5a (100 nM) or control buffer at 37°C for 90 minutes. S1.

Supplementary Table S3

Sequences of miR-E based BRD4-and control shRNAs used for knockdown studies in human mast cell lines*

Abbreviations: shRNA, short hairpin ribonucleic acid; T, thymine; G, guanine; A, adenine; C, cytosine; BRD4, bromodomain-containing protein 4.

*Shown are 97mer oligos used for standard cloning of miR-E based shRNAs. Cloning of miR-E based shRNAs was peformed as described in Fellmann et al. 9

Supplementary Table S4 Sequences of oligonucleotide primers used for qPCR --------------------------------------------------------------------------------------------------------Gene Sequence - ------------------------------------------------------------------------------------------------------ -------------------------------------------------------------------------------------------------------Abbreviations: qPCR, quantitative polymerase chain reaction; BRD4, bromodomaincontaining protein 4; fwd, forward; rev, reverse; MYC, myelocytomatosis oncogene MCL-1, myeloid cell leukemia sequence 1; BCL-XL, B-cell lymphoma-extra-large; BCL-2, B-cell leukemia/lymphoma 2; ABL, Abelson murine leukemia virus oncogene homolog 1.

Supplementary Figures