Allan Beke

Lucie Laplane

Julie Riviere

Qin Yang

Miguel Torres- Martin

Thibault Dayris

Philippe Rameau

Veronique Saada

Chrystèle Bilhou-Nabera

Ana Hurtado

Larissa Lordier

William Vainchenker

Maria E Figueroa

Nathalie Droin

Eric Solary
email: eric.solary@gustaveroussy.fr

Multilayer intraclonal heterogeneity in chronic myelomonocytic leukemia

T he functional diversity of cells that compose myeloid malignancies, i.e., the respective roles of genetic and epigenetic heterogeneity in this diversity, remains poorly understood. This question is addressed in chronic myelomonocytic leukemia, a myeloid neoplasm in which clinical diversity contrasts with limited genetic heterogeneity. To generate induced pluripotent stem cell clones, we reprogrammed CD34 + cells collected from a patient with a chronic myelomonocytic leukemia in which whole exome sequencing of peripheral blood monocyte DNA had identified 12 gene mutations, including a mutation in KDM6A and two heterozygous mutations in TET2 in the founding clone and a secondary KRAS(G12D) mutation. CD34 + cells from an age-matched healthy donor were also reprogrammed. We captured a part of the genetic heterogeneity observed in the patient, i.e. we analyzed five clones with two genetic backgrounds, without and with the KRAS(G12D) mutation. Hematopoietic differentiation of these clones recapitulated the main features of the patient's disease, including overproduction of granulomonocytes and dysmegakaryopoiesis. These analyses also disclosed significant discrepancies in the behavior of hematopoietic cells derived from induced pluripotent stem cell clones with similar genetic background, correlating with limited epigenetic changes. These analyses suggest that, beyond the coding mutations, several levels of intraclonal heterogeneity may participate in the yet unexplained clinical heterogeneity of the disease.

Introduction

Intratumoral heterogeneity is a major tenet of cancer biology. A tumor clone emerges from a single cell that has acquired one or several somatic mutations. Additional driver events that occur in individual daughter cells generate tumor subclones, each being endowed with specific functional properties and fitness. [START_REF] Mcgranahan | Clonal heterogeneity and tumor evolution: past, present, and the future[END_REF] This intraclonal genetic heterogeneity may not explain all the functional heterogeneity among individual cells within a tumor clone. Epigenetic variation also contributes to the heterogeneity of cells that form a tumor. [START_REF] Landau | Locally disordered methylation forms the basis of intratumor methylome variation in chronic lymphocytic leukemia[END_REF][START_REF] Li | Distinct evolution and dynamics of epigenetic and genetic heterogeneity in acute myeloid leukemia[END_REF] Chronic myelomonocytic leukemia (CMML) is a neoplastic disease whose limited genetic heterogeneity contrasts with its clinical diversity. [START_REF] Ball | When clinical heterogeneity exceeds genetic heterogeneity: thinking outside the genomic box in chronic myelomonocytic leukemia[END_REF] CMML is defined by a persistent clonal monocytosis, with or without dysplasia. [START_REF] Deininger | Turning the tide in myelodysplastic/myeloproliferative neoplasms[END_REF] The mutational landscape contains a small number of somatic mutations in DNA methylation, histone modifier, splicing factor and signaling genes. [START_REF] Merlevede | Mutation allele burden remains unchanged in chronic myelomonocytic leukaemia responding to hypomethylating agents[END_REF] Mapping of CMML clonal architecture identified early clonal dominance, intratumor heterogeneity in the hematopoietic stem and progenitor cell compartment in which mutations accumulate mostly linearly, and growth advantage to the most mutated cells. [START_REF] Itzykson | Clonal architecture of chronic myelomonocytic leukemias[END_REF] Hypomethylating agents, which are commonly used in severe dysplastic forms of the disease, can restore a balanced hematopoiesis. [START_REF] Solary | How I treat chronic myelomonocytic leukemia[END_REF] The response to these drugs, which correlates with DNA demethylation, can occur in the absence of any decrease in mutation allele burden measured in circulating monocytes, [START_REF] Merlevede | Mutation allele burden remains unchanged in chronic myelomonocytic leukaemia responding to hypomethylating agents[END_REF] arguing for a role of epigenetic alterations in disease expression and outcome. [START_REF] Meldi | Specific molecular signatures predict decitabine response in chronic myelomonocytic leukemia[END_REF] One of the main limitations in studying CMML pathophysiology is the lack of appropriate models, either patient-derived cell lines or genetically modified animals, which faithfully reproduce disease features. Currently, the best available CMML preclinical model is xenotransplantation of CMML cells in immunocompromised mice, especially those with transgenic expression of human cytokines including granulocyte-macrophage colony-stimulating factor. [START_REF] Zhang | Engraftment of chronic myelomonocytic leukemia cells in immunocompromised mice supports disease dependency on cytokines[END_REF][START_REF] Yoshimi | Robust patient-derived xenografts of MDS/MPN overlap syndromes capture the unique characteristics of CMML and JMML[END_REF] The modeling of myeloid malignancies by generating patient-derived induced pluripotent stem cells (iPSC) recently appeared as another opportunity to model these diseases and, although challenging, capture their genetic heterogeneity. [START_REF] Chao | Human AML-iPSCs reacquire leukemic properties after differentiation and model clonal variation of disease[END_REF][START_REF] Kotini | Stagespecific human induced pluripotent stem cells map the progression of myeloid transformation to transplantable leukemia[END_REF][START_REF] Taoka | Using patient-derived iPSCs to develop humanized mouse models for chronic myelomonocytic leukemia and therapeutic drug identification, including liposomal clodronate[END_REF][START_REF] Papapetrou | Modeling myeloid malignancies with patient-derived iPSCs[END_REF] In CMML, we previously demonstrated that intraclonal heterogeneity was rarely detected in mature cells of the clone as a consequence of a growth advantage to most mutated cells with differentiation but was preserved in stem and progenitor cells. [START_REF] Merlevede | Mutation allele burden remains unchanged in chronic myelomonocytic leukaemia responding to hypomethylating agents[END_REF][START_REF] Itzykson | Clonal architecture of chronic myelomonocytic leukemias[END_REF] Therefore, we sorted CD34 + cells from a CMML patient and reprogrammed these cells to capture some intraclonal genetic heterogeneity and characterize hematopoiesis derived from genetically close but distinct iPSC.

Methods

Generation, characterization and maintenance of induced pluripotent stem cells

CD34 + cells collected from a healthy donor and a CMML patient, with informed consent and approval of the Ethics Committee (DC-2014-209), were infected with non-integrated Sendai virus encoding Klf4, Oct4, Sox2 and c-Myc to generate iPSC. An additional iPSC (Co6) was kindly provided by Dr Weiss. [START_REF] Mills | Clonal genetic and hematopoietic heterogeneity among human induced pluripotent stem cell lines[END_REF] iPSC were passaged once a week to yield a cell suspension of small colonies (3-10 cells). Intracellular and extracellular pluripotency markers were detected by flow cytometry and teratoma formation was evaluated by intramuscular injection of iPSC into NOD/SCID/IL2rγ -/-mice. Karyotyping and comparative genomic hybridization were performed. The procedures are detailed in the Online Supplementary Material.

Hematopoietic cell differentiation

A two-dimensional monolayer system was used to differentiate iPSC into CD34 + CD43 + hematopoietic progenitor cells (HPC). Clonogenic assays were performed by mixing HPC in serum-free medium with MethoCult H4434 classic (Stem Cell Technologies, Grenoble, France) before plating the cell suspension in 35-mm dishes. Colonies were scored after 14 days and analyzed on a BD LSRFortessa X-20. HPC mixed with serum-free fibrin clots were seeded for 10 days in the presence of thrombopoietin and stem cell factor before measuring colony-forming unit-megakaryocyte (CFU-Mk) colonies. HPC were also suspended in serum-free liquid medium with growth factors for 10 days before flow analysis of cell surface markers and May-Grünwald-Giemsa staining of cytospins. More details are provided in the Online Supplementary Material.

Flow cytometry and cell sorting

The antibodies used are listed in Online Supplementary Table S1. Cells were analyzed using a BD LSRFortessa™ X-20 and Kaluza analysis software. HPC, monocytes and megakaryocytes were sorted on a BD Influx™ Cell sorter. Details are provided in the Online Supplementary Material.

Whole exome sequencing

We collected genomic DNA from sorted monocytes and CD3 + T cells and iPSC to perform whole exome sequencing. Raw reads were aligned to the reference human genome hg19 (Genome Reference Consortium GRCh37) using BWA 0.5.9 (Burrows-Wheeler Aligner) backtrack algorithm with default parameters. A mutation was reported as present if the variant allele frequency was ≥4%. More details are provided in the Online Supplementary Material.

Genome-wide DNA methylation detected by enhanced reduced representation bisulfite sequencing

High-molecular weight DNA was sequenced on a HiSeq3000 Illumina sequencer and 50 bp reads were aligned against a bisulfite-converted human genome (hg19). Differentially methylated regions (DMR) identified an absolute methylation difference ≥40% with a false discovery rate <5% and were annotated using the ChIPenrich R package, [START_REF] Welch | ChIP-Enrich: gene set enrichment testing for ChIP-seq data[END_REF] which was also used for gene ontology and pathway analysis. For correspondence analysis and hierarchical clustering, tiles with the highest standard deviation (SD >0.03) were used. More details are provided in the Online Supplementary Material.

Statistical analysis

Statistical analysis was performed with GraphPad Prism software, using an unpaired t test and Mann-Whitney test, depending on distribution, similarity of variance, and sample number. The Kruskal-Wallis test was used for multiple comparisons. haematologica | 2020; 105(1) those from control iPSC-derived CFU-M (Figure 3C). A similar defect in the generation of CD14 + cells expressing both CD16 and CD163 was detected when iPSC-derived hematopoietic cells were induced to differentiate in liquid culture (Figure 3C). Since the patient demonstrated megakaryocytic hyperplasia and dysplasia, we performed coagulum assays in the presence of stem cell factor and thrombopoietin to analyze the generation of megakaryocytes and platelets (Figure 3D). All iPSC generated a similar number of colonies (Figure 3E) but those generated by CMML iPSC were much larger (Figure 3F-H). We observed a decrease in the fraction of CD42 + cells among CD41 + cells (Figure 3I) and the fraction of CMML iPSC-derived megakaryocytes that produced platelets was decreased (Figure 3J,K).

CD34 + CD43 + cells generated from iPSC were also cultured in liquid medium in the presence of stem cell factor, interleukin-3, interleukin-6, erythropoietin, granulocytemacrophage colony-stimulating factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, granulocyte colonystimulating factor and monocyte colony-stimulating factor for 10 days (Figure 4A). The quantity of cells generated by each clone and the number of viable cells after 10 days were not significantly different, except for clone A1 that demonstrated more dead cells (Figure 4B,C). Multiparameter flow cytometry analysis was used to measure each cell population obtained in culture (Online Supplementary Figure S2B). CMML iPSC generated a majority (~40%) of CD14 + cells (Figure 4D,E). Although they both had a KRAS(G12D) mutation, the A5 clone generated more CD14 + cells (~80%) than the A3 clone. In fact, monocyte production by the A3 clone was not significantly different from that of KRAS wildtype clones (Figure 4E, insert). Compared to control clones, KRAS(G12D) CMML iPSC generated fewer CD33 + , CD14 -, CD41 -, CD235a - cells (Figure 4F) and KRAS wildtype CMML clones generated fewer CD123 + , CD14 -, CD41 -, CD235a -cells (Figure 4G). The generation of CD235a + erythroid cells was more heterogeneous and higher in KRAS wildtype compared to KRAS(G12D) CMML clones (Figure 4H). The generation of CD41 + cells was not significantly different between control and patient-derived iPSC (Figure 4I). These liquid cultures also revealed the defective differentiation of monocytes into macrophages (Figure 3C) and the defective generation of platelets (Figure 3D).

As expected, patient-derived clones showed a bias in their differentiation towards monocyte production. However, hematopoietic differentiation of CMML iPSC also demonstrated significant intraclonal heterogeneity that could not be explained by the sole genetic alterations detected in coding regions. The A1, A2 and A4 clones, which have the same mutations in coding regions, showed heterogeneous differentiation into CD235a + and CD14 + cells whereas A3 and A5, which are KRAS-mutated clones, showed a marked difference in their monocytic differentiation in liquid culture. A summary of this clonal heterogeneity is shown in Figure 4J.

While the viability of cells generated by control iPSC was high in all but one experiment, the viability of CMML iPSC, especially KRAS wildtype CMML iPSC, was much more heterogeneous than that of control clones, suggesting a higher sensitivity to small variations in culture conditions (Figure 5A). Of note, the number of generated cells (indicated by the diameter of the circles in Figure 5A) could remain high in cultures in which the cell death rate was elevated. In contrast, a decrease in cell viability was associated with an increase in the fraction of CD235a + cells and a decrease in the fraction of CD14 + cells generated by KRAS wildtype CMML iPSC (Figure 5A). By eliminating this culture condition-related variability in cell production, using a cut-off of 90% viability, we observed a much more robust trend in the differentiation of A1, A2 and A4 clones (Online Supplementary Figure S4). In contrast, even with this cut-off on viability, the A5 clone consistently produced more CD14 + cells than the A3 clone (Figure 5B).

A. Beke et al.

Epigenetic heterogeneity among induced pluripotent stem cell clones from the patient with chronic myelomonocytic leukemia

Epigenetic intraclonal heterogeneity has been reported in hematologic malignancies [START_REF] Landau | Locally disordered methylation forms the basis of intratumor methylome variation in chronic lymphocytic leukemia[END_REF][START_REF] Oakes | Evolution of DNA methylation is linked to genetic aberrations in chronic lymphocytic leukemia[END_REF] as well as in solid tumors, [START_REF] Mazor | Intratumoral heterogeneity of the epigenome[END_REF] and co-dependency between epigenetic and genetic evolution has been questioned, e.g., in large B-cell lymphoma [START_REF] Pan | Epigenomic evolution in diffuse large B-cell lymphomas[END_REF] and acute myeloid leukemia. [START_REF] Li | Distinct evolution and dynamics of epigenetic and genetic heterogeneity in acute myeloid leukemia[END_REF] We performed DNA methylation analysis on CD34 + CD43 + cells generated from iPSC to investigate their epigenetic state. Unsupervised analysis by correspondence analysis separated control-from CMML-derived cells. The latter showed a much greater diversity (Figure 6A). We then used a β binomial model implemented in methylSig [START_REF] Park | MethylSig: a whole genome DNA methyla-tion analysis pipeline[END_REF] to perform a supervised analysis of differentially methylated regions (DMR). We identified 5,651 hypermethylated DMR in CMML-derived cells compared to controls. In contrast, only 874 DMR were identified as more methylated in control iPSC-derived cells (Figure 6B,C). Genomic annotation revealed that, in CMML samples, DMR were depleted at promoter regions and CpG islands while being enriched at gene body and intergenic enhancers (P<0.001 in all cases) (Figure 6D). DMR detected at CpG islands, gene body enhancers and intergenic enhancers were significantly more often hypermethylated regions (P<0.001) (Online Supplementary Figure S5A). In accordance with changes in DNA methylation affecting enhancers, we observed significant enrichment of DMR within enhancers [START_REF] Akalin | Base-pair resolution DNA methylation sequencing reveals profoundly divergent epigenetic landscapes in acute myeloid leukemia[END_REF] compared to background (36.8% at total DMR vs. 15.2% background, P-value <2.2x10 -16 ). Motif enrichment analysis suggested that the main sequences targeted by DMR were motifs recognized by transcription factors of the ETS family (Online Supplementary Figure S5B). Gene ontology analysis of biological processes of DMR hypermethylated in CMMLderived cells showed enrichment of differentiation and hematopoietic development categories (Figure 6E), suggesting that DNA methylation differences between CMML-and control-derived cells may capture an epigenetic memory related to the biology of the disease, still present after having been reprogrammed. Finally, focusing on CMML iPSC-derived hematopoietic cells, unsupervised hierarchical clustering based on their DNA methylation profiles revealed high concordance with their phylogenetic background (Figure 6F).

In order to correlate changes in DNA methylation with those seen at the expression level, we first looked at the expression status of genes closest to DMR using a nearest gene annotation approach. Using this approach, only 114 genes showed overlapping changes in expression and DNA methylation. However, since focusing on DMRnearest gene correlations may not correctly capture the three-dimensional nature of gene regulation, we next explored the role of DMR within specific topologically associated domains (TAD) identified using publicly available coordinates. We thus localized each DMR into a given TAD. For every gene within a TAD, we correlated gene expression and methylation levels across the samples using Pearson correlation. With this method, of 196 DMR identified in 66 TAD, we detected changes in gene expression in 72 genes (Online Supplementary Table S2).

To further explore the epigenetic differences between subclones of the original disease and the potential contribution of specific mutations to the epigenetic programming, we also compared DNA methylation profiles in KRAS wildtype (A1, A2, A4) and KRAS(G12D) (A3, A5) clones (Figure 7). Globally, clones that had acquired KRAS(G12D) seemed to be relatively hypomethylated compared to the KRAS wildtype clones (Figure 7A,B). Acquisition of KRAS(G12D) correlated with a new repartition of DMR, including a significant decrease in methylation at intronic regions (P<0.001), CpG shores (P<0.001), gene body (P<2. 2 10 -16 ) and intergenic enhancers (P<0.001) and a significant increase in methylation at promoter regions (P<0.001) and CpG islands (P<0.001) (Figure 7C). Importantly, using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, KRAS(G12D) acquisition was shown to induce significant changes in Wnt and Hedgehog signaling pathways, which may indicate a differential role for these pathways in the malignant transformation of the different clones (Figure 7D).

Discussion

The reprogramming of CD34 + cells from a CMML patient generated iPSC whose hematopoietic differentiation recapitulated the main features of the disease while demonstrating functional heterogeneity between clones (see graphical abstract in Figure 8).

Discrepancies between the functional properties of clones sharing a similar genetic background have been reported in organoids derived from colorectal cancer cells. [START_REF] Roerink | Intratumour diversification in colorectal cancer at the single-cell level[END_REF] We cannot exclude an effect of recurrent somatic mutations in non-coding regions, as described in some solid tumors, 25 but such events have not been identified yet in CMML cells. [START_REF] Merlevede | Mutation allele burden remains unchanged in chronic myelomonocytic leukaemia responding to hypomethylating agents[END_REF] A role for cell reprogramming [START_REF] Cahan | Origins and implications of pluripotent stem cell variability and heterogeneity[END_REF][START_REF] Nishizawa | Epigenetic variation between human induced pluripotent stem cell lines is an indi-cator of differentiation capacity[END_REF] in this heterogeneity is also unlikely as control iPSC established independently from two healthy donors had a similar and reproducible behavior, in contrast to the differences observed between genetically identical CMML iPSC (Figure 4J). In addition differences sometimes observed between individual clones derived from the same genotype were shown to pre-exist in the tissue of origin rather than being induced by reprogramming. [START_REF] Kwon | iPSCs and fibroblast subclones from the same fibroblast population contain comparable levels of sequence variations[END_REF][START_REF] Yoshizato | Somatic mutations and clonal hematopoiesis in aplastic anemia[END_REF] The distinct behavior of CMML-derived iPSC could also reflect intrinsic heterogeneity in CD34 + cell-priming for differentiation. [START_REF] Naik | Diverse and heritable lineage imprinting of early haematopoietic progenitors[END_REF][START_REF] Velten | Human haematopoietic stem cell lineage commitment is a continuous process[END_REF] If such an intrinsic heterogeneity of CD34 + cell-priming is a general property of CD34 + cells, discrepancies should also have been observed between control iPSC. An alternative explanation is that heterogeneous priming is a specific feature of CMML progenitor cells, which could be related to intraclonal epigenetic het-erogeneity. Our epigenetic analyses indicate that, in patient-derived clones, the global pattern of DNA methylation correlates with genetic alterations. However, limited differences in their methylation profiles could possibly account for their functional heterogeneity.

iPSC allow the combined investigation of all levels of intratumoral heterogeneity, from genetic, to epigenetic, to phenotypic and functional properties associated with the disease, offering unique opportunities to study diseases in which functional heterogeneity exceeds genetic heterogeneity, such as CMML. [START_REF] Ball | When clinical heterogeneity exceeds genetic heterogeneity: thinking outside the genomic box in chronic myelomonocytic leukemia[END_REF] These benefits do, however, come at a cost. As recently reviewed, [START_REF] Papapetrou | Modeling myeloid malignancies with patient-derived iPSCs[END_REF] the derivation of iPSC from patients with a myeloid malignancy to model their disease has to face the relative refractoriness of malignant progenitors to reprogramming, which is in part related to their genetic background and could preclude the capture of intraclonal heterogeneity as some subclones may be reprogrammed more easily than others. The dysplastic nature of these cells, which often correlates with an increased sensitivity to apoptosis, is another challenge to overcome. Lastly, in the patients, the heterogeneous behavior of individual cells may be further influenced by clonal interference and non-cell-autonomous factors, [START_REF] Marusyk | Non-cellautonomous driving of tumour growth supports sub-clonal heterogeneity[END_REF] contributing to the diversity of CMML phenotypic traits, and accounting for the current failure of treatments aiming at eradicating the malignant clone.

Figure 1 .BFigure 2 .

 12 Figure 1. Generation and genetic characterization of chronic myelomonocytic leukemia-and control-induced pluripotent stem cells. (A) CD34 + cells from a patient with chronic myelomonocytic leukemia and an age-matched healthy donor were reprogrammed through infection with Sendai virus encoding the transcription factors Sox2, Klf4, Oct4, and c-Myc (SKOM) before characterization and selection of indicated induced-pluripotent stem clones (iPSC). An additional control iPSC (Co6) was kindly provided by Dr. Weiss. (B) Whole exome sequencing of DNA collected from sorted peripheral blood monocytes (in black) and from five iPSC selected from patient A (each color indicates a specific clone), showing the detection of two genotypes with nine and 12 somatic variants, respectively.

Figure 3 .

 3 Figure 3. Defective maturation of chronic myelomonocytic leukemia induced pluripotent stem cell-derived hematopoietic cells into macrophages and platelet-forming megakaryocytes. (A) Representative morphology of colony-forming unit -macrophage (CFU-M) generated by hematopoietic cells derived from control (Co6) and chronic myelomonocytic leukemia (CMML) (clone A5) induced pluripotent stem cells (iPSC) cultured in methylcellulose as described in Figure 2A. Light microscopy visualization; scale bar indicates magnification. The rectangle is a zoom on the fibroblast-like shape of macrophages in the Co6 culture, which was not observed in A5 colonies. (B) Fraction of cells with a fibroblast-like shape in CFU-M generated from hematopoietic cells derived from four control and five CMML iPSC; unpaired t test. (C) Flow cytometry analysis of cells expressing both CD16 and CD163 in CD14 + cells collected from colonies generated by the five control iPSC-and the five CMML iPSC-derived hematopoietic cells plated in methylcellulose or in liquid medium, Kruskal-Wallis test. (D) Control iPSC and CMML iPSC-derived CD34 + CD43 + hematopoietic cells were sorted and plated in coagulum for 10 days in the presence of 50 ng/mL stem cell factor (SCF) and 10 ng/mL thrombopoietin (TPO) to generate colony-forming unitmegakaryocyte (CFU-Mk). (E) Total number of colonies generated by plating 1,500 hematopoietic cells derived from the indicated iPSC. (F) Representative experiments showing the differential morphology of CFU-Mk generated by plating healthy donor (Co1 clone) or CMML (A2 clone) iPSC-derived hematopoietic cells (scale bar, 100 mm). (G) Fractions of large colonies, >50 cells, among the total number of colonies shown in panel E; Kruskal-Wallis test. (H) Fractions of intermediate colonies, 10-50 cells, among the total number of colonies shown in panel E; Kruskal-Wallis test. (I) Fractions of CD41 + megakaryocytes generated in liquid culture with all cytokines for 10 days and expressing the cell surface marker CD42, as detected by flow cytometry; Kruskal-Wallis test. (J) Upper panels show May-Grünwald-Giemsa-stained cytospins of CD41 + cells generated in liquid culture, 5 days after cell sorting, with a normal (Co3) or dysplastic (A3) morphology. Lower panels: representative images of plateletproducing megakaryocytes generated by hematopoietic cells derived from indicated clones; scale bars, 50 mm. (K) Fractions of platelet-producing megakaryocytes in CD41 + cells sorted from liquid culture of CD34 + CD43 + cells with all cytokines for 10 days, then cultured for 6 days with SCF and TPO; Kruskal-Wallis test. Mk: megakaryocytes. Colors as in Figure 1. Bars: mean ± standard deviation. *P<0.05; **P<0.01; ****P<0.0001.

Figure 4 .

 4 Figure 4. Functional heterogeneity of the patient's induced pluripotent stem cell-derived hematopoietic cells. (A) Control and chronic myelomonocytic leukemia (CMML) induced pluripotent stem cell (iPSC)-derived CD34 + CD43 + cells were cultured in liquid medium for 10 days in the presence of 50 ng/mL stem cell factor (SCF), 10 ng/mL Fms-like tyrosine kinase 3 ligand (FLT3L), 10 ng/mL interleukin-3 (IL-3), 10 ng/mL interleukin-6 (IL-6), 50 ng/mL thrombopoietin (TPO), 1 U/mL erythropoietin (EPO), 10 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 10 ng/mL granulocyte colony-stimulating factor (G-CSF), and 10 ng/mL monocyte colony-stimulating factor (M-CSF). (B) Total number of hematopoietic cells generated by 5,000 CD34 + CD43 + cells cultured in liquid medium for 10 days. (C). Fractions of viable, DAPI-negative cells measured on day 10; Kruskal-Wallis test. (D) Representative flow cytometry analysis of CD33 + CD14 + cells generated in liquid culture by the Co3 and A2 clones. (E-I) Fractions of CD33 + CD14 + cells (E and insert), CD33 + CD14 -CD41 -cells (F), CD123 + CD33 -CD235a -CD14 -CD41 -cells (G), CD235a + CD14 -CD41 -cells (H), and CD41 + CD14 -cells (I) generated in liquid culture by the indicated clones; Kruskal-Wallis test. (J) Radar representation of the differentiation potential of CD34 + CD43 + hematopoietic cells derived from control iPSC (green), KRAS wildtype CMML iPSC (blue) and KRAS(G12D) CMML iPSC (red). Bars: mean ± standard deviation. *P<0.05; ***P<0,001 ****P<0.0001

Figure 5 .BFigure 6 .

 56 Figure 5. Impact of viability on differentiation pattern analysis. (A) Percentages of DAPI-negative viable cells (x axis) and numbers of hematopoietic cells generated (dots size) are plotted against the fraction of CD14 + and CD235a + cells generated in liquid culture. The vertical hatched line is an arbitrary cut-off value established at 90% viable cells; the horizontal hatched line emphasizes the discrepancies between samples with <90% viable cells and the others. (B) Fractions of CD33 + CD14 + cells for the indicated clones after removing experiments in which cell viability was below 90%; Kruskal-Wallis test. *P<0.05.

Figure 7 .

 7 Figure 7. Differential methylation profile of KRAS-wildtype and KRAS-mutated chronic myelomonocytic leukemia clones. (A) Hypermethylated regions detected by enhanced reduced representation bisulfite sequencing in KRAS wildtype (A1, A2, A4) compared to KRAS mutated (A3, A5) chronic myelomonocytic leukemia (CMML)derived induced pluripotent stem cells (iPSC). Arrows, percentage of methylation required to be considered as a differentially methylated region (DMR). Red dots, tiles that fulfill the criteria [absolute methylation difference ≥40% and false discovery rate (FDR) <5%]. (B) Heatmap of DMR based on the Euclidean distance matrix. Red, hypomethylation; blue, hypermethylation; (C) Repartition of DMR that are hypo-or hyper-methylated in KRAS wildtype compared to KRAS(G12D) clones. Asterisk, significance according to the binomial test (P<0.001). (D) Gene ontology of DMR methylated on KRAS(G12D) clones. Bar chart: processes related to hematopoiesis with a FDR <0.1. X-axis, -log 10 of the FDR.

Figure 8 .

 8 Figure 8. Graphical summary of intratumor heterogeneity layers detected by analysis of five patient-derived induced pluripotent stem cells.

Acknowledgments

The authors would like to thank Dr Weiss for kindly providing a control iPSC clone. This work was supported by grants from the Ligue Nationale Contre le Cancer (équipe labelisée), Institut National du Cancer (INCA_8073; PRT-K 16-047), Fondation

Data availability

Accession numbers for enhanced reduced representation bisulfite sequencing, whole exome sequencing and RNA sequencing data are GSE114115, E-MTAB-7917 and E-MTAB-7850, respectively.

Results

Reprogramming of CD34 + cells from a patient with chronic monomyelocytic leukemia captures a part of the disease's genetic heterogeneity

We reprogrammed CD34 + cells collected from a CMML patient whose monocyte DNA whole exome sequencing had identified 12 mutations, including two mutations in TET2 (S1691fs and R1516X) and heterozygous mutations in KRAS(G12D) and KDM6A(R61X). The clinical and biological features of the patient's disease are depicted in the Online Supplementary Material. Reprogramming of CD34 + cells collected from an age-matched healthy donor generated control clones (Figure 1A,B). We selected nine clones (5 from the patient; 4 from the healthy donor) demonstrating pluripotency features, including morphology (Online Supplementary Figure S1A), expression of markers (Online Supplementary Figure S1B) and formation of teratomas in immunodeficient mice (Online Supplementary Figure S1C), without reprogramming-induced cytogenetic abnormalities (Online Supplementary Figure S1D). We also used an additional, independently generated control clone (Figure 1A). [START_REF] Mills | Clonal genetic and hematopoietic heterogeneity among human induced pluripotent stem cell lines[END_REF] Whole exome sequencing and Sanger sequencing of patient-derived iPSC indicated that we had captured a part of the genetic heterogeneity of her leukemic clone, i.e., three clones (A1, A2, A4) recapitulated the founding clone while the other two (A3, A5) were reprogrammed from a KRAS(G12D) subclone (Figure 1A, B and Online Supplementary Figure S1E). In contrast with other studies, [START_REF] Chang | Dissecting the contributions of cooperating gene mutations to cancer phenotypes and drug responses with patient-derived iPSCs[END_REF] we did not reprogram any wildtype CD34 + cells, probably due to the early clonal dominance that characterizes CMML clonal architecture, with very few residual wildtype cells in the stem cell compartment. 7

Hematopoietic cells derived from chronic myelomonocytic leukemia induced pluripotent stem cells recapitulate the disease features

iPSC were induced to differentiate into CD34 + CD43 + hematopoietic progenitors (Online Supplementary Figure S2A), which were plated for 10 days in methylcellulose in the presence of stem cell factor, interleukin-3, erythropoietin, and granulocyte-macrophage colony-stimulating factor (Figure 2A). The total numbers of colonies generated by healthy donor-and CMML iPSC-derived hematopoietic progenitors were similar (Figure 2B and Online Supplementary Figure S3A). The fraction of clusters (colonies <50 cells) generated by KRAS wildtype CMML iPSC was significantly higher than that generated by KRAS(G12D)-mutated CMML iPSC and control clones (Figure 2C and Online Supplementary Figure S3B,C). KRAS(G12D) clones produced larger granulocytemacrophage (CFU-GM) and macrophage (CFU-M) colonies (Figure 2D) as well as a higher proportion of CFU-M colonies (Figure 2E). Compared to control clones, CMML-derived clones generated fewer granulocytic and multipotent progenitor colonies (Figure 2E, F and Online Supplementary Figure S3D) and more granulocytemacrophage colonies (Figure 2E) whereas the proportions of erythroid colonies were not significantly different (Figure 2F and Online Supplementary Figure S3D). Colonies derived from CMML iPSC also demonstrated an increased fraction of CD14 + cells (Figure 2G) at the expense of CD33 + , CD123 + , CD235a + or CD41 + populations (Online Supplementary Figure S3E, summary in Figure 2H).

Cells that formed CFU-M generated by CMML iPSC did not show the typical, fibroblast-like shape of macrophages generated by healthy donor-derived iPSC (Figure 3A,B


Assessment of mesh adaptation algorithms for LES and DES simulation of detached flows B. Sauvage * , † , F. Miralles ¶ , S. Wornom ¶ , B. Koobus ¶ , F. Alauzet and A. Dervieux †, ‡ † Université Côte d'Azur, INRIA, Sophia-Antipolis, France, bastien.sauvage@inria.fr ¶ IMAG, Univ. Montpellier, CNRS, France, florian.miralles@umontpellier.fr, bruno.koobus@umontpellier.fr, stephen.wornom@inria.fr INRIA Saclay Ile-de-France, Palaiseau, France, frederic.alauzet@inria.fr ‡ Lemma, Biot, France, alain.dervieux@inria.fr

ABSTRACT

The proposed communication, discusses the adaptation of a transient fixed point adaptation algorithm to the computation of detached flows with LES of VMS type [START_REF] Moussaed | Simulation of the flow past a circular cylinder in the supercritical regime by blending RANS and variational-multiscale LES models[END_REF][START_REF] Itam | Combining a DDES model with a dynamic variational multiscale formulation[END_REF] and DDES (Delayed Detached Eddy Simulation) models.

The Transient Fixed Point adaptation algorithm relies on a representation of the mesh by a Riemannian metric [START_REF] Dervieux | Mesh Adaptation for Computational Fluid Dynamics 1 and 2[END_REF]. We seek for an optimal metric according to a criterion prescribed by the user. In the Transient Fixed Point, the metric, and then the mesh, is frozen for the timesteps of a subinterval of the time interval. Several strategies are possible in the choice of the subinterval size, from a rather small interval allowing to follow many details to an interval of one or several main periods, and will be discussed during the presentation. Iterative remeshings are applied in order to reach some fixed point, see [START_REF] Alauzet | 3D transient fixed point mesh adaptation for time-dependent problems: Application to CFD simulations[END_REF]. Refinement criteria involve Mach number fields for resolution boundary layers and pressure time derivatives in order to better capture acoustics.

Flows around cylinder with Reynolds number from 3900 to 1 million, airfoils with mean and high angle of attack and a Caradonna rotor will be considered for the evaluation, with comparison with experiments and calculations with taylored fixed meshes.


F Miralles

B Sauvage

S Wornom

Bruno Koobus

Alain Dervieux

Assessment of turbulence hybrid models with transition modeling for the simulation of massively separated flows

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Assessment of turbulence hybrid models with transition modeling for the simulation of massively separated flows F. Miralles * , ¶ , B. Sauvage † , S. Wornom ¶ , B. Koobus ¶ and A. Dervieux †, ‡ ¶ IMAG, Univ. Montpellier, CNRS, France, florian.miralles@umontpellier.fr, bruno.koobus@umontpellier.fr, stephen.wornom@inria.fr † Université Côte d'Azur, INRIA, Sophia-Antipolis, France, bastien.sauvage@inria.fr, alain.dervieux@inria.fr ‡ Lemma, Biot, France, alain.dervieux@inria.fr

ABSTRACT

In the proposed communication, several hybrid turbulence models, possibly equipped with transition modeling, are evaluated on the simulation of the flow around a circular cylinder in the supercritical regime and over an airfoil at incidence. The flow through the Caradonna-Tung rotor will complete the benchmarks considered in this study.

The first hybrid approach investigated in this work is the classical Delayed Detached Eddy Simulation (DDES) model. The two other hybrid models combine either a RANS model or the DDDES approach with a dynamic variational multiscale large eddy simulation (DVMS) model. A smooth blending function, which is based on the value of a blending parameter, is used for switching from RANS to DVMS in the RANS/DVMS strategy [START_REF] Moussaed | Simulation of the flow past a circular cylinder in the supercritical regime by blending RANS and variational-multiscale LES models[END_REF]. In the DDES/DVMS approach [START_REF] Itam | Combining a DDES model with a dynamic variational multiscale formulation[END_REF], the DVMS model is preferentially activated in the wake in order to more accurately predict this region of the flow thanks to the low dissipation introduced by this model. With the aim of improving the prediction of the targeted flows, a new kεγ transition model based on the model of Akhter [START_REF] Akhter | Numerical Simulation of Heat Transfer Coefficient on Turbine Blade using Intermittency Factor Equation[END_REF] is also developped and used in these hybrid approaches.

Results are compared to those of other RANS, LES and hybrid simulations in the literature and with experimental data, and highlight the overall good prediction capabilities of the proposed hybrid strategies for the simulation of such massively separated flows, with often a significant improvement when the transition model is activated.


Loïc Sauvée

Mantiaba Coulibaly

The Interorganizational Dynamics of Brand Alliances

The objective of the research is to put in evidence the interorganizational dynamics of brand alliances. More specifically the aim of the paper is to identify the business-to-business interactions within brand alliances through the governance adaptations that occur during a period of time. We show that these governance adaptations result from external (competitive pressure, value perception by consumers and customers) as well as internal forces (objectives and expectations of the partners, network positions, resources of the partners). Consequently, the level of stability in the long run of brand alliances can be linked to organizational factors. For our demonstration, we propose an analytical framework that combines IMP concepts with theoretical works on dynamics of strategic alliances. The methodology follows the case study approach, with an empirical application to two examples of brand alliances: A certification brand with a banana brand on the Fair Trade market and an association brand with a processed pork brand on the health food market

Introduction

The objective of the research is to put in evidence the interorganizational dynamics of brand alliances, defined as "a combination of two or more existing brands into a joint product or marketed together" [START_REF] Keller | Strategic Brand Management[END_REF]. More specifically, the aim of the paper is to identify the business-to-business interactions within brand alliances through the phenomenon of governance adaptations. We show that these governance adaptations result from external as well as internal forces. To do so, we propose an analytical framework that combines IMP concepts with theoretical works on dynamics of strategic alliances. In previous works [START_REF] Sauvée | Value Creation in Brand Alliances: A Network Approach[END_REF], we have shown that the value creation process is crucial to understand and analyze brand alliances from a network perspective. Indeed, the value creation process of brand alliances sends back the resources that are mobilized by the partners. Thus, the in-depth study of the network from where these resources are embedded, is an initial step for the research. But, following numerous authors, we suggest that a complementary analysis of dynamic processes is necessary. Indeed, the value creation process in brand alliances is not given in itself. It is a dynamic building, affected by external (competitive pressure, value perception by consumers and customers) as well as internal forces (objectives and expectations of the partners, network positions, resources of the partners). In consequence, the partners involved in the brand alliance will constantly redefine governance mechanisms in order to mitigate risks and hazards and maximize value.

The paper is organized as follows. In a first part [START_REF] Coughlan | Channel Management: Structure, Governance, and Relationship Management[END_REF], following the literature in the IMP tradition and works on strategic alliances, we identify some key elements to be acknowledged in the analysis. Then [START_REF] Blois | The Market form Concept in B2B Marketing[END_REF], we propose an analytical framework crafted to study the dynamics of brand alliances from an interorganizational perspective. In the third part [START_REF] Das | Instabilities of Strategic Alliances: An Internal Tensions Perspective[END_REF] we apply this framework to two case studies of brand alliances and their afferent organizational forms. We identify the main elements that explain the level of stability and the long run dynamics of brand alliances. Finally, we develop the managerial implications and we propose some organizational and business-to-business key success factors for partners involved in brand alliances [START_REF] Das | The Dynamics of Alliance Conditions in the Alliance Development Process[END_REF].

Theoretical Backgrounds: Combining IMP Concepts with Literature on Strategic Alliances Brand alliances, seen from an inter-organizational perspective, necessitate the mobilization of complementary research streams. In the first section (1-1), we will show the relevance of the internal tension approach. In the second section (1-2) we will see that the concept of interaction is also an important theoretical contribution. Finally, in the third section (1-3), we identify the specificity of the concept of governance in strategic alliance settings.

The Internal Tension Approach

We will follow [START_REF] Das | Instabilities of Strategic Alliances: An Internal Tensions Perspective[END_REF], 2002and 2003) in their view of strategic alliances in a dynamic perspective. We will consider that "strategic alliances are the sites in which conflicting forces develop" (Das and Teng, 2000, p. 84). Thus, the starting point of our analysis of brand alliances from an interorganizational point of view is, thus, the idea of forces that will shape the on-going process of alliance transformation, from initiation to termination. For [START_REF] Das | Instabilities of Strategic Alliances: An Internal Tensions Perspective[END_REF], there are several explanations of strategic alliances instability and these explanations can be applied, mutatis mutandis, to brand alliances: transaction-cost economics (with the role of opportunistic behaviors), game theory, resource dependence/bargaining power, agency theory. Another stream of literature on strategic alliance is mobilized: the Das and Teng conceptualization of an alliance seen as an 'internal tensions' system [START_REF] Das | Instabilities of Strategic Alliances: An Internal Tensions Perspective[END_REF]. Following [START_REF] Selnes | A Dynamic Customer Portfolio Management Perspective on Marketing Strategy[END_REF] insights, we will consider that "effective marketing strategies are found when there is a good match with resource allocation and organization and one type of value creation" (Selnes and Johnson, 2004, p. 126). In a longitudinal approach, several features may affect this good match and therefore modify the equilibrium of the network form. Therefore, the tension within brand alliances is to be found in the intrinsic instability of this match.

We will consider, following Ebers and Grandori (1999) that inter-organizational relationships are inherently unstable and from that point of view alliances based on brands are not different. [START_REF] Ebers | The Forms, Costs and Development Dynamics of Inter-Organizational Networking[END_REF] characterized these "evolutionary forces as outcome-driven feedback loops". This unstability is to be related to changes in actors' resource base, to changes in actors' information base and to actors' expectations of their network partners' behavior and actions [START_REF] Ebers | The Forms, Costs and Development Dynamics of Inter-Organizational Networking[END_REF]. The changes in actors' resource base, following the resource-based view of the firm, reflect the fact that the partners "reduce the diversity of their resource bases and thus gradually destroy the foundations on which heir-relationships rests" (Ebers and Grandori, 1999, p. 277). The changes in actors' information base and actors' expectations also reflect the idea of evolving initial conditions inherent to any partners involved in a network.

Interaction Processes within Brand Alliances

Being a value differentiation strategy, brand alliances are prone, like any other type of brand differentiation strategy, to value erosion. One could say that, unlike other types of alliances, the volatility of the benefits of a brand alliance is a key characteristic. The changing environmental conditions like changing consumer behaviors, market conditions and technology shifts will affect greatly the evolution of the brand alliances. All these changes are factors of instability and may affect the value of the differentiation but, more importantly, it also may affect the value creation process developed by the partners during the initial stage of the brand alliance. Consequently, it will be necessary to focus the analysis upon any types of interactions within and between the business network and its environment. The importance of interaction processes has been widely acknowledged in the IMP literature (Håkansson et al., 2004). Following [START_REF] Ritter | Interaction Between Suppliers and Customers in Business Markets[END_REF] we suggest, classifying the interacting process into three categories:

• Interaction within existing relationships: As Ritter and Ford argue, "a certain level of confrontation (and thus conflict) is needed in relationships to allow them to develop further, to reinvest themselves." Inside the existing network, one of the partner or even the two of them will develop/ identify new opportunities that could affect the existing relationships.

• Choices about position in the network: This question is especially crucial in brand alliances. The value of a brand is highly dependent upon the value for the direct or indirect customers. Being able to modify this value, for instance, in developing new marketing channels through e-commerce will raise the perceived value of the brand and consequently improve the network position of the owner. In the words of [START_REF] Ritter | Interaction Between Suppliers and Customers in Business Markets[END_REF], the company has the choice between "consolidate by stabilizing and strengthening its existing network position" or "create a new position by changing the combination of its existing relationships or developing new ones".

• Choices about how to network: The partners have many possibilities between ranges of modes of control, from total ownership to informal agreements. In other words, the interaction process is also concerned with how to concede and how to coerce.

We will broaden the perspective on interactions in business networks with the work of [START_REF] Olkkonen | The Role of Communication in Business Relationships and Networks[END_REF]. Indeed, brand alliances are often loosely coupled business networks, or, in the term of [START_REF] Blois | The Market form Concept in B2B Marketing[END_REF] close to 'market forms'. For this author, the market form (unlike a business network) takes as its focus a firm's product family. A brand alliance is a situation where different organizations will impact the creation of value without real exchanges. For instance, considering the role of informal communication exchanges in brand alliances, there is an important role of broad communication processes. These communication channels are widely based on interpersonal links ("the lower level of interpersonal communication processes", in the words of [START_REF] Olkkonen | The Role of Communication in Business Relationships and Networks[END_REF]. Network forms in brand alliances are complex organizational forms with a focal net surrounded by direct and indirect partners. In a dynamic perspective, it is thus necessary to identify, within this 'market form', what are the different categories of interactions.

Governance

In a comparison between the classical strategic management literature with the network approach, [START_REF] Håkansson | Strategic Alliances in a Network Perspective[END_REF] suggest that a major difference between the two "lies in their emphasis on the governance structure in strategic alliances" (Håkansson and Deo, 1996, p. 123). For the strategic management literature, the legal structure (formal contract) will define the respective contributions of the partners. For the tenants of the network approach, informal exchanges, mainly due to cognitive limitations of the human brain and the complex environment, are necessary to develop and "appropriate process to relate and coordinate activities and resources with the counterpart firm" (Håkansson and Deo, 1996, p. 124). Consequently we will consider the status of interactions as the basic line of governance modes. We will combine the two approaches: formal and informal governance mechanisms. Following [START_REF] Wilke | Levels of Analysis in Business-to-Business Marketing[END_REF] and Ritter (2007), we will thus consider the question of governance in relation with that of level of analysis in business-to-business marketing. This question is of tremendous importance and not only for analytical reasons. Indeed, we will see that important evolutions are observed on the ground in the distribution of governance functions and contents between macro, meso and micro levels. Usually devoted to the dyads, the basic element of inter-organizational research has progressively shifted towards firm's net and network [START_REF] Ritter | A Framework for Analyzing Relationship Governance[END_REF]. We will consider in our analysis, the three levels of analysis: the dyad constituting the two brand owners, the network form that may surround the brand owners (and especially when this brand owner is a complex organizational form such as an association or a co-operative, for instance), and the macro level of the global market/sector, especially norms and standards defined at the macro level institutions. As stated by [START_REF] Ritter | A Framework for Analyzing Relationship Governance[END_REF], "the different levels (…) do not exist in isolation nor are they researched separately. Rather, the interplay between the different levels is most often reported in studies." The interaction processes is thus part of the analysis. But we follow Wilke and Ritter, "The different levels of analysis must be treated as quasiisolated but as complementary connected in our efforts to understand the overall picture" (Wilke and Ritter, 2007, p. 51).

The strategic management literature also gives other interesting complementary insights on governance. For instance, the works of [START_REF] Reuer | Post-Formation Dynamics in Strategic Alliances[END_REF] with their approach of governance change in interorganizational alliances will be mobilized. These authors propose an analysis of "alliance adjustments in collaborative agreements (…) by studying the occurrence and determinants of post-formation governance changes in alliances" (Reuer et al., 2002, p. 138). The question is then the nature of these governance changes: what types of structural characteristics or evolving mechanisms should be studied? The authors distinguished three types of ex-post governance changes: contract alterations, joint board or committee, monitoring mechanisms. For instance, contractual changes (or contract alterations) may include modification of royalty percentages paid by the licensee, joint boards may evolve in size and/or composition, monitoring mechanisms such as liaison desks may be institutionalized. These changes reflect the fact that "the firm's accumulation of experience with alliances is seen as providing the firm with different types of expertise and capabilities in forming alliances and managing their evolution" (Reuer et al., 2002, p. 138).

Analytical Framework

From these seminal works on alliance instability, interactions and governance, we develop a theoretical model for the study of inter-organizational dynamics of brand alliances. It is based on three components:

(1) The identifications of the main changes to be observed in resource base, actors' information, actors' expectation [START_REF] Blois | The Market form Concept in B2B Marketing[END_REF] The induced modifications of internal tensions: more or less co-operation/competition; more or less rigidity/ flexibility; more or less short-term/long-term orientation; and (3) The identification of ex-post governance changes (monitoring mechanisms, contractual provisions, boards, etc.).

The global conception of this analytical grid is in the line of reasoning with Poole and Van de Ven's (1989) conception of a process. Brand alliances in dynamics, from an interorganizational perspective, have no particular life-cycle and their future is unpredictable. Doing so, we then develop the idea of a dialectic evolution of brand alliances base on a few structural components of their afferent network form of organization.

Following research antecedents on inter-organizational dynamics, we will adapt our analytical grid to the contextual aspects of brand alliances detailed above: changes in internal and external conditions that have affected more or less the value creation process of brand alliances, the interaction processes between partners in the brand alliance (inter individual as well as interorganizational processes), the tangible results of these interaction processes: governance adaptations and changes. Let us detail these components.

• The change in internal initial conditions (resource base, actors' information, actors' expectations) and external initial conditions (market changes, consumers' expectations …). Following [START_REF] Ebers | The Forms, Costs and Development Dynamics of Inter-Organizational Networking[END_REF], we consider first that internal initial conditions are a necessary step to study inter-organizational dynamics. An alliance is generally crafted to exploit differences in resources. For instance, in brand alliances, complementary attributes may induce spill-over effects. Market access, contact with new customers or alternative marketing channels could also be opportunities. This is the same with actors' information, for instance market predictions. Ebers and Grandori linked this question of information to that of mutual adaptation and learning. They suggest, for instance, that "the discovery of greater goal differences and cultural diversity among partners may lead to the premature termination of a relationship". Expectations are not stable either: they may evolve through acquisition of information or because of internal changes (a new brand manager, for instance). The study of changes in external conditions is directly linked to the value-creation process. We suggest that phenomenon such as a higher competitive pressure, changing behaviors from consumers or new legal rules may affect greatly the brand alliances. Indeed the value of differentiation is extremely fragile and these external factors need to be incorporated in the framework.

• The modifications of internal tensions between partners through permanent interaction processes: identification of co-operation/competition evolutions, rigidity/flexibility continuum, short/long-term orientations of partners. For [START_REF] Das | Instabilities of Strategic Alliances: An Internal Tensions Perspective[END_REF], 2002and 2003) the role of an internal tensions framework is to identify the main conflicting forces that will explain the 'intrinsic vulnerability of alliances'. But alliances are not only unstable they are also unpredictable. In the vein of Van de Ven research works (Van de Ven and Walker, 1984; Van de Ven and Poole, 1995; and Dooley and Van de Ven, 1999), Das and Teng consider that the logic of alliance dynamics is dialectic. An important consequence of their conception is the focus of their analytical framework on processes and the resulting effects, without any claim about what could be the future of the alliance. They distinguish three types of output or elementary 'pairs' of conflicting forces: rigidity vs. flexibility, short-term vs. long-term orientation and cooperation vs. competition. The rigidity vs. flexibility pair reflects the "degree of connectedness of members with each other in an on-going relationship". Then "constituencies within formal organizations are rigidly linked with each other". The problem of strategic alliances is the blurred frontier between formal and informal mechanisms. In order to avoid these difficulties, we will consider the degree of flexibility in co-ordination mechanisms. Formal contracts, centralized decision rights, financial integration will decrease the flexibility of these co-ordination mechanisms. On the contrary, informal and/or interpersonal agreements, decentralized decision devices, autonomy of decision-centers about finance or strategic decisions will increase the flexibility. We find here the classical opposition between hierarchies on one hand and market institutional forms on the other.

• The identification of ex-post governance changes: monitoring devices (boards, pilots, formal consortiums, in other words 'who is in charge of what'), governance mechanisms (contractual provisions such as control schemes, price premiums, rewards, fees and royalties), governance levels (linked to the level of analysis). The changes in governance, called in the grid 'governance adaptations' refer to the well known modifications in monitoring and governance mechanisms [START_REF] Heide | Interorganizational Governance in Marketing Channels[END_REF][START_REF] Ghosh | Governance Value Analysis and Marketing Strategy[END_REF][START_REF] Coughlan | Channel Management: Structure, Governance, and Relationship Management[END_REF][START_REF] Reuer | Post-Formation Dynamics in Strategic Alliances[END_REF]. We add the category of governance level. In brand alliances, shifts in product parameters can modify the governance level: this is especially the case in the context of certification schemes with their certification brands.

The Figure 1 summarizes the main components of the analytical framework.

Empirical Research

We will firstly present our research methodology based upon a qualitative approach of case studies (4-1). Then we will give a detailed description of the two cases of brand alliances (4-2). We will finally apply the framework of the cases (4-3)

Research Methodology

The empirical part is based on two case studies of brand alliances and the evolution of their network forms over one period of time. In these two case studies of brand alliances, actors in relation mobilized resources, implemented governance mechanisms (such as ownership, contracts, incentives, internal or external controls) and defined strategies to mutually defend their position both on the market and within the partnership. Thus, beyond the combination of their brands (strong or weak or of equal force) at initial time, the evolution of the global value creation process in an alliance is conditioned by the interorganizational dynamics.

The methodology set up for these case studies is based upon previous theoretical works on longitudinal approaches [START_REF] Eisenhardt | Theory Building from Cases: Opportunities and Challenges[END_REF]) and of their application in industrial marketing cases [START_REF] Dubois | Systematic Combining: An Abductive Approach to Case Research[END_REF]Dubois and[START_REF] Dubois | Research Methods in Industrial Marketing Studies[END_REF][START_REF] Ritter | A Framework for Analyzing Relationship Governance[END_REF]. Other research works on longitudinal studies must be acknowledged. [START_REF] Pettigrew | Longitudinal Field Research on Change: Theory and Practice[END_REF] and Van de Ven and Huber (1990) made a distinction of methods when studying organizational changes: some approaches are centered on antecedents and consequences of changes and other approaches are centered on the emergence and evolution of organizational change. For Van de Ven and Huber, the second approach "requires ...'process theory' explanation of the temporal order and sequence in which a discrete set of events occurred based on a story or historical narrative. In terms of causality (…) this approach explains an observed sequence of events in terms of some underlying generative mechanisms or laws that have the power to cause events to happen in the real world and the particular circumstances or contingencies when these mechanisms operate" (Van de Ven and Huber, 1990, p. 213). Our research methodology will follow this process view of organizational change.

The case study research protocol is done in the vein of Yin (2003). The selection of cases is done according to previous research works on brand alliances. We have adopted a qualitative approach with the use of semi-directive interviews to collect data. According to [START_REF] Eisenhardt | Theory Building from Cases: Opportunities and Challenges[END_REF], interview is a very rich source of information that facilitates data collection, especially when the phenomenon is highly occasional. This is the case of brand alliances which have products with short life-cycles. We have carried out interviews principally in face-to-face because it established trust between the interviewer and the interviewees. The latter is also more likely to give detailed information to the interviewer in comparison to requests by telephone or e-mail. Through the case study method, we have obtained a great number of details concerning the circumstances in which the various types of brand alliances occur. In order to succeed with the interview methodology, Eisenhardt and Graebner (2007) advise to carry out discussions with the organizational actors at various hierarchical levels, in different sectors, different groups and geographical places, actors of other organizations like external observers, etc. Thus, we interviewed different actors on the two markets concerned by the alliance, with owners of the brands, marketing directors and persons in charge of association or company. Also we interviewed partners who are indirectly related to alliance. Thus, to collect primary data, we did 15 semi-directive face-to-face interviews, 6 interviews by telephone (that lasted on average one hour and a half) and sent 4 questionnaires to get additional details. These primary data were supplemented by annual reports of activity, the reports of the board of directors, our participation in conferences organized on the markets where alliances take place. Regarding the data processing, we used the method of content analysis. After recording the interviews, we progressively transcribed them (after each interview) to ensure the safety of information and notes during interviews. For the data processing, we used qualitative data processing software 'QSR Nvivo 2.0' to identify the topics of our analytical framework in the interviews. This software also enabled us to make classification without moving away from paper and traditional method of information analysis (topics). To do the coding, we used a dictionary which presents topics of our analytical grid. According to our framework, these topics are changes in factors' situation during one period of time, interaction processes and governance adaptations.

Presentation of the Cases Case 1

Our first case study relates to the alliance between the private certification brand Fair Trade (Max Havelaar association or hereafter MH) and a banana brand Oké (AgroFair's company). MH is an association with no lucrative goals which offers outlets for trade to producers. The brand Fair Trade is a promise of ethical value releasing a strong image for the product and positive attributes on the market. AgroFair is a banana importer that buys bananas to producers in developing countries, to sell them to developed countries (indirectly to final customer via hypermarkets) [START_REF] Sauvée | Value Creation in Brand Alliances: A Network Approach[END_REF].

Indeed the sales turnover of MH's labelized products increases because a great part of consumers (78% in France) consider that MH's products resulting from the Fair Trade have a good quality. Among nine labelized products, banana is growing in the market. An alliance between MH and AgroFair aims to promote and commercialize Fair Trade bananas through an equal distribution of the benefits between actors.

MH and AgroFair are thus, in relation with producers, distributors, organizations of control (like FLO Cert, Fair Trade Labeling Organization, a certifying organization) and final consumers. FLO Cert defines the rules of operation and writes the schedule of conditions. FLO Cert grants certification and ensures control. By granting its license to AgroFair realizing the payment of a royalty, MH checks that information on Fair Trade bananas is in conformity with the standards of the market without being responsible for quality. Thus in the relationship, MH seeks to protect its reputation and image of its strong brand via the Fair Trade concept. AgroFair permanently wants to improve its image in putting in front of access its brand Oké. This potential rivalry fuels the interorganizational dynamics between the partners [START_REF] Sauvée | Value Creation in Brand Alliances: A Network Approach[END_REF].

Case 2

The second case study is an alliance between the brand of health nutrition Omega 3 from Bleu-Blanc-Coeur's association (hereafter BC) and the private brand Fleury Michon belonging to the company Fleury Michon (hereafter FM), a food company which sells processed pork products. BC, an association with no lucrative goals, promotes the use of flax in animal and human food with Oméga 3 [START_REF] Sauvée | Value Creation in Brand Alliances: A Network Approach[END_REF]). BC's concept from Omega 3 consists to give feed to animal (mainly flax seeds): The animals (for instance pigs) concentrate Omega 3 and make them available for consumers in a context where problems of obesity and cardiovascular diseases have increased. BC success is thus strongly related to the characteristics of the nutrition health market with very specific products' attributes. This association is composed of members such as pork producers, retailers, food companies, consumers and their associations, feed companies. In the alliance, BC animates the scientific step (clinical studies) and contributes to the communication around the Omega 3 ingredient. FM takes care of the distribution of its products with the brand Fleury Michon and the logo BC Omega 3. The presence of this logo is conditioned by a payment of royalty to BC. FM is committed to respect like all the other members (producers, feed companies, distributors ...) the requirement specifications and the rules of procedure set up by BC. The control committee of the association checks the application of the user requirement specifications.

The scientific committee defines the research orientations as well as publications, takes part in the development of the nutritional speech and ensures the bond with the research institutes. This committee is composed of specialists who are members of BC. Then the marketing commission manages the communication budget, defines the communication contents and the market and consumer studies. Considering the importance of the topics for members, a need for a tighter coordination has progressively emerged [START_REF] Sauvée | Value Creation in Brand Alliances: A Network Approach[END_REF].

Results of the Case Study Research

We will apply our analytical grid in following the three-step level: identification of internal and external changes between T1 and T2, modification of internal tensions between partners, governance adaptation and changes within the network forms.

But in order to apply properly our analytical grid, the internal tensions perspective will be focused on two or three leading facts. These facts reflect the major changes that have been observed during the period of time, either concerning the internal factors (such as resource base or actors' expectations about the alliance) or concerning the external ones (market conditions, legal environment, etc.). These facts will act as major drivers of change in the brand alliance. These major drivers of change will modify the internal tensions between the partners in the brand alliance. Following [START_REF] Das | Instabilities of Strategic Alliances: An Internal Tensions Perspective[END_REF], 2002and 2003) in their approach, we will put the stress on the results of internal tensions between the two partners in the alliance. Consequently the governance structures will evolve, and this will be identified through three components: monitoring procedures; control and incentive mechanisms, governance levels (Figure 1).

Case Study 1: Max Havelaar/Oké-AgrofFair Initial conditions in this case differ greatly: A well established certification brand, Max Havelaar has provided its image of Fair Trade products to AgroFair and its brand Oké. After a few years, some major changes in these initial conditions have occurred and can be summarized in two major points.

Firstly, the market for Fair Trade products has grown rapidly for several product categories, including bananas. Considering the internal conditions, this rapid evolution has changed the communication policy of Max Havelaar. The organization decided to improve its links with final consumers through a development of market communication. But at the same time and for the same reasons, AgroFair tried to extend its product range from bananas to tropical fruit (mangoes, citrus for instance) and also decided to develop its communication policy. The global evolution of the market is therefore a way for the pa.rtners in the brand alliance to widen their influence. So we can say that actors' expectations about the alliance have somewhat changed, with a real internal evolution of their financial resources.

A second important evolution in the case study is the legal environment that will necessitate a formal separation between the certifying organization FLO Cert on one hand, FLO who defines the rules and Max Havelaar who markets the products indirectly through its license, on the other hand. In doing so, the role of Max Havelaar, as an organization, is to refocus its activity on the communication policy towards final consumers. But without real production activities in the agrifood chain, its position may be weakened.

Table 1 gives the main features of this evolution for the three components of the framework with this example of Case 1.

Case Study 2: Bleu-Blanc-Coeur/Fleury Michon In the second case study, three facts seem particularly relevant. There is also a rapid market expansion for the products with nutritional allegations. So, the number of members has increased significantly since the year 2000. The resource of BC being related to the amount of fees, the association has increased its financial possibilities. Consequently, the expectations of the association have been enlarged and have moved towards new objectives. Initially, the objectives of BC were centered on the knowledge about nutritional benefits. The objective is now to promote the use of flax in more and more agrifood chains, thus reflecting the necessity to increase the market. Simultaneously, the resource base of the association has grown significantly, mainly in human resources.

A second fact in external environment has also affected the situation: The new legal aspects concerning nutritional allegations. Created in 2007, the legal constraints concerning nutritional allegations have two consequences. The first one is the fact that this it is likely to be more difficult, in the future, to maintain the price premium for products with Omega 3 allegations and the competitive pressure from other products and markets will be heightened. The evolution of legal rules encourages BC to undertake complete clinical studies. A recent study is related to the metabolic syndrome (overweight and obesity). BC also carried out several researches with hospital and veterinary research centers to show positive impacts of Omega 3 in human and animal nutrition.

The second consequence stems from the first one: The BC association must clearly develop its differentiation on other aspects than nutritional allegations, for instance sustainable development, local production, etc.

As shown in Table 1, in Case Study 2 the internal tension situation between the partners is clearly oriented towards increased co-ordination mechanisms and more cooperation between the partners. But, while the orientation for the BC association brand is clearly stated, the Fleury Michon orientation is less clear.

Governance Changes and Adaptations

In the two case studies, we have put in evidence, important evolutions of the initial conditions during a limited period of time. We have shown that brand alliances, like any other form of strategic alliances are unstable by nature. Due to internal and/or external evolutions, the changing equilibrium between the partners will modify the relationships between them. Let us now consider the consequences of these facts on governance changes and adaptations.

In the Case Study 1, the main feature that has been observed is the development of different levels of governance with a complementarity of functions. Mainly due to an important market expansion for Fair Trade products, it shows that, to maintain their efficiency, the partners try to improve the way the alliance is conducted. Consequently, the internal tension equilibrium is somewhat contrasted. While the two direct partners, Max Havelaar and AgroFair, benefit from this expansion and increase their co-operation policy, the links between Max Havelaar and the certifying organization could diminish in the long run. Concerning the governance adaptations, we highlight the development of external enforcement mechanisms such as Fair Trade certification schemes. This multilevel governance may affect in the long run the differentiation strategy followed by AgroFair and jeopardize the situation of Max Havelaar with regards to its direct clients.

In Case Study 2, the situation is quite different. The BC association has clearly extended its role towards several directions. Unlike Max Havelaar, the reputation of the logo was very limited and, through a rapid market and expansion in the number of members, the objectives have changed. The governance of the BC network has thus evolved with a specialization of governance functions. The scientific, communication, sales force and control functions are the main monitoring authorities. The decision processes within the association are thus highly integrated. BC association acts like a quasi-firm or more precisely like a club (on clubs and club goods, cf. Torre, 2006) and has been able to develop its reputation. This fact is corroborated by the importance in the number of the new comers and in some cases by the reputation of their brands.

Our analytical framework of inter-organizational dynamics is thus applied to the case studies. In these two case studies, created sometime ago, it is possible to identify significant evolutions in the value creation processes. According to this framework, we identify the main changes that fall into three broad categories: main changes in resource base, in actors' information and in actors' expectation. It is shown for instance that the expectations of the partners have drastically changed, notably because of modifications in market positions. Internal tensions have somewhat arisen in one of the two case studies, while the other seems to develop a long-term orientation with a clear co-operation strategy for all the partners who are members of the association. Finally, the governance changes that are identified show the main evolutions within the network forms, with the creation of new devices, new contractual provisions, complementary enforcement procedures, new roles for the committee boards.

Managerial Implications and Concluding Comments

The brand alliance seen in an inter-organizational dynamics perspective has many interesting implications for the managers. First of all, the partners in the alliance have different level of relational experience. This experience effect will impact the performance of the brand alliance. We have seen in particular the role of market information in the constitution of this experience. We suggest that the relative scope of market positions will directly influence the benefits from the alliance.

The question of initial conditions and the role of the alliance formation stage have also to be acknowledged. We have seen in the case studies that differences in brand reputations between the two brands in the alliances at the initial stage have organizational consequences. This asymmetrical situation necessitates from the partners resource adaptations. The ability to adapt through resource acquisition linked to the value creation process is thus critical. The characteristics of contributed resources will significantly affect alliance conditions. For example, as suggested by [START_REF] Das | Partner Analysis and Alliance Performance[END_REF], "partners with imitable resources are likely to lose their mutual dependence rather quickly".

Our research puts in light the interests of cumulative knowledge and the links with alliance performance. In a longitudinal approach, it is possible to identify some features such as the reputation of the brands, the possible conflicts occurring between the partners and the interdependencies leading to opportunism and to co-relate these features with the outcomes and benefits of the alliances. Some preliminary results emphasized the importance of some characteristics of brand alliances for their stability: the nature of collective resources linked to the alliance, the effects of opportunism, the balance of power and the rise of a symmetric partnership, especially in considering the level of the spillover effect between brands (and its evolution overtime).

The main result of this research is to show that, in line of reasoning with our previous works on brand alliances, the understanding of brand alliance evolution towards expansion, termination or stability cannot be separated from inter-organizational aspects. Following Van de Van and Poole and Das and Teng analysis of organizational dynamics, we think that there are no life-cycles in brand alliances. The logic of this dynamic is dialectic and mainly found in organizational components. In other words, the evolution of brand alliance is unpredictable and is the results of contradictory forces between the partners that will modify permanently the network form. But being unpredictable is different from having no rationality. To be intelligible, we suggest that the study of brand alliances dynamics should focus on a few key organizational aspects.