D Bordereaux

Martins

Lehmann-Che

Martinez-Climent Ja

Copie-Bergman

Tesson, B 12 , Jais, JP Tilly H 11

Leroy

Molina

The alternative RelB NF-κB subunit is a novel critical player in diffuse large B-cell lymphoma

It's not the size of the dog in the fight, it's the size of the fight in the dog.

ACKNOWLEDGMENTS

To begin, I would like to state the honor that presenting my work of 5 intense years is and how proud I am for the opportunity to complete a Ph.D. at Université de Paris. It represents the end of a journey and the beginning of a new one, which I will gladly embrace with courage and joy.

First, I would like to thank my Ph.D. supervisor, Dr. Véronique Baud, for having the chance to explore several exciting projects and topics. Under her supervision, I had no choice but constantly pursue scientifical and intellectual precision. She provided the tough conditions for me to learn and grow.

Thanks to my collaborators who through convivial exchanges helped me significantly in my investigations, especially Benjamin Bonsang, Gildas Bertho and Martial Caly.

I would also like to thank the distinguished researchers who accepted to be part of my jury, it is an honor to be judged by them.

For granting me the scholarships to accomplish this PhD, I would like to thank the association Prolific, Clinisciences and the ARC association.

A special thanks to the members of my research team, especially Didier and Clara, with whom I had a lot of fun between the harsh realities of a lab, Davi, for our profound and absurd discussions, and Stéphanie for the laughter and shared love of good food. I would also like to thank members of surrounding teams, Ingrid, Stéphanie, Eric, Jean-Claude and all the others.

They played an important role in those 5 years.

Finally, I would like to thank my friends and my family. Especially parents, they indefectibly supported me throughout those years: I would not have acccomplished it without them.

Baptiste Eluard

ABSTRACT (english version)

Impact of RelB NF-κB subunit in diffuse large B cell lymphoma and breast cancer aggressiveness

Although mortality rates have declined, the majority of cancers remains incurable and the challenge to improve knowledge of cancer biology is evident. NF-κB transcription factors have been described to play many roles in the malignant phenotype of numerous cancers.

Nonetheless, the impact of RelB, the subunit mainly controlled by the NF-κB alternative pathway, remains largely unexplored in cancer. In that perspective, the discovery of new RelBdependent cancer vulnerabilities and functions is emerging as a promising field of investigation that I focused on during my PhD.

First, we evaluated in breast cancer RelB phosphorylation on serine 472 (pS472-RelB), a post-translational modification previously demonstrated in our laboratory to be involved in primary fibroblast migration. Using a specific antibody developed in our laboratory (Patent EP2793026-A1; WO2014170487-A1, 2014), we revealed by immunohistochemistry that nuclear pS472-RelB is associated with poor prognosis and metastasis development in a cohort of 234 breast cancer (BC) patients. Mechanistic study in triple-negative BC (TNBC) cell line revealed that pS472-RelB drives a pro-metastatic and pro-invasive phenotype and showed that pS472-RelB exerts its pro-invasive functions through direct control of metalloproteinase MMP13 expression, and subsequent activity. Our results demonstrate for the first time a pivotal role of a post-translational modification of RelB in BC patients aggressiveness and identify a new mechanism for RelB pro-malignant role in BC cells, paving the way for a novel approach to assess RelB regulation and function in cancer.

Unexpectedly, we also observed a direct RelB-dependent regulation of glucose transporter GLUT3 expression in BC cell lines, leading to an increased rate of glucose metabolism. We demonstrated that this RelB-dependent increase in glucose metabolism confers a resistance to genotoxic and metabolic stress of BC cells in vitro. We also observed that a high expression of GLUT3 is associated with poor prognosis in BC patients. These results demonstrate for the first time, to our knowledge, a role for RelB in the upregulation of glucose metabolism in cancer and could help deciphering the role of breast cancer metabolism deregulation in resistance to genotoxic and metabolic treatment.

Finally, we unveiled a frequent DNA-binding activity of RelB in DLBCL cell lines and patients, and we found that RelB activation is associated with poor prognosis in diffuse large B cell lymphoma (DLBCL) patients. Further, we defined a gene expression signature (GEP) and a unique mutational profile specific to RelB activation in patients. Mechanistically, we showed that RelB plays an important role in the resistance of DLBCL cell lines to doxorubicin-induced DNA damage and apoptosis, the main drug in DLBCL treatment. Moreover, we discovered that RelB activation correlates with expression of the anti-apoptotic protein cIAP2 in DLBCL cell lines and patients, suggesting that control of cIAP2 expression could participate in the RelBdependent resistance to doxorubicin. These results strongly suggest that RelB activation is a potential new biomarker for DLBCL resistance to anthracycline-based treatment and that its specific GEP and mutatome could participate in a better therapeutic selection of DLBCL patients.

Altogether, my PhD has highlighted new functions for RelB in BC and DLBCL biology that could contribute to a better patient stratification and pave the way for studying the impact of RelB in other cancer types.

Keywords: Breast cancer -metastasis -post-translational modifications -RelB -metabolism -DLBCLapoptosis -biomarker

ABSTRACT (version francais)

Impact de la sous-unité de NF-κB RelB dans l'agressivité des lymphomes diffus à grande cellules B et des cancers du sein Bien que les taux de mortalité aient chuté, la majorité des cancers restent incurables et il est évident que l'approfondissement des connaissances de la biologie des cancers reste un défi. Les facteurs de transcription NF-κB ont été décrits comme jouant plusieurs rôles dans différents cancers. L'impact de RelB, la sous-unité principalement contrôlée par la voie de signalisation alternative de NF-κB, reste cependant largement inconnu dans les cancers. Donc, la découverte de nouvelles fonctions dépendantes de RelB émerge comme un champ de recherche prometteur auquel je me suis intéressé au cours de ma thèse.

Nous avons d'abord étudié la phosphorylation de RelB sur la sérine 472 (pS472-RelB), une modification post-traductionnelle préalablement démontrée comme étant impliquée dans la migration des fibroblastes primaires. En utilisant un anticorps spécifique développé par notre laboratoire (brevet EP2793026-A1 ; WO2014170487-A1, 2014), nous avons révélé par immunohistochimie que pS472-RelB nucléaire est associé au mauvais pronostic et au développement métastatique d'une cohorte de 234 patientes atteintes de cancers du sein (CS).

L'étude mécanistique dans des lignées de CS triple-négatifs a révélé que pS472-RelB promeut un phénotype pro-métastatique et pro-invasif à travers la régulation directe de l'expression et l'activité de la métalloprotéinase MMP13. Nos résultats démontrent pour la première fois un rôle crucial d'une modification post-traductionnelle de RelB dans l'agressivité des CS et identifient un nouveau mécanisme pro-tumoral de RelB dans les cellules de CS, proposant une nouvelle approche d'évaluer la régulation de RelB et de ses fonctions dans les cancers.

De manière inopinée, nous avons également observe la régulation directe de l'expression du transporteur de glucose GLUT3 par RelB dans des lignées de CS, induisant l'augmentation du métabolisme du glucose. Nous avons démontré in vitro que cette augmentation du métabolisme du glucose confère une résistance aux stress génotoxiques et métaboliques aux cellules de CS. Nous avons également observé que l'expression élevée de GLUT3 est associée à un mauvais pronostic des patientes atteintes de CS. Ces résultats démontrent pour la première fois, à notre connaissance, un role de RelB dans l'augmentation du métabolisme du glucose dans le cancer et pourrait participer à résoudre le rôle des dérégulations du métabolisme dans la résistance aux traitements génotoxiques et métabolique des CS.

Enfin, nous avons révélé une fréquente activité de liaison de RelB dans des lignées cellulaires de lymphomes diffus à grande cellules B (DLBCL) et de patients, et nous avons découvert que l'activation de RelB est associée à un mauvais pronostic des patients atteints de DLBCL. De plus, nous avons défini une signature d'expression génique et une signature mutationnelle unique spécifiques à l'activation de RelB chez les patients. Mecanistiquement, nous avons montré que RelB joue un rôle important dans la résistance des lignées de DLBCL aux dommages à l'ADN et à l'apoptose induits par la doxorubicine, la drogue principale dans le traitement du DLBCL. De plus, nous avons découvert que l'activation de RelB correle avec l'expression de la proteine anti apoptotique cIAP2 dans les lignées et chez les patients, suggerant que le contrôle de l'expression de cIAP2 pourrait participer à la resistance à la doxorubicine induite par RelB. Ces résultats suggerent que l'activation de Rel Best un biomarqueur potentiel de la résistance du DLBCL aux traitements avec anthracycline, et que les signatures géniques et mutationnelles pourraient mener à une meilleure sélection thérapeutique des patients atteints de DLBCL.

En conclusion, ma thèse a permis d'identifier de nouvelles fonctions de RelB dans la

Table of illustrations

INTRODUCTION I. NF-B family of transcription factors and the two main activation pathways

The NF-κB family contains five members in vertebrates: NF-κB1 (p50 and its precursor p105), NF-κB2 (p52 and its precursor p100), c-Rel, RelA (p65) and RelB 1 (Fig. 1). Two main pathways regulate the transcriptional activity of these proteins that bind to DNA as homo-and heterodimers. The first one, better known as the classical pathway, applies mainly to RelA/p50 dimers and relies on the cellular distribution of these dimers (Fig. 2). In the absence of activation, the binding to inhibitory proteins IκB confine RelA/p50 dimers in the cytoplasm.

In response to stimuli such as cytokines, mitogenic agents, double-strand RNA, growth factors, viral or bacterial products and various forms of cellular stress (oxidative stress, radiations, and genotoxic agents), IκB kinase (IKK) phosphorylate IκB proteins on specific serines, which then undergo ubiquitination and finally degradation by the proteasome 2 . When released from their inhibitors, RelA/p50 dimers can enter the nucleus where they regulate the transcription of specific target genes (Fig. 2). This pathway plays a crucial role in innate immunity, inflammatory response and cell survival. A second pathway, which principally relies on the specific activation of RelB, has recently been characterised. A smaller number of cytokines, all members of the TNF superfamily (such as CD40L lymphotoxin β or BAFF), viral proteins (LMP1 from EBV, Tax from HTLV1, etc.) and bacterial strain Gram -Helicobacter pylori induce this pathway, also known as the alternative pathway [START_REF] Baud | Is NF-κB a good target for cancer therapy? Hopes and pitfalls[END_REF] . It involves the kinase NIK that activates IKKα, which then induces the phosphorylation of the main RelB inhibitor p100 and its subsequent cleavage by the proteasome, eventually leading to the nuclear transport of RelB/p50 and RelB/p52 3 (Fig. 3).

Worthy of note, most inducers of the alternative pathway also activate the classical pathway, and a reciprocal regulation between RelA and RelB has been underlined 4 . The alternative pathway has a pivotal function in lymphoid organogenesis, B cell maturation and adaptive immunity (Fig. 3).

A. RelB functions in normal cells

RelB is the only NF-κB subunit that has not been demonstrated to homodimerize, and its transcriptional activity is only activated through dimerization with p50 or p52 [START_REF] Bours | A novel mitogen-inducible gene product related to p50/p105-NF-kappa B participates in transactivation through a kappa B site[END_REF][START_REF] Bours | Human RelB (I-Rel) functions as a kappa B site-dependent transactivating member of the family of Rel-related proteins[END_REF][START_REF] Ryseck | a new Rel family transcription activator that can interact with p50-NF-kappa B[END_REF][START_REF] Dobrzanski | Both N-and C-terminal domains of RelB are required for full transactivation: role of the N-terminal leucine zipper-like motif[END_REF] . Aside of the regulation by the alternative NF-κB signaling pathway, RelB DNA-binding activity was shown to be negatively regulated via several mechanisms such as nuclear inhibitory sequestration by RelA or p100 [START_REF] Jacque | RelA repression of RelB activity induces selective gene activation downstream of TNF receptors[END_REF][START_REF] Yilmaz | RelB is required for Peyer's patch development: differential regulation of p52-RelB by lymphotoxin and TNF[END_REF][START_REF] Derudder | RelB/p50 Dimers Are Differentially Regulated by Tumor Necrosis Factor-α and Lymphotoxin-β Receptor Activation: CRITICAL ROLES FOR p100[END_REF] , and post-translational modifications. Beyond the different in signaling molecules required for their activation, the transcriptional activity cross-talk between RelBcontaining dimers and classical NF-κB pathway dimers is also different and complex: one hand RelB activation can increase the positive regulation of canonical NF-κB pathway target genes, and on the other hand it can downregulate the expression of other positively canonical NF-κB pathway target genes [START_REF] Dejardin | The lymphotoxin-beta receptor induces different patterns of gene expression via two NF-kappaB pathways[END_REF][START_REF] Saccani | Modulation of NF-κB Activity by Exchange of Dimers[END_REF] . Thus, both pathways can induce different and sometimes opposites transcriptional programs. Moreover, RelB activation can also exhibit DNA-binding specificity depending on its dimerization [START_REF] Bonizzi | Activation of IKKα target genes depends on recognition of specific κB binding sites by RelB:p52 dimers[END_REF][START_REF] Fusco | NF-kappaB p52:RelB heterodimer recognizes two classes of kappaB sites with two distinct modes[END_REF][START_REF] Natoli | Shaping alternative NF-κB-dependent gene expression programs: new clues to specificity[END_REF] .

Phenotype examination of RelB-deficient mice has revealed that RelB is crucial for the development of medullary epithelium, mature dendritic cell function, and secondary lymphoid tissue organization [START_REF] Wu | RelB Is Essential for the Development of Myeloid-Related CD8α-Dendritic Cells but Not of Lymphoid-Related CD8α+ Dendritic Cells[END_REF][START_REF] Weih | Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-κB/Rel family[END_REF][START_REF] Weih | Multifocal defects in immune responses in RelBdeficient mice[END_REF][START_REF] Weih | Essential Role of RelB in Germinal Center and Marginal Zone Formation and Proper Expression of Homing Chemokines[END_REF] . These observations suggest that RelB regulates positively essential developmental mechanisms. RelB deficiency in mice also caused a spontaneous lasting noninfectious multi-organ inflammatory syndrome, leading to mouse lethality [START_REF] Weih | Both multiorgan inflammation and myeloid hyperplasia in RelB-deficient mice are T cell dependent[END_REF] . Additionally, RelB has been demonstrated to participate in dendritic cell development and in the immune sensitivity in response to inflammation [START_REF] Zhu | Tolerogenic dendritic cells generated by RelB silencing using shRNA prevent acute rejection[END_REF][START_REF] Vogel | Aryl hydrocarbon receptor signaling regulates NF-κB RelB activation during dendritic-cell differentiation[END_REF] . RelB also downregulates the expression of proinflammatory proteins in the establishment of endotoxin tolerance in circulating leukocytes like monocytes [START_REF] Yoza | Induction of RelB Participates in Endotoxin Tolerance[END_REF][START_REF] Gazzar | Epigenetic Silencing of Tumor Necrosis Factor α during Endotoxin Tolerance[END_REF][START_REF] Chen | The NF-κB Factor RelB and Histone H3 Lysine Methyltransferase G9a Directly Interact to Generate Epigenetic Silencing in Endotoxin Tolerance[END_REF] . More recently, the role of RelB in the regulation of non-hematopoietic functions has come to light. RelB was demonstrated to have an key function in the regulation of lipopolysaccharide-induced proinflammatory response in human fibroblasts [START_REF] Xia | RelB regulation of chemokine expression modulates local inflammation[END_REF][START_REF] Xia | RelB Modulation of IκBα Stability as a Mechanism of Transcription Suppression of Interleukin-1α (IL-1α), IL-1β, and Tumor Necrosis Factor Alpha in Fibroblasts[END_REF] , subsequently participating in the control of acute inflammation ending. RelB was also demonstrated to play a part in the circadian rhythm in murine fibroblasts [START_REF] Bellet | The RelB subunit of NFκB acts as a negative regulator of circadian gene expression[END_REF] and to participate in the detoxificating response to xenobiotics of lung fibroblasts [START_REF] Vogel | a New Partner of Aryl Hydrocarbon Receptor-Mediated Transcription[END_REF][START_REF] Baglole | The Aryl Hydrocarbon Receptor Attenuates Tobacco Smoke-induced Cyclooxygenase-2 and Prostaglandin Production in Lung Fibroblasts through Regulation of the NF-κB Family Member RelB[END_REF] . RelB also supports RANKLinduced osteoclastogenesis [START_REF] Vaira | RelB is the NF-κB subunit downstream of NIK responsible for osteoclast differentiation[END_REF][START_REF] Soysa | The Pivotal Role of the Alternative NF-κB Pathway in Maintenance of Basal Bone Homeostasis and Osteoclastogenesis[END_REF][START_REF] Taniguchi | RelB-induced Expression of Cot, an MAP3K Family Member, Rescues RANKL-induced Osteoclastogenesis in Alymphoplasia Mice by Promoting NF-κB2 Processing by IKKα[END_REF] .

B. Regulation of RelB by post-translational modifications

Post-translational modifications occur after the process of translation required for protein biosynthesis, either by adding covalently a tag to the peptide, or during the proteolytic and folding mechanisms. These modifications can alter the protein 3D structure and start a complete new set of functions through new signaling cascade opportunities. In recent years, posttranslational modifications have been recognized as one of the main mechanisms for the finetuned regulation of NF-κB. Post-translational modifications such as phosphorylation, acetylation, methylation, ubiquitinylation, SUMOylation, and isomerization of specific amino acid residues, have been reported for the regulation of the IKKs, the IκBs, the NF-κB subunits, and NF-κB-mediated factors [START_REF] Perkins | Post-translational modifications regulating the activity and function of the nuclear factor kappa B pathway[END_REF][START_REF] Huang | Posttranslational modifications of NF-κB: Another layer of regulation for NF-κB signaling pathway[END_REF][START_REF] Calao | A pervasive role of histone acetyltransferases and deacetylases in an NF-κB-signaling code[END_REF][START_REF] Lu | Regulation by Methylation[END_REF][START_REF] Schmukle | No one can whistle a symphony alone -how different ubiquitin linkages cooperate to orchestrate NF-B activity[END_REF][START_REF] Sasaki | Roles of linear ubiquitinylation, a crucial regulator of NF-κB and cell death, in the immune system[END_REF][START_REF] Mabb | SUMO and NF-κB ties[END_REF] . These structural changes depend on the specificity of stimulus and participate in the activation and continuation of NF-κB response, and for its initiation of specific signaling pathway activation to initiate the transcription of a determined st of genes. Of note, these modifications can either activate or inhibit NF-κB activity depending on cell type and stimulus specificity [START_REF] Perkins | Post-translational modifications regulating the activity and function of the nuclear factor kappa B pathway[END_REF][START_REF] Huang | Posttranslational modifications of NF-κB: Another layer of regulation for NF-κB signaling pathway[END_REF][START_REF] Smale | Hierarchies of NF-κB target-gene regulation[END_REF] . Among those involving NF-κB transcription subunits, site-specific modifications of RelA is by far the most well known [START_REF] Huang | Posttranslational modifications of NF-κB: Another layer of regulation for NF-κB signaling pathway[END_REF][START_REF] Calao | A pervasive role of histone acetyltransferases and deacetylases in an NF-κB-signaling code[END_REF][START_REF] Lu | Regulation by Methylation[END_REF][START_REF] Msaki | The role of RelA (p65) threonine 505 phosphorylation in the regulation of cell growth, survival, and migration[END_REF][START_REF] Lu | Negative regulation of RelA phosphorylation: Emerging players and their roles in cancer[END_REF] .

Since RelB human protein is composed of 46 serine, 24 threonine, 22 lysine and 10 tyrosine residues, the potential for the identification of RelB post-translational modification is evident.

So far, phosphorylation, ubiquitinylation and SUMOylation of only a few residues have been demosntrated to have an effect on RelB activity, either enhancing or weakening it (Fig. 4),

indicating that these post-translational modifications could either induce RelB to display an inhibitory or activation function.

Serine 552 and Threonine 84

RelB post-translational phosphorylations were first described on threonine 84 and serine 552 sites using in vitro kinase assays [START_REF] Marienfeld | Signal-specific and phosphorylation-dependent RelB degradation: a potential mechanism of NF-κB control[END_REF] . Moreover, it was described by in vivo labeling in murine EL-4 T cells that the phosphorylation of these two sites is induced by T cell receptor or TPA/ionomycin stimulation. T cell receptor or TPA/ionomycin stimulation decreased RelB protein expression in human peripheral blood T cells and Jurkat cells. However, TNFα did not alter RelB expression levels. Strikingly, stabilization of RelB was induced by defective mutation for phosphorylation of serine 552 to cysteine and threonine 84 to alanine (S552C/T84A). These results suggest that T cell receptor or TPA/ionomycin stimulation of S552 and T84 phosphorylation participate in RelB degradation. Interestingly, a cleaved form of RelB was observed following pretreatment with proteasome inhibitors in T-cells, indicating that RelB processing probably happens before its degradation by the proteasome. Of note, processing of RelB in the N-terminus section of the peptide (after arginine 85) by the paracaspase MALT1 was observed [START_REF] Hailfinger | Malt1-dependent RelB cleavage promotes canonical NF-kappaB activation in lymphocytes and lymphoma cell lines[END_REF] . Nonetheless, RelB cleavage by MALT1 in 293T cells did not depend on serine 552 and threonine 84 potential phosphorylation, suggesting that these two sites do not seem to participate in MALT1-dependent RelB processing [START_REF] Hailfinger | Malt1-dependent RelB cleavage promotes canonical NF-kappaB activation in lymphocytes and lymphoma cell lines[END_REF] .

Serine 368

RelB serine 368 was discovered in the C-terminal part of the Rel Homology domain (RHD) as a conserved amino acid in human and drosophila NF-κB subunits [START_REF] Maier | Critical role of RelB serine 368 for dimerization and p100 stabilization[END_REF] . Mutations of serine 368 with a non-phosphorylable residue (S368A) or with a phosphomimetic residue (S368E) decreased significantly RelB transcriptional activity in RelB-defective murine S107 plasmacytoma cells after reintroduction of mutants, compared to reintroduction of wild-type (WT) RelB. These results indicate serine 368 alone is crucial for the control of RelB transcriptional activity. Moreover, mutations of serine 368 strongly altered RelB ability to dimerize with its interacting partners p50, p52, RelA and p100. Interestingly, loss of serine 368 was associated with a marked decrease in p100 half-life and a correlated increase of p52. Of note, it was not the case for p105. Innate phosphorylation of RelB serine 368 is still unknown.

Serine 472

Even though RelB DNA-binding activity is not stimulated by TNFα treatment in fibroblasts, it is still known to promote a huge nuclear accumulation of RelB [START_REF] Jacque | RelA repression of RelB activity induces selective gene activation downstream of TNF receptors[END_REF] . My laboratory has recently unraveled an important pro-migratory function of RelB in fibroblasts persistently stimulated with TNFα stimulation. Outstandingly, the induction of serine 472 phosphorylation has been demonstrated to drive this pro-migratory role [START_REF] Authier | IKK phosphorylates RelB to modulate its promoter specificity and promote fibroblast migration downstream of TNF receptors[END_REF] (Fig. 5). Moreover, my lab discovered that TNFα stimulation activate the two kinases IKKα and IKKβ to interact with RelB and induce partners its phosphorylation on serine 472. In addition to TNFα, my team has observed that specific RelB phosphorylation on serine 472 is also induced in fibroblasts in response to PDGFβ [START_REF] Authier | IKK phosphorylates RelB to modulate its promoter specificity and promote fibroblast migration downstream of TNF receptors[END_REF] . Phosphorylation of RelB on serine 472 in the nucleus promotes its release from inhibitory interaction with protein IκBα, allowing to be recruited to the promoter of essential pro-migration genes, such as the metalloproteinase matrix metallopeptidase 3 (MMP3) (Fig. 5).

Indeed, my team also demonstrated that RelB serine 472 phosphorylation directly regulates MMP3 expression and pro-migration activity [START_REF] Authier | IKK phosphorylates RelB to modulate its promoter specificity and promote fibroblast migration downstream of TNF receptors[END_REF] (Fig. 5). Interestingly, it is the second phosphorylation of an NF-κB subunit to participate in fibroblast migration since phosphorylation of RelA on threonine 505, was shown to inhibit it [START_REF] Msaki | The role of RelA (p65) threonine 505 phosphorylation in the regulation of cell growth, survival, and migration[END_REF] .

Serine 151

Recent evidence that RelB phosphorylation on serine 151 by serine/threonine p21-activated kinase 4 (PAK4) has been suggested to regulate RelB DNA-binding capacity, possibly due to electrostatic repulsion with the DNA phosphate backbone [START_REF] Fusco | NF-kappaB p52:RelB heterodimer recognizes two classes of kappaB sites with two distinct modes[END_REF] , and C/EBPβ expression [START_REF] Costa | PAK4 suppresses RELB to prevent senescence-like growth arrest in breast cancer[END_REF] .

Furthermore, PAK4 -RELB -C/EBPβ axis overcome oncogene H-RAS-V12-induced senescence of Hs 578T BC cells [START_REF] Costa | PAK4 suppresses RELB to prevent senescence-like growth arrest in breast cancer[END_REF] .

Other Putative Phosphorylation Sites

Mass spectrometry analysis of RelB have point out several other putative amino acids for phosphorylation, such as serine 20, serine 37, serine 116, serine 139, serine 217, tyrosine 293, serine 425, and threonine 579 (http://www.phosphosite.org). This observation reinforces the idea the possibilities for the regulation of RelB by phosphorylation, and also raises the question of the functional relevance of such modifications in vivo.

Polyubiquitination of RelB

.RelB transcriptional activity was also reported to be increased via ubiquitination dependentmechanisms, but it was not the case for RelB nuclear localization nor DNA binding activity [START_REF] Leidner | Identification of lysine residues critical for the transcriptional activity and polyubiquitination of the NF-κB family member RelB[END_REF] .

RelB serine 368, serine 552 or threonine 84 have not been described yet to participate in the polyubiquitination of RelB non-degradative-ubiquitin chains [START_REF] Zinngrebe | Ubiquitin in the immune system[END_REF] . Indeed, various lysine residues in RelB protein have be reported and could act as ubiquitination sites. Of note, Lysine 273/274 and Lys305/308 seem to be important for the overall ubiquitination-dependent upregulation of RelB transcriptional activity, even though the specificity of these ubiquitin-chain is yet to be determined. In the same manner, the identification of the co-activators or released co-repressors involved in this ubiquitination-dependent control of RelB activity still need to be pursued.

SUMOylation of RelB

NF-κB has also been reported to be susceptible for the covalent bond with SUMO peptides at lysine residues, a mechanism called SUMOylation [START_REF] Mabb | SUMO and NF-κB ties[END_REF] . SUMOylation process requires several enzymes similar to the ubiquitination cascade: SUMO-activating protein (E1), the SUMO conjugating protein UBC 9 (E2) and a panel of SUMO ligases (E3). However, SUMOylation and ubiquitination often show opposing effects for a specific function of a protein [START_REF] Gill | SUMO and ubiquitin in the nucleus: different functions, similar mechanisms?[END_REF] . Indeed, SUMOylation was reported to decrease RelB transcriptional activity, but no change in RelB nuclear localization or its DNA-binding ability was observed [START_REF] Leidner | SUMOylation Attenuates the Transcriptional Activity of the NF-κB Subunit RelB: SUMOYLATION CONTROLS ACTIVITY OF NF-ΚB SUBUNIT RELB[END_REF] . The mutational study of lysine residues in RelB protein showed that numerous sites can be modified by SUMOylation, and inactivation of seven lysine residues-positions 387, 388, 390, 411, 414, 415, and 416-is necessary to alter RelB SUMOylation. However, the machinery behind the decrease of RelB transcriptional activity Another post-translational modification reported to modulate the functionality of via SUMOylation currently have not been described yet.

Because RelB is a transcriptional factor that can activate or repress the transcription of target genes, this suggests that different cofactors could be recruited to RelB depending on its posttranslational-modifications, leading to specific and precise RelB-dependent transcriptional responses. Similarly, several post-translational modifications could induce the recruitment of a same cofactor but with different genes targeted, thereby producing different RelB functions.

Since these modifications could alter RelB degradation, they could also determine the timelength of RelB-dependent activation in response to several inductors. However, RelB cleavage and following release resulting from classical NF-κB activation pathway have not been associated with specific post-translational modifications yet. Discovering and describing RelB post-translational modifications will improve to understand the fine-tuned regulation of RelB and the alternative NF-κB pathway and specificity of its reponses to different stimuli.

Furthermore, unraveling these modifications would be also crucial to decipher the molecular mechanisms involved in the deregulated activity and the pathological consequences of RelB and the alternative NF-κB pathway. All these possibilities, considered together with all those of other NF-κB family members that interact with and regulate RelB, could explain the versatility of this factor.

II. NF-B and cancer

More than twenty-five years after their discovery, the arousing interest for the NF-κB transcription factors emanates from the pleiotropy of cellular responses these factors are associated with. Indeed, they play a key role in inflammation, innate and adaptive immunity, cell death/survival balance as well as tumorigenesis [START_REF] Baldwin | Regulation of cell death and autophagy by IKK and NF-κB: critical mechanisms in immune function and cancer: Regulation of cell death and autophagy by IKK/NF-κB[END_REF][START_REF] Naugler | NF-κB and cancer-identifying targets and mechanisms[END_REF] .

Through the generation of NF-κB abnormal forms or from defects in their transcriptional activities, the disruption of NF-κB transcription factors activity is found in both several haematological diseases (e.g. lymphoma and leukaemia) and many solid tumours [START_REF] Baud | Is NF-κB a good target for cancer therapy? Hopes and pitfalls[END_REF][START_REF] Rayet | Aberrant rel/nfkb genes and activity in human cancer[END_REF] (Fig. 6).

A. Alternative NF-κB signaling and Cancer

While canonical NF-κB pathway has been largely examined in many types of cancer, alternative NF-κB pathway remains unexplored. Abnormal activation of alternative NF-κB signaling pathway has been reported in various solid cancers (Fig. 8) and appears to be associated with tumor burden [START_REF] Tegowski | Noncanonical NF-κB in Cancer[END_REF][START_REF] Sun | The noncanonical NF-κB pathway: The noncanonical NF-κB signaling pathway[END_REF][START_REF] Cildir | Noncanonical NF-κB Signaling in Health and Disease[END_REF] .

Multiple myeloma

Alternative NF-κB pathway is crucial for B-cell homeostasis and germinal center establishment [START_REF] Miyawaki | A new mutation,aly, that induces a generalized lack of lymph nodes accompanied by immunodeficiency in mice[END_REF][START_REF] Koike | The splenic marginal zone is absent in alymphoplasticaly mutant mice[END_REF][START_REF] Franzoso | Mice Deficient in Nuclear Factor (NF)-κB/p52 Present with Defects in Humoral Responses, Germinal Center Reactions, and Splenic Microarchitecture[END_REF][START_REF] Caamaño | Nuclear Factor (NF)-κB2 (p100/p52) Is Required for Normal Splenic Microarchitecture and B Cell-mediated Immune Responses[END_REF][START_REF] De Silva | Transcription factors of the alternative NF-κB pathway are required for germinal center B-cell development[END_REF] . Multiple myeloma (MM) is caused by deregulated proliferation of center mature B cells in bone marrow [START_REF] Victora | Germinal centers[END_REF] . Strikingly, around 15-20% of multiple myeloma patients harbor NF-κB-activating mutations. Moreover, mutations of alternative pathway regulators such as NIK, TRAFs, cIAPs, CD40, LTβR, CYLD, p50/p105, and p52/p100 have been observed, with TRAF3 inactivating mutations being the most recurrent genetic features.

Abnormal activation of the alternative NF-κB pathway due to persistent cleavage of p100 to p52 by NIK stabilization has also been observed in several TRAF3 mutated MM cell lines [START_REF] Keats | Promiscuous Mutations Activate the Noncanonical NF-κB Pathway in Multiple Myeloma[END_REF][START_REF] Annunziata | Frequent Engagement of the Classical and Alternative NF-κB Pathways by Diverse Genetic Abnormalities in Multiple Myeloma[END_REF] .

Moreover, activation of the NF-κB alternative pathway depends on proteasome degradative function for p100 processing to p52. Dexamethasone and proteasome inhibitors, which have been clinically approved for MM treatment, can inhibit NF-κB activation [START_REF] Scheinman | Role of Transcriptional Activation of IkappaBalpha in Mediation of Immunosuppression by Glucocorticoi ds[END_REF][START_REF] Auphan | Immunosuppression by Glucocorticoids: Inhibition of NF-kappaB Activity Through Induction of IkappaB Sy nthesis[END_REF][START_REF] Raab | Multiple myeloma[END_REF] . Remarkably, constitutive activation of NF-κB alternative pathway by TRAF3 inactivating mutations drives on one hand resistance to dexamethasone but on the other hand increased to proteasome inhibition [START_REF] Keats | Promiscuous Mutations Activate the Noncanonical NF-κB Pathway in Multiple Myeloma[END_REF] . RelB DNA-binding activity was also demonstrated to be constitutively activated in MM patients and to exert a crucial anti-apoptotic activity in MM cell lines by regulation of anti-apoptotic cIAP2 expression [START_REF] Cormier | Frequent Engagement of RelB Activation Is Critical for Cell Survival in Multiple Myeloma[END_REF] .

Prostate cancer

Alternative NF-κB signaling has been also correlated with androgen receptor (AR) activity, suggested to play an important role in promoting prostate cancer. While prostate cancer can be treated with surgical or chemical androgen deprivation therapy [START_REF] Tan | Androgen receptor: structure, role in prostate cancer and drug discovery[END_REF] , development of acquired resistance often due to AR ligand-independent activity has proven to be challenging [START_REF] Harris | Androgen deprivation therapy: progress in understanding mechanisms of resistance and optimizing androgen depletion[END_REF]

Pancreatic cancer

Activation of alternative NF-κB signaling, partially through NIK stabilization and constant p100 processing, has also been detected in pancreatic cancer cell lines [START_REF] Prabhu | Critical role of NF-κB in pancreatic cancer[END_REF][START_REF] Wharry | Constitutive non-canonical NFkappaB signaling in pancreatic cancer cells[END_REF] . Protein deregulation of several regulators of the alternative NF-κB signaling has been reported to play an important role for the tumorigenicity of pancreatic cancer cells. Indeed, NIK stabilization, due to constitutive degradation of its negative regulator TRAF2, is suggested to be crucial for pancreatic cancer cell proliferation [START_REF] Döppler | Downregulation of TRAF2 Mediates NIK-Induced Pancreatic Cancer Cell Proliferation and Tumorigenicity[END_REF] . Moreover it has been shown that glycogen synthase kinase-3 (GSK3α) activity induces p100 processing and nuclear accumulation of p52 in MiaPaCa-2 pancreatic cancer cell line to promote cell proliferation and cell survival [START_REF] Bang | GSK-3α promotes oncogenic KRAS function in pancreatic cancer via TAK1-TAB stabilization and regulation of noncanonical NF-κB[END_REF] . It has also been reported that induction of RelB expression by activated Nuclear Protein 1 (Nupr1) in KrasG12D premalignant lesions and PDAC cell lines. Subsequently, RelB overexpression protects cells from nutrient deprivation apoptosis through regulation of immediate early response 3 (Ier3) [START_REF] Hamidi | Nuclear protein 1 promotes pancreatic cancer development and protects cells from stress by inhibiting apoptosis[END_REF] . In addition to this function, RelB:p52 heterodimers have been demonstrated

to control human PDAC cell cycle progression through the S-phase kinase associated protein 2 (Skp2) and p27Kip1 [START_REF] Schneider | IKKalpha controls p52/RelB at the skp2 gene promoter to regulate G1-to S-phase progression[END_REF] .

Ovarian cancer

Activation of the alternative NF-κB signaling pathway has been correlated with ovarian cancer stem cell (CSC) maintenance and self-renewal [START_REF] House | NFκB Promotes Ovarian Tumorigenesis via Classical Pathways That Support Proliferative Cancer Cells and Alternative Pathways That Support ALDH+ Cancer Stem-like Cells[END_REF] . Indeed, SKOV3 cell line expressing CD44 (CSC marker) showed and increased RelB and IKKα protein levels compared to SKOV3 cells not expressing CD44. Strikingly, IKKα inhibition blocked CD44+ SKOV3 cell line colonyforming capacity in vitro and tumorigenicity in vivo [START_REF] House | NFκB Promotes Ovarian Tumorigenesis via Classical Pathways That Support Proliferative Cancer Cells and Alternative Pathways That Support ALDH+ Cancer Stem-like Cells[END_REF][START_REF] Gonzalez-Torres | NF-κB Participates in the Stem Cell Phenotype of Ovarian Cancer Cells[END_REF] . RelB expression was also observed to be increased in other CSC OC cell lines [START_REF] House | NFκB Promotes Ovarian Tumorigenesis via Classical Pathways That Support Proliferative Cancer Cells and Alternative Pathways That Support ALDH+ Cancer Stem-like Cells[END_REF] . In addition, inhibition of RelB in OC cell xenografts noticeably lowered tumor burden [START_REF] House | NFκB Promotes Ovarian Tumorigenesis via Classical Pathways That Support Proliferative Cancer Cells and Alternative Pathways That Support ALDH+ Cancer Stem-like Cells[END_REF] . Even though similar results were obtained with canonical NF-κB pathway subunit RelA, RelB had a superior expression level and function than RelA in CSC OC cell lines [START_REF] House | NFκB Promotes Ovarian Tumorigenesis via Classical Pathways That Support Proliferative Cancer Cells and Alternative Pathways That Support ALDH+ Cancer Stem-like Cells[END_REF][START_REF] Gonzalez-Torres | NF-κB Participates in the Stem Cell Phenotype of Ovarian Cancer Cells[END_REF] . It has been also observed that RelB activation by secreted ECMinteracting protein Transglutaminase (TG2) promotes the induction of CD44 expression and downregulates E-cadherin, suggesting that RelB is crucial in TG2-dependent OC metastasis development [START_REF] Yakubov | Extracellular tissue transglutaminase activates noncanonical NF-κB signaling and promotes metastasis in ovarian cancer[END_REF] . Alternative NF-κB signaling is also important for CSC maintenance in endometrioid endometrial cancer (EEC). Expression of RelB was elevated in EEC cell lines and cocomitent elevated expression of c-Myc, cyclins D1 and E, and Bcl-2 and Bcl-xl [START_REF] Ge | RelB/NF-κB links cell cycle transition and apoptosis to endometrioid adenocarcinoma tumorigenesis[END_REF] .

Inhibition of RelB activation in EEC cell lines affected cell proliferation in vitro tumor burden in vivo [START_REF] Ge | RelB/NF-κB links cell cycle transition and apoptosis to endometrioid adenocarcinoma tumorigenesis[END_REF][START_REF] Campbell | Cisplatin mimics ARF tumor suppressor regulation of RelA (p65) nuclear factor-kappaB transactivation[END_REF] .

Glioma and glioblastoma

It has been also observed that RelB promotes glioma tumor growth and invasion partially due to matrix metalloproteinase 9 (MMP-9) Olig2, a regulator of cancer stem cell proliferation in glioma. These results highlight the therapeutic potential of targeting NF-κB alternative signaling to treat theis aggressive disease [START_REF] Lee | The NF-κB RelB protein is an oncogenic driver of mesenchymal glioma[END_REF][START_REF] Cherry | Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) promotes glioma cell invasion through induction of NF-κB-inducing kinase (NIK) and noncanonical NF-κB signaling[END_REF] . More recently, two cytokines interleukin-1β (IL-1) and oncostatin M (OSM) were observed to negatively correlate with glioblastoma multiforme (GBM) patient survival and to activate RelB in GBM cells. RelB was also shown to interact with transcription factor Yin Yang 1 (YY1) in cell lines, driving a constant cytokines release to create feedforward inflammatory loop 89 .

Breast cancer

Alternative NF-κB pathway has been demonstrated to regulate mammary gland development [START_REF] Cao | IKKα Provides an Essential Link between RANK Signaling and Cyclin D1 Expression during Mammary Gland Development[END_REF][START_REF] Demicco | RelB/p52 NF-kappaB complexes rescue an early delay in mammary gland development in transgenic mice with targeted superrepressor IkappaB-alpha expression and promote carcinogenesis of the mammary gland[END_REF] . Since the discovery of NF-κB activation in breast cancer (BC) more than twenty years ago [START_REF] Sovak | Aberrant nuclear factor-kappaB/Rel expression and the pathogenesis of breast cancer[END_REF] , RelB expression and activity investigation in the promotion of breast cancer has been mainly focused on its correlation with estrogen receptor (ER) expression. Expression of RelB was found to be inversely correlated with ER expression in breast tumors negative [START_REF] Wang | Oestrogen signalling inhibits invasive phenotype by repressing RelB and its target BCL2[END_REF] .

Indeed, RelB expression was demonstrated to be negatively regulated by ERα signaling in human BC cell lines [START_REF] Wang | Oestrogen signalling inhibits invasive phenotype by repressing RelB and its target BCL2[END_REF] . RelB was also demonstrated to induce epithelial to mesenchymal transition (EMT) by a direct upregulation of Bcl-2 synthesis in ERα negative BC cell lines [START_REF] Wang | Oestrogen signalling inhibits invasive phenotype by repressing RelB and its target BCL2[END_REF] .

Further, RelB was shown to reciprocally inhibit ERα expression in BC cell line via the regulation of the zinc finger repressor protein Blimp1 (B-lymphocyte-induced maturation protein) [START_REF] Wang | RelB NF-kappaB represses estrogen receptor alpha expression via induction of the zinc finger protein Blimp1[END_REF] . High mRNA co-expression of RelB and p100/p52 was more recently demonstrated to be associated with poor prognosis in ER positive breast cancer patients [START_REF] Rojo | Non-canonical NF-κB pathway activation predicts outcome in borderline oestrogen receptor positive breast carcinoma[END_REF] . FoxA1, an important regulator of ER transcriptional activity, has also been shown to control RelB synthesis in a subset of ER negative BC cell lines [START_REF] Naderi | Cross-regulation between FOXA1 and ErbB2 signaling in estrogen receptor-negative breast cancer[END_REF] . Interestingly, RelB-containing dimers have been demonstrated to regulate gene expression by co-activation with histone methyltransferase enhancer of zeste homolog 2 (EZH2) in triple-negative breast cancer (TNBC) cells [START_REF] Lee | Context-specific regulation of NF-κB target gene expression by EZH2 in breast cancers[END_REF] . In addition, EZH2 can also induce RelB expression via a methyltransferase-independent function, promoting TNBC tumor-initiating cells (TIC) ability for self-renewal [START_REF] Kendellen | Canonical and non-canonical NF-κB signaling promotes breast cancer tumor-initiating cells[END_REF] . It was recently shown that RelB stimulates EMT and tumorsphere formation of BC TICs by upregulating inflammatory cytokines IL-1β and IL-6 [START_REF] Kendellen | Canonical and non-canonical NF-κB signaling promotes breast cancer tumor-initiating cells[END_REF] . Inhibition of RelB expression lowered survival in response to γ-irradiation and the chemotherapeutic agent doxorubicin in TBC cell lines while introduction of RelB in a low-RelB BC cell line protected these cells from radiation [START_REF] Mineva | Inhibition of RelB by 1,25-dihydroxyvitamin D3 promotes sensitivity of breast cancer cells to radiation[END_REF] .

Additionally, high serum levels of 25-hydroxyvitamin D, associated with lower breast cancer risk and mortality, decreased RelB expression [START_REF] Mineva | Inhibition of RelB by 1,25-dihydroxyvitamin D3 promotes sensitivity of breast cancer cells to radiation[END_REF] . Recent evidence that RelB phosphorylation on serine 151 by serine/threonine p21-activated kinase 4 (PAK4) has been suggested to regulate

RelB DNA-binding capacity, possibly due to electrostatic repulsion with the DNA phosphate backbone [START_REF] Fusco | NF-kappaB p52:RelB heterodimer recognizes two classes of kappaB sites with two distinct modes[END_REF] , and C/EBPβ expression [START_REF] Costa | PAK4 suppresses RELB to prevent senescence-like growth arrest in breast cancer[END_REF] . Furthermore, PAK4 -RELB -C/EBPβ axis overcome oncogene H-RAS-V12-induced senescence of Hs 578T BC cells [START_REF] Costa | PAK4 suppresses RELB to prevent senescence-like growth arrest in breast cancer[END_REF] .

B. Targeting NF-B in oncology

The activation of both NF-κB pathways contains many key steps such as ligand-specific cell surface receptor binding, cytoplasm signal transduction, nuclear translocation of NF-κB complexes and ultimately their binding to specific regions of genomic DNA leading to the transcriptional regulation of specific target genes. Hence, the therapeutic approaches to inhibit NF-κB constitutive abnormal activity in oncology rely on the inhibition of one of those key steps (Fig. 9).

The abundant number of studies demonstrating the pivotal role of NF-κB abnormal activation in the development and the progression of cancer, as well as in acquired resistance to conventional anti-cancer therapies are the foundation of the emergence of NF-κB-targeted therapies are based upon. Therefore, the pharmaceutical industry has deployed tremendous efforts in order to develop such anti-NF-κB therapies, many of which are currently being tested in clinical trials. DNA binding inhibition.

Antagonists of ligand-specific cell surface receptor binding

Interaction between receptor RANK and its ligand RANKL activates both classical and alternative NF-κB pathways and play a major part in the bone resorption activity of osteoclasts 100,101 . It is also involved in the occurrence of skeletal-related events associated with certain types of cancers (e.g. multiple myeloma) 102,103 , and with a few antitumor treatment (e.g.

aromatase inhibitors for women suffering from breast cancer) 104 , but also involved with bone metastases appearance (e.g. prostate cancer) 105 . Circulating tumour cells secret osteoblast activation factors (PHTrP, IL-1, IL-11) inducing RANKL production (Fig. 10). Subsequently, RANKL binds to its receptor RANK on pre-osteoclasts, inducing their differentiation in osteoclasts, and on osteoclasts, activating their osteolytic function 106 . Denosumab (Prolia  ), is a human IgG2 monoclonal antibody that targets RANKL, approved by the FDA for the prevention of skeletal-related events for postmenopausal women suffering from breast cancer and receiving aromatase inhibition therapy (2010) 107 , for men suffering from castrate-resistant prostate cancer receiving androgen deprivation therapy (2011) 108 and for patients suffering from multiple myeloma (2018) 109 . human monoclonal RANKL antagonistic antibody, prevents RANK/RANKL-induced bone resorption and bone metastases appearance in cancer patients (e.g. multiple myeloma, breast cancer, prostate cancer). Denosumab has been approved for the prevention of cancer skeletalrelated events and the treatment of bone metastases in cancer patients.

Antagonists of cytoplasm transduction cascade a. IAPs antagonists

The overexpression of cIAP1 and cIAP2, both part of the family of IAPs proteins containing 8 known members in man 110 , has been linked to tumour cell survival. Genetic amplification of cIAP1 and cIAP2, identified in several types of tumours (e.g. oesophageal squamous cell carcinoma 111 , pancreatic cancer 112 ) and chromosomal rearrangement of cIAP2 (especially observed in MALT lymphoma [mucosa-associated lymphoid tissue] 113 ) correlate with a poor prognosis in patients and contribute to tumour resistance against conventional therapies 114,115 .

Considering the high expression level of cIAPs in various types of cancer and their function in tumour cell survival, small molecules mimicking a mitochondrial inhibitor of cIAPs, SMAC, were developed 116 . These antagonists induce cIAP1/2 degradation by the proteasome, thus activating both NF-B signalling pathways through the stabilization of NIK (Fig. 9) 117 leading to TNFα production and thereby to the formation of a cell death complex, the ripoptosome 118 .

These IAPs antagonists also provoke TNFα-induced cell death by hindering the inhibitory activity of cIAP1/2 and XIAP on the caspase activation cascade 119 . Although these IAPs antagonists have a moderate effect on tumour cell death, in vitro and in vivo studies have described that these IAPs antagonists best potentiate cancer cell death when combined with other antitumor treatments such as chemotherapy or oncolytic virus therapy 120,121 . More recently, it has also been described that SMAC mimetics in combination with oncolytic viruses can promote anticancer T-cell immunity 122 . These molecules are currently in phase I/II clinical trials.

b. IKK complex activity inhibition

The activation of the classical NF-κB pathway (RelA) and the alternative NF-κB pathway relies on the phosphorylation of IκB inhibitory proteins by IKK (IκBα for the classical pathway and p100 for the alternative pathway) (Fig. 9). IKK is a multi-protein complex composed of two catalytic subunits, IKKα and IKKβ, and the regulatory subunit NEMO/IKKγ 123,124 . Hence, Inhibiting IKK complex activity has represented a leading strategy for the past years. In order to do so, several approaches were developed either by targeting IKKα and/or IKKβ activity (e.g. with the compounds MLN120B and BMS345-541) 125 or the regulatory subunit NEMO/IKKγ (with the help of NEMO peptides blocking its dimerization 126 and NBD interfering with NEMO-IKKβ interaction 1 ), either by aiming at the IKK activation cofactors (such as HSP90 inhibitor 17AAG, a derivative of the antibiotic geldanamycin) [37]. To our knowledge, despite the continuous efforts supported by pharmaceutical industry in the field of oncology, these inhibitors exhibit no therapeutic benefit in clinical trials probably due to in vivo toxicity, lack of specificity and poor bioavailability.

c. Inhibition IκBs inhibitory proteins degradation by proteasome inhibitors

Proteasome-mediated degradation of IκBs is a key step of NF-κB activation 1 (Fig. 9).

Bortezomib (Velcade  ), a reversible 26S proteasome inhibitor approved by the FDA for multiple myeloma, proved its clinical efficacy alone or in combination in several phase III clinical trials 127 . Nevertheless, its clinical activity might not systematically be due to NF-κB inhibition. FDA approved the proteasome inhibitor carfilzomib (Krypolis  ) in the treatment of multiple myeloma in 2012 128 . Approved in 2015 by the FDA in the treatment of multiple myeloma, ixazomib (Ninlaro  ) is the first oral reversible 20S proteasome inhibitor 129,130 .

Compared to bortezomib, ixazomib promotes a stronger antitumor activity in vivo and is active in bortezomib resistant mice models. Neither ixazomib nor carfilzomib induce severe peripheral neuropathy, one of the main limiting effects of bortezomib 130 . Although promising, these drugs remain highly harmful because of the many other signalling pathways connected to the ubiquitin-proteasome system that are essential to sustain a normal cell functions, limiting then the therapeutic index of such inhibitors.

Nuclear translocation inhibition

Once Henceforth, the major challenge for clinicians and researchers will be not only to identify the patients who will benefit from an anti-NF-κB therapy, but also which of the classical and alternative pathway has to be targeted, and what step of NF-κB signalling cascade must be inhibited. Besides, reinforced focus should be on the development of NF-κB inhibitors that do not alter other signalling pathways and that are more effective on cancer cells than normal cells.

Moreover, although a highly potent inhibition of NF-κB is required during treatment, this should be a temporary and reversible effect in order to limit the long-term risks of immunosuppression. Since this field of research is making huge strides and constitutes a major challenge, it is highly likely that such compounds will be used in clinics in a near future.

In the past two decades, much effort has been made by the pharmaceutical industry to target the NF-κB pathways in cancer. Proteasome inhibitors such as Bortezomib (Velcade  ) or Ixazomib (Ninlaro  ) were approved for the treatment of multiple myeloma, but have a low therapeutic index and cause systemic toxicity due to the pleiotropic function of the proteasome 127,130 .

Similarly, despite extensive effort, IKK inhibitors have failed to enter clinical trials due to in vivo toxicity, low bioavailability, and lack of specificity [START_REF] Baud | Is NF-κB a good target for cancer therapy? Hopes and pitfalls[END_REF] . In regards to recent outstanding progress in the understanding of NF-B regulation -including post-translational modifications [START_REF] Authier | IKK phosphorylates RelB to modulate its promoter specificity and promote fibroblast migration downstream of TNF receptors[END_REF]138,139 , and function 140,141 , new classes of NF-B inhibitors will undoubtedly emerged and there is hope that it will be learnt how to use these compounds in the clinic.

C. NF-κB in the New Era of Cancer Therapy

As the development of precision and personalized medicine remains a major goal for optimal cancer treatment, improving the knowledge on molecular pathogenesis of cancers and deciphering the different malignant behavior acquired by cancer cells are two key elements of the puzzle to tailored therapies (Fig. 11). On the one hand, molecular profiling of cancers through cutting-edge technologies is providing access to a large array of information on cancer genetics and cancer genome for each patient.

This allows the identification of novel druggable vulnerabilities and biomarkers predicting response to treatment. On the other hand, the variety of properties that cancer cells exhibit to survive and thrive, well-known as cancer hallmarks, is also a source of targetable features to prevent cancer development and progression along with resistance to cancer treatment 142 .

Beside the first generation of cancer hallmarks 143 , a second generation has emerged that includes reprogramming of energy metabolism and escape from immune destruction and has recently been recognized as being equally critical for cancer development and progression 142 (Fig. 11).

Strengthening the efficiency of immune checkpoint inhibitors through NF-B

Even though immune checkpoint blockade has had great clinical success through the targeting of immune checkpoints programmed cell death and its ligand 1 (PD-1/PD-L1) or cytotoxic T lymphocyte antigen 4 (CTLA-4), a substantial number of patients do not respond to these immunotherapies 144 . Thus, the identification of T cell immunosuppression mechanisms and pathways enhancing the efficiency of immune checkpoint inhibitors is required to optimize their clinical use. Very interestingly, the classical and alternative NF-κB pathways have recently been highlighted as promising molecular targets to promote the efficacy of anti-tumor activity of checkpoint inhibitors [145][146][147] (Fig. 13). Combination of anti-PD-1 therapy with activation of the alternative NF-B pathway (using either agonistic CD40 mAbs or SMAC mimetics) leads to complete and durable anti-tumor response that was not seen using each monotherapy in

NF-B and CAR T cell efficacy

Breakthrough chimeric antigen receptor (CAR) T cell immunotherapy, promoted as the "advance of the year" in 2018 by the American Society of Clinical Oncology (ASCO), could change the landscape of cancer therapy. However, even though it shows remarkable clinical results in several B-cell malignancies [153][154][155] , many challenges arise from the complexity of CAR T cell procedures and anti-tumor efficacy (Fig. 14). signaling prevented tonic CAR signaling-dependent T cell toxicity 160 . These observations are reminiscent of the pro-apoptotic role of NF-B in T cells 161,162 . Further studies will be required to better understand the role and specificity of action of each TRAF molecules and the NF-κB signaling pathways in 4-1BB CAR T cells functions under antigen-dependent and independent stimulation to design optimal CARs and generate potent and longer-lasting strategies for CAR T cell efficacy [163][164][165][166] .

NF-B activation by RANK/RANKL in bone cancer complications and BRCA1 driven breast cancer

Bone metastasis and skeletal-related events (SREs) are major hurdles for survival and quality of life of many cancer patients, particularly breast cancer and multiple myeloma 167 . Therefore, much effort has been made to prevent and delay SRE appearance and bone metastasis development in cancer patients. NF-B activation mediated through receptor activator of NF-B (RANK)/RANK ligand (RANKL) has been demonstrated to play a major role in these clinical manifestations [START_REF] Vaira | RelB is the NF-κB subunit downstream of NIK responsible for osteoclast differentiation[END_REF]103,[168][169][170] (Fig. 16A). The primary cellular mechanism responsible for bone metastasis and SREs is osteoclastic activation and associated bone resorption. Circulating tumor cells secret osteoblast activation factors (PHTrP, IL-1, IL-11) inducing RANKL production. Subsequently, RANKL binds to its receptor RANK not only at the surface of preosteoclasts inducing their differentiation in osteoclasts, but also at the membrane of osteoclasts, activating their osteolytic function. RANK/RANKL has also been involved in SREs induced by anticancer agents such as aromatase inhibitors in women with breast cancer 171 and by androgen deprivation therapy in castration-resistant prostate cancer 172,173 . Denosumab (Xgeva  , Prolia  ) is a human IgG2 monoclonal RANKL antagonistic antibody approved for the prevention of SREs and the treatment of bone metastases for cancer patients 106,107,[174][175][176] (Fig. 16A).

Recent studies also demonstrated that the RANK/RANKL/NF-κB axis is a major element of breast cancer in BRCA1 mutant patients [177][178][179] . High RANK expression has been observed in

NF-B genetic profiling to define new clinical subgroups of cancer patients: highlights on B-cell lymphoma and anti-BTK ibrutinib responsiveness

Genetic defects in several components of the classical and alternative NF-B signalling are now frequently reported in B cell lymphomas. Very importantly, those NF-B-linked mutations appeared to enhance the clinician's ability to predict which gene mutation is the cause of these lymphomas and assist them to select the patients more prone to respond to a specific treatment.

Diffuse large B-cell lymphoma (DLBCL), the most common form of non-Hodgkin lymphoma, is classified in two distinct molecular subtypes: germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Chronic active BCR signaling has been demonstrated as a pathogenetic mechanism linked to NF-B activation in ABC DLBCL [181][182][183] (Fig. 17). Chronic BCR activation is also involved in other B-cell malignancies, such as Mantle Cell Lymphoma, (MCL) or chronic lymphocytic leukemia, through abnormal activation of NF-B 184-188 .

Bruton's Tyrosine Kinase (BTK) is a key link between the BCR activating cascade and the chronic activation of NF-B 182,183 . Ibrutinib is a first-in-class BTK inhibitor approved for clinical use in relapse/refractory MCL and is also very promising as a new treatment for ABC-DLBCL. Activating mutations that target the gene encoding the BCR subunits CD79A and CD79B, the BCR pathway adapter caspase recruitment domain family member 11 (CARD11)

and Toll-like receptor (TLR) signaling adapter MYD88 promote NF-B activity in ABC DLBCL [181][182][183]189 . Loss-of-function mutations also occurred in genes encoding negative regulators of both the classical and alternative NF-B pathways, including TNFAIP3 (negative regulator of the classical NF-B pathway) and TRAF3 (negative regulator of the alternative NF-B pathway) [190][191][192][193] . Chronic activation of BCR signaling by autoantigens and mutations of BCR subunits CD79A and CD79B drives the IKK kinase activation through the CARD11-BCL10-MALT1 complex (CBM complex) 183,194 . Survival of ABC-DLBCL cell lines is driven by a constitutive activation of NF-B induced by chronic active BCR signaling and activating mutations of MYD88 (the most prevalent mutation being MYD88 L265P) 181,183,189 .

In refractory/relapsed ABC DLBCL patients, the most striking response rate (up to 80%) upon ibrutinib treatment was observed in patients bearing both CD79B and MYD88 mutations 195 .

These observations suggest that not only CD79A/B but also MYD88 status should be taken into In relapsed/refractory MCL, ibrutinib induces response rates of 70%, whereas 30% of patients are refractory to ibrutinib therapy. MCL cells that are resistant to ibrutinib tend to have somatic mutations in inhibitors of the alternative NF-B pathway, such as BIRC2/3, TRAF2/3 184,185 .

These mutations cause ibrutinib insensitive cell lines to depend on the deregulated alternative NF-B pathway for survival and proliferation 184,185 , thus highlighting a direct link between alternative NF-B activation and ibrutinib resistance in MCL. of metabolic deregulation as one of the hallmarks of cancer 142 is recent, only a small number of studies have clearly demonstrated an important and complex role of NF-B in cancer metabolism, we will therefore describe them with scrutiny. Indeed, it has been reported that depending on the status of p53, RelA can either promote or repress both glycolysis or oxidative phosphorylation (Fig. 19). expressing cancer cells, RelA interacts with the heat shock protein mortalin, thus leading to its translocation to the mitochondria where RelA represses Oxphos.

RelB and cell metabolism

As the interest on the roles of NF-κB in cancer metabolism is rising, further investigations need to been conducted o meticulously describe and characterize the part of the other NF-κB subunits.

Recent studies have led to believe that RelB could also be implicated in several metabolic pathways in cancer cells.

B. Breast cancer metabolism

Breast cancer, the most frequent cancer in women, is a highly heterogeneous disease classically defined by immunohistochemistry in 4 major subtypes: luminal A, luminal B, HER2 + , and triple-negative tumors. Moreover, transcriptomic characterization of breast cancer classifies tumors into five subtypes: luminal A, luminal-B, HER2-enriched, basal-like, and a normal breast-like group 209,210 (Fig. 21). Although all intrinsic subtypes can correlate with the subtypes defined by immunohistochemistry, basal-like tumors exhibit the greatest overlap with triple negative breast cancer (TNBC). Between 50-75% of TNBC have a basal transcriptomic profiling and approximately 80% of basal-like tumors are TNBC (Fig. 21). Although breast cancer mortality rates have reduced due to iproved systemic adjuvant therapy, most breast cancer deaths are still caused by distant metastasis. BC molecular heterogeneity is also demonstrated in its variety of metabolic reprogramming and functions. Understanding BC cell metabolism appears of great interest for the identification of novel potential therapeutic targets.

Glucose metabolism

Glucose is the one of most important fuel sources for cells' energy production, processed by glycolysis is a pathway almost commonly shared amongst all prokaryotic and eukaryotic cells.

As glucose processing in glycolysis is a multistep enzymatic process producing many byproducts that are at the source of other metabolic pathways 212,213,214,215 (Fig. 22). In 1956, Warburg first postulated that cancer cells had a significantly higher uptake of glucose as than normal tissues. Warburg also described that cancer cells preferentially used anaerobic glycolysis rather than oxidative phosphorylation, the pyruvate produced by a full cycle of glycolysis is converted into lactate rather than converted into acetyl-CoA by the mitochondrial metabolic pathways such as the tricarboxylic acid (TCA) cycle and oxidative phosphorylation.

Warburg attributed this process, known today as the "Warburg Effect", due to impaired mitochondrial functions in most cancer cells 217,218 (Fig. 23). This effect has been updated since increase of anaerobic glycolysis can also occur even in the presence of functional mitochondria, and upregulation of mitochondrial metabolic pathways have also been described also to play a crucial role in many malignant cancer behaviours 217 .

Many human cancer cells exhibit elevated rate of glycolysis and other metabolic pathways starting with from glycolytic-produced metabolites. These branching metabolic pathways are involved in the synthesis of essential cellular macromolecules to meet the demands for highly proliferative cancer cells 220 .

a. Deregulation of glucose transporter expression in BC

Glucose enters the cell through plasma glucose transporters (GLUT) 221 . The GLUT family are facilitator transporters involved in the transport of glucose in the direction of its concentration gradient. This family of transporters is composed of 14 members. Each of the GLUT transport proteins has different affinities for glucose and other hexoses such as fructose. GLUT1, GLUT3

and GLUT4 have a strong affinity for glucose, allowing glucose transport at a high rate under normal physiological conditions 222 . It has been described that altered expression of several glucose transporters such as GLUT1, GLUT2, GLUT3, GLUT4, GLUT5 and GLUT 12 have been found on the membrane of cancer cells to play a role in the grade and the prognosis of BC 223 (Fig. 23).

Overexpression of GLUT1 is critical for the uptake of glucose in breast cancer cell lines 222 . The overexpression of GLUT1 induces a change in the metabolism of breast epithelial cells which precedes the morphological malignant changes into breast cancer cells, and therefore may be a fundamental part of the neoplastic process. In addition There is a strong correlation between the expression of the GLUT1 gene and higher-grade breast cancers with a proliferative index and a lower degree of differentiation and a higher malignant potential, invasion and therefore a poorer prognosis 222,[224][225][226] . In addition GLUT1 and GLUT3 appear to be important markers in the differentiation of endometrial and breast tumors 227 . GLUT3 is also overexpressed in human malignant tissue. The expression of the GLUT3 protein level would GLUT3 plays a role in cell differentiation and the invasion of in breast cancer cell lines 227,228 . Involvement of GLUT12, GLUT2 and GLUT5 in the deregulation of glucose metabolism of various cancer cells including BC have been described in cancer cell lines, therefore its potential as a new therapeutic target for the treatment of BC 229 . The potential of targeting GLUT deregulation in breast cancer is currently under intense investigation 218 . An inhibitor of GLUT1 activity has shown synergistic anticancer effects in combination with chemotherapeutic agents cisplatin or paclitaxel in a luminal BC cell line 226,230 .

The energy yield from glucose in cancer cells is lower and therefore glucose absorption must be increased. Two families of glucose transporters mediate glucose uptake in cells, sodiumdependent glucose co-transporters (SGLT) and glucose-enhancing transporters (GLUT).

The SGLT family are secondary active membrane transporters which transfer glucose coupled with Na + transport. Na+ gradient is maintained by the Na + / K + ATPase pump. For every glucose molecule transported, two Na + are also transported into the cell. SGLT transporters have a strong affinity for glucose. Overexpression of SGLT1 and SGLT2 is present in some types of cancer, such as pancreatic, prostate, lung, liver and ovarian cancer, but these transporters have not been described in breast cancer 231,232 .

b. Glycolytic enzymes and Pentose Phosphate Pathway

Due to the heterogeneity of BC, looking at the metabolic differences between molecular subtypes in order to identify new treatment strategies specific to each subtype is essential. It has been reported that glycolysis rates increase through with the aggressivity of BC [233][234][235] .

Among all BC subtypes, TNBC have a higher glycolytic gene profile than the other BC subtypes 236,237 . Deregulation of many glycolytic enzymes has been reported in TNBC. Pyruvate kinase 2 (PKM2) isoenzyme is crucial for the increased glycolytic levels and tumor growth of TNBC 238 . Lactate dehydrogenase A (LDH-A) plays a key role in the maintenance of the TNBC cell lines and LDH-A expression is higher in human breast tumor tissue than in normal tissue 239,240 . Glucose-6-Phosphate and fructose-6-Phosphate concentrations appear to be significantly lower in TNBC compared to the other BC subgroups. Fructose-1,6-bisphosphate (F1,6BP) intracellular levels, on the other hand, is significantly higher in TNBC, and has been demonstrated to plays a very important role in tumor growth by directly increasing lactate excretion and the activity of the epidermal growth factor receptor (EGFR) 233,241 .

The pentose phosphate pathway (PPP) is a major pathway in glucose catabolism which branches out from glucose 6-phosphate (G6P), the first glucose intermediates produced by glycolysis (Fig. 23). The PPP significantly regulates homeostasis and the biosynthesis nucleotides and of cellular reduction-oxidation (redox). PPP enzymes are reported to play an important role in breast cancer [242][243][244] . G6PD, the PPP rate-limiting enzyme, drives the production of NADPH. The regulation of cell proliferation and transformation appears to be related to the cellular concentration of G6PD. Overexpression of G6PD is associated with a higher risk of recurrent metastasis and poor survival of BC [245][246][247] . Moreover, PPP is an important pathway for NADPH production, a key component required by the glutathione system and other ROS-reactive oxygen species scavengers to maintain redox homeostasis nucleotide biosynthesis, which is essential for DNA replication and repair of DNA damage 242,248 . Elevated levels NADPH and nucleotides by high rates of PPP have been demonstrated

to participate in the resistance to DNA-damage-induced treatments of cancer cells, suggesting that targeting PPP is becoming a possible strategy to overcome treatment resistance 249 .

Lipid metabolism

Lipids are processed in cells beta-oxidative phosphorylation and TCA cycle to promote the synthesis of ATP, NADPH and macromolecules as well as regulate many signaling pathways [250][251][252][253] . Lipids are generally classified into two groups lipoid (phospholipids, choline glycolipids and sterols) and fat like triglycerides, sterol (cholesterol). Because of their central role in cells, the importance of lipid metabolism in cancer has shown a growing interest. Diet and obesity are important risk factors for breast cancer development, as the accumulation of fat tissue appears to be a risk factor for developing breast cancer. Moreover, obese women are more likely to have recurrence of their cancer than non-obese women [254][255][256] . Increase in fatty acids, cholesterol and triglycerides intake were found in several studies to be positively correlated with the progression of BC [257][258][259] . The deregulation of lipid metabolism has been demonstrated to play an important role proliferation, migration, invasion and therapy resistance of breast cancer cells [260][261][262] . Deregulation of lipid metabolism can be modulated by various processes intrinsic to cancer cells, such as an abnormal expression of genes, proteins, deregulation of cytokines 251,263 .

a. Fatty acid metabolism

Cholesterol is a fundamental nutrient for many body functions. Indeed, it is the precursor of steroid hormones, in particular sex hormones, the deregulation of which can be at the origin of certain cancers such as breast cancer, prostate cancer, testicular cancer or ovarian cancer 264,265 .

Cholesterol metabolism is a major contributor to cancer progression, including cell In addition, unsaturated FAs (oleate and linoleate) support cell growth, while saturated FAs such as tend palmitate are toxic to cells, suggesting that each type of fatty acid play an important and specific role. Extracellular lipid environment of breast cancer cell lines showed that the accumulation of fatty acid in cancer cells was not specific for the species of fatty acid 281 .

Adipose tissue is thought to play a role in hormone production, especially sex hormones and growth hormones. It would seem very interesting to be interested in their metabolism to possibly better understand the implication in hormone-depend breast cancers and potentially to define new targeted therapies 277 .

b. Cholesterol metabolism

Cholesterol is a fundamental nutrient for many body functions. Indeed, it is the precursor of steroid hormones, in particular sex hormones, the deregulation of which can be at the origin of certain cancers such as breast cancer, prostate cancer, testicular cancer or ovarian cancer 264,265 .

Cholesterol metabolism is a major contributor to cancer progression, including cell proliferation, migration and invasion 266 . Sterol regulatory element-binding protein 2 (SREBP2)

and RAR-related orphan receptor γ (RORγ), transcription factors regulating cholesterol metabolism, activates the cholesterol bio-synthesis program and facilitates the progression of TNBC 267 . In addition, TP53-mediated activation of cholesterol synthesis via the SREBP pathway appears to induce proliferation and self-renewal of cancer cells of the breast 268

The Caveolin-1 and Caveolin-2(CAV1 and CAV2, respectively), a protein binding with cholesterol to form plasma membrane caveolae, has been proposed to play a role in intracellular cholesterol trafficking. CAV1 expression levels also higher in BC cell lines compared to normal cells. CAV1

and CAV2 have been identified as markers for basal-like breast tumors. CAV1 and CAV2

expression is associated with highly breast aggressive tumors 269

c. Cholin metabolism

Choline (Cho) is an essential nutrient for cells. It is a precursor of membrane phospholipids, phospholipids that make up breast milk, as well as certain neurotransmitters, the best known of which is acetylcholine. Choline appears that in addition to being a fundamental nutrient for the synthesis of membranes of rapidly proliferating cancer cells, choline and its intermediate metabolites could be markers of aggressive cancer and poor prognosis 282,283 (Fig. 25).

Several observations highlight the role of choline in breast cancer. Choline appears to be a useful surrogate for monitoring response to treatment 275 . Total Cho level is not significantly changed in breast cancer cells. However, basal-like BC the have significantly higher glycerophosphorylcholine (GPC) levels than phosphatidylcholine (PC levels while the opposite is observed in luminal-like BC. Moreover, high GPC levels is associated with aggressivity for TNBC patients, and decrease of GPC levels in BC patients after treatment has been associated

with longer survival 284 . GPC therefore seems to be an interesting and promising treatment target; however its metabolism needs more insights. While it has been demonstrated that intracellular tCho levels are similar amongst breast cancer subtype, it is not the case for GPC and PC, suggesting that choline metabolism could be differentially altered depending on BC subtype. Indeed, several genes are responsible for these differences between basal-like and luminal-like BC. In particular, genes involved in membrane transport such as the SLC44A1 family, the expression of which is significantly different between the two subtypes. Expression of these transporter, also known as choline transporter protein 1 (CTL1) is lower in concentration in basal-like BC than luminal-like BC xenografts 285 . This transporter could prove to be a therapeutic target of interest. Moreover, choline is converted in PC by choline kinase A (CHKA) and choline kinase B (CHKB). Expression of both these two enzymes (CHKA and CHKB) have been demonstrated to be higher in luminallike BC than in basal-like BC 275 . Studies also showed that downregulation of CHKA dramatically reduces proliferation of BC cells. These observations suggest that liminal-like and basal-like cancer differ in the regulatory mechanisms of choline metabolism

In addition, phospholipase A2 (PLA2), an enzyme involved in the conversion of PC to Acyl-GPC, expression is in basal-like BC. This could explain why the concentration of PC is lower in basal-like BC, as well as that of GPC is higher 285 .

Other genes such as glycerol-3-phosphate dehydrogenase 1 (GPD1L), Phospholipase C Delta 4 (PLCD4) and Glycerophosphodiester Phosphodiesterase Domain Containing 3 (GDPD3) are involved in GPC levels, adding to the complexity of its levels. Estrogen receptors also play an important regulatory role on choline metabolism, proposing a functional difference of GPC metabolism in BC subtypes 275,285 .

Phospholipase D1 (PLD1) is also upregulated in various human cancers, including breast cancers. An association between the expression of PLD1 and CHKA in malignant tumors of breast cancer has recently been observed, showing that the concentration of PLD1 was higher in cancer cells than in normal cells. In addition, the analysis was also performed in vitro this time showing no significant difference between tumor cells and normal cells. This suggests that this increase may be caused by an influence of the tumor microenvironment or vascularity 250,286 .

Introduction

Breast cancer, the most frequent cancer in women, is a highly heterogeneous disease classically defined in 4 major subtypes: luminal A, luminal B, HER2 + , and triple-negative tumors.

Although breast cancer mortality rates have reduced due to improved systemic adjuvant therapy, most breast cancer deaths are still caused by distant metastasis. Metastasis is a coordinated process of multiple steps such as cancer cell dissemination, invasion, migration, intravasation, extravasation and colonization [287][288][289][290] . The identification of predictive biomarkers for distant metastasis development could help therapeutic choices and offer new targets for potential drug developpement 291 .

NF-κB transcription factors are major players in the control of cell proliferation and survival, as well as in the pathophysiology of various cancers [START_REF] Baldwin | Regulation of cell death and autophagy by IKK and NF-κB: critical mechanisms in immune function and cancer: Regulation of cell death and autophagy by IKK/NF-κB[END_REF][START_REF] Naugler | NF-κB and cancer-identifying targets and mechanisms[END_REF][START_REF] Nakanishi | Nuclear factor-κB inhibitors as sensitizers to anticancer drugs[END_REF][292][293][294] . Mammals express five NF-κB members, including c-Rel, RelB, RelA (p65), p50, and p52, which can form either homoor heterodimers [START_REF] Oeckinghaus | The NF-B Family of Transcription Factors and Its Regulation[END_REF][START_REF] Karin | Phosphorylation Meets Ubiquitination: The Control of NF-κB Activity[END_REF] . In response to a wide variety of cellular stimuli, NF-κB becomes active via one of two pathways: the classical NF-κB activation pathway, a second NF-κB pathway has more recently been discovered, the alternative NF-κB pathway. This pathway mainly controls the activity of RelB, a member of the NF-κB family [START_REF] Sun | The noncanonical NF-κB pathway: The noncanonical NF-κB signaling pathway[END_REF]123,292 .

Post-translational modifications have recently emerged as a strategy for the fine-tuned regulation of NF-κB [START_REF] Costa | PAK4 suppresses RELB to prevent senescence-like growth arrest in breast cancer[END_REF]295,296 . We have previously demonstrated that phosphorylation of RelB on Serine 472 (pS472-RelB) as an important positive regulatory mechanism of TNFα-induced fibroblast migration through specific binding to the promoter of matrix metallopeptidase 3 (MMP3) 296 . RelB expression has been reported to be inversely correlated with oestrogen receptor in BC and was shown to participate to BC cell lines invasion and tumor-initiating renewal [START_REF] Wang | Oestrogen signalling inhibits invasive phenotype by repressing RelB and its target BCL2[END_REF][START_REF] Wang | RelB NF-kappaB represses estrogen receptor alpha expression via induction of the zinc finger protein Blimp1[END_REF][START_REF] Kendellen | Canonical and non-canonical NF-κB signaling promotes breast cancer tumor-initiating cells[END_REF] . However, the clinical outcome associated with elevated levels of RelB remains unclear [START_REF] Rojo | Non-canonical NF-κB pathway activation predicts outcome in borderline oestrogen receptor positive breast carcinoma[END_REF]297 .

In this study, we have revealed that the presence of nuclear pS472-RelB is associated with poor prognosis of BC patients via distant metastasis development. We have found that pS472-RelB drives a pro-metastatic and pro-invasive phenotype in triple-negative BC cells (TNBC) and showed that pS472-RelB exerts its pro-invasive functions through direct regulation of metalloproteinase MMP13 expression and successive activity. Our results demonstrate for the first time a pivotal role of a post-translational modification of RelB in BC patient's aggressiveness and identify a new mechanism for RelB protumoral activity in TNBC cells.

Results

Nuclear presence of pS472-RelB correlates with worse outcome of BC patients

We first examined the role of pS472-RelB in the clinical outcome of BC patients by analysis of tissue microarray-based IHC (immunohistochemistry) in 210 BC tissues from a Institut Curie cohort using a custom antiphospho serine 472-specific RelB antibody previously validated 296 .

Patients were divided into two subgroups depending on the nuclear presence status of pS472-RelB (figure 1A). Nuclear presence of pS472-RelB was found to be significantly associated with reduced overall survival (OS) (figure 1B). In addition, patients with nuclear pS472-RelB presence had a higher probability of developing metastases overtime (figure 1C) and a reduced distant metastasis-free survival (DMFS) (figure 1D). Moreover, a multivariate analysis including 8 known poor prognostic variables showed that nuclear presence of pS472-RelB is an independent prognostic predictor for poor OS (Hazard ratio (HR) = 4,195, p = 0.003), DMFS (HR = 6,125, p = 0.001) and for high cumulative incidence of metastasis overtime (HR = 3,478, p = 0.002). Remarkably, this multivariate analysis also showed that nuclear presence of pS472-RelB was of worst prognosis than any of the other 8 variables included (Table S1). These indicate that nuclear presence of pS472-RelB is significantly and strongly associated with poor prognosis of BCs.

Legend to

Figure 26. Nuclear Presence of pS472-RelB Correlates with Distant Metastasis and Poor Prognosis of Human BCs

Phosphorylation of RelB serine 472 promotes metastasis development in vivo

To evaluate the metastatic capacity of pS472-RelB in vivo, we injected intracardially bioluminescent highly metastatic MDA-MB-231-D3H2LN breast cancer cells transduced with lentiviruses encoding either a cDNA for ectopic expression of WT RelB (WT RelB), a cDNA for ectopic expression of the non-phosphorylable RelB mutant S472A (S472A RelB) or a scrambled control (Control) in mice (two independent experiments, n=13 mice/per group).

Using bioluminescent imaging, we followed metastatic development from day 7 to day 41 by quantifying the number photon/sec for each mouse. Mice injected with WT RelB-transduced D3H2LN cells showed an increase in metastasis development overtime compared to the other groups (figure 2A and2B). Time to appearance of the first metastasis was also 3 times shorter in the WT RelB cells-injected mice compared to the other groups (figure 2C). Moreover, the number of metastasis per mouse and the number of mice with metastatic burden were higher in the group injected with the WT-RelB cells (figure 2D and2E). These results demonstrate that pS472-RelB drives a metastasis development of TNBC cells in vivo.

Legend to

Figure 27. Phosphorylation of RelB serine 472 promotes metastasis development in vivo

High levels of RelB serine 472 phosphorylation correlates with lethal metastatic TNBC

To begin to decipher the involvement of pS472-RelB in BC invasiveness, we first examined the levels of pS472-RelB in a panel of 12 BC cells lines (figure 3A). As previously observed [START_REF] Wang | Oestrogen signalling inhibits invasive phenotype by repressing RelB and its target BCL2[END_REF] , we confirmed an increase of RelB protein expression in TNBC cell lines compared to the other subgroups of BC (figure 3A). pS472-RelB levels were also higher in most of the TNBC cell lines, with a subset of high-expressing RelB cell lines with low levels of phosphorylation on serine 472 (figure 3A). Remarkably, TNBC patients with positive nuclear pS472-RelB staining showed a percentage of death with metastatic burden almost doubled compared to the non-TNBC with positive nuclear pS472-RelB staining (figure 3B), while negative nuclear pS472-RelB TNBC and negative nuclear pS472-RelB non-TNBC had a similar percentage of death with metastatic burden (figure 3B). Interestingly, even though the number of patients was small, TNBC patients with nuclear pS472-RelB presence had a significantly reduced DMFS than TNBC patients without nuclear pS472-RelB (figure 3C). To gain further insights on the impact of the phosphorylation of RelB on serine 472 in TNBC, we first developed a stable ectopic expression approach by introducing either an empty lentivirus or WT RelB or RelB S472A in TNBC cell line MDA-MB-231. We confirmed by immunochemistry and immunoblot that the introduction of WT RelB indeed increased the level of pS472-RelB but not the introduction of RelB S472A, even though RelB expression levels were similarly elevated (figure 3D andE). We also developed a stable RelB knockdown approach by RNA interference to study RelB endogenous role in order to prevent artefactual interpretations of the results obtained with our ectopic expression of RelB model. Knockdown of endogenous RelB also decreased pS472-Relb levels in MDA-MB-231 (figure 3E). Importantly, RelB S472A mutation did not alter RelB DNA-binding activity, while RelB knockdown did (figure 3F). We also confirmed that S472A mutation does not affect regulation of BCL2 and PRDM1, demonstrated to be RelB-dependent genes in BC [START_REF] Wang | Oestrogen signalling inhibits invasive phenotype by repressing RelB and its target BCL2[END_REF][START_REF] Wang | RelB NF-kappaB represses estrogen receptor alpha expression via induction of the zinc finger protein Blimp1[END_REF] (figure S1A

Figure 29. pS472-RelB promotes migration and invasion of TNBC in vitro.

RelB serine 472 phosphorylation-mediated pro-invasive capacity requires direct regulation of MMP13 expression and activity in TNBC cells

Next we sought to determine the mechanism controlled by pS472-RelB promoting TNBC cells migration and invasion. We examined RelB serine 472 phosphorylation status impact on the

Figure 30. RelB serine 472 phosphorylation-mediated pro-invasive capacity requires direct regulation of MMP13 expression and activity in TNBC cells

Discussion

The emerging role of post-translational modifications for the fine-tuned regulation of RelB transcriptional activity [START_REF] Costa | PAK4 suppresses RELB to prevent senescence-like growth arrest in breast cancer[END_REF]295,296 and its direct impact on BC patient prognosis remains unclear.

In the study presented here, we have uncovered a critical function of RelB phosphorylation at serine 472 as key promoter of BC metastasis development, correlating with dismal prognosis in patients. We also demonstrated a critical role for RelB in inducing TNBC cells migration and invasion via its phosphorylation at serine 472 and subsequent selective gene expression of MMP13. Our findings constitute a significant advance in understanding the fine-tuned regulation of RelB in BC, suggesting that pS472-RelB could become a prognostic marker and therapeutic target for BC metastasis.

Abnormal elevated expression levels of RelB has been reported in BC cancers and are suggested to participate to tumor aggressiveness in BC [START_REF] Sovak | Aberrant nuclear factor-kappaB/Rel expression and the pathogenesis of breast cancer[END_REF][START_REF] Wang | Oestrogen signalling inhibits invasive phenotype by repressing RelB and its target BCL2[END_REF][START_REF] Wang | RelB NF-kappaB represses estrogen receptor alpha expression via induction of the zinc finger protein Blimp1[END_REF][START_REF] Kendellen | Canonical and non-canonical NF-κB signaling promotes breast cancer tumor-initiating cells[END_REF][START_REF] Mineva | Inhibition of RelB by 1,25-dihydroxyvitamin D3 promotes sensitivity of breast cancer cells to radiation[END_REF] . Our study demonstrates that the nuclear presence of a RelB phosphorylation of serine 472 correlates with poor prognosis and development of metastasis in BC patients. These results were shown to be particularly robust as our multivariate analysis revealed that out of the 8 variables of poor BC outcome tested, nuclear presence of pS472-RelB exhibited the worst one. While RelB mRNA expression has been demonstrated in several studies to be inversely correlated with ER expression in BC tumors [START_REF] Wang | Oestrogen signalling inhibits invasive phenotype by repressing RelB and its target BCL2[END_REF] , the connection between high RelB expression levels and BC patients survival remains controversial [START_REF] Rojo | Non-canonical NF-κB pathway activation predicts outcome in borderline oestrogen receptor positive breast carcinoma[END_REF]297 . Indeed, measuring RelB expression does not necessarily determines its DNA-binding activity nor following regulation of transcriptional activity, which could explain unclear RelB clinical relevance. Even though we show that phosphorylation of RelB serine 472

does not alter RelB DNA-binding capacity in BC cells, assessment of RelB expression levels and associated-DNA-binding activity of the patients from our cohort would still provide meaningful indications on the fine regulation of RelB, Moreover, evaluating RelB DNAbinding activity in tumor samples could prove to be challenging for clinical applicability because of the quality of material needed and the time to analysis required. These results suggest that nuclear presence of pS472-RelB detection by IHC could represent the most suited approach for evaluating the clinical outcome of RelB in BC patients.

Triple-negative breast cancer (TNBC) remains the most challenging BC subtype to treat a deeper characterization of TNBC tumors could help for a better patient stratification and therapeutical decisions 291 . We demonstrate that nuclear presence of pS472-RelB distinguish two subsets of TNBC patients associated with significant worse clinical outcome and metastasis appearance, even though the number of TNBC patients evaluated was small.

Promotion of epithelial-mesenchymal transition and cell migration and invasion constitute key features for cancer progression through distant metastasis burden development 288,301 . It's been suggested that RelB participate in such aggressive phenotypes in BC cells by regulating the expression of several genes such as BCL2, Blimp1 and MMP1 [START_REF] Wang | Oestrogen signalling inhibits invasive phenotype by repressing RelB and its target BCL2[END_REF][START_REF] Wang | RelB NF-kappaB represses estrogen receptor alpha expression via induction of the zinc finger protein Blimp1[END_REF]298 . We confirm that these genes are indeed modulated by the expression levels of RelB in BC cells, however we show that the non phosphorylable RelB S472 mutant does not alter RelB-dependent regulation of these genes. We demonstrate that pS472-RelB promotes cell migration and invasion of TNBC cells in a serine 472-dependent manner via the specific induction of MMP13 and subsequent activity. Our results indicate that pS472-RelB acts through another regulating layer of RelBdependent transcriptional activity, highlighting that it may also need the recruitment of other transcriptional coactivators and/or repressors to promote of specific target genes and following associated-aggressive behavior of BC.

In recent years, MMP13 has been proposed to be associated with poor prognosis and metastasis spread in BC patients 300,[302][303][304][305] . Evaluating MMP13 expression in the patients of our cohort and establishing nuclear pS472-RelB a dependent-gene expression profiling would provide significant advances to decipher the regulation context of RelB impact on BC patient impact.

In addition, induction of RelB phosphorylation at serine 472 by platelet-derived growth factor receptor-β (PDGFβ) and tumor necrosis factor α (TNFα) has been demonstrated in fibroblasts 296 . TNFα and PDGF signaling have been suggested to be involved in BC tumor microenvironment [306][307][308] . Evaluating tumor microenvironment composition could contribute to understand the regulating mechanisms controlling RelB phosphorylation on serine 472 in BC.

The recent increase in the description of abnormal expression of RelB in several cancers such as multiple myeloma, prostate, ovarian, glioma or pancreatic cancer [START_REF] Cormier | Frequent Engagement of RelB Activation Is Critical for Cell Survival in Multiple Myeloma[END_REF][START_REF] Xu | RelB Enhances Prostate Cancer Growth: Implications for the Role of the Nuclear Factor-B Alternative Pathway in Tumorigenicity[END_REF][START_REF] Wharry | Constitutive non-canonical NFkappaB signaling in pancreatic cancer cells[END_REF][START_REF] Gonzalez-Torres | NF-κB Participates in the Stem Cell Phenotype of Ovarian Cancer Cells[END_REF][START_REF] Waters | RelB acts as a molecular switch driving chronic inflammation in glioblastoma multiforme[END_REF] highlights the relevance of assessing the phosphorylation of RelB at serine 472 in these cancers.

In conclusion, we demonstrate for the first time a significant role of a post-translational modification of RelB in BC outcome, suggesting that pS472-RelB could become a predictive biomarker for metastasis burden and a potential therapeutic target for BC.

Intracardiac experimental mouse model of metastasis.

Experimental metastasis was conducted as described 309,310 . Following intracardiac injection of

In vivo Bioluminescence and Computed Tomography Imaging

Images were acquired using IVIS SpectrumCT (Perkin Elmer). This multimodality imaging system allows the detection of tumors and metastases in X-ray tomography coregistered with optical images of tagged tumor cells without image adjustment for anatomical correspondence.

As light is only emitted by tumor cells without any background signal, bioluminescence is a highly specific and sensitive methodology for tumor detection and follow-up over time 311 . For optical detection, mice were injected intraperitoneally with 150 mg/kg of D-luciferin (Beetle luciferin, Promega, Charbonnières, France) and then anesthetized with 3% isoflurane. For primary tumors as for metastases, acquisition parameters were automatically computed by the SpectrumCT software to optimize bioluminescence signals (photons per second [p/s]) detection.

Statistical Analysis

Kaplan-Meyer estimate of overall survival were performed using the log-rank test (Mantel-Cox). Multivariate overall survival analyses were performed using Cox proportional-hazards model. Overall survival was evaluated from the date of enrolment to the date of death. Cases were censored on the date the patient was last known to be alive or, for patients in complete response dying as a result of causes unrelated to the breast cancer or treatment. Statistical significance was accessed using unpaired t tests (Prism 5.0c, GraphPad Software). A value of p=0.05 was considered as statistically significant with the following degrees: * p<0.05; **p<0.01; ***p<0.001.

Cells Culture and reagents.

MDA-MB-231 cells were grown in DMEM with 10% (vol/vol) FBS and 1% antibiotics.

Antibodies and Reagents.

The antibodies were purchased from Santa Cruz (RelB C19 # sc-226, RelA C20 # sc-372 BCL-

Immunoblotting

Immunoblotting were performed as previously described 296 .

Electrophoretic mobility shift assays for NF-κB

NF-κB activation was analyzed by electrophoretic mobility shift assay (EMSA) using the human immunodeficiency virus long terminal repeat tandem κB oligonucleotide as κB probe as previously described [START_REF] Jacque | RelA repression of RelB activity induces selective gene activation downstream of TNF receptors[END_REF] .

Scratch-Wound Assays.

Cells were grown for 36 h in DMEM with 10% FBS to reach a 80% confluent monolayer, cells were then pre-treated 2h in DMEM with 5μg/ml mitomycin C before scratchs were realised using a 0.5-mm-diameter tip. Scratch area was monitored over 24 h in DMEM with 0.1% FBS.

Scratchs assays were then quantified using the MRI wound healing tool of ImageJ (http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool).

Transwell Invasion Assays.

Cells (1×10 6 cells per dish) in DMEM with 0.1% FBS were seeded to pre-coated with 0.1%

Corning Matrigel matrix FluoroBlok systems of 8-μM pore size whereas the lower chamber was filled with 10% (vol/vol) FBS in DMEM. The chambers were incubated at 37 °C in a 5%

CO2 atmosphere for 8h, and photos were taken. Number of fluorescent invading cells was then calculated cells using ImageJ.

Displacement in 3D matrix Assays.

Cells (1500 cells per well) in DMEM 10% FBS + 4% Corning Matrigel matrix were seeded on 24 plate well coated with 100% Corning Matrigel matrix. Pictures were taken at X200 and displacement of single cells after 7 days were calculated using Image. All values were normalized to the diameter of each cell.

Quantitative RT-PCR.

Total RNA extraction and RT were performedusing TRIzol (Life Technologies) and SuperScript VILO reverse transcriptase (Life Technologies), respectively. Real-time PCR analysis was carried outwith LightCycler FastStart DNAMasterplus SYBR Green I on a LightCycler 1.5 (Roche Applied Science). All values were normalized to the level of hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA by use of the 2-ΔCt calculation method (Ct indicates cycle threshold).

MMP13 Activity Assays.

Cell culture supernatant were collected, incubated 2 hours in wells coated with monoclonal anti human MMP-13 antibody, at RT on a plate shaker (40 rpm). Wells were then incubated with 1mM of 4-aminophenylmercuric acetate for 40min at 37 °C, and MMP13 activity was measured 16h hours later by using the SensoLyte 520 MMP13 Fluorometric Assay Kit (AnaSpec).

Fluorescent Gelatin Degradation Assays.

Rhodamine-labeled gelatin was obtained according to the protocol of the Invadosome Consortium (Buccione Lab.), by addition of gelatin (G1890-100G, Sigma) at the concentration of 0,2mg/mL, dissolved in 50 mM of sodium borat, 62mM of NaCl and 0,036 mg/L of NHS-Rhodamine (ThermoScientific #46406) which was after dialysed in PBS. Then, 2% sucrose was dissolved into the gelatin and aliquots of the solution were store at -20°C. This assay was performed as described previously with minor modifications 312 . Coverslips (18-mm diameter) were coated with 0,5µg/mL poly-L-lysine (Sigma-Aldrich) for 20 min at RT, washed with PBS, and fixed with 0,5% glutaraldehyde (Electron Microscopy Sciences) for 15 min. After three washes in PBS, the coverslips were inverted on an 80µL drop of fluorescent gelatin and incubated for 10 min at RT. After washing with PBS, coverslips were incubated in 5mg/mL sodium borohydride (Sigma Aldrich) for 3 min, washed three times in PBS, and finally incubated in 1mL of complete medium for a minimum of 30 min before adding the cells. To assess the ability of the cells to degrade the matrix, 7 x 10 4 cells per wells were plated on fluorescent gelatin-coated coverslips and incubated at 37°C for 5h to 8h. Cells were then fixed with PFA 4% for 20 min at RT, quenched with 50mM NH4CL for 10 min, permeabilized with 0,5%Triton X-100 and processed for intracellular IF labelling with AlexaFluor 647-phalloidin

(1/1000ème,). Cells were imaged with a 63x objective of a fluorescent microscope (200 M,

II. RelB drives glucose metabolism through GLUT3 regulation in breast cancer

RelB promotes metabolic reprograming of BC cells.

Observation of RelB-dependent differences in growth medium color of MDA-MB-231 and MCF7 BC cell lines after reaching confluence led to investigate their pH levels. Strikingly, RelB knockdown in MDA-MB-231 increased growth medium basicity, while RelB ectopic expression in MDA-MB 231 and MCF7 promoted the acidification of the growth medium.

(figure 1A). Since MCF7 cell line displays extremely low endogenous expression levels of RelB, I did not inhibit its endogenous expression. As pH modifications in medium are often consequences of altered metabolism, I investigated RelB impact on BC cellular energy. Indeed, the inhibition of RelB expression lowered by almost 25% MDA-MB-231 ATP content, while ectopic expression of RelB in both in MCF7 and MDA-MB-231 boosted significantly by 30% their ATP content (figure 1B). Further analysis by Nuclear Magnetic Resonance (NMR)-based metabolic profiling displayed a striking RelB-dependent reprogramming of MDA-MB-231 metabolism (figure 1C). These results suggest that RelB participate in BC metabolism. RelB drives BC cell growth via glucose metabolism in physiological glucose conditions.

Next it was important to assess the functional role of RelB-dependent metabolic reprogramming in BC cells. Since our observation of medium pH modifications and our NMR analysis support a pro-glycolysis role of RelB in BC cells, and considering that RelB does not alter BC cell lines growth in recommended glucose concentrations (4.5 g.L -1 ), I studied cell growth in physiological glucose concentrations (1 g.L -1 ). Surprisingly, RelB knockdown in MDA-MB-231 slowed cell growth only in medium with 1 g.L -1 of glucose, and RelB ectopic expression in MDA-MB 231 and MCF7 promoted cell growth at only at 1 g.L -1 glucose. (figure 2A). To assess which RelB-dependent metabolic pathway is responsible for cell growth at phyiological conditions, I evaluated the impact of key nutrients of which levels were observed to be altered by RelB, glucose, pyruvate and glutamin (figure 1C). Remarkably, glucose deprivation prevented RelB-dependent cell growth observed in a 1 g.L -1 glucose concentration medim(figure 2B). Even though glutamine deprivation decreased overall cell growth, it did not inhibit the significant difference induced by RelB knockdown or ectopic expression in BC cell lines at 1 g.L -1 of glucose (figure 2C). However, pyruvate deprivation did not alter at all BC cell growth (figure 2D). Of interesting note, lowering glucose concentrations had a much higher impact on MDA-MB-231 cell line than MCF7. These results demonstrate a key role of RelB in BC cell growth in physiological conditions. through regulation of glucose metabolism.

SLC2A3 (GLUT3) is a novel RelB target gene that correlates with BC overall survival.

To gain further insights into RelB-dependent regulation of glucose metabolism, transcriptomic analysis of MDA-MB-231 was performed and showed that glucose transporter GLUT3 mRNA is overexpressed in cells with ectopic expression of RelB (data not shown). We confirmed that knockdown of RelB in MDA-MB-231 decreased GLUT3 mRNA and protein expression (figure 3A), and ectopic expression of RelB in MDA-MB-231 and MCF7 increased GLUT3 expression (figure 3B and3C). Additionally, chromatin IP (ChIP) analysis showed that RelB is recruited to GLUT3 promoter, suggesting a direct regulation of GLUT3 by RelB in MDA-MB-231

(figure 5D). To extend the analysis of RelB and GLUT3 correlating expressions, I collected published GLUT3 mRNA expression levels of 17 BC cell lines for which we quantified RelB expression 314 . Spectacularly, a strong positive correlation between RelB and GLUT3 expressions was observed in BC cell lines (figure .3E). Moreover, high expression of GLUT3 in BC patients from NKI cohort 315,316 was found to be significantly associated with reduced overall survival (figure 3F). Altogether, those results suggest that GLUT3 expression is dependent of RelB in BC cell lines and is of poor prognosis in BC patients. Wether RelBdependent expression of GLUT3 and increased glucose metabolism in BC to promote cell growth remains unknown and will need further investigations. Published data for GLUT3 mRNA expression of BC cell lines were extracted using CellExpress software 314 . http://cellexpress.cgm.ntu.edu.tw/

Statistical Analysis

Kaplan-Meyer estimate of overall survival were performed using the log-rank test (Mantel-Cox). Overall survival was evaluated from the date of enrolment to the date of death using.PROGgeneV2 http://genomics.jefferson.edu/proggene/ 315

Statistical significance was accessed using unpaired t tests (Prism 5.0c, GraphPad Software). A value of p=0.05 was considered as statistically significant with the following degrees: * p<0.05; **p<0.01; ***p<0.001. Statistical differences between all other parameters were determined using Pearson correlation.

1H-NMR spectroscopy

The preprocessing of the 1H-NMR spectra and analysis was performed as described 317

III. Frequent activation of

Introduction

Diffuse Large B-cell Lymphoma (DLBCL) is the most common lymphoma in adults 318,319 .

Even though cure rates have significantly improved since the introduction of the immunochemotherapy R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone), refractory/relapse cases reach up to 40% 320 . DLBCL is a highly heterogeneous disease and additional complexity still remains to be defined for deeper DLBCL stratification and tailored therapies.

NF-κB transcription factors are major players in the control of cell proliferation and survival, as well as in the pathophysiology of numerous cancers [START_REF] Baldwin | Regulation of cell death and autophagy by IKK and NF-κB: critical mechanisms in immune function and cancer: Regulation of cell death and autophagy by IKK/NF-κB[END_REF][START_REF] Naugler | NF-κB and cancer-identifying targets and mechanisms[END_REF][START_REF] Nakanishi | Nuclear factor-κB inhibitors as sensitizers to anticancer drugs[END_REF][292][293][294] . The NF-κB transcription factor family is composed of five members in mammals: RelA (p65), RelB, cRel (Rel), NF-κB1 (p50 and its precursor p105) and NF-κB2 (p52 and its precursor p100) 321 . These proteins form various homo-and heterodimeric complexes, the activity of which is regulated by two main pathways. The first one, known as the canonical NF-κB activation pathway, mainly applies to RelA and/or cRel containing complexes [START_REF] Karin | Phosphorylation Meets Ubiquitination: The Control of NF-κB Activity[END_REF] The second one, the alternative NF-κB activation pathway, leads to the activation of RelB containing dimers 292,[START_REF] Sun | The noncanonical NF-κB pathway: The noncanonical NF-κB signaling pathway[END_REF]123 .

Gene expression profiling identified two prominent cell-of-origin (COO) DLBCL subtypes, the germinal center B-cell-like (GCB) and activated B-cell-like (ABC), this latter being associated with worse outcome among patients treated with immunochemotherapy 322-324 . More recently, major advances were made to capture the genetic heterogeneity of DLBCL [325][326][327][328][329][330][331] and have elucidated oncogenic mechanisms in DLBCL. Frequent mutations in regulators of the classical NF-κB pathway (MYD88, TNFAIP3, CD79A/B, CARD11) are recognized as a hallmark of ABC DLBCL patients [332][333][334][335][336] , and inhibition of the classical NF-κB pathway induces cell death in ABC DLBCL cell lines 337 . In contrast, the involvement of the alternative RelB NF-κB subunit in DLBCL pathogenesis and how it may impact on DLBCL cell survival remain a central unanswered question.

In the study presented here, we reveal a frequent RelB DNA-binding activity in newly diagnosed DLBCL patients, regardless of their ABC or GCB subtypes, that predicted worse overall survival. Further, we found that RelB activation defines a new subset of DLBCL patients with a distinct gene expression profile and genetic characteristics from that of the BCRdependent classical NF-κB signaling. Functional studies demonstrated that RelB confers resistance to DNA damage-induced apoptosis in DLBCL cells, both ABC and GCB subtypes.

Altogether, our data highlight previously unrecognized function for the alternative NF-κB RelB subunit in DLBCL, and provide the framework for the development of new drugs targeting RelB to overcome chemoresistance in DLBCL.

Results

RelB is frequently activated in DLBCL patients irrespectively of their ABC or GCB subtypes and is associated with worse overall survival

To directly assess the activation status of RelB alternative NF-κB subunit in DLBCL patients, we performed EMSA combined with supershift analysis using an anti-RelB antibody to evaluate RelB DNA-binding activity in 66 de novo DLBCL patients (41 ABC and 25 GCB, Table 1) included in clinical trials from the Lymphoma Study Association (LYSA clinical trial LNH03) 338 . A constitutive binding of RelB was observed in 44 DLBCL patients (66,6%) (Fig. 1a,b). Importantly, the RelB positive subset includes both ABC and GCB cases (Fig. 1c), indicating that RelB is capturing largely different aspects than the DLBCL cell-of-origin.

Stronger RelA activity compared to the other NF-κB subunits tended to be preferentially present in ABC cases (30/37; 81%, p= 0.0003). Inversely, those with stronger cRel activity tended to be of GCB type (10/13; 77%, p= 0.0012) (Fig. 1d). In a next step, it was important to assess how RelB activation impacted on outcome. Remarkably, RelB activation as evaluated by EMSA was significantly associated with worse overall survival (p=0.0276) (Fig. 1e).

Legend to

Identification of a RelB gene expression signature in DLBCL patients

To go one step further and generate a molecular portrait of RelB activation in DLBCL patients, we further explored whether gene expression profiling could subdivide DLBCL patients based on their status of RelB activation. A hierarchical clustering algorithm was used to group genes on the basis of RelB positive vs RelB negative cases as evaluated by EMSA. We identified a set of 140 probesets corresponding to 118 genes associated with RelB binding activity (Supplemental Fig. 1). In a next step, the RelB gene expression signature was used to clusterize the GSE87371 cohort of 202 DLBCL de novo patients (83 ABC, 85 GCB and 34 unclassified) . 56% of DLBCL cases exhibited a RelB gene expression profile, (Fig. 2a). Just as what was seen for RelB positive cases evaluated by EMSA, the frequency of RelB cases evaluated by GEP was not significantly different between ABC and GCB subtypes (Fig. 2b,c). Most importantly, we confirmed that this RelB gene signature predicted RelB activation with high confidence among the DLBCL patients that we have evaluated by EMSA (97,14% of positive predictive value, 95,45% of specificity, 77,27% of sensitivity), indicating that the follow-up of these gene mRNA expression constitutes a new tool to evaluate the activity of RelB. A more detailed analysis of the RelB signature revealed increased expression of genes linked to cell death and survival, metabolism, immune cell trafficking, inflammation, and proliferation (Fig. 2d).

Further, it was important to examine whether the previously reported NF-κB characteristics in DLBCL gene expression signatures reflected the activation status of the alternative RelB NF-κB subunit. Remarkably, RelB activation was not associated with any reported NF-κB signatures in normal and malignant lymphoid cells, including DLBCL 339 (Fig. 2e), indicating that RelB activity did not correlate with classical NF-κB gene expression features of DLBCL.

There was no association between RelB activation and either BCL2, MYC or double expression BCL2/MYC as evaluated by immunohistochemistry (Fig. 2f). Next, we evaluated the impact of RelB activation as evaluated by its newly identified gene expression signature, on the survival of R-CHOP treated patients from the GSE87371 cohort (n=98) (Fig. 2g). RelB positive cases had significantly an inferior overall survival (p=0,0320). Multivariate analysis including IPI low/high risk group and COO classification, showed that RelB activation was an independent predictor for worse overall survival (p=0,004, HR=2,95) (Fig. 2h). In conclusion,

we have developed a gene expression signature that accurately predicts RelB activation status in DLBCL patients. Moreover, our study brings up RelB as a potential new independent marker of worse prognosis among patients with DLBCL treated with R-CHOP.

Legend to

Figure 35Identification of a RelB gene expression signature associated with worse outcome of R-CHOP treated DLBCL patients

Genetic attributes of RelB activation in DLBCL

Since RelB activation in ABC and GCB DLBCL patient samples was so far overlooked, we next examined the genetic composition associated with RelB activation. We cataloged genetic alterations associated with the newly defined RelB signature using the Lymphopanel Next-Generation Sequencing to identify mutations in 34 genes important for lymphomagenesis. RelB positive DLBCLs were characterized by an enrichment of genetic alterations of ITPKB, a regulator of B-cell survival and response to antigen presentation, as well as with those of the immune escape molecule B2M (Fig. 3a,b). We also identified that mutation of IRF4, a gene involved in B-cell terminal differentiation, was significantly associated with RelB activation (Fig. 3a,b). In contrast, RelB positive DLBCLs rarely exhibited mutations in GNA13, an indirect modifier of BCR/Pi3K frequently found mutated in germinal center (GC)-derived Bcell lymphomas (Fig. 3a,b). There is no association between the RelB positive subgroup and either BCL2 or MYC breaks, whereas there is a co-exclusion tendency of RelB activation with Bcl6 genetic alterations (8/52 RelB positive vs 21/55 RelB negative), as evaluated by FISH (Fig. 3c).

Next, it was important to examine whether the reported NF-κB-linked genetic alterations in DLBC reflected the activation status of the alternative RelB NF-κB subunit. First, we analyzed the concurrent mutations of MYD88 L265P and CD79B that cooperatively activate classical NF-κB via the My-T-BCR supercomplex involving MYD88, TLR9 and the BCR 340 . Remarkably, RelB positive subset appeared to be significantly co-exclusive with MYD88 L265P and CD79B double mutations (Fig. 3a,b). Similarly, there is no significant enrichment in mutations targeting CARD11, reported to be associated with the BCR-dependent NF-κB pathway (Fig. 3a). Further, we examined how RelB activation segregates with the genetic subtypes that the publicly accessible LymphGen probabilistic classification tool classifies 327 (Fig. 3d). RelB activation did not correlate with the MCD genetic subtype (enriched for ABC tumors carrying MYD88 L265P and CD79B mutations that cooperatively activate NF-κB), neither with the EZB subtype (enriched for GCB tumors carrying EZH2 mutations, BCL2 translocation and REL amplification) (Fig. 3d). Interestingly, strong RelA binding as evaluated by EMSA correlates with MCD and strong cRel correlates with EZB (Fig. 3e and Supplemental Fig. 2). These observations indicate that the current genetic tools to evaluate NF-κB activity do not provide information on the alternative NF-κB RelB activation status. staining, was markedly increased upon knockdown of RelB expression in all three DLBCL cell lines (Fig. 5b). This effect was associated with greater cleavage of caspase 3 (Fig. 5c). Upon doxorubicin treatment, RelB expression knockdown greatly increased H2AX protein expression levels (Fig. 5d) and H2AX foci (Fig. 5e). Altogether, these data indicate that RelB exerts a critical pro-survival function to protect DLBCL cells from DNA-damage induced apoptosis. To further assess underlying mechanisms, we determined whether RelB constitutive activation in DLBCL cells upregulates the expression of endogenous anti-apoptotic NF-κB responsive genes, such as cIAP2, Bcl-xL, Bcl2, XIAP and TRAF2. Knockdown of RelB expression markedly and significantly decreased cIAP2 mRNA levels (Fig. 5f). In contrast, no differences in Bcl-xL, Bcl2, XIAP and TRAF2 mRNA levels were observed upon RelB knockdown (Supplemental Fig. 2). In concert with these results, the amount of cIAP2 protein was markedly and significantly decreased upon RelB knockdown in DLBCL cells (Fig. 5g).

Legend to

Most importantly, a significant accumulation of cIAP2 protein was observed in DLBCL patients with constitutive activation of RelB (Fig. 5h). Collectively, we have uncovered that

Figure 38. RelB protects ABC and GCB DLBCL cells from induction of apoptosis upon doxorubicin treatment

Discussion

Although recurrent genetic lesions were associated with activation of the canonical NF-κB pathway in human DLBCL, predominantly the ABC subtype 329,[333][334][335] , whether the alternative NF-κB pathway is functionally activated in DLBCL patients is still debatable. In the study presented here, we conducted the first comprehensive analysis of the alternative NF-κB DNAbinding activity (i.e. RelB) by EMSA, the gold standard method to evaluate NF-κB DNA binding activity, on a large cohort of DLBCL patient samples and cell lines. We have revealed that constitutive activation of RelB is frequent (approximately 66%) in newly diagnosed DLBCL patients, almost equally distributed among ABC and GCB, indicating that it represents a major event in DLBCL independent of the cell-of-origin. Moreover, we uncovered a crucial role for RelB in promoting DLBCL cell survival upon DNA damage. These results have important implications for the role of RelB in DLBCL pathogenesis and therapy.

Loss-of-function genetic alterations in genes of the alternative NF-κB pathway have been reported in a subset of human DLBCLs. Inactivating genetic alterations of TRAF3 occur in around 15% of human DLBCL, irrespectively of their ABC or GCB subtypes 342 . Loss-offunction genetic alterations in TRAF2 and BIRC2/3, two other negative regulators of the alternative NF-κB pathway (but also positive regulators of the canonical NF-κB) have been reported in about 12% and 10% of DLBCL cases, respectively 329,335,343,344 . TRAF2 mutations are preferentially associated with GCB and BIRC2/3 with ABC. In regards to the frequency of RelB activation in human DLBCL that we report here, one can hypothesized that important regulators of the alternative NF-κB pathway remain to be identified in the context of DLBCL Indeed, although cIAP2 was originally characterized by its ability to inhibit cell death through suppression of caspase activity 345 , cIAP2 is also involved in many aspects of innate and adaptive immune cell function through the ubiquitin-mediated regulation of the NF-κB pathways 117,346 . Altogether, our data point to RelB as a promising target to sensitize DLBCL cells to pro-apoptotic stimuli to circumvent resistance to treatment.

In conclusion, we established that RelB is a crucial positive regulator of DLBCL cell survival upon genotoxic agent exposure that is frequently activated in both ABC and GCB DLBCL patients. Our data are of great functional importance because they constitute a significant advance in the understanding of RelB function in DLBCL and provide a strong rationale for the development of new molecules targeting RelB for therapeutic intervention in DLBCL.

METHODS

Determination of RelB DNA-binding status

Sixty-six DLBCL patients (41 ABC and 25 GCB) enrolled in the prospective, multicenter, and randomized LNH-03B LYSA trials with available frozen tumor samples 338 and centralized histopathologic review were selected. The COO molecular classification was obtained with HGU133+2.0 AffymetrixGeneChip arrays (Affymetrix). Cases were considered "RelB positive" when they presented detectable RelB DNA binding activity regardless of intensity using electrophoretic mobility shift assays (EMSA) combined with supershift. Cases were "RelB negative" when this activity was not detected. Cases were analyzed and classified independently by 3 senior researchers. A consensus was achieved for the discordant cases.

Electrophoretic mobility shift assays for NF-κB

NF-κB activation was analyzed by electrophoretic mobility shift assay (EMSA) using the human immunodeficiency virus long terminal repeat tandem κB oligonucleotide as κB probe as previously described [START_REF] Jacque | RelA repression of RelB activity induces selective gene activation downstream of TNF receptors[END_REF] . For supershift assays, total protein extracts were incubated with specific antibodies for 30 min on ice before incubation with the labeled probe. Beyond RelB DNA-binding status described above, an analysis of DNA binding activity of the two canonical NF-κB subunits RelA and cRel was performed. RelA binding activity being observed in almost all cases, RelA vs cRel scoring was established according to the NF-κB subunit that presented the strongest DNA-binding activity.

Determination of RelB gene expression signature

Differential gene expression analysis of the 66 DLBCL patients included in the RelB DNAbinding study by EMSA was performed according to RelB positive vs RelB negative status using LIMMA, and identified a set of 140 probesets corresponding to 118 genes differentially expressed with a fold change of two and a p-value <0.05 (see supplementary Fig. 1). A hierarchical clustering based on the EMSA-based RelB gene expression signature was then performed using 202 DLBCL patients from the GSE87371 cohort defining two distinct subgroups corresponding to RelB positive and RelB negative DLBCL patients.

Transcriptomic Analysis

Microarray experiments were performed on Affymetrix Human Genome HGU133plus2.0 GeneChips (a genome wide array with 54674 probe sets targeting 19418 transcripts). Gene expression levels were normalized using the GC-RMA algorithm and flags were computed using MAS5. Quality assessment of the chips has been performed with affyQCReport R package. MAS5 algorithm produces a flag "P" for "Present", "M" for "Marginal" or "A" for "Absent" associated to each intensity measure. This flag is an estimation of the statistical difference between PM (Perfect Match) and MM (Mismatch). Three probe lists have been used

for each comparison according to flagged measurement in the relevant chips. The "PP" list is made of probes only flagged as "Present" for all chips involved in the comparison. The "P50" list has been created filtering probes flagged as "Present" for at least half of the chips. The "All" list is made of all probes without any filter. Three groups of two biologically independent samples were compared. The group comparisons were done using Student's t test. To estimate the false discovery rate we filtered the resulting pvalues at 5% and used the Benjamini and Hochberg (BH), Bonferroni (B) or without correction (SC). Cluster analysis was performed by hierarchical clustering using the Spearman correlation similarity measure and average linkage algorithm. Data were submitted to Ingenuity Pathway Analysis (IPA) to model relationships among genes and proteins and to construct putative pathways and relevant biological processes.

NGS experiments and data analysis

Patients were sequenced as previously described 338 using the Lymphopanel, which was designed to identify mutations in 34 genes important for lymphomagenesis, based on literature data 347 and WES of relapsed/refractory DLBCL sequencing 348 .

Immunohistochemistry

Immunohistochemistry was performed on 3μm TMA tissue sections for BCL6, BCL2 and MYC as described previously 349,350 . Standard cutoff levels were applied for Bcl6, Bcl2 and Myc, i.e. 30%, 50% and 40%, respectively, in accordance with previous publications 349,350 .

FISH analysis

FISH analysis was performed on 3μm TMA tissue sections using break-apart FISH DNA probes for BCL2/18q21, and BCL6/3q27 (probes Y5407, and Y5408; Dako A/S) as previously described 351 . For MYC/8q24, two different MYC break-apart FISH DNA probes were used:

MYC FISH DNA probe Split Signal (Y5410, Dako A/S) and Vysis LSI MYC dual color, BA rearrangement probe (Abbott Laboratories, Chicago, IL), as previously described 352

LymphGen genetic subtype classification

The genetic subtypes of DLBCL were established using the LymphGen probabilistic classifier using the publicly accessible server (https://explore.openaire.eu) 327 (ref wright Staudt Cancer Cell 2020).

Statistical analysis

Kaplan-Meyer estimate of overall survival were performed using the log-rank test (Mantel-Cox). Multivariate overall survival analyses were performed using Cox proportional-hazards model. Overall survival was evaluated from the date of enrolment to the date of death. Cases were censored on the date the patient was last known to be alive or, for patients in complete response dying as a result of causes unrelated to the lymphoma or treatment. AnnexinV/DAPI was assessed using unpaired t tests (Prism 5.0c, GraphPad Software). A value of p=0.05 was considered as statistically significant with the following degrees: * p<0.05; **p<0.01; ***p<0.001. Statistical differences between all other parameters were determined using

Pearson correlation or Fisher exact tests when appropriate. P values or false discovery rates (FDR) < 0.05 were considered statistically significant.

Human DLBCL cell lines

Cell lines derived from DLBCL patient samples were grown in RPMI-1640 medium (Gibco glutamax®) supplemented with 10 % heat-inactivated fetal bovine serum (HyClone), 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen) 353 .

Antibodies

The antibodies were purchased from Santa Cruz (RelB, RelA, cRel, and GAPDH), Sigma (βactin), Abcam (γ-H2AX), and Cell Signaling (cleaved caspase 3 (Asp175), and cIAP2).

Immunoblotting

Immunoblotting were performed as previously described 296 .

Lentiviral production and transduction

Production of infectious recombinant lentiviruses was performed by transient transfection of 293T cells as previously described 313 . For infections, cells were incubated overnight with recombinant lentiviruses. An equal amount of fresh culture medium was added 24 hours later and after 48 hours, cells were washed and seeded in fresh culture medium. GFP positive cells were sorted with FACSAria™ sorter (Becton Dickinson).

Annexin V binding assay

Cells were harvested and washed twice with cold PBS. Cells were resuspended in 1X binding buffer containing Annexin V-APC (BD Biosciences Pharmingen) and 4',6-diaminidino-2phenylindole (DAPI, Molecular Probes) following the manufacturer's instructions. The samples were subjected to cytometric analysis with a MACSQuant cytometer (Miltenyi Biotec) and the data was statistically evaluated using the Flowjo v10.2 software.

γH2AX foci

Cells were fixated in formalin 4%, permeabilized in SDS 1%, and then stained with an anti-γH2AX antibody. Samples were incubated with secondary anti-body Alexa Fluor 647® and then analyzed using an ImageStream X Mark II Imaging Flow Cytometer. Data were acquired at a X60 magnification with EDF using the 642 nm laser at 150 mW and INSPIRE software.

At least 8000 events of cells per sample were analyzed. Acquired data were analyzed using the IDEAS analysis software (v6.1; Merck-Millipore). Cells were gated for focused cells using the Gradient RMS feature. Cells were gated for single cells using the aspect ratio and area features.

The spot counting analysis wizard was used.

RT-qPCR

Total RNA extraction and reverse transcription were performed as previously described

GENERAL DISCUSSION

Altough this work has led to the identification and demonstration of RelB activation and regulation as a new marker for BC and DLBCL aggressivity, many questions still arise from the results, leading to exciting future directions.

A. RelB and BC glucose metabolism

Deregulation of metabolic pathways in cancer has been discovered for almost a century and is now emerging as a vital field for its therapeutic potential. In that perspective, our first observations of RelB as a metabolic regulator in BC cells leads to many interrogations and possibilities of application.

BC cell growth

The observation of glucose metabolism regulation by RelB in BC cells was realized in "standard" high glucose concentrations (4,5 g.L -1 ), which was then confirmed by NMR metabolomic analysis. Intriguingly, we did not observe any specific basal phenotype driven by Combining fasting or fasting-mimicking diets (FMDs) with BC treatment (chemotherapy or immunotherapy) are currently under intense study and show great promises [355][356][357] . Indeed, fasting and FMDs drive major change in metabolite levels and growth drivers, allowing to drastically lower the capacity of cancer cells to proliferate, and adapt and resist to classical therapies 357 . However, fasting or FMDs can be a risk for weak patients as this type of regimen could increase sarcopenia, cachexia and chemotherapy-induced anorexia, suggesting that defining a metabolic profile of tumors would help to choose the most adapted FMD for clinical application, if necessary 358,359 . Since our results also demonstrate that glucose deprivation (0 g.L -1 ) inhibits RelB-dependent BC cell growth, testing different FMDs in BC PDX mouse models characterized for RelB analysis (DNA-binding activity, regulation by pS472, transcriptional activity) would determine the most fitted regimen to hinder RelB-dependent metabolic reprogramming. These results could lead to a better identification of RelB as a potential metabolic biomarker to assess the capacity of BC cells to utilize glucose metabolism for optimal growth and participate in precision therapy.

BC resistance to chemotherapy

Incidentally, metabolic alterations are also increasingly demonstrated to actively and strongly influencing cancer cell resistance to chemotherapeutic agents, encouraging specific targeting of metabolic pathways in combination with standard anticancer treatments 360,361 . Since alterations in glucose metabolism has been linked to antrahcyclin-based therapies 361 , we wanted to test whether RelB-dependent increase of glucose metabolism in BC cell lines would alter doxorubicin-induced cell death. We first observed an increase of cell death after doxorubicin treatment in MDA-MB-231 cell line with inhibited expression of RelB, and conversely, we observed a decrease of doxorubicin-induced cell death in MCF7 cell line with ectopic expression of RelB. Moreover, early evidence suggest that the resistance of BC cell lines is driven RelB-dependent regulation of glucose metabolic pathways, as inhibition of glycolysis using 2-Deoxy-D-glucose (2-DG) completely hampered RelB effect on cell survival after doxorubicin treatment (data not shown). Of note, these results were obtained in a medium with a glucose concentration of 4.5 g.L -1 . Further experiments should be conducted i) to test whether RelB influence on doxorubicin-induced BC cell death is strengthen in physiological glucose concentration (1 g.L -1) and ii) confirm that glucose is the nutrient at the source of RelBdependent resistance to doxorubicin using a 0 g.L -1 medium. To help identify RelB-dependent metabolome associated with RelB-dependent resistance to doxorubicin, NMR-based metabolic profiling of cells treated with doxorubicin should be performed as well. These results could lead to the identification of specific metabolites involved in anthracycline resistance to propose rational therapeutic combination with doxorubicin rather than complete inhibition of glucose metabolism. This would also avoid unpredicted cancer cell metabolic reprogramming following compete deprivation of glucose, that could induce unknown mechanisms of resistance.

Using the BC PDX mouse models described earlier, we could assess in vivo the role of RelB in anthracycline resistance by following tumor recurrence and metastasis development with [START_REF] Weih | Multifocal defects in immune responses in RelBdeficient mice[END_REF] FDG-PET combined with computational tomography ( 18 FDG-PET/CT). [START_REF] Weih | Multifocal defects in immune responses in RelBdeficient mice[END_REF] FDG-PET/CT analysis would also add valuable information concerning glucose metabolism fluctuations in response to doxorubicin, according to RelB characterization. Depending on these results and those described before, either FMDs or specific targeting of the metabolic pathway involved in resistance could be combined with anthracyclin-therapy. Such treatments could also be tested on established PDX mouse models from residual breast tumors 362 after RelB characterization.

Survival and metastasis development would also be important to follow before and after treatments, as well as in vivo NMR metabolic profiling of tumors and fluids.

All these informations would help comprehend RelB role on BC metabolism and potential associated therapeutic liabilities for patients. Starvation or FMDs in combination with chemotherapeutic agents holds great promises also because it has been demonstrated in vitro and in vivo that such regimen is protective for normal cells in response to chemotherapy 363 .

Since PDX mouse models from breast tumors have been extensively established and studied for some of the experiments described above, retrospective analysis of these data could be performed once RelB characterization would have been assessed by WB and quantitative RT-PCR (RelB expression), IHC (pS472-RelB localization) and EMSA (RelB DNA-binding activity).

BC invasive capacities

In our study about the role of pS472-RelB in BC, we described through several assays that this post-translational modification drives pro-migratory and pro-invasive capacities of TNBC cells via the direct regulation of MMP13, which we demonstrated in further experiments using a specific inhibitor of MMP13. One of these assays consisted in following the rate and the speed of degradation of a fluorescent gelatin by cancer cells. Intriguingly, even though MMP13 inhibitor managed to completely hinder pS472-RelB effect in all the experiments, it was not entirely the case in the gelatin degradation assay. Indeed, although MMP13 inhibitor decreased almost by half pS472-RelB-dependent degradation index (figure 5F), MDA-MB-231 with ectopic expression of WT RelB still showed a degradation index two-times higher than the control cells. Sucrose, a disaccharide composed of two monosaccharides (glucose and fructose), is part of the composition of the fluorescent gelatin, at a high concentration of 20 g.L -1 . These results raise two questions: i) the significance of RelB-dependent glucose metabolism in the pro-invasive capacity of BC cells and ii) the importance of pS472-RelB on glucose metabolism regulation, which will be discussed later.

The hypothesis that RelB increases BC metabolism via increased glucose uptake speculates that glucose availability in medium is the limiting factor for cells expressing high levels of RelB,

and not the machinery able to internalize and process it. According to this theory, only WT RelB expressing cells in this experiment had the capacity to internalize the excess of sucrose present in the gelatin, that may have boosted its pro-degradative effect beyond MMP13 activity control. A possible explanation of these intriguing results could be found in the acidification driven by RelB resulting from altered glucose metabolism. Indeed, low pH is known to induce for the activation of proteases like MMPs and cathepsins in the tumoral microenvironment other than MMP13, thus promoting extracellular matrix (ECM) degradation and favor cancer cell invasion 364 . Performing this experiment with glycolysis inhibitor 2-DG could answer the question of RelB-dependent altered metabolism role in its degradative capacity, as well as following sucrose uptake using labelled [START_REF] Bonizzi | Activation of IKKα target genes depends on recognition of specific κB binding sites by RelB:p52 dimers[END_REF] C sucrose.

Still, increasing glucose metabolism could participate to RelB role in migration and invasion via multiple other processes, such as EMT promotion, anoikis resistance or colonization 364,365 .

GLUT3 in BC

GLUT3 has been demonstrated to be a RelA target gene in p53 -/-MEFs 203 . Since we unveiled GLUT3 regulation by RelB in a p53 WT BC cell line (MCF7), this suggests that it is a mechanism of control which is independent of RelA. Since RelB is also a RelA target gene, assessment of RelA should no matter what be performed by EMSA and WB, to determine if GLUT3 expression control is specifically related to RelB in a RelA dependent manner.

Moreover, since we found out that RelB promotes GLUT3 expression in BC cells, further experiments would be needed to assess if GLUT3 regulation drives RelB-dependent increase of glucose metabolism. Both inhibition of GLUT3 expression by RNAi in WT RelB and ectopic introduction of GLUT3 in shRelB in BC cells would be required to answer that question. Then, in vitro assays described earlier to assess the RelB function in BC metabolism (ATP content, NMR analysis, glucose uptake) and on the phenotype under metabolic stress (1g. L -1 cell growth) and genotoxic stress (cell death upon doxorubicin treatment).

In vivo assessment of GLUT3 expression in the PDX mouse models described earlier would also provide meaningful information, especially in regards to the different classifications of RelB (DNA binding activity, expression levels) to determine the correlation of GLUT3 expression and RelB parameters.

Could GLUT3 be an interesting target for BC treatment? Specific chemical inhibition has not

been demonstrated yet in vitro nor in vivo, even though the interest for such inhibitors is growing. Our analysis could provide additive arguments to assess and target these receptors and their impact on the pro-tumoral effects of glucose metabolism.

Phosphorylation of RelB at serine 472

Final question is related to the impact of serine 472 phosphorylation to RelB-dependent regulation of glucose metabolism.

B. RelB and DLBCL metabolism

Since we reported earlier a RelB-dependent metabolic reprogramming in BC cells, the next question is if RelB regulates DLBCL metabolism.

In the study on the frequent RelB DNA-binding activity in newly diagnosed DLBCL patients predicting worse overall survival, we found that RelB activation defines a new subset of DLBCL patients with a distinct gene expression profile. As we unraveled several cell key functions associated with this gene expression profile in patients, we also discovered several genes that were involved in cell metabolism. However, most of those genes were found to be involved in several metabolic pathways mainly lipid metabolism and glucose metabolism, indicating that further analysis of RelB in DLBCL metabolism must be performed. Of note, lipid metabolism and glucose metabolism were also both observed to be regulated by RelB in BC. We also suggest in our that TRAF3 mutation could be a causal mutation of RelB activity in DLBCL. Interestingly, it has been also reported that TRAF3 deficiency promotes metabolic reprogramming in B cells by boosting glucose metabolism and oxidative phosphorylation 366 , adding a supposition of RelB activity in the control of DLBCL metabolism.

NMR analysis of blood, urine and tumor samples in patients could be very interesting in regard to RelB DNA-binding activity classification and GEP classification. Since we also described that RelB activity is associated with worse prognosis in patients treated with R-CHOP, NRM analysis could also be performed before and after RCHOP treatment to determine if RelB correlates with a specific metabolic signature in patients not responding to RCHOP treatment.

Such results would also inform if a potential metabolic reprogramming associated with RelB

  biologie des CS et du DLBCL, qui pourraient contribuer à une meilleure stratification des patients et à de nouvelles études de RelB dans d'autres cancers. Mots-clés : cancers du sein -métastase -modifications post-traductionnelles -RelBmétabolisme -DLBCLapoptose -biomarqueur a. Deregulation of glucose transporter expression in BC ...................................................... b. Glycolytic enzymes and Pentose Phosphate Pathway ....................................................... 2. Lipid metabolism ................................................................................................................... a. Fatty acid metabolism........................................................................................................ b. Cholesterol metabolism ..................................................................................................... c. Cholin metabolism............................................................................................................. RESULTS ................................................................................................................................. I. Phosphorylation of RelB on S472 promotes breast cancer metastasis and cancer cell invasion via the control of MMP-13. ............................................................................................................... II. RelB drives glucose metabolism through GLUT3 regulation in breast cancer ................... III. Frequent activation of RelB NF-κB alternative pathway subunit and its associated gene expression signature predict worse prognosis in diffuse large B-cell lymphoma. .......................... GENERAL DISCUSSION ....................................................................................................... A. RelB and BC glucose metabolism ........................................................................................... 1. BC cell growth .....................................................................................................................

2 .

 2 BC resistance to chemotherapy ........................................................................................... 3. BC invasive capacities ......................................................................................................... 4. GLUT3 in BC ...................................................................................................................... 5. Phosphorylation of RelB at serine 472 ................................................................................ B. RelB and DLBCL metabolism ................................................................................................ ........................................................................................................................

Figure 1 :

 1 Figure 1: The NF-kB transcription factor family. ....................................................................

Figure 2 :

 2 Figure 2: The classical NF-kB pathway.. .................................................................................

Figure 3 :

 3 Figure 3: The alternative NF-kB pathway.. ..............................................................................

Figure 4 :

 4 Figure 4: RelB post-translational modifications.. ....................................................................

Figure 5 :

 5 Figure 5: RelB serine-472 phosphorylation acts as an activator of inflammation-mediated cell migration in fibroblasts.. ..........................................................................................................

Figure 6 :

 6 Figure 6: Human cancers that have been linked to constitutive NF-κB activation.. ................

Figure 7 :

 7 Figure 7: NF-κB target genes involved in cancer development and progression. ...................

Figure 8 :

 8 Figure 8:: Human cancers that have been linked to RelB activation. ......................................

Figure 9 :

 9 Figure 9:: Main NF-B pathways and the six strategies pre-clinical and clinical studied underway to inhibit its activity in oncology. ............................................................................

Figure 10 :

 10 Figure 10: NF-B activation by RANK/RANKL in bone cancer complications. ...................

Figure 11 :

 11 Figure 11:Personalized medicine for cancer patients. ..............................................................

Figure 12 :

 12 Figure 12: Emerging Hallmarks of cancer from Hanahan and Weinberg, Cell, 2011 .............

Figure 13 :

 13 Figure 13: The role of classical and alternative NF-B subunits in the efficacy of immune checkpoint inhibitors.. ..............................................................................................................

Figure 14 :

 14 Figure 14: Mechanisms of resistance to CAR T cell therapy. .................................................

Figure 15 :

 15 Figure 15: The role of NF-B on CAR T cell efficacy. ...........................................................

Figure 16 :

 16 Figure 16: NF-B activation by RANK/RANKL in bone cancer complications and BRCA1 driven breast cancer.. ................................................................................................................

Figure 17 :

 17 Figure 17: NF-B gain and loss-of-function mutations in ABC DLBCL: Impact on chronic active BCR signaling and ibrutinib responsiveness.. ...............................................................

Figure 18 :

 18 Figure 18: NF-κB in the new era of cancer therapy. ................................................................

Figure 19 :

 19 Figure 19: NF-κB integrating metabolism. ..............................................................................

Figure 20 :

 20 Figure 20: The role of NF-B on cancer cell energy metabolism............................................

Figure 21 :

 21 Figure 21: Breast cancer subtypes. ...........................................................................................

Figure 22 :

 22 Figure 22: Glucose metabolism. ...............................................................................................

Figure 23 :

 23 Figure 23: Warburg effect. .......................................................................................................

Figure 24 :

 24 Figure 24: Overview of cellular fatty acid metabolism. ...........................................................

Figure 25 :

 25 Figure 25: The major enzymes involved in choline phospholipid metabolism in the cell.. .....

Figure 1 :

 1 Figure 1: The NF-kB transcription factor family. The five members of the NF-kB family are presented with their main domains. Proteins p100/p52 and p105/p50 are shown in their full length. DD, death domain; GRR, glycine-rich region; LZ, leucine-zipper; RHD, Rel homology domain; TAD, transactivation domain. From Oeckinghaus and Ghosh, Cold Spring Harb Perspect Biol, 2009.

Figure 2 :

 2 Figure 2: The classical NF-kB pathway. The classical (or canonical) pathway: Activation of various receptors, such as TNFRs, induces phosphorylation of the inhibitory IκB proteins by the IKK complex, leading to their phosphorylation and degradation by the proteasome. This RelA-and cRel-containing dimers to translocate to the nucleus where they activate the transcription of specific NF-κB target genes.

Figure 3 :

 3 Figure 3: The alternative NF-kB pathway. The alternative pathway: activation of a more restricted set of receptors (e.g., BAFF, lymphotoxin β), causes the degradation of TRAF3 by the cIAP1/2 E3 ligases, leading to the activation of NIK that activates IKKα, subsequently leading to the phosphorylation and proteasome-dependent processing of p100 and ultimately resulting in the release of either RelB/p50 or RelB/p52 dimers.

Figure 4 :

 4 Figure 4: RelB post-translational modifications. The site(s) involved and the functional effect of the different published post-translational modifications of RelB.

Figure 5 :

 5 Figure 5: RelB serine-472 phosphorylation acts as an activator of inflammation-mediated cell migration in fibroblasts. The IκB kinase (IKK) complex constitutively interacts with the RelB subunit of NF-κB. Activation of IKK following TNFα treatment induces phosphorylation of RelB on serine 472. Phosphorylation of serine 472 drives RelB to bind to the promoter of pro-migration genes such as MMP3, subsequently promoting fibroblast migration. TNFαinduced IKK-driven RelB serine-472 phosphorylation is required for efficient cell migration in an MMP3-dependent manner.

Figure 6 :

 6 Figure 6: Human cancers that have been linked to constitutive NF-κB activation. Various hematological malignancies and solid tumors that exhibit constitutive NF-κB activation. From Baud and Karin, Nat Rev Drug Discov, 2009.

Figure 7 :

 7 Figure 7: NF-κB target genes involved in cancer development and progression. NF-κB activation affects all first six hallmarks of cancer through the transcription of genes involved in cell proliferation, angiogenesis, metastasis, inflammation and suppression of apoptosis as identified in both cell lines and tissue samples. From Baud and Karin, Nat Rev Drug Discov, 2009 3 .

Figure 8 :

 8 Figure 8: Human cancers that have been linked to RelB activation.

Figure 9 :

 9 Figure 9: Main NF-B pathways and the six strategies pre-clinical and clinical studied underway to inhibit its activity in oncology. Classical and alternative NF-B are presented. Six key steps allowing to target NF-B activity in oncology are indicated and represented by six classes of inhibitor that have been reported: 1. Antagonists of ligand-specific cell surface receptor binding; 2. IAPs antagonists 3. IKK complex activity inhibition 4 Inhibition IκBs inhibitory proteins degradation by proteasome inhibitors 5. Nuclear translocation inhibition 6.

Figure 10 :

 10 Figure 10: NF-B activation by RANK/RANKL in bone cancer complications. Circulating tumor cells release PTHrP, IL-1 and IL-11 that drives RANKL secretion by osteoblasts, inducing RANKL interaction with its receptor RANK at the surface of pre-osteoclasts and osteoclasts. This binding of RANKL to RANK promotes the differentiation of pre-osteoclasts to osteoclasts, relocation to the bone, and activation of their osteolytic function. Denosumab, a

4 .

 4 uncovered by the proteosomal degradation of IκB proteins, nuclear translocation signals of NF-κB subunits are recognized by the importin α family of proteins, thereby allowing NF-κB complexes to translocate into the nucleus 131,132 . DHMEQ is derivative of epoxyquinomicin C that greatly reduces importins α affinity NF-κB complexes 133 . Treatment by DHMEQ significantly decreases tumour size of a xenograft mouse model of acquired platinum-resistant bladder cancer and is associated with a diminution of RelA nuclear localization 134 . DNA binding inhibition Inhibition of NF-κB complexes binding to specific DNA κB sites consequently drives the blockade of NF-κB target genes transcriptional regulation. Therefore, PBS-1086 hinders DNA binding of NF-κB complexes of both classical and alternative pathways 135 . As demonstrated in pre-clinical studies, PBS-1086 causes cell death of bortezomib-resistant MM cell lines and inhibits osteoclastic differentiation 136 . Moreover, it overcomes the NF-κB-dependent acquired resistance to erlotinib of a non-small-cell lung cancer patient-derived xenograff mouse model 137 .

Figure 11 :

 11 Figure 11:Personalized medicine for cancer patients. Personalized medicine takes into account cancer genetics, genomics and any other biological information in order to make a wellinformed decision regarding the diagnosis, prevention and the design of tailored ttherapy for the treatment of cancer of each patient.

Figure 12 :

 12 Figure 12: Emerging Hallmarks of cancer. from Hanahan and Weinberg, Cell, 2011 142

  murine xenograft models of melanoma and colorectal tumors 145 . The beneficial effect of the alternative NF-B pathway is exerted through the amplification of IL-12 + tumor infiltrating dendritic cells, thereby increasing anti-tumor T cell immunity 145 . Anti-PD-1 therapy promotes the secretion of IFN-γ by cytotoxic CD8 + T cells, inducing the upregulation of alternative NF-B pathway-regulated genes in tumor-infiltrating dendritic cells DCs, and subsequent production and release of IL-12, thus enabling CD8 + T cell anti-tumor activity. Altogether, this study suggests that PD-1 immune checkpoint inhibitors require NF-B alternative pathwaydependent IL-12 production by a subset of tumor-infiltrating dendritic cells to increase efficacy of anti-PD-1 immunotherapy. Agonistic CD40 mAbs and SMAC mimetics are under clinical trials in combination with anti-PD-1 checkpoint inhibitors for several solid and hematological cancers (clinicaltrials.gov: NCT03123783, NCT02376699 and NCT03270176). A recent breakthrough study demonstrated that the classical cRel NF-B subunit is immunosuppressive through the maintenance of activating Tregs, a tumor-infiltrating Treg subset inhibiting antitumor effector CD8 + T cells 146 (Fig. 13). cRel specific inhibition either by genetic ablation or by the specific inhibitor pentoxifylline not only decreased the expression of activating Tregs identity markers such as Foxp3, CD25, and Helios, but also increased IFN-γ production by activating Tregs, thereby promoting CD8 + T cells tumoricidal activity. cRel inhibition also increased CD8 + T cells number and IFN-γ production in the tumor, thereby resulting in a drastic reduction of tumor growth in a murine xenograft model of melanoma 146 . Importantly, tumor volume reduction was much more pronounced by combination of anti-PD-1 therapy with cRel inhibition compared to what was seen with anti PD-1 treatment alone 146 , indicating that optimal efficacy of anti PD-1 inhibitors requires inhibition of cRel activity. Strikingly, RelA is mostly dispensable for activating Treg 146 . Thus, even though the two classical RelA and cRel NF-B subunits were shown to both play an important role for Treg cells thymic development, cell activity and maintenance of immune tolerance 148-151 , only cRel appears to exert its immunosuppressive function through the activating Treg maintenance. Pro-inflammatory cytokines produced in the tumor microenvironment lead to eradication of anti-tumor immunity and enhanced tumor cell survival. Thus, understanding the complex relation between cancer cells and immune response during chronic inflammation should help to optimize anti-tumor immunity for cancers accompanied by inflammation, such as breast cancer, liver and colorectal cancers 152 . The classical NF-B pathway activation induced by inflammatory signals was shown to be detrimental to the efficacy of anti-tumor immunotherapy (Fig. 13). Upon TNF stimulation, activation of RelA NF-B subunit mediates T cell immunosuppression, through increased expression of PD-L1 147 . Mechanistic studies indicate that RelA regulates the transcription of CSN5, a deubiquitinase enzyme that interacts with PDL-1, leading to PD-L1 stabilization upon inflammation. Inhibition of CSN5 and NF-B by curcumin sensitized cancer cells to anti-CTLA4 therapy 147 . Importantly, high CSN5 and RelA expression is associated with poor prognosis in a cohort of 261 human breast tumor samples.

Figure 13 :

 13 Figure 13: The role of classical and alternative NF-B subunits in the efficacy of immune checkpoint inhibitors. (A) The alternative NF-B pathway (i.e. RelB) is beneficial through the amplification of tumor infiltrating dendritic cells, their increased production of IL-12 and subsequent stimulation of CD8 + T cell antitumor immunity. Anti-PD-1 therapy stimulates alternative NF-B pathway activity through IFN-γ secretion by CD8 + T cells. (B) cRel classical NF-B subunit is immunosuppressive through the increased maintenance of activated Tregs, a tumor-infiltrating Treg subset inhibiting antitumor effector CD8 + T cells. (C) RelA classical NF-B subunit activation by inflammatory signals in cancer cells mediates T cell immunosuppression through increased stabilization of PD-L1 linked to increased expression of the deubiquitin enzyme CSN5.

Figure 14 :

 14 Figure 14: Mechanisms of resistance to CAR T cell therapy. from Shah and Fry, Nat. Rev. Clin. Oncol, 2019 156

Figure 15 :

 15 Figure 15: The role of NF-B on CAR T cell efficacy. Second-generation CAR contains a costimulatory endodomain, such as 4-1BB. 4-1BB recruits the adaptor protein TRAF2 through its TRAF binding domain, thereby leading to NF-B activation. Upon CAR stimulation by tumor-associated antigen, 4-1BB-dependent NF-B activation leads to CAR T cell persistence (A). Upon self-clustering of CARs, 4-1BB-dependent NF-B activation increases the expression of pro-apoptotic genes such as Fas, FasL and TRAIL, thus leading to CAR T cell apoptosis (B).

  premalignant lesions and breast cancer samples of patients carrying a BRCA1 mutation 177 . It was recently discovered that genetic inactivation of RANK prevented BRCA1 mutation-driven tumorigenesis via defective proliferation of human mammary progenitor cells and that RANK genome variations are significantly associated with risk of developing breast cancer in women with BRCA1 mutations 177 (Fig 16B). Remarkably, long-term pharmacological inhibition with Denosumab in mice abolished the occurrence of BRCA1 mutation driven preneoplastic lesions 178 . Very interestingly, the alternative NF-B pathway is activated in BRCA1-deficient mammary progenitors and is essential for BRCA1 deficient-hormone independent progenitor cell growth 180 (Fig 16B).

Figure 16 :

 16 Figure 16: NF-B activation by RANK/RANKL in bone cancer complications and BRCA1 driven breast cancer. (A) Circulating tumor cells release PTHrP, IL-1 and IL-11 that drives RANKL secretion by osteoblasts, inducing RANKL interaction with its receptor RANK at the surface of pre-osteoclasts and osteoclasts. This binding of RANKL to RANK promotes the differentiation of pre-osteoclasts to osteoclasts, relocation to the bone, and activation of their osteolytic function. Denosumab, a human monoclonal RANKL antagonistic antibody, prevents RANK/RANKL-induced bone resorption and bone metastases appearance in cancer patients (e.g. multiple myeloma, breast cancer, prostate cancer). Denosumab has been approved for the prevention of cancer skeletal-related events and the treatment of bone metastases in cancer patients. (B) The alternative NF-B pathway (i.e. RelB) is activated in BRCA1-deficient mammary progenitors and is essential for BRCA1 deficient-hormone independent progenitor cell growth. Genetic inactivation of RANK or RANKL inhibition by denosumab prevented BRCA1 mutation-driven tumorigenesis via defective proliferation of human mammary progenitor cells.

  account to better predict ibrutinib responsive DLBCL patients. Very importantly, a breakthrough study from L. Staudt lab demonstrated a new mode of oncogenic BCR signaling coordinated by a multiprotein supercomplex formed by MYD88, TLR9 and the BCR, named the My-T-BCR supercomplex 196 (Fig 17). This My-T-BCR supercomplex is mainly present in ABC DLBCL, but not GC DLBCL. My-T-BCR interacts with the CBM complex to activate IKK/NF-B. Ibrutinib disrupt the formation of the My-T-BCR supercomplex in DLBCL cell lines and patient biopsies 196 (Fig 17). A recent phase III trial (PHOENIX) combining conventional immune-chemotherapy R-CHOP (anti-CD20 rituximab combined with cyclophosphamide, doxorubicin, vincristine, and prednisone) with ibrutinib on a cohort of ABC DLBCL patients did not reach its primary endpoint 197 . Altogether, it indicates that the BCR/NF-B mutational status and the presence of the My-T-BCR supercomplex should be evaluated for a better stratification of DLBCL patients more prone to respond to ibrutinib treatment.

Figure 17 :

 17 Figure 17: NF-B gain and loss-of-function mutations in ABC DLBCL: Impact on chronic active BCR signaling and ibrutinib responsiveness. The two modes of oncogenic BCR activation are represented. The first one through BTK (left) and the second one through the My-T-BCR complex (right). Arrows indicate activating steps and bars indicate inhibitory steps. Mutations in positive and negative regulators of NF-B are colored in yellow. Either type of mutation leads to NF-B constitutive activation and survival of ABC DLBCL cells. The BTK inhibitor ibrutinib blocks the two modes of BCR activation.

Figure 18

 18 Figure 18: NF-κB in the new era of cancer therapy

Figure 19 :

 19 Figure 19: NF-κB integrating metabolism. from Tornatoreet al, Trends in Cell Biol, 2012 199

Figure 20 :

 20 Figure 20: The role of NF-B on cancer cell energy metabolism. (A) In WT p53 expressing cancer cells, RelA increases the expression of the mitochondrial gene SCO2, thereby leading to sustained Oxphos. RelA inhibition renders tumors highly susceptible to metformin, an antitype II diabetes drug that has been shown to have an anti-tumor effect. (B) In inactive p53

Figure 21 :

 21 Figure 21: Breast cancer subtypes. from Heng et al, Oncotarget, 2016 211

Figure 22 :

 22 Figure 22: Glucose metabolism. from from Hay, Nat Rev Cancer, 2016 216

Figure 23 :

 23 Figure 23: Warburg effect. From Vander Heiden, Cantley and Thompson, Science, 2009 219

  proliferation, migration and invasion266 . Sterol regulatory element-binding protein 2 (SREBP2) and RAR-related orphan receptor γ (RORγ), transcription factors regulating cholesterol metabolism, activates the cholesterol bio-synthesis program and facilitates the progression of TNBC 267 . In addition, TP53-mediated activation of cholesterol synthesis via the SREBP pathway appears to induce proliferation and self-renewal of cancer cells of the breast 268The Caveolin-1 and Caveolin-2(CAV1 and CAV2, respectively), a protein binding with cholesterol to form plasma membrane caveolae, has been proposed to play a role in intracellular cholesterol trafficking. CAV1 expression levels also higher in BC cell lines compared to normal cells. CAV1 and CAV2 have been identified as markers for basal-like breast tumors. CAV1 and CAV2 expression is associated with highly breast aggressive tumors269 . In basal-like breast cancers, gene amplification of CAV1 and CAV2 could be explained by epigenetic modulation of CAV1 and CAV2 promoters 269 . Moreover ACAT1, the enzyme regulating the conversion of cholesterol into cholesterol ester, appears to decrease the LDL-induced proliferation of basal-like BC cell lines 270 . Overexpression of ACAT1 and CAV1 are found in basal-type BC cells compared to normal epithelial cells and luminal-like BC cells 270 . Accumulation of cholesterol ester as a potential indicator of proliferation in human breast cancer. Overexpression of LDLR and Scavenger receptor class B type 1 (SCARB1) in addition to activation of Acetyl-CoA acetyltransferase 1 (ACAT1) may be crucial players causing intratumoral accumulation of cholesterol ester (CE). These results thus open the door to new anticancer therapeutic strategies 271,272 .Fatty acids (FAs), along with glucose, are the most important fuel sources for cells' energy production. FA are catabolized mainly in ATP and NADPH, NADH and FADH mechanisms by several pathways such as fatty acid β-oxidation (FAO), the TCA cycle (or krebs cycle) and oxidative phosphorylation223,273 (Fig.24).

Figure 24 :

 24 Figure 24: Overview of cellular fatty acid metabolism. from Currie et al, cell Metabolism, 2013 274

  . In basal-like breast cancers, gene amplification of CAV1 and CAV2 could be explained by epigenetic modulation of CAV1 and CAV2 promoters 269 . Moreover ACAT1, the enzyme regulating the conversion of cholesterol into cholesterol ester, appears to decrease the LDL-induced proliferation of basal-like BC cell lines 270 . Overexpression of ACAT1 and CAV1 are found in basal-type BC cells compared to normal epithelial cells and luminal-like BC cells 270 . Accumulation of cholesterol ester as a potential indicator of proliferation in human breast cancer. Overexpression of LDLR and Scavenger receptor class B type 1 (SCARB1) in addition to activation of Acetyl-CoA acetyltransferase 1 (ACAT1) may be crucial players causing intratumoral accumulation of cholesterol ester (CE). These results thus open the door to new anticancer therapeutic strategies 271,272 .

Figure 25 :

 25 Figure 25: The major enzymes involved in choline phospholipid metabolism in the cell. from Glunde et al, Nat. Rev. Cancer, 2011 283 .

Figure 1 .

 1 Nuclear Presence of pS472-RelB Correlates with Distant Metastasis and Poor Prognosis of Human BCs Tissue microarray based IHC study (immunohistochemistry) of nuclear presence of pS472-RelB in 210 BCs tissues from an Institut Curie cohort with available treament information was performed. (A) Representative images of immunohistochemical (IHC) staining of positive nuclear pS472-RelB. Scale bars, 50 μm. Kaplan-Meier model of (B) overall survival, (C) cumulative incidence of metastases and (D) distant metastasis free survival, according to nuclear pS472-RelB presence status. P-value was calculated using a Log-rank (Mantel-Cox) test.

Figure 2 .

 2 Phosphorylation of RelB serine 472 promotes metastasis development in vivo Following intracardiac injection of MDA-MB-231-luc D3H2LN cell line transduced with the indicated lentivirus: (A) Representative images of bioluminescence (6/13 mice) at day 33 (B) Number of photons/s per mouse visualized in ventral position (n = 13 per group) at days 7 to 41, (C) cumulative incidence of metastases of mice (n = 13 per group) up to 41 days, (D) number of ventral metastasis per mouse (n = 13 per group) and (E) number of mice with metastasis (n = 13 per group).

  and S1B). Moreover, neither the inhibition of RelB expression nor the introduction of WT RelB and RelB S472A affected NF-κB canonical subunit RelA DNA-binding capacity and expression (figure 3F). These results demonstrate that phosphorylation of RelB on serine 472 boost TNBC metastasis development without affecting RelB DNA-binding capacity. Whole cell extracts from 12 BC cell lines were analyzed for RelB and pS472-RelB protein expression by immunoblotting for the indicated proteins. (B) Incidence of death depending on the nuclear presence of pS472-RelB in BC patients. (C) distant metastasis free survival in TNBC, according to nuclear pS472-RelB presence status. P-value was calculated using a Logrank (Mantel-Cox) test. (D) Whole cell extracts from MDA-MB-231 cell line transduced with the indicated lentiviruses were analyzed by immunoblotting for RelB and pS472-RelB expression. Left panel: lentiviruses encoding either a shRNA targeting RelB (shRNA RelB) or a scrambled control (shRNA control). Right panel: lentiviruses encoding either a cDNA for ectopic expression of WT RelB (WT RelB), a cDNA for ectopic expression of the nonphosphorylable RelB mutant S472A (S472A RelB) or a scrambled control (Control). (E) pS472-RelB immunohistochemical (IHC) staining of MDA-MB-231 cell line transduced with lentiviruses encoding for WT RelB, S472A RelB and control. (F) Whole cell extracts from MDA-MB-231 cell line transduced with the indicated lentiviruses were analyzed by EMSA (top panels) and by immunoblotting for the indicated proteins (bottom panels). RelA-and RelBcontaining complexes are indicated by arrows.

Figure 28 .

 28 Figure 28. High levels of RelB serine 472 phosphorylation correlates with lethal metastatic TNBC

  RelB-mediated gene expression program. As we and others have demonstrated a close regulation of several members of matrix metalloproteinase family by RelB296,298,299 , we evaluated the expression of the MMPs have been identified as poor prognosis markers for breast cancer patients300 . Strikingly, we revealed that only MMP13 mRNA expression is upregulated by ectopic expression of WT RelB but not S472A mutant in MDA-MB-231 cell line (figure5Aand S3A). In accordance with these results, inhibition of endogenous RelB knockdown decreased the MMP13 levels in MDA-MB-231 (figure5A and S3B). Further, we demonstrated a direct regulation of MMP13 by pS472-RelB using chromatin IP (ChIP) analysis as WT RelB but not S472A mutant is recruited to MMP13 promoter (figure5B). MMP13 activity was also markedly and significantly increased in WT RelB but not S472A-expressing MDA-MB-231 cells, and decreased by inhibition of RelB expression (figure 5C). Because we have characterized MMP13 as a serine 472-dependent RelB target gene in MDA-MB-2321 cell line, we examined the direct contribution of MMP13 in RelB serine 472-mediated cell migration and cell invasion by a specific chemical approach of MMP13 inhibition. Stunningly, MMP13 inhibition significantly decreased MDA-MB-231 migration, invasion and degradation capacities of WT RelB-expressing cells (figure 5D, 5e and 5F). Taken together, our data suggest that serine 472 phosphorylation of RelB promotes TNBC cell migration and invasion by upregulating the expression of selective NF-κB target genes, such as MMP13. Legend to Figure 5. RelB serine 472 phosphorylation-mediated pro-invasive capacity requires direct regulation of MMP13 expression and activity in TNBC cells Cell extracts from MDA-MB-231 cell line transduced with WT RelB, S472A RelB and Control (left panel) and shRNA RelB or shRNA control (right panel) lentiviruses were analyzed: (A) for MMP13 mRNA expression by quantitative RT-PCR. Quantitative RT-PCR was performed with specific primer pairs for MMP13 Results are means ± SD of three independent experiments line normalized to the level of HPRT mRNA. P-values by unpaired t test *** P<0.001, ** P<0.01, * P<0.05, (B) Recruitment to MMP13 promoter was examined by ChIP assays followed by qPCR analysis normalized to inputs; n = 3, Error bars are SD of 3 independent experiments. P-values by unpaired t test *** P<0.001, ** P<0.01, * P<0.05 and (C) MMP13 activity is represented as relative fluorescence units (RFUs); n = 3, Error bars are SD of 3 independent experiments. P-values by unpaired t test *** P<0.001, ** P<0.01, * P<0.05. (D) MDA-MB-231 cell line transduced with the indicated lentiviruses were analyzed for cell migration by scratch-wound assays for 24h with or without MMP13 specific inhibitor (CL-82198). Error bars are SD of 3 independent experiments. *** P<0.001, ** P<0.01, * P<0.05. (E) MDA-MB-231 cell line transduced with the indicated lentiviruses were analyzed for cell invasion after 8h with or without MMP13 specific inhibitor (CL-82198). Error bars are SD of 3 independent experiments. P-values by unpaired t test *** P<0.001, ** P<0.01, * P<0.05. (F) MDA-MB-231 cell line transduced with the indicated lentiviruses were analyzed for fluorescent gelatin degradation index after 6h with or without MMP13 specific inhibitor (CL-82198). Error bars are SD of 3 independent experiments. P-values by unpaired t test *** P<0.001, ** P<0.01, * P<0.05.

  control (Control). (A) Whole cell extracts from MDA-MB-231 cell line transduced with the indicated lentiviruses encoding either a shRNA targeting RelB (shRNA RelB) or a scrambled control (shRNA control) were analyzed by immunoblotting for BCL2 expression. (B) Whole cell extracts from MDA-MB-231 cell line transduced with the indicated lentiviruses encoding either WT RelB, S472A RelB or Control were analyzed for PRDM1 mRNA expression by quantitative RT-PCR. Quantitative RT-PCR was performed with specific primer pairs for MMP13 Results are means ± SD of three independent experiments normalized to the level of HPRT mRNA. P-values by unpaired t test *** P<0.001, ** P<0.01, * P<0.05. Cell growth of MDA-MB-231 cell line transduced with the indicated lentiviruses encoding either WT RelB, S472A RelB or Control was measured by counting cells each day for 3 days. Results for each are normalized with the number of cells seeded at day 0 and are of three independent experiments. (B) Cell growth of MDA-MB-231 cell line transduced with the indicated lentiviruses encoding either shRelB or shControl was measured by counting cells each day for 3 days. Results for each are normalized with the number of cells seeded at day 0 and are of three independent experiments. (C) MDA-MB-231 cell line transduced with the indicated lentiviruses encoding WT RelB, S472A RelB or Control were analyzed for cell displacement in 4% Matrigel 7 days after seeding. All values were normalized to the diameter of each cell. Representative images are presented on the left panel. Error bars are SD of 3 independent experiments. P-values by unpaired t test *** P<0.001, ** P<0.01, * P<0.05. (D) MDA-MB-231 cell line transduced with the indicated lentiviruses encoding shRelB or shControl were analyzed for cell displacement in 4% Matrigel 7 days after seeding. All values were normalized to the diameter of each cell. Representative images are presented on the left panel. Error bars are SD of 3 independent experiments. P-values by unpaired t test *** P<0.001, ** P<0.01, * P<0.05. Samples of primary breast tumors surgically removed prior to any radiation, hormonal or chemotherapy treatment were collected at Institut Curie from 2005 to 2006. Analyses of human samples were performed in accordance with the French Bioethics Law 2004-800, the French National Institute of Cancer (INCa) Ethics Charter, and after approval by the Institut Curie review board and ethics committee (Comité de Pilotage du Groupe Sein).Patients were informed of the research use of their tissues and did not declare any opposition for such research. Data were analyzed anonymously.Tumor breast molecular subtypes were defined as follows according to the guidelines of the American Society of Clinical Oncology (ASCO)/College of American Pathologists (Wolff,JCO, 2007; Prat, JCO, 2013): Luminal A: estrogen-receptor (ER)≥10%, progesterone-receptor (PR)≥20%, Ki-67<14%; Luminal B: ER≥10%, PR<20%, Ki-67≥14%; ER-/PR-/HER2+: ER<10%, PR<10%, HER2 2+ amplified or 3+; ER-/PR-/HER2-(Triple-negative breast cancers, TNBC): ER<10%, PR<10%, HER2 0/1+ or 2+ non-amplified.Preparation of TMA and immunohistochemistryTMA consisted of replicate of 1mm diameter tumor core selected from whole-tumor tissue section in the most representative tumor areas (high tumor cell density) of each tumor sample and a matched tissue core from adjacent non tumoral breast epithelium.

  stable RelB-negative, WT (wild-type), or S472A (Serine 472-substituted phosphorylation-null mutant) MDA-MB-231-Luc-D3H2LN cell clones. All injected cells showed similar viability as measured by Annexin V apoptosis kit (Beckman Coulter). Animal experiments were approved by the CEEA26, CEEA PdL No 6 Ethics Committee, and the Ministry of Agriculture (approval number: APAFIS#1648-2015090713516480) and performed under the conditions established by the European Community (Directive 2010/63/UE).

  2 C-2 # sc-7382), Sigma-Aldrich (β-actin clone AC-15 # A5441) and Clinisciences (mouse monoclonal antibody anti-RelB phosphorylated S472 Cat# NB-19-0001 clone RA3-AF3).

Figure 1 .Figure 31 .

 131 Figure 1. RelB promotes metabolic reprogramation of BC cells.

Figure 2 .

 2 Figure 2. RelB enhances BC cell growth via glucose metabolism in physiological glucose

Figure 32 .

 32 Figure 32. RelB enhances BC cell growth via glucose metabolism in physiological glucose

Figure 3 .

 3 Figure 3. SLC2A3 (GLUT3) is a novel RelB target gene that correlates with BC overall

Figure 33 .

 33 Figure 33. SLC2A3 (GLUT3) is a novel RelB target gene that correlates with BC overall

  Collares D* (co-first authors), Faumont N, Nuan-Aliman S, Bordereaux D, Cagnard N, Martins I, Riviere P, Lordello L, Taoui O, Lehmann-Che J, Jardin F, Copie-Bergman C, Mauiri C, Jais JP, Leroy K, Kroemer G, Pasqualucci, L, Molina T, Feuillard J*(cocorresponding), Baud V*. Submitted manuscript (21/10/2020) -Nature Cancer

Figure 1 .

 1 Activation of Rel B is frequent in de novo DLBCL patients irrespectively of their ABC or GCB subtypes and is associated with worse overall survival. Whole cell extracts from 66 de novo DLBCL cases frozen samples (from the LYSA LNH03 trial) were analyzed by EMSA for the alternative RelB NF-κB subunit DNA-binding activity Four representative RelB positive cases are presented (2 ABC and 2 GCB). For supershifts, extracts were incubated with either anti-RelB or anti-RelA antibodies prior to incubation with the labeled probe. RelA-and RelB-containing complexes are indicated. (b) Distribution of RelB DNA binding activity as defined by EMSA among the GCB and ABC DLBCL patients (n=66) (c) Top: prevalence of RelB activation as defined by EMSA in ABC and GCB DLBCLs (n=66). Bottom: prevalence of ABC and GCB DLBCLs within the positive RelB binding activity subgroup. (d) Top: prevalence of dominant activation of the two canonical NF-κB subunit RelA and cRel as defined by EMSA in ABC and GCB DLBCLs (n=66). Bottom: prevalence of ABC and GCB DLBCLs within the two dominant canonical NF-κB subunit RelA and cRel activation subgroups. (e) Kaplan-Meier plot of overall survival according to RelB DNA-binding status (LYSA LNH03 trial, EMSA cases, n=66). P-value by Log-rank (Mantel-Cox) test.

Figure 34 .

 34 Figure 34. Activation of Rel B is frequent in de novo DLBCL patients irrespectively of their ABC or GCB subtypes and is associated with worse overall survival.

Figure 2 .

 2 Identification of a RelB gene expression signature associated with worse outcome of R-CHOP treated DLBCL patients (a) Hierarchical clustering of 202 DLBCL patients from the GSE87371 cohort with the 140 differentially expressed probesets identified based on RelB DNA-binding gene expression signature (see Supplementary fig. 1). Lines and columns of the heatmap correspond to probesets and patients respectively. Upregulated genes are coded in green, downregulated genes are coded in red. COO classification of each case is represented on top of the heatmap. (b) Distribution of RelB activity as defined by GEP among GCB and ABC DLBCL patients from the GSE 87371 cohort (n=202). (c) Top: prevalence of RelB activation as defined by GEP in ABC and GCB DLBCLs (n=202). Bottom: prevalence of ABC and GCB DLBCLs within the RelB positive cluster as defined by GEP (n=202). (d) RelB signature analysis by the Ingenuity Pathway Analysis software (www.ingenuity.com). (e) Volcano plot depicting the enrichment score of ten NF-κB signatures involved in regulatory processes in normal and malignant blood (https://lymphochip.nih.gov/signaturedb/) within the RelB positive subset (x axis, log2 enrichment; y axis: log10 p-value <1). P value by Fisher's exact test. (f) Absence of correlation between Myc (cut off ≥ 40%, n=114 patients), Bcl2 (cut off ≥ 50%, n=95 patients), Myc/Bcl2 coexpression (n=87 patients), and Bcl6 (cut off ≥ 30%, n=108 patients) as evaluated by IHC with the RelB gene-expression signature. P value by Fisher's Exact test. (g) Kaplan-Meier plot of overall survival of the GSE87371 cohort (R-CHOP treated ABC and GCB DLBCL patients, n=98), according to RelB GEP clusters. P value by Log-rank (Mantel-Cox) test. (h) Multivariate analysis of the indicated risk factors for overall survival (GSE87371 cohort, R-CHOP treated patients, n=98) using Cox regression.

Figure 3 .

 3 Genetic features associated with RelB activation in DLBCL (a) The mutational heatmap indicates twenty-six recurrently altered genes in ABC and GCB DLBCL cases (GSE87371, n=156) with frequency > 5%. Right to the mutational heatmap is shown the prevalence of the indicated mutated genes in RelB-positive vs RelB negative DLBCL cases as defined by GEP. P value by Fisher's exact test. Color-codes indicate the presence or absence of the corresponding feature. RelB activation status and ABC vs GCB DLBCL subtypes are indicated above the mutational map. (b) Volcano plot depicting the differences of gene mutations between RelB positive and RelB negative DLBCL as defined by GEP (x axis, log2 odds ratio; y axis: -log10 p-value>1). P value by Fisher's exact test. (c) Correlation of either Bcl6, Bcl2 or Myc fusion as evaluated by FISH with RelB gene-expression signature, n=107 patients. P value by Fisher's exact test. Color codes indicate the presence or absence of the corresponding feature. (d) Top: prevalence of each genetic subtype defined by the LymphGen algorithm (https://explore.openaire.eu) within the RelB positive and RelB negative clusters as defined by GEP (n=168). Bottom: prevalence of RelB binding activity as evaluated by GEP within each LymphGen genetic subtype (n=168). (e) Top: prevalence of each genetic subtype defined by the LymphGen algorithm within each canonical NF-κB DNA-binding subgroup (RelA and cRel). Bottom: prevalence of canonical NF-κB DNA-binding subgroups (RelA and cRel) within each LymphGen genetic subtype.

Figure 36 .

 36 Figure 36. Genetic features associated with RelB activation in DLBCL

Figure 37 .

 37 Figure 37. Frequent RelB-DNA binding activity in ABC and GCB DLBCL cell lines

  RelB protects DLBCL cells (both ABC and GCB) from DNA damage accumulation and subsequent apoptosis upon doxorubicin treatment.Legend toFigure. 5. RelB protects ABC and GCB DLBCL cells from induction of apoptosis upon doxorubicin treatment (a) RelB protein levels (top) and RelB DNA binding activity (bottom) are efficiently knocked down by stable RNA interference in ABC and GCB DLBCL cell lines. Whole cell extracts from MD901 (ABC), K231 (GCB) and OCI-ly8 (GCB) cell lines transduced with lentiviruses encoding either a shRNA targeting RelB (shRNA RelB) or a scrambled control (shRNA control) were analyzed by immunoblotting (top) and by EMSA (bottom) for the indicated proteins. (b) RelB knockdown induces ABC and GCB DLBCL cell apoptosis upon doxorubicin treatment. MD901, K231 and OCI-ly8 cell lines transduced as in (a) were treated with 2µM doxorubicin for 16h (MD901 and K231) or 24h (OCI-ly8), and monitored for apoptosis by annexin V-APC and DAPI staining followed by FACS analysis. Error bars are means ±SD of 3 independent experiments. P-values by unpaired t test, *** P<0.001, ** P<0.01. (c,d) Whole cell extracts of MD901 cells transduced as in (a) were treated with doxorubicin 2µM for the indicated periods of time, and analyzed by immunoblotting for cleaved caspase 3 (c) and -H2AX (d). GAPDH (c) and -actin (d) were also analyzed, n=3. (e) RelB knockdown induces -H2AX foci in DLBCL cells upon doxorubicin treatment. Left: % of high γH2AX foci positive MD901 cells. Right: γH2AX foci count mean in response to doxorubicin normalized to foci count mean of untreated MD901 cells. Data of a representative experiment are shown (f) RelB knockdown decreases cIAP2 expression at mRNA level in ABC and GCB DLBCL cell lines. Quantitative RT-PCR was performed with specific primer pairs for cIAP2 using total RNAs prepared from three DLBCL cell lines (1 ABC and 2 GCB) upon RelB knockdown as in (a). Results are means ± SEM of three independent experiments for each cell line normalized to the level of HPRT mRNA. P-values by unpaired t test, **** P<0.0001, *** P<0.001. (g) RelB knockdown decreases cIAP2 protein expression levels in ABC and GCB DLBCL cell lines. Whole cell extracts prepared as in (a) were analyzed by immunoblotting for the indicated proteins, n=2. (h) RelB activation as evaluated by EMSA is associated with increased cIAP2 protein expression in DLBCL patients. Whole cell extracts from twenty-four R-CHOP treated DLBCL patients out of the 66 DLBCL patients used in the EMSA study were analyzed for cIAP2 protein expression levels by immunoblotting. Left: one representative immunoblot is presented. Right: cIAP2 protein expression levels normalized to β-actin, n=24. P-values by Pearson correlation test, ** P<0.01.

  pathogenesis, and thereby their putative mutations being overlooked. The present work shows that RelB positive DLBCLs are significantly associated with/characterized by an enrichment of genetic alterations of ITPKB, B2M and IRF4. Whether mutations affecting these genes could be functionally involved in RelB activation is currently unknown but worth further investigation. Alternatively, additional mechanisms might contribute to the regulation of RelB activity in DLBCL cells independently of intrinsic mutations, such as stimuli originating from the tumor microenvironment and loss of nuclear inhibitory mechanisms.Evasion of cell death is a characteristic feature of DLBCL and represents a key cause of resistance to current treatment approaches. Therefore, reactivation of cell death programs in DLBCL cells is a promising strategy to overcome resistance to treatment. Our study clearly shows that RelB protects DLBCL cells from DNA damage induced by doxorubicin, a genotoxic agent used in front-line treatment for DLBCL, thereby leading to increased resistance of DLBCL cells to doxorubicin. First, we observed an increase in H2AX expression and foci induced by doxorubicin upon RelB expression inhibition. Second, RelB knockdown induces a marked increase in DLBCL cell apoptosis upon doxorubicin. Third, RelB activity is required for optimal expression of the anti-apoptotic gene cIAP2 in DLBCL cells, although it remains unknown if the pro-survival activity of RelB is exerted through the control of cIAP2 expression.

  RelB in those cells in 2D conditions. Only by reducing glucose concentrations to physiological concentration did we reveal that RelB, via control of glucose metabolic pathways, boosts BC cell growth. These results suggest that RelB could confer a survival advantage to BC cells based on the availability of glucose which would confirm by following glucose uptake in cells, depending on RelB expression. Since the cell lines used for modifications of RelB expression are accustomed to grow in an elevated glucose concentration, such models could mislead our interpretation of RelB role in BC metabolism and associated phenotypes, thereby prompting for in vivo evaluation of RelB in physiological conditions.To confirm our hypothesis in vivo, we could first characterize RelB DNA-binding activity, regulation and expression in established BC patient-derived xenografts mouse model(PDX) and correlating these profiles of RelB with tumor glucose metabolism. First, we could evaluate glucose uptake and glycolysis rate in tumors by following glucose analogue, 2-18 fluoro-2deoxyglucose imaging by positron emission tomography ( 18 FDG-PET), and then we could follow the NMR-based metabolic profiling of these tumors and blood samples. Further, we could evaluate the glucose metabolic pathways associated with RelB assessment using[START_REF] Bonizzi | Activation of IKKα target genes depends on recognition of specific κB binding sites by RelB:p52 dimers[END_REF] Cglucose labeling with combined with dynamic contrast-enhanced MRI (DCE-MRI)354 , providing deeper understanding of RelB function in metabolic reprogramming.

  activity has been established by cancer cells in response R-CHOP.It has been recently hypothesized that combination of inhibitors (L-asparaginase, mTOR inhibitor, and metformin) targeting different metabolic pathways could offer new therapeutic opportunities to DLBCL patients with a low response to R-CHOP367 . Studying RelB-associated metabolic reprogramming in DLBCL with our RelB-modified DLBCL cell lines (using the in vitro assays described sooner) and analyze RelB-dependent cell death in response to metabolic drugs could help improve the selection of metabolic drug in low R-CHOP responsive patients.Reporting a RelB-dependent metabolic vulnerability involved in R-CHOP resistance could also rationalize the combination of R-CHOP with specific metabolic inhibitors as a first line of treatment.Moreover, recent observation that high baseline total metabolic tumor volume (TMTV) to be negatively impact survival of DLBCL patients increases the interest to study DLBCL metabolism368 . TMTV is computed on baseline positron emission tomography/computed tomography. Correlation between TMTV and RelB DNA-binding activity could provide remarkably interesting information concerning RelB impact on DLBCL metabolism in patients.CONCLUSIONDuring my Ph.D. I had the opportunity to tackle the role of NF-κB alternative pathway subunit RelB in two cancer models, with a variety of approaches including in vitro, in vivo and patient's extensive data analysis. Hence, these investigations have allowed me to access to a general observation of RelB in cancer and to the possible future directions for its study. I was also able to extensively examine NF-κB signaling pathways and their potential vulnerabilities for therapeutic intervention. In summary, first I demonstrated a novel mechanism of aggressivity in breast cancer driven by RelB-through the fine-tuned regulation of its transcriptional activity. It also constitutes the first time that the specific detection of a RelB post-translational modification in patients correlates with a poor clinical outcome. Of all BC subtypes, TNBC remains the one with the greatest therapeutical challenge, requiring a thorough profiling of tumors for molecular signaling and associated-phenotypic features. Extensive comprehension of RelB and its specific regulation via phosphorylation might have great potential for therapeutic innovation, and further analysis of RelB S472 phosphorylation could determine an alternative vision of RelB function in cancer. I also contributed to the establishment of a RelB gene expression profiling (GEP) based on the its frequent DNA-binding activity in diffuse large B cell lymphoma patients associated with poor prognosis, constituting a novel model of investigating RelB in cancer malignancy. In adddition, I unveiled a unique RelB mutational profile specific associated with RelB GEP and I participated in the demonstration of RelB control DLBCL cell lines resistance to the main drug in DLBCL treatment. This study establishes a strong case for RelB DNA-binding activation analysis in cancer and its extension via characterization of transcriptional and mutational specific signatures. Lastly, I unexpectadly observed an original function controlled by RelB in the regulation of glucose metabolic reprogramming in cancer cells. This constitutes, to my knowledge, the first observation of the implication of RelB in cancer metabolism. Furthermore, RelB-dependent glucose metabolism reprogramming function in cell growth has allowed to identify an underappreciation for the contribution of RelB in cancer, as in vitro experiments are usually led in out-of physiological conditions evidence of RelB in metabolism in other cancers might have slipped from scientifical observation because of it. This suggests that there might be a whole new field of exploration for RelB in cancer. I also determined that RelB protected BC cells from chemotherapeutic drug doxorubicin through this glucose metabolism regulation (data not shown). These results modestly add to the increasing number of studies exhibiting a potential strong benefit for therapeutic intervention by targeting metabolism in combination with chemotherapy. Overall during my Ph.D., not only did I observed news insights of RelB crucial role in two very distinct cancers via distinct mechanisms, but I was also able to benefit from a large range of tools for the analysis of RelB. This might provide a new research apparatus for the study of RelB in cancer. "Dans la vie, rien n'est à craindre, tout est à comprendre » Marie Skłodowska-Curie

  [START_REF] Leidner | Identification of lysine residues critical for the transcriptional activity and polyubiquitination of the NF-κB family member RelB[END_REF]

  It was first described that RelB regulates mitochondrial biogenesis and subsequent ATP production in immortalized C2C12 myoblast cells to promote myotube homeostasis204 . The same group also showed that RelB promotes oxidative phosphorylation in skeletal muscles through the transcriptional regulation of mitochondrial regulator PGC-1β 205 .

Similar results were observed in murine osteoclasts mitochondrial respiration, but through a distinct mechanism than RelB-dependent osteoclast differentiation and bone resorption 206 , and in monocytes where induction of RelB by SIRT1 after acute inflammation stimulation leads to transcriptional expression of SIRT3 and increase of mitochondrial bioenergetics in human blood monocytes and mouse splenocytes 207 . Interestingly, RelB deficient mice also showed an increase of lactate in muscles and increased oxygen consumption, suggesting that loss of RelB could induce a metabolic shift towards glycolysis 205 . Because RelA function in cancer metabolism is intimately connected to p53 status, investigation of RelB in cancer metabolism will have to assess p53. RelB has already been demonstrated to inhibit proliferation of MEFs and tumor growth in a p53-dependent manner 208 , suggesting that metabolic reprogramming could also be in play.

of glucose, with or without

2mM of Sodium Pyruvate. (D) growth of MDA-MB-231 cell line

Acknowledgements

We thank the Centre d'Histologie, d'Imagerie Cellulaire et de Cytométrie, Centre de Recherche des Cordeliers UMRS1138, Paris, France for cell sorting and FACS analysis. We are grateful to Laura Pasqualucci for valuable genetic data in DLBCL cell lines. This work was supported by grants from Institut National du Cancer, Agence Nationale pour la Recherche, the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 766214, Université de Paris (to V.B.), Instituto de Salud Carlos III-FIS-CIBERONC (CB16/12/00489) (to J.A.M.-C.), and doctoral funding from IDEX doctoral international program (to D.C.), Prolific (to B.E.), Fondation Nelia et Amadeo Barletta, Switzerland (to S.N.A), the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 766214, Université de Paris (to L.L.), and Societé Française d'Hémathologie (to D.C.) and Fondation ARC pour la Recherche sur le Cancer (to B.E.).

RESULTS

I. Phosphorylation of RelB on S472 promotes breast cancer metastasis and cancer cell invasion via the control of MMP-13.

Eluard B 1 , Benatar C 1 , Bonsang B 2 , Bordereaux D 1 , Tanguy ML [START_REF] Baud | Is NF-κB a good target for cancer therapy? Hopes and pitfalls[END_REF] , Riviere P 1 , Billiot K 1 , Caly Table S 1: multivariate analysis Legend to Supplemental Figure 1 Whole cell extracts from MDA-MB-231 cell line transduced with the indicated lentiviruses encoding either a cDNA for ectopic expression of WT RelB (WT RelB), a cDNA for ectopic expression of the non-phosphorylable RelB mutant S472A (S472A RelB) or a scrambled Axiovert) equipped with a color camera (ICC1). For the quantification of degradation, the area of degraded matrix (black pixels) of each degradating cells were measured using threshold command of Image J software. Then the total of cell's area of degraded matrix was divided by the total number of degradating cells to define a degradation index.

Legends to Supplementary Figures and Tables

Chromatin Immunoprecipitation.

Chromatin immunoprecipitation (IP) assays were performed as described 296 . Samples were analyzed by real-time PCR. Sequences of promoter-specific primers are as follow for MMP13.

Lentiviral production and transduction

Production of infectious recombinant lentiviruses was performed by transient transfection of 293T cells as previously described 313 . For infections, cells were incubated overnight with recombinant lentiviruses. An equal amount of fresh culture medium was added 24 hours later and after 48 hours, cells were washed and seeded in fresh culture medium. GFP positive cells were sorted with FACSAria™ sorter (Becton Dickinson).

Materials and methods

Cells Culture and reagents.

MCF7 and MDA-MB-231 cells were grown in DMEM with 10% (vol/vol) FBS and 1%

antibiotics. For Nutrient deprivation/starvation, cells were grown in a no glucose, no glutamine and no pyruvate DMEM with 10% (vol/vol) FBS and 1% antibiotics and with the adjusted concentrations of glutamine, sodium pyruvate and glucose.

Antibodies and Reagents.

The antibodies were purchased from Santa Cruz (RelB C19 # sc-226, GLUT3 B-6 # sc-74497)

and Sigma-Aldrich (β-actin clone AC-15 # A5441).

Immunoblotting

Immunoblotting were performed as previously described 296 .

Quantitative RT-PCR.

Total RNA extraction and RT were performedusing TRIzol (Life Technologies) and SuperScript VILO reverse transcriptase (Life Technologies), respectively. Real-time PCR analysis was carried outwith LightCycler FastStart DNAMasterplus SYBR Green I on a Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoid malignancy affecting adults. A marked elevation of canonical NF-B signaling has been documented in DLBCL.

However, alternative NF-B activation and its role in DLBCL pathogenesis remain elusive.

Here we report that activation of RelB, the transcriptionally active alternative NF-B subunit,

Competing Interests statement

The authors declare no conflict of interests.

Even though we observed in MDA-MB 231 cell line that glucose metabolism is indeed increased by the phosphorylated form of RelB only, we still have many interrogations concerning the regulation of GLUT3. Indeed, analysis of GLUT3 mRNA expression in BC cell lines obtained from published data (http://cellexpress.cgm.ntu.edu.tw/) revealed that 2 cell lines express high levels of GLUT3 and RelB, but very low levels of pS472-RelB (data not shown).

Moreover, MCF7 cell line transduced with WT RelB and S472A RelB both displayed a similar increase of GLUT3 expression levels, questioning the impact of pS472-RelB on GLUT3 expression regulation.

Interestingly, we observed that pS472-RelB was endogenously increased by insulin treatment in 3 TNBC cell lines (data not shown). Since insulin is a major regulator of glucose transporters expression, it would be very interesting if GLUT3 is induced by such treatment and if RelB knockdown-inhibits this insulin-dependent regulation. These results suggest that RelB, through its phosphorylation on serine 472, may control glucose metabolism, but not just through GLUT3 expression alone.

Establishing a nuclear pS472-RelB gene expression profile in the BC patient cohort that was analysed in our first study would help identify if some pS472-RelB-associated genes are involved in cell metabolism and answer the question of pS472-RelB potential role in BC metabolism. In the meantime, NMR analysis of blood, urine and tumor samples of these patients could also be performed, as well as retrospective analysis of 18 FDG-PET. -Main organizer of ADELIS international congress 4 th edition, Paris, October 2021 "Metabolism in cancer: from reprogramming to therapeutical vulnerability"

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