Adaptateurs de Signalisation en Hématologie

BACKGROUND: Interactions with the microenvironment, such as bone marrow mesenchymal stromal cells and nurse-like cells, protect chronic lymphocytic leukemia cells from spontaneous and drug-induced apoptosis. This protection is partially mediated by the chemokine SDF-1α (CXCL12) and its receptor CXCR4 (CD184) present on the chronic lymphocytic leukemia cell surface. DESIGN AND METHODS: Here, we investigated the ability of AMD3100, a CXCR4 antagonist, to sensitize chronic lymphocytic leukemia cells to chemotherapy in a chronic lymphocytic leukemia/mesenchymal stromal cell based or nurse-like cell based microenvironment co-culture model. RESULTS: AMD3100 decreased CXCR4 expression signal (n=15, P=0.0078) and inhibited actin polymerization/migration in response to SDF-1α (n=8, P<0.01) and pseudoemperipolesis (n=10, P=0.0010), suggesting that AMD3100 interferes with chronic lymphocytic leukemia cell trafficking. AMD3100 did not have a direct effect on apoptosis when chronic lymphocytic leukemia cells were cultured alone (n=10, P=0.8812). However, when they were cultured with SDF-1α, mesenchymal stromal cells or nurse-like cells (protecting them from apoptosis, P<0.001), chronic lymphocytic leukemia cell pre-treatment with AMD3100 significantly inhibited these protective effects (n=8, P<0.01) and decreased the expression of the anti-apoptotic proteins MCL-1 and FLIP. Furthermore, combining AMD3100 with various drugs (fludarabine, cladribine, valproïc acid, bortezomib, flavopiridol, methylprednisolone) in our mesenchymal stromal cell co-culture model enhanced drug-induced apoptosis (n=8, P<0.05) indicating that AMD3100 could mobilize chronic lymphocytic leukemia cells away from their protective microenvironment, making them more accessible to conventional therapies. CONCLUSIONS: Taken together, these data demonstrate that interfering with the SDF-1α/CXCR4 axis by using AMD3100 inhibited chronic lymphocytic leukemia cell trafficking and microenvironment-mediated protective effects. Combining AMD3100 with other drugs may, therefore, represent a promising therapeutic approach to kill chronic lymphocytic leukemia cells.

Mast cell degranulation requires N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and mammalian uncoordinated18 (Munc18) fusion accessory proteins for membrane fusion. However, it is still unknown how their interaction supports fusion. In this study, we found that small interfering RNA-mediated silencing of the isoform Munc18-2 in mast cells inhibits cytoplasmic secretory granule (SG) release but not CCL2 chemokine secretion. Silencing of its SNARE-binding partner syntaxin 3 (STX3) also markedly inhibited degranulation, whereas combined knockdown produced an additive inhibitory effect. Strikingly, while Munc18-2 silencing impaired SG translocation, silencing of STX3 inhibited fusion, demonstrating unique roles of each protein. Immunogold studies showed that both Munc18-2 and STX3 are located on the granule surface, but also within the granule matrix and in small nocodazole-sensitive clusters of the cytoskeletal meshwork surrounding SG. After stimulation, clusters containing both effectors were detected at fusion sites. In resting cells, Munc18-2, but not STX3, interacted with tubulin. This interaction was sensitive to nocodazole treatment and decreased after stimulation. Our results indicate that Munc18-2 dynamically couples the membrane fusion machinery to the microtubule cytoskeleton and demonstrate that Munc18-2 and STX3 perform distinct, but complementary, functions to support, respectively, SG translocation and membrane fusion in mast cells.

CD9, a member of the tetraspanin family, has been implicated in hematopoietic and leukemic stem cell homing. We investigated the role of CD9 in the dissemination of B acute lymphoblastic leukemia (B-ALL) cells, by stably downregulating CD9 in REH and NALM6 cells. CD9 expression was associated with higher levels of REH cell adhesion to fibronectin and C-X-C motif chemokine receptor 4 (CXCR4)-mediated migration. Death occurred later in NOD/SCID mice receiving REH cells depleted of CD9 for transplantation than in mice receiving control cells. After C-X-C motif chemokine ligand 12 (CXCL12) stimulation, CD9 promoted the formation of long cytoplasmic actin-rich protrusions. We demonstrated that CD9 enhanced RAC1 activation, in both REH cells and blasts from patients. Conversely, the overexpression of a competing CD9 C-terminal tail peptide in REH cytoplasm decreased RAC1 activation and cytoplasmic extension formation in response to CXCL12. Finally, the inhibition of RAC1 activation decreased migration in vitro, and the depletion of RAC1 protein from transplanted REH cells increased mouse survival. Furthermore, a testis-conditioned medium induced the migration of REH and NALM6 cells, and this migration was impeded by an anti-CD9 antibody. The level of CD9 expression also influenced the homing of these cells in mouse testes. These findings demonstrate, for the first time, that CD9 plays a key role in the CXCR4-mediated migration and engraftment of B-ALL cells in the bone marrow or testis, through RAC1 activation.

Activating mutations in signaling molecules, such as JAK2-V617F, have been associated with myeloproliferative neoplasms (MPNs). Mice lacking the inhibitory adaptor protein Lnk display deregulation of thrombopoietin/thrombopoietin receptor signaling pathways and exhibit similar myeloproliferative characteristics to those found in MPN patients, suggesting a role for Lnk in the molecular pathogenesis of these diseases. Here, we showed that LNK levels are up-regulated and correlate with an increase in the JAK2-V617F mutant allele burden in MPN patients. Using megakaryocytic cells, we demonstrated that Lnk expression is regulated by the TPO-signaling pathway, thus indicating an important negative control loop in these cells. Analysis of platelets derived from MPN patients and megakaryocytic cell lines showed that Lnk can interact with JAK2-WT and V617F through its SH2 domain, but also through an unrevealed JAK2-binding site within its N-terminal region. In addition, the presence of the V617F mutation causes a tighter association with Lnk. Finally, we found that the expression level of the Lnk protein can modulate JAK2-V617F-dependent cell proliferation and that its different domains contribute to the inhibition of multilineage and megakaryocytic progenitor cell growth in vitro. Together, our results indicate that changes in Lnk expression and JAK2-V617F-binding regulate JAK2-mediated signals in MPNs.

BACKGROUND: We previously showed that B-cell receptor (BCR) signaling pathways are important for in vitro survival of mantle cell lymphoma (MCL) cells. To further identify early BCR-activated signaling pathways involved in MCL cell survival, we focused our study on BCR-proximal kinases such as LYN whose dysregulations could contribute to the aggressive course of MCL. METHODS: Primary MCL cells were isolated from 14 leukemic patients. Early BCR-induced genes were identified by qRT-PCR array. The basal and BCR-induced phosphorylation of LYN and JNK were evaluated by immunoblottting. Cell survival signals were evaluated by apoptosis using flow cytometry. RESULTS: We showed that LYN was constitutively phosphorylated in MCL cell lines and in 9/10 leukemic MCL cases. Treatment with dasatinib or with a specific inhibitor of Src kinases such as PP2 suppressed constitutive LYN activation and increased in vitro spontaneous apoptosis of primary MCL cells. BCR engagement resulted in an increase of LYN phosphorylation leading to activation of c-JUN NH2-terminal kinase (JNK) and over-expression of the early growth response gene-1 (EGR-1). Inhibition of JNK with SP600125 induced apoptosis and reduced level of basal and BCR-induced expression of EGR-1. Furthermore, decreasing EGR1 expression by siRNA reduced BCR-induced cell survival. Treatment with PP2 or with dasatinib suppressed BCR-induced LYN and JNK phosphorylation as well as EGR-1 upregulation and is associated with a decrease of cell survival in all cases analysed. CONCLUSIONS: This study highlights the importance of BCR signaling in MCL cell survival and points out to the efficiency of kinase inhibitors in suppressing proximal BCR signaling events and in inducing apoptosis.

Only a minority of chronic lymphocytic leukemia (CLL) patients harboring a positive direct antiglobulin test (DAT) will develop autoimmune hemolytic anemia (AIHA). In a single institution cohort of 378 CLL patients, 56 patients (14.8%) had at least one positive DAT during the course of the disease, either at diagnosis or later. We found no relationship between the time of the first positive DAT and overall survival (OS). However, patients with a positive DAT who did not develop AIHA had the same adverse outcome as patients who developed AIHA. Of the patients who were in Binet stage A at diagnosis, those with a positive DAT had a significantly shorter OS, regardless of their IGHV mutational status, however, there was a strong association with VH1-69. By multivariate analysis, a positive DAT was found to be an independent adverse prognostic factor for OS. Thus, DAT represents a strong adverse prognostic factor and its determination should be repeated during follow-up.

The prognosis of patients with chronic lymphocytic leukaemia (CLL) is highly variable, from a normal life span without any treatment to short survival after several lines of therapy. For eligible patients with high risk or refractory CLL, allogeneic haematopoietic stem cell transplantation (HSCT) has been considered as a standard option until recently. This paradigm is being challenged by the introduction of novel classes of drugs, such as the B-cell receptor (BCR) signalling inhibitors ibrutinib and idelalisib. These new drugs have shown impressive results in high risk or refractory CLL patients, even if the proportion of complete response and negative minimal residual disease are low (Byrd et al, 2014; Furman et al, 2014) and survival short after drug withdrawal (Maddocks et al, 2015). Thus, HSCT may remain an option in high-risk CLL, although new guidelines for high-risk CLL patients are needed (Dreger et al, 2014). Another concern is the outcome of patients after transplantation. Non-relapse mortality has significantly decreased over the last years, but disease relapse after transplantation, occurring in about 30% of the patients at 5 years post-transplantation, remains a major problem. Mixed chimerism has been associated with treatment failure, and donor lymphocyte infusions (DLI) have been proposed to recover full donor chimerism (Richardson et al, 2013). More recently, a study demonstrated that most CLL patients relapsing after HSCT remained sensitive to salvage therapies (Rozovski et al, 2015), and novel agents seem of particular interest in this setting (Coutre et al, 2014). We report herein the case of a CLL patient who relapsed after HSCT and was subsequently treated with ibrutinib for more than 1 year. Not only did ibrutinib induce a complete response but it also allowed the recovery of full donor chimerism, which had been lost at the time of relapse. A 44-year-old man was diagnosed in 2000 with asymptomatic Binet stage A CLL. At the time of diagnosis, his white blood cell (WBC) count was 18 × 109/l with 80% CD19/CD5 positive kappa-restricted lymphocytes, Royal Marsden Hospital score 5. Haemoglobin and platelet count were normal. There was no lymphadenopathy and no treatment was initiated. The disease progressed in 2005 with peripheral lymphadenopathies, bulky retroperitoneal mass and marked splenomegaly. WBC count was 120 × 109/L with 92% lymphocytes, while haemoglobin level and platelet count remained normal. Immunoglobulin heavy chain variable (IGHV) genes were unmutated and fluorescence in situ hybridization (FISH) analysis revealed a 17p deletion in 94% of interphase nuclei. He received two courses of FCCam (fludarabine, cyclophosphamide, alemtuzumab) and was subsequently switched to high-dose methylprednisolone because of insufficient response and immune thrombocytopenia. A partial response was reached. Allogeneic HSCT from a sibling donor was performed in January 2007, after a reduced intensity conditioning with rituximab, fludarabine and 2 Gy total body irradiation. Quantitative analysis of chimerism by polymerase chain reaction amplification of microsatellites was regularly assessed on blood total leucocytes and CD3 positive T-cells. Shortly after transplantation, the patient was in complete response and had achieved a full donor chimerism. He developed a limited chronic oral graft-versus-host disease (GvHD), which was controlled by prolonged immunosuppressive therapy with both mycophenolic acid and low-dose corticosteroids. In January 2014, seven years after transplantation, the patient complained of fatigue and abdominal pain. Computerized tomography (CT) scan showed massive mesenteric and retroperitoneal lymphadenopathies (13 cm × 6 cm). There was no sign of chronic GvHD and immunosuppressive agents were withdrawn. At this time, lymphocyte count was normal and lymphocytosis reccurred a few weeks later. Evaluation of chimerism showed a loss of full donor chimerism. Richter transformation was ruled out on the basis of clinical, biological and scintigraphic features. Treatment with ibrutinib was delayed until July 2014 due to the occurrence of pneumocystosis. A CT scan, performed in July 2014 prior to starting ibrutinib, showed an increase of the abdominal nodal mass (17 × 10 cm). At the same time, the lymphocyte count increased to 62 × 109/l with a lymphocyte doubling time of 1 month. Cytogenetics revealed a complex karyotype with add(3), del(3), -5, del(5), add(17p) and add(19). TP53 deletion was still present by FISH. Abdominal pain resolved rapidly and the lymphocyte count transiently increased before it decreased. Treatment was well tolerated. After 9 months of therapy, a partial response was achieved with normal blood counts (lymphocytes 1·6 × 109/l) and a 60% reduction of lymph nodes size. Limited labial chronic GvHD reappeared, requiring topical corticosteroids. At the same time, we observed a recovery of full donor chimerism, on both total leucocytes and CD3 positive T-cells (Fig 1). After 15 months of follow-up, the patient is still on ibrutinib therapy and has now reached complete response but with detectable minimal residual disease in the blood. Chronic GvHD is still limited to oral mucosa. Our observation suggests that ibrutinib is not only highly effective on CLL cells but might also have the ability to enhance the activity of donor T cells in the context of relapse after HSCT. Of note, the patient remained off therapy for 6 months when he developed infectious complications between terminating immunosuppressive treatment and starting ibrutinib, suggesting that stopping immunosuppression by itself was not helpful to control the disease. Ibrutinib was thus able to restore a full donor chimerism concomitantly with the appearance of moderate GvHD, directly related with the graft-versus-leukaemia effect. Previous studies demonstrated that ibrutinib also inhibited Interleukin-2 inducible T cell kinase (ITK), another member of the TEC family of kinases, and was able to target Th2 CD4 T-cells and drive a Th1 selective pressure on T-cell lymphocytes (Dubovsky et al, 2013, 2014). Complete responses are not frequent during ibrutinib therapy, particularly for patients with complex karyotype (Thompson et al, 2015). This emphasizes the importance of the recovery of an efficient donor-mediated anti-tumoural immunity. These data provide a rationale for further studies of ibrutinib in the particular setting of CLL patients relapsing after allogeneic HSCT, not only for its anti-tumoural effect, but also to stimulate donor T cells which, to the best of our knowledge, has never been yet reported. Further studies are needed to better understand the underlying biological mechanisms of these observations. Conception and design: AQ, GS, AD, RPL. Provision of study material and patient: AQ, ED, GS, AD, RPL. Data collection and assembly: AQ, FS, GS, AD, RPL. Data analysis and interpretation: AQ, MP, GS, AD, RPL. Manuscript: AQ, GS, AD, RPL. Final approval of manuscript: AQ, FS, ED, MP, DM, GS, AD, RPL. The authors declare no competing conflicts of interest.

Le facteur de transcription STAT1 est un effecteur majeur de la réponse à l’interféron exerçant ainsi un rôle clé dans l’immunité innée. Il est également un suppresseur de tumeur et régule des effets antiprolifératifs et pro-apoptotiques. Afin de mettre en évidence de nouvelles implications anti-tumorales de STAT1, ce projet de doctorat a porté sur son implication fonctionnelle i) dans l’expression des immunoglobulines (Ig), processus essentiel de l’immunité adaptative et ii) dans la réponse cellulaire aux traitements par les agents génotoxiquesNotre étude cinétique, après induction de l’expression de STAT1 dans des cellules initialement totalement déficientes, a montré son implication dans l’expression des IgG membranaires. Le mécanisme régulateur est indirect : il impliquerait une inhibition de l’activation de STAT3 conduisant à l’inhibition de l’expression de BLIMP1, un acteur essentiel de la différenciation plasmocytaire. La capacité de STAT1 à pouvoir se fixer sur le promoteur de BLIMP1, de même que STAT3 n’exclut pas une implication transcriptionnelle.Au cours d’un traitement génotoxique par le MNNG, nous avons décrit la participation de STAT1 à un complexe de réparation de l’ADN. Seule la présence de STAT1 permet l’intégration au complexe MLH1/p53 de la kinase c-Abl. Dans ce contexte, on observe une cytotoxicité sous le contrôle de l’activité kinase de c-Abl, une réparation rapide et efficace de l’ADN, un arrêt seulement transitoire du cycle et une orientation vers la survie cellulaire équivalent à une résistance à ce traitement.Ces résultats mettent en évidence deux nouveaux rôles anti-tumoraux de STAT1 par sa contribution à des complexes régulateurs essentiels et la désigne comme une potentielle cible thérapeutique.

Chronic lymphocytic leukemia (CLL) is a common B-cell malignancy with a remarkably heterogeneous course,ranging from indolent disease to rapidly progressive disease associated with therapeutic resistance. Recentsequencing efforts in the field have deciphered the genomic landscape of the disease and uncovered new putativecancer drivers such as the IKZF3 L162R mutation. IKZF3 encodes a lymphoid transcription factor with all mutationsoccurring at a single hotspot located within the DNA binding domain. Here we show that B cell-restrictedconditional knock-in of this mutation skews B cell development and induces CLL-like disease in elderly mice (35%penetrance), confirming its role as a CLL driver. Using ChIP-seq, we showed that the mutation induces subtlemodifications of the recognition of IKAROS family DNA motif, yet with a broad impact on the specificity of IKZF3 fortarget genes selection. Mutant IKZF3 is enriched in promotor regions of BCR, NF-κB and migration pathway genes,and functions mostly as a transcriptional activator. RNA-seq studies evidenced enrichment in BCR signaling andmigration pathway signatures associated with mut-IKZF3 B cells, mediating direct functional consequences on thesetwo functions. Aside to this novel mutation, TP53 abnormalities are more frequent and associated with adverseprognosis and resistance to chemotherapy. In this study, we show that, unlike the IKZF3 mutation, TP53abnormalities are acquired during the disease course and promote clonal evolution and acquisition.

La leucémie lymphoïde chronique (LLC) est un syndrome lymphoprolifératif résultant de la prolifération de lymphocytes B matures CD19+CD5+. Elle est caractérisée par une accumulation des cellules tumorales dans le sang, la moelle osseuse et les ganglions lymphatiques. Dans la LLC comme dans tous les cancers le dialogue entre la cellule tumorale et son micro-environnement joue un rôle majeur dans la survie et la prolifération des cellules tumorales. L’objectif de notre étude est d’évaluer le rôle des cellules du micro-environnement ganglionnaire dans la rétention des lymphocytes B tumoraux.L’établissement d’une cartographie du micro-environnement nous a permis de confirmer la désorganisation de l’architecture du ganglion de LLC, caractérisée par une infiltration diffuse des lymphocytes B tumoraux exprimant le récepteur CCR7 de façon constitutive, une perte de la structuration des zones corticales folliculaires et para corticales due à la disparition des cellules dendritiques folliculaires (FDC), et une désorganisation du réseau des cellules réticulaires fibroblastiques (FRC).Nous avons mis en évidence une présence plus importante des cellules positives à la fois pour le CD68 et pour le CCL21, ligand de CCR7, dans les ganglions provenant de patients atteints de LLC en comparant avec des ganglions non tumoraux. Le CCL21 est une chimiokine qui joue un rôle important dans le trafic des lymphocytes et qui est normalement produite par les FRCPour mieux comprendre le rôle des macrophages CD68+retrouvés dans les ganglions, nous avons utilisé un système qui permet de différencier in vitro des cellules NLC CD68+à partir de PBMC issus de patients atteints de LLC.Les NLC correspondraient aux macrophages associés aux tumeurs (TAM) de la LLC. Nous avons confirmé la production de CCL21 par ces cellules en utilisant une nouvelle approche de détection de l’ARN basée sur la cytométrie en flux.En conclusion, nos travaux nous ont permis d’identifier une population cellulaire CD68+ qui pourrait être une source alternative de CCL21 et qui pourrait jouer un rôle important dans la rétention des lymphocytes B exprimant le récepteur CCR7. Les interactions cellulaires entre NLC et cellules leucémiques pourraient représenter une cible thérapeutique intéressante.

The LNK adaptor protein is a key negative regulator of signalling pathways, such as JAK/STAT, important in the development of the hematopoietic system. Its implication in chronic blood diseases, such as Myeloproliferative Neoplasms (MPN) has been confirmed by studies on Lnk-deficient mice, as well as the identification of LNK mutations in MPN patients. However, the LNK mechanism of regulation on its partners and the functional implication of LNK mutations in MPN pathogenesis, are still unclear. Therefore, my PhD project covers the structural and functional analysis of theLNK/JAK2 signalling complex and the development of a molecular strategy to use LNK as a therapeutic tool for the treatment of MPN patients. Our study showed, for the first time, the inhibitory function of the N-terminal region and the pleckstrin homology domain of LNK on JAK2 activity, which occurs more importantly on JAK-V617F than JAK2 wild type form. Moreover, our study provided evidence on how LNK mutations located in this LNK region could contribute to these haematological diseases and has allowed us to propose a model for LNK regulatory function on JAK2activity. Furthermore, we developed a cell penetrating peptide-based strategy to deliver this regulatory region of LNK in hematopoietic cells to specifically inhibit JAK2-V617F oncogenic form. The finalaim is to use this region as a therapeutic molecule to treat JAK2-V617F-positive MPN patients.

Chronic Lymphocytic Leukemia (CLL) is a clonal B cell malignancy of the elderly. This neoplasm is characterized by a heterogeneous clinical course from indolent chronic disease to progressive lymphadenopathy that remains incurable. The aim of my PhD was to undertake a comprehensive phenotypic and functional analysis of the B cell subpopulations responsible for their survival advantage. CLL B cell purification from 30 patients indicate the presence in various extents of leukemic B cell subpopulations producing immune-regulatory cytokines, notably IL-10 and TGFβ1. Remarkably, these CLL subpopulations express the FOXP3 transcription factor, a marker of regulatory T cells. These subpopulations present a phenotypic signature, distinguishable from regulatory B cells populations, with specific markers of activated memory B cells. Functional studies prove their regulatory capacities on T cell differentiation, proliferation and secretion, contributing to the T cell dysfunction observed in CLL. Finally, statistical analysis combining IL-10, TGFβ1 and FOXP3 expressions for these subpopulations allows generating a poly-functional index correlated with two major risk factors of CLL progression. Our data characterize novel CLL B cell subpopulations that are involved in disease progression and give a rational to CLL B cell survival in secondary lymphoid organs.

Des altérations de la signalisation en aval du récepteur à l’antigène (BCR) jouent un rôle clé dans la physiopathologie de la Leucémie Lymphoïde Chronique (LLC). Notre laboratoire a montré qu’une stimulation antigénique ex-vivo des cellules B-LLC conduit à une survie et une migration cellulaires différentielles qui distingue deux groupes de patients. Sur la base de ces résultats, nous avons montré que l’avantage de survie cellulaire en réponse à une stimulation antigénique observé dans un groupe est dépendant 1) d’un seuil imposé par les niveaux d’expression des effecteurs précoces (BCR, Syk et Zap70), de la capacité des cellules leucémiques à répondre en termes de phosphorylation de Syk, d’activation de la PLCƳ2, de mobilisation calcique et d’activation du facteur de transcription NFAT2 ; l’activation de la voie BCR/NFAT mesurée par la survie des cellules B-LLC ex-vivo étant corrélée à la survie globale des patients ; 2) de l’augmentation des niveaux de phosphorylation globale et spécifique de Syk, de la distribution subcellulaire de phospho-Syk, de la capacité de Syk à interagir avec des effecteurs positifs et négatifs et à les activer. De plus, notre étude sur la diminution de la migration des cellules B de LLC en réponse à une stimulation du BCR montre qu’elle dépend du taux d’internalisation du CXCR4 qui est régulé par l’activation des PI3Ks situées en amont des PKDs ; ces dernières activées phosphorylent le CXCR4 qui est alors internalisé. L’ensemble de ces données nous a permis de mieux définir les mécanismes moléculaires sous-jacents à une survie accrue et à une migration diminuée des cellules BLLC en réponse à une stimulation antigénique, de mettre en évidence d’éventuels biomarqueurs fonctionnels de stratification (pSyk et pPLCƳ2), de pointer de potentielles cibles thérapeutiques (NFATs et PKDs) et d’expliquer en partie l’action de drogues, comme le Fostamatinib et l’Idelalisib, utilisées en thérapie dans la LLC.

The transcription factor STAT1, as a major effector of interferon, plays a key role in innate immunity. Through its strong anti-proliferative and pro-apoptotic properties STAT1 is also considered as a tumor suppressor. The aim of this project was to delineate new potential tumor suppressor properties for STAT1 in two signaling mechanisms: i) Ig expression in plasmacytoid cells and ii) cellular response to genotoxic stress.Our kinetic experiments, upon “de novo” expression of STAT1 in a rare STAT1-deficient cell line showed its modulation of membrane IgG expression. The underlying mechanism involves a STAT1-dependent inactivation of STAT3 and subsequently a decreased expression of BLIMP1, a major contributor to plasma cell differentiation. Since STAT1, like STAT3, is able to bind to the BLIMP1 promoter elements a transcriptional interference cannot be excluded.During alkylating agent treatment with MNNG we have observed the presence of STAT1 in DNA repair complex. STAT1 expression allows the recruitment into a MLH1/p53 complex of the kinase c-Abl. This complex leads to cytotoxic dependence on c-Abl kinase activity, an efficient DNA repair with a transient cell cycle arrest and to signaling mechanisms toward cell survival. At longer term of exposure STAT1 also lead to cellular resistance to treatment.These results provide evidences for new anti-tumor roles of STAT1 in two major regulatory systems and indicate STAT1 as a potential therapeutic target.

Altered B-cell antigen receptor (BCR) signaling pathways play a key role in chronic lymphocytic leukemia(CLL) pathophysiology. Our lab has previously shown that ex-vivo antigenic stimulation of CLL-B cells led todifferential cell survival and cell migration, which allowed the distinction between two groups of patients. Basedon these results, we evidenced that the cell survival advantage in response to BCR engagement from one groupdepends on 1) a critical threshold mediated by the early effector expression levels (BCR, Syk et Zap70), a BCR competency of the leukemic cells translated by Syk phosphorylation, PLCƳ2 activation, intracellular Ca2+mobilization and the transcription factor NFAT2 activation; this activated BCR/NFAT signaling cascade, which is reflected by the ex-vivo measurement of CLL cell survival, was correlated to the overall survival from CLLpatients; 2) increased levels of global and specific Syk phosphorylation, phospho-Syk subcellular distribution, Sykability to interact with positive and negative effectors and to activate them. Moreover, study of BCR stimulation mediated decreased migration in CLL B cells showed that it relied on CXCR4 internalization levels that were regulated by activated PI3Ks acting upstream of the PKDs; activation of the latters allowed CXCR4 phosphorylation and then its endocytosis. Altogether, these data allowed us to better understand the molecular mechanisms underlying the survival advantage and the decreased migration of CLL B cells in response to antigenic stimulation, to evidence eventual functional biomarkers of stratification (pSyk and pPLCƳ2), to point out potential therapeutic targets (NFATs and PKDs), and to partially explain how Fostamatinib and Idelalisib function as therapeutic drugs in CLL.

L’adaptateur LNK est un régulateur négatif des voies de signalisation, dont la voie JAK/STAT,essentielle au développement du système hématopoïétique. Son implication dans les hémopathies chroniques, notamment les Néoplasmes Myéloprolifératifs (NMP), a été mise en évidence par l’analyse de souris invalidées pour cet adaptateur et l’identification de mutations de LNK chez les patients atteints de ces pathologies. Toutefois, le mécanisme permettant la régulation de ses partenaires, dont la kinase JAK2, et l’implication fonctionnelle des mutations de LNK dans les NMP, restent à définir. Ainsi, mon projet de thèse a porté sur l’analyse structurale et fonctionnelle des complexes de signalisation LNK/JAK2 et sur le développement d’une stratégie moléculaire pour l’utilisation thérapeutique de LNK dans les NMP. Nos résultats ont montré pour la première fois, la fonction inhibitrice de la région N-terminale incluant le domaine d’homologie à la Pleckstrine deLNK sur JAK2 normale et de manière plus importante, sur la forme mutée JAK2-V617F, retrouvée chez les patients atteints de NMP. De plus, nos études sur les mutations de LNK localisées dans cette région régulatrice, ont permis de comprendre leur contribution dans le développement de ces hémopathies et de proposer un mécanisme d’inhibition de l’activation de JAK2 par LNK. Nos résultats permettent d’utiliser le ciblage de la région N-terminale de LNK comme stratégie moléculaire inhibant spécifiquement la forme oncogénique JAK2-V617F à l’aide de peptides pénétrants (CPP). A long terme, cette approche pourrait être utilisée comme outil thérapeutique dans le traitement de patients atteints de NMP positifs pour JAK2-V617F.

La leucémie lymphoïde chronique (LLC) est une hémopathie B qui se caractérise par une évolution clinique hétérogène et un dysfonctionnement du système immunitaire. Des travaux antérieurs du laboratoire ont mis en évidence l’existence de sous-populations lymphocytaires B CD5⁺/CD19⁺ qui sécrètent des facteurs solubles immuno-régulateurs, tels que l’IL10 et le TGFβ1 et, qui expriment le facteur de transcription FOXP3. Mon travail de thèse a consisté à poursuivre la caractérisation phénotypique et fonctionnelle de ces sous populations,et notamment celle sécrétant de l’IL10, tout en évaluant son évolution chez des patients en cours de traitement par ibrutinib, un inhibiteur de Btk. Sur le plan fonctionnel,mes travaux démontrent que les lymphocytes B de LLC ont la capacité d’inhiber la prolifération lymphocytaire T CD4⁺, d’induire une différenciation lymphocytaire T régulatrice et d’inhiber la différenciation Th1. Mes résultats démontrent également que la sous population IL10⁺ présente des caractéristiques phénotypiques et d’activation communes aux cellules de la fraction proliférative. De plus, la fréquence de la population IL10⁺, plus élevée chez les patients évolutifs, révèle que la capacité de sécrétion de l’IL10 par les cellules B de LLC est corrélée à la progression. Enfin, le traitement par ibrutinib induit une diminution rapide de la sous-population IL10+ qui se comporte comme celle de la sous-population CXCR4ˡᴼᵂ, un marqueur de la fraction proliférative. L’ensemble de ces données souligne l’importance des sous-populations immuno-régulatrices dans l’évolutivité de la LLC et apporte de nouveaux éléments sur les mécanismes d’action de l’ibrutinib.

Chronic lymphocytic leukemia (CLL) is a lympho-proliferative disorder resulting from the proliferation of mature B cells CD19+ CD5+. CLL is characterized by an accumulation of tumor cells in the blood, bonemarrow and lymph nodes. My project aims to analyze the crosstalk between tumor B cells and microenvironment cells in the lymph node, in order to understand the mechanisms affecting the retention and survival of tumor cells in secondary lymphoid organs. The cartography of the microenvironment showed the disorganization of the cellular architecture, characterized by diffuse infiltration of tumor B cells, loss of follicular dendritic cells and disruption of the fibroblastic reticular cells network.I detected the presence of a population of CD68+ macrophages expressing the CCL21 chemokine. These macrophages could play a major role in the retention in lymph node of tumor cells constitutively expressing there ceptor for CCL21.In order to better understand the role of these macrophages CD68+, I established a system of celldifferentiation in vitro from PBMCs of CLL patients. NLCs would correspond to tumor-associated macrophages (TAMs) of CLL. We confirmed the production of CCL21 by these cells using a novel approach to RNA detection based on flow cytometry. In conclusion, our work identified a CD68 + cell population which could be an alternative source of CCL21 and which could play an important role in the retention of B cells expressing the CCR7 receptor. Cellular interactions between NLC and leukemic cells could be an interesting therapeutic target.

L’ion calcium est un second messager qui intervient dans de nombreux processuscellulaires dont la proliferation, la differenciation et l’apoptose. Ainsi, l’homeostasiecalcique constitue un point central de regulation de la signalisation cellulaire. En effet, laconcentration calcique cytosolique de calcium subit des oscillations, qui suivant leuramplitude ou leur frequence, vont etre capables d’activer specifiquement certains facteursde transcription. La regulation de ces oscillations implique entre autres les ATPases de typeSERCA (Sarco/Endoplasmic Reticulum Calcium ATPase) qui accumulent le calcium dansle reticulum endoplasmique. L’objectif de ce travail de these a ete l’etude des SERCAs aucours de la differenciation lymphocytaire B et dans l’epithelium du plexus choroide ; ceci,afin de mieux comprendre le profil d’expression de ces pompes et les mecanismes deregulation impliques.Au cours de la differenciation de lignees de leucemie aigue lymphoblastique (LAL) nousavons observe que l’expression de l’isoforme SERCA2 restait stable ou augmentaitlegerement alors que celle de l’isoforme SERCA3 etait toujours fortement induite, pouvantatteindre des niveaux observes dans les cellules lymphoides matures. Nous avons egalementobserve que l’inhibition de l’activite des SERCAs altere la differenciation cellulaire qui estdependante de la voie des PKC. Ces donnees indiquent que SERCA3 pourrait etre utiliseecomme marqueur de la differenciation lymphocytaire B. Une regulation de l’expression desSERCAs a egalement ete mise en evidence au cours de la differenciation de l’epithelium duplexus choroide normal ou tumoral. SERCA3 est fortement exprimee dans l’epitheliumnormal, mais on retrouve une baisse ou une perte de son expression dans l’epitheliumtumoral, cette baisse est correlee a la perte de la differenciation selon le grade des tumeurs.L’etude de l’expression des SERCAs dans les cellules primaires du plexus choroide traitepar des agents cyto-differenciateurs (acides gras a chaine courte), montre que ladifferenciation est associee a une surexpression de SERCA3. SERCA3 peut donc egalementetre un marqueur de la differenciation de l’epithelium du plexus choroide.L’ensemble de ce travail a montre que la differenciation cellulaire est associee a la regulationde proteines impliquees dans la regulation de l’homeostasie calcique : les SERCAs. On peutainsi proposer SERCA3 comme un nouveau marqueur phenotypique utile pour l’analyse dela differenciation du plexus choroide normale et neoplasique, ainsi que pour celle de ladifferenciation lymphoide pre-B leucemique.