Orientadores: João Agostinho Machado Neto, Sara Teresinha Olalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T20:37:02Z (GMT). No. of bitstreams: 1 MachadoNeto_JoaoAgostinho_D.pdf: 13204649 bytes, checksum: a00ab26c9097cb10817c14fe2bdb2356 (MD5) Previous issue date: 2015 / Resumo: A identificação de genes/proteínas diferencialmente expressos em neoplasias hematológicas e a investigação funcional destas proteínas no fenótipo neoplásico são essenciais para o entendimento da biologia da doença. ANKHD1 é altamente expressa em células de leucemia aguda e tem potencial para regular vários processos celulares através de seus domínios de repetição de anquirina. Nosso grupo de pesquisa havia previamente identificado a interação entre ANKHD1 e SIVA através de ensaio de duplo híbrido. Outras proteínas potencialmente relacionadas à ANKHD1 que foram de interesse deste trabalho foram a Stathmin 1 que interage com SIVA e a YAP1 que interage com ANKHD1 em células não hematológicas. O objetivo principal do presente trabalho foi investigar a participação funcional de ANKHD1 e proteínas relacionadas em neoplasias hematológicas. Utilizamos modelos de linhagens leucêmicas humanas e células hematopoéticas primárias provenientes de indivíduos normais (n=52) e de pacientes com diagnóstico de síndrome mielodisplásica (SMD; n=65), neoplasia mieloproliferativa (NMP; n=82), leucemia mieloide aguda (LMA; n=60) e leucemia linfoide aguda (LLA; n=19). Técnicas para avaliação de expressão gênica (qPCR), expressão e interação proteica (western blotting), ensaios funcionais de migração (ensaio transwell), proliferação (MTT e clonogenicidade in vitro, formação de tumor in vivo) e apoptose (Anexina V/IP, TUNEL), e inibição de proteínas de interesse através do uso de inibidores farmacológicos ou ferramentas de terapia gênica foram utilizados. A interação entre ANKHD1 e SIVA foi confirmada por ensaios de coimunoprecipitação. Em linhagens celulares leucêmicas U937 e Jurkat, o silenciamento de ANKHD1 reduziu a proliferação celular, migração e o crescimento tumoral, enquanto que a inibição de SIVA aumentou a migração e o crescimento tumoral e reduziu a sensibilidade à apoptose induzida por radiação UV de células U937. A inibição de ANKHD1 reduziu a atividade de Stathmin 1 e a interação entre SIVA/Stathmin 1. Grande parte das linhagens leucêmicas e amostras primárias de pacientes com neoplasias hematológicas não apresentou a expressão de YAP1, e o silenciamento de ANKHD1 não modulou a atividade de YAP1 em células U937. A expressão de Stathmin 1 foi significativamente elevada em amostras de células hematopoéticas de pacientes com neoplasisa hematológicas (SMD AREB-1/AREB-2, LMA, LLA e mielofibrose primária) quando comparada às amostras de doadores saudáveis. O silenciamento de Stathmin 1 reduziu a proliferação celular e clonogenicidade em células leucêmicas U937, Namalwa e células HEL JAK2V617F. Especificamente em células HEL JAK2V617F, a inibição de Stathmin 1 ou o tratamento com o agente estabilizador de microtúbulos paclitaxel teve um efeito potencializador ao tratamento com ruxolitinib, inibidor de JAK1/2, na indução de apoptose. Em conclusão, propomos que a ANKHD1 participa do fenótipo neoplásico de células leucêmicas através de sua interação com SIVA, inibição de SIVA e ativação de Stathmin 1. Os nossos resultados indicam que a função de ANKHD1 na leucemogênese independe de sua interação com YAP1. Em células humanas com ativação constitutiva da via JAK2/STAT, identificamos a relevância funcional da participação de Stathmin 1 no fenótipo neoplásico / Abstract: The identification of genes/proteins differentially expressed in hematopoietic neoplasms and the functional investigation of these proteins in the neoplastic phenotype are essential for the understanding of disease biology. ANKHD1 is highly expressed in acute leukemia cells and has a potential role in regulating many cellular processes through its ankyrin repeat domains. Our research group has previously identified the interaction between ANKHD1 and SIVA through two-hybrid assay. Other proteins potentially related to ANKHD1 that were of interest of this research are Stathmin 1 that interacts with SIVA, and YAP1 that interacts with ANKHD1 in non hematologic cells. The aim of this study was to investigate the functional role of ANKHD1 and its related-proteins in hematologic malignancies. We used human leukemia cell lines and primary hematopoietic cells from healthy donors (n=52) and patients with myelodysplastic syndromes (MDS; n=65), myeloproliferative neoplasms (MPN; n=82), acute myeloid leukemia (AML; n=60) and acute lymphoid leukemia (ALL; n=19). Techniques for the evaluation of gene expression (qPCR), protein expression and interaction (western blotting), functional assays of migration (transwell assay), proliferation (in vitro MTT and clonogenicity, in vivo tumor formation) and apoptosis (Annexin V/PI, TUNEL), and inhibition of proteins of interest through pharmacologic agents or gene therapy tools were used. The interaction between ANKHD1 and SIVA was confirmed by co-immunoprecipitation assays. In leukemia cell lines U937 and Jurkat, ANKHD1 silencing reduced cell proliferation, migration and tumor growth, while SIVA inhibition increased migration and tumor growth, and reduced sensitivity to UV-induced apoptosis of U937 cells. Inhibition of ANKHD1 reduced Stathmin 1 activity and the interaction between SIVA/Stathmin 1. A large proportion of leukemia cell lines and primary samples from patients with hematologic malignancies did not present YAP1 expression, and ANKHD1 silencing did not modulate YAP1 activity in U937 cells. Stathmin 1 expression was significantly higher in primary hematopoietic cells from patients with hematological malignancies (MDS RAEB-1/RAEB-2, AML, ALL and primary myelofibrosis) compared to samples from healthy donors. Stathmin 1 silencing reduced cell proliferation and clonogenicity of U937 and Namalwa leukemia cells, and HEL JAK2V617F cells. Specifically in HEL JAK2V617F cells, Stathmin 1 silencing or treatment with the microtubule-stabilizing agent paclitaxel had a potentiating effect to treatment with the JAK1/2 inhibitor ruxolitinib on apoptosis induction. In conclusion, we propose that ANKHD1 participates in the leukemia phenotype through its interaction with SIVA, SIVA inhibition, and Stathmin 1 activation. Our results indicate that ANKHD1 plays a role in leukemogenesis independently of its interaction with YAP1. In human cells with a constitutive activation of the JAK2/STAT pathway, we identified the relevant role of Stathmin 1 in the malignant phenotype / Doutorado / Clinica Medica / Doutor em Clínica Médica

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Cables1 est impliqué dans la régulation du cycle cellulaire et la survie. Par QPCR et western blot, Cables1 est fortement exprimé dans les cellules souches hématopoïétiques (CSH), les progéniteurs, les cellules de la niche médullaire et les mégakaryocytes. En utilisant un modèle de souris Cables1-/-, nous avons démontré que Cables1 est un régulateur clé de la maintenance homéostatique des CSH àl’état basal et sous stress hématopoïétique. Chez les souris jeunes dépourvues de Cables1, les progéniteurs hématopoïétiques sont hyperprolifératifs et ont un avantage compétitif de repeuplement. La surexpression lentivirale et la déplétion par shRNA de la protéine Cables1 ont respectivement entraîné une régulation positive et négative de p21, indiquant que l'effet de Cables1 sur la prolifération des progéniteurs est partiellement médiée par la régulation de p21. Avec l’âge, les souris déficientes en Cables1 présentent des anomalies du nombre de globules blancs accompagnées d'une réduction significative du compartiment CSH associée à une mobilisation accrue des progéniteurs. De plus, les souris Cables1-/-présentent une sensibilité accrue à un agent myélotoxique à l’irradiation due à des défauts dumicroenvironnement médullaire. Dans les mégacaryocytes, la diminution de Cables1 par shRNA entraîne un défaut de prolifération et unediminution du pourcentage de MK matures. De plus, un défaut de la capacité de formation de proplaquette a été observé après la diminution de Cables1. Ces effets peuvent s’expliquer par une apoptose accrue. En conclusion, Cables1 régule à la fois les progéniteurs et la mégacaryopoïèse. Cables1 donc est essentiel pour l'homéostasie des CSH et le contrôle du stress des CSH. La manipulation del’expression de Cables1 pourrait représenter une opportunité pour optimiser les schémas de chimiothérapie. / Cables1 has been described to be involved in cell cycle regulation and survival. Using QPCR and western blot, we demonstrate for the first time that Cables1 in highly expressed in hematopoietic stem cells, in niche cells and megakaryocytes. Using the Cables1-/- mouse model, we demonstrate that Cables1 is a key regulator of homeostatic HSC maintenance and under hematopoietic stress. Young mice lacking Cables1 showed hyper proliferation within the hematopoietic progenitor and stem cell (HSPC) compartment. Loss ofCables1 conferred increased competitive repopulating capacity to the HSPCs. Lentiviral mediated overexpression and shRNA mediated depletion of Cables1 protein resulted in p21 up and down regulation, respectively, indicating that the effect of Cables1 on HSPC proliferation is partially mediated through regulating p21. By 1,5 to 2 years of age, Cables1 deficient mice displayed anomalies in whiteblood cell counts accompanied by a significant a reduction in the HSC compartment coupled with increased mobilization of HPC. In addition, Cables1-/- mice displayed increased sensitivity to myelotoxic agent and irradiation. These defects are related to abnormal microenvironment. We also investigated Cables1 function during megakaryopoiesis. Down regulation of Cables1 in CD41+CD42- megakaryocytic progenitors resulted in proliferative defect and decreased percentage of mature MKs, which were accompanied by p21(cyclin dependent kinase inhibitor) and Bax (an apoptosis related gene) up-regulation. Moreover, defect of proplatelet forming capacity was observed after Cables1 knockdown, which can also be explained by elevated apoptosis induced by Bax protein. In conclusion, Cables1 regulate both HSPCs and the process of megakaryopoiesis. It represents a opportunities to optimize chemotherapy schemes.

Hematopoiesis is a vital process in which red blood cells and cells of the immune system are formed. It is initiated during early embryonic development when we find hematopoietic progenitors in separate anatomical sites. Embryonic hematopoiesis comprises three successive and partly overlapping waves of progenitors with a different hematopoietic potential. The primary anatomical place where hematopoiesis takes place shortly before the birth is the bone marrow (BM). Since at this time point of development BM is already populated by hematopoietic stem cell (HSCs) progenitors, it becomes also the site of hematopoiesis in adulthood. However, the bone marrow is not the only place where hematopoietic progenitors emerge and develop. The Yolk sac (YS) and the Aorta-Gonad-Mesonephros (AGM) region are the initial sites of the appearance of the three waves of progenitors in the early embryogenesis. These progenitors and their descendants play an indispensable role during the development of an individual. Because there are no specific markers that would unambiguously characterize progenitors of these individual waves, their physical separation and hence also functional characterization is still incomplete. Recent studies have shown that Toll-like receptors (TLRs) are expressed on adult HSCs. The stimulation of...

Understanding the transcriptional mechanisms that control hematopoiesis and the interaction between hematopoietic stem cells and the bone marrow (BM) microenvironment in vivo is of considerable interest. The calcineurin-dependent transcription factor NFAT (Nuclear Factor of Activated T cells) is known as master regulator of cytokine production in T lymphocytes and therefore central for T cell-dependent immune reactions, but has also been shown to regulate a process of differentiation and tissue adaptation in various cell types. The activation of NFAT is dependent on the calcium level within the cell. In resting cells, calcium levels are low and NFAT is cytoplasmic and inactive. A sustained increase in the internal calcium concentration within an external stimuli leads to activation of the calcium-dependent calcineurin, followed by dephosphorylation and nuclear translocation of NFAT. We have previously shown that NFATc2, a member of the NFAT family, is expressed in CD34+ hematopoietic stem cells (HSC). A mouse model harboring NFATc2 deficiency provides the opportunity for in vivo investigation of the role of NFATc2 in hematopoiesis. Our recent observations showed that aged mice lacking the transcription factor NFATc2 develop peripheral blood anemia and thrombocytopenia, BM hypoplasia and extramedullary hematopoiesis in spleen and liver. The proliferation and differentiation of NFATc2-deficient hematopoietic stem cells ex vivo, however, was found to be intact. It remained therefore unclear whether the disturbed hematopoiesis in NFATc2-deficient mice was caused by the hematopoietic or the stroma component of the BM hematopoietic niche. In the current study we dissected the relative contribution of hematopoietic and stroma cells to the phenotype of the NFATc2-deficent mice by transplanting immuno-magnetically purified NFATc2-deficient (KO) HSCs to lethally irradiated wild type (WT) mice, and vice versa. After a post-transplantation period of 6-8 months, peripheral blood, BM as well as spleen and liver of the transplanted animals were analyzed and compared to WT and KO mice transplanted with control cells. Transplantation of NFATc2-deficient HSCs into WT recipients (KO WT) induced similar hematological abnormalities as those occurring in non-transplanted KO mice or in KO mice transplanted with KO cells (KO KO). Compared to WT mice transplanted with WT cells (WT WT), KO WT mice showed evidence of anemia, thrombocytopenia and a significantly reduced number of hematopoietic cells in their BM. Likewise, KO WT mice developed clear signs of extramedullary hematopoiesis in spleen and liver, which was not the case in WT WT control animals. In addition to the hematopoietic abnormalities, transplantation of NFATc2-deficient HSC also induced osteogenic abnormalities such as BM sclerosis and fibrosis in WT mice. This phenomenon was rather subtle and of incomplete penetrance, but never seen in mice transplanted with WT cells. These data demonstrate for the first time, that the NFATc2 transcription factor directly regulates the intrinsic function of hematopoietic stem cells in vivo. However, the transcriptional targets for NFAT in these cells are yet unknown. In addition to hematopoietic stem cells, NFATc2 has been shown to be expressed in a lineage-specific manner during myeloid differentiation and, notably, is maintained during megakaryopoiesis while it is suppressed during the differentiation of neutrophils. Bone marrow megakaryocytes are the precursors of peripheral blood platelets and therefore constitute an integral part of primary hemostasis, thrombosis and wound healing. The biological role of NFAT in megakaryocytes is unknown. We have recently shown that NFATc2 is not necessary for megakaryocytic differentiation. On the other hand, recent evidence suggests that NFATc2 is required for the transcription of specific megakaryocytic genes. In this study, we showed that activation of the calcineurin/NFAT pathway in either primary megakaryocytes or CMK megakaryocytic cells forces the cells to go into apoptosis. Cell death in megakaryocytes is induced by treating the cells with the calcium ionophore ionomycin and suppressed by either the pan-caspase inhibitor zVAD or the calcineurin inhibitor cyclosporin A (CsA). Ionomycin stimulation of megakaryocytes leads to the expression of Fas Ligand (FASLG), a pro-apoptotic member of the tumor necrosis factor superfamily. Expression of FASLG was detectable as early as four hours after stimulation on the membrane of ionomycin-treated megakaryocytes, was augmented in cells stably overexpressing NFATc2, and was suppressed in cells either pretreated with CsA or expressing the specific peptide inhibitor of NFAT, VIVIT. To investigate the physiological relevance of FASLG expression on megakaryocytes, we performed co-cultures of megakaryocytes with Fas-expressing T-lymphocytes, in which CMK cells were left either unstimulated or pre-stimulated with ionomycin and then added to Jurkat cells. The presence of ionomycin-stimulated CMK cells, but not of unstimulated cells or cells stimulated in the presence of CsA, significantly induced apoptosis in Jurkat cells. Overexpression of NFATc2 in CMK cells enhanced their potency to induce apoptosis in Jurkat cells, while cells expressing VIVIT were less effective. Apoptosis induction of Jurkat cells by stimulated CMK cells was partially blocked by the presence of either a neutralizing antibody against FASLG or an antagonistic antibody to Fas during the co-culture period, indicating involvement of the FASLG/Fas apoptosis pathway. These results represent the first clear evidence for a biological function of the calcineurin/NFAT pathway in megakaryocytes, namely the regulation of Fas/FASLG-dependent apoptosis. Second, they underline that the biological role of megakaryocytes is not restricted to the production of proteins and other cellular structures for platelet assembly, but that this population of cells fulfills an independent regulatory function in the context of the surrounding tissue. Finally, we have identified by RNA sequencing analysis of NFATc2-expressing and -deficient cells, the entire set of genes which is induced by NFATc2 in stimulated megakaryocytes. Functional pathway analysis suggests an involvement of NFATc2 in pro-inflammatory pathways in these cells. The significance of these findings has to be addressed in further studies.

L’hématopoïèse est initiée à partir de cellules souches hématopoïétiques et aboutit à la production continue et contrôlée des cellules sanguines matures. Runt-related transcription factor 1 (RUNX1) code pour un facteur de transcription qui joue un rôle clé dans l'hématopoïèse. Des dérégulations de RUNX1 sont fréquemment associées à des cancers hématologiques, en particulier dans les leucémies aiguës lymphoblastiques à précurseurs B chez l'enfant (LAL-B). De plus, son activité transcriptionnelle est contrôlée par le recrutement de cofacteurs. Pour comprendre le mécanisme impliqué dans le contrôle de l’activité transcriptionnelle de RUNX1 nous avons réalisé des immunoprécipitations de RUNX1 couplées à de la spectrométrie de masse et identifié Far Upstream Element binding protein 1 (FUBP1) comme un partenaire protéique potentiel de RUNX1. FUBP1 est un régulateur multifonctionnel impliqué dans divers processus cellulaires. Il a notamment été décrit récemment comme essentiel à l’expansion et à l’auto-renouvellement des cellules souches hématopoïétiques. Par ailleurs, une surexpression du gène et des mutations potentiellement oncogéniques ont été décrits dans des cas de leucémies lymphoblastiques. Nous avons montré, grâce à la technique de PLA (Proximity Ligation Assay) que nous avons optimisé sur cellules non-adhérentes, que FUBP1 est un nouveau cofacteur de RUNX1 et qu’ils peuvent faire partie d’un même complexe régulateur dans les lymphoblastes pré-B humains. Par des expériences de ChIP couplées à du séquençage, nous avons localisé les régions de la chromatine sur lesquelles étaient fixées RUNX1 et FUBP1 de façon commune. Nous avons identifié l’oncogène c-KIT comme un gène cible commun et nous avons caractérisé deux régions régulatrices fixées par RUNX1, FUBP1 et des marques d’activation de la chromatine, au niveau du premier intron de c-KIT : une à +700 pb et l’autre au niveau d’un enhancer à +30 kb. De plus, nous avons déterminé les motifs de liaison de RUNX1 et FUBP1 essentiels pour l'activation de l’enhancer. Enfin nous avons démontré que la surexpression de FUBP1 et RUNX1 conduit à une augmentation de l’expression de c-KIT en ARNm et en protéine, augmente une des voies activées par c-KIT en présence de son ligand, favorise la prolifération cellulaire in vitro et in vivo et rend les cellules moins sensibles à un inhibiteur de c-KIT. En conclusion, nous avons démontré que FUBP1 est un nouveau partenaire de RUNX1 et qu’ensembles, ils activent la transcription de l’oncogène c-KIT en se fixant sur un enhancer commun, favorisant ainsi la prolifération cellulaire. Par conséquent, puisque FUBP1 et RUNX1 sont surexprimés dans certains types de leucémie, des altérations de cette régulation pourraient participer à l'apparition ou au maintien de leucémies. Nos résultats ouvrent donc de nouvelles perspectives sur la compréhension du contrôle de l’activité transcriptionnelle de RUNX1 et sur les hémopathies malignes associées à des dérégulations de RUNX1, FUBP1 ou c-KIT. / Hematopoiesis is initiated from hematopoietic stem cells and results in the continuous and controlled production of mature blood cells. RUNX1 (Runt-related transcription factor 1) encodes a transcription factor playing a key role in hematopoiesis. Abnormal functions of this protein are implicated in blood cancer, notably in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Moreover, its transcriptional activity is controlled by the recruitment of cofactors. To unravel the mechanisms behind the regulation of RUNX1 transcriptional activity, we performed RUNX1 specific immunoprecipitation experiments followed by mass spectrometry and identified the Far Upstream Element Binding Protein 1 (FUBP1) as a potential cofactor of RUNX1. FUBP1 is a multifunctional regulator involved in diverse cellular processes. FUBP1 has recently been described to be essential for expansion and self-renewal of hematopoietic stem cells and to function as a potential cancer driver gene in lymphoblastic leukemia. We have shown, with a proximity ligation assay that we have optimized in non-adherent cells, that FUBP1 is a new cofactor of RUNX1 and that these two proteins can be part of the same regulatory complex in human pre-B lymphoblasts. By chromatin immunoprecipitation combined with sequencing, we have localized common chromatin regions bound by RUNX1 and FUBP1. We have identified the oncogene c-KIT as a common target gene, and characterized two regulatory regions bound by both RUNX1, FUBP1 and active histone marks, within the first c-KIT intron: one at +700 bp and the other on an enhancer at +30 kb. Moreover, we have determined RUNX1 and FUBP1 binding sites essential for the enhancer activation. Finally, we have demonstrated that RUNX1 and FUBP1 overexpression in a pre-B cell line increases the expression of c-KIT both at mRNA and protein levels, exacerbates one of the c-KIT downstream pathways, promotes cell proliferation in vitro and in vivo and renders cells more resistant to a c-KIT inhibitor. In conclusion, we have demonstrated that FUBP1 is a new cofactor of RUNX1 and that they activate the transcription of the c-KIT oncogene by binding on a common enhancer, thus promoting cell proliferation. Therefore, since FUBP1 and RUNX1 are overexpressed in some types of leukemia, alterations in this regulation may contribute to the onset or maintenance of leukemias. These new findings open new perspectives on understanding the control of RUNX1 transcriptional activity, and on leukemias related to RUNX1, FUBP1 or c-KIT deregulations.

Maintenance of the hematopoietic system is dependent upon the proper regulation and orchestrated functions of the hematopoietic stem cell (HSC) pool. A number of extrinsic signaling pathways and intrinsic regulators have been found to regulate HSC processes. However a full understanding of the ability of HSC to balance the processes of self-renewal, quiescence, and lineage specification is not yet clear. We therefore set out to identify novel HSC regulators by comparative gene expression analysis of whole genome transcriptomes generated for long-term (LT)-HSC (Hoechst low/- Lin- Sca1+ cKit+ CD34-), short-term (ST)-HSC (Hoechst low/- Lin- Sca1+ cKit+ CD34+), and non-HSC (Hoechst+) of the bone marrow. These studies identified IGF2 as one of the most differentially expressed genes within LT-HSC, suggesting a potential role for IGF2 in the regulation of HSC. Using a combination of lentiviral-mediated overexpression and knockdown experiments, we found IGF2 to confer enhanced self-renewal in vitro and in vivo. Overexpression of IGF2 resulted in an increased percentage of multi-lineage colonies within colony-forming unit (CFU) assays without affecting lineage specification. In vivo, serial bone marrow transplantation revealed that IGF2 within HSC enhances short-term and long-term donor contribution. Analysis of the expression of key cell cycle regulators revealed that IGF2 induced upregulation of p57 expression specifically within HSC. This upregulation could be attributed to differences in the methylation status of the p57 promoter in HSC compared to other progenitor and mature blood cell populations. p57, a member of the Cip/Kip family of cyclin dependent kinase inhibitors, has recently been shown to be required for the regulation of HSC quiescence and long-term self-renewal. Analysis of bone marrow obtained from primary and secondary transplant recipients showed that overexpression of IGF2 resulted in an increased percentage of quiescent HSC. Treatment of HSC overexpressing IGF2 with LY294002, a PI3K-Akt inhibitor, prevented IGF2-mediated upregulation of p57 expression. These findings demonstrate that within HSC, IGF2 induces p57 expression through activation of the PI3K-Akt pathway to regulate HSC quiescence. We have identified a novel role for IGF2 in HSC function, providing new insights into the biology of HSC and opening potential platforms for the development of better therapies involving HSC-mediated hematopoietic reconstitution.

Le couple CXCL12/CXCR4 joue un rôle essentiel dans le maintien de l’homéostasie des cellules souches et progéniteurs hématopoïétiques (CSPHs) et constitue un axe clé par lequel les niches et les CSPHs communiquent au sein de la moelle osseuse (MO). Des mutations hétérozygotes du gène CXCR4, qui tronquent le domaine C-terminal de la protéine et entraînent un défaut de désensibilisation homologue de CXCR4 et une hypersensibilité à CXCL12, ont été identifiées dans le Syndrome WHIM (SW), une immunodéficience rare caractérisée notamment par une lymphopénie. Les mécanismes sous-jacents de cette anomalie restaient inconnus. Grâce à un modèle murin porteur d’une mutation gain de fonction de Cxcr4 identifiée chez certains patients et phénocopiant la lymphopénie du SW, nous avons exploré la possibilité qu’un défaut de domiciliation, de différenciation ou d’expansion des CSPHs dans la MO soit à l’origine de la lymphopénie circulante. Nous avons mis en évidence que la désensibilisation de Cxcr4 régule la balance quiescence/cycle des CSHs à court terme ainsi que leur différenciation en progéniteurs multipotents et progéniteurs engagés vers le lignage lymphoïde. Nos travaux révèlent donc que la désensibilisation de Cxcr4 est requise à la différenciation lymphoïde des CSPHs et suggèrent que l’absence de ce processus soit à l’origine de la lymphopénie observée chez les souris mutantes et, par extrapolation, chez les patients. Ces altérations lymphoïdes impliquaient à la fois des défauts intrinsèques (CSPHs) et extrinsèques (stroma), ce qui nous a conduit à considérer l’impact de la mutation gain de fonction de Cxcr4 sur le stroma médullaire. Dans ce contexte, l’objectif principal de mon projet de thèse a consisté à investiguer à l’aide du modèle murin du SW le rôle de la désensibilisation de Cxcr4 dans le maintien des composantes mésenchymateuses au sein de la MO. Nos données ont permis de mettre en lumière que la désensibilisation de Cxcr4 est intrinsèquement requise à la régulation de l’équilibre quiescence/cycle des cellules souches mésenchymateuses (CSMs), ainsi qu’à la préservation de leur potentiel ostéogénique en contrôlant l'expression et la biodisponibilité de Cxcl12 de manière autocrine. Par conséquent, nos travaux suggèrent que les actions autocrines et paracrines de l’axe de signalisation Cxcl12/Cxcr4 au sein des CSMs régulent leur différenciation en ostéoblastes tout en contribuant au maintien des niches des CSPHs et au processus d’hématopoïèse. / The CXCL12/CXCR4 signaling axis plays an essential role in the maintenance of hematopoietic stem and progenitor cell (HSPC) homeostasis and constitutes a key pathway through which the niches and HSPCs communicate in the bone marrow (BM). Heterozygous gain-of-function mutations of CXCR4, which engender a truncated receptor and affect its homologous desensitization in response to CXCL12, have been reported in the WHIM Syndrome (WS); a rare immunodeficiency notably characterized by lymphopenia. The mechanisms underpinning this remain obscure. Using a mouse model harboring a naturally occurring WS-linked Cxcr4 gain-of-function mutation, we explored the possibility that the lymphopenia in WS arise from defects at the HSPC level in the BM. We showed that Cxcr4 desensitization is required for proper quiescence/cycling balance of short-term HSCs as well as their differentiation into multipotent progenitors and downstream lymphoid-biased progenitors. Thus, our results suggest that efficient Cxcr4 desensitization is critical for lymphoid differentiation of HSPCs, and its impairment is a key mechanism underpinning the lymphopenia observed in WS mice. The role of Cxcr4 desensitization in regulating such lympho-hematopoiesis process implicated both intrinsic and extrinsic properties, thus raising the question of the impact of a gain-of-Cxcr4-function mutation on BM stroma. Therefore, the main part of my PhD project was dedicated to evaluate using this relevant knock-in model the impact of Cxcr4 desensitization on maintenance of BM mesenchymal elements. We have found unexpectedly that such regulatory mechanism is intrinsically required for regulating quiescence/cycling balance of mesenchymal stem cells (MSCs) and preserving their osteogenic potential through the control of Cxcl12 expression and availability in an autocrine manner. Therefore, these findings support autocrine and paracrine actions of the Cxcl12/Cxcr4 signaling axis within MSCs to regulate osteoblast differentiation while contributing to HSPC niches and hematopoiesis.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18906号 / 医科博第62号 / 新制||医科||4(附属図書館) / 31857 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 山下 潤, 教授 野田 亮, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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