Implication de la glutamine dans l'activation de mTORC1 dans les leucémies aiguës myéloïdes et inhibition ciblée

Dans les leucémies aiguës myéloïdes (LAM), l activation anormale de nombreuses voies de signalisation intracellulaires favorise la croissance et la survie des cellules tumorales. L amélioration des connaissances biologiques de ces pathologies hétérogènes, dont le pronostic est réservé, devrait permettre le développement de thérapies ciblées. La kinase oncogénique mTOR est présente au sein de deux complexes, parmi lesquels mTORC1, activé constitutivement dans la majorité des blastes primaires de patients porteurs de LAM, qui contrôle la synthèse protéique, et mTORC2 activé constitutivement dans 50% des LAM. Les inhibiteurs allostériques de mTORC1 (la rapamycine et ses dérivés) n inhibent pas la phosphorylation du répresseur traductionnel 4E-BP1, ne diminuent pas la traduction et induisent peu d apoptose in vitro dans les LAM. Utilisés en monothérapie, leur effet est décevant. De plus ces inhibiteurs n agissent pas sur le complexe mTORC2. J ai étudié l effet d un inhibiteur catalytique de mTOR, l AZD8055, actif sur les deux complexes. In vitro, l AZD8055 inhibe efficacement la signalisation en aval de mTORC1 et de mTORC2, dont les sites de phosphorylation de 4E-BP1 résistants à la rapamycine, ainsi que la synthèse protéique. Il diminue la prolifération, bloque le cycle cellulaire en phase G0G1 et induit une apoptose caspase-dépendante dans les blastes primaires de LAM. Il diminue également la clonogénicité des progéniteurs leucémiques, sans affecter celle des cellules CD34+ normales. Dans un modèle murin de xéno-transplantation, l AZD8055 inhibe la croissance tumorale et améliore la survie des souris traitées. Je me suis également intéressée à la régulation de l activité de mTORC1 par les acides aminés. Dans les cellules de mammifères, l activation de mTORC1 nécessite la présence de glutamine et de leucine qui agissent en coopération via deux transporteurs membranaires, SLC1A5 et SLC7A5/SLC3A2. J ai montré que la privation en glutamine, obtenue par l activité glutaminase de la drogue L-asparaginase ou par l utilisation de milieux de culture spécifiques dépourvus sélectivement en acides aminés, inhibe l activation de mTORC1 et induit de l apoptose dans diverses lignées leucémiques et dans les blastes primaires de LAM. La L-asparaginase inhibe la synthèse protéique et ses effets fonctionnels sont liés à son activité glutaminase. J ai pu également constater une augmentation de l expression protéique de la glutamine synthase induite par la Lasparaginase, dont l inhibition majore l apoptose induite par la L-asparaginase dans certaines lignées leucémiques. J ai également étudié l effet de l inhibition spécifique par un shARN inductible du transporteur SLC1A5, qui permet l import de glutamine. L inhibition de SLC1A5 bloque la réactivation de mTORC1 par l association leucine/glutamine après privation et induit de l apoptose dans la lignée leucémique MOLM14. Cette inhibition diminue la croissance tumorale dans un modèle de xénogreffeAcute myeloid leukaemias (AML) are heterogeneous diseases associated with poor prognosis. In AML, aberrant activation of many signaling pathways enhances proliferation and survival of leukemic blast cells. Understanding the mechanisms underlying survival of tumoral cells should allow the development of targeted therapies. The oncogenic kinase mTOR belongs to two distinct multimeric complexes. MTORC1 that controls protein translation, is constitutively activated in most of primary blast cells at AML diagnosis, while mTORC2 is constitutively activated in about half of AML samples. In AML, some phosphorylation events of the translational repressor 4E-BP1, are resistant to allosteric inhibitors of mTORC1 including rapamycin and its analogs. These first generation inhibitors of mTORC1 have only few effects on AML and do not induce significant apoptosis in vitro. I have tested a second generation mTOR kinase inhibitor active on both mTORC1 and mTORC2 complexes. In vitro, AZD8055 blocked mTORC1 and mTORC2 signaling, including 4E-BP1 rapamycin resistant phosphorylation events and protein synthesis. This compound decreased AML blast cells proliferation and cell cycle progression, reduced the clonogenic growth of leukemic progenitors and induced caspase-dependant apoptosis in leukemic cells but not in normal immature CD34+ cells. Finally, AZD8055 reduced tumor growth and improved survival in xenografted mouse model. In the second part of this work, I have studied the regulation of mTORC1 by amino acids in AML. In mammalian cells, activation of mTORC1 requires the presence of glutamine and leucine acting together via two membrane transporters, SLC1A5 and SLC7A5/SLC3A2. I showed that glutamine deprivation, obtained by L-asparaginase glutaminase activity or specific alpha-MEM use, inhibited mTORC1 and induced apoptosis in AML cell lines and primary AML blasts. L-asparaginase also inhibited protein synthesis and I have observed a correlation between the functional effects of L-asparaginase and its glutaminase activity. L-asparaginase induced an up-regulation of glutamine synthase (GS) protein and shRNA-induced GS inhibition increased L-asparaginase-dependant apoptosis in the MV4-11 AML cell line. I have also studied the effects of SLC1A5 inhibition with an inducible shRNA expressed in MOLM14 cells. Inhibition of this high afffinity transporter for glutamine blocked mTORC1 stimulation by leucine and glutamine after deprivation and induced apoptosis in MOLM-14 cell line. SLC1A5 inhibition reduced tumor growth and improved survival in transplanted micePARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

The epigenetic regulator RINF (CXXC5) maintains SMAD7 expression in human immature erythroid cells and sustains red blood cells expansion

The gene CXXC5, encoding a Retinoid-Inducible Nuclear Factor (RINF), is located within a region at 5q31.2 commonly deleted in myelodysplastic syndrome (MDS) and adult acute myeloid leukemia (AML). RINF may act as an epigenetic regulator and has been proposed as a tumor suppressor in hematopoietic malignancies. However, functional studies in normal hematopoiesis are lacking, and its mechanism of action is unknow. Here, we evaluated the consequences of RINF silencing on cytokineinduced erythroid differentiation of human primary CD34+ progenitors. We found that RINF is expressed in immature erythroid cells and that RINF-knockdown accelerated erythropoietin-driven maturation, leading to a significant reduction (~45%) in the number of red blood cells (RBCs), without affecting cell viability. The phenotype induced by RINF-silencing was TGFβ-dependent and mediated by SMAD7, a TGFβ- signaling inhibitor. RINF upregulates SMAD7 expression by direct binding to its promoter and we found a close correlation between RINF and SMAD7 mRNA levels both in CD34+ cells isolated from bone marrow of healthy donors and MDS patients with del(5q). Importantly, RINF knockdown attenuated SMAD7 expression in primary cells and ectopic SMAD7 expression was sufficient to prevent the RINF knockdowndependent erythroid phenotype. Finally, RINF silencing affects 5’-hydroxymethylation of human erythroblasts, in agreement with its recently described role as a Tet2- anchoring platform in mouse. Altogether, our data bring insight into how the epigenetic factor RINF, as a transcriptional regulator of SMAD7, may fine-tune cell sensitivity to TGFβ superfamily cytokines and thus play an important role in both normal and pathological erythropoiesis

Vitamin D Receptor Controls Cell Stemness in Acute Myeloid Leukemia and in Normal Bone Marrow.

Vitamin D (VD) is a known differentiating agent, but the role of VD receptor (VDR) is still incompletely described in acute myeloid leukemia (AML), whose treatment is based mostly on antimitotic chemotherapy. Here, we present an unexpected role of VDR in normal hematopoiesis and in leukemogenesis. Limited VDR expression is associated with impaired myeloid progenitor differentiation and is a new prognostic factor in AML. In mice, the lack of Vdr results in increased numbers of hematopoietic and leukemia stem cells and quiescent hematopoietic stem cells. In addition, malignant transformation of Vdr-/- cells results in myeloid differentiation block and increases self-renewal. Vdr promoter is methylated in AML as in CD34+ cells, and demethylating agents induce VDR expression. Association of VDR agonists with hypomethylating agents promotes leukemia stem cell exhaustion and decreases tumor burden in AML mouse models. Thus, Vdr functions as a regulator of stem cell homeostasis and leukemic propagation

L'inhibition de mTORC1 active la voie P13K/Akt en augmentant la signalisation IGF-1/IGF-1R dans les leucémies aiguës myéloïdes

PARIS-BIUP (751062107) / SudocSudocFranceF

Activation des voies PI3K/AKT et mTOR dans les LAM (thérapeutiques ciblées et Implication des facteurs de transcription FOXO dans la leucémogenèse)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Targeting glutamine uptake in AML.

International audienceCancer cells require nutrients and energy to adapt to increased biosynthetic activity and depend on mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis. Whereas they exhibit a pronounced Warburg effect, their TCA cycle remains intact and becomes more dependent on glutamine metabolism through glutaminolysis[1]. Besides this role, intracellular glutamine is also essential for mTORC1 activation by leucine[2]. Many upstream signals regulate mTORC1 activation. Among them, a major process is the availability of leucine, which is required to activate the Rag (for Ras-related GTPases) proteins that enable the proper localization of mTORC1 at the lysosome surface close to its activator Rheb[3]. Leucine uptake into the cells is regulated by the bidirectional transporter SLC7A5/3A2, in exchange for glutamine. The level of leucine thereby depends on the intracellular glutamine concentrations, which is mainly mediated by the high affinity transporter SLC1A5. Thus, the cellular uptake and subsequent rapid efflux of glutamine in the presence of leucine make glutamine availability a limiting step for the activation of mTORC1. MTORC1 positively regulates protein translation through phosphorylation of protein S6 Kinase (P70S6K) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). Protein synthesis is controlled by the translational repressor 4E-BP1 whose phosphorylation at serine 65 is required to initiate the formation of the translation initiation complex. The dependence of acute myeloid leukemia (AML) cells to glutamine is little studied. In a recent work, we have tested the effects of glutamine depletion in AML cells[4]: leukemic cells are sensitive to glutamine removal leading to mTORC1 inhibition and apoptosis. The drug L-asparaginase (L-ase) also inhibits mTORC1 activity in AML cells, suppresses protein synthesis and induces apoptosis. The anti-leukemic effects of the two clinically available forms of L-ase, E Coli L-ase (Kidrolase®) and E. Chrysanthemi L-ase (Erwiniase®) are not mediated by the asparaginase activity of the enzyme. L-ases have also a glutaminase activity and transform extracellular glutamine into glutamate. Both L-ases induce dose and time-dependent mTORC1-inhibition which correlates with extra-cellular glutamine depletion[4]. Downstream of mTORC1, L-ase suppresses 4E-BP1 phosphorylation and inhibits [S 3

Dérégulation des voies de signalisation P13K/Akt et mTOR dans les Leucémies Aigües Myéloïdes

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Ferroptose et cancer

La ferroptose est un processus conduisant à la mort de la cellule avec, pour évènement final, l’accumulation létale de lipides peroxydés. Le fer libre intracellulaire est au centre des réactions entraînant la formation de ces lipides peroxydés. Un système antioxydant dédié à la détoxification de ces lipides permet de prévenir la mort cellulaire. Le processus de ferroptose est impliqué dans un grand nombre de maladies, notamment dans la pathogénie des maladies neurodégénératives et infectieuses et du cancer. Nous présentons dans cette revue les principaux acteurs cellulaires qui contrôlent la ferroptose et proposons une synthèse des données actuelles impliquant ce processus dans le cancer