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

Abstract : Acute 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 mice
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Lise Willems. Implication de la glutamine dans l’activation de mTORC1 dans les leucémies aiguës myéloïdes et inhibition ciblée. Médecine humaine et pathologie. Université René Descartes - Paris V, 2012. Français. ⟨NNT : 2012PA05S016⟩. ⟨tel-00868018⟩

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