Dual MTORC2/mTORC1Inhibitor Induces Autophagic Cell Death Of MDS/AML And Its Underlying Mechanism

Part Ⅰ. Dual mTORC2/mTORCl inhibitor induces autophagic cell death of MDS/AML cellsObjective:To investigate the influence of the dual mTORC1/mTORC2inhibitor on the proliferation of MDS/AML cells, and further explore the mechanism of inhibition of cell proliferation in apoptosis and autophagy.Methods:Myelodysplastic syndrome (MDS) transformed acute myeloid leukemia cell line SKM-1cells were collected, To detect the influence of different concentrations of mTORC1inhibitor Rapamycin and dual mTORC2/mTORC1inhibitor OSI-027respectively on the proliferation of SKM-1cells by CCK-8. To determine the induced apoptosis by the AnnexinV-FITC/PI double staining flow cytometry and Western-blot for detection PARP activity. Flow cytometry was used to detect the influence of the cell cycles; Autophagic vacuoles and autophagosome in the cells were observed by transmission electron microscope; after treatment SKM-1cells were stained with acridine orange, and the autophagic cells (acridine orange positive cells) was analyzed by flow cytometry. SKM-1cells were left untreated or were treated with various concentrations of OSI-027in the presence or absence of5μM chloroquine for48h; Relative amounts of viable cells were detected by CCK-8. RT-PCR method was use for detecting autophagy related genes mRNA expression of Beclin-1and LC3B; Western-blot method was used to detect the protein expression of Beclin-1and LC3.Results:Dual mTORC2/mTORC1inhibitor OSI-027exhibits more potent cytotoxicity effects than mTORC1inhibitor Rapamycin on MDS/AML cells. For the mode of cell death, we found OSI-027induced a higher percent of dead cells and a lower percent of apoptotic cell and hypodiploid cells, suggesting non-apoptotic cell death in OSI-027treated SKM-1cells. Massive autophagic vacuoles and autophagosome in OSI-027treated cells were observed by transmission electron microscope and autolysosome formation (acridine orange positive cells) was detected after OSI-027treatment; Moreover, OSI-027-induced autophagy in SKM-1cells was confirmed by the up-regulation of autophagy-related gene Beclin-1and LC3B, the up-regulation of the expression of autophagy-related protein Beclin-1and LC3-Ⅱ conversion.Conclusion:Dual mTORC2/mTORC1inhibitor can inhibit the proliferation of SKM-1cells and we clarified the mechanism of action of dual mTORC2/mTORC1inhibitor OSI-027against human SKM-1cells in the mode of autophagic cell death. Part Ⅱ. The underlying mechanisms of dual mTORC2/mTORCl inhibitor induces autophagic cell death of MDS/AML cellsObjective:To explore the potent mechanisms of dual mTORC2/mTORC1inhibitor OSI-027induces autophagic cell death of MDS/AML cells.Methods:MDS/AML cell line SKM-1cells were incubated with OSI-027and Rapamycin for12h, Western blot was used to detect the expression of p-mTOR ser2448, p-mTOR ser2481, p-P70S6K, P70S6K, p-4E-BP1Thr37/46,4E-BP1, p-Akt ser473, Akt, p-FoxO3a Th32, FoxO3a; Lentivrus transfection was carried out for silence the expression of FOXO3a, the protein expression of FOX03a was assessed by western blot further OSI-027incubated with the siFOXO3a cells for48h and real-time PCR and western blot were used to examine the expression of mRNA of LC3B and Beclin-1and the protein. The formation of4E-BP1-elF4E complexes or the formation of e1F4E-eIF4G complexes were determined by co-immunoprecipitation (co-IP).Results:The complexes of mTORC1contain mTOR phosphorylated on Ser2448and mTOR phosphorylated on Ser2481is a maker of the mTORC2complexes; we firstly determined that mTORC1complexes and mTORC2complexes are present in MDS/AML cells. Treatment of cells with either rapamycin or OSI-027respectively resulted in suppression of phosphorylated mTOR on Ser2448, demonstrating that two agent both can inhibit the activity of mTORC1. However, only OSI-027inhibited phosphorylated mTOR on Ser2481, demonstrating that OSI-027selectively target mTORC2. The downstream protein of p-P70S6K apparently decreased after the treatment of rapamycin and OSI-027respectively. Additionally, the protein of phosphorylated4E-BP1on Thr37/46only decreased after OSI-027treatment. The treatment of OSI-027can effectively induce the formation of4E-BP1-elF4E complexes that suppress cap-dependent translation and block the formation of e1F4E-eIF4G complexes by co-immunoprecipitation test. For the downstream of mTORC2, rapamycin negatively enhanced phosphorylated Akt on Ser473, reflecting potent induction of mTORC2activity. However, dual mTORC2/mTORC1ihibitor OSI-027suppressed phosphorylated Akt on ser473, further the suppression of phosphorylated Akt of Ser473inhibit the FoxO3phosphorylation, accordingly the decreasing of phosphorylation in Thr32of FOXO3a results in the upregulation of the transcription of autophagy-related genes.Conclusion:Treatment with rapamycin resulted in inhibition of mTORCl, but negative enhancement of Akt phosphorylation/activation, reflecting potent induction of mTORC2activity. Dual mTORC2/mTORC1ihibitor OSI-027promoted the autophagy of SKM-1cells mainly depend on suppressing mTORC2and down-regulation of the phosphorylation of Akt on ser473and FOXO3a on Thr32.