Oncogenic Role Of NOTCH1 Target Gene DEPTOR In T Cell Lymphoblastic Leukemia

Objective:Acute T-cell lymphoblastic leukemia(T-ALL),an aggressive hematologi-cal malignant disorder of T cell progenitors,is one of the intractable tumors in pediatrics.Recent reports show that aberrant activation of NOTCH1 signaling plays a vital role in the pathogenesis of T-ALL.Yet the molecular events downstream of NOTCH1 that drive T-cell leukemogenesis remain incompletely understood.The goal of the present study is to identify the crucial role of DEPTOR in T-ALL pathology and the mechinism of transcriptional regulation of DEPTOR by NOTCH 1 pathway in T-ALL.Methods:We employed genome-wide gene expression profiling in a murine T-ALL cell to seek important NOTCH1 transcriptional targets,and then performed chromatin immunoprecipitation(ChIP)assay and luciferase reporter assay to validate if DEPTOR as a direct NOTCH1 downstream target in T-ALL.To ensure the NOTCH1 regulation of DEPTOR across species,we performed Western blot assay and RT-qPCR assay to evaluate the DEPTOR expression in human T-ALL cell lines and primary samples associated with oncomine database analysis,as well as the correlation between DEPTOR and NOTCH 1 or its activity.We next sought to determine potential role of DEPTOR in T-ALL cell proliferation,viability and glucose metabolism by using the short-hairpin RNA(shRNA)-mediated DEPTOR ablation in loss-of-function assays or exogenous overexpression of DEPTOR in gain-of-function assays in vitro,and to validate if DEPTOR overexpression could rescue the growth or glucose inhibition caused by the NOTCH inhibitor.In addition,the immunodeficient mice model of leukemia was established to investigate the effects of DEPTOR deletion or overexpression on the onset of leukemia and the infiltration of leukemia cells in vivo.Finally,the effects of DEPTOR ablation,overexpression or mutation on mTOR pathway and AKT phosphorylation were analyzed by Western blot assays to seek the potential molecular mechanism of DEPTOR in T-ALL.Results:We found that DEPTOR was up-regulated by NOTCH1 in T-ALL,and the expression of DEPTOR in primary patients was positively correlated with the NOTCH1 activity.NOTCH1 binds specifically to the DEPTOR promoter region and directly activates its transcription activity.shRNA-mediated DEPTOR depletion abolished cellular proliferation,attenuated glycolytic metabolism and enhanced cell death,while ectopically expressed DEPTOR significantly accelerated cell growth and glycolysis.Moreover,DEPTOR depletion in a human T-ALL xenograft model significantly prolonged mice lifespans(p<0.01),alleviated splenomegaly,reduced leukemia cells in the peripheral blood,spleen,bone marrow as well as liver(p<0.001),and caused a substantial decrease of AKT activation in leukemic blasts.We further showed that DEPTOR depletion inhibited while its overexpression enhanced AKT activation in T-ALL cells.Importantly,AKT inhibition by MK 2206 completely abrogated DEPTOR-mediated cell growth advantages.The results show that DEPTOR promotes the activation of mTORC2 pathway by inhibiting mTORC1 acitivity and further increases AKT phosphorylation,promotes T-ALL cell growth,survival and glycolysis.Conclusion:Our data thus provide strong evidence that NOTCH1 directly activates DEPTOR transcription,DEPTOR-mediated AKT activation accounts for accelerated cell expansion.We thus reveal a novel mechanism underlying crosstalk of the NOTCH1 and AKT pathways.Overall,we identify transcriptional control of DEPTOR and its regulation of AKT as additional key elements of the leukemogenic program activated by NOTCH1,suggesting high DEPTOR expression may be a potential clinical biomarker and therapeutic targeting of DEPTOR may benefit T-ALL patients.