Targeting the mTOR Pathway Suppresses the Growth of Acute Myeloid Leukemia (AML) Progenitors and Enhances Arsenic Trioxide Induced Antileukemic Responses.

Abstract

-Background: A limiting factor for the use of arsenic trioxide (As2O3) in the treatment of non-APL subtypes of AML is the requirement of very high concentrations for the induction of leukemic cell death. The mammalian target of rapamycin (mTOR) is a key cellular regulator of multiple signaling events that control initiation of mRNA translation in leukemia cells. We examined the effects of As2O3 on mTOR-dependent cellular pathways in AML cells and determined whether modulation of the mTOR pathway sensitizes them to the antileukemic properties of As2O3.Methods: The human AML cell lines KG1, U937, or MM6 were treated with increasing concentrations of As2O3. Activation of p70 S6 kinase (p70S6K) was evaluated by immune-complex kinase assays in anti-p70S6K immunoprecipitates, while phosphorylation of various downstream mTOR effectors was assessed by immunoblotting with specific antibodies against phosphorylated S6 ribosomal protein (rpS6) and the translational repressor 4E-BP1. MTT assays were used to assess proliferation of the various cell lines, while apoptosis was determined by annexin V-propidium iodide staining. Finally, bone marrow mononuclear cells from patients with AML were used in clonogenic assays in methylcellulose to assess the effects of As2O3 and/or rapamycin on leukemic progenitor cell growth.Results: Treatment of AML cells with As2O3 resulted in activation of the p70 S6K and phosphorylation of its downstream effector, rpS6, on Ser240/244, and such events were mTOR-dependent. In addition, the translational repressor 4E-BP1 was phosphorylated on Thr37/46 and Thr70, sites whose phosphorylation is required for 4E-BP1 de-activation and dissociation from eIF4E. Pharmacological inhibition of mTOR activity blocked the growth of AML cell lines as determined by MTT assays, while the addition of As2O3 to the cultures further enhanced the inhibitory effects of rapamycin. On the other hand, AS2O3-dependent induction of apoptosis was not enhanced by rapamycin in AML cells, suggesting selective effects on cell cycle and growth inhibition. Rapamycin also inhibited leukemic CFU-GM progenitor colony formation from 6 AML patients and enhanced the suppressive effects of AS2O3 on leukemic progenitor cell growth.Conclusions: Paradoxically, As2O3 activates the pro-growth and pro-survival mTOR pathway in AML cells. Such activation appears to occur in a negative feedback regulatory manner to compensate for AS2O3 responses. Thus, combinations of AS2O3 with mTOR inhibitors may be an effective approach to overcome AS2O3-resistance in AML cells.