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

-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.