mTOR-generated signals play critical roles in growth of leukemic cells by controlling mRNA translation of genes that promote mitogenic responses. Despite extensive work on the functional relevance of rapamycin-sensitive mTORC1 complexes, much less is known on the roles of rapamycin-insensitive (RI) complexes, including mTORC2 and RI-mTORC1, in BCR-ABL-leukemogenesis. We provide evidence for the presence of mTORC2 complexes in BCR-ABL-transformed cells and identify phosphorylation of 4E-BP1 on Thr37∕46 and Ser65 as RI-mTORC1 signals in primary chronic myelogenous leukemia (CML) cells. Our studies establish that a unique dual mTORC2∕mTORC1 inhibitor, OSI-027, induces potent suppressive effects on primitive leukemic progenitors from CML patients and generates antileukemic responses in cells expressing the T315I-BCR-ABL mutation, which is refractory to all BCR-ABL kinase inhibitors currently in clinical use. Induction of apoptosis by OSI-027 appears to negatively correlate with induction of autophagy in some types of BCR-ABL transformed cells, as shown by the induction of autophagy during OSI-027-treatment and the potentiation of apoptosis by concomitant inhibition of such autophagy. Altogether, our studies establish critical roles for mTORC2 and RI-mTORC1 complexes in survival and growth of BCR-ABL cells and suggest that dual therapeutic targeting of such complexes may provide an approach to overcome leukemic cell resistance in CML and Phþ ALL.mRNA translation | cell proliferation | cellular signaling | kinase | OSI-027

Arsenic trioxide (As 2 O 3 ) exhibits potent antitumor effects in vitro and in vivo, but the precise mechanisms by which it generates such responses are not well understood. We provide evidence that As 2 O 3 is a potent inducer of autophagy in leukemia cells. Such induction of autophagy by As 2 O 3 appears to require activation of the MEK/ERK pathway but not the AKT/mammalian target of rapamycin or JNK pathways. In efforts to understand the functional relevance of arsenic-induced autophagy, we found that pharmacological inhibitors of autophagy or molecular targeting of beclin 1 or Atg7 results in reversal of the suppressive effects of As 2 O 3 on leukemic cell lines and primary leukemic progenitors from acute myelogenous leukemia patients. Altogether, our data provide direct evidence that autophagic cell death is critical for the generation of the effects of As 2 O 3 on acute myelogenous leukemia cells and raise the potential of modulation of elements of the autophagic machinery as an approach to enhance the antitumor properties of As 2 O 3 and possibly other heavy metal derivatives. Arsenic trioxide (As 2 O 3 )2 is a metalloid that exhibits potent antineoplastic effects in vitro and in vivo (1-3). This arsenic derivative induces apoptosis and suppresses the growth of various types of malignant cells of diverse origin in vitro (1-3). Different mechanisms by which As 2 O 3 promotes cell death of target cells have been extensively studied and described (reviewed in Refs. 1-3). As 2 O 3 -dependent generation of reactive oxygen species leads to activation of pro-apoptotic pathways in different types of cells (1-3). In addition, there is evidence for mechanisms involving As 2 O 3 -dependent cell typespecific targeting of malignant cells with distinct molecular abnormalities (reviewed in Ref.3). For instance, there is evidence for As 2 O 3 -dependent specific targeting of cells expressing AML1/MDS1/EVI1 involving degradation of the abnormal fusion protein (4), evidence for arsenic-dependent BCR-ABL ubiquitination and proteasomal degradation (5), and eradication of leukemia-initiating cells in acute promyelocytic leukemia via As 2 O 3 -inducible PML-RAR␣ degradation (6).Despite extensive research over the years, the precise mechanisms of action of arsenic trioxide in malignant cells are not well understood. In particular, the specific cellular events that account for differential sensitivity of malignant cells to As 2 O 3 remain to be precisely defined. Notably, arsenic trioxide treatment induces responses in patients with acute promyelocytic leukemia in vivo (7,8), and it is an agent approved by the United States Food and Drug Administration for the treatment of this leukemia. The unusually high sensitivity of acute promyelocytic leukemia cells to the effects of arsenic trioxide likely reflects the requirement of lower As 2 O 3 concentrations for induction of leukemic cell differentiation seen in these cells versus apoptotic cell death (3), but other mechanisms may be involved as well.Autophagy is a cell death mechani...

Purpose To determine whether mTORC2 and RI-mTORC1 complexes are present in AML cells and to examine the effects of dual mTORC2/mTORC1 inhibition on primitive AML leukemic progenitors. Experimental Design Combinations of different experimental approaches were used, including immunoblotting to detect phosphorylated/activated forms of elements of the mTOR pathway in leukemic cell lines and primary AML blasts; cell proliferation assays; direct assessment of mRNA translation in polysomal fractions of leukemic cells; and clonogenic assays in methylcellulose to evaluate leukemic progenitor colony formation. Results mTORC2 complexes are active in AML cells and play critical roles in leukemogenesis. Rapamycin insensitive (RI) mTORC1 complexes are also formed and regulate the activity of the translational repressor 4E-BP1 in AML cells. OSI-027, blocks mTORC1 and mTORC2 activities and suppresses mRNA translation of cyclin D1 and other genes that mediate proliferative responses in AML cells. Moreover, OSI-027 acts as a potent suppressor of primitive leukemic precursors from AML patients and is much more effective than rapamycin in eliciting antileukemic effects in vitro. Conclusions Dual targeting of mTORC2 and mTORC1 results in potent suppressive effects on primitive leukemic progenitors from AML patients. Inhibition of the mTOR catalytic site with OSI-027 results in suppression of both mTORC2 and RI-mTORC1 complexes and elicits much more potent antileukemic responses than selective mTORC1 targeting with rapamycin.