Abstract 4536: Simultaneous blockade of MEK and mTOR kinase signaling synergizes pro-apoptotic effects in AML cells, which is further potential by interfering with dimerization of BH3 domains
Authors: Zhang Weiguo , Konopleva Marina , Tsao Twee , Schober Wendy D. , Andreeff Michael 
Published: February 2011

Abstract
Simultaneous activation of PI3K/Akt/mTOR and Raf/MEK/ERK signaling pathways is common in acute myeloid leukemia (AML) and is associated with worse prognosis(1). Several small molecule inhibitors targeting mTOR or MEK/ERK signaling are being evaluated in clinical trials in AML, including allosteric mTOR inhibitors (CCI-779, RAD001) and MEK inhibitors (CI-1040, AZD6244 [ARRY-142886]), and demonstrated encouraging pre-clinical results(2-4). However, the efficacy of these agents as single agents is variable in AML(3, 5, 6). The predominant effects of these inhibitors are cytostatic rather than cytotoxic in AML. AZD8055 is a novel mTOR kinase inhibitor, acting against both mTORC1 and mTORC2, and is currently in Phase 1 clinical trials in solid tumors.
In the present study, we investigated the anti-leukemia efficacy of the AZD8055, alone or in combination with AZD6244 in human leukemic cells and in primary AML samples. AZD8055 potently suppressed p-S6K and p-4E-BP1 in AML cells (70-85% inhibition), significantly blocked p-AKT(473) in OCI-AML3 and MOLM13 cells, and inhibited both, p-AKT(473) and p-AKT(308) in PTEN-mutant U937 cells. This resulted in profound inhibition of cell growth at lower IC50s (40-147nM) in all these AML cells. However, AZD8055 did not induce apoptosis in the cells even up to 10μM concentrations. The combination of AZD8055 with AZD6244 triggered synergistic pro-apoptotic responses in U937 (CI = 0.22 ± 0.04) and MOLM13 (CI = 0.66 ± 0.06) cells, but not in OCI/AML3 and KG-1 cells. MEK inhibition resulted in partial blockade of S6K phosphorylation in OCI/AML3 and KG-1 cells, suggesting that S6K activity can also be regulated by MAPK pathways in some AML cell lines. Further analysis demonstrated that sensitive cells (U937 and MOLM13) express lower basal levels of Bcl-2 protein compared to resistant cell lines (OCI/AML3 and KG-1), and showed pronounced upregulation of pro-apoptotic Bim after AZD6244 and AZD8055/AZD6244 combination treatment. Furthermore, interference with dimerization of Bcl2 family proteins by BH3 mimetic ABT-737 dramatically enhanced the pro-apoptotic responses of the combination in resistant cells even when all three agents are used at sub-micromolar concentrations. Depleting Bim levels by siRNA rescued sensitive cells from AZD8055/AZD6244-induced apoptosis, indicating a key role of downstream Bim in cytotoxicity of these agents. In primary AML samples AZD8055/AZD6244 induced apoptosis in an additive fashion, while simultaneous use of ABT-737 significantly increased the pro-apoptotic effects.
In summary, our results indicate that parallel blockade of PI3K/MAPK signaling and manipulation of the mitochondrial Bcl-2 rheostat potently enhances apoptosis induction in AML cells and may provide a viable therapeutic strategy for resistant/refractory AML patients.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4536.