Combination of a MEK Inhibitor, AZD6244, and Dual PI3K/mTOR Inhibitor, NVP-BEZ235: An Effective Therapeutic Strategy for Acute Myeloid Leukemia

Abstract 3978

It has been demonstrated that MEK/MAPK and PI3K/Akt are constitutively activated in the majority of AML cases and that their aberrant expression is associated with a poor prognosis. Targeted inhibition of either the MEK/MAPK or the PI3K/Akt pathway alone has only demonstrated mild to modest clinical activity, possibly due to feedback activation of compensatory pathways. Thus, preclinical studies have recently turned to targeted inhibition of both of these pathways simultaneously. In the current study, the efficacy of the combination of two orally available inhibitors to MEK (AZD6244, Astra Zeneca) and PI3K/mTOR (NVP-BEZ235, Novartis) was evaluated in AML cell lines and in primary AML patient samples. In MV 4;11 AML cells (harboring both the MLL re-arrangement and FLT3 internal tandem mutation), AZD6244 or BEZ235 alone moderately decreased viable cell numbers by 30–40% as measured by the MTS assay, a colorimetric assay for cellular growth and survival, but the combination of these two had a dramatic additive effect with a decrease of viable cell numbers by 70–80%. Similar effects were observed in AML cell lines with different cytogenetic and molecular abnormalities including THP-1 [t (6;11)], HL-60, KG-1 [del(5q)], and Kasumi-1 [t(8;21)]. Similar results were also obtained in leukemia cells from 3 patients with AML with different recurring cytogenetic abnormalities. Apoptotic cell death was determined by detection of <2N DNA using 7AAD staining, and the cell cycle was measured using BrdU incorporation followed by flow cytometric analysis. The combination therapy additively induced apoptotic cell death up to 50–60% and cell cycle arrest, whereas either inhibitor alone resulted in only mild apoptotic cell death (∼15-30%). Although dual pathway inhibition was efficacious in all AML cell lines, no additive effect of dual inhibition was observed in Jeko-1, a mantle cell lymphoma cell line. To evaluate the underlying mechanisms of apoptosis, flow cytometry was used to detect phospho-protein and apoptosis-associated proteins. Interestingly, inhibition of MEK/MAPK alone with AZD6244 resulted in decreased pErk level, but increased pmTOR and anti-apoptotic Mcl-1 levels. These results suggest a feedback activation of PI3K/Akt/mTOR pathway, which could be abrogated by the addition of BEZ235. Similarly, inhibition of PI3K/Akt/mTOR resulted in increased pErk and pJNK which could be abrogated by adding AZD6244. AZD6244 also resulted in increased expression of pro-apoptotic Bim, and anti-apoptotic Bcl-2 in AML cell lines, which could not be abrogated by inhibition of PI3K/Akt/mTOR by BEZ235, suggesting that the modulation of these two proteins is independent of the PI3K/Akt/mTOR pathway. Taken together, these findings suggest that inhibition of Bcl-2 might further sensitize AML cells to apoptotic cell death induced by the combination of AZD6244 and BEZ235. In conclusion, these data provide a strong rationale for drug combination targeting of PI3K/Akt/mTOR and MEK pathways for the treatment of AML. Furthermore, inhibition of BCl-2 anti-apoptosis family members may, in part, explain the efficacy of dual signaling blockade in AML cells and suggests an additional therapeutic targeting strategy. Single agent small molecule inhibitors of PI3K/Akt/mTOR, MEK and BCL-2 are already being tested in early phase clinical trials in solid tumors and in hematological malignancies; thus, feasibility studies of combinations of these small molecule inhibitors should be designed to test their efficacy in patients with AML.