MEK Inhibitor AZD6244 Induces Cell Growth Arrest and Synergizes Nutlin-3a-Mediated Cell Death by Upregulating p53 and PUMA Levels in Acute Myelogenous Leukemia.

Raf/MEK/Erk signaling is constitutively activated in the majority of acute myeloid leukemias (AML), providing rationale for targeting this pathway with therapeutic intent. We investigated growth-inhibitory and pro-apoptotic effects of AZD6244, a small molecule MEK1/2 inhibitor, as well as potential synergistic effects with the MDM2 antagonist Nutlin-3a in AML. AZD6244 inhibited proliferation of AML cells by arresting cells in G1 phase, which resulted from inhibition of cell cycle checkpoint proteins cyclin E and cdk2, and was associated with the modulation of cyclin D1, cyclin A, cyclin B1, p27 and Rb phosphorylation levels. AZD6244 also induced apoptotic cell death in AML cell lines with constitutive activation of MEK/ERK signaling (OCI/AML3, HL60 and MOLM13) via decrease of anti-apoptotic Mcl-1 protein expression and upregulation of pro-apoptotic Bax, Bak, Bad and Bim proteins. Next we tested the hypothesis that activation of p53 signaling with a small molecule MDM2 inhibitor Nutlin-3a would enhance the pro-apoptotic effects of MEK inhibition in AML. Combinations of AZD6244 and Nutlin-3a triggered profound apoptotic responses in AML cells with wild-type p53 (OCI/AML3, MOLM13 and Baf3-FLT3-ITD), with combination indices (averaged ED50=0.37, ED75=0.38, ED90=0.43) suggesting strong synergism. In contrast, no synergism was seen in p53 null cell HL60 (ED50=1.27, ED75=1.38, ED90=1.66) or in OCI-AML3 cells stably transduced with p53 shRNA. Mechanistically, combined blockade of MEK pathway and activation of p53 signaling resulted in marked upregulation of p53 expression and of the p53 target protein PUMA, while only partially suppressing Nutlin-induced p21 upregulation. This resulted in loss of the mitochondrial membrane potential, cytochrome c release and cleavage of caspase-9 and -3. Importantly, combined AZD6244 and Nutlin-3a treatment potently induced apoptosis in blasts from primary AML patients. In summary, AZD6244 induced growth-inhibitory and pro-apoptotic effects in AML cells with constitutive activation of MEK/ERK signaling. These effects were synergistically enhanced by simultaneous activation of p53 signaling by Nutlin-3a, resulting in the activation of the intrinsic apoptotic pathway. Altogether, these results strongly suggest potential activity of AZD6244 alone or in combination with MDM2 antagonists as novel mechanism-based therapeutic agents in AML therapy.