Pro-apoptotic synergistic interactions between ERK1/2 and Bcl-2 inhibitors in acute myeloid leukemia cells.

We previously reported that activation of the Ras/MEK/ERK signaling pathway alone or in combination with high levels of Bcl-2 confers poor prognosis in patients with acute myeloid leukemias (AML). In this study, we investigated whether the simultaneous disruption of these two pathways by pharmacological inhibition of ERK1/2 and Bcl-2 utilizing novel small molecule inhibitors would induce pro-apoptotic activity in myeloid leukemic cells. For inhibition of ERK1/2 signaling, we utilized the MEK inhibitor CI-1040 (Pfizer Global Research and Development). Functional Bcl-2 inhibition was achieved by the novel BH3 peptide binding domain inhibitor A438744.7 (Abbott Laboratories). A dose-dependent increase in apoptosis as assessed by an increase in phosphatidylserine externalization was observed in OCI-AML3 cells after 24h incubation with CI-1040 or A438744.7 (both in a range between 0.25 and 2 µM). When these agents were used in combination at a fixed 1:1 ratio, a dramatic enhancement of cell killing was observed especially at lower concentrations (70.63%±9.95 Annexin V+ cells at 0.25 µM of each compound). Isobologram analysis (Chou and Talalay method) revealed a Combination Index (CI) < 1 (CI=0.114 and 0.436 at 0.25 µM and 2 µM, respectively) suggesting the strongly synergistic nature of these interactions. In addition, combined treatment with CI-1040 and A438744.7 resulted in a substantial increase of mitochondrial damage and caspase cleavage. Pre-incubation (1-hour) with a pan-caspase inhibitor (IDN-1965) was able to completely abrogate sensitivity to the inhibitors. We further demonstrated that bcl-2 overexpression prevented induction of apoptosis by low doses of both, MEK and BH3 inhibitors, whereas enforced Bcl-XL expression essentially abrogated the lethal effects of A438744.7, but not of CI-1040. Finally, to evaluate whether the combined strategy of ERK1/2 and Bcl-2 inhibition would be potentially applicable to leukemia patients, primary cells were isolated from AML samples and exposed to CI-1040 and A438744.7, as single agents or in combination at a 1:1 ratio for up to 96 hours. In each sample, drugs administrated individually were minimally toxic. Co-administration of CI-1040 and BH3 inhibitors displayed strong synergistic lethal effects toward primary cells from AML patients (CI=0.45±0.42 at 0.25 µM and CI=0.20±0.22 at 0.5 µM after 48h incubation). In conclusion, simultaneous exposure to nanomolar concentrations of MEK and bcl-2 inhibitors induced mitochondrial dysfunction, caspases activation and striking synergistic pro-apoptotic activity in myeloid leukemic cells. Similar synergistic interactions occured in primary AML samples. Together, these data strongly suggest that therapeutic strategies combining MEK and Bcl-2 inhibitors warrant further examination in AML.