Abstract
Introduction: ABT‐737 is a BH3 mimetic that selectively inhibits Bcl‐2, Bcl‐xL and Bcl‐w and has been shown to induce apoptosis in several tumor types. Mcl‐1 expression has been proposed as a potential mechanism of resistance to Mcl‐1. Sorafenib is an oral multikinase inhibitor that targets VEGFR2, PDGFR, B‐Raf and has also been shown to down modulate Mcl‐1. PLX4720 is a potent inhibitor of the V600E mutant B‐Raf which is an oncogenic activating mutation that is found in approximately 40–60% of patients with cutaneous melanoma. PLX4032, a similar and more bioavailable molecule than PLX4720, is currently in clinical development and was associated with clinical responses in patients with malignant melanoma in a phase I trial. Since we have previously shown that sorafenib has a synergistic effect when used in combination with ABT‐737, we set out to evaluate and compare the effects of sorafenib and PLX4720 alone and in combination with ABT‐737 on melanoma cell lines.
Methods: The B‐Raf V600E mutant melanoma cell lines A375, A2058 and SKMEL5 were treated with DMSO, sorafenib (10 uM) or PLX4720 (1 uM) alone or in combination with ABT‐737 (5 uM). Cell lysates were prepared after 4 hours and probed with specific antibodies to detect the presence of vinculin, PARP, Mcl‐1, total Erk, and phosphorylated Erk (Thr202/Tyr204) using standardWestern Blot technique. In addition, cells treated for 24 hours were assessed for cytotoxic activity using propidium iodide staining followed by flow cytometeric analysis.
Results: Results were broadly similar in all three cell lines tested. Both sorafenib and PLX4720, but not ABT‐737, were found to down‐modulate Mcl‐1 and the phosphorylation of Erk (pErk). The effect of PLX4720 on Erk phosphorylation was greater than the effect of sorafenib while, conversely, sorafenib‐downmodulation of Mcl‐1 was more profound than that of PLX4720. PARP cleavage was identified in ABT‐737 treated cells at a low level, however this was substantially enhanced in combination with either sorafenib or PLX4720. Cytotoxicity was greater in sorafenib‐treated cells than those treated with PLX4720. The effect of each treatment was enhanced, however, in combination with ABT‐737, which exhibited little to no single‐agent activity across all cell lines. The combination of sorafenib and ABT‐737 had the strongest cytotoxic effect of the treatments evaluated in each cell line.
Conclusion: Both the multitargeted TKI sorafenib and the more specific V600E mutant B‐raf inhibitor PLX4720 enhance the cytotoxicity of the BH3‐mimetic ABT‐737 in melanoma cell lines. Our results suggest that ABT‐737‐mediated cytotoxicity is dependent on Mcl‐1 downmodulation. This is supported by the finding that the combination of sorafenib and ABT‐737 is more toxic than PLX4720 and ABT‐737 and is associated with a more effective reduction of Mcl‐1 levels in 4 hour lysates. Xenograft testing of these combinations is planned with the goal of translating the most successful combination into an early phase clinical trial.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B89.
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