Abstract B89: The effects of raf kinase inhibition with either sorafenib or PLX-4720 on Mcl-1 expression and cytotoxicity alone and in combination with ABT-737 in melanoma cell lines
Authors: Curtis Lauren M. , Panka David J. , Cho Daniel C. , Mier James W. , Sullivan Ryan J. 
Published: June 2012

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.