Abstract 4881: Induction of Bim by the BRAF inhibitor PLX4720 sensitizes human melanoma cells to the BH3 mimetic ABT-737

Once melanoma has metastasized to distant sites it has proved difficult to treat by a variety of chemotherapeutic and biological agents. Significant advances have been made in identifying subgroups of patients with activating mutations in signalling pathway proteins. In particular, the finding that approximately 50% of melanoma have V600E mutations in the BRAF serine/threonine kinase has resulted in the generation of a number of new drugs which target the mutated active form of BRAF. One of these drugs, Vemurafenib (PLX4032/RG704) has shown significant promise in prolonging survival compared to standard chemotherapy, however reports of patient relapse has prompted investigation into means of overcoming acquired resistance to the agent. We report here that efficacy of the PLX4032 analogue, PLX4720, is greatly enhanced by co-treatment with the BH3 mimetic, ABT-737. Treatment with either agent is able to induce apoptosis in melanoma cells, however the kinetics are typically slow, with cell death occurring 48 or 72 hours post treatment. In this study, while either agent was largely ineffective as a single treatment 24 hours following treatment, addition of the combination of drugs rapidly induced significant killing. We show that apoptosis was caspase-dependent, in that inhibition of caspase activation almost totally blocked induction of cell death by the combination of agents. Apoptosis was also associated with activation of the intrinsic apoptotic pathway, with reduction of mitochondrial membrane potential and activation of caspase-9. Exposure of melanoma cells to PLX4720 rapidly decreased phosphorylation of the pro-apoptotic BH3-only protein Bim, resulting in strong upregulation as early as 6 hours after addition of the compound. Strong upregulation of Bim was shown by RNAi knockdown to play an important role in sensitizing cells to co-treatment with ABT-737, by assisting in the inhibition of anti-apoptotic Bcl-2 family proteins. Further, the combination of agents induced significant levels of apoptosis in melanoma cells derived from patients harbouring the BRAFV600E mutation. Collectively, these results show that the BRAF inhibitor PLX4720 sensitizes melanoma cells to apoptosis by the BH3 mimetic ABT-737 in a Bim- and caspase-dependent manner. Additionally, these studies identify a potential clinical application for BH3 mimetics in overcoming resistance of melanoma to treatment with BRAF inhibitors, increasing efficacy of this class of agent and improving