Abstract 1790: Apoptotic response of mutant B-RAF human melanoma cells to a B-RAF inhibitor involves increased splicing production of BimS

Treatment of metastatic melanoma with systemic agents such as chemotherapy and biological agents has in general been unrewarding. Results from phase I studies with small molecule inhibitors of mutated BRAF have been encouraging and promise to provide a much needed breakthrough in treatment of melanoma with the mutant form of BRAF. However, the mechanism(s) of the cytocidal effect of inhibition of mutant B-RAF on melanoma cells has not been well established, even though dephosphorylation of BimEL has been shown to play a part in induction of apoptosis by inhibition of B-RAF. We show in this report that the B-RAF inhibitor PLX4720 preferentially inhibits proliferation and induces apoptosis in B-RAF mutant melanoma cells, and that induction of apoptosis by PLX4720 involves transcriptional up-regulation of Bim and preferential splicing production of BimS. The relative selectivity of PLX4720 on mutant B-RAF melanoma cells were confirmed in a panel of 31 melanoma cell lines, of which, 13 (41.9%) carried active BRAF mutations with 12 being the V600E mutation and another one, the K601E mutation. Activation of the mitochondrial apoptotic pathway appeared essential in PLX-4720-induced apoptosis, but intriguingly, there was little, or no, reduction in mitochondrial membrane potential and activation of Bak. The caspase cascade is involved, but induction of apoptosis could only partially be inhibited by the general caspase inhibitor z-VAD-fmk. PLX4720 caused up-regulation of the Bim isoforms, BimEL, BimL, and BimS at the protein level, with the increase in BimS most prominent and sustained. Subsequent studies revealed that the three Bim mRNA species were also increased by transcriptional mechanisms by PLX4720. Significantly, the ratios of BimS mRNA to BimEL and BimL mRNA after treatment with PLX4720 were markedly higher than those before treatment, suggesting that PLX4720 may trigger preferential splicing production of BimS. In support, enforced expression of BRAFV600E inhibits BimS expression in melanocytes, and blocked up-regulation of Bims by the histone deacetylase inhibitor SBHA that is known to increase Bim at the transcriptional level. Specific inhibition of BimEL and BimS by siRNA demonstrated that, although both isoforms were involved in induction of apoptosis by PLX4720, BimS plays a greater part than BimEL. Taken together, our results indicate that apoptosis of B-RAF mutant melanoma cells induced by PLX4720 involves transcriptional up-regulation of Bim, and