Apoptosis of human melanoma cells induced by inhibition of B-RAF ^V600E involves preferential splicing of bim _S

Abstract

Bim is known to be critical in killing of melanoma cells by inhibition of the RAF/MEK/ERK pathway. However, the potential role of the most potent apoptosis-inducing isoform of Bim, Bim _S, remains largely unappreciated. Here, we show that inhibition of the mutant B-RAF ^V600E triggers preferential splicing to produce Bim _S, which is particularly important in induction of apoptosis in B-RAF ^V600E melanoma cells. Although the specific B-RAF ^V600E inhibitor PLX4720 upregulates all three major isoforms of Bim, Bim _EL, Bim _L, and Bim _S, at the protein and mRNA levels in B-RAF ^V600E melanoma cells, the increase in the ratios of Bim _S mRNA to Bim _EL and Bim _L mRNA indicates that it favours Bim _S splicing. Consistently, enforced expression of B-RAF ^V600E in wild-type B-RAF melanoma cells and melanocytes inhibits Bim _S expression. The splicing factor SRp55 appears necessary for the increase in Bim _S splicing, as SRp55 is upregulated, and its inhibition by small interfering RNA blocks induction of Bim _S and apoptosis induced by PLX4720. The PLX4720-induced, SRp55-mediated increase in Bim _S splicing is also mirrored in freshly isolated B-RAF ^V600E melanoma cells. These results identify a key mechanism for induction of apoptosis by PLX4720, and are instructive for sensitizing melanoma cells to B-RAF ^V600E inhibitors.