Apoptosis of human melanoma cells induced by inhibition of B-RAFᵛ⁶⁰⁰ᴱ involves preferential splicing of bimS

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, BimS, remains largely unappreciated. Here, we show that inhibition of the mutant B-RAFᵛ⁶⁰⁰ᴱ triggers preferential splicing to produce BimS, which is particularly important in induction of apoptosis in B-RAFᵛ⁶⁰⁰ᴱ melanoma cells. Although the specific B-RAFᵛ⁶⁰⁰ᴱ inhibitor PLX4720 upregulates all three major isoforms of Bim, BimEL, BimL, and BimS, at the protein and mRNA levels in B-RAFᵛ⁶⁰⁰ᴱ melanoma cells, the increase in the ratios of BimS mRNA to BimEL and BimL mRNA indicates that it favours BimS splicing. Consistently, enforced expression of B-RAFᵛ⁶⁰⁰ᴱ in wild-type B-RAF melanoma cells and melanocytes inhibits BimS expression. The splicing factor SRp55 appears necessary for the increase in BimS splicing, as SRp55 is upregulated, and its inhibition by small interfering RNA blocks induction of BimS and apoptosis induced by PLX4720. The PLX4720-induced, SRp55-mediated increase in BimS splicing is also mirrored in freshly isolated B-RAFᵛ⁶⁰⁰ᴱ melanoma cells. These results identify a key mechanism for induction of apoptosis by PLX4720, and are instructive for sensitizing melanoma cells to B-RAFᵛ⁶⁰⁰ᴱ inhibitors