Identification of PLX4032-resistance mechanisms and implications for novel RAF inhibitors.

BRAF inhibitors improve melanoma patient survival, but resistance invariably develops. Here we report the discovery of a novel BRAF mutation that confers resistance to PLX4032 employing whole-exome sequencing of drug-resistant BRAF ^V600K melanoma cells. We further describe a new screening approach, a genome-wide piggy Bac mutagenesis screen that revealed clinically relevant aberrations ( N-terminal BRAF truncations and CRAF overexpression). The novel BRAF mutation, a Leu505 to His substitution ( BRAF ^L505H), is the first resistance-conferring second-site mutation identified in BRAF mutant cells. The mutation replaces a small nonpolar amino acid at the BRAF- PLX4032 interface with a larger polar residue. Moreover, we show that BRAF ^L505H, found in human prostate cancer, is itself a MAPK-activating, PLX4032-resistant oncogenic mutation. Lastly, we demonstrate that the PLX4032-resistant melanoma cells are sensitive to novel, next-generation BRAF inhibitors, especially the 'paradox-blocker' PLX8394, supporting its use in clinical trials for treatment of melanoma patients with BRAF-mutations. [ABSTRACT FROM AUTHOR]