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 piggyBac 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.