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

Pigment Cell Melanoma Res. 2014 Mar;27(2):253-62. doi: 10.1111/pcmr.12197. Epub 2014 Jan 6.


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.

Keywords: BRAF; PLX4032; drug resistance; melanoma; paradox blockers.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • DNA Transposable Elements / genetics
  • Drug Resistance, Neoplasm / drug effects*
  • Humans
  • Indoles / pharmacology*
  • MAP Kinase Signaling System / drug effects
  • Melanoma / enzymology
  • Melanoma / pathology
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Insertional / genetics
  • Mutant Proteins / metabolism
  • Mutation / genetics
  • Protein Kinase Inhibitors / pharmacology*
  • Proto-Oncogene Proteins B-raf / antagonists & inhibitors*
  • Proto-Oncogene Proteins B-raf / metabolism
  • Sulfonamides / pharmacology*
  • Vemurafenib


  • DNA Transposable Elements
  • Indoles
  • Mutant Proteins
  • Protein Kinase Inhibitors
  • Sulfonamides
  • Vemurafenib
  • Proto-Oncogene Proteins B-raf