Identification and characterization of a BRAF fusion oncoprotein with retained autoinhibitory domains

Oncogene. 2020 Jan;39(4):814-832. doi: 10.1038/s41388-019-1021-1. Epub 2019 Sep 26.

Abstract

Fusion proteins involving the BRAF serine/threonine kinase occur in many cancers. The oncogenic potential of BRAF fusions has been attributed to the loss of critical N-terminal domains that mediate BRAF autoinhibition. We used whole-exome and RNA sequencing in a patient with glioblastoma multiforme to identify a rearrangement between TTYH3, encoding a membrane-resident, calcium-activated chloride channel, and BRAF intron 1, resulting in a TTYH3-BRAF fusion protein that retained all features essential for BRAF autoinhibition. Accordingly, the BRAF moiety of the fusion protein alone, which represents full-length BRAF without the amino acids encoded by exon 1 (BRAFΔE1), did not induce MEK/ERK phosphorylation or transformation. Likewise, neither the TTYH3 moiety of the fusion protein nor full-length TTYH3 provoked ERK pathway activity or transformation. In contrast, TTYH3-BRAF displayed increased MEK phosphorylation potential and transforming activity, which were caused by TTYH3-mediated tethering of near-full-length BRAF to the (endo)membrane system. Consistent with this mechanism, a synthetic approach, in which BRAFΔE1 was tethered to the membrane by fusing it to the cytoplasmic tail of CD8 also induced transformation. Furthermore, we demonstrate that TTYH3-BRAF signals largely independent of a functional RAS binding domain, but requires an intact BRAF dimer interface and activation loop phosphorylation sites. Cells expressing TTYH3-BRAF exhibited increased MEK/ERK signaling, which was blocked by clinically achievable concentrations of sorafenib, trametinib, and the paradox breaker PLX8394. These data provide the first example of a fully autoinhibited BRAF protein whose oncogenic potential is dictated by a distinct fusion partner and not by a structural change in BRAF itself.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aged
  • Antineoplastic Agents / pharmacology
  • Cell Line, Tumor
  • Chloride Channels / genetics
  • Chloride Channels / metabolism
  • Female
  • Glioblastoma / drug therapy
  • Glioblastoma / genetics*
  • Glioblastoma / metabolism
  • Glioblastoma / pathology*
  • Heterocyclic Compounds, 2-Ring / pharmacology*
  • Humans
  • MAP Kinase Signaling System / drug effects
  • Oncogene Proteins, Fusion*
  • Phosphorylation
  • Protein Domains
  • Proto-Oncogene Proteins B-raf / antagonists & inhibitors
  • Proto-Oncogene Proteins B-raf / genetics*
  • Proto-Oncogene Proteins B-raf / metabolism
  • Pyridones / pharmacology*
  • Pyrimidinones / pharmacology*
  • Signal Transduction
  • Sulfonamides / pharmacology*

Substances

  • Antineoplastic Agents
  • Chloride Channels
  • Heterocyclic Compounds, 2-Ring
  • Oncogene Proteins, Fusion
  • PLX8394
  • Pyridones
  • Pyrimidinones
  • Sulfonamides
  • TTYH3 protein, human
  • trametinib
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf