Abstract LB-B08: Identification and characterization of an unusual BRAF fusion oncoprotein with retained autoinhibitory domains
Authors: Weinberg Florian , Herr Ricarda , Fröhlich Martina , Heining Christoph , Braun Sandra , Spohr Corinna , Iconomou Mary , Hollek Viola , Röring Michael , Horak Peter , Kreutzfeldt Simon , Warsow Gregor , Hutter Barbara , Uhrig Sebastian , Neumann Olaf , Reuss David , Heiland Dieter Hendrik , Kalle Christof von , Weichert Wilko , Stenzinger Albrecht , Brors Benedikt , Glimm Hanno , Fröhling Stefan , Brummer Tilman 
Published: March 2020

Fusion proteins involving the BRAF serine/threonine kinase occur in many cancers. Oncogenic BRAF fusion proteins usually consist of the BRAF kinase domain and an N-terminal fusion partner that replaces the critical domains required for BRAF autoinhibition. We applied whole-exome and RNA sequencing in a patient with glioblastoma multiforme (GBM) 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 structural prerequisites for BRAF autoinhibition. Indeed, the BRAF moiety of the fusion protein alone, which represented near-full-length BRAF without the amino acids encoded by exon 1 (BRAFΔE1), did not induce MEK/ERK phosphorylation or cellular transformation. Similarly, 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 BRAFΔE1 to the (endo)membrane system. Consistent with this mechanism, a synthetic approach in which BRAFΔE1 was localized 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 relies on an intact BRAF dimer interface and forms homo- and heterodimers with RAF1 or ARAF. Moreover, the MEK phosphorylation and transformation potential of TTYH3-BRAF requires the activation loop phosphorylation sites T599 and S602. Various cell line models, including primary human astrocytes and a GBM stem cell line, expressing TTYH3-BRAF exhibited increased MEK/ERK signaling. TTYH3-BRAF-induced MEK/ERK phosphorylation 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.

Citation Format: Florian Weinberg, Ricarda Herr, Martina Fröhlich, Christoph Heining, Sandra Braun, Corinna Spohr, Mary Iconomou, Viola Hollek, Michael Röring, Peter Horak, Simon Kreutzfeldt, Gregor Warsow, Barbara Hutter, Sebastian Uhrig, Olaf Neumann, David Reuss, Dieter Hendrik Heiland, Christof von Kalle, Wilko Weichert, Albrecht Stenzinger, Benedikt Brors, Hanno Glimm, Stefan Fröhling, Tilman Brummer. Identification and characterization of an unusual BRAF fusion oncoprotein with retained autoinhibitory domains [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B08. doi:10.1158/1535-7163.TARG-19-LB-B08