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

Oncogene volume 39, pages 814–832 (2020)Cite this article

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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.

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Acknowledgements

This work was supported by the German Research Foundation (DFG) by BR3662/4–1, SFB 850 B04, and EXC 294 BIOSS to TB, and by grant 021 from the DKFZ-Heidelberg Center for Personalized Oncology to HG and SF, TB is the recipient of a Heisenberg Professorship from the DFG. CS is supported in part by the DFG-funded Spemann Graduate School of Biology and Medicine (SGBM, GSC 4). We thank the DKFZ-HIPO Sample Processing Laboratory, the DKFZ Genomics and Proteomics Core Facility, the Omics IT and Data Management Core Facility for technical support. We also thank D. Richter, K. Beck, K. Willmund, R. Eils, and P. Lichter for infrastructure and program development within DKFZ-HIPO.

Author information

Author notes

  1. These authors share first authorship: Florian Weinberg, Ricarda Griffin

  2. These authors contributed equally: Hanno Glimm, Stefan Fröhling, Tilman Brummer

Authors and Affiliations

  1. Institute of Molecular Medicine and Cell Research, Faculty of Medicine, University of Freiburg, Freiburg, Germany

    Florian Weinberg, Ricarda Griffin, Sandra Braun, Corinna Spohr, Mary Iconomou, Viola Hollek, Michael Röring & Tilman Brummer

  2. Centre for Biological Signalling Studies BIOSS, University of Freiburg, Freiburg, Germany

    Florian Weinberg, Sandra Braun & Tilman Brummer

  3. Division of Applied Bioinformatics, German Cancer Research Center (DKFZ) and National Center for Tumor Diseases (NCT) Heidelberg, Heidelberg, Germany

    Martina Fröhlich, Barbara Hutter, Sebastian Uhrig & Benedikt Brors

  4. Department of Translational Medical Oncology, NCT Dresden, Dresden, and DKFZ, Heidelberg, Germany

    Christoph Heining & Hanno Glimm

  5. University Hospital Carl Gustav Carus, Technical University Dresden, Dresden, Germany

    Christoph Heining & Hanno Glimm

  6. German Cancer Consortium (DKTK), Dresden, Germany

    Christoph Heining & Hanno Glimm

  7. Faculty of Biology, University of Freiburg, Freiburg, Germany

    Corinna Spohr

  8. Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany

    Corinna Spohr

  9. Department of Translational Medical Oncology, NCT Heidelberg and DKFZ, Heidelberg, Germany

    Peter Horak, Simon Kreutzfeldt & Stefan Fröhling

  10. DKTK, Heidelberg, Germany

    Peter Horak, Simon Kreutzfeldt, Olaf Neumann, David Reuss, Albrecht Stenzinger, Benedikt Brors & Stefan Fröhling

  11. Omics IT and Data Management Core Facility, DKFZ, Heidelberg, Germany

    Gregor Warsow

  12. Division of Theoretical Bioinformatics, DKFZ, Heidelberg, Germany

    Gregor Warsow

  13. Faculty of Biosciences, Heidelberg University, Heidelberg, Germany

    Sebastian Uhrig

  14. Institute of Pathology, Heidelberg University Hospital, Heidelberg, Germany

    Olaf Neumann & Albrecht Stenzinger

  15. Department of Neuropathology, Institute of Pathology, Heidelberg University Hospital, Heidelberg, Germany

    David Reuss

  16. Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany

    David Reuss

  17. Department of Neurosurgery, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

    Dieter Henrik Heiland

  18. Translational NeuroOncology Research Group, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

    Dieter Henrik Heiland

  19. Department of Translational Oncology, NCT Heidelberg and DKFZ, Heidelberg, Germany

    Christof von Kalle

  20. Institute of Pathology, Technical University Munich, Munich, Germany

    Wilko Weichert

  21. DKTK, Munich, Germany

    Wilko Weichert

  22. Comprehensive Cancer Centre Freiburg, University of Freiburg, Freiburg, Germany

    Tilman Brummer

  23. DKTK Partner Site Freiburg and DKFZ, Heidelberg, Germany

    Tilman Brummer

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  1. Florian Weinberg
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Contributions

FW, RH, SB, CS, MI, VH, and TB performed all wet lab experiments. MF, MR, GW, BH, SU, BB, SF, and TB analyzed the complex rearrangements of the TTYH3 and BRAF loci. CH, PH, SK, ON, DR, CvK, WW, AS, HG, and SF collected and analyzed clinical data. DHH provided GBM cell lines and important scientific input. FW, SF, and TB conceived the project and wrote the paper with MF.

Corresponding authors

Correspondence to Stefan Fröhling or Tilman Brummer.

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Weinberg, F., Griffin, R., Fröhlich, M. et al. Identification and characterization of a BRAF fusion oncoprotein with retained autoinhibitory domains. Oncogene 39, 814–832 (2020). https://doi.org/10.1038/s41388-019-1021-1

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  • DOI: https://doi.org/10.1038/s41388-019-1021-1

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