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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Turski ML, Vidwans SJ, Janku F, Garrido-Laguna I, Munoz J, Schwab R, et al. Genomically driven tumors and actionability across histologies: BRAF-mutant cancers as a paradigm. Mol Cancer Ther. 2016;15:533–47.
Ross JS, Wang K, Chmielecki J, Gay L, Johnson A, Chudnovsky J, et al. The distribution of BRAF gene fusions in solid tumors and response to targeted therapy. Int J Cancer. 2016;138:881–90.
Dankner M, Rose AAN, Rajkumar S, Siegel PM, Watson IR. Classifying BRAF alterations in cancer: new rational therapeutic strategies for actionable mutations. Oncogene. 2018;37:3183–99.
Chen SH, Zhang Y, Van Horn RD, Yin T, Buchanan S, Yadav V, et al. Oncogenic BRAF deletions that function as homodimers and are sensitive to inhibition by RAF dimer inhibitor LY3009120. Cancer Discov. 2016;6:300–15.
Lavoie H, Therrien M. Regulation of RAF protein kinases in ERK signalling. Nat Rev Mol Cell Biol. 2015;16:281–98.
Terrell EM, Morrison DK. Ras-mediated activation of the Raf family kinases. Cold Spring Harb Perspect Med. 2019;9. pii: a033746. https://doi.org/10.1101/cshperspect.a033746.
Eisenhardt AE, Sprenger A, Roring M, Herr R, Weinberg F, Kohler M, et al. Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles. Oncotarget. 2016;7:26628–52.
Röring M, Herr R, Fiala GJ, Heilmann K, Braun S, Eisenhardt AE, et al. Distinct requirement for an intact dimer interface in wild-type, V600E and kinase-dead B-Raf signalling. EMBO J. 2012;31:2629–47.
Freeman AK, Ritt DA, Morrison DK. Effects of Raf dimerization and its inhibition on normal and disease-associated Raf signaling. Mol Cell. 2013;49:751–8.
Hu J, Stites EC, Yu H, Germino EA, Meharena HS, Stork PJ, et al. Allosteric activation of functionally asymmetric RAF kinase dimers. Cell. 2013;154:1036–46.
Köhler M, Röring M, Schorch B, Heilmann K, Stickel N, Fiala GJ, et al. Activation loop phosphorylation regulates B-Raf in vivo and transformation by B-Raf mutants. EMBO J. 2016;35:143–61.
Diedrich B, Rigbolt KT, Roring M, Herr R, Kaeser-Pebernard S, Gretzmeier C, et al. Discrete cytosolic macromolecular BRAF complexes exhibit distinct activities and composition. EMBO J. 2017;36:646–63.
Yuan J, Ng WH, Lam PYP, Wang Y, Xia H, Yap J, et al. The dimer-dependent catalytic activity of RAF family kinases is revealed through characterizing their oncogenic mutants. Oncogene. 2018;37:5719–34.
Sievert AJ, Lang SS, Boucher KL, Madsen PJ, Slaunwhite E, Choudhari N, et al. Paradoxical activation and RAF inhibitor resistance of BRAF protein kinase fusions characterizing pediatric astrocytomas. Proc Natl Acad Sci USA. 2013;110:5957–62.
Karoulia Z, Gavathiotis E, Poulikakos PI. New perspectives for targeting RAF kinase in human cancer. Nat Rev Cancer. 2017;17:676–91.
Yao Z, Torres NM, Tao A, Gao Y, Luo L, Li Q, et al. BRAF mutants evade ERK-dependent feedback by different mechanisms that determine their sensitivity to pharmacologic inhibition. Cancer Cell. 2015;28:370–83.
Palanisamy N, Ateeq B, Kalyana-Sundaram S, Pflueger D, Ramnarayanan K, Shankar S, et al. Rearrangements of the RAF kinase pathway in prostate cancer, gastric cancer and melanoma. Nat Med. 2010;16:793–8.
Jones DT, Kocialkowski S, Liu L, Pearson DM, Ichimura K, Collins VP. Oncogenic RAF1 rearrangement and a novel BRAF mutation as alternatives to KIAA1549:BRAF fusion in activating the MAPK pathway in pilocytic astrocytoma. Oncogene. 2009;28:2119–23.
Cin H, Meyer C, Herr R, Janzarik WG, Lambert S, Jones DT, et al. Oncogenic FAM131B-BRAF fusion resulting from 7q34 deletion comprises an alternative mechanism of MAPK pathway activation in pilocytic astrocytoma. Acta Neuropathol. 2011;121:763–74.
Jain P, Fierst TM, Han HJ, Smith TE, Vakil A, Storm PB, et al. CRAF gene fusions in pediatric low-grade gliomas define a distinct drug response based on dimerization profiles. Oncogene. 2017;36:6348–58.
Jones DT, Kocialkowski S, Liu L, Pearson DM, Backlund LM, Ichimura K, et al. Tandem duplication producing a novel oncogenic BRAF fusion gene defines the majority of pilocytic astrocytomas. Cancer Res. 2008;68:8673–7.
Selt F, Hohloch J, Hielscher T, Sahm F, Capper D, Korshunov A, et al. Establishment and application of a novel patient-derived KIAA1549:BRAF-driven pediatric pilocytic astrocytoma model for preclinical drug testing. Oncotarget. 2017;8:11460–79.
Tien AC, Tsai HH, Molofsky AV, McMahon M, Foo LC, Kaul A, et al. Regulated temporal-spatial astrocyte precursor cell proliferation involves BRAF signalling in mammalian spinal cord. Development. 2012;139:2477–87.
Galabova-Kovacs G, Catalanotti F, Matzen D, Reyes GX, Zezula J, Herbst R, et al. Essential role of B-Raf in oligodendrocyte maturation and myelination during postnatal central nervous system development. J Cell Biol. 2008;180:947–55.
Berghoff AS, Preusser M. BRAF alterations in brain tumours: molecular pathology and therapeutic opportunities. Curr Opin Neurol. 2014;27:689–96.
Behling F, Schittenhelm J. Oncogenic BRAF alterations and their role in brain tumors. Cancers (Basel). 2019;11. pii: E794. https://doi.org/10.3390/cancers11060794.
Horak P, Klink B, Heining C, Groschel S, Hutter B, Frohlich M, et al. Precision oncology based on omics data: the NCT Heidelberg experience. Int J Cancer. 2017;141:877–86.
Frattini V, Trifonov V, Chan JM, Castano A, Lia M, Abate F, et al. The integrated landscape of driver genomic alterations in glioblastoma. Nat Genet. 2013;45:1141–9.
Brennan CW, Verhaak RG, McKenna A, Campos B, Noushmehr H, Salama SR, et al. The somatic genomic landscape of glioblastoma. Cell. 2013;155:462–77.
Suzuki M, Mizuno A. A novel human Cl(-) channel family related to Drosophila flightless locus. J Biol Chem. 2004;279:22461–8.
Han YE, Kwon J, Won J, An H, Jang MW, Woo J, et al. Tweety-homolog (Ttyh) family encodes the pore-forming subunits of the swelling-dependent volume-regulated Anion channel (VRACswell) in the brain. Exp Neurobiol. 2019;28:183–215.
He Y, Hryciw DH, Carroll ML, Myers SA, Whitbread AK, Kumar S, et al. The ubiquitin-protein ligase Nedd4-2 differentially interacts with and regulates members of the Tweety family of chloride ion channels. J Biol Chem. 2008;283:24000–10.
He Y, Ramsay AJ, Hunt ML, Whitbread AK, Myers SA, Hooper JD. N-glycosylation analysis of the human Tweety family of putative chloride ion channels supports a penta-spanning membrane arrangement: impact of N-glycosylation on cellular processing of Tweety homologue 2 (TTYH2). Biochem J. 2008;412:45–55.
Mercer KE, Pritchard CA. Raf proteins and cancer: B-Raf is identified as a mutational target. Biochim Biophys Acta. 2003;1653:25–40.
Chmielecki J, Hutchinson KE, Frampton GM, Chalmers ZR, Johnson A, Shi C, et al. Comprehensive genomic profiling of pancreatic acinar cell carcinomas identifies recurrent RAF fusions and frequent inactivation of DNA repair genes. Cancer Discov. 2014;4:1398–405.
Hutchinson KE, Lipson D, Stephens PJ, Otto G, Lehmann BD, Lyle PL, et al. BRAF fusions define a distinct molecular subset of melanomas with potential sensitivity to MEK inhibition. Clin Cancer Res. 2013;19:6696–702.
Nicolaides TP, Li H, Solomon DA, Hariono S, Hashizume R, Barkovich K, et al. Targeted therapy for BRAFV600E malignant astrocytoma. Clin Cancer Res. 2011;17:7595–604.
Jung E, Osswald M, Blaes J, Wiestler B, Sahm F, Schmenger T, et al. Tweety-homolog 1 drives brain colonization of gliomas. J Neurosci. 2017;37:6837–50.
Stefaniuk M, Swiech L, Dzwonek J, Lukasiuk K. Expression of Ttyh1, a member of the Tweety family in neurons in vitro and in vivo and its potential role in brain pathology. J Neurochem. 2010;115:1183–94.
International Cancer Genome Consortium PedBrain Tumor P. Recurrent MET fusion genes represent a drug target in pediatric glioblastoma. Nat Med. 2016;22:1314–20.
Kordes M, Röring M, Heining C, Braun S, Hutter B, Richter D, et al. Cooperation of BRAF(F595L) and mutant HRAS in histiocytic sarcoma provides new insights into oncogenic BRAF signaling. Leukemia. 2016;30:937–46.
Poulikakos PI, Persaud Y, Janakiraman M, Kong X, Ng C, Moriceau G, et al. RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E). Nature. 2011;480:387–90.
Heidorn SJ, Milagre C, Whittaker S, Nourry A, Niculescu-Duvas I, Dhomen N, et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell. 2010;140:209–21.
Rushworth LK, Hindley AD, O'Neill E, Kolch W. Regulation and role of Raf-1/B-Raf heterodimerization. Mol Cell Biol. 2006;26:2262–72.
Ritt DA, Monson DM, Specht SI, Morrison DK. Impact of feedback phosphorylation and Raf heterodimerization on normal and mutant B-Raf signaling. Mol Cell Biol. 2010;30:806–19.
Littman DR, Thomas Y, Maddon PJ, Chess L, Axel R. The isolation and sequence of the gene encoding T8: a molecule defining functional classes of T lymphocytes. Cell. 1985;40:237–46.
Hennecke S, Cosson P. Role of transmembrane domains in assembly and intracellular transport of the CD8 molecule. J Biol Chem. 1993;268:26607–12.
Iodice L, Sarnataro S, Bonatti S. The carboxyl-terminal valine is required for transport of glycoprotein CD8 alpha from the endoplasmic reticulum to the intermediate compartment. J Biol Chem. 2001;276:28920–6.
Ramis G, Thomas-Moya E, Fernandez de Mattos S, Rodriguez J, Villalonga P. EGFR inhibition in glioma cells modulates Rho signaling to inhibit cell motility and invasion and cooperates with temozolomide to reduce cell growth. PLoS One. 2012;7:e38770.
Fan QW, Cheng CK, Gustafson WC, Charron E, Zipper P, Wong RA, et al. EGFR phosphorylates tumor-derived EGFRvIII driving STAT3/5 and progression in glioblastoma. Cancer Cell. 2013;24:438–49.
Ricklefs FL, Maire CL, Reimer R, Duhrsen L, Kolbe K, Holz M, et al. Imaging flow cytometry facilitates multiparametric characterization of extracellular vesicles in malignant brain tumours. J Extracell Vesicles. 2019;8:1588555.
Marshall CJ. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 1995;80:179–85.
Masilamani AP, Ferrarese R, Kling E, Thudi NK, Kim H, Scholtens DM, et al. KLF6 depletion promotes NF-kappaB signaling in glioblastoma. Oncogene. 2017;36:3562–75.
Aoki Y, Niihori T, Narumi Y, Kure S, Matsubara Y. The RAS/MAPK syndromes: novel roles of the RAS pathway in human genetic disorders. Hum Mutat. 2008;29:992–1006.
Girotti MR, Lopes F, Preece N, Niculescu-Duvaz D, Zambon A, Davies L, et al. Paradox-breaking RAF inhibitors that also target SRC are effective in drug-resistant BRAF mutant melanoma. Cancer Cell. 2015;27:85–96.
Zhang C, Spevak W, Zhang Y, Burton EA, Ma Y, Habets G, et al. RAF inhibitors that evade paradoxical MAPK pathway activation. Nature. 2015;526:583–6.
Phuchareon J, McCormick F, Eisele DW, Tetsu O. EGFR inhibition evokes innate drug resistance in lung cancer cells by preventing Akt activity and thus inactivating Ets-1 function. Proc Natl Acad Sci USA. 2015;112:E3855–3863.
Herr R, Halbach S, Heizmann M, Busch H, Boerries M, Brummer T. BRAF inhibition upregulates a variety of receptor tyrosine kinases and their downstream effector Gab2 in colorectal cancer cell lines. Oncogene. 2018;37:1576–93.
Pratilas CA, Taylor BS, Ye Q, Viale A, Sander C, Solit DB, et al. (V600E)BRAF is associated with disabled feedback inhibition of RAF-MEK signaling and elevated transcriptional output of the pathway. Proc Natl Acad Sci USA. 2009;106:4519–24.
Strumberg D, Richly H, Hilger RA, Schleucher N, Korfee S, Tewes M, et al. Phase I clinical and pharmacokinetic study of the Novel Raf kinase and vascular endothelial growth factor receptor inhibitor BAY 43-9006 in patients with advanced refractory solid tumors. J Clin Oncol. 2005;23:965–72.
Karajannis MA, Legault G, Fisher MJ, Milla SS, Cohen KJ, Wisoff JH, et al. Phase II study of sorafenib in children with recurrent or progressive low-grade astrocytomas. Neuro Oncol. 2014;16:1408–16.
Isaacson AL, Guseva NV, Bossler AD, Ma D. Urothelial carcinoma with an NRF1-BRAF rearrangement and response to targeted therapy. Cold Spring Harb Mol Case Stud. 2019;5. pii: a003848. https://doi.org/10.1101/mcs.a003848.
Fangusaro J, Onar-Thomas A, Young Poussaint T, Wu S, Ligon AH, Lindeman N, et al. Selumetinib in paediatric patients with BRAF-aberrant or neurofibromatosis type 1-associated recurrent, refractory, or progressive low-grade glioma: a multicentre, phase 2 trial. Lancet Oncol. 2019;20:1011–22.
Kim HS, Jung M, Kang HN, Kim H, Park CW, Kim SM, et al. Oncogenic BRAF fusions in mucosal melanomas activate the MAPK pathway and are sensitive to MEK/PI3K inhibition or MEK/CDK4/6 inhibition. Oncogene. 2017;36:3334–45.
McEvoy CR, Xu H, Smith K, Etemadmoghadam D, San Leong H, Choong DY. et al. Profound MEK inhibitor response in a cutaneous melanoma harboring a GOLGA4-RAF1 fusion. J Clin Investig. 2019;129:1940–5.
Caunt CJ, Sale MJ, Smith PD, Cook SJ. MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road. Nat Rev Cancer. 2015;15:577–92.
Yao Z, Gao Y, Su W, Yaeger R, Tao J, Na N, et al. RAF inhibitor PLX8394 selectively disrupts BRAF dimers and RAS-independent BRAF-mutant-driven signaling. Nat Med. 2019;25:284–91.
Dankort D, Curley DP, Cartlidge RA, Nelson B, Karnezis AN, Damsky WE Jr., et al. Braf(V600E) cooperates with Pten loss to induce metastatic melanoma. Nat Genet. 2009;41:544–52.
Robinson JP, VanBrocklin MW, Guilbeault AR, Signorelli DL, Brandner S, Holmen SL. Activated BRAF induces gliomas in mice when combined with Ink4a/Arf loss or Akt activation. Oncogene. 2010;29:335–44.
Zhang Y, Kwok-Shing NgP, Kucherlapati M, Chen F, Liu Y, Tsang YH, et al. A pan-cancer proteogenomic atlas of PI3K/AKT/mTOR pathway alterations. Cancer Cell. 2017;31:820–32 e823.
Ng PK, Li J, Jeong KJ, Shao S, Chen H, Tsang YH, et al. Systematic functional annotation of somatic mutations in cancer. Cancer Cell. 2018;33:450–62 e410.
Sun M, Hillmann P, Hofmann BT, Hart JR, Vogt PK. Cancer-derived mutations in the regulatory subunit p85alpha of phosphoinositide 3-kinase function through the catalytic subunit p110alpha. Proc Natl Acad Sci USA. 2010;107:15547–52.
Eisenhardt AE, Olbrich H, Roring M, Janzarik W, Anh TN, Cin H, et al. Functional characterization of a BRAF insertion mutant associated with pilocytic astrocytoma. Int J Cancer. 2011;129:2297–303.
Terai K, Matsuda M. The amino-terminal B-Raf-specific region mediates calcium-dependent homo- and hetero-dimerization of Raf. Embo J. 2006;25:3556–64.
Jang JS, Lee A, Li J, Liyanage H, Yang Y, Guo L, et al. Common oncogene mutations and novel SND1-BRAF transcript fusion in lung adenocarcinoma from never smokers. Sci Rep. 2015;5:9755.
Lu H, Villafane N, Dogruluk T, Grzeskowiak CL, Kong K, Tsang YH, et al. Engineering and functional characterization of fusion genes identifies novel oncogenic drivers of cancer. Cancer Res. 2017;77:3502–12.
Papin C, Denouel-Galy A, Laugier D, Calothy G, Eychene A. Modulation of kinase activity and oncogenic properties by alternative splicing reveals a novel regulatory mechanism for B-Raf. J Biol Chem. 1998;273:24939–47.
Leevers SJ, Paterson HF, Marshall CJ. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature. 1994;369:411–4.
Fehrenbacher N, Bar-Sagi D, Philips M. Ras/MAPK signaling from endomembranes. Mol Oncol. 2009;3:297–307.
Selt F, Deiss A, Korshunov A, Capper D, Witt H, van Tilburg CM, et al. Pediatric targeted therapy: clinical feasibility of personalized diagnostics in children with relapsed and progressive tumors. Brain Pathol. 2016;26:506–16.
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.
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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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