Approximately 200 BRAF mutant alleles have been identified in human tumours. Activating BRAF mutants cause feedback inhibition of GTP-bound RAS, are RAS-independent and signal either as active monomers (class 1) or constitutively active dimers (class 2)1. Here we characterize a third class of BRAF mutants—those that have impaired kinase activity or are kinase-dead. These mutants are sensitive to ERK-mediated feedback and their activation of signalling is RAS-dependent. The mutants bind more tightly than wild-type BRAF to RAS–GTP, and their binding to and activation of wild-type CRAF is enhanced, leading to increased ERK signalling. The model suggests that dysregulation of signalling by these mutants in tumours requires coexistent mechanisms for maintaining RAS activation despite ERK-dependent feedback. Consistent with this hypothesis, melanomas with these class 3 BRAF mutations also harbour RAS mutations or NF1 deletions. By contrast, in lung and colorectal cancers with class 3 BRAF mutants, RAS is typically activated by receptor tyrosine kinase signalling. These tumours are sensitive to the inhibition of RAS activation by inhibitors of receptor tyrosine kinases. We have thus defined three distinct functional classes of BRAF mutants in human tumours. The mutants activate ERK signalling by different mechanisms that dictate their sensitivity to therapeutic inhibitors of the pathway.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Heparanase modulates the prognosis and development of BRAF V600E-mutant colorectal cancer by regulating AKT/p27Kip1/Cyclin E2 pathway
Oncogenesis Open Access 21 September 2022
Molecular Cancer Open Access 04 August 2022
Cell Death & Disease Open Access 18 May 2022
Subscribe to Nature+
Get immediate online access to Nature and 55 other Nature journal
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Yao, Z. et al. BRAF mutants evade ERK-dependent feedback by different mechanisms that determine their sensitivity to pharmacologic inhibition. Cancer Cell 28, 370–383 (2015)
Wan, P. T. et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116, 855–867 (2004)
Drosten, M. et al. Genetic analysis of Ras signalling pathways in cell proliferation, migration and survival. EMBO J. 29, 1091–1104 (2010)
Heidorn, S. J. et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 140, 209–221 (2010)
Cancer Genome Atlas Network. Genomic classification of cutaneous melanoma. Cell 161, 1681–1696 (2015)
Chang, M. T. et al. Identifying recurrent mutations in cancer reveals widespread lineage diversity and mutational specificity. Nat. Biotechnol. 34, 155–163 (2016)
Hall, R. D. & Kudchadkar, R. R. BRAF mutations: signaling, epidemiology, and clinical experience in multiple malignancies. Cancer Contr. 21, 221–230 (2014)
Zheng, G. et al. Clinical detection and categorization of uncommon and concomitant mutations involving BRAF. BMC Cancer 15, 779 (2015)
Nieto, P. et al. A Braf kinase-inactive mutant induces lung adenocarcinoma. Nature http://dx.doi.org/10.1038/nature23297 (2017)
Chen, S. H. et al. Oncogenic BRAF deletions that function as homodimers and are sensitive to inhibition by RAF dimer inhibitor LY3009120. Cancer Discov. 6, 300–315 (2016)
Garnett, M. J., Rana, S., Paterson, H., Barford, D. & Marais, R. Wild-type and mutant B-RAF activate C-RAF through distinct mechanisms involving heterodimerization. Mol. Cell 20, 963–969 (2005)
Poulikakos, P. I., Zhang, C., Bollag, G., Shokat, K. M. & Rosen, N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 464, 427–430 (2010)
Karoulia, Z. et al. An integrated model of RAF inhibitor action predicts inhibitor activity against oncogenic BRAF signaling. Cancer Cell 30, 485–498 (2016)
Poulikakos, P. I. et al. RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E). Nature 480, 387–390 (2011)
Yang, H. et al. RG7204 (PLX4032), a selective BRAFV600E inhibitor, displays potent antitumor activity in preclinical melanoma models. Cancer Res. 70, 5518–5527 (2010)
Cheng, D. T. et al. Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): a hybridization capture-based next-generation sequencing clinical assay for solid tumor molecular oncology. JMD 17, 251–264 (2015)
Hembrough, T. et al. Application of selected reaction monitoring for multiplex quantification of clinically validated biomarkers in formalin-fixed, paraffin-embedded tumor tissue. JMD 15, 454–465 (2013)
Catenacci, D. V. et al. Absolute quantitation of Met using mass spectrometry for clinical application: assay precision, stability, and correlation with MET gene amplification in FFPE tumor tissue. PLoS One 9, e100586 (2014)
We are grateful to P. Lito, Y. Gao, W. Su, L. Desrochers, D. Santamaria and O. Abdel-Wahab for useful discussions. We thank S. Lowe for the vectors of the retrovirus-based inducible expression system and M. Baccarini for the Raf1-knockout MEFs. We thank Novartis for supplying INC280. We also thank I. Maruani Ryan for her help on this work. This research was supported by grants to N.R. from the National Institutes of Health (NIH) (P01 CA129243; R35 CA210085); the Melanoma Research Alliance (237059 and 348724); The Commonwealth Foundation for Cancer Research and The Center for Experimental Therapeutics at Memorial Sloan Kettering Cancer Center; and the Stand Up To Cancer – American Cancer Society Lung Cancer Dream Team Translational Research Grant (SU2C-AACR-DT17-15). Support was also received from the NIH MSKCC Cancer Center Support Grant P30 CA008748. Additional funding was provided by a Career Development Award from the Conquer Cancer Foundation of the American Society of Clinical Oncology (R.Y.); and from the NIH (R01 CA204749 and R01 CA180037), the Sontag and Josie Robertson Foundations and the Cycle For Survival (B.S.T.). Additional NIH funding was received by E.d.S. (U54 ODSS020355). We would like to acknowledge the support of the Arlene and Joseph Taub Foundation and of Paula and Thomas McInerney, without which this work would not have been possible. M.T.C. was supported in part by the NIH training grant T32 GM007175. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by grants from the European Research Council (ERC-AG/250297-RAS AHEAD), EU-Framework Programme (HEALTH-F2-2010-259770/LUNGTARGET and HEALTH-2010-260791/EUROCANPLATFORM) and Spanish Ministry of Economy and Competitiveness (SAF2011-30173 and SAF2014-59864-R) to M.B. M.B. is the recipient of an Endowed Chair from the AXA Research Fund.
N.R. is on the scientific advisory board of and receives research funding from Chugai, and is on the scientific advisory board of and owns stock in Beigene, Wellspring and Kura. N.R. is also on the scientific advisory board of Daiichi-Sankyo, Astra-Zeneca and Takeda, and is a consultant to Novartis. V.S.R.-O. is now an employee of Novartis Pharmaceutical Corporation as Precision Medicine Associate Director. F.C. is an employee at NantOmics, LLC. T.H. is the President of Proteomics at NantOmics and owns the stock.
Reviewer Information Nature thanks L. Garraway and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Figure 1 Activation of MEK/ERK signalling by hypoactive BRAF mutants is RAS-dependent.
a, V5-tagged wild-type (WT) or mutant BRAF kinases were expressed in 293H cells that stably express NRAS(Q61K). These BRAF protein kinases were isolated with anti-V5 agarose. The in vitro kinase assay was performed with kinase-dead MEK1(K97R). The phosphorylation of MEK1 was determined by western blot. For gel source data, see Supplementary Fig. 4. b, Western blot analysis for components of the RAS/RAF/ERK signalling pathway in a panel of cancer cell lines harbouring the indicated mutations. Cellular RAS–GTP levels were determined by the active RAS pull-down assay. For gel source data, see Supplementary Fig. 4. c, In vitro kinase activity of the indicated BRAF proteins which were isolated from 293H cells that stably express NRAS(Q61K) was assessed as described in a. For gel source data, see Supplementary Fig. 4. d, MEK/ERK activation mediated by indicated BRAF proteins was assayed as described in Fig. 1d. For gel source data, see Supplementary Fig. 4. e, NIH3T3 cells expressing the indicated BRAF proteins were stimulated with 100 ng ml−1 EGF for 15 min, serum deprived for 6 h, or left untreated. RAS/RAF/MEK/ERK signalling activation of these cells was examined by western blot. Cellular RAS–GTP levels were determined by the active RAS pull-down assay. For gel source data, see Supplementary Fig. 5. f, Cells as indicated in Fig. 1a were cultured in doxycycline (30 ng ml−1) containing medium for 24 h and then treated with 500 nM ERK inhibitor SCH772984 for 8 h. Whole-cell lysates were then prepared and examined by western blot. RAS–GTP levels were determined using the active RAS pull-down assay. For gel source data, see Supplementary Fig. 5. g, The frequency distribution of the three classes of BRAF mutants in human BRAF-mutant melanoma tumours, or colorectal or non-small cell lung carcinomas. The data were collected from http://cbioportal.org. h, The frequency of coexistent RAS or NF1 alterations in human BRAF mutant melanomas compared to that in NSCLC and colorectal cancers.. The calculation was based on the sample sets as shown in Fig. 1e. The P values were calculated by using a paired t-test. N.S., not significant. i, The phosphorylation of multiple RTKs in the indicated cell lines was assayed using the Human Phospho-RTK Array Kit. Phosphorylated RTKs are highlighted with boxes in different colours. j, Cells were treated with increasing concentrations of cetuximab for 4 h. Levels of ERK signalling intermediates were determined by western blot. Cellular RAS–GTP levels were determined by the active RAS pull-down assay. For gel source data, see Supplementary Fig. 6. k, Cells were cultured and exposed to cetuximab at concentrations of 0, 0.3, 1, 3, 10, 30, 100 and 300 nM for 3 days. The effects of drug on cell growth were quantified using the ATP-Glo assay. Graphs were generated using Prism 6 (mean ± s.d. are represented by the dots and error bars, n = 8). l, GFP, wild-type NRAS or NRAS(Q61K) were stably expressed in H1666 cells. Then the indicated cells were treated with cetuximab at increasing doses for 2 h. Cells were collected and cell lysates were examined by western blot. RAS–GTP levels were determined using the active RAS pull-down assay. For gel source data, see Supplementary Fig. 7. m, The effects of cetuximab or trametinib on the growth of the cells described in l was determined by ATP-Glo assay after 3 days treatment. Graphs were generated using Prism 6 (mean ± s.d., n = 8). n, NRAS(Q61K) or vector was stably expressed in H508 cells. The cells were treated with cetuximab at increasing doses for 2 h. Cells were collected and cell lysates were examined by western blot. For gel source data, see Supplementary Fig. 7. o, Growth inhibition of the cells described in n after three days exposure to varying doses of cetuximab on day 3 was determined by ATP-Glo assay. Graphs were generated using Prism 6 (mean ± s.d., n = 8)
Extended Data Figure 2 Enhanced binding of hypoactive BRAF mutants to RAS increases activated mutant BRAF–wild-type CRAF heterodimers and amplifies ERK signalling.
a, The levels of p-MEK1/2 and p-ERK1/2 in each transfectant in Fig. 2a were quantified by densitometry analysis using ImageJ software. The columns represent the relative levels of p-MEK/12 and p-ERK1/2 as normalized to the levels in cells transfected with vector plasmid and the expression levels of V5-tagged BRAF proteins (mean ± s.d. calculated on the basis of four independent experiments). b, Flag-tagged wild-type BRAF was co-expressed with the indicated V5-tagged BRAF proteins in 293H cells that were induced to express NRAS(Q61K). CRAF-bound BRAF proteins were determined by immunoprecipitation followed by western blot analysis. The results show RAS-dependent enhanced binding of CRAF to hypoactive BRAF mutants compared to their binding to wild-type BRAF. For gel source data, see Supplementary Fig. 8. c, V5-tagged wild-type or mutant BRAF kinases were expressed in Raf1-knockout cells with or without V5-tagged CRAF expression. For gel source data, see Supplementary Fig. 8. d, 293H cells were transfected with plasmids that encode the indicated BRAF proteins. After 24 h, the cells were collected. Cell lysates were then analysed by western blot with the indicated antibodies. For gel source data, see Supplementary Fig. 8. e, 293H cells stably expressing Flag-tagged NRAS(Q61K) were transfected with pcDNA3 plasmids expressing indicated proteins. The interaction between active RAS and the indicated BRAF proteins was determined by immunoprecipitation with anti-Flag beads. For gel source data, see Supplementary Fig. 9. f, 293H cells stably expressing Flag-tagged NRAS(Q61K) were treated with indicated RAF inhibitors for 1 h. The mutant RAS-bound BRAF and CRAF proteins were pulled down by immunoprecipitation with anti-Flag antibody and examined by western blot with indicated antibodies. For gel source data, see Supplementary Fig. 9. g, V5 tagging identified BRAF or CRAF proteins that were expressed in 293H cells stably expressing Flag-tagged NRAS(Q61K). BRAF(T529M) and CRAF(T421N) gatekeeper mutants that do not bind inhibitors were used as controls. NRAS-bound mutant BRAF and CRAF proteins were pulled down by anti-FLAG antibody from cells treated with or without dabrafenib (1 μM, 1 h). The immunoprecipitated proteins were assayed by western blot. For gel source data, see Supplementary Fig. 9.
a, Inhibition of ERK signalling in a panel of cell lines exposed trametinib for 1 h at indicated doses. For gel source data, see Supplementary Fig. 10. b, The cell lines as indicated in a were treated with different concentrations of trametinib for 3 days. Cell viability was determined by ATP-Glo assay. Dose-dependent inhibition curves were generated using Prism 6 (mean ± s.d., n = 8).
Extended Data Figure 4 In hypoactive BRAF-mutant tumours with wild-type RAS/NF1 ERK signalling is sensitive to upstream inhibition of RAS.
a, Cells isolated from the patient-derived xenograft (PDX) models as described (Fig. 4b) were treated with increasing doses of vemurafenib for 1 h or cetuximab for 4 h. Levels of indicated proteins were examined by western blot. For gel source data, see Supplementary Fig. 10. b, RTK phosphorylation profile of the cells isolated from the ovarian metastatisis derived PDX (BRCC-OVA) was assessed with Human Phospho-RTK arrays. The elevated p-EGFR and p-MET bands are within the red and green rectangles, respectively. c, Cells isolated from BRCC-OVA were treated with indicated drugs for 4 h and cell lysates were then analysed. For gel source data, see Supplementary Fig. 11. d, e, The BRCC-OVA cells were treated with indicated drugs or combinations thereof over a range of drug concentrations for 4 h. INC280 is a selective inhibitor of MET activity. The ERK signalling was assayed by western blot. The cellular RAS–GTP level was determined using the active RAS pull-down assay. For gel source data, see Supplementary Fig. 11. f, The growth inhibition of BRCC-OVA cells with indicated drugs at 0, 1, 3, 10, 30, 100, 300 and 1,000 nM of trametinib or 0, 10, 30, 100, 300, 1,000, 3,000 and 10,000 nM of INC280 or combination of increasing dose of trametinib with 100 nM INC280 was determined by ATP-Glo assay after 3 days of treatment. Graphs were generated using Prism 6 (mean ± s.d., n = 8).
About this article
Cite this article
Yao, Z., Yaeger, R., Rodrik-Outmezguine, V. et al. Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS. Nature 548, 234–238 (2017). https://doi.org/10.1038/nature23291
This article is cited by
Molecular Cancer (2022)
British Journal of Cancer (2022)
Nature Communications (2022)
Nature Communications (2022)
Cell Death & Disease (2022)