Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50–70% of malignant melanomas1. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma2,3,4,5,6—an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials7,8,9. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance10,11,12. Identification of resistance mechanisms in a manner that elucidates alternative ‘druggable’ targets may inform effective long-term treatment strategies13. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.
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.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Davies, H. et al. Mutations of the BRAF gene in human cancer. Nature 417, 949–954 (2002)
Hoeflich, K. P. et al. Antitumor efficacy of the novel RAF inhibitor GDC-0879 is predicted by BRAFV600E mutational status and sustained extracellular signal-regulated kinase/mitogen-activated protein kinase pathway suppression. Cancer Res. 69, 3042–3051 (2009)
McDermott, U. et al. Identification of genotype-correlated sensitivity to selective kinase inhibitors by using high-throughput tumor cell line profiling. Proc. Natl Acad. Sci. USA 104, 19936–19941 (2007)
Solit, D. B. et al. BRAF mutation predicts sensitivity to MEK inhibition. Nature 439, 358–362 (2006)
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)
Wellbrock, C. et al. V599EB-RAF is an oncogene in melanocytes. Cancer Res. 64, 2338–2342 (2004)
Flaherty, K. T. et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N. Engl. J. Med. 363, 809–819 (2010)
Infante, J. R. et al. Safety and efficacy results from the first-in-human study of the oral MEK 1/2 inhibitor GSK1120212. J. Clin. Oncol. 28 (suppl.),. 2503 (2010)
Schwartz, G. K. et al. A phase I study of XL281, a selective oral RAF kinase inhibitor, in patients (Pts) with advanced solid tumors. J. Clin. Oncol. 27 (suppl.),. 3513 (2009)
Engelman, J. A. et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316, 1039–1043 (2007)
Gorre, M. E. et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 293, 876–880 (2001)
Heinrich, M. C. et al. Molecular correlates of imatinib resistance in gastrointestinal stromal tumors. J. Clin. Oncol. 24, 4764–4774 (2006)
Daub, H., Specht, K. & Ullrich, A. Strategies to overcome resistance to targeted protein kinase inhibitors. Nature Rev. Drug Discov. 3, 1001–1010 (2004)
Tsai, J. et al. Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc. Natl Acad. Sci. USA 105, 3041–3046 (2008)
Emery, C. M. et al. MEK1 mutations confer resistance to MEK and B-RAF inhibition. Proc. Natl Acad. Sci. USA 106, 20411–20416 (2009)
Hatzivassiliou, G. et al. RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 464, 431–435 (2010)
Heidorn, S. J. et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 140, 209–221 (2010)
Karreth, F. A., DeNicola, G. M., Winter, S. P. & Tuveson, D. A. C-Raf inhibits MAPK activation and transformation by B-RafV600E . Mol. Cell 36, 477–486 (2009)
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)
Edlundh-Rose, E. et al. NRAS and BRAF mutations in melanoma tumours in relation to clinical characteristics: a study based on mutation screening by pyrosequencing. Melanoma Res. 16, 471–478 (2006)
Seth, R. et al. Concomitant mutations and splice variants in KRAS and BRAF demonstrate complex perturbation of the Ras/Raf signalling pathway in advanced colorectal cancer. Gut 58, 1234–1241 (2009)
George, D. et al. Discovery of thieno[2,3-c]pyridines as potent COT inhibitors. Bioorg. Med. Chem. Lett. 18, 4952–4955 (2008)
Hirata, K. et al. Inhibition of tumor progression locus 2 protein kinase suppresses receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis through down-regulation of the c-Fos and nuclear factor of activated T cells c1 genes. Biol. Pharm. Bull. 33, 133–137 (2010)
Lee, K. M., Lee, K. W., Bode, A. M., Lee, H. J. & Dong, Z. Tpl2 is a key mediator of arsenite-induced signal transduction. Cancer Res. 69, 8043–8049 (2009)
Tsatsanis, C., Patriotis, C. & Tsichlis, P. N. Tpl-2 induces IL-2 expression in T-cell lines by triggering multiple signaling pathways that activate NFAT and NF-κB. Oncogene 17, 2609–2618 (1998)
Barbie, D. A. et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature 462, 108–112 (2009)
Beroukhim, R. et al. The landscape of somatic copy-number alteration across human cancers. Nature 463, 899–905 (2010)
Boehm, J. S. et al. Integrative genomic approaches identify IKBKE as a breast cancer oncogene. Cell 129, 1065–1079 (2007)
Lundberg, A. S. et al. Immortalization and transformation of primary human airway epithelial cells by gene transfer. Oncogene 21, 4577–4586 (2002)
We thank members of the Broad Institute/Novartis Cancer Cell Line Encyclopedia (CCLE) for contributing cell line genomic data, expression data and pharmacological cell line sensitivity data; J. Thibault and A. Shipway for CCLE related tissue culture; R. Depinho, G. Dunn, S. Ethier, H. Greulich, A. Henderson, D. Kaplan, R. Levine, C. Miller, H. Piwnica-Worms, H. Suzuki, M. Vigny, D. Vollrath and the Harvard Institute of Proteomics for contributing templates for the kinase collection; J. Du and D. B. Wheeler for assistance with functional testing of kinases; D. A. Barbie for helpful discussions, S. E. Moody and H. W. Cheung for technical assistance, and J. K. Grenier and S. J. Silver for compiling and annotating the list of kinases. This work was supported by the NIH Director’s New Innovator Award (L.A.G.), grants from the Novartis Institutes for Biomedical Research (L.A.G.), Melanoma Research Alliance (L.A.G.), Starr Cancer Consortium (L.A.G.) the US National Cancer Institute (R33 CA128625, RC2 CA148268) (W.C.H.), NIH (CA134502) (J.J.Z) the Swiss National Foundation (310040-103671) (R.D.), the Gottfried and Julia Bangerter Rhyner Stiftung (R.D.) the Ellison Foundation (M.V. and CCSB) and institute sponsored research funds from the DFCI Strategic Initiative (M.V. and CCSB).
C.M.E., G.C., S.K., J.L.H., C.J.W., V.E.M., P.M.F., B.W., W.R.S. and R.S. are employees of Novartis Pharmaceuticals, Inc. J.J.Z., T.M.R., W.C.H. and L.A.G. are consultants for Novartis Pharmaceuticals, Inc. L.A.G. is a consultant and shareholder of Foundation Medicine. All other authors declare no competing financial interests.
About this article
Cite this article
Johannessen, C., Boehm, J., Kim, S. et al. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 468, 968–972 (2010). https://doi.org/10.1038/nature09627
American Journal of Clinical Dermatology (2021)
Journal of Experimental Medicine (2021)
Pflügers Archiv - European Journal of Physiology (2021)