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COT drives resistance to RAF inhibition through MAP kinase pathway reactivation


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.

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Figure 1: An ORF-based functional screen identifies COT and C-RAF kinases as drivers of resistance to B-RAF inhibition
Figure 2: Resistance to B-RAF inhibition via MAPK pathway activation
Figure 3: COT expression predicts resistance to B-RAF inhibition in cancer cell lines
Figure 4: COT-expressing B-RAF(V600E) cell lines exhibit resistance to allosteric MEK inhibitors

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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).

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Authors and Affiliations



C.M.J., J.S.B. and L.A.G. designed the experiments, with help from C.M.E. C.M.J., S.Y.K. and L.W. performed the primary screen, supervised by W.C.H. L.A.J. helped perform drug sensitivity profiling. J.S.B. designed and created the CCSB/Broad Institute Kinase ORF Collection in collaboration with S.R.T., H.H., R.R.M., K.S.-A., J.J.Z., M.V., T.M.R., T.G., D.E.H. and W.C.H. Additional help with ORF experiments was provided by X.Y., D.E.R. and O.A. Clinical samples were collected or experiments performed by C.M.J., A.P.C., K.T.F., R.D. and J.A.W. Large scale cell genomic and expression profiling along with pharmacological screening efforts were designed and data analysed by N.S., J.B., G.C., S.K., J.H., C.J.W., V.E.M., P.M.F., B.L.W., W.R.S., R.S. and L.A.G. C.M.J. and L.A.G. wrote the manuscript. All authors discussed results and edited the manuscript.

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Correspondence to Levi A. Garraway.

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Competing interests

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.

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Johannessen, C., Boehm, J., Kim, S. et al. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 468, 968–972 (2010).

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