COT drives resistance to RAF inhibition through MAP kinase pathway reactivation

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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

References

  1. 1

    Davies, H. et al. Mutations of the BRAF gene in human cancer. Nature 417, 949–954 (2002)

    ADS  CAS  Article  Google Scholar 

  2. 2

    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)

    CAS  Article  Google Scholar 

  3. 3

    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)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Solit, D. B. et al. BRAF mutation predicts sensitivity to MEK inhibition. Nature 439, 358–362 (2006)

    ADS  CAS  Article  Google Scholar 

  5. 5

    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)

    CAS  Article  Google Scholar 

  6. 6

    Wellbrock, C. et al. V599EB-RAF is an oncogene in melanocytes. Cancer Res. 64, 2338–2342 (2004)

    CAS  Article  Google Scholar 

  7. 7

    Flaherty, K. T. et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N. Engl. J. Med. 363, 809–819 (2010)

    CAS  Article  Google Scholar 

  8. 8

    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)

    Article  Google Scholar 

  9. 9

    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)

  10. 10

    Engelman, J. A. et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316, 1039–1043 (2007)

    ADS  CAS  Article  Google Scholar 

  11. 11

    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)

    CAS  Article  Google Scholar 

  12. 12

    Heinrich, M. C. et al. Molecular correlates of imatinib resistance in gastrointestinal stromal tumors. J. Clin. Oncol. 24, 4764–4774 (2006)

    CAS  Article  Google Scholar 

  13. 13

    Daub, H., Specht, K. & Ullrich, A. Strategies to overcome resistance to targeted protein kinase inhibitors. Nature Rev. Drug Discov. 3, 1001–1010 (2004)

    CAS  Article  Google Scholar 

  14. 14

    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)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Emery, C. M. et al. MEK1 mutations confer resistance to MEK and B-RAF inhibition. Proc. Natl Acad. Sci. USA 106, 20411–20416 (2009)

    ADS  CAS  Article  Google Scholar 

  16. 16

    Hatzivassiliou, G. et al. RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 464, 431–435 (2010)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Heidorn, S. J. et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 140, 209–221 (2010)

    CAS  Article  Google Scholar 

  18. 18

    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)

    CAS  Article  Google Scholar 

  19. 19

    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)

    ADS  CAS  Article  Google Scholar 

  20. 20

    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)

    CAS  Article  Google Scholar 

  21. 21

    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)

    CAS  Article  Google Scholar 

  22. 22

    George, D. et al. Discovery of thieno[2,3-c]pyridines as potent COT inhibitors. Bioorg. Med. Chem. Lett. 18, 4952–4955 (2008)

    CAS  Article  Google Scholar 

  23. 23

    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)

    CAS  Article  Google Scholar 

  24. 24

    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)

    CAS  Article  Google Scholar 

  25. 25

    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)

    CAS  Article  Google Scholar 

  26. 26

    Barbie, D. A. et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature 462, 108–112 (2009)

    ADS  CAS  Article  Google Scholar 

  27. 27

    Beroukhim, R. et al. The landscape of somatic copy-number alteration across human cancers. Nature 463, 899–905 (2010)

    ADS  CAS  Article  Google Scholar 

  28. 28

    Boehm, J. S. et al. Integrative genomic approaches identify IKBKE as a breast cancer oncogene. Cell 129, 1065–1079 (2007)

    CAS  Article  Google Scholar 

  29. 29

    Lundberg, A. S. et al. Immortalization and transformation of primary human airway epithelial cells by gene transfer. Oncogene 21, 4577–4586 (2002)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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).

Author information

Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Levi A. Garraway.

Ethics declarations

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.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-19 with legends and Supplementary Tables 1-3. (PDF 17722 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

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

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing