RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF (original) (raw)

Nature volume 464, pages 427–430 (2010)Cite this article

Subjects

Abstract

Tumours with mutant BRAF are dependent on the RAF–MEK–ERK signalling pathway for their growth1,2,3. We found that ATP-competitive RAF inhibitors inhibit ERK signalling in cells with mutant BRAF, but unexpectedly enhance signalling in cells with wild-type BRAF. Here we demonstrate the mechanistic basis for these findings. We used chemical genetic methods to show that drug-mediated transactivation of RAF dimers is responsible for paradoxical activation of the enzyme by inhibitors. Induction of ERK signalling requires direct binding of the drug to the ATP-binding site of one kinase of the dimer and is dependent on RAS activity. Drug binding to one member of RAF homodimers (CRAF–CRAF) or heterodimers (CRAF–BRAF) inhibits one protomer, but results in transactivation of the drug-free protomer. In BRAF(V600E) tumours, RAS is not activated, thus transactivation is minimal and ERK signalling is inhibited in cells exposed to RAF inhibitors. These results indicate that RAF inhibitors will be effective in tumours in which BRAF is mutated. Furthermore, because RAF inhibitors do not inhibit ERK signalling in other cells, the model predicts that they would have a higher therapeutic index and greater antitumour activity than mitogen-activated protein kinase (MEK) inhibitors, but could also cause toxicity due to MEK/ERK activation. These predictions have been borne out in a recent clinical trial of the RAF inhibitor PLX4032 (refs 4, 5). The model indicates that promotion of RAF dimerization by elevation of wild-type RAF expression or RAS activity could lead to drug resistance in mutant BRAF tumours. In agreement with this prediction, RAF inhibitors do not inhibit ERK signalling in cells that coexpress BRAF(V600E) and mutant RAS.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 51 print issues and online access

$199.00 per year

only $3.90 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

Similar content being viewed by others

References

  1. Solit, D. B. et al. BRAF mutation predicts sensitivity to MEK inhibition. Nature 439, 358–362 (2006)
    Article ADS CAS Google Scholar
  2. 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)
    Article ADS CAS Google Scholar
  3. Wellbrock, C. et al. V599EB-RAF is an oncogene in melanocytes. Cancer Res. 64, 2338–2342 (2004)
    Article CAS Google Scholar
  4. Chapman, P. et al. Early efficacy signal demonstrated in advanced melanoma in a phase I trial of the oncogenic BRAF-selective inhibitor PLX4032. Eur. J. Cancer 7 (suppl.). 5 (2009)
    Article Google Scholar
  5. Flaherty, K. et al. Phase I study of PLX4032: Proof of concept for V600E BRAF mutation as a therapeutic target in human cancer. J. Clin. Oncol. 27 (suppl.), abstr. 9000 (2009)
  6. 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)
    Article ADS CAS Google Scholar
  7. Wellbrock, C., Karasarides, M. & Marais, R. The RAF proteins take centre stage. Nature Rev. Mol. Cell Biol. 5, 875–885 (2004)
    Article CAS Google Scholar
  8. Young, A. et al. Ras signaling and therapies. Adv. Cancer Res. 102, 1–17 (2009)
    Article CAS Google Scholar
  9. Konecny, G. E. et al. Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells. Cancer Res. 66, 1630–1639 (2006)
    Article CAS Google Scholar
  10. Weber, C. K., Slupsky, J. R., Kalmes, H. A. & Rapp, U. R. Active Ras induces heterodimerization of cRaf and BRaf. Cancer Res. 61, 3595–3598 (2001)
    CAS PubMed Google Scholar
  11. 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)
    Article CAS Google Scholar
  12. Rushworth, L. K., Hindley, A. D., O’Neill, E. & Kolch, W. Regulation and role of Raf-1/B-Raf heterodimerization. Mol. Cell. Biol. 26, 2262–2272 (2006)
    Article CAS Google Scholar
  13. Cutler, R. E., Stephens, R. M., Saracino, M. R. & Morrison, D. K. Autoregulation of the Raf-1 serine/threonine kinase. Proc. Natl Acad. Sci. USA 95, 9214–9219 (1998)
    Article ADS CAS Google Scholar
  14. Okuzumi, T. et al. Inhibitor hijacking of Akt activation. Nature Chem. Biol. 5, 484–493 (2009)
    Article CAS Google Scholar
  15. Cameron, A. J., Escribano, C., Saurin, A. T., Kostelecky, B. & Parker, P. J. PKC maturation is promoted by nucleotide pocket occupation independently of intrinsic kinase activity. Nature Struct. Mol. Biol. 16, 624–630 (2009)
    Article CAS Google Scholar
  16. Karreth, F. A., DeNicola, G. M., Winter, S. P. & Tuveson, D. A. C-Raf inhibits MAPK activation and transformation by B-Raf(V600E). Mol. Cell 36, 477–486 (2009)
    Article CAS Google Scholar
  17. Blair, J. A. et al. Structure-guided development of affinity probes for tyrosine kinases using chemical genetics. Nature Chem. Biol. 3, 229–238 (2007)
    Article CAS Google Scholar
  18. Sun, Y. et al. Growth inhibition of nasopharyngeal carcinoma cells by EGF receptor tyrosine kinase inhibitors. Anticancer Res. 19, 919–924 (1999)
    CAS PubMed Google Scholar
  19. Rajakulendran, T., Sahmi, M., Lefrancois, M., Sicheri, F. & Therrien, M. A dimerization-dependent mechanism drives RAF catalytic activation. Nature 461, 542–545 (2009)
    Article ADS CAS Google Scholar
  20. Hall-Jackson, C. A. et al. Paradoxical activation of Raf by a novel Raf inhibitor. Chem. Biol. 6, 559–568 (1999)
    Article CAS Google Scholar
  21. King, A. J. et al. Demonstration of a genetic therapeutic index for tumors expressing oncogenic BRAF by the kinase inhibitor SB-590885. Cancer Res. 66, 11100–11105 (2006)
    Article CAS Google Scholar
  22. 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)
    Article CAS Google Scholar
  23. Heidorn, S. J. et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 140, 209–221 (2010)
    Article CAS Google Scholar
  24. Dummer, R. et al. AZD6244 (ARRY-142886) vs temozolomide (TMZ) in patients (pts) with advanced melanoma: An open-label, randomized, multicenter, phase II study. J. Clin. Oncol. 26 (suppl.), abstr. 9033 (2008)
    Article Google Scholar
  25. Montagut, C. et al. Elevated CRAF as a potential mechanism of acquired resistance to BRAF inhibition in melanoma. Cancer Res. 68, 4853–4861 (2008)
    Article CAS Google Scholar
  26. Bankston, D. et al. A scaleable synthesis of BAY 43-9006: A potent Raf kinase inhibitor for the treatment of cancer. Org. Process Res. Dev. 6, 777–781 (2002)
    Article CAS Google Scholar

Download references

Acknowledgements

We are grateful to W. Kolch for the BRAF plasmids and M. Baccarini for the RAF knockout MEFs. We thank J. Blair for synthesis of JAB compounds and A. Dar, S. Chandarlapaty and D. Solit for discussions. This work has been funded by the Melanoma Research Alliance, the Starr Cancer Consortium, an NIH/NCI P01 grant (1P01CA129243-02) and by Joan’s Legacy: United Against Lung Cancer Foundation (P.I.P., N.R.). K.M.S. would like to thank NIH-2R01EB001987, The Children’s Tumor Foundation and the Waxman Foundation for funding.

Author Contributions P.I.P. and C.Z. designed research, performed experiments, analysed data and co-wrote the paper. G.B. provided reagents, analysed data and co-wrote the paper. K.M.S. and N.R. designed research, analysed experiments and co-wrote the paper.

Author information

Authors and Affiliations

  1. Program in Molecular Pharmacology and Chemistry and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA,
    Poulikos I. Poulikakos & Neal Rosen
  2. Howard Hughes Medical Institute & Department of Cellular and Molecular Pharmacology, University of California San Francisco, California 94158, USA,
    Chao Zhang & Kevan M. Shokat
  3. Plexxikon Inc., 91 Bolivar Drive, Berkeley, California 94710, USA,
    Gideon Bollag

Authors

  1. Poulikos I. Poulikakos
  2. Chao Zhang
  3. Gideon Bollag
  4. Kevan M. Shokat
  5. Neal Rosen

Corresponding author

Correspondence toNeal Rosen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

PowerPoint slides

Rights and permissions

About this article

Cite this article

Poulikakos, P., Zhang, C., Bollag, G. et al. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF.Nature 464, 427–430 (2010). https://doi.org/10.1038/nature08902

Download citation

This article is cited by

Editorial Summary

Mixed signals from RAF

Abnormal activation of the RAS-RAF-MEK-ERK signalling pathway is a feature of many human cancers, making it an attractive target for antitumour therapy. Several RAF and MEK inhibitors are in clinical trials, but an unexpected complication has emerged. Although selective BRAF inhibitors are effective in treating mutant BRAF melanoma, in which they potently suppress RAF-MEK-ERK signalling, the same inhibitors are ineffective against tumours that carry an oncogenic mutation in the KRAS gene. Two groups now report that the reason for this dramatic difference is that RAF 'inhibitors' have dual activity, functioning as either inhibitors or activators of RAF, depending on the cellular context and mutational status of RAF. In News & Views, Karen Cichowski and Pasi Jänne discuss the mechanistic and clinical implications of these findings and similar work reported in Cell.

Associated content