Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

RAF inhibitor PLX8394 selectively disrupts BRAF dimers and RAS-independent BRAF-mutant-driven signaling

Nature Medicine volume 25, pages 284–291 (2019)Cite this article

Subjects

Abstract

Activating BRAF mutants and fusions signal as RAS-independent constitutively active dimers with the exception of BRAF V600 mutant alleles which can function as active monomers1. Current RAF inhibitors are monomer selective, they potently inhibit BRAF V600 monomers but their inhibition of RAF dimers is limited by induction of negative cooperativity when bound to one site in the dimer1,2,3. Moreover, acquired resistance to these drugs is usually due to molecular lesions that cause V600 mutants to dimerize4,5,6,7,8. We show here that PLX8394, a new RAF inhibitor9, inhibits ERK signaling by specifically disrupting BRAF-containing dimers, including BRAF homodimers and BRAF–CRAF heterodimers, but not CRAF homodimers or ARAF-containing dimers. Differences in the amino acid residues in the amino (N)-terminal portion of the kinase domain of RAF isoforms are responsible for this differential vulnerability. As a BRAF-specific dimer breaker, PLX8394 selectively inhibits ERK signaling in tumors driven by dimeric BRAF mutants, including BRAF fusions and splice variants as well as BRAF V600 monomers, but spares RAF function in normal cells in which CRAF homodimers can drive signaling. Our work suggests that drugs with these properties will be safe and useful for treating tumors driven by activating BRAF mutants or fusions.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: Effects of RAF inhibitors on p-ERK in cell lines with wild type or mutant RAS and BRAF.
Fig. 2: PLX8394 selectively disrupts BRAF homodimers and BRAF/CRAF heterodimers by binding to only one protomer in the dimers.
Fig. 3: PLX8394 inhibits ERK signaling driven by BRAF monomers or dimers, but activates ERK signaling driven by CRAF homodimers.
Fig. 4: PLX8394 preferentially inhibits ERK signaling and cell growth in tumors driven by RAS-independent BRAF mutants.

Similar content being viewed by others

Data availability

Source data of all the western blotting in this work are provided in Supplementary Figs. 7–15. The data that support the findings of this study are available from the corresponding author upon request.

References

  1. 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).

    Article  CAS  Google Scholar 

  2. Karoulia, Z. et al. An integrated model of RAF inhibitor action predicts inhibitor activity against oncogenic BRAF signaling. Cancer Cell 30, 485–498 (2016).

    Article  CAS  Google Scholar 

  3. Yang, H. et al. RG7204 (PLX4032), a selective BRAFV600E inhibitor, displays potent antitumor activity in preclinical melanoma models. Cancer Res. 70, 5518–5527 (2010).

    Article  CAS  Google Scholar 

  4. Nazarian, R. et al. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature 468, 973–977 (2010).

    Article  CAS  Google Scholar 

  5. Poulikakos, P. I. et al. RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E). Nature 480, 387–390 (2011).

    Article  CAS  Google Scholar 

  6. 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).

    Article  CAS  Google Scholar 

  7. Shi, H. et al. Melanoma whole-exome sequencing identifies (V600E)B-RAF amplification-mediated acquired B-RAF inhibitor resistance. Nat. Commun. 3, 724 (2012).

    Article  Google Scholar 

  8. Yaeger, R. et al. Mechanisms of acquired resistance to BRAF V600E inhibition in colon cancers converge on RAF dimerization and are sensitive to its inhibition. Cancer Res. 77, 6513–6523 (2017).

    Article  CAS  Google Scholar 

  9. Zhang, C. et al. RAF inhibitors that evade paradoxical MAPK pathway activation. Nature 526, 583–586 (2015).

    Article  CAS  Google Scholar 

  10. Basile, K. J., Le, K., Hartsough, E. J. & Aplin, A. E. Inhibition of mutant BRAF splice variant signaling by next-generation, selective RAF inhibitors. Pigment Cell Melanoma Res. 27, 479–484 (2014).

    Article  CAS  Google Scholar 

  11. Jain, P. et al. CRAF gene fusions in pediatric low-grade gliomas define a distinct drug response based on dimerization profiles. Oncogene 36, 6348–6358 (2017).

    Article  CAS  Google Scholar 

  12. Okimoto, R. A. et al. Preclinical efficacy of a RAF inhibitor that evades paradoxical MAPK pathway activation in protein kinase BRAF-mutant lung cancer. Proc. Natl Acad. Sci. USA 113, 13456–13461 (2016).

    Article  CAS  Google Scholar 

  13. Sievert, A. J. et al. Paradoxical activation and RAF inhibitor resistance of BRAF protein kinase fusions characterizing pediatric astrocytomas. Proc. Natl Acad. Sci. USA 110, 5957–5962 (2013).

    Article  CAS  Google Scholar 

  14. Nakamura, A. et al. Antitumor activity of the selective pan-RAF inhibitor TAK-632 in BRAF inhibitor-resistant melanoma. Cancer Res. 73, 7043–7055 (2013).

    Article  CAS  Google Scholar 

  15. Peng, S. B. et al. Inhibition of RAF isoforms and active dimers by LY3009120 leads to anti-tumor activities in RAS or BRAF mutant cancers. Cancer Cell. 28, 384–398 (2015).

    Article  CAS  Google Scholar 

  16. Fabian, J. R., Daar, I. O. & Morrison, D. K. Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase. Mol. Cell. Biol. 13, 7170–7179 (1993).

    Article  CAS  Google Scholar 

  17. Stokoe, D. & McCormick, F. Activation of c-Raf-1 by Ras and Src through different mechanisms: activation in vivo and in vitro. EMBO J. 16, 2384–2396 (1997).

    Article  CAS  Google Scholar 

  18. Takahashi, M., Li, Y., Dillon, T. J., Kariya, Y. & Stork, P. J. S. Phosphorylation of the C-Raf N-region promotes Raf dimerization. Mol. Cell. Biol. 37, e00132-17 (2017).

    Article  Google Scholar 

  19. Chong, H., Lee, J. & Guan, K. L. Positive and negative regulation of Raf kinase activity and function by phosphorylation. EMBO J. 20, 3716–3727 (2001).

    Article  CAS  Google Scholar 

  20. Yao, Z. et al. Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS. Nature 548, 234–238 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Nieto, P. et al. A Braf kinase-inactive mutant induces lung adenocarcinoma. Nature 548, 239–243 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 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 

  23. Karoulia, Z., Gavathiotis, E. & Poulikakos, P. I. New perspectives for targeting RAF kinase in human cancer. Nat. Rev. Cancer 17, 676–691 (2017).

    Article  CAS  Google Scholar 

  24. 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. J. Mol. Diagn. 17, 251–264 (2015).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to L.M. Desrochers, I. Mellinghoff, and A. Ventura for useful discussions. We thank S. Lowe for the vectors of the retrovirus-based inducible expression system. 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 and T32 CA009207 (to J.T.). Additional funding was provided by a Conquer Cancer Foundation of the American Society of Clinical Oncology Young Investigator Award (to J.T.) and Career Development Award (to R.Y.). We would like to acknowledge the support of the Arlene and Joseph Taub Foundation and of P. and T. McInerney, without whom this work would not have been possible. 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 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.

Author information

Author notes

  1. These authors contributed equally: Zhan Yao, Yijun Gao, Wenjing Su

Authors and Affiliations

  1. Program in Molecular Pharmacology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Zhan Yao, Yijun Gao, Wenjing Su, Na Na, Neilawattie M. Timaul, Rory Mcgriskin, Nathaniel A. Outmezguine, HuiYong Zhao, Qing Chang, Besnik Qeriqi, Elisa de Stanchina & Neal Rosen

  2. Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Zhan Yao, Rona Yaeger, Jessica Tao, David M Hyman & Neal Rosen

  3. Weill Cornell Medical College, New York, NY, USA

    Rona Yaeger & David M Hyman

  4. Plexxikon Inc., Berkeley, CA, USA

    Ying Zhang, Chao Zhang, Andrey Rymar & Gideon Bollag

  5. Center for Neural Science, College of Arts and Sciences, New York University, New York, NY, USA

    Anthony Tao

  6. Molecular Oncology Programme, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain

    Mariano Barbacid

  7. Center for Mechanism-Based Therapeutics, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Neal Rosen

Authors
  1. Zhan Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yijun Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wenjing Su
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rona Yaeger
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jessica Tao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Na Na
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ying Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Chao Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Andrey Rymar
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Anthony Tao
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Neilawattie M. Timaul
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Rory Mcgriskin
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Nathaniel A. Outmezguine
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. HuiYong Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Qing Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Besnik Qeriqi
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Mariano Barbacid
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Elisa de Stanchina
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. David M Hyman
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Gideon Bollag
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Neal Rosen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Z.Y. and N.R. conceived the project and designed the experiments. Z.Y. and N.R. wrote the manuscript. Y.G., W.S., R.Y., C.Z., M.B., D.M.H., and G.B. provided critical revisions of the manuscript. Z.Y., Y.G., W.S., R.Y., J.T., N.N., Y.Z., C.Z., A.R., A.T., N.M.T., R.M., N.A.O., H.Z., Q.C., B.Q., E.d.S., and D.M.H. established the in vitro and in vivo experimental systems, performed the laboratory experiments, and analyzed the results.

Corresponding author

Correspondence to Neal Rosen.

Ethics declarations

Competing interests

N.R. is on the scientific advisory board (SAB) and receives research funding from Chugai, on the SAB and owns equity in Beigene, and Fortress. N.R. is also on the SAB of Daiichi-Sankyo, Astra-Zeneca-MedImmune, and F-Prime, and is a past SAB member of Millenium-Takeda, Kadmon, Kura, and Araxes. N.R. is a consultant to Novartis, Boehringer Ingelheim, Tarveda, and Foresight and consulted in the last three years with Eli Lilly, Merrimack, Kura Oncology, Araxes, and Kadman. N.R. owns equity in ZaiLab, Kura Oncology, Araxes, and Kadman. N.R. collaborates with Plexxikon. R.Y. has received research support from Array BioPharma, Genentech, GlaxoSmithKline, and Norvatis; received travel expenses from Array BioPharma; and served on the advisory board for GlaxoSmithKline. D.M.H. has consulted for Atara Biotherapeutics, Chugai Pharma, CytomX Therapeutics, Boehringer Ingelheim, AstraZeneca, Pfizer, Bayer, Debiopharm Group, and Genetech and has received research funding from AstraZeneca, Puma Biotechnology, and Loxo Oncology. Y.Z., C.Z., A.R., and G.B. are employees of Plexxikon Inc.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15 and Supplementary Table 1–4

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yao, Z., Gao, Y., Su, W. et al. RAF inhibitor PLX8394 selectively disrupts BRAF dimers and RAS-independent BRAF-mutant-driven signaling. Nat Med 25, 284–291 (2019). https://doi.org/10.1038/s41591-018-0274-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41591-018-0274-5

This article is cited by

Search

Advanced 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