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⇱ RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF | Nature

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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.

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Figure 1: RAF inhibitors rapidly activate MEK/ERK in cells with wild-type BRAF.
Figure 2: MEK/ERK activation requires binding of drug to the catalytic domain of RAF.
Figure 3: RAF inhibitor induces the active, phosphorylated state of wild-type and kinase-dead RAF.
Figure 4: MEK/ERK activation occurs via transactivation of RAF dimers.

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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 to Neal Rosen.

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The authors declare no competing financial interests.

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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

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