Abstract
Activating mutations in KRAS and BRAF are found in more than 30% of all human tumours and 40% of melanoma, respectively, thus targeting this pathway could have broad therapeutic effects1. Small molecule ATP-competitive RAF kinase inhibitors have potent antitumour effects on mutant BRAF(V600E) tumours but, in contrast to mitogen-activated protein kinase kinase (MEK) inhibitors, are not potent against RAS mutant tumour models, despite RAF functioning as a key effector downstream of RAS and upstream of MEK2,3. Here we show that ATP-competitive RAF inhibitors have two opposing mechanisms of action depending on the cellular context. In BRAF(V600E) tumours, RAF inhibitors effectively block the mitogen-activated protein kinase (MAPK) signalling pathway and decrease tumour growth. Notably, in KRAS mutant and RAS/RAF wild-type tumours, RAF inhibitors activate the RAF–MEK–ERK pathway in a RAS-dependent manner, thus enhancing tumour growth in some xenograft models. Inhibitor binding activates wild-type RAF isoforms by inducing dimerization, membrane localization and interaction with RAS–GTP. These events occur independently of kinase inhibition and are, instead, linked to direct conformational effects of inhibitors on the RAF kinase domain. On the basis of these findings, we demonstrate that ATP-competitive kinase inhibitors can have opposing functions as inhibitors or activators of signalling pathways, depending on the cellular context. Furthermore, this work provides new insights into the therapeutic use of ATP-competitive RAF inhibitors.
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Acknowledgements
We would like to thank the chemists at Genentech and Array BioPharma for providing GDC-0879. We thank Oncotest GmbH, L. Murray, S. Gould, B. Alicke, W. Voegtli, K. Bock, H. Stern and the immunohistochemistry core laboratory for technical support. We thank B. Vogelstein for HCT116 isogenic BRAF+/+ and BRAF-/- cell lines, and T. Waldman for the HEC1A BRAF+/+ and BRAF-/- cell lines. Special thanks goes to A. Bruce for help with artwork.
Author Contributions G.H. and S.M. designed the studies, interpreted the data and wrote the manuscript. K.S., I.Y., B.L., S.S. and D.S. conducted cellular experiments and dimerization assays. D.J.A., M.J.C.L. and R.A. conducted microscopy experiments. B.J.B., G.V., T.M. and I.A. conducted crystallography and provided structural input. S.L.G. conducted enzymology. K.P.H. and H.K. conducted in vivo experiments and immunohistochemistry. B.S.J. and S.S. generated inducible shRNA cell lines. M.B. and L.S.F. interpreted the data and wrote the manuscript.
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G.H., K.S., I.Y., D.J.A., R.A., M.J.C.L., D.S., I.A., B.L., S.S., K.P.H., B.S.J., S.S., H.K., M.B., L.S.F. and S.M. are employees of Genentech, Inc. B.J.B., S.L.G., T.M. and G.V. are employees of Array BioPharma.
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Hatzivassiliou, G., Song, K., Yen, I. et al. RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 464, 431–435 (2010). https://doi.org/10.1038/nature08833
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DOI: https://doi.org/10.1038/nature08833
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