RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF
Poulikos I Poulikakos, Chao Zhang, Gideon Bollag, Kevan M Shokat, Neal Rosen, Poulikos I Poulikakos, Chao Zhang, Gideon Bollag, Kevan M Shokat, Neal Rosen
Abstract
Tumours with mutant BRAF are dependent on the RAF-MEK-ERK signalling pathway for their growth. 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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References
- Solit DB, et al. BRAF mutation predicts sensitivity to MEK inhibition. Nature. 2006;439:358–62.
- 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 U S A. 2007;104:19936–41.
- Wellbrock C, et al. V599EB-RAF is an oncogene in melanocytes. Cancer Res. 2004;64:2338–42.
- 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 Suppl. 2009;7:5.
- 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. 2009;27 Abstract 9000.
- Tsai J, et al. Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc Natl Acad Sci U S A. 2008;105:3041–6.
- Wellbrock C, Karasarides M, Marais R. The RAF proteins take centre stage. Nat Rev Mol Cell Biol. 2004;5:875–85.
- Young A, et al. Ras signaling and therapies. Adv Cancer Res. 2009;102:1–17.
- Konecny GE, et al. Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells. Cancer Res. 2006;66:1630–9.
- Weber CK, Slupsky JR, Kalmes HA, Rapp UR. Active Ras induces heterodimerization of cRaf and BRaf. Cancer Res. 2001;61:3595–8.
- Wan PT, et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell. 2004;116:855–67.
- Rushworth LK, Hindley AD, O’Neill E, Kolch W. Regulation and role of Raf-1/B-Raf heterodimerization. Mol Cell Biol. 2006;26:2262–72.
- Cutler RE, Jr., Stephens RM, Saracino MR, Morrison DK. Autoregulation of the Raf-1 serine/threonine kinase. Proc Natl Acad Sci U S A. 1998;95:9214–9.
- Okuzumi T, et al. Inhibitor hijacking of Akt activation. Nat Chem Biol. 2009;5:484–493.
- Cameron AJ, Escribano C, Saurin AT, Kostelecky B, Parker PJ. PKC maturation is promoted by nucleotide pocket occupation independently of intrinsic kinase activity. Nat Struct Mol Biol. 2009;16:624–30.
- Karreth FA, DeNicola GM, Winter SP, Tuveson DA. C-Raf inhibits MAPK activation and transformation by B-Raf(V600E) Mol Cell. 2009;36:477–86.
- Blair JA, et al. Structure-guided development of affinity probes for tyrosine kinases using chemical genetics. Nat Chem Biol. 2007;3:229–38.
- Sun Y, et al. Growth inhibition of nasopharyngeal carcinoma cells by EGF receptor tyrosine kinase inhibitors. Anticancer Res. 1999;19:919–24.
- Rajakulendran T, Sahmi M, Lefrancois M, Sicheri F, Therrien M. A dimerization-dependent mechanism drives RAF catalytic activation. Nature. 2009;461:542–5.
- Hall-Jackson CA, et al. Paradoxical activation of Raf by a novel Raf inhibitor. Chem Biol. 1999;6:559–68.
- King AJ, et al. Demonstration of a genetic therapeutic index for tumors expressing oncogenic BRAF by the kinase inhibitor SB-590885. Cancer Res. 2006;66:11100–5.
- Hoeflich KP, 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. 2009;69:3042–51.
- Heidorn SJ, et al. Kinase-Dead BRAF and Oncogenic RAS Cooperate to Drive Tumor Progression through CRAF. Cell. 2010;140:209–221.
- 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. 2008;26 Abstract 9033.
- Montagut C, et al. Elevated CRAF as a potential mechanism of acquired resistance to BRAF inhibition in melanoma. Cancer Res. 2008;68:4853–61.
- Bankston D, et al. A Scaleable Synthesis of BAY 43-9006: A Potent Raf Kinase Inhibitor for the Treatment of Cancer. Organic Process Research and Development. 2002;6:777–781.
Source: PubMed