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CRAF inhibition induces apoptosis in melanoma cells with non-V600E BRAF mutations

Abstract

Here, we identify a panel of melanoma lines with non-V600E mutations in BRAF. These G469E- and D594G-mutated melanomas were found to exhibit constitutive levels of phospho-extracellular signal-regulated kinase (pERK) and low levels of phospho-mitogen-activated protein kinase/ERK kinase (pMEK) and were resistant to MEK inhibition. Upon treatment with the CRAF inhibitor sorafenib, these lines underwent apoptosis and associated with mitochondrial depolarization and relocalization of apoptosis-inducing factor, whereas the BRAF-V600E-mutated melanomas did not. Studies have shown low-activity mutants of BRAF (G469E/D594G) instead signal through CRAF. Unlike BRAF, CRAF directly regulates apoptosis through mitochondrial localization where it binds to Bcl-2 and phosphorylates BAD. The CRAF inhibitor sorafenib was found to induce a time-dependent reduction in both BAD phosphorylation and Bcl-2 expression in the D594G/G469E lines only. Knockdown of CRAF using a lentiviral shRNA suppressed both Bcl-2 expression and induced apoptosis in the D594G melanoma line but not in a V600E-mutated line. Finally, we showed in a series of xenograft studies that sorafenib was more potent at reducing the growth of tumors with the D594G mutation than those with the V600E mutation. In summary, we have identified a group of melanomas with low-activity BRAF mutations that are reliant upon CRAF-mediated survival activity.

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Acknowledgements

We thank the Bayer Corporation for providing the BAY 43-9006 (sorafenib).

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Correspondence to K S M Smalley or M Herlyn.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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Smalley, K., Xiao, M., Villanueva, J. et al. CRAF inhibition induces apoptosis in melanoma cells with non-V600E BRAF mutations. Oncogene 28, 85–94 (2009). https://doi.org/10.1038/onc.2008.362

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