title: 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.
authors.affiliation: The Wistar Institute, Philadelphia, PA, USA. k.smalley@mac.com
authors.forename: K S M
authors.initials: KS
authors.lastname: Smalley
authors.forename: M
authors.initials: M
authors.lastname: Xiao
authors.forename: J
authors.initials: J
authors.lastname: Villanueva
authors.forename: T K
authors.initials: TK
authors.lastname: Nguyen
authors.forename: K T
authors.initials: KT
authors.lastname: Flaherty
authors.forename: R
authors.initials: R
authors.lastname: Letrero
authors.forename: P
authors.initials: P
authors.lastname: Van Belle
authors.forename: D E
authors.initials: DE
authors.lastname: Elder
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: K L
authors.initials: KL
authors.lastname: Nathanson
authors.forename: M
authors.initials: M
authors.lastname: Herlyn
mesh_terms: D017209:Apoptosis / Q000235:genetics; D001557:Benzenesulfonates / Q000494:pharmacology; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D055785:Gene Knockdown Techniques; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008545:Melanoma / Q000201:enzymology* / Q000235:genetics; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D009154:Mutation; D009536:Niacinamide / Q000031:analogs & derivatives; D010671:Phenylurea Compounds; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors* / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D000077157:Sorafenib; D014633:Valine / Q000235:genetics / Q000378:metabolism
chemical_list: D001557:Benzenesulfonates; D010671:Phenylurea Compounds; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D034741:RNA, Small Interfering; D009536:Niacinamide; D018698:Glutamic Acid; D000077157:Sorafenib; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D020929:Mitogen-Activated Protein Kinase Kinases; D014633:Valine
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