1. ¹The Wistar Institute, Philadelphia, PA, USA
2. ²Division of Hematology–Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
3. ³Division of Medical Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
4. ⁴Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
5. ⁵Department of Otorhinolaryngology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
6. ⁶The Abramson Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA, USA

Correspondence: Dr KSM Smalley, Department of Molecular Oncology, The Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, SRB, Tampa, FL 33612, USA. E-mail: k.smalley@mac.com; M Herlyn, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA, USA. E-mail: herlynm@wistar.org

⁷Current address: Department of Molecular Oncology, The Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA.

Received 13 May 2008; Revised 6 August 2008; Accepted 18 August 2008; Published online 15 September 2008.

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.