CRAF R391W is a melanoma driver oncogene.

Mohammad Atefi,Antoni Ribas,Charles Ng,Allison Le,Thomas G Graeber,Amanda Lassen,Anastasia Lomova,Michael Friedman,Jennifer Tsoi,Bjoern Titz,Earl Avramis,Bartosz Chmielowski

doi:10.1038/srep27454

Abstract

Approximately 75% of melanomas have known driver oncogenic mutations in BRAF, NRAS, GNA11 or GNAQ, while the mutations providing constitutive oncogenic signaling in the remaining melanomas are not known. We established a melanoma cell line from a tumor with none of the common driver mutations. This cell line demonstrated a signaling profile similar to BRAF-mutants, but lacked sensitivity to the BRAF inhibitor vemurafenib. RNA-seq mutation data implicated CRAF R391W as the alternative driver mutation of this melanoma. CRAF R391W was homozygous and over expressed. These melanoma cells were highly sensitive to CRAF, but not BRAF knockdown. In reconstitution experiments, CRAF R391W, but not CRAF WT, transformed NIH3T3 cells in soft-agar colony formation assays, increased kinase activity in vitro, induced MAP kinase signaling and conferred vemurafenib resistance. MAP kinase inducing activity was dependent on CRAF dimerization. Thus, CRAF is a bona fide alternative oncogene for BRAF/NRAS/GNAQ/GNA11 wild type melanomas.

Highlights

75% of melanomas have known driver oncogenic mutations in BRAF, NRAS, GNA11 or GNAQ, while the mutations providing constitutive oncogenic signaling in the remaining melanomas are not known
We identified a CRAF R391W missense mutation in a melanoma metastasis biopsy and its derived cell line
This CRAF mutation shows the critical aspects of an oncogenic driver mutation: homozygosity and highly amplified expression of this locus indicates strong positive selection pressure[25]; the derived melanoma cell line is strictly dependent on CRAF (R391W) expression for activity of MAPK signaling and proliferation; exogenous CRAF R391W expression strongly enhances kinase activity, homodimerization and induces downstream MAP kinase signaling; and CRAF R391W induces anchorage independent growth and can functionally replace BRAF V600E in a melanoma cell line

Summary

Introduction

75% of melanomas have known driver oncogenic mutations in BRAF, NRAS, GNA11 or GNAQ, while the mutations providing constitutive oncogenic signaling in the remaining melanomas are not known. While BRAF was found to be mutated in 8% of all cancers, CRAF demonstrated a significantly lower mutation frequency of 0.7% in cancer cell lines[14] Both BRAF and CRAF are expressed and active in melanoma signaling processes, only BRAF shows a high mutation frequency (approximately 50% of melanomas), whereas CRAF mutations are rare and so far have not been demonstrated to generate an alternative activated oncogene. Explanations for this striking difference have implicated the additional levels of negative regulation acting on CRAF, which – unlike BRAF – requires more than one mutation for the activation of an independent high kinase activity with MAPK inducing abilities[14]. For both resistance mechanisms the in vivo occurrence has not been demonstrated

Methods

Results

Conclusion