Data from Coexpression of NRAS<sup>Q61R</sup> and BRAF<sup>V600E</sup> in Human Melanoma Cells Activates Senescence and Increases Susceptibility to Cell-Mediated Cytotoxicity

Activating mutations in BRAF and NRAS oncogenes in human melanomas are mutually exclusive. This finding has suggested an epistatic relationship but is consistent even with synthetic lethality. To evaluate the latter possibility, a mutated NRAS^Q61R oncogene was expressed, under a constitutive or a doxycycline-regulated promoter, in a metastatic melanoma clone (clone 21) harboring an activated BRAF^V600E oncogene. After the first 10 to 12 in vitro passages, the constitutive NRAS^Q61R transfectant displayed progressive accumulation in G₀-G₁ phase of the cell cycle and stained for the senescence-associated β-galactosidase activity (SA-β-Gal). Inducible expression of NRAS^Q61R, by the Tet-Off system, in clone 21 cells (21NRAS^61ON) led to overactivation of the RAS/RAF/mitogen-activated protein kinase signaling pathway and, after the 10th in vitro passage, led to promotion of senescence. This was documented by reduced proliferation, flattened cell morphology, reduced growth in Matrigel, positive staining for SA-β-Gal, and expression of AMP-activated protein kinase and of the cell cycle inhibitor p21^waf1/Cip1. These effects were detected neither in 21 cells with silenced NRAS^Q61R (21NRAS^61OFF) nor in cells transfected with an inducible wild-type NRAS gene (21NRAS^WTON). In addition, when compared with parental 21 cells, or with 21NRAS^61OFF, 21NRAS^61ON and constitutive NRAS^Q61R transfectants cells showed increased susceptibility to cytotoxicity by both HLA class I antigen–restricted and nonspecific T cells and up-regulation of several MHC class I antigen processing machinery components. These results suggest a relationship of synthetic lethality between NRAS and BRAF oncogenes, leading to selection against “double-mutant” cells. (Cancer Res 2006; 66(13): 6503-11)