We have extensively studied the phenotypic heterogeneity of patient-derived melanoma cells. Here, whole-exome sequencing revealed novel variants of genes associated with the MAPK, NOTCH, Hippo, cell-cycle, senescence, and ubiquitin-dependent pathways, which could contribute to the observed phenotypic diversity between cell lines. Focusing on mutations in the MAPK pathway-associated genes, we found BRAF (BRAFV600E ) and RAS subtypes, including NRASQ61R and the rare HRASQ61R variant, and additional alterations potentially leading to different ERK1/2 activity. Both RASQ61R cell lines harbored a MEK1P124S variant and exerted a low level of phospho-MEK1/2. Activity of the MAPK pathway was further attenuated in NRASQ61R /MEKP124S cells by trametinib, and this effect was also shown in HRASQ61R /MEKP124S melanoma cells. The observed variability in doubling time might be a consequence of diverse MAPK and PI3K/AKT pathway activities, but not exclusively, as a senescence program was also executed to different extent in distinct melanoma cell lines. Low percentages of senescent cells might result from mutations in CDKN2A, E2F3, and EZH2, and a high c-MYC expression. Vemurafenib and trametinib induced senescence concomitantly with c-MYC downregulation and irrespectively of CDKN2A mutation, but the EZH2S412C variant might limit senescence induction. Damaging alterations in Hippo pathway-associated genes were accompanied with variability in the phosphorylation level of YAP1/TAZ and CTGF expression. Our study also suggests opposite activity of NOTCH2F1209V and NOTCH2N2002S variants. Additionally, we found a novel FBXW7V418M variant that retained its function in melanoma cells. The obtained molecular data might be further exploited in genotype-phenotype relationship studies and in identifying novel biomarkers and therapies for melanomas.
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