Deciphering the Role of Oncogenic MITF^E318K in Senescence Delay and Melanoma Progression

Background: MITF encodes an oncogenic lineage-specific transcription factor in which a germline mutation (MITF^E318K) was identified in human patients predisposed to both nevus formation and, among other tumor types, melanoma. The molecular mechanisms underlying the oncogenic activity of MITF^E318K remained uncharacterized. Methods: Here, we compared the SUMOylation status of endogenous MITF by proximity ligation assay in melanocytes isolated from wild-type (n=3) or E318K (n=4) MITF donors. We also used a newly generated Mitf^E318K knock-in (KI) mouse model to assess the role of Mitf^E318K (n=7 to 13 mice per group) in tumor development in vivo and performed transcriptomic analysis of the tumors to identify the molecular mechanisms. Finally, using immortalized or normal melanocytes (wild-type or E318K MITF, n=2 per group), we assessed the role of MITF^E318K on the induction of senescence mediated by BRAF^V600E. All statistical tests were two-sided. Results: We demonstrated a decrease in endogenous MITF SUMOylation in melanocytes from MITF^E318K patients (mean of cells with hypoSUMOylated MITF, MITF^E318K vs MITF^WT, 94% vs 44%, difference=50%, 95% CI=21.8% to 67.2%, P=.004). The Mitf^E318K mice were slightly hypopigmented (mean melanin content Mitf^WT vs Mitf^E318K/+, 0.54 arbitrary units [AU] vs 0.36 AU, difference=-0.18, 95% CI=-0.36 to -0.007, P=.04). We provided genetic evidence that Mitf^E318K enhances BRaf^V600E-induced nevus formation in vivo (mean nevus number for Mitf^E318K, BRaf^V600E vs Mitf^WT, BRaf^V600E, 68 vs 44, difference=24, 95% CI=9.1 to 38.9, P=.006). Importantly, although Mitf^E318K was not sufficient to cooperate with BRaf^V600E alone in promoting metastatic melanoma, it accelerated tumor formation on a BRaf^V600E, Pten-deficient background (median survival, Mitf^E318K/+=42 days, 95% CI=31 to 46 vs Mitf^WT=51 days, 95% CI=50 to 55, P<.001). Transcriptome analysis suggested a decrease in senescence in tumors from Mitf^E318K mice. We confirmed this hypothesis by in vitro experiments, demonstrating that Mitf^E318K impaired the ability of humanmelanocytes to undergo BRAF^V600E-induced senescence. Conclusions: We characterized the functions of melanoma-associated MITF^E318K mutations. Our results demonstrate that MITF^E318K reduces the program of senescence to potentially favor melanoma progression in vivo.