Targeting negative regulation of p53 by MDM2 and WIP1 as a therapeutic strategy in cutaneous melanoma

Background:Cutaneous melanoma is the most serious skin malignancy and new therapeutic strategies are needed for advanced melanoma. TP53 mutations are rare in cutaneous melanoma and hence activation of wild-type p53 is a potential therapeutic strategy in cutaneous melanoma. Here, we investigated the WIP1 inhibitor, GSK2830371, and MDM2-p53 binding antagonists (nutlin-3, RG7388 and HDM201) alone and in combination treatment in cutaneous melanoma cell lines and explored the mechanistic basis of these responses in relation to the genotype and induced gene expression profile of the cells.Methods:A panel of three p53 WT (A375, WM35 and C8161) and three p53 MUT (WM164, WM35-R and CHL-1) melanoma cell lines were used. The effects of MDM2 and WIP1 inhibition were evaluated by growth inhibition and clonogenic assays, immunoblotting, qRT-PCR gene expression profiling and flow cytometry.Results:GSK2830371, at doses (≤10 μM) that alone had no growth-inhibitory or cytotoxic effects on the cells, nevertheless significantly potentiated the growth-inhibitory and clonogenic cell killing effects of MDM2 inhibitors in p53 WT but not p53 MUT melanoma cells, indicating the potentiation worked in a p53-dependent manner. The siRNA-mediated knockdown of p53 provided further evidence to support the p53 dependence. GSK2830371 increased p53 stabilisation through Ser15 phosphorylation and consequent Lys382 acetylation, and decreased ubiquitination and proteasome-dependent degradation when it was combined with MDM2 inhibitors. These changes were at least partly ATM mediated, shown by reversal with the ATM inhibitor (KU55933). GSK2830371 enhanced the induction of p53 transcriptional target genes, cell cycle arrest and apoptosis.Conclusions:GSK2830371, a WIP1 inhibitor, at doses with no growth-inhibitory activity alone, potentiated the growth-inhibitory and cytotoxic activity of MDM2 inhibitors by increasing phosphorylation, acetylation and stabilisation of p53 in cutaneous melanoma cells in a functional p53-dependent manner.