PO-450 MEK inhibition synergizes with MDM2 inhibitors through DUSP6 suppression

Abstract

IntroductionMAPK and ARF-MDM2-p53 pathways are critical in cutaneous melanomas. This study examined the combination treatment of MEK inhibitor, trametinib, with MDM2 inhibitors, nutlin-3/RG7388/HDM201, in BRAFV600E mutant and p53 wild-type (p53WT) melanoma cell lines and explored the mechanistic basis of these responses.Material and methodsTwo BRAFV600E and p53WT melanoma cell lines, A375 and WM35, were treated with either trametinib or MDM2 inhibitors, or combinations.Results and discussionsThe combination treatment in both A375 and WM35 showed additive to synergistic effects by Chou-Talalay median effect analysis and combination index scores. The combination treatments induced higher levels of p53 target gene transcripts (CDKN1A(p21), MDM2, BAX, BBC3(PUMA), TP53I3(PIG-3)) and protein products (p21, MDM2), resulting in increased cell cycle arrest and apoptosis compared with MDM2 inhibitors alone, suggesting trametinib synergized with MDM2 inhibitors via upregulation of p53-dependent pathways. In addition, DUSP6 phosphatase mRNA and protein were down-regulated following pERK reduction by trametinib, but did not change after p53 activation by MDM2 inhibitors. Furthermore, suppression of DUSP6 by siRNA, or inhibition with the small molecule inhibitor, BCI, potentiated the effect of MDM2 inhibitors through increased ATM-dependent p53 phosphorylation, as demonstrated by complete reversal with the ATM inhibitor, KU55933.ConclusionTrametinib synergizes with MDM2 inhibitors through a DUSP6 mechanism in BRAFV600E and p53WT melanoma cells. DUSP6 regulation of p53 phosphorylation via ATM, could be a therapeutic target for combination with treatments involving activation of the ATM/p53 pathway.