76P - Targeting p63 upregulation may prevent the development of MAPK inhibitor resistance in melanoma

Abstract

Background: Melanoma progression is often characterized by mutations in the mitogen-activated protein kinase (MAPK) and phosphoinositol-3-kinase (PI3K) pathways. Understanding regulation of these pathways has led to the development of novel targeted therapies which show high response rates. However, many patients relapse with ensuing resistant disease. P63, a p53 homologue, carries a poorer prognosis when overexpressed. In keratinocytes, degradation could be regulated by two ubiquitin ligases, MDM2 and FBXW7. This project explored the expression of p63, MDM2 and FBXW7 in MAPK-inhibitor (MAPKi) sensitive and resistant melanoma cell lines. Methods: Morphology change associated with the development of resistance was assessed by actin immunofluorescence experiments. Western blot and qRT-PCR enabled analysis of protein and RNA expression of p63, FBXW7 and MDM2, complemented by immunofluorescent staining and confocal microscopy data. Finally, a novel technique of flow cytometry enabled analysis of cell death in MAPKi-resistant melanoma cells treated with Nutlin-3A. Results: MAPKi resistance in melanoma cells is associated with increased p63 expression. Resistance is furthermore associated with reduced FBXW7 expression and enrichment of nuclear MDM2, suggesting a potential nuclear interaction between MDM2 and FBXW7. The resultant inactivation of FBXW7 explains the increased p63 expression upon MAPKi- resistance. Furthermore, by treating MAPKi-resistant cells with the MDM2-inhibitor Nutlin-3A, we were able to restore FBXW7 expression and promote p63-degradation, with resulting increased melanoma cell death. Conclusions: This project identifies FBXW7 and MDM2 as regulators of p63 expression in MAPKi-resistant melanoma and proposes a possible role for Nutlin-3A in treating advanced MAPKi-resistant melanoma. Legal entity responsible for the study: Queen Mary University of London (Blizzard Institute) - Cutaneous Research. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.