Abstract 4900: Reactive oxygen species scavenger extends the efficacy of BRAF inhibitors in BRAF-mutant melanoma

Abstract

Abstract We aim to identify whether reactive oxygen species (ROS) targeted therapy using a ROS scavenger, A100, will inhibit tumor growth and evade BRAF inhibitor induced resistance in melanoma cells harboring the endogenous BRAFV600E mutation. We also propose to investigate the mechanism by which A100 sensitizes the dabrafenib-mediated resistance. Dabrafenib-resistant (DR) melanoma cells (WM-115DR, SK-MEL-24DR and A375DR) were generated using increasing doses of dabrafenib. We conducted flow cytometry experiments to measure ROS levels before and after acquisition of resistance. DCF-DA assay indicated upregulation of hydrogen peroxide levels in dabrafenib resistant cell lines versus parental cell lines. Furthermore, dabrafenib resistant cell lines demonstrated elevated superoxide levels as analyzed by MitoSOX. Crystal violet and three-dimensional matrigel assays were performed to analyze the effect of A100 and dabrafenib on cell growth of dabrafenib resistant and parental melanoma cell lines. We observed a statistically significant decrease in cell proliferation and colony formation of dabrafenib resistant cells when subjected to the combination of A100 and dabrafenib compared to single agent dabrafenib or A100. We also noted a trend of inhibition of cell proliferation in response to combined treatment of A100 and dabrafenib in parental cell lines versus the single agent. There was significant reduction in colony formation under similar condition in parental cells. Dabrafenib as a single agent had similar effect in reducing phosphorylated ERK1/2 as compared to co-treatment with A100 in A375, SK-MEL-24 and WM-115 parental cells. As expected, there was no alteration of MAPK signaling in resistant WM-115DR, SK-MEL-24DR and A375DR cells upon treatment with dabrafenib. A100 has been reported to cause DNA damage in leukemia cells. We analyzed the effect of A100 in the presence or absence of dabrafenib on markers of DNA damage. The data suggested that DNA damage is induced by A100 as assessed by increased levels of p-ATM, p-ATR and γ-H2AX. Further, A100 in combination with dabrafenib increased levels of p-ATM, p-ATR and γ-H2AX in parental and dabrafenib resistant cell lines. Proteomics analysis on WM-115 versus WM-115DR cell line was performed to determine alterations in mitochondrial protein expression upon resistance to BRAF inhibitors. The data indicated upregulation of proteins with antioxidant functions including SOD2 (superoxide dismutase [Mn]) and Peroxiredoxin-1 (PRDX1) in WM-115DR cells compared to WM-115 parental which was confirmed by immunoblotting analysis. Overall, this study suggested that the combination of ROS quenching agent (A100) with BRAF inhibitor (dabrafenib) could be a potential strategy to treat BRAF-mutant melanoma patients. Citation Format: Long Yuan, Rosalin Mishra, Hima Patel, Samar Alanazi, Joan Garrett. Reactive oxygen species scavenger extends the efficacy of BRAF inhibitors in BRAF-mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4900.