Abstract B09: High-throughput RNAi sensitization screening identifies a novel drug combination for metastatic melanoma

Abstract

Abstract Metastatic malignant melanoma is an incurable disease with a poor 5-year survival rates. Despite the recent development of several novel therapeutic agents, there remains an urgent need to improve the effectiveness of conventional chemotherapeutics such as temozolomide (TMZ). In this study, we developed RNA-interference-based (RNAi) TMZ chemosensitization assays in melanoma cell lines in order to better understand how to pharmacologically augment TMZ response. Initially 65 melanoma cell lines were surveyed for siRNA optimization, with 18 selected to carry out extensive assay optimization across a multitude of assay conditions. Then we applied these optimized RNAi screening parameters and performed high-throughput RNAi screens with a 7,000 gene siRNA library targeting the druggable genome. Five melanoma cell lines demonstrating various response in the presence of TMZ were selected for the primary screens: two primary screens were performed using two siRNAs per target, followed by three screens using four siRNAs per target. The sensitizer screen was conducted with a drug dose-response methodology using five different concentrations of TMZ (targeting IC10 to IC80), and cell viability was measured 120 hour post-treatment. The IC50 was calculated for each siRNA and individual siRNA hits were selected when their IC50 values were significantly different from that of the scrambled siRNA control. Genes that had two or more siRNAs demonstrating TMZ sensitization were selected for confirmation and further validation in a panel of 15 total melanoma cell lines. 137 genes were validated as TMZ sensitizers in two or more melanoma cell lines. Because multiple members of the CHEK1/CHEK2 pathway were among these validated sensitizers, we chose to focus on this pathway and evaluate checkpoint kinase inhibition as a potential means of augmenting TMZ response. We assessed four CHEK1 inhibitors for synergistic effects when combined with TMZ. Notably, we observed measurably synergistic effects for four CHEK1 inhibitors in each of the most TMZ-resistant cell lines, with only moderate/marginal effects in the most TMZ-sensitive lines. These synergistic effects on cell viability were accompanied by an increase in apoptotic events, and the synergy between TMZ and CHEK1 inhibition was further validated in a mouse xenograft model. Our findings suggest that targeting the CHEK1/CHEK2 pathway may augment TMZ response in melanoma, and lay the foundation for the testing of novel combination therapies for melanoma patients who are vulnerable to conventional treatment. Citation Format: Hongwei Holly Yin, Donald Chow, Chris Sereduk, Meraj Aziz, Jeff Kiefer, Mai Xu, Michael Garside, Nanyun Tang, Chao Sima, Jianping Hua, Michelle Kassner, Mike Bittner, Nick Hayward, Kevin Brown, Trent Jeff. High-throughput RNAi sensitization screening identifies a novel drug combination for metastatic melanoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B09.