Abstract B25: An unbiased functional screen identifies kinases essential to ovarian cancer cell survival

Abstract

Abstract Ovarian cancer is the most lethal gynecologic cancer in USA. Many efforts including gene expression profiling, SNP analysis, comparative genomic hybridization, and exome sequencing have revealed that ovarian cancer is extremely heterogeneous. This implies that therapeutic strategy should be specifically designed based on molecular characteristics. Previously, we focused on investigating the functional role of NF-κB pathway in ovarian cancer initiation, propagation, and dissemination, performing two independent shRNA screens in the context of NF-κB signaling. In this study, we have re-focused our efforts towards an unbiased loss-of-function screen across 4 ovarian cancer cell lines Caov3, Igrov1, Ovcar5, and A2780. Under the most stringent cut-off of 4 out of 4 cell lines with a p value of less than 0.05 and fold change less than 0.7, five genes including GUCY2F, MKNK2, PDK3, PIK3AP1, and WEE1 were identified as essential for ovarian cancer cell viability. When the stringency of analysis was relaxed to allow 3 out of 4 cell lines affected, a total of 55 genes were included. The most significant networks among these 55 genes were those of cell cycle, and cancer cell death and survival, as determined by Ingenuity Pathway Analysis. In order to validate and prioritize candidate genes, we focused siRNA lethality screening to the 55 genes in an expanded set of 6 ovarian cancer cell lines additionally including Skov3 and Ovcar8. Two siRNAs per gene (Qiagen) were tested in 384-well format. Transient transfection protocols such as seeding cell numbers, lipid to siRNA ratio, and incubation time were optimized in each individual cell line using positive and negative siRNA controls. Based on cell viability, 9 genes including EPHB1, FER, MAP3K7, MAP3K8, MGC42105 (NIM1), PRKCA, PLK1, ERBB2, and WEE1 were confirmed as essential in the initial shRNA screened 4 ovarian cancer cell lines, while six genes (EPHB1, FER, MAP3K7, PLK1, ERBB2, and WEE1) were identified in all 6 cell lines. To move these findings towards the clinic, we next investigated whether pharmacological inhibitors could recapitulate the siRNA effect. We tested 4 inhibitors: Oxozeaenol (for MAP3K7/TAK1), BI6727 (PLK1), MK1775 (WEE1), and Lapatinib (ERBB2). Using 2-fold serial dilutions in 6 cell lines, ranges of IC50 were determined based on cell proliferation and calculated by CompuSyn software. The ranges of IC50 were 1- 6 uM, 10 - 35 nM, and 0.2 - 0.6 uM for Oxozeaenol, BI6727, and MK1775, respectively. All 6 cell lines were resistant to Lapatinib at 10 uM, suggesting a kinase independent function of ERBB2 in ovarian cancer cells. Single agent therapies often result in resistance and relapse. As such, our future work will include identification of predictive biomarkers for selecting drugs and the efficacy of sequential versus simultaneous treatment with these drugs in different combinations. In summary, a loss-of-function screening followed by validation using clinically relevant inhibitors helps us to identify essential targets in ovarian cancer and to systematically refine therapeutic strategies in ovarian cancer. Citation Format: Marianne K. Kim, Sirisha Chakka, Natasha Caplen, Christina Annunziata. An unbiased functional screen identifies kinases essential to ovarian cancer cell survival. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B25.