Abstract C222: The identification of kinase targets in Ewing sarcoma cell lines using RNAi and high-throughput investigational agents screens.

Abstract

Abstract Ewing sarcoma (EWS), the second most frequent bone cancer, is an aggressive primitive neuroectodermal tumor that occurs primarily in children, adolescents and young adults. A pathognomonic translocation involving the EWS gene on chromosome 22 and the ETS-type gene FLI1 on chromosome 11 occurs in about 85% of cases. The EWS/FLI1 fusion protein product of this translocation is a potent transcription factor that functions as an oncoprotein. The current study uses complementary approaches of high-throughput siRNA mediated RNAi and Investigational Agents screening to identify new therapeutic strategies for Ewing sarcoma. For RNAi screening the TC32 cell line, a well-characterized EWS cell line that expresses the EWS-FLI1 fusion transcript, was assayed for cell viability (CellTiterGlo, Promega) 72 hrs post siRNA transfection using a library of siRNAs corresponding to the genes encoding proteins with kinase function, three siRNA per gene (Ambion). Fifteen kinase genes were identified as inducing a significant decrease in the survival of TC32 cells when silenced by RNAi. These genes were chosen for validation in further EWS lines and two non-EWS cell lines. Using a cut off of a 40% or greater decrease in cell viability induced by two or more siRNAs per gene in at least at one EWS cell line eleven genes of interest were identified: PLK1, AURKB, AKT1, CDC2L1, CRKRS, ATP6VOB, CDC2L5, AURKA, CHEK1, NRBP2 and CDK4. The importance of several of these kinases in the survival of EWS cell lines was confirmed by an independent high-throughput screen of 63 human pediatric and adult sarcoma lines, including 19 EWS lines, with the Approved Oncology Drugs (AOD) library of compounds and an Investigational Agents library (350 agents) using inhibition of proliferation as an endpoint. Cells were exposed to compounds at varying concentrations (10μM to 1.5nM) for 96 h and the effect of compound on cell viability was monitored using Alamar Blue. The IC50 (50% growth inhibitory) concentrations of each agent on the cell lines were determined using curve fitting algorithms. Compared to the other sarcoma lines, the EWS lines were: more sensitive to eight Aurora kinase inhibitors (IC50 ∼0.05 µM) (IC50 ∼2.5 µM);more sensitive (IC50 ∼0.1 µM) to five checkpoint kinase (CHK) inhibitors (IC50 about 1.3 µM); and more sensitive to eight polo-like kinase (PLK1) inhibitors. These results demonstrate the power of integrating RNAi and compound inhibition studies to identify molecular targets for specific cancer types. We are currently further evaluating the inhibition of the mitosis-associated proteins identified by these complementary screening approaches as a strategy for the treatment EWS. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C222. Citation Format: Amy McCalla, Patrick Grohar, Scott Martin, Eugen Buehler, Suntae Kim, Natasha Caplen, Joel Morris, Mark Kunkel, David Evans, Anne Monks, Beverly Teicher, Lee Helman. The identification of kinase targets in Ewing sarcoma cell lines using RNAi and high-throughput investigational agents screens. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C222.