Abstract 2174: A high-throughput, high-content assay for the discovery of new inhibitors of DNA double-strand break repair

Abstract

Abstract DNA repair pathways are being intensively investigated as targets for chemotherapeutic interventions. Despite the immense interest in this area, few whole-cell screens for the discovery of DNA repair inhibitors have been described. We have developed an automated, whole-cell, high-throughput assay for the unbiased discovery of inhibitors of the non-homologous end joining (NHEJ) and homologous recombination HR) repair pathways, the two primary pathways that ameliorate DNA double-strand breaks. Our assay enables indirect measurement of DNA repair activity by monitoring the kinetics of γH2AX and 53BP1 foci resolution after treatment with ionizing radiation (IR) and candidate DNA damage response (DDR) inhibitor. We also have developed a unique platform of secondary assays to rank-order the potency of lead compounds and gain insights into their mechanism of action. In addition to hit validation and rigorous exclusion of false-positive hits, our follow-up platform includes orthogonal assays for measuring mutagenic NHEJ and HR repair activity, as well as high-throughput assays for quantitative analysis of cell viability and cell cycle. Notably, both our primary and secondary assays are fully-automated and may be applied to screen large libraries of compounds. We validated our high-throughput assay using known DDR and DNA damage checkpoint inhibitors, and applied this screen toward the evaluation of 2,366 structurally-diverse small molecules with known bioactivity. Vorinostat and quercetin, compounds known to possess DDR inhibitory activity, were identified in the screen, supporting the sensitivity of our approach. Notably, the screen also lead to the identification of cardiac glycosides, natural products in clinical use for the treatment of heart failure and atrial arrhythmia, as potent inhibitors of DNA double-strand break repair. Many studies have noted the anticancer properties of the cardiac glycosides, but the basis of this activity has remained a “black box”. Our data suggest an explanation for this long-standing unsolved problem, and argue that this activity should be taken into consideration when evaluating other clinical applications of these agents. Because these compounds are already approved for use in humans, we believe they are excellent candidates for repurposing as chemo and radiosensitizers. Citation Format: Yulia Surovtseva, Vikram Jairam, Ranjini Sundaram, Ranjit Bindra, Seth Herzon. A high-throughput, high-content assay for the discovery of new inhibitors of DNA double-strand break repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2174.