Abstract PO-023: Impact of a novel Ku-DNA binding inhibitor on the IR-induced DNA damage response

Abstract

Abstract The DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) double stand break (DSB) repair pathway and the DNA damage response (DDR). Consequently, blocking DNA-PK kinase activity is a novel anti-cancer therapeutic strategy in combination with ionizing radiation (IR). Towards developing a series of a new class of DNA-PK inhibitors, our laboratory has exploited the mechanism of DNA-PK activation which requires binding to DNA termini via the Ku 70/80 heterodimer. We recently reported the development of Ku-DNA binding inhibitors (Ku-DBi’s) that act via this novel mechanism of action to inhibit DNA-PK catalytic kinase activity. Ku-DBi’s display nanomolar activity in vitro, possess cellular DNA-PK and NHEJ inhibitory activity and sensitize non-small cell lung cancer (NSCLC) cells to chemotherapeutics bleomycin and etoposide. In this study, we demonstrate that pre-incubation of our novel Ku-DBi’s can potentiate the cellular effects of IR. We demonstrate that the increase in IR sensitivity is preceded by a decrease in DNA-PKcs autophosphorylation events at the S2056 (pS2056) cluster. Using a combination of Western blot and immunofluorescence assays in the NSCLC NCI-H460 cell line, Ku-DBi 245 treatment in combination with IR showed a significant reduction of phosphorylation of DNA-PKcs at S2056 compared to IR alone. Analysis of gH2A.X also showed an inhibition of the DDR as a function of 245 treatment, evidenced by reduction of S139 phosphorylation. These results demonstrate that Ku-DBi 245 blocks DNA-break dependent DNA-PKcs autophosphorylation, resulting in an increased in radiation sensitivity. These data are consistent with Ku-DBi’s possessing a novel mechanism of action that abrogates autophosphorylation of DNA-PKcs and DDR signaling as part of an anticancer therapeutic strategy in combination with IR. Citation Format: Pamela Mendoza-Munoz, Navnath S. Gavande, Pamela S. VanderVere-Carozza, Katherine S. Pawelczak, Joseph R. Dynlacht, Joy Garrett, John J. Turchi. Impact of a novel Ku-DNA binding inhibitor on the IR-induced DNA damage response [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-023.