Abstract A020: Deciphering DNA damage repair in ATM mutant prostate cancers
Authors: Hofstad Mia E. , Yu Lan , Woods Andrea , Sychev Zoi E. , Mazzagatti Alice , Huo Xiaofang , Kittler Ralf , Ly Peter , Drake Justin M. , Raj Ganesh V. 
Published: January 2024

Abstract
Background: Mutations in DNA Damage Response (DDR) genes, including those resulting in loss of Ataxia-Telangiectasia, Mutated (ATM) expression are common in advanced prostate cancers (PC). Poly (ADP-ribose) polymerase (PARP) inhibitors are approved in this setting but demonstrate limited clinical efficacy in ATM-mutant PCs. In this project, we sought to define the impact of ATM loss on DDR pathways in PC, with the goal of identifying alternate therapeutic vulnerabilities. Methods: ATM-KO PC cell lines were generated via CRISPR-Cas9. ATM loss and attenuation of downstream kinase activity were confirmed via western blot. Phosphoproteomic evaluation in parental and ATM-KO cells was performed following ionizing radiation (IR). Clonogenic survival assays were performed after treatment with inhibitors and/or IR. Kinetics of DDR foci recruitment and resolution were interrogated with immunofluorescence (IF). DNA fiber assays were utilized to assess replication stress. Results: ATM-KO PC cells were able to effectively repair DNA damage following IR, as measured by resolution of yH2ax, 53BP1, and MDC1 foci. Phosphoproteomic studies indicated that ATM-KO cells maintained canonical DDR pathways through activation of ATR and DNA-PKcs kinases. Treatment of ATM-KO cells with either the ATR inhibitor VX970 or the DNA-PKcs inhibitor M3814 incrementally affected DDR in ATM-KO cells compared to ATM-proficient parental control cells. Importantly, combination treatment with VX970 and M3814 was needed to prevent downstream DDR foci recruitment in ATM-KO cells and was more effective in radiosensitizing ATM-KO PC than either parental control cells or single drug treatment. These data indicate that the activity of any one of the trinities of these kinases is sufficient to mediate DDR and that blockade of both ATR and DNA-PKcs is needed to effectively prevent DDR in ATM-KO PC. Further, DNA fiber assays showed significantly decreased fork length with dual treatment compared to control or single drug, indicating increased replication stress. We then leveraged a RUVBL1 ATPase inhibitor Compound B, which affects the expression of these three kinases. In ATM-KO PC cells, Compound B attenuated ATR and DNA-PKcs expression and radiosensitized the cells as shown by clonogenic survival assays. DNA fiber assays indicated that Compound B induced replication stress in ATM-KO PC cells to the same extent as combination therapy. In combination with IR, Compound B also significantly attenuated the growth of ATM-KO PC xenografts in vivo. Conclusions: Our unbiased phosphoproteomic approach indicated that ATR and DNA-PKCs mediate DDR following IR in ATM-KO PC cells, and that dual targeting of both ATR and DNA-PKcs is necessary to block DDR in ATM-KO PC cells. We have identified that Compound B effectively depletes ATR and DNA-PKcs, sensitizes to DNA damage, and induces replication stress in ATM-KO PCs. Our studies suggest that Compound B may be a novel therapeutic strategy in ATM-mutant PC.
Citation Format: Mia E. Hofstad, Lan Yu, Andrea Woods, Zoi E. Sychev, Alice Mazzagatti, Xiaofang Huo, Ralf Kittler, Peter Ly, Justin M. Drake, Ganesh V. Raj. Deciphering DNA damage repair in ATM mutant prostate cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A020.