Abstract B22: Combined disruption of ATM and CHK1 functionalities reveals redundancies in the DNA damage response pathways and results in synthetic growth inhibition following γ-irradiation

Exposure of proliferating cells to genotoxic stresses activates a cascade of signaling events termed the DNA damage response (DDR). The DDR preserves genetic stability by detecting DNA lesions, activating cell cycle checkpoints, and promoting DNA damage repair. The phosphoinositide 3-kinase related kinases (PIKKs) Ataxia Telangiectasia-Mutated (ATM), ATM and Rad 3-related kinase (ATR), and DNA-dependent Protein Kinase (DNA-PK) are crucial for lesion sensing and signal transduction. The checkpoint kinase 1 (CHK1) is a traditional ATR target involved in DDR and normal cell cycle progression and represents a pharmacological target for anti-cancer regimens. We hypothesized that ATM-deficient cells depend on signaling through CHK1 for survival after genotoxic exposures. The purpose of this study was to develop isogenic cell lines that are stably depleted for CHK1, ATM, or both and examine cross-talk and compensatory effects on cellular growth in general and the G2/M checkpoint in particular following treatment with γ-irradiation (IR). Human epithelial cell cultures (HME-CC) were stably transduced with either control or CHK1-targeting lentiviral shRNA particles in a wild-type or ATM-deficient background. Cell line validation and characterization was performed with cellular assays in conjunction with real-time quantitative PCR and Western blotting. Cell cycle profiling was done by flow cytometry. In addition, pharmacological approaches were used to functionally disrupt the proteins of interest for this study. We show that individual depletion of CHK1 and ATM results in augmented cellular proliferation as reflected in shortened cell population doubling times. Notably, a high level of depletion of CHK1 (HEK293 and HME-CC cells) or ATM (HME-CC) renders cells radiosensitive without abrogating their IR-mediated G2/M checkpoint arrest. Our data indicates that following IR, CHK1-deficient cells display enhanced ATM phosphorylation compared to their wild-type counterparts and conversely, ATM-depleted cells exhibit enhanced CHK1 phosphorylation, thus revealing a redundancy in the G2/M checkpoint enforcement after DNA damage. We provide evidence that this ATM-CHK1 cooperation is mediated at least in part by a feedback regulatory loop with the phosphatase PP2A. CHK1-depleted cells exhibit a lower nuclear abundance of the PP2A catalytic subunit, and a concurrent increase in the inhibitory phosphorylated state of the PP2A catalytic subunit compared to CHK1-proficient cells. Intriguingly, stable depletion of CHK1 in an ATM-deficient background showed only a 50% reduction from wild-type CHK1 protein expression levels, suggesting that loss of CHK1 by ATM-deficient cells may be synthetically lethal. Combined ATM depletion (10% of wild-type level) and CHK1-deficiency (50%) resulted in an additive attenuation of the G2/M checkpoint response compared to the individual knockdown, whereas pharmacological ATM inhibition in conjunction with 90% CHK1 depletion abrogated the early G2/M checkpoint and precluded the cells from mounting an efficient compensatory response to IR at later time-points. Furthermore, at 48 h post-IR the individually-depleted cell lines continued to proliferate, whereas the double-deficient cell line ATM-CHK1 halted proliferation. We conclude that dual targeting of ATM and CHK1 functionalities disrupts the compensatory response to DNA damage, abrogates the checkpoint arrest, and leads to synthetic growth inhibition. A combined targeting of ATM and CHK1 functionalities merits further investigation for developing more efficient anti-neoplastic treatments. Citation Format: Stela S. Palii, Richard S. Paules. Combined disruption of ATM and CHK1 functionalities reveals redundancies in the DNA damage response pathways and results in synthetic growth inhibition following γ-irradiation. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B22.