Abstract ATM is a master regulator of the DNA damage response (DDR) elicited by double-strand breaks (DSB). After DNA DSB, ATM controls cell fate via cell cycle arrest, apoptosis, and DNA damage repair. Numerous tumor types have ATM gene loss-of-function mutations, resulting in genomic instability. Most knowledge on the effect of ATM loss on DDR and tumor therapeutic response is from ataxia-telangiectasia (A-T) patients and ATM knockout models. A distinct and more deleterious phenotype has been proposed for kinase-dead (KD) ATM mutants that retain ATM protein expression. Inhibition of ATM activity does not fully mimic ATM loss; therefore, KD ATM mutants may be better predictors of response to ATM inhibitors and chronic effects of ATM inhibition. Using an inducible lentiviral CRISPR/Cas9 genome-editing system, we generated in-frame ATM mutated clonal lines derived from a bladder cancer PDX model (BLX1), via targeting of ATM exon 57, which maps to the second exon in the kinase domain. CRISPR-edited clones consistently exhibited reduced phosphorylation of Kap1 (S824) but not CHK2, p53 or NBS1 when challenged with DSB-inducing agents such as ionizing radiation (IR) and etoposide. Similar results were obtained in unedited cells treated with the ATM inhibitor KU55933, confirming that changes observed are ATM-dependent and suggesting that reduced Kap1 phosphorylation is a robust readout of compromised ATM activity in response to DSB-inducing therapies. Radiosensitivity is a hallmark of the A-T syndrome and the CRISPR-edited clones had increased sensitivity to DSB-inducing agents including IR, bleomycin, etoposide, and doxorubicin but not to S-phase drugs gemcitabine and camptothecin in a 10-day colony formation assay. ATM deficiency increased the dependency of cancer cells on ATM-independent repair mechanisms. Indeed, short-term (4-day) proliferation assays demonstrated the synthetic lethality of KD ATM with ATR inhibitors (AZD6738, VE821), Chk1 inhibitors (MK8776, LY2606368), as well as high concentrations of PARP inhibitors (Olaparib, BMN673) but not the DNAPK inhibitor (NU7441). KD ATM clones did not have increased response to cisplatin as a single agent except at high concentrations (>10 µM); the combination of low-concentration cisplatin (<1 µM) and an ATR inhibitor had a greater effect in KD ATM clones compared to unedited cells, demonstrating increased dependence on ATR. In summary, CRISPR-Cas9 editing of the ATM kinase domain was a useful tool for characterizing ATM loss-of-function effects, identifying targets to resensitize ATM mutants and elucidating compensatory mechanisms driven by chronic ATM loss. Funded by NCI Contract No. HHSN261200800001E. This research was supported, in part, by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Citation Format: Lara H. El Touny, John Connelly, Curtis Hose, Anne Monks, Dianne Newton, Luke Stockwin, Melinda Hollingshead, Ralph Parchment, James H. Doroshow, Beverly A. Teicher, Annamaria Rapisarda. Modeling ATM loss of function via CRISPR-Cas9 as a predictive tool for therapeutic responses in cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A117.