ATR inhibition is selectively cytotoxic to both ATM-defective and TP53-defective CLL cells in vitro and in vivo. (A) CII-GFPsh, CII-ATMsh (ATM-deficient), and Mec1 (p53-defective) cells were treated with AZD6738 for 4 days, and viability was measured using the CellTiter-Glo assay. Surviving fraction is expressed relative to untreated controls. AZD6738 induced significantly greater dose-dependent cytotoxicity with significantly lower AZD6738 EC₅₀ in CII-ATMsh and Mec1 cells compared with CII-GFPsh cells. (B) Mec1 cells transfected with either wild-type TP53 (Mec1-p53-pcDNA3.1) or GFP (Mec1-GFP-pcDNA3.1, as control) were treated with AZD6738 for 4 days, and viability was measured using the CellTiter-Glo assay. Surviving fraction is expressed relative to untreated controls. AZD6738-induced cytotoxicity was reduced with significantly higher AZD6738 EC₅₀ in Mec1-p53-pcDNA3.1 cells compared with Mec1-GFP-pcDNA3.1 cells. (C) Carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled primary CLL cells with or without ATM/TP53 defects and healthy donor peripheral blood mononuclear cells (PBMCs) cocultured with CD40L/IL-21 were treated with AZD6738 for 4 days. Viability was measured by propidium iodide exclusion of the proliferating cell population that was identified by reduction in CFSE fluorescence intensity as shown in supplemental Figure 2. Surviving fraction is expressed relative to untreated controls. AZD6738 induced significantly greater dose-dependent cytotoxicity in ATM/TP53-defective CLL cells than either ATM/TP53 wild-type CLL cells or healthy donor PBMCs. (D) The EC₅₀ of AZD6738 was significantly lower for ATM/TP53-defective primary CLL samples than both ATM/TP53 wild-type samples and healthy donor PBMCs. A list of CLL samples assessed and their respective EC₅₀ values are provided in supplemental Table 1. (E) There was no significant difference in the EC₅₀ of AZD6738 between subgroups of CLL samples stratified according to Binet stage, prior treatment, and IGHV mutational status. (F) Primary CLL samples (CLL29, CLL31, CLL25, CLL26) with biallelic TP53 or ATM defects were engrafted into NOD/Shi-scid/IL-2Rγ^null mice and treated with vehicle (n = 11) or AZD6738 (n = 10). Collectively, AZD6738 treatment significantly reduced tumor load compared with vehicle treatment in TP53/ATM-defective xenografts. (G) When analyzed separately, both TP53 and ATM defective xenografts showed significant reduction in tumor load following AZD6738 treatment compared with vehicle-treated controls. (H) Fluorescence in situ hybridization probes for 11q were applied on human CD45⁺ CD19⁺ cells collected from the spleens of CLL26 xenografts (harboring del(11q) and L3013Q ATM mutation) treated with AZD6738 (n = 3) or vehicle (n = 3). Two hundred cells were analyzed per mouse. The proportion of CLL cells with del(11q) was significantly reduced following AZD6738 treatment compared with vehicle-treated controls. All data are displayed as mean ± standard error of the mean (SEM). Statistical significance was determined using 2-way analysis of variance (ANOVA) with Bonferroni post hoc analysis (A,B,C,G), 1-way ANOVA (A,D), or Student t test (B,F,H). Statistical significance vs CII-GFPsh (A), Mec1-GFP-pcDNA3.1 (B), ATM/TP53 wild-type samples (*) or healthy donor PBMCs (^†) (C,D), or vehicle (F-H) is indicated by *^,†P < .05, **^,††P < .01, and ***^,†††P < .001. Nonsignificant results are denoted by n.s.