ATR inhibition leads to increased replication stress and ATM/p53 dependent G1/S cell cycle arrest in CLL cells. (A) AZD6738 (1 μM) treatment of 1 hour led to increased replication stress in Mec1 (p53-defective) and CII-ATMsh (ATM-deficient) cells as demonstrated by DNA fiber analysis. Replicating DNA in cycling cells was sequentially labeled with CldU and IdU for 20 min each, after which DNA fibers were analyzed by immunofluorescence microscopy. Representative images are displayed. (B-C) AZD6738 (1 μM) treatment of 1 hour significantly reduced interorigin distance (B) and fork progression rate (C) in Mec1 and CII-ATMsh cells. (D-F) Cell cycle analyses on CII-GFPsh (D), CII-ATMsh (E), and Mec1 (F) cells were carried out following 24-hour treatment with either AZD6738 (1 μM or 3 μM) or RPMI media. AZD6738 treatment induced G1/S cell cycle arrest in CII-GFPsh cells but not CII-ATMsh or Mec1 cells. (G-H) Cell cycle analyses on TP53/ATM-wt CLL samples (CLL03, CLL06, CLL07) (G) and TP53-defective samples (CLL32, CLL35, CLL36) (H) were carried out following 48-hour treatment with AZD6738 (3 μM or 10 μM) or RPMI media. Analyses in panels G-H were performed on the viable cell population by expressing the G0/G1, S, and G2/M populations as a percentage of the total viable cells. Data are displayed as mean ± SEM of triplicate experiments. Statistical significance was determined using Student t test (B), Mann-Whitney U test (C), or 2-way ANOVA with Bonferroni post hoc analysis (D-H). Statistical significance vs dimethylsulfoxide (DMSO)–treated controls (B) or untreated controls (D-H) is indicated by *P < .05 and **P < .01.