ATR inhibition results in accumulation of DNA damage and mitotic catastrophe in CLL cells with ATM or p53 deficiency. CII cells pretreated for 2 hours with ATMi (10 μM), CII cells without ATMi pretreatment, and Mec1 (p53-defective) cells were exposed to AZD6738. (A-B) Cells treated with AZD6738 (1 μM) for 24 hours were colabeled with anti-γH2AX and anti-pH3 antibodies and analyzed by immunofluorescence microscopy using a ×60 lens. A cell was considered γH2AX positive if >5 γH2AX foci were present. At least 200 mitotic (phosphohistone H3 ser-10 [pH3]–positive) cells were analyzed in each sample. AZD6738 induced γH2AX foci in Mec1 and ATMi pretreated CII cells, but not in CII cells without ATMi pretreatment. (C) The pattern of 53BP1 labeling distinguishes 53BP1 bodies from 53BP1 foci. 53BP1 bodies are characterized by robust blocklike staining and indicate underreplicated DNA. 53BP1 foci are characterized by discrete punctate staining and indicate DNA damage. A cell was considered 53BP1 foci positive if >5 53BP1 foci were present. (D-F) Cells treated with AZD6738 (1 μM) for 48 hours were labeled with anti-53BP1 antibodies, and at least 200 cells were then analyzed in each sample using a ×60 lens. AZD6738 treatment led to an accumulation of 53BP1 foci in Mec1 and ATMi pretreated CII cells and an accumulation of 53BP1 bodies in CII cells without ATMi pretreatment. (G) Colabeling with anti-lamin B and anti-pH3 antibodies allows apoptotic CLL cells to be distinguished from cells undergoing mitotic catastrophe. At least 200 cells were analyzed in each sample. (H) AZD6738 exposure for 72 hours resulted in significantly elevated levels of mitotic catastrophe in Mec1 and ATMi pretreated CII cells. Data are displayed as mean ± SEM of triplicate results from a representative experiment. Statistical significance was determined using Student t test. Statistical significance vs untreated controls is indicated by *P < .05, ** P < .01, and ***P < .001.