ATR signaling is activated in response to replication stress in proliferating primary CLL cells and is inhibited by AZD6738. (A) Stimulation of primary CLL cell proliferation by coculture with CD40L-expressing murine embryonic fibroblasts in the presence of IL-21 (CD40L/IL-21) for 4 days resulted in induction of ATR expression in primary CLL cells irrespective of ATM or TP53 status. Cyclin A expression is a marker of proliferating cells. Actin is the loading control. (B) Cryopreserved or fresh primary CLL cells cultured with or without CD40L/IL-21 (lanes 1-4 and 9 and 10, and lanes 5-8, respectively) were treated with HU. Cryopreserved samples not cocultured with CD40L/IL-21 were resuspended and preincubated in culture media for 24 hours prior to treatment (lanes 5 and 6), whereas fresh cells were treated immediately upon isolation from peripheral blood without preincubation (lanes 7 and 8). Exposure to HU (1 mM), which induces replication stress, led to Chk1 phosphorylation in primary CLL cells cocultured with CD40L/IL-21 (lanes 2, 3, and 10). (C) TP53/ATM wild-type (TP53/ATM-wt) primary CLL cells cocultured with CD40L/IL-21 (C) and CII cells (D), both CII-GFPsh and CII-ATMsh, were treated with AZD6738 (1 μM) and/or the ATM inhibitor KU-55933 (ATMi; 10 μM) for 2 hours, or left untreated, prior to exposure to HU (1 mM) or IR (6 Gy) for a further 5 hours. AZD6738 treatment inhibited ATR signaling as indicated by a reduction in HU-induced Chk1 phosphorylation (panel C, lane 4 vs 6; panel D, lanes 3 vs 4 and 9 vs 10). In ATM-proficient CLL cells, this also led to ATM activation as evidenced by ATM phosphorylation and Chk2 phosphorylation (panel C, lane 4 vs 6; panel D, lane 3 vs 4). In panels B-C, representative blots from experiments on 3 CLL samples are shown.