AEB071 induces selective cytotoxicity in CLL cells and inhibits proliferation. (A-B) CD19⁺ cells from CLL patients (N = 11) were incubated with or without increasing concentrations of AEB071 for up to 72 hours. Viability was determined by MTS assay and was calculated relative to time-matched untreated controls. Dark lines represent averages. (C) Whole blood from CLL patients (N = 5) was incubated with AEB071 (2 μM) for 24 hours. Viability was determined by flow cytometry as described in “Methods.” Dark lines represent averages. (D) Whole blood from normal subjects (N = 5) was incubated without AEB071 (2 μM) for 24 hours. Viability was determined by flow cytometry as described in “Methods.” Dark lines represent averages. (E) CD19⁺ cells from CLL patients (N = 3) were treated with AEB071 (2 μM) and stimulated with CpG685 oligonucleotides (3.2 μM) for 24 hours. AKT phosphorylation at Ser473, GSK3-β phosphorylation at Ser9, and ERK phosphorylation at Thr202/Tyr204 was assessed by immunoblot. A representative blot with band quantification is presented; black line indicates cropped regions wherein only relevant bands are shown. (F) CD19⁺ cells from CLL patients (N = 17) were incubated with AEB071 (1 μM) and CpG685 (3.2 μM) for 72 hours. Viability was determined by MTS. Dark lines represent averages. (G) CD19⁺ cells from CLL patients (N = 10) were incubated with CpG685 (3.2 μM) and treated with AEB071 (1 or 2 μM) or PCI-32765 (1 μM) and proliferation was assessed 120 hours later by tritiated thymidine. Dark lines represent averages.