Synergistic effects of ibrutinib (IB) plus GSK126 in primary CLL cells. (A) Negatively selected CD19⁺ cells from 3 CLL cases were assessed for cell viability after prestimulation with CpG for 24 hours, followed by single IB or GSK126 treatment (1, 5, and 10 μM) or combined treatment with these drugs in a 1:1 ratio at the aforementioned doses for a total of 72 hours. In the graph, the y-axis depicts the fraction affected values and the x-axis depicts either the single drug dose treatment (blue and red labels) or the total drug dose after combinational treatment (green label). All dose–response curves plotted in the graph concern 1 representative case. (B) The interaction between IB and GSK126 at a 1:1 ratio in graded concentrations (1-10 μM) was synergistic (combination index [CI] values <1) in primary CLL cells. Scatter plots in the graph present the mean values of the CI of 3 CLL cases. Effects of incubation with CpG for 24 hours after single or combined treatment of 5 μM IB and/or 10 μM GSK126 for 3 days on CLL cell viability (C) and H3K27me3 levels (D) as measured by using flow cytometry. Connected points in the graphs represent the percentage of viable cells or H3K27me3 levels for each case in all conditions described, normalized to control cells (dimethyl sulfoxide [DMSO]–treated). The bars in the graphs show the mean values. Asterisks above bars indicate significant differences compared with CpG-treated cells (n = 7). (E) Each bar in the graph shows the mean values of FC of Bcl-2, Bcl-xl, Mcl-1, cleaved PARP, and cleaved caspase-3 (Casp-3) of stimulated cells with CpG for 24 hours after treatment with 10 μM GSK126 for 3 days, compared with CpG-stimulated control cells. Asterisks indicate significant differences compared with the CpG-stimulated control cells (n = 4; FC = –1.7, P < .05; FC = –1.6, P < .05; FC = –1.9, P < .05; FC = 1.8; and FC = 1.6, respectively). Effects of incubation with 10 μM of the EZH2 inhibitor GSK126 for 3 days on levels of H3K27me3 (F) and on CLL cell viability (G) using flow cytometry. Connected points in the graphs represent the percentage of H3K27me3 or viable cells treated with GSK126 or DMSO-treated control cells (n = 6). (H) Each bar in the graph shows the mean values of FC of Bcl-2, Mcl-1, cleaved PARP, and cleaved caspase-3 (Casp-3) of cells treated with 10 μM GSK126 for 3 days compared with DMSO-treated cells. Asterisks indicate significant differences compared with control cells (DMSO-treated) (n = 4; FC = –1.2, P < .05; FC = –1.4, P < .05; FC = 2; and FC = 2.5). *P < .05, **P < .01.