Synergism between BTK and PI3Kδ inhibition in ibrutinib-resistant GCB lymphoma cells. (A) Schematic representation BCR signaling. Ibrutinib and idelalisib block 2 effectors downstream of the BCR. (B) Percentage of viable cells in the indicated GCB lymphoma cell lines, treated with DMSO, ibrutinib, and idelalisib as single agents or in combination, at the indicated concentrations, for 72 hours. Each cell line was analyzed in triplicate, and data are shown as a bar graph corresponding to the mean ± SD. (C) Induction of apoptosis in SU-DHL-10 cell lines treated with ibrutinib and idelalisib as single agents or in combination at the indicated concentrations. Data are shown as a bar graph corresponding to the mean ± SD of 3 replicates. P values were calculated using 2-tailed Student t test. Significant changes between DMSO-treated and ibrutinib- and/or idelalisib-treated cells were labeled with **P ≤ .01; ****P ≤ .0001. (D) Phosphoflow cytometry analysis and quantification of pBTK (Tyr223), pCD19 (Tyr 531), and pGSK3β (Ser 9) in stimulated SU-DHL-10 cells pretreated with DMSO or ibrutinib (0.5 μM) and/or idelalisib (1 μM) for 6 hours. Data are represented as bar plots corresponding to the mean ± SD of 3 replicates, normalized to the stained, unstimulated controls. Significant changes between stimulated cells (orange bars) and stimulated cells treated with ibrutinib (blue bars), idelalisib (light blue bars), or a combination of the 2 (red bars) are labeled with *P ≤ .05; **P ≤ .01; ***P ≤ .001. Unlabeled bars indicate not statistically significant changes. (E) Western blot analysis of the indicated cell lines treated with DMSO or ibrutinib (0.5 μM) and/or idelalisib (1 μM) for 24 hours. Signal quantification was performed using Image Studio Lite and normalized to the DMSO-treated control. CTR, control; IBR, ibrutinib; ID, idelalisib.