Drug screen performed using patient PBMCs cultured in an ex vivo microenvironment model identifies novel venetoclax combinations that exhibit selective cytotoxicity in multidrug-resistant CLL cells. (A) Diagram presenting the experimental approach for combination drug screening in microenvironmentally induced multidrug-resistant CLL cells ex vivo. PBMCs of patients with CLL were preincubated with the combination of CpG-ODN, sCD40L, and IL-10 (agonist mix) for 12 hours. Then, samples were treated with venetoclax (25 nM) in combination with increasing concentrations of the IKKα/β inhibitor BMS345541 (2, 4, 6, or 8 µM), the MALT1 inhibitor MI-2 (0.18, 0.37, 0.75, or 1.5 µM), the proteasome inhibitor bortezomib (1, 2, 4, or 8 nM), or the Nedd8 activating enzyme inhibitor pevonedistat/MLN4924 (0.25, 0.5, 0.75, or 1 µM), as well as a second dose of agonist mix for 24 hours. (B) The cleaved PARP in CD5⁺/CD19⁺ (CLL) cells was analyzed by FCM. (C) Bar graphs presenting Bliss predicted and actual (ie, observed) cytotoxicity for the combination of venetoclax (25 nM) with BMS345541 (8 µM), MI-2 (1.5 µM), bortezomib (8 nM), or pevonedistat/MLN4924 (1 µM) in CLL cells. The Bliss predicted cytotoxicity was determined as described earlier.^16,44-46  Synergistic benefit is expected when the actual cytotoxicity is more than Bliss-predicted cytotoxicity. (D) The cleaved PARP in CD3⁺/CD5⁺ cells (normal T lymphocytes) was analyzed by FCM. The data in panels B and D are presented after subtracting spontaneous apoptosis values from DMSO treatment controls. Statistical significance was determined by ANOVA with Sidak’s post-hoc test for multiple comparisons. *P < .05; **P < .01; ***P < .001; ****P < .0001. Data are presented as mean ± SD.