Enzastaurin-induced cytotoxicity is enhanced in combination with the novel agent bortezomib, the anti-CD20 monoclonal antibody, rituximab, and fludarabine. (A) ADCC realized on BCWM.1. BCWM.1 cells were treated with enzastaurin (7.5 μM), rituximab (10 μg/mL), and the combination in absence and presence of activated effector cells. Results are reported in terms of mean percentage of specific lysis characterized by measurement of release of calcein-AM upon different effector-target ratios (E/T ratio). The following controls showed minimum released of calcein-AM and were not added to the figure: medium alone, BCWM.1 or activated effector cells alone, BCWM.1 incubated with activated effector cells in absence of rituximab, activated effector cells with rituximab in absence of target cells. (B) BCWM.1 cells were cultured with enzastaurin (2.5 and 5 μM) in the absence and presence of bortezomib (5 and 10 nM). Cytotoxicity was assessed with MTT assay. (C) BCWM.1 cells were cultured with enzastaurin (5 and 7.5 μM) in the absence and presence of fludarabine (5 and 10 μg/mL). Cytotoxicity was assessed with MTT assay. (D) Representative isobologram of enzastaurin associated to fludarabine with CalcuSyn software demonstrating synergy for the combinations 1 to 4: (1) enzastaurin 5 μM plus fludarabine 5 μg/mL; (2) enzastaurin 7.5 μM plus fludarabine 5 μg/mL; (3) enzastaurin 5 μM plus fludarabine 10 μg/mL; (4) enzastaurin 7.5 μM plus fludarabine 10 μg/mL.