EZH2 mutations prevent EZH2 inhibitor binding. (A) SU-DHL-10 cells overexpressing either EZH2^WT, EZH2^C663Y, EZH2^E720G, EZH2^Y726F, or empty vector were treated with the specified concentrations of GSK126 for 72 hours and assayed for cell viability using an MTT assay. The cell viability compared with untreated cells is shown. (B) SU-DHL-10 cells overexpressing either EZH2^WT, EZH2^C663Y, EZH2^E720G, EZH2^Y726F, or an empty vector were treated with GSK126 (1 μM) for 48 hours and analyzed for EZH2 and H3K27me3 by immunoblotting. Histone H3 and ACTINB were used as loading controls. The densitometric quantitation for H3K27me3 is presented below the respective blots and were normalized to total histone H3. (C) SU-DHL-10 cells overexpressing either EZH2^WT, EZH2^C663Y, EZH2^E720G, EZH2^Y726F, or an empty vector were treated with GSK126 (5 μM) for 3 or 10 days and apoptosis was analyzed by annexin V–FITC staining. (D-E) Athymic nude mice were subcutaneously injected with KARPAS-422 cells expressing either empty vector, EZH2^WT, EZH2^Y726F (D) or with empty vector or PI3KCA (E) and treated with GSK126 (50 mg/kg) every alternate day for 31 days. Mean tumor volume ± SEM (n = 5) and representative tumor images for the experiments presented in panels D and E under the indicated conditions are shown. AUC was calculated to allow for the comparison between the 2 curves and the P values were calculated using a Student t test. Data are presented as mean ± SEM. **P < .01; ***P < .001.