Pharmacologic inhibition of EZH2 in WT1 mutated AML promotes myeloid differentiation. (A) Time course of inhibition of H3K27 trimethylation by GSK-126 in WT1 mutation–positive CTS cells. Western blots were performed on CTS cells after treatment with 1 μM GSK-126 for the time intervals as shown. Whole cell extracts were blotted for anti-H3K27me3, anti-histone H3 (total), and anti-actin. (B) CTS cells were treated with dimethylsulfoxide (DMSO), 1 μM ATRA, or 10 μM GSK-126 and 1 μM ATRA for 72 hours, after which the percentage of cells expressing CD11b was determined by flow cytometry. Bars represent mean of 3 replicates plus standard deviation. A representative of 3 independent experiments in shown. *P < .05, Student t test, 2-tailed. (C) Flow plots showing upregulation of CD15 and CD11c after 72 hours of GSK-126 treatment. Primary AML blasts were obtained from an adult patient (SU383) with a 2-bp insertion mutation in exon 7 of WT1 at P381 and sorted for blasts expressing CD117 and CD34 and cultured in either DMSO or 1 μM GSK-126 for 72 hours. (D) Summary of fold increase in CD15 antigen expression after GSK-126 treatment in vitro compared with DMSO for WT1mut+ AMLs SU359, SU383, and SU480, and 9 WT1 wild-type AMLs as shown, including 1 acute promyelocytic leukemia, SU600. *P < .05; **P < .001, Student t test unpaired, 2-tailed. Statistical significance is only indicated for AMLs that responded with upregulation of CD15 after treatment. (E-G) The fold increase in CD11c (E), CD11b (F), and CD14 antigen expression (G) as measured by flow cytometry in cultured primary AML WT1mut+ blasts after 72 hours of GSK-126 treatment is presented. *P < .05; **P < .001, Student t test unpaired, 2-tailed.