EZH2 is upregulated in WT1 mutated AML and contributes to myeloid differentiation block. (A) Box-and-whisker plots showing log2-normalized RNA Seq expression data for EZH2, SUZ12, and EED in NK AML with and without mutation in WT1 from TCGA patient data. Bar represents Student t test was used to determined statistical significance. (B) Heatmap of β methylation values of WT1me CpG sites in primary AML sample SU359, CTS cells, 11 AML TCGA patients with WT1 mutation, and NK AML TCGA patients without WT1 mutations. (C) CTS cells with endogenous mutation in WT1 were treated with ATRA for 72 hours, and CD11b expression was measured by flow cytometry. Cumulative data from 3 independent experiments is presented with statistical significance determined by unpaired 2-tailed Student t test. (D) CTS cells were transduced with scrambled or EZH2-specific shRNAs and RFP using lentivirus. RFP-positive cells were sorted after 5 days and whole cell lysates were blotted with anti-EZH2, anti-H3K27me3, and anti-actin–specific antibodies. (E) Sorted RFP-positive CTS cells expressing scrambled or EZH2 targeting shRNAs #2 or #3 were passaged for 7 days and stained with anti-CD11b antibody. The percentage of CD11b-positive cells compared with isotype control (left); geometric mean fluorescence intensity (right). Bars indicate standard deviations of a representative experiment performed in triplicate. **P < .001, Student t test unpaired, 2-tailed.
