WT1 mutation is associated with repression of hematopoietic PRC2 targets and blocks myeloid differentiation. (A) Heatmap showing normalized U133A microarray expression of H3K27me3-marked genes (identified from the K562 H3K27me3 chromatin immunoprecipitation ENCODE data) that are normally upregulated in maturing myeloid cells in WT1mut AML (total of 26 patient samples, 11 from TCGA and 15 from Cancer and Leukemia Group B data sets) and normal CD34⁺CD38⁻ hematopoietic stem and progenitor cells, common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), mature granulocytes (Gran), and mature monocytes (Mo). (B) Gene set enrichment plot assessing the degree of PRC2 target gene repression in the WT1mut AML subgroup with respect to other NK AML subtypes (TCGA and CALBG). Genes were ranked according to the degree of downregulation in WT1mut AML compared with rest of NK AML based on the t test. A positive enrichment score (normalised enrichment score = 1.75, nominal P value = 0) indicates repression of H3K27me3 genes that are normally upregulated during myeloid differentiation in WT1mut AML when compared with other NK AML. (C) Human CD34⁺ cord blood stem/progenitor cells were transduced with lentiviruses encoding WT1mut–IRES-GFP, WT1 wild type–IRES-GFP, or GFP empty vector and cultured in myeloid differentiation media supplemented with IL-3, SCF, FLT3L, and GM-CSF. At day 6, GFP-positive cells were analyzed for expression of myeloid markers CD33, CD11b, and CD14. Flow cytometry analysis shows the percentage of CD14- and CD11b-positive cells within the GFP-positive populations, as well as untransduced cells from 1 representative experiment out of 3 independent experiments. (D) Summary of data from 3 independent experiments showing the percentage of CD33⁺/CD14⁺ and CD33⁺/CD11b⁺ cells compared with GFP control. Unpaired Student t test was used to determine statistical significance between GFP and WT1mut populations.