Decreased H3K27me3 enrichment caused by PRC2 disruption strikingly enhances the activation of FLT3 and therefore the downstream pathway. (A) Hierarchical clustering profile of the top 60 differentially expressed genes (P < .001) by whole-transcriptome sequencing. Columns indicate genes, and rows represent PRC2^Mut patients. The normalized expression level for each gene (z-score) is indicated by a color (red for overexpression and blue for underexpression). PRC2 genetic makeup and T-ALL subtype of each patient are also shown. (B-D) FLT3 messenger RNA level verified in T-ALL, ETP-ALL, and non-ETP-ALL patients. (E) FLT3 expression of leukemic cells from 4 T-ALL patients: TJCT003 (EZH2^Mut-P1), TJCT006 (EZH2^Mut-P2), TJCT021 (EZH2^WT-P2 [PRC2 WT]), and TJCT037 (EZH2^WT-P1 [PRC2 WT]) detected by flow cytometry. (F-G) FLT3 expression and phosphorylation in EZH2-knockout (EZH2-KO) (Δ/Δ and WT/Δ) and EZH2^WT Jurkat cell clones. (H) EZH2 expression, H3K27me3 modification, and FLT3 downstream signaling pathway analyzed by western blotting in EZH2-KO (Δ/Δ and WT/Δ) and EZH2^WT Jurkat cell clones. (I-J) H3K27me3 (I) mark and (J) POLII enrichment at the transcriptional start site of FLT3 measured by chromatin immunoprecipitation quantitative PCR in EZH2 KO cells (JE043 and JE010 clones). Normal rabbit immunoglobulin G (IgG) was used as the control. (K) FLT3 expression levels and downstream pathway activities assessed in EZH2 WT (Jurkat-Cas9), heterozygous KO (JE043), and homozygous KO (JE010) Jurkat cells after being treated with vehicle (dimethyl sulfoxide [DMSO]), sorafenib (10 nM), and quizartinib (10 nM) for 24 hours. All the WT and EZH2 KO Jurkat cells were cultured with normal medium (RPMI-1640 and 10% fetal bovine serum [FBS]) and supplied with FLT3 ligand (5 ng/mL). All experiments were performed in triplicate. Data are presented as the mean ± standard deviation (SD). Error bars: SD of 3 independent experiments. ***P < .001. ns, not significant.