The BCAA metabolism activity is required for the maintenance of core PRC2 components, including EZH2 and EED in human acute leukemia. (A) Enrichment plots of genes positively regulated by EZH2 in HSCs (KAMMINGA_EZH2_TARGETS) from THP-1, Kasumi-9, and Jurkat cells cultured in control or BCAA-free DMEM/F12 media supplemented with 10% FBS (upper panels). Heat map for the expression of HSC-related EZH2-target genes in THP-1, Kasumi-9, and Jurkat cells (lower panels). (B) Quantitative real-time PCR analysis of EZH2 and EED messenger RNA (mRNA) in primary CD34⁺ AML cells (n = 7) and CD34⁺ ALL cells (n = 6) cultured in control or BCAA-free media supplemented with 10% FBS for 48 hours. Relative expression of control samples is adjusted to 1.0. (C) Quantitative real-time PCR analysis of EZH2 and EED mRNA in primary CD34⁺ AML cells (n = 7) and CD34⁺ ALL cells (n = 6) treated with or without 20 mM of gabapentin for 24 hours. (D) A representative result of intracellular FCM analysis for quantification of EZH2 and EED levels in primary ALL cells treated with 1.0 IU/ml of L-asparaginase or 20mM of gabapentin for 24h. (E) Summary of the intracellular FCM analysis of EZH2 and EED levels in primary AML cells (n = 6) and ALL cells (n = 5) treated with L-asparaginase or gabapentin. The MFI of the control sample is set to 1.0. (F) A representative result of intracellular FCM analysis to quantify EZH2 and EED levels in AML cells cultured in Thr/Phe/Lys-free or BCAA-free media supplemented with 10% FBS for 48h. (G) Summary of the intracellular FCM analysis of EZH2 and EED levels in primary AML cells (n = 8) and ALL cells (n = 7) cultured in the control, the Thr/Phe/Lys-free, and the BCAA-free media supplemented with 10% FBS for 24 to 48 hours. The MFI of the control sample is set to 1.0. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, panels B, C, E, and G, mean ± SEM. EED, embryonic ectoderm development.