Acetate prevents myeloid differentiation of AMPK-deficient AML cells through Acss2-mediated production of acetyl-CoA. (A) Giemsa staining (upper panels) and flow cytometry to detect Gr-1 expression (lower panels) of AML cells cultured with or without acetate (n = 3). (B) Quantitative PCR to detect Myc, Meis1, and Hoxa9 expression in AML cells cultured with or without acetate (n = 3). (C) A schematic illustration of acetate conversion to acetyl-CoA by Acss. (D) Measurement of intracellular acetyl-CoA in AMPK KO AML cells by acetate supplementation (n = 3). (E) Immunoblotting of ACSS1, ACSS2, and ACSS3 after deleting the corresponding genes in MLL-AF9-induced AML cells with CRISPR/Cas9. Two sgRNA were used individually or in combination. (F) WT and AMPK KO AML cells were ablated for Rosa26 (control), Acss1, Acss2, or Acss3 and cultured in the presence (Ac) or absence (P: phosphate-buffered saline) of acetate, and cell proliferation was monitored (n = 3). (G) Acetyl-coA levels of MLL-AF9-induced AML cells after deleting Rosa26, Acss1, Acss2, or Acss3 (n = 3). (H) Survival of mice after transplanting Rosa26- or Acss2-deleted AML cells (n = 6). (I) Quantitative PCR of Myc and Hoxa9 transcripts in Acss2-deleted AML cells (red bars) compared with Rosa26-deleted cells (black bars) (n = 3). All data represent mean ± standard deviation. *P < .05; **P < .01; ***P < .001 by Student’s t-test or 1-way ANOVA, except for comparison of the survival curves, in which the significance was accessed by a log-rank test.