Myb and Etv5 expression were indispensable for ASXL1-MT induced myeloid leukemogenesis. (A) Levels of Myb protein in cSAM/Cas9 and cRAM/Cas9 cells transduced with NT or 2 independent Myb-targeting sgRNAs. Transduction of sgMyb #1 or sgMyb #2 resulted in profound reduction of Myb protein level in cSAM/Cas9 cells and cRAM/Cas9 cells. (B) Depletion of Myb promoted apoptosis in cSAM/Cas9 and cRAM/Cas9 cells at day 4 (n = 4). (C) Depletion of Myb reduced colony-forming activity in cSAM/Cas9 and cRAM/Cas9 cells (n = 5). (D) Levels of Etv5 protein in cSAM/Cas9 and cRAM/Cas9 cells transduced with NT or 2 independent Etv5-targeting sgRNAs. Transduction of sgEtv5 #1 or sgEtv5 #2 resulted in profound reduction of Myb protein level in cSAM/Cas9 cells and cRAM/Cas9 cells. (E) Depletion of Etv5 reduces colony-forming activity in cSAM/Cas9 and cRAM/Cas9 cells (n = 4). (F) Depletion of Etv5 promoted differentiation in cSAM/Cas9 and cRAM/Cas9 cells at day 4 (n = 4). (G) Schematic presentation of experiments in panels H and I. cSAM/Cas9 cells were transduced with vector/MYB/ETV5 (coexpressing KuO). Forty-eight hours after transduction, KuO⁺ cells were sorted by FACS. Sorted KuO⁺ cells were transduced with sgNT or Hhex-targeting sgRNAs (coexpressing puromycin resistance gene). After puromycin selection, cells were subjected to a colony-forming assay. (H) Cell lysates extracted from vector/MYB/ETV5-transduced cSAM/Cas9 cells were subjected to immunoblotting with anti-MYB (left), anti-ETV5 (right), and anti-GAPDH antibodies. $, Ectopically expressed MYB. #, Endogenous MYB. (I) Ectopic expression of MYB and ETV5 ameliorated colony-forming activity in Hhex-depleted cSAM/Cas9 cells (left; n = 4) and representative images of the colonies (scale bars, 1 mm). Data are shown as mean ± SEM. Statistical analyses were performed by 1-way ANOVA with Tukey’s multiple comparisons test. *P < .05; **P < .01; ***P < .001; ****P < .0001.