Endogenous HHEX promoted survival of leukemia cells expressing ASXL1-MT. (A) Schematic presentation for experimental procedure for panels B-F. (B) Levels of Hhex protein in cSAM/Cas9 and cRAM/Cas9 cells transduced with NT or 2 independent Hhex-targeting sgRNAs. (C, D) Depletion of endogenous Hhex promoted differentiation (C; n = 4 each) and apoptosis (D; n = 5 each) in cSAM/Cas9 cells and cRAM/Cas9 cells at day 4. One-way ANOVA with Tukey’s multiple comparisons test. (E) Depletion of endogenous Hhex reduced colony-forming activity in cSAM/Cas9 cells (n = 4) and cRAM/Cas9 cells (n = 6). (F) Depletion of endogenous Hhex abrogated leukemogenicity in cSAM/Cas9 cells and cRAM/Cas9 cells. Kaplan-Meier curve for the survival of the transplanted mice in each group (n = 6-8; log-rank test). (G) Levels of Hhex protein in ASXL1-mutated human leukemia cell lines (MEG-01, Kasumi-1, TS9;22, and NOMO-1) transduced with NT or 2 independent HHEX-targeting sgRNAs. (H) Four ASXL1-mutated leukemia cell lines were transduced with NT or HHEX-targeting sgRNAs (coexpressing tRFP657). The cells were cultured in vitro to monitor the changes of the frequency of tRFP657-positive cells in duplicated wells. Results are normalized to the frequency of tRFP657-positive cells of each population on day 4, set to 1. (I) Representative cytospin preparation of control or sgHHEX-transduced MEG-01 and Kasumi-1 cells (scale bars, 20 μm). (J) Representative FACS histogram of CD11b expression of control or sgHHEX-transduced MEG-01 cells at day 7 (left), and the frequency of CD11b-positive cells are shown (right; n = 4). One-way ANOVA with Tukey’s multiple comparisons test. (K) The frequencies of Annexin V–positive cells of control or sgHHEX-transduced MEG-01 and Kasumi-1 cells at day 4 (n = 4). Statistical analyses were performed by 1-way ANOVA with Tukey’s multiple comparisons test. Data are shown as mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001.