ASXL1-MT and HHEX cooperatively blocked differentiation and enhanced the susceptibility of leukemic transformation in vivo. (A) Schematic presentation of BM transplantation model. (B) Kaplan-Meier curve for the survival of the mice transplanted with control/HHEX transduced c-Kit⁺ BM cells derived from control or ASXL1-MT-KI mice (n = 12 each group, log-rank test). (C) Representative hepatosplenomegaly in leukemic mice transplanted with HHEX-expressing c-Kit⁺ ASXL1-MT-KI BM cells. (D) Cytospin preparation of PB, BM, and spleen cells from leukemic mice induced by ASXL1-MT, and HHEX (upper, scale bars: 20 μm) and representative FACS plots (lower). (E) Distribution of the indicated surface markers in leukemic cells infiltrating BM induced by ASXL1-MT and HHEX (n = 5). (F-J) Analyses of nonleukemic mice of the indicated groups at the end-stage (control+EV, n = 4; ASXL1-MT+EV, n = 5; control+HHEX, n = 4; ASXL1-MT+HHEX, n = 5). (F) Blood counts of peripheral white blood cells (WBC), red blood cells (RBC), hemoglobin (Hb), mean corpuscular volume (MCV), and platelets (PLT). (G) BM chimerism of NGFR⁺ cells at the end-stage of nonleukemic mice and leukemic mice by ASXL1-MT and HHEX expression. (H, I) Lineage composition of the NGFR-positive cells from PB (H) and BM (I). (J) The frequency of HSPCs of the NGFR⁺ cells. (K) Cytospin preparation of NGFR⁺ BM cells from the indicated recipient mice (left) and the distribution of lymphocytes and mature, immature, and blast-like myeloid cells (right; n = 3). (F-K) 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. CMP, common myeloid progenitor; LK, Lineage⁻, Sca-1⁻, c-Kit⁺; LSK, Lineage⁻, Sca-1⁺, c-Kit⁺; MEP, megakaryocyte-erythroid progenitor.