Mutant KI increased susceptibility to leukemic transformation. (A) Proportion of RUNX1 mutations among ASXL1-mutated (left, n = 64) or ASXL1-WT (right, n = 304) MDS patients and types of RUNX1 mutations in each group. Statistical significance was evaluated by chi-square test. (B) Kaplan–Meier analysis for survival (left) and cumulative incidence of leukemic transformation (right) depending on mutational status of ASXL1 and RUNX1 in MDS patients. (C) Enumeration of peripheral WBCs, RBCs, hemoglobin (Hb), and MCV in mice transplanted with RUNX1-S291fsX–transduced Vav-Cre–negative Asxl1-MT^fl/wt (control + RUNX1-S291fsX, white circle, n = 6) or Vav-Cre–positive Asxl1-MT^fl/wt 3 mo after transplantation (KI + RUNX1-S291fsX, black circle, n = 6). (D) Representative hepatosplenomegaly in the KI group. (E) Kaplan–Meier analysis for the survival of transplanted mice (n = 6 each). (F) Cytospin preparations of BM (top) and spleen (middle) and pathological analysis of liver (bottom) from control and KI group. Bars: 20 µm (top and middle) and 50 µm (bottom). (G) Morphological abnormality of PB cells found in KI group 3 or 4 mo after transplantation. Howell–Jolly bodies are indicated by arrows (right). Bars, 10 µm. (H) Proportion of LSK in Lin⁻ cells and CMPs, GMPs, and MEPs in Lin⁻c-Kit⁺ BM cells of the recipient mice transplanted with control (Vav-Cre–negative Asxl1-MT^fl/fl) plus empty vector (white, n = 3), KI (Vav-Cre–positive Asxl1-MT^fl/fl) plus empty vector (black, n = 5), control plus RUNX1-S291fsX (blue, n = 6), or KI plus RUNX1-S291fsX mutation (red, n = 6). (I) Proportion of LT-HSC and MPP2 (CD150⁺CD48⁺LSK), MPP3/4 (CD150⁻CD48⁺LSK) in Lin⁻ BM cells of the recipient mice transplanted with control (Vav-Cre–negative Asxl1-MT^fl/fl) plus empty vector (white, n = 3), KI (Vav-Cre–positive Asxl1-MT^fl/fl) plus empty vector (black, n = 4), control plus RUNX1-S291fsX (blue, n = 4), or KI plus RUNX1-S291fsX mutation (red, n = 4). All mice were euthanized 3 mo after transplantation. (J) Absolute number of colonies (BFU-Es, CFU-Es, and CFU-GMs) using control or KI BM cells infected with or without the RUNX1-S291fsX mutation. For the CFU-E and BFU-E assay, 10⁵ and 2 × 10⁴ BM cells per well were used, respectively. For the CFU-GM assay, 2 × 10⁴ BM cells per well were used and replated similarly every week. The color of bars represents the same group shown in I. Duplicate samples were plated. (K) Kaplan–Meier analysis for the survival after injection of MOL4070A (control, white circle, n = 8 and KI, black circle, n = 8). (L) Representative hepatosplenomegaly in KI mice plus MOL4070A group. (M) Cytospin preparations of spleen (top) and BM (bottom) cells derived from mice with KI mice plus MOL4070A. Bars, 20 µm. (N) Asxl1-MT KI accelerated MLL-AF9 induced leukemia. Kaplan–Meier curve for the survival of the mice transplanted with MLL-AF9–transduced control (Vav-Cre–negative Asxl1-MT^fl/wt) and KI (Vav-Cre–positive Asxl1-MT^fl/wt) BM cells (n = 6 each) are shown. Data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student’s t test). Log-rank test was used to compare the survival distributions of two samples.