Increased RUNX1A:RUNX1C ratio induces malignant ML-DS phenotype. (A-C) Neonatal (top) and fetal (bottom) CD34⁺ HSPCs were lentivirally transduced with RUNX1A, RUNX1C, or EV control. (A) Colonies after plating CD34⁺ HSPCs from 2 independent donors in methylcellulose-based colony-forming unit (CFU) assays (mean ± standard deviation [SD], n = 2, 1-way ANOVA). Percentage of immature (CD41⁺CD61⁺/CD42⁻) and mature megakaryocytes (CD41⁺CD61⁺/CD42⁺) (B) and immature CD34⁺ cells after 7 days in media promoting megakaryocytic differentiation (mean ± SD, n = 5, 2-way ANOVA) (C). (D-F) Cells derived from patients with ML-DS were lentivirally transduced with RUNX1A, RUNX1C, or EV control. (D) Percentage of GFP⁺ transduced cells normalized to day 0 after transduction (mean ± SD, n = 3, 1-way ANOVA, ∗∗P < .01). Bar graphs show the percentage of CD41⁺CD117⁺ and CD41⁺CD117⁻ megakaryocytic cells (E) and CD33⁺CD117⁺ and CD33⁺CD117⁻ myeloid cells (F) 5 days after transduction (mean ± SD, n = 3, 2-way ANOVA). (G) Experimental setup for evaluating RUNX1A:RUNX1C restoration in vivo. ML-DS blasts from 2 patients were transduced with RUNX1A (GFP⁺), RUNX1C (GFP⁺), or EV control (GFP⁺) and mixed 1:1 with EV control–transduced blasts (dTomato⁺), before transplantation into sublethally irradiated recipient mice. (H) Ratio of GFP⁺ to dTomato⁺ cells in the bone marrow of mice euthanized 4 to 8 weeks after transplantation (n = 5, Kruskal-Wallis test). dT, dTomato; ns, not significant.