Deletion of Gadd45a enhances oncogenic potential and LSC activity on serial transplantation while inducing mutations. (A) Schematic overview of the experimental procedure. (B-E) Kaplan-Meier mouse survival curves of primary AML (B), secondary AML (C), tertiary AML (D), and quaternary AML (E). Primary AML was generated by transplanting 1 × 10⁶Gadd45a^−/− vs Gadd45a^+/+ preleukemic cells, induced by MLL-AF9-GFP, into sublethally irradiated C57BL/6 (BL6) recipient mice (n = 6 for each group). Secondary AML, tertiary AML, and quaternary AML were generated by transplanting 1 × 10⁴ GFP⁺ leukemic bone marrow (BM) cells flow sorted from mice with primary, secondary, and tertiary AML, respectively, into recipient mice (n = 6 for each group). P value was determined by the log-rank test. (F) In vivo limiting dilution transplantation assay showing a 10-fold increase in LSC frequency in Gadd45a^−/− vs Gadd45a^+/+ mice (1/142 vs 1/1650; P = .00114). LSC frequency was calculated using L-Calc software (StemCell Technologies). Kaplan-Meier survival curves of mice receiving the indicated number of GFP⁺ leukemic BM cells (n = 6 for each group), and P value was determined by the log-rank test. (G) Scatter plots with a bar graph depicting the percentage of Gr-1^-/lowc-Kit^high population in total GFP⁺ leukemic BM cells from Gadd45a^−/− vs Gadd45a^+/+ mice (n = 5 for each group). Data are given as mean ± SD. ∗∗∗P < .0005. Unpaired t-test. (H) Stacked bar plots displaying the number of coding mutations identified by whole-genome sequencing (WGS) of Gadd45a^−/− vs Gadd45a^+/+ LSCs from primary (1^st) and tertiary (3^rd) AML. Green, yellow, and silver stacked bars represent synonymous, missense, and others (inframe insertion/deletion, frameshit, splice donor variant in coding sequence, start lost, and stop retained/gained), respectively. (I) Distribution of the coding mutations in Gadd45a^−/− vs Gadd45a^+/+ LSCs from primary AML. (J) Flow cytometry histograms showing decreased intracellular ROS in Gadd45a^−/− LSCs compared with Gadd45a^+/+ LSCs. (K) The number of colonies following treatment of Gadd45a^−/− vs Gadd45a^+/+ AML LSCs with dimethyl sulfoxide (DMSO) control or doxorubicin (DOX: 2.5 or 5 ng/mL) for 5 days in methylcellulose (n = 4). Data are given as mean ± SD. ∗P < .05, ∗∗P < .005, ∗∗∗P < .0005; NS, not significant (P > .05). One-way ANOVA. (L) In vivo BrdU proliferation assay with dot plots depicting BrdU⁺ GFP⁺ leukemic cells engrafted in the mouse BM at 12 days posttransplantation. Gadd45a^−/− or Gadd45a^+/+ AML LSCs were pretreated ex vivo with DMSO vs 5 ng/mL DOX for 48 hours, and 1 × 10⁵ treated cells were transplanted into BL6 mice for leukemic cell engraftment and subsequent BrdU proliferation assay. Data are presented as the mean percentage relative to DMSO ± SD (n = 5 mice per group). ∗∗∗∗P < .0001; NS, not significant (P > .05). Unpaired t-test. (M) Flow cytometry histograms showing intracellular ROS levels in Gadd45a^−/− vs Gadd45a^+/+ AML LSCs treated with DMSO vs DOX (2.5 or 5 ng/mL) for 5 days, or pretreated with 1 mM N-acetylcysteine (NAC) for 1 hour, followed by treatment with 100 μM menadione for an additional 1 hour.