Knockdown of Kdm1a reduces leukemic burden in vivo. (A) Setup of mouse experiments. Top: for survival analyses, leukemic mice (WBCs ∼50 to 70 × 10⁹/L) were treated with Dox to induce Kdm1a-KD and observed until the clinical endpoints (defined in supplemental Materials). Blood was taken every week to monitor leukemic load and changes in other blood parameters. Bottom: for RNA-seq and cryosectioning, leukemic mice (WBCs ∼30 × 10⁹/L) were exposed to Dox for 2 weeks. For ChIP experiments, mice were treated with Dox for 2 weeks when WBCs reached 80 to 100 × 10⁹/L. (B) Significantly longer survival (Log-rank test) of Dox-treated leukemic iKdm1a^KD;Eμ-TCL1A (red, 12 females, median 67 days) vs Dox-exposed leukemic Eμ-TCL1A mice (blue, 12 females, median 39 days, P = .018) or vs non-Dox–exposed leukemic iKdm1a^KD;Eμ-TCL1A mice (green, 9 females, median 41 days, P = .036). (C) Significantly lower WBCs in iKdm1a^KD;Eμ-TCL1A (N = 8, red) leukemic mice under Dox treatment as compared with Eμ-TCL1A (N = 7, blue, P = .01, 2-way analysis of variance [ANOVA]). (D) Lower total number of PB leukemic CD19⁺CD5⁺ B cells (flow cytometry) in iKdm1a^KD;Eμ-TCL1A mice (N = 6, red) in comparison with those of Eμ-TCL1A (N = 6, blue, P = .013, Mann Whitney test, mean ± SEM). End: end point of survival experiment. (E) Lower percentage of CD19⁺CD5⁺ leukemic B cells in spleens and BM of iKdm1a^KD;Eμ-TCL1A vs Eμ-TCL1A mice under short term (≤2 weeks, left) or long term (≥4 weeks, right) in vivo Dox treatment (Mann Whitney test, mean ± SEM). (F) Top: representative images (taken at 60× original magnification) of immunofluorescent B220/KDM1A/Hoechst staining of spleen sections from Eμ-TCL1A (left) and iKdm1a^KD;Eμ-TCL1A (right) mice under Dox treatment for 2 weeks (B220 green, KDM1A red, Hoechst blue). Bottom: quantification of B220/KDM1A signals of spleen sections by StrataQuest showing reduced population of KDM1A⁺ cells (left, P < .001), B220⁺ B cells (middle, P = .002), and KDM1A⁺B220⁺ cells (right, P = .009) in spleens of iKdm1a^KD;Eμ-TCL1A (red) than that of Eμ-TCL1A (blue) mice (Mann Whitney test, mean ± SD). (G) Spleen volumetry after magnetic resonance imaging scanning of Eμ-TCL1A (left) and iKdm1a^KD;Eμ-TCL1A (right) mice (WBCs ∼50 × 10⁹/L) treated with Dox for 6 weeks. Representative images of abdominal region (red dashed line highlights splenic circumference). Bar chart displays splenic volumes before and under Dox treatment at 2, 4, and 6 weeks. Both groups presented significantly enlarged spleens at 6 weeks (N = 5, P < .05, Student t test, mean ± SEM). (H) Tissue cytometer analysis of B220/Ki-67 immunofluorescent signals of spleen sections by StrataQuest. Left: representative tissue cytometry plots show B220⁺/Ki-67⁺ B cells in spleens (Eμ-TCL1A blue, iKdm1a^KD;Eμ-TCL1A red). Right: quantified signal implicates decreased Ki-67⁺ B cells in spleens of iKdm1a^KD;Eμ-TCL1A vs Eμ-TCL1A animals (N = 4 of each group, P = .029, Mann Whitney test, mean ± SD). (I) Flow cytometric analysis of apoptotic leukemic B cells in spleens (annexin V staining). Left: representative density plots and histograms illustrate a smaller population of leukemic CD19⁺CD5⁺ B cells and a higher percentage of apoptotic leukemic cells in iKdm1a^KD;Eμ-TCL1A mice after 2 weeks of Dox treatment (comparison to Eμ-TCL1A). Right: higher percentages of apoptotic CD19⁺CD5⁺ leukemic B cells in spleens of iKdm1a^KD;Eμ-TCL1A compared with that of Eμ-TCL1A mice (N = 5 of each group, P = .008, Mann Whitney test, mean ± SD). BMMCs, bone marrow MCs.