MKD reduces proliferation and clonogenicity of B-ALL cells in vitro and tumorigenicity in vivo. (A) Knockdown efficiency of MondoA by doxycycline-inducible shRNA against MondoA compared with control shRNA (shCTRL, Tet-On) by qRT-PCR after treatment with doxycycline for 48 hours. Data are mean and SEM of n = 6 experiments, P < .05 (Reh) and P < .001(Nalm6). (B) Western blot of knockdown efficiency after 48 hours of doxycycline. (C) Contact-independent growth in Nalm6 and 697 cells stably transfected with doxycycline-inducible shRNA either against MondoA or control. Data are mean of n = 2 experiments; P < .01; Mann Whitney test. (D-F) Long-term proliferation measured by BrdU assay. Control and MKD cells pretreated with doxycycline for 48 hours (n = 6 for each time point; P < .001; 2-way ANOVA). (G-H) Xenograft transplantation of Nalm6 and 697 cells stably transfected with doxycycline-inducible shRNA infectants in Rag2^−/−yc^−/− mice. MKD clones and control Nalm6 and 697 cells were injected into the tail veins of immunocompromised mice. Serial sectioning of liver and spleen tissue, including measurements of liver and spleen weights as well as the relative amount of human CD10⁺ leukemic blasts in blood, BM, and spleen were then assessed. The reduced number of leukemic cells was also confirmed by hematoxylin-and-eosin (H&E) staining of formalin-fixed paraffin-embedded spleen and liver tissue. H&E staining of formalin-fixed paraffin-embedded liver and spleen (scale bar, 1.0 mm). Images captured from H&E staining of the 4-µm sections of paraffin blocks by Axioplan2 microscopy (Carl Zeiss) and AxioVs40 V.4.8.2.0 software. (I) Weight of spleen in grams. Cell lines either expressing shRNA against MondoA or control, as indicated (n = 5 in each group; P < .05). (J) Weight of liver in grams. Cell lines either expressing shRNA against MondoA or control, as indicated (n = 5 in each group, P < .01, results are presented as mean plus or minus SEM; Mann-Whitney test). MKD was associated with a significant reduction in average size and weights of spleen and liver (P < .05 and P < .01). CD10⁺ leukemic blasts in BM (K), spleens (L), and blood (M) in cell lines either expressing shRNA against MondoA or control, as indicated (P < .001 [Nalm6] and P < .01 [697]; results are presented as mean plus or minus SEM; Mann Whitney test). ddCt, ΔΔ cycle threshold; ns, not significant. *P < .05; **P < .01; ***P < .001.