Figure 5.

DNMT1 inhibition and CpG/TLR9 stimulation synergize to stimulate CMM cell differentiation and leukemia regression in vivo

C57BL/6 mice with established, disseminated CMM leukemia were treated i.v. using azacitidine (1 mg/kg), CpG oligonucleotide (1 mg/kg), a combination thereof, or PBS every day for six times. Two days after the last treatment, mice were euthanized to analyze AML burden by measuring spleen weight (A) and size (B), bone marrow appearance (C), and the percentages of proliferating GFP⁺/c-Kit⁺ leukemic cells in spleen and bone marrow (D and E). (F) The combined azacitidine/CpG treatment induced expression of IRF8 in splenic leukemic cells as assessed using flow cytometry (F) and western blotting (G). (H and I) The combination of DNMT1 inhibitor and TLR9 stimulation promoted (H) differentiation and maturation of CMM cells to antigen-presenting phenotype (CD11b⁺/MHC-II⁺/CD86⁺) with (I) the increased recruitment of predominantly CD8 T cells with smaller percentages of regulatory T cells; means ± SEM (n = 5). Shown are results representative of two independent experiments. Statistically significant p values were indicated as follows: ∗∗∗∗, p < 0.0001; ∗∗∗, p < 0.001; ∗∗, p < 0.01 and ∗, p < 0.05.