Systemic CpG-STAT3dODN treatment induces regression of syngeneic Cbfb/Myh11/Mpl leukemia in mice. C57BL/6 mice were injected IV with 250 000 fresh CMM cells. After 7 to 10 days when tumors were engrafted (ranging from 1% to 5% of AML cells in blood), mice were injected 6 times with CpG(1668)-STAT3dODN or control CpG(1668)-scrODN (5 mg/kg) every other day and euthanized a day after the last treatment. (A) Reduction of leukemia burden in spleen (top) or bone marrow (bottom) after treatment using CpG-STAT3dODN, STAT3dODN alone, or control CpG-scrODN. (Left) The representative flow cytometric results of GFP⁺c-Kit⁺ AML cells from various groups of mice. Shown are combined results from 6 mice per group; means ± SEM. Statistically significant differences between groups are indicated with asterisks. (B) Spleen sizes in various group of mice treated as described in panel A. (Left) Representative image of spleens harvested from mice at the end of experiment. (Right) Combined results showing spleen weight for each treatment group. (C) STAT3 inhibition by CpG-STAT3dOND results in decreased pSTAT3 and total STAT3 levels as well as in reduction of BCL-X_L. The western blot analysis of bone marrow from CpG-STAT3dODN– or control CpG-scrODN–treated mice. Similar results were derived from 3 independent experiments. (D) Band intensities were quantified densitometrically using ImageJ version 1.46 software based on identically exposed images. Statistically significant differences between CpG-STAT3 dODN and CpG-scrODN–treated groups are indicated by asterisks (**P < .003; ***P < .0001). (E) Systemic administration of CpG-STAT3dODN reduces leukemia-initiating potential in the CMM model. CMM cells were isolated from bone marrow of primary recipient mice treated as described in panel A. Magnetically enriched c-Kit⁺ AML cells pooled from CpG-STAT3dODN, CpG-scrODN, or untreated mice were combined and counted, and identical cell numbers were injected into secondary recipient mice. Long-term survival of the secondary transplant recipients is shown; n = 10 for each group. (F) Systemic STAT3 inhibition using a small-molecule Jak2/STAT3 inhibitor (SAR302503/TG101348) fails to improve survival of AML-bearing mice. Mice engrafted with CMM leukemia as described in panel A were treated 6 times every other day using CpG-STAT3dODN (5 mg/kg by IV injections), SAR302503 alone (100 mg/kg by oral gavage), or SAR302503 combined with CpG ODN (5 mg/kg by IV injections) vs control mice (treated with PBS); n = 6 in each treatment group.