SRA

Cultured acute myeloid leukemia (AML) blasts can differentiate into antigen-presenting cells but achieving leukemic cell immunogenicity proved challenging in clinical setting. Despite expressing multiple immunostimulatory receptors, such as Toll-like Receptor 9 (TLR9), AML cells resist immunostimulation. We previously demonstrated that eliminating Signal Transducer and Activator of Transcription 1 and 3 (STAT1/3) signaling in AML cells using decoy DNA strategy (CpG-STAT3dODN) synergized with TLR9 stimulation and resulted in T cell-mediated leukemia regression. Here, we interrogated the molecular underpinnings of CpG-STAT3dODN-driven differentiation and immunogenicity of mouse Cbfb/MYH11/Mpl AML. The transcriptional profiling of AML cells isolated from mice after inducible Stat3 silencing plus TLR9 stimulation or CpG-STAT3dODN treatment, revealed comparable upregulation of genes related to myeloid cell differentiation (Irf8, Cebpa) and immune stimulation (Gadd45a, Ciita, Il12a, Ifng) but decreased expression of leukemia-promoting Runx1 and Run1t1. The conditional Irf8 silencing in AML cells almost completely abrogated TLR9-driven leukemia cell differentiation. The AML cell reprogramming by CpG-STAT3dODN was likely regulated epigenetically as revealed by reduced global promoter hypomethylation of crucial myeloid cell differentiation and immune activation genes. These changes were associated with reduced expression of known STAT3 targets, DNMT1 and DNMT3a/b. Finally, we provide initial evidence of anti-leukemic effects of CpG-STAT3 inhibitor against human AML model in humanized mice. Our studies suggest that, beyond oncogenic functions, STAT3 in AML cells may be a primary checkpoint in control of immune-stimulatory and differentiation driving effects. These findings support further development of CpG-STAT3 inhibitors as a new bi-functional agents for AML immunotherapy. Overall design: To unravel the molecular mechanism underlying leukemic cell differentiation in response to combined STAT3-inhibition/TLR9-stimulation, we compared global gene expression in sorted GFP+/c-Kit+CMM cells derived from spleens of mice treated three times every other dayin vivousing CpG-STAT3dODN, CpG-scrODN or PBS.