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STAT3 inhibition permits epigenetic reprogramming and TLR9/IRF8-driven differentiation of inv(16) acute myeloid leukemia [RNASEQ_AML_DECSCR]

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE233096 | GEO | 2024/07/23

REPOSITORIES: GEO

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Project description:Myeloid differentiation is blocked in acute myeloid leukemia (AML), but the molecular mechanisms are not well characterized. MN1 is overexpressed in some AML patients and confers resistance to all-trans retinoic acid (ATRA)-induced differentiation. To understand the role of MN1 as a transcriptional regulator in myeloid differentiation, we fused transcriptional activation (VP16) or repression (M33) domains with MN1 and characterized these cells in vivo. Transcriptional activation of MN1 target genes induced myeloproliferative disease with long latency and differentiation potential to mature neutrophils. A large proportion of differentially expressed genes between leukemic MN1 and differentiation-permissive MN1VP16 cells belonged to the immune response pathway like Irf8 and Ccl9. As MN1 is a co-factor of MEIS1 and RARA, we compared chromatin occupancy between MN1, MEIS1 and RARA. Immune response genes that were upregulated in MN1VP16 cells were co-targeted by MN1 and MEIS1, but not RARA, suggesting that myeloid differentiation is blocked through transcriptional repression of shared target genes of MN1 and MEIS1. Constitutive expression of Irf8 or its target gene Ccl9 identified these genes as potent inhibitors of MN1-induced leukemia. Our data show that MN1 prevents activation of the immune response pathway, and suggest that restoration of Irf8 signalling as a novel therapeutic target in AML. C57BL/6J bone marrow cells were harvested from mice treated for 4 days with 150 mg 5-fluorouracil/kg and stimulated for 48 hours in DMEM supplemented with 15% FBS, 10 ng/mL hIL6, 6ng/mL mIL3, and 20ng/mL mSCF. The cells were infected with MN1 or MN1VP16 retroviral constructs by cocultivation with irradiated E86 viral producer cells in the presence of 5μg/mL protamine sulfate (Sigma) for 48 hours and then transplanted into lethally irradiated syngeneic recipient mice. 4 weeks after transplantation MN1, MN1VP16 GFP+ cells and Gr1+/CD11b+ bone marrow cells were FACS-sorted and analyzed by Affymetrix GeneChip Mouse 430 2.0 (43.000 probes) microarrays

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STAT3 inhibition permits epigenetic reprogramming and TLR9/IRF8-driven differentiation of inv(16) acute myeloid leukemia [RNASEQ_AML_DECSCR]

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