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Background Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by uncontrolled clonal proliferation of myeloid cells and potent immune tolerance. Our recent studies demonstrated the critical role of STAT3/DNMT1 signalling axis in the epigenetic suppression of leukemic cell differentiation and antigen-presentation. 1 2 Methods Here, we have developed a new strategy for direct DNMT1 inhibition in TLR9 + human and mouse AML cells using a specific RNA aptamer linked to CpG-oligodeoxynucleotide (ODN) as a targeting and immunostimulatory moiety.3 The double-stranded design of the conjugate facilitated efficient intracellular delivery of DNMT1-specific aptamer into a panel of human and mouse AML cells, including leukemia progenitor and stem cells (LPC/LSC).Results CpG-DNMT1apt oligonucleotides almost completely abrogated DNMT1 activity and leukemic cell proliferation while upregulating markers of immunogenic cell death (calreticulin) as well as myeloid cell differentiation (CD11b, HLA-DR) on AML cells both in vitro and in vivo. Compared to azacitidine as the benchmark hypomethylating agent, the intravenous injections of CpG-DNMT1apt conjugate inhibited progression of xenotransplanted human MOLM13 leukemia in immunodeficient mice and significantly extended animal survival. In immunocompetent mice, in, TLR9-targeted DNMT1 inhibition resulted in regression of mouse inv(16) AML with the evidence of leukemic cell differentiation, CD8 T cell activation and recruitment into spleen and bone marrow. Finally, we assessed the effect of CpG-DNMT1apt on a panel of patient-derived AML blasts with diverse genetics, including FLT3ITD, IDH1 and/or NPM1 mutations. CpG-DNMT1apt induced cytotoxicity to AML cells and specifically LPC/LSC subsets in 4 out of 8 tested specimens. In contrast, both azacitidine and venetoclax (Bcl2 inhibitor) showed none or only moderate cytotoxic activity on human LPC/LSC in vitro.Conclusions The myeloid cell-targeted DNMT1 inhibition combined with TLR9-driven immunostimulation offers potentially safer and more effective therapeutic approach for patients with therapy-resistant and high-risk AML compared to standard hypomethylating agents. The myeloid cell-targeted DNMT1 inhibition combined with TLR9-driven immunostimulation offers potentially safer and more effective therapeutic approach for patients with therapy-resistant and high-risk AML compared to standard hypomethylating agents.References Zhang Q, Hossain DM, Duttagupta P, Moreira D, Zhao X, Won H, Buettner R, Nechaev S, Majka M, Zhang B, Cai Q, Swiderski P, Kuo YH, Forman S, Marcucci G, Kortylewski M. Serum-resistant CpG-STAT3 decoy for targeting survival and immune checkpoint signaling in acute myeloid leukemia. Blood. 2016. PMID: 26796361; PMCID: PMC4817311.Esposito CL, Autiero I, Sandomenico A, Li H, Bassal MA, Ibba ML, Wang D, Rinaldi L, Ummarino S, Gaggi G, Borchiellini M, Swiderski P, Ruvo M, Catuogno S, Ebralidze AK, Kortylewski M, de Franciscis V, Di Ruscio A. Targeted systematic evolution of an RNA platform neutralizing DNMT1 function and controlling DNA methylation. Nat Commun. 2023. PMID: 36609400; PMCID: PMC9823104.Wang D, Kaniowski D, Jacek K, Su YL, Yu C, Hall J, Li H, Feng M, Hui S, Kaminska B, DeFranciscis V, Esposito CL, DiRuscio A, Zhang B, Marcucci G, Kuo YH, Kortylewski M. Bi-functional CpG-STAT3 decoy oligonucleotide triggers multilineage differentiation of acute myeloid leukemia in mice. Mol Ther Nucleic Acids. 2024. PMID: 39171140; PMCID: PMC11338104.