Bi-functional thiopurine-based oligonucleotides for AML cell-targeted telomere damage and immunostimulation

Telomerase (TERT) is an enzyme commonly activated in human cancers and critical for maintaining cell survival. TERT expression and activation correlates with more aggressive, treatment-resistant disease, with the highest telomerase activity in relapsed acute myeloid leukemia (AML) patients. Telomerase inhibitors showed promise in preclinical studies on AML leading to leukemic stem cells (LSC) eradication. However, TERT inhibition in cancer cells faced challenges, such as the delayed clinical responses and on-target/off-tumor toxicities to hematopoietic stem cells (HSCs) or to activated T cells that result in cytopenias or immunosuppression, respectively. We previously developed clinically-relevant strategy for targeted delivery of therapeutic molecules into TLR9 + positive myeloid cells, such as AML cells including LSCs, using synthetic CpG oligodeoxynucleotides (CpG-ODNs) as a targeting domain. Here, we report generation of new CpG-conjugates for the delivery of a synthetic TERT substrate, 6-thio-2'-deoxy-guanosine (6tdG) into AML cells. CpG-6tdG oligonucleotides (CpG-6tdGOs) comprise multiple (5-10) 6tdG nucleosides in the 3' end of CpG-ODN. The CpG-6tdGOs retain serum stability, while allowing for slow release of the 6tdG nucleotides after uptake into target leukemic cells. In vitro , CpG-6tdG-oligonucleotides (CpG-6tdGOs) were selectively cytotoxic to human TLR9 + /TERT + AML cells without affecting activated TLR9 - /TERT + T-cells, HSCs or non-malignant TERT - cells. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed that 6tdG was effectively incorporated into chromatin of target cancer cells after in vitro and in vivo treatment using CpG-6tdGO. Repeated intravenous injections of CpG-6tdGO, but not 6tdG nucleoside, within days induced cytotoxic effects against xenotransplanted models of primary human AML with diverse genomic background in immunodeficient NSG mice and inhibited leukemia progression. CpG-6tdGO showed enhanced antitumor activity when tested in vivo against syngeneic Cbfb/MYH11/Mpl (CMM) and C1498 mouse AML models. In immunocompetent mice, treatment with CpG-6tdGO induced systemic, cancer cell-selective and CD8 T-cell-mediated antitumor immune responses that were at least partly dependent on TLR9- and STING-mediated signaling in response to CpG-6tdGO-induced cancer cell death. Importantly, the repeated treatments were well-tolerated in humanized hCD34/NOG mice. Except for the reduced percentage of human B-cells, CpG-6tdGO did not decrease the numbers of HSCs, myeloid cells, or T-cells. Overall, CpG-6tdGO offers an effective and safer strategy against aggressive TERT + hematologic malignancies and potentially certain solid tumors.