Abstract 1484: 5-Aza-4'-thio-2'-deoxycytidine induces C to G transversions in a specific trinucleotide context and leads to acute lymphoid leukemia
Authors: Bertoli Ryan Matthew , Chung Yang Jo , Difilippantonio Michael , Wokasch Anthony , Marasco Madison R. , Klimaszewski Haley , Garber Susannah , Zhu Yuelin Jack , Walker Robert L. , Cao Dengchao , Doroshow James , Meltzer Paul , Aplan Peter D. 
Published: March 2024

Abstract
Myelodysplastic syndromes (MDS) are a diverse group of blood cancers characterized by peripheral blood cytopenias and dysplastic blood cell morphology. Up to 40% of patients with high risk MDS will transform to acute myeloid leukemia (AML), an aggressive blood cancer with significant morbidity and an overall 5-year survival of less than 35%. Both MDS and AML have been linked to hypermethylation of cytosine residues. Hypermethylation in the 5’ regulatory region can result in epigenetic downregulation of gene expression, and it has been speculated that hypermethylation of tumor suppressor genes can contribute to malignant transformation. DNA methyltransferase inhibitors (DNMTi), most commonly cytidine analogs, are compounds that are used clinically to decrease 5’-cytosine methylation, with the aim of inducing the re-expression of tumor suppressor genes resulting in a halt of tumor progression. 5-Aza-4’-thio-2’-deoxycytidine (Aza-TdCyd or ATC) is a cytidine analog that incorporates an aza modification (nitrogen in place of carbon) of the cytosine base and a thioether modification of the deoxyribose sugar. ATC is a promising new DNMT1i that has demonstrated efficacy in preclinical studies of solid tumors. Based on these prior studies, we sought to characterize the potential therapeutic effect of ATC in MDS.Mice that express a NUP98::HOXD13 (NHD13) transgene develop an MDS that closely recapitulates all of the key features of human MDS, including pancytopenia, dysplastic morphology, and transformation to AML. We transplanted NHD13 and wild-type (WT) hematopoietic stem and progenitor cells into WT recipients, and used this model of MDS to investigate the therapeutic potential of ATC. We found that ATC treatment led to both T and B lineage lymphoid leukemia (ALL) originating from the NHD13 cells. Surprisingly, ATC treatment also led to T and B lineage ALL in the WT cells, and further experiments demonstrated that non-transplanted, healthy WT mice developed T and B cell ALL after treatment with ATC. In sum, all mice that received at least 8 cycles of ATC therapy at the 1.0 mg/kg dose developed leukemia. Whole exome sequencing of tumors revealed thousands of acquired mutations, almost all of which were C&gt;G transversions in a previously unrecognized, specific 5’-NCG-3’ context. These mutations involved a number of genes well-known to be involved in human lymphoid leukemia, such as Notch1, Pten, Pax5, Trp53, and Nf1. Treatment of the human U937 and CEM cell lines in vitro showed thousands of acquired C&gt;G transversions in a similar context. Taken together, these findings demonstrate that ATC can be a potent mutagen in human and mouse cells, both in vitro and in vivo.
Citation Format: Ryan Matthew Bertoli, Yang Jo Chung, Michael Difilippantonio, Anthony Wokasch, Madison R. Marasco, Haley Klimaszewski, Susannah Garber, Yuelin Jack Zhu, Robert L. Walker, Dengchao Cao, James Doroshow, Paul Meltzer, Peter D. Aplan. 5-Aza-4'-thio-2'-deoxycytidine induces C to G transversions in a specific trinucleotide context and leads to acute lymphoid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1484.