Abstract
5-Azacytidine (5AZA) is a DNA methyltransferase inhibitor (DNMTi) used clinically to treat myelodysplastic neoplasm (MDS), and is used off-label for a number of malignancies including acute myeloid leukemia. This cytidine analog depletes intracellular DNMT1, and it has been hypothesized that DNMT1 depletion leads to hypomethylation and de-repression of methylated tumor suppressor genes. We used a pre-clinical model of MDS to investigate the efficacy of 5-azacytidine. Unexpectedly, we found an increased frequency of acute lymphoid leukemia (ALL) in 5AZA treated mice. Whole exome sequencing (WES) revealed a large number of C > G transversions in 5AZA treated mice, including genes known to be important for ALL such as Chd4, Ikzf1, and Trp53. Single base substitution (SBS) profiling revealed increased C > G mutations in the ALL cells, with a mutation signature similar to the previously described SBS39 signature. An in vitro GEMINI (Genotoxic Mutational Signature Identified After Clonal Expansion In vitro) assay recapitulated the finding of increased C > G mutations in both murine and human cell lines. Furthermore, similar GEMINI assays revealed induction of C > G mutations in cells treated with decitabine. Taken together, these findings demonstrate that azanucleosides induce C > G mutations both in vitro and in vivo, and are linked to leukemic transformation in murine cells.