Activation of SIRT6 by DNA hypomethylating agents and clinical consequences on combination therapy in leukemia

The FDA-approved DNA hypomethylating agents (DHAs) like 5-azacytidine (5AC) and decitabine (DAC) demonstrate efficacy in the treatment of hematologic malignancies. Despite previous reports that showed histone acetylation changes upon using these agents, the exact mechanism underpinning these changes is unknown. In this study, we investigated the relative potency of the nucleoside analogs and non-nucleoside analogs DHAs on DNA methylation reversal using DNA pyrosequencing. Additionally, we screened their effect on the enzymatic activity of the histone deacetylase sirtuin family (SIRT1, SIRT2, SIRT3, SIRT5 and SIRT6) using both recombinant enzymes and nuclear lysates from leukemia cells. The nucleoside analogs (DAC, 5AC and zebularine) were the most potent DHAs and increased the enzymatic activity of SIRT6 without showing any significant increase in other sirtuin isoforms. ChIP-Seq analysis of bone marrow cells derived from six acute myeloid leukemia (AML) patients and treated with the nucleoside analog DAC induced genome-wide acetylation changes in H3K9, the physiological substrate for SIRT6. Data pooling from the six patients showed significant acetylation changes in 187 gene loci at different chromosomal regions including promoters, coding exons, introns and distal intergenic regions. Signaling pathway analysis showed that H3K9 acetylation changes are linked to AML-relevant signaling pathways like EGF/EGFR and Wnt/Hedgehog/Notch. To our knowledge, this is the first report to identify the nucleoside analogs DHAs as activators of SIRT6. Our findings provide a rationale against the combination of the nucleoside analogs DHAs with SIRT6 inhibitors or chemotherapeutic agents in AML due to the role of SIRT6 in maintaining genome integrity and DNA repair. Overall design: We compared the relative potency of different DHAs in reversing DNA methylation and screened their activity against the NAD+-dependent HDAC sirtuins (SIRT1, SIRT2, SIRT3, SIRT5 and SIRT6). The nucleoside analogs DAC, 5AC and zebularine were more potent than the non-nucleoside analogs in reversing DNA methylation. Moreover, they significantly increased the activity of SIRT6 isoform in both recombinant enzyme assay and in nuclear lysates. DAC induced genome-wide changes in the acetylation of H3K9, the physiological substrate of SIRT6. This dataset includes H3K9ac ChIP-seq of DAC- or DMSO-treated bone marrow mononuclear cells from AML patients.