DNA methyltransferase inhibitors (DNMTi) approved for older AML patients are clinically tested in combination with histone deacetylase inhibitors (HDACi). The mechanism of action of these drugs is still under debate. In colon cancer cells, 5-aza-2′-deoxycytidine (DAC) can downregulate oncogenes and metabolic genes by reversing gene body DNA methylation, thus implicating gene body methylation as a novel drug target. We asked whether DAC-induced gene body demethylation in AML cells is also associated with gene repression, and whether the latter is enhanced by HDACi.
Transcriptome analyses revealed that a combined treatment with DAC and the HDACi panobinostat or valproic acid affected significantly more transcripts than the sum of the genes regulated by either treatment alone, demonstrating a quantitative synergistic effect on genome-wide expression in U937 cells. This effect was particularly striking for downregulated genes. Integrative methylome and transcriptome analyses showed that a massive downregulation of genes, including oncogenes (e.g., MYC) and epigenetic modifiers (e.g., KDM2B, SUV39H1) often overexpressed in cancer, was associated predominantly with gene body DNA demethylation and changes in acH3K9/27. These findings have implications for the mechanism of action of combined epigenetic treatments, and for a better understanding of responses in trials where this approach is clinically tested.
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We thank Rainer Claus (Augsburg, Germany), Justus Duyster (Freiburg, Germany), Martin Schumacher (Freiburg, Germany) and Peter Jones (Grand Rapids, MN, US) for continuous helpful discussions. We thank the microarray unit of the DKFZ Genomics and Proteomics Core Facility for providing the Illumina Human Methylation arrays and related services. We thank Ruhtraut Ziegler, Carmen Strittmatter and Tobias Ma for the technical support provided. The authors acknowledge the support of the Freiburg Galaxy Team: Björn Grüning and Rolf Backofen, Bioinformatics, University of Freiburg, Germany funded by Collaborative Research Centre 992 Medical Epigenetics (DFG grant SFB 992/1 2012) and German Federal Ministry of Education and Research (BMBF grant 031 A538A RBC (de.NBI)).
NBD designed and performed experiments, analyzed and interpreted data, drafted and revised the manuscript. PS performed the statistical and bioinformatic analysis, interpreted data, drafted and revised the manuscript. GG performed experiments, analyzed and interpreted data, drafted and revised the manuscript. DP performed transcriptome analyses, interpreted data and revised the manuscript. RM performed experiments and analyzed data. AB performed the ChIP-seq experiments. DB performed the analyses of the ChIP-seq experiments (U937) and the TINATs data (NCI-H1299), and drafted the methods section on ChIP-seq and TINATs. CP designed the TINAT study, interpreted ChIP-seq data and revised the manuscript. ML designed the study, interpreted data, drafted and revised the manuscript. All authors read and approved the final manuscript.
This work was supported by the DFG (SPP 1463, CRC 992/MEDEP and FOR 2674) and the German José Carreras Leukaemia Foundation (DJCLS R 14/25).
Conflict of interest
The authors declare that they have no conflict of interest.
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Blagitko-Dorfs, N., Schlosser, P., Greve, G. et al. Combination treatment of acute myeloid leukemia cells with DNMT and HDAC inhibitors: predominant synergistic gene downregulation associated with gene body demethylation. Leukemia 33, 945–956 (2019). https://doi.org/10.1038/s41375-018-0293-8
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