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Acute myeloid leukemia

Combination treatment of acute myeloid leukemia cells with DNMT and HDAC inhibitors: predominant synergistic gene downregulation associated with gene body demethylation

Leukemia volume 33, pages 945–956 (2019)Cite this article

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Abstract

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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References

  1. Dawson MA, Kouzarides T. Cancer epigenetics: from mechanism to therapy. Cell 2012;150:12–27.

    Article  CAS  Google Scholar 

  2. Baylin SB, Jones PA. A decade of exploring the cancer epigenome—biological and translational implications. Nat Rev Cancer. 2011;11:726–34.

    Article  CAS  Google Scholar 

  3. Maunakea AK, Nagarajan RP, Bilenky M, Ballinger TJ, D’Souza C, Fouse SD, et al. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature. 2010;466:253–7.

    Article  CAS  Google Scholar 

  4. Shenker N, Flanagan JM. Intragenic DNA methylation: implications of this epigenetic mechanism for cancer research. Br J Cancer. 2012;106:248–53.

    Article  CAS  Google Scholar 

  5. Dombret H, Seymour JF, Butrym A, Wierzbowska A, Selleslag D, Jang JH, et al. International phase 3 study of azacitidine vs conventional care regimens in older patients with newly diagnosed AML with >30% blasts. Blood. 2015;126:291–9.

    Article  CAS  Google Scholar 

  6. Kantarjian HM, Thomas XG, Dmoszynska A, Wierzbowska A, Mazur G, Mayer J, et al. Multicenter, randomized, open-label, phase III trial of decitabine versus patient choice, with physician advice, of either supportive care or low-dose cytarabine for the treatment of older patients with newly diagnosed acute myeloid leukemia. J Clin Oncol. 2012;30:2670–7.

    Article  CAS  Google Scholar 

  7. Lübbert M, Suciu S, Baila L, Rüter BH, Platzbecker U, Giagounidis A, et al. Low-dose decitabine versus best supportive care in elderly patients with intermediate- or high-risk myelodysplastic syndrome (MDS) ineligible for intensive chemotherapy: final results of the randomized phase III study of the European Organisation for Research and Treatment of Cancer Leukemia Group and the German MDS Study Group. J Clin Oncol. 2011;29:1987–96.

    Article  Google Scholar 

  8. Cameron EE, Bachman KE, Myöhänen S, Herman JG, Baylin SB. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet. 1999;21:103–7.

    Article  CAS  Google Scholar 

  9. Prebet T, Sun Z, Figueroa ME, Ketterling R, Melnick A, Greenberg PL, et al. Prolonged administration of azacitidine with or without entinostat for myelodysplastic syndrome and acute myeloid leukemia with myelodysplasia-related changes: results of the US Leukemia Intergroup Trial E1905. J Clin Oncol. 2014;32:1242–8.

    Article  CAS  Google Scholar 

  10. Gore SD. New agents for the treatment of AML recent study findings. Clin Adv Hematol Oncol. 2008;6:6–8.

    PubMed  Google Scholar 

  11. Kirschbaum M, Gojo I, Goldberg SL, Bredeson C, Kujawski LA, Yang A, et al. A phase 1 clinical trial of vorinostat in combination with decitabine in patients with acute myeloid leukaemia or myelodysplastic syndrome. Br J Haematol. 2014;167:185–93.

    Article  CAS  Google Scholar 

  12. Wouters BJ, Delwel R. Epigenetics and approaches to targeted epigenetic therapy in acute myeloid leukemia. Blood. 2016;127:42–52.

    Article  CAS  Google Scholar 

  13. Tan P, Wei A, Mithraprabhu S, Cummings N, Liu HB, Perugini M, et al. Dual epigenetic targeting with panobinostat and azacitidine in acute myeloid leukemia and high-risk myelodysplastic syndrome. Blood Cancer J. 2014;4:e170.

    Article  CAS  Google Scholar 

  14. Flotho C, Claus R, Batz C, Schneider M, Sandrock I, Ihde S, et al. The DNA methyltransferase inhibitors azacitidine, decitabine and zebularine exert differential effects on cancer gene expression in acute myeloid leukemia cells. Leukemia. 2009;23:1019–28.

    Article  CAS  Google Scholar 

  15. Schmelz K, Sattler N, Wagner M, Lübbert M, Dörken B, Tamm I. Induction of gene expression by 5-Aza-2’-deoxycytidine in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) but not epithelial cells by DNA-methylation-dependent and -independent mechanisms. Leukemia. 2005;19:103–11.

    Article  CAS  Google Scholar 

  16. Yang X, Han H, De Carvalho DD, Lay FD, Jones PA, Liang G. Gene body methylation can alter gene expression and is a therapeutic target in cancer. Cancer Cell. 2014;26:577–90.

    Article  CAS  Google Scholar 

  17. Tost J, Dunker J, Gut IG. Analysis and quantification of multiple methylation variable positions in CpG islands by pyrosequencing. Biotechniques. 2003;35:152–6.

    Article  CAS  Google Scholar 

  18. Brocks D, Schmidt CR, Daskalakis M, Jang HS, Shah NM, Li D, et al. DNMT and HDAC inhibitors induce cryptic transcription start sites encoded in long terminal repeats. Nat Genet. 2017;49:1052–60.

    Article  CAS  Google Scholar 

  19. Karahoca M, Momparler RL. Pharmacokinetic and pharmacodynamic analysis of 5-aza-2′-deoxycytidine (decitabine) in the design of its dose-schedule for cancer therapy. Clin Epigenetics. 2013;5:3.

    Article  CAS  Google Scholar 

  20. Lübbert M, Kuendgen A. Combining DNA methyltransferase and histone deacetylase inhibition to treat acute myeloid leukemia/myelodysplastic syndrome: achievements and challenges. Cancer . 2015;121:498–501.

    Article  Google Scholar 

  21. Pandiyan K, You JS, Yang X, Dai C, Zhou XJ, Baylin SB, et al. Functional DNA demethylation is accompanied by chromatin accessibility. Nucleic Acids Res. 2013;41:3973–85.

    Article  CAS  Google Scholar 

  22. Ramos M-P, Wijetunga NA, McLellan AS, Suzuki M, Greally JM. DNA demethylation by 5-aza-2′-deoxycytidine is imprinted, targeted to euchromatin, and has limited transcriptional consequences. Epigenetics Chromatin. 2015;8:11.

  23. Ueda T, Nagamachi A, Takubo K, Yamasaki N, Matsui H, Kanai A, et al. Fbxl10 overexpression in murine hematopoietic stem cells induces leukemia involving metabolic activation and upregulation of Nsg2. Blood. 2015;125:3437–46.

    Article  CAS  Google Scholar 

  24. Lai Y-S, Chen J-Y, Tsai H-J, Chen T-Y, Hung W-C. The SUV39H1 inhibitor chaetocin induces differentiation and shows synergistic cytotoxicity with other epigenetic drugs in acute myeloid leukemia cells. Blood Cancer J. 2015;5:e313.

    Article  Google Scholar 

  25. Reed-Inderbitzin E, Moreno-Miralles I, Vanden-Eynden SK, Xie J, Lutterbach B, Durst-Goodwin KL, et al. RUNX1 associates with histone deacetylases and SUV39H1 to repress transcription. Oncogene. 2006;25:5777–86.

    Article  CAS  Google Scholar 

  26. Stopa N, Krebs JE, Shechter D. The PRMT5 arginine methyltransferase: many roles in development, cancer and beyond. Cell Mol Life Sci Cmls. 2015;72:2041–59.

    Article  CAS  Google Scholar 

  27. Baldwin RM, Haghandish N, Daneshmand M, Amin S, Paris G, Falls TJ, et al. Protein arginine methyltransferase 7 promotes breast cancer cell invasion through the induction of MMP9 expression. Oncotarget. 2015;6:3013–32.

    Article  Google Scholar 

  28. Almstedt M, Blagitko-Dorfs N, Duque-Afonso J, Karbach J, Pfeifer D, Jäger E, et al. The DNA demethylating agent 5-aza-2′-deoxycytidine induces expression of NY-ESO-1 and other cancer/testis antigens in myeloid leukemia cells. Leuk Res. 2010;34:899–905.

    Article  CAS  Google Scholar 

  29. Kulis M, Queirós AC, Beekman R, Martín-Subero JI. Intragenic DNA methylation in transcriptional regulation, normal differentiation and cancer. Biochim Biophys Acta. 2013;1829:1161–74.

    Article  CAS  Google Scholar 

  30. Varley KE, Gertz J, Bowling KM, Parker SL, Reddy TE, Pauli-Behn F, et al. Dynamic DNA methylation across diverse human cell lines and tissues. Genome Res. 2013;23:555–67.

    Article  CAS  Google Scholar 

  31. Klco JM, Spencer DH, Lamprecht TL, Sarkaria SM, Wylie T, Magrini V, et al. Genomic impact of transient low-dose decitabine treatment on primary AML cells. Blood. 2013;121:1633–43.

    Article  CAS  Google Scholar 

  32. Lund K, Cole JJ, VanderKraats ND, McBryan T, Pchelintsev NA, Clark W, et al. DNMT inhibitors reverse a specific signature of aberrant promoter DNA methylation and associated gene silencing in AML. Genome Biol. 2014;15:406.

  33. Cervoni N, Szyf M. Demethylase activity is directed by histone acetylation. J Biol Chem. 2001;276:40778–87.

    Article  CAS  Google Scholar 

  34. Gu S, Tian Y, Chlenski A, Salwen HR, Lu Z, Raj JU, et al. Valproic acid shows a potent antitumor effect with alteration of DNA methylation in neuroblastoma. Anticancer Drugs. 2012;23:1054–66.

    Article  CAS  Google Scholar 

  35. Kalac M, Scotto L, Marchi E, Amengual J, Seshan VE, Bhagat G, et al. HDAC inhibitors and decitabine are highly synergistic and associated with unique gene-expression and epigenetic profiles in models of DLBCL. Blood. 2011;118:5506–16.

    Article  CAS  Google Scholar 

  36. Liang G, Gonzales FA, Jones PA, Orntoft TF, Thykjaer T. Analysis of gene induction in human fibroblasts and bladder cancer cells exposed to the methylation inhibitor 5-aza-2′-deoxycytidine. Cancer Res. 2002;62:961–6.

    CAS  PubMed  Google Scholar 

  37. Gius D, Cui H, Bradbury CM, Cook J, Smart DK, Zhao S, et al. Distinct effects on gene expression of chemical and genetic manipulation of the cancer epigenome revealed by a multimodality approach. Cancer Cell. 2004;6:361–71.

    Article  CAS  Google Scholar 

  38. Falk H, Foitzik RC, Allan E, deSilva M, Yang H, Bozikis YE, et al. Abstract 5371: PRMT5 inhibitors as novel treatment for cancers. Cancer Res. 2015;75(15 Supplement):5371.

    Google Scholar 

  39. van den Boom V, Maat H, Geugien M, Rodríguez López A, Sotoca AM, Jaques J, et al. Non-canonical PRC1.1 targets active genes independent of H3K27me3 and is essential for leukemogenesis. Cell Rep. 2016;14:332–46.

    Article  Google Scholar 

  40. He J, Nguyen AT, Zhang Y. KDM2b/JHDM1b, an H3K36me2-specific demethylase, is required for initiation and maintenance of acute myeloid leukemia. Blood. 2011;117:3869–80.

    Article  CAS  Google Scholar 

  41. Janzer A, Stamm K, Becker A, Zimmer A, Buettner R, Kirfel J. The H3K4me3 histone demethylase Fbxl10 is a regulator of chemokine expression, cellular morphology, and the metabolome of fibroblasts. J Biol Chem. 2012;287:30984–92.

    Article  CAS  Google Scholar 

  42. Greve G, Schiffmann I, Pfeifer D, Pantic M, Schüler J, Lübbert M. The pan-HDAC inhibitor panobinostat acts as a sensitizer for erlotinib activity in EGFR-mutated and -wildtype non-small cell lung cancer cells. BMC Cancer. 2015;15:947.

    Article  Google Scholar 

  43. Duque-Afonso J, Yalcin A, Berg T, Abdelkarim M, Heidenreich O, Lübbert M. The HDAC class I-specific inhibitor entinostat (MS-275) effectively relieves epigenetic silencing of the LAT2 gene mediated by AML1/ETO. Oncogene. 2011;30:3062–72.

    Article  CAS  Google Scholar 

  44. Yao L, Shen H, Laird PW, Farnham PJ, Berman BP. Inferring regulatory element landscapes and transcription factor networks from cancer methylomes. Genome Biol. 2015;16:105.

  45. Ptasinska A, Assi SA, Mannari D, James SR, Williamson D, Dunne J, et al. Depletion of RUNX1/ETO in t(8;21) AML cells leads to genome-wide changes in chromatin structure and transcription factor binding. Leukemia. 2012;26:1829–41.

    Article  CAS  Google Scholar 

  46. Srivastava P, Paluch BE, Matsuzaki J, James SR, Collamat-Lai G, Blagitko-Dorfs N, et al. Induction of cancer testis antigen expression in circulating acute myeloid leukemia blasts following hypomethylating agent monotherapy. Oncotarget. 2016;7:12840–56.

  47. Li H, Chiappinelli KB, Guzzetta AA, Easwaran H, Yen R-WC, Vatapalli R, et al. Immune regulation by low doses of the DNA methyltransferase inhibitor 5-azacitidine in common human epithelial cancers. Oncotarget. 2014;5:587–98.

    PubMed  PubMed Central  Google Scholar 

  48. Yang H, Bueso-Ramos C, DiNardo C, Estecio MR, Davanlou M, Geng Q-R, et al. Expression of PD-L1, PD-L2, PD-1 and CTLA4 in myelodysplastic syndromes is enhanced by treatment with hypomethylating agents. Leukemia. 2014;28:1280–8.

    Article  CAS  Google Scholar 

  49. Lübbert M, Bertz H, Rüter B, Marks R, Claus R, Wäsch R, et al. Non-intensive treatment with low-dose 5-aza-2′-deoxycytidine (DAC) prior to allogeneic blood SCT of older MDS/AML patients. Bone Marrow Transplant. 2009;44:585–8.

    Article  Google Scholar 

  50. Steinmann J, Bertz H, Wäsch R, Marks R, Zeiser R, Bogatyreva L, et al. 5-Azacytidine and DLI can induce long-term remissions in AML patients relapsed after allograft. Bone Marrow Transplant. 2015;50:690–5.

    Article  CAS  Google Scholar 

  51. Bug G, Burchert A, Wagner E-M, Kröger N, Berg T, Güller S, et al. Phase I/II study of the deacetylase inhibitor panobinostat after allogeneic stem cell transplantation in patients with high-risk MDS or AML (PANOBEST trial). Leukemia. 2017;31:2523–5.

    Article  CAS  Google Scholar 

  52. Chiappinelli KB, Strissel PL, Desrichard A, Li H, Henke C, Akman B, et al. Inhibiting DNA methylation causes an interferon response in cancer via dsRNA including endogenous retroviruses. Cell. 2015;162:974–86.

    Article  CAS  Google Scholar 

  53. Roulois D, Loo Yau H, Singhania R, Wang Y, Danesh A, Shen SY, et al. DNA-Demethylating Agents Target Colorectal Cancer Cells by Inducing Viral Mimicry by Endogenous Transcripts. Cell 2015;162:961–73.

    Article  CAS  Google Scholar 

  54. Grishina O, Schmoor C, Döhner K, Hackanson B, Lubrich B, May AM, et al. DECIDER: prospective randomized multicenter phase II trial of low-dose decitabine (DAC) administered alone or in combination with the histone deacetylase inhibitor valproic acid (VPA) and all-trans retinoic acid (ATRA) in patients > 60 years with acute myeloid leukemia who are ineligible for induction chemotherapy. BMC Cancer. 2015;15:430.

    Article  Google Scholar 

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Acknowledgements

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)).

Author contributions

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.

Funding

Funding

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).

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  1. These authors contributed equally: Nadja Blagitko-Dorfs, Pascal Schlosser, Gabriele Greve

Authors and Affiliations

  1. Division of Hematology, Oncology and Stem Cell Transplantation, Department of Internal Medicine, Faculty of Medicine and Medical Center, University of Freiburg, Freiburg, Germany

    Nadja Blagitko-Dorfs, Gabriele Greve, Dietmar Pfeifer, Ruth Meier & Michael Lübbert

  2. Center for Chronic Immunodeficiency (CCI), Medical Center – University of Freiburg, Freiburg, Germany

    Nadja Blagitko-Dorfs

  3. Institute of Genetic Epidemiology, Faculty of Medicine and Medical Center, University of Freiburg, Freiburg, Germany

    Pascal Schlosser

  4. Division of Epigenomics and Cancer Risk Factors, DKFZ, Heidelberg, Germany

    Annika Baude, David Brocks & Christoph Plass

  5. DKTK, German Consortium for Translational Cancer Research, Freiburg, Germany

    Michael Lübbert

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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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Key Words

  • cancer-testis antigen (CTA)
  • TINAT

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