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Azacitidine induces profound genome-wide hypomethylation in primary myelodysplastic bone marrow cultures but may also reduce histone acetylation

Leukemia volume 28, pages 411–413 (2014)Cite this article

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5-Azacitidine (azacitidine) is approved for treatment of myelodysplastic syndromes (MDSs) and mixed MDS/MPN (myeloproliferative neoplasm).1 Its clinical superiority to the related substance cytarabine2 and the slow onset of response has been attributed to its DNA hypomethylating effects. Although cytarabine induced S-phase arrest in cell line experiments, azacitidine did not interfere with cell cycle progression.3 Azacitidine has been shown to induce DNA demethylation in myeloid cell lines and to induce re-expression of genes silenced by aberrant promoter methylation.4, 5 Other studies show that its mechanisms of action are complex and involve induction of apoptosis,6 immunomodulation,7 differentiation of blasts,4 histone modifications8 and inhibition of RNA methylation.9 Sequential samples from patients under treatment show reduced CDKN2B promoter methylation in complete remission and reappearance of methylation before relapse,10, 11 but this may also reflect replacement of cancer cells with normal hematopoiesis and vice versa. The effect of azacitidine on MDS bone marrow (BM) progenitors in vitro has not been investigated in detail.

We assessed the direct effects of azacitidine on BM progenitors from patients with high-risk MDS eligible for azacitidine treatment according to label and from healthy controls (normal BM=NBM), respectively. The clinical characteristics of the eight patients included in the main experiment, methylation array (Illumina, San Diego, CA, USA) and corresponding western blot analysis, are shown in Supplementary Table 1. Mononuclear cells (MNCs) were sorted from freshly harvested BM by density gradient technique (Lymphoprep, Axis-Shield, Oslo, Norway). CD34+ cells were then purified by magnetic bead sorting (Progenitor Cell Isolation kit, Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured in RPMI Glutamax with 10% fetal bovine serum, 10 ng/ml granulocyte colony-stimulating factor (G-CSF), 10 ng/ml interleukin-3 and 24 ng/ml stem cell factor with varying concentrations of azacitidine. DNA was extracted using Gene Elute genomic DNA extraction kit (Sigma, St Louis, MO, USA) and bisulfite conversion was carried out using EZ methylation gold kit (Zymo Research, Irvine, CA, USA).

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Authors and Affiliations

  1. Department of Medicine, Center for Hematology and Regenerative Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden

    M Grövdal, M Karimi, M Tobiasson, M Jansson, J Ungerstedt & E Hellström-Lindberg

  2. Department of Bioscience and Nutrition, Karolinska Institutet, Stockholm, Sweden

    L Reinius, J Kere & D Greco

  3. Center for Bioscience, Karolinska Institutet, Stockholm, Sweden

    K Ekwall & J Kere

  4. Molecular Medicine Research Program, Biomedicum, University of Helsinki and Folkhälsan Institute of Genetics, Helsinki, Finland

    J Kere & D Greco

Authors
  1. M Grövdal
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  2. M Karimi
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  10. E Hellström-Lindberg
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Correspondence to M Grövdal.

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Grövdal, M., Karimi, M., Tobiasson, M. et al. Azacitidine induces profound genome-wide hypomethylation in primary myelodysplastic bone marrow cultures but may also reduce histone acetylation. Leukemia 28, 411–413 (2014). https://doi.org/10.1038/leu.2013.265

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