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Concomitant inhibition of DNA methyltransferase and BCL-2 protein function synergistically induce mitochondrial apoptosis in acute myelogenous leukemia cells

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Abstract

DNA methylation and BLC-2 are potential therapeutic targets in acute myeloid leukemia (AML). We investigated pharmacologic interaction between the DNA methyltransferase inhibitor 5-azacytidine (5-AZA) and the BCL-2 inhibitor ABT-737. Increased BCL-2 expression determined by reverse phase protein analysis was associated with poor survival in AML patients with unfavorable cytogenetics (n = 195). We found that 5-AZA, which itself has modest apoptotic activity, acts synergistically with ABT-737 to induce apoptosis. The 5-AZA/ABT-737 combination enhanced mitochondrial outer membrane permeabilization, as evidenced by effective conformational activation of BAX and ∆ψm loss. Although absence of p53 limited apoptotic activities of 5-AZA and ABT-737 as single agents, the combination synergistically induced apoptosis independent of p53 expression. 5-AZA down-regulated MCL-1, known to mediate resistance to ABT-737, in a p53-independent manner. The 5-AZA/ABT-737 combination synergistically induced apoptosis in AML cells in seven of eight patients. 5-AZA significantly reduced MCL-1 levels in two of three samples examined. Our data provide a molecular rationale for this combination strategy in AML therapy.

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Grant support

This study is supported by the National Institutes of Health Lymphoma SPORE (CA136411), P01 “The Therapy of AML” (CA55164), Leukemia SPORE (CA100632), and the Paul and Mary Haas Chair in Genetics (M. Andreeff).

Disclosure of potential conflicts of interest

The authors declare no competing financial interests.

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Authors and Affiliations

  1. Department of Leukemia, The University of Texas M. D. Anderson Cancer Center, 1400 Holcombe Blvd, Unit 425, Houston, TX, 77030, USA

    Twee Tsao, Yuexi Shi, Steven Kornblau, Hongbo Lu, Ansu Antony, Vivian Ruvolo, Yi Hua Qiu, Michael Andreeff, Kensuke Kojima & Marina Konopleva

  2. Department of Hematopathology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA

    Sergej Konoplev

  3. Department of Bioinformatics and Computational Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA

    Ninaxiang Zhang & Kevin R. Coombes

Authors
  1. Twee Tsao
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  2. Yuexi Shi
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  3. Steven Kornblau
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  4. Hongbo Lu
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  5. Sergej Konoplev
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  6. Ansu Antony
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  7. Vivian Ruvolo
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  8. Yi Hua Qiu
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  9. Ninaxiang Zhang
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  10. Kevin R. Coombes
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  11. Michael Andreeff
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  12. Kensuke Kojima
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  13. Marina Konopleva
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Corresponding author

Correspondence to Marina Konopleva.

Additional information

Kensuke Kojima and Marina Konopleva contributed equally to this project.

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Supplemental Fig. S1

5-AZA, ABT-737 and their combination did not affect levels of BCL-2 family proteins BAX, BAK, BCL-XL, and BCL-2, or p53. Expression of total BAX, BAK, BCL-2, BCL-XL, and p53 in OCI-AML-3 cells treated for 72 h with indicated concentrations of 5-AZA and ABT-737, either as individual agents or in combination. Two membranes were used to detect BCL-2 family proteins, as they exhibit similar protein mass. (DOC 62 kb)

Supplemental Fig. S2

5-AZA and ABT-737 synergistically induce mitochondrial apoptosis in AML cells irrespective of p53 expression. MOLM-13 cells transduced with retroviruses encoding either scrambled shRNA (shC) or p53-specific shRNA (shp53), were treated for 72 h with 250, 500, 1,000, or 2,000 nM 5-AZA and ABT-737, either as individual agents or in combination. The concentration ratio of ABT-737 to 5-AZA was 1:10. Annexin V-positive fractions were measured by flow cytometry. Results are expressed as mean ± SD. (DOC 199 kb)

Supplemental Table S1

Clinical data and mutations in NPM1 and FLT3 (DOC 43 kb)

Supplemental Table S2

Percentages of circulating human CD45+ cells (DOC 33 kb)

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Tsao, T., Shi, Y., Kornblau, S. et al. Concomitant inhibition of DNA methyltransferase and BCL-2 protein function synergistically induce mitochondrial apoptosis in acute myelogenous leukemia cells. Ann Hematol 91, 1861–1870 (2012). https://doi.org/10.1007/s00277-012-1537-8

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  • DOI: https://doi.org/10.1007/s00277-012-1537-8

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