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

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 ∆ψ(m) 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.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

The authors declare no competing financial interests.

Figures

Figure 1. Increased BCL-2 expression predicts for poor survival in AML patients with unfavorable cytogenetics

(a) Histogram of BCL-2 expression in all AML patients relative to normal CD34+ cells. (b) BCL-2 expression levels in patients with AML classified according to FAB classification. (c) BCL-2 expression levels in patients with AML classified according to cytogenetic abnormalities. (d) Kaplan Meier curves for overall survival in patients with unfavorable cytogenetics.

Figure 2. 5-AZA and ABT-737 synergistically induce apoptosis in AML cells

(a) OCI-AML-3 and MOLM-13 cells were treated with a range of concentrations of 5-AZA and ABT-737 for 72 hours, either as individual agents or in combination, and the Annexin V-positive fractions were measured by flow cytometry. Results are expressed as mean ± SD. A minority of untreated cells (6.5 ± 0.9% in OCI-AML-3, 5.3 ± 0.6% in MOLM-13 and 4.8 ± 2.0% in HL-60 cells) was positive for Annexin V. (b) OCI-AML-3 cells were treated for 24 hours with 2 µM 5-AZA and 6 hours with 2 µM ABT-737, and BAX conformational change was determined. To block the caspase activation-mediated conformational change of BAX, cells were preincubated for 1 hour with 50 M Z-VAD-FMK. Results are expressed as mean SD of triplicate measurements. Asterisk (*) indicates significance at P < 0.01.

Figure 3. 5-AZA and ABT-737 synergistically induce mitochondrial apoptosis in AML cells irrespective of p53 expression

(a) OCI-AML-3 cells transduced with retroviruses encoding either scrambled shRNA (shC) or p53-specific shRNA (shp53), were treated for 72 hours with 250, 500, 1000 or 2000 nM 5-AZA and ABT-737, either as individual agents or in combination. The concentration ratio of ABT-737 to 5-AZA was 1:1. Annexin V–positive fractions were measured by flow cytometry. Results are expressed as mean ± SD. (b) OCI-AML-3 cells expressing scrambled shRNA (shC) or p53-specific shRNA (shp53), were treated for 18 hours with indicated concentrations of 5-AZA and ABT-737, either as individual agents or in combination. Δψm loss was determined. Results are expressed as mean ± SD.

Figure 4. 5-AZA reduces MCL-1 levels in a p53-independent manner

(a) OCI-AML-3 cells transduced with retroviruses encoding either scrambled shRNA (shC) or p53-specific shRNA (shp53) and were treated for 48 hours with 1 µM 5-AZA. (b) Primary AML cells from 3 patients were incubated for 72 hours with the indicated concentrations of 5-AZA.

Figure 5. 5-AZA and ABT-737 decrease the infiltrated CD45+ human leukemic cells in spleen

Mouse spleen sections were stained with hematoxylin and eosin (H.E.) and anti-human CD45. The frequency of CD45+ cells enumerated by pathologist was as following: control, 30%; 5-AZA, 3%; ABT-737, 10%; 5-AZA+ABT-737, 0%.