The Synergistic Effect of DNA Methylation Inhibitor 5-Azacytidine and Bcl-2 Signaling Blockade ABT-737 Combination Treatment Induce Apoptosis In AML

Abstract 3311

Bcl-2 family proteins are key regulators of apoptosis. Aberrations in Bcl2 levels are known to promote tumorigenesis and chemoresistance. Thus strategies to target Bcl2 will likely provide effective therapies for malignancies such as acute myeloid leukemia (AML). ABT-737 is a small molecule BH3 mimetic that binds tightly to a hydrophobic cleft on Bcl-2 and Bcl-XL and exerts its proapoptotic function by preventing antiapoptotic Bcl-2 family members from sequestering activating BH3 proteins (Oltersdorf et al., Nature 2005). We have reported that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induces the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway (Konopleva et al., Cancer Cell 2006). The ABT-737 related clinical compound, ABT-263, is undergoing Phase I/II studies in chronic lymphocytic leukemia, with initial signs of clinical activity. However, the main side effect is thrombocytopenia resulting from inhibition of Bcl-XL. Hence, combinations of ABT-737 with non-cytotoxic agents are desirable to take full advantage of ABT236's unique spectrum of biophysical and preclinical activities.

In this project, we studied pharmacologic interactions between ABT-737 and the DNA methyltransferase inhibitor 5-azacytidine (5-azaC). 5-azaC is a cytidine analog with clinical activity in myelodysplastic syndromes (MDS) and in AML. Since recent studies indicate that 5-azaC induce DNA damage in p53-dependent fashion, we tested the hypothesis that the pro-apoptotic effects of 5-azaC/ABT-737 combination are related to non-redundant activation of BH3-only proteins and mitochondrial apoptosis in AML cells with wt p53.

In vitro, 5-azaC and ABT-737 in combination for 72 hrs induced growth inhibition and apoptosis in AML cell lines OCI-AML3, MOLM-13 and U937 in a highly synergistic, dose-dependent fashion, with combination indices (CIs) ranging from 0.1 to 0.22. These effects were observed even at low concentrations (5-azaC 100nM and ABT-737 10nM, at 10:1 ratio). In contrast, no synergistic apoptosis was seen in p53-null HL-60 cells. Likewise, ABT-737/5-azaC induced apoptosis in a synergistic fashion in OCI-AML3 cells infected with vector control shRNA (CI=0.04) but failed to induce cell death in OCI-AML3 p53 shRNA cells, indicating critical p53-dependent mechanisms of cell death. In primary AML samples sensitive to ABT-737 alone (n=3), synergistic and additive effects were seen. The combined effects of 5-azaC and ABT-737 were further investigated in NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ mice injected with cells from a patient with primary refractory AML. Seven days after leukemia transplantation, mice were treated with vehicle, ABT-737 (intraperitoneally (IP), 75mg/kg/day for 10 days), 5-azacytidine (IP, 4mg/kg/day for 5 days) or with the combination. Engraftment of patient leukemia cells was analyzed by the immunohistochemical detection of CD45-positive cells in bone marrow and spleen seven weeks after transplantation. Both, ABT-737 and 5azaC each exerted anti-leukemic effects, as evidenced by significant reduction in leukemia cells in bone marrow and spleen (10% and 3% CD45⁺ cells detectable in 1/3 mice in ABT-737 and 5-azaC groups, respectively), no CD45+ AML cells were detected in organs of 3/3 mice treated with the combination. No overt hemorrhage was detected in the animals.

In summary, the combination of 5-azaC and ABT-737 induces synergistic cells death in AML cell lines and in a subset of primary AML samples in a p53-dependent fashion. The mechanisms of this pharmacologic interactions including the p53-dependent upregulation of BH3-only proteins, described by us for ABT-737/MDM2 inhibitor combinations, are currently under investigation. Results suggest that this therapeutic strategy can be successfully utilized in AML patients with low mutation rate and unimpaired signaling of p53.