Disruption of Bcl-2 Heterodimerization by the BH3 Mimetic ABT-737 Restores Spontaneous Apoptosis and Induces Differentiation in High Risk MDS and AML.

Abstract

Background : Increased spontaneous apoptosis is a hallmark of low risk MDS and its suppression by secondary events favors progression to AML. The ratio of Bcl-2 family members is related to progression in MDS, and high expression of proapoptotic Bax is associated with favorable outcome while high expression of antiapoptotic Bcl-2 is linked to poor prognosis.Methods : we investigated the potential of ABT-737 - known to disrupt the Bcl-2/Bax heterodimer - on cell death, cell cycle progression and differentiation of AML and MDS cells, including AML and MDS cell lines (HL 60, U 937, P39) and cells from MDS and AML patients.Results : In the cell lines studiedi.ABT-737 induced cell death in a time- (from 24 to 72 hours) and dose-dependent (from 100 nM to 1 mM) manner as evidenced by the loss of the mitochondrial transmembrane potential and expression of phosphatidylserine residues on the cell surface. In addition, incubation of HL60 and P39 cell lines with ABT-737 increased apoptosis (up to 80% at 48h with 500 nM of ABT 737) while U937 cells were resistant ;ii.ABT-737 promoted concomittant release of mitochondrial protein cytochrome c and subsequent activation of caspase-3iii.ABT-737 caused caspase-dependent cell death, which was prevented by the pan-caspase inhibitor zVAD-fmk.Evaluating the underlying molecular mechanisms by small interfering RNA, we found that knockdown of Bcl-2 recapitulated the apoptosis-inducing effect of ABT-737, whereas abrogation of its proapoptotic counterpart Bax remained without effect. Moreover, down-regulation of Mcl-1 rendered otherwise ABT-737-resistant blasts (U937 cells) sensitive to the drug. Noteworthy, although ABT-737 had no impact on the cell cycle distribution of blasts, it nevertheless enabled them to overcome the differentiation block. Indeed, incubation of HL60, P39 cells with ABT-737 increased cell surface expression of the differentiation marker CD11b, an event that could also be prevented by caspase inhibition. Finally, CD34-positive cells from 12 MDS and AML patients were treated ex vivo with ABT-737. ABT-737 induced apoptosis in the 8 samples of high risk MDS and AML (20–60% increase in cell death with 200 nM of ABT737, as compared to controls), whereas the 4 samples of low risk MDS proved to be resistant.Conclusion, we provide novel evidence that ABT-737 is able to restore apoptosis and to overcome the differentiation block that characterizes malignant cells of high risk MDS and AML, suggesting potential therapeutic activity in those disorders.