Activation of p53 Signaling Is Synergistically Enhanced by Bcl-2 Inhibition through Induction of Noxa and Bak/Bax Heterodimers Resulting in Apoptosis of AML Stem Cells

Disruption of Mdm2-p53 interaction by the small molecule Nutlin-3a activates p53 signaling and induces apoptosis in acute myeloid leukemia (AML) (Kojima et al., Blood 2005).  ABT-737 binds to the hydrophobic BH3-binding groove of the Bcl-2 family members Bcl-X_L, Bcl-2, Bcl-w but not Mcl-1 and exerts its pro-apoptotic function by preventing anti-apoptotic Bcl-2 family members from sequestering activating BH3 proteins (Konopleva et al., Cancer Cell 2006). We have recently reported that Nutlin-3a and ABT-737 induced Bax conformational change and mitochondrial apoptosis in AML cells in a strikingly synergistic fashion (Kojima et al., Cell Cycle 2006).  In this study, we investigated molecular mechanisms of this synergism. Nutlin-3a and ABT-737 synergistically induced apoptosis in OCI-AML3 and MOLM13 cells with wt-p53, but not in p53-null HL-60 cells or in OCI-AML3 stably transfected with p53 shRNA. Both, Nutlin-3a and ABT-737 induced Noxa protein levels resulting in Noxa/Mcl-1 heterodimers and therefore inactivation of Mcl-1. Lentiviral transfection of MOLM13 cells with Noxa shRNA protected cells from ABT-737/Nutlin-induced apoptosis. As reported by us, ABT-737 disrupted Bcl-2/Bak leading to formation of pro-apoptotic Bak/Bax heterodimers. Despite increased p53 levels there was no change in p53/Bcl-2 or p53/Bak association; however, combined exposure of cells to Nutlin-3a/ABT-737 induced massive Bak/Bax heterodimers and facilitated direct binding of p53 to Bax. HCT-116 cells deficient in Bax were largely protected from pro-apoptotic effects of the Nutlin-3a/ABT-737 combination. Taken together, these findings indicate that in leukemic cells p53-dependent apoptosis is executed through pro-apoptotic Bcl-2 family proteins, which are also targeted by BH3 mimetics. Bcl-2 is critical for AML leukemogenesis (Padua et. al., ASH 2008) and is overexpressed in AML stem cells.  In primary AML samples, the Nutlin-3a/ABT-737 combination synergistically depleted CD34⁺CD38^-CD123⁺ AML stem cells (combination index 0.5). Since high Bcl-2 and increased Bcl-2/Bax ratios are associated with poor clinical response in AML, the combination strategy proposed here may have considerable therapeutic promise in targeting AML stem cells.