Abstract 4090: The role of caspase-2 in apoptotic signaling in AML cells

We are investigating signaling molecules involved in apoptotic cell death in acute myeloid leukemia (AML) cells after treatment with the targeted drug gemtuzumab ozogamicin (GO) and the conventional drug daunorubicin, with focus on caspase-2. New effective therapeutic drugs are highly desirable in the treatment of AML. Although a majority of treated patients initially respond to conventional treatment, most patients will relapse within 1-2 years. Also, the treatment is associated with severe off-target effects. GO is a novel therapeutic approach, consisting of a monoclonal CD33 antibody coupled to calicheamicin, which cause DNA double strand breaks (DSB). We demonstrated that activation of Bak and Bax is critical for GO- and daunorubicin- induced apoptotic signaling in AML cells. The activation was followed by mitochondrial depolarization, caspase-3 activation and nuclear fragmentation. We continued to examine potential proapoptotic signaling events upstream of mitochondria in HL60 AML cells in vitro. We identified caspase-2 and the pro-apoptotic BH3-only protein Bid as two candidates. By analyzing caspase-2 by western blotting after GO treatment we found that GO caused cleavage of caspase-2 after 24h, which was prior to mitochondria-mediated signaling. Also, full-length Bid was cleaved to its truncated, active form tBid. The importance of caspase-2 activation in the signaling cascade after GO treatment was demonstrated using z-VDVAD-fmk, a caspase-2 inhibitor. Preincubation with this inhibitor reduced caspase-3 activation with around 50%, suggesting that caspase-2 is an important molecule, located upstream of caspase-3, in the signaling between DNA double stranded breaks and apoptosis signaling in AML cells. In conclusion, we demonstrate that GO and daunorubicin, at clinically applicable concentrations, induce pro-apoptotic signaling which involve activation of caspase-2, and activation of the BH3-only protein Bid. Blocking caspase-2 activity reduced caspase-3 activation with 50%. This suggests multiple signaling pathways capable of activating caspase-3. The caspase-2 pathway appears central for downstream caspase-3 activity, after GO treatment in AML. Our findings may highlight possible resistance mechanisms in AML, which might have profound therapeutic implications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4090. doi:10.1158/1538-7445.AM2011-4090