Abstract B25: Apoptotic signaling in AML cells after therapy with the monoclonal antibody-therapeutic gemtuzumab ozogamicin

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Abstract The aim of this study was to identify essential signaling molecules involved in apoptotic cell death in acute myeloid leukemia (AML) cells after treatment with the targeted drug gemtuzumab ozogamicin (GO). 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 a DNA damaging toxin, calicheamicin. GO enters the cell after binding to the CD33 antigen, preferentially expressed on myeloid cells, and cause DNA double strand breaks (DSB). We have previously demonstrated that activation of Bak and Bax is critical for GO-induced apoptotic signaling, followed by downstream mitochondrial depolarization and caspase-3 activation. As a continuation, we have now examined potential proapoptotic signaling events induced by GO 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. At the same time, 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, at clinically applicable concentrations (100–1000ng/ml), induce pro-apoptotic signaling which involve activation of caspase-2, and cleavage of the BH3-only protein Bid to the truncated, active protein tBid. Blocking caspase-2 activity reduced caspase-3 activation to only half. 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 Information: Mol Cancer Ther 2009;8(12 Suppl):B25.