Abstract
The antibody conjugate gemtuzumab ozogamicin (GO; Mylotarg®) provides targeted therapy of acute myeloid leukemia (AML), with recent approvals for patients with CD33-positive disease at diagnosis or relapse, as monotherapy or combined with chemotherapeutics. While its clinical efficacy is well documented, the molecular routes by which GO induces AML cell death warrant further analyses. We have earlier reported that this process is initiated via mitochondria-mediated caspase activation. Here we provide additional data, focusing on the involvement of caspase-2 in this mechanism. We show that this enzyme plays an important role in triggering apoptotic death of human AML cells after exposure to GO or its active moiety calicheamicin. Accordingly, the caspase-2 inhibitor z-VDVAD-fmk reduced GO-induced caspase-3 activation. This finding was validated with shRNA and siRNA targeting caspase-2, resulting in reduced caspase-3 activation and cleavage of poly [ADP-ribose] polymerase 1 (PARP-1). We previously demonstrated that GO-induced apoptosis included a conformational change of Bax into a pro-apoptotic state. Present data reveal that GO-treatment also induced Bid cleavage, which was partially reduced by caspase-2 specific inhibition while the effect on GO-induced Bax conformational change remained unaltered. In mononuclear cells isolated from AML patients that responded to GO treatment in vitro, processing of caspase-2 was evident, whereas in cells from an AML patient refractory to treatment no such processing was seen. When assessing diagnostic samples from 22 AML patients, who all entered complete remission (CR) following anthracycline-based induction therapy, and comparing patients with long versus those with short CR duration no significant differences in baseline caspase-2 or caspase-3 full-length protein expression levels were found. In summary, we demonstrate that GO triggers caspase-2 cleavage in human AML cells and that the subsequent apoptosis of these cells in part relies on caspase-2. These findings may have future clinical implications.