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 switch remained unaltered. 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 obtaining 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 exhibited that GO-induced apoptosis included a conformational switch 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 switch remained unaltered. In mononuclear cells isolated from AML patients that responded to GO treatment in vitro, processing of caspase-2 was obvious, whereas in cells from an AML patient refractory to treatment no such processing was seen. When assessing Vatalanib (PTK787) 2HCl diagnostic samples from 22 AML patients, who all joined total remission (CR) following anthracycline-based induction therapy, and comparing patients with long versus those with short CR period 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. and . Given this, GO offers Vatalanib (PTK787) 2HCl a way to more specifically target leukemic blasts [2, 4]. GO was, when granted accelerated approval for relapsed AML by the FDA in 2000, used as single agent and provided second remission rates of approximately 25% in aggregate phase 2 studies . When subsequently used in combination with daunorubicin and ara-C (DA), GO-induced toxicity, in particular adverse liver events, raised concerns, leading to the withdrawal of the FDA approval in 2010 2010 . However, additional phase 3 studies combining lower doses of GO with DA showed improved event-free and overall survival, particularly in patients with favorable- and intermediate-risk AML karyotypes [8, 9]. These data motivated FDA to renew their approval for GO in September 2017, this time for CD33-positive AML both in front collection and in relapsed settings . The following 12 months GO was also approved by the European Vatalanib (PTK787) 2HCl Medicines Agency (EMA) to be used in Europe in adult patients with newly diagnosed CD33+ AML . GO is also approved in the US to be used in combination with all-retinoic acid (ATRA) and arsenic trioxide (ATO) , and as standard treatment for core-binding factor positive AML (CBF-AML) [10, 11]. Thus, the clinical efficacy and usefulness of GO is usually obvious in CD33-positive AML, but the more precise molecular mechanisms behind GOs ability to selectively kill AML blasts remain to be further elucidated. So far, the mechanisms involve binding of GO to the CD33 antigen followed by cellular uptake of the created complex and redistribution from endosomes to lysosomes where the calicheamicin moiety is set free from the CD33-antibody carrier via cleavage. The free calicheamicin subsequently intercalate with DNA in the nucleus resulting in the formation of DNA double-strand breaks (DSBs), induction of the cell cycle arrest and activation of cell death [4, 5]. Thus, we and others previously reported that GO-induced cell death in part entails activation of apoptosis via the intrinsic mitochondrial route, leading to caspase-3 activation and cleavage of its substrates [12, 13]. In this work we focused on caspase-2 and its role in GO-induced apoptotic cell death. Caspase-2 has been shown to be important for the initiation and execution of apoptosis in response to DNA damaging drugs, e.g., cisplatin, etoposide, and doxorubicin [14C18]. Caspase-2 activation entails the AURKB dimerization of inactive monomers.