Apoptosis by leukemia cell-targeted diphtheria toxin occurs via receptor-independent activation of Fas-associated death domain protein

Purpose: We examined the mechanism of action of a targeted fusion toxin consisting of diphtheria toxin fused to granulocyte macrophage colony stimulating factor (GMCSF) (DT₃₈₈-GMCSF), which was designed to selectively kill acute myeloid leukemia cells. Experimental design and Results: U937 cells treated with DT₃₈₈-GMCSF underwent apoptosis as shown by chromatin degradation and cellular and nuclear fragmentation. This apoptosis was prevented by a general caspase inhibitor. DT₃₈₈-GMCSF treatment resulted in activation of the initiator caspases 8 and 9 and effector caspases. A selective caspase 8 inhibitor prevented activation of caspase 9, whereas a selective caspase 9 inhibitor did not prevent activation of caspase 8, indicating that caspase 8 activation is the proximal event in DT₃₈₈-GMCSF-induced apoptosis. Caspase 8 was activated through a Fas-associated death domain protein (FADD)-dependent mechanism as demonstrated by inhibition of DT₃₈₈-GMCSF-induced apoptosis on expression of a dominant negative FADD molecule. However, unlike most FADD-dependent apoptosis, this pathway may not involve death receptors, including Fas, tumor necrosis factor receptor 1, or tumor necrosis factor-related apoptosis-inducing ligand receptors, because inhibitors of the receptors did not prevent DT₃₈₈-GMCSF-induced apoptosis. Conclusions: These data indicate that targeted toxins induce apoptosis by activating components of the death receptor pathway in a receptor-independent manner.