Hypoxia Activated Pro-Drug TH-302 Induces Hypoxia-Dependent Anti-leukemia Activity in vitro and in vivo

Caspase-8 mutant Jurkat cells (JurkatI9.2) were completely resistant to birinapant, further supporting the critical role of caspase-8 in birinapant-mediated apoptosis. Birinapant synergistically enhanced apoptosis when combined with various nucleoside analogues clinically used in AML therapy and demethylating agents even in the cells co-cultured with bone marrow-derived stromal cells (MSCs). Mechanistic studies showed that decitabine and 5-azacytidine increased and activated caspase-8; induced XAF-1, a XIAP antagonist known to be hypermethylated in various malignant cells; and inhibited canonical and non-canonical NF-kB signaling. In addition, birinapant was effective against blasts from primary AML samples cocultured with MSCs with no toxicity in normal CD34 cells. Importantly, birinapant not only induced apoptosis in bulk AML blast but also in CD3438 AML stem/progenitor cells and prolonged mouse survival which was further enhanced by demethylating agents in a xenograft human AML model. Collectively, we showed that cIAP1 and caspase-8 are overexpressed and SMAC underexpressed in AML stem/progenitor cells and that inhibition of IAPs by birinapant synergistically enhances drug induced-death of AML cells in vitro and in vivo and has the potential to eliminate AML stem/progenitor cells.