The prognosis for patients with acute myeloid leukaemia (AML) is still poor, thus calling for novel treatment strategies. Here, we report that the small-molecule Smac mimetic BV6, which antagonizes Inhibitor of Apoptosis (IAP) proteins, acts in concert with cytarabine (AraC) to trigger cell death in AML cells in a highly synergistic manner (combination index 0.02-0.27). Similarly, BV6 cooperates with AraC to trigger cell death in primary AML samples, underscoring the clinical relevance of our findings. Molecular studies reveal that the TNFα-blocking antibody Enbrel significantly reduces BV6/AraC-induced cell death, demonstrating that an autocrine/paracrine TNFα loop mediates cell death. Furthermore, BV6 and AraC synergize to induce loss of mitochondrial membrane potential, caspase activation and DNA fragmentation, consistent with apoptotic cell death. Nevertheless, the caspase inhibitor zVAD.fmk fails to protect against BV6/AraC-induced cell death. Intriguingly, this cell death upon caspase inhibition is significantly reduced by pharmacological inhibition of two key components of necroptosis signaling, i.e. by RIP1 kinase inhibitor Necrostatin-1 or MLKL inhibitor NSA. Thus, BV6 sensitizes AML cells to AraC-induced cell death and overcomes apoptosis resistance by triggering necroptosis as alternative form of cell death. These findings have important implications for Smac mimetic-based strategies to bypass apoptosis resistance of AML.
Keywords: AML; Apoptosis; AraC; BIR; Baculovirus IAP Repeat; CBF; CI; Cytarabine; FACS; FADD; FAS-associated death domain protein; IAP; IAP proteins; Inhibitor of apoptosis; Leukaemia; MLKL; MMP; N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; NF-κB; Nec; Necroptosis; Necrostatin-1; Nuclear Factor kappaB; PI; RING; RIP1; Really Interesting New Gene; Receptor-Interacting Protein 1; Smac; TNF; TNFR1; X-linked inhibitor of apoptosis; XIAP; acute myeloid leukaemia; cIAP1; cellular inhibitor of apoptosis protein 1; combination index; core-binding factor; fluorescence-activated cell-sorting; mitochondrial membrane potential; mixed lineage kinase domain-like protein; propidium iodide; second mitochondria-derived activator of caspases; tumour necrosis factor; tumour necrosis factor receptor 1; zVAD.fmk.
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