Abstract
Evasion of apoptosis, for example, by inhibitor of apoptosis (IAP) proteins, contributes to treatment resistance and poor outcome in acute myeloid leukemia (AML). Here we identify a novel synergistic interaction between the small-molecule second mitochondria-derived activator of caspases (Smac) mimetic BV6, which antagonizes X-linked IAP, cellular IAP (cIAP)1 and cIAP2, and the demethylating agents 5-azacytidine or 5-aza-2′-deoxycytidine (DAC) to induce cell death in AML cells, including apoptosis-resistant cells. Calculation of combination index (CI) confirms that this drug combination is highly synergistic (CI 0.02–0.4). In contrast, BV6 and DAC at equimolar concentrations do not cause synergistic toxicity against normal peripheral blood lymphocytes, pointing to some tumor cell selectivity. Molecular studies reveal that BV6 and DAC cooperate to trigger the activation of caspases, mitochondrial perturbations and DNA fragmentation, consistent with apoptotic cell death. However, the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to protect against BV6/DAC-induced cell death and even significantly increases the percentage of Annexin-V/propidium iodide double-positive cells. Importantly, BV6/DAC-induced cell death in the presence of zVAD.fmk is significantly reduced by pharmacological inhibition of key components of necroptosis signaling, that is, receptor-interacting protein (RIP) 1 using necrostatin-1 or mixed lineage kinase domain-like protein (MLKL) using necrosulfonamide. This indicates a switch from BV6/DAC-induced cell death from apoptosis to necroptosis upon caspase inhibition. Thus, BV6 cooperates with demethylating agents to induce cell death in AML cells and circumvents apoptosis resistance via a switch to necroptosis as an alternative mode of cell death. The identification of a novel synergism of BV6 and demethylating agents has important implications for the development of new treatment strategies for AML.
Highlights
BV6/DAC-induced cell death in the presence of zVAD.fmk is significantly reduced by pharmacological inhibition of key components of necroptosis signaling, that is, receptor-interacting protein (RIP) 1 using necrostatin-1 or mixed lineage kinase domain-like protein (MLKL) using necrosulfonamide
We previously reported that small-molecule IAP inhibitors enhance tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), Fas ligand- or chemotherapy-induced apoptosis in childhood acute lymphoblastic leukemia (ALL).[24,25,26]
Searching for novel combination regimens with Smac mimetic, we investigated the effect of the small-molecule Smac mimetic BV6, which neutralizes X-linked IAP (XIAP), cIAP1 and cIAP2,21 in the presence and absence of the DNA methyltransferase inhibitors 5-azacytidine or 5-aza-20 -deoxycytidine against several acute myeloid leukemia (AML) cell lines with different molecular characteristics (Supplementary Table 1)
Summary
BV6/DAC-induced cell death in the presence of zVAD.fmk is significantly reduced by pharmacological inhibition of key components of necroptosis signaling, that is, receptor-interacting protein (RIP) 1 using necrostatin-1 or mixed lineage kinase domain-like protein (MLKL) using necrosulfonamide. Evasion of programmed cell death belongs to the hallmarks of human cancers, including leukemia.[5] There are several forms of programmed cell death, including apoptosis and necroptosis.[6] Apoptosis signals via two key pathways, that is, the death receptor (extrinsic) and the mitochondrial (intrinsic) pathway.[4] Stimulation of death receptors, for example, CD95 or tumor necrosis factor (TNF) receptor 1 (TNFR1), by their respective ligands, for example, CD95 ligand or TNFa, leads to caspase-8 activation, which in turn can cleave downstream effector caspases, such as caspase-3.7 The activation of the mitochondrial pathway results in caspase-3 activation via the release of mitochondrial intermembrane space proteins into the cytosol, including cytochrome c and Smac.[8] Necroptosis is another regulated mode of cell death that typically occurs under circumstances in which caspase
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