Smac mimetic and demethylating agents synergistically trigger cell death in acute myeloid leukemia cells and overcome apoptosis resistance by inducing necroptosis

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.

USP11-dependent selective cIAP2 deubiquitylation and stabilization determine sensitivity to Smac mimetics

“…29,43 Recently, combination treatment with a demethylating agent largely improved the tumor-suppressive effects of Smac mimetics in several cancer models. 27,44 In addition to these, numerous new compounds including LCL161 and birinapant are currently in phase I and II clinical trials. 5,14 As our data suggest, cIAP2 sensitivity against Smac mimetics in relation to USP11 expression should be considered in the abovementioned combination therapies.…”

“…25,26 While several studies have supported hypotheses for how cIAP2 survives in the presence of Smac mimetics, numerous independent studies have also shown that cIAP2 can be efficiently degraded by Smac mimetics in various cell lines. [27][28][29][30][31][32] These observations suggest the existence of other factors that specifically regulate cIAP2 stability upon Smac mimetic treatment. In this study, we propose a new mechanism involving USP11-mediated cIAP2 regulation.…”

Targeting inhibitor of apoptosis proteins by Smac mimetic elicits cell death in poor prognostic subgroups of chronic lymphocytic leukemia

“…We previously reported that Smac mimetics can act as chemosensitizers in combination with anticancer drugs, 30,46 demethylating agents or glucocorticoids 45,47 and as radiosensitizer. 31,48,49 Our findings showing that BV6 cooperates together with Obatoclax to induce cell death in CLL cells, but not together with ABT-263 may be related to the differential activity profiles of these BH3 mimetics, as Obatoclax antagonizes BCL-2, BCL-xL, BCL-W and MCL-1, whereas ABT-263 targets BCL-2, BCL-xL and BCL-W, but not MCL-1.…”

Promises and Challenges of Smac Mimetics as Cancer Therapeutics

“…These results provide the basis for a randomized phase II study comparing birinapant in combination with 5-Aza against 5-Aza alone in the first-line setting for patients with MDS (70). Consistently, recent evidence from preclinical studies revealed synergistic antileukemic effects of Smac mimetics plus 5-Aza against AML cells, including AML stem/progenitors (55,56).…”

“…Smac mimetics were also shown to enhance the sensitivity toward radiotherapy both in vitro and in preclinical in vivo models of human cancers (42)(43)(44)(45)(46). Furthermore, synergistic induction of cell death was reported for Smac mimetics in combination with novel agents, including TRAIL receptor agonists (47)(48)(49)(50)(51)(52)(53)(54), epigenetic drugs (55)(56)(57), or immunotherapies (58-61). Together, these preclinical studies underscore the potential of Smac mimetics to sensitize cancer cells for cytotoxic therapies by lowering the threshold to trigger cell death.…”