Apoptosis Repressor with Caspase Recruitment Domain Is Regulated by the cIAP1-NIK Axis and Confers Resistance to SMAC Mimetic Birinapant-Induced Cell Death in AML

Abstract 534

The inhibitors of apoptosis (IAPs), including cIAP1, cIAP2, and XIAP are a family of anti-apoptotic proteins that play important roles in regulating cell survival. SMAC, a mitochondrial protein, is a natural cellular inhibitor of IAPs. SMAC mimetics, mimicking the IAP-binding site in the N-terminal AVPI peptide sequence of SMAC, are a new class of anticancer agents that degrade cIAPs and suppress XIAP activity. ARC (Apoptosis repressor with caspase recruitment domain) is an anti-apoptotic protein that inhibits the activation of caspase-8. We previously reported that the SMAC mimetic birinapant (TL32711; Tetralogic Pharmaceuticals, Malvern, PA) degrades cIAP1 and promotes apoptosis via the death receptor/caspase-8-mediated extrinsic pathway in primary AML cells and in AML cell lines in the presence of death receptor ligands (Carter BZ et al., ASH 2011). High ARC levels also predict adverse outcome in patients with AML (Carter BZ et al., Blood 2011). Here we report that birinapant-induced reduction in cIAP1 is accompanied by increased ARC levels.

cIAPs are known E3 ligases for NF-κB-inducing kinase (NIK), an upstream kinase of non-canonical NF-κB. SMAC mimetics, including birinapant cleave cIAPs, leading to stabilization of NIK and activation of non-canonical NF-κB signaling and its downstream targets. To determine whether ARC is regulated via the cIAP1-NIK axis, we knocked down NIK in OCI-AML3 and Molm13 cells by siRNAs and found that inhibition of NIK decreased ARC RNA and protein levels in these cells and suppressed birinapant-induced increases of ARC, suggesting that ARC is regulated via the cIAP1/NIK/NF-κB cascade. We determined levels of ARC and cIAP1 by reverse-phase protein array in 511 samples obtained from patients with newly diagnosed AML and found that cIAP1 and ARC were inversely correlated (R = −0.225, P< 0.0001) further supporting the negative regulation of ARC by cIAP1 in primary AML samples.

Data indicate that birinapant induces caspase-8-mediated cell death, but increases levels of ARC in AML cells which inhibits caspase-8 activation, suggesting that ARC is a resistance factor for birinapant-induced cell death. To further investigate this mechanism, we generated stable ARC-knock down (K/D) OCI-AML3 and Molm13 cells and stable ARC-overexpressing (O/E) KG-1 cells and treated these cells with birinapant or birinapant plus TNFα. We found what ARC-K/D OCI-AML3 and Molm13 cells were more sensitive and ARC-O/E KG-1 cells were more resistant to birinapant- or birinapant plus TNFα-induced apoptosis than their control cells. We reported previously that demethylating agents can enhance birinapant-induced apoptosis induction in AML cells. Examination of NIK and ARC levels in decitabine or 5-azacytidine treated AML cells showed that the demethylating agents indeed decreased NIK and ARC protein levels.

Leukemia cells are in close contact with the bone marrow (BM) microenvironment in vivo that protects them from cell death induced by various therapeutic agents. Leukemia cells were co-cultured with BM-derived mesenchymal stromal cells (MSCs) in vitro to mimic in vivo conditions. We found that birinapant decreased cIAP1 and increased ARC levels also in MSCs co-cultured with AML cells. We generated stable ARC-K/D MSCs and treated KG-1, OCI-AML3, and Molm13 cells co-cultured with ARC-K/D or vector control MSCs with birinapant plus TNFα and primary AML patient samples co-cultured with ARC-K/D or vector control MSCs with birinapant. ARC-K/D MSCs provided AML cells with less protection than control MSCs against birinapant plus TNFα- or birinapant-induced apoptosis.

Collectively, data demonstrate that ARC is regulated via the cIAP1/NIK signaling pathway and is a resistance factor for SMAC mimetic birinapant-induced cell death. ARC K/D sensitizes AML cells to SMAC mimetic-induced cell death and also suppresses MSC-mediated protection of AML cells against drug-induced apoptosis.