Abstract
The acquisition of apoptosis resistance is a fundamental event in cancer development. Among the mechanisms used by cancer cells to evade apoptosis is the dysregulation of inhibitor of apoptosis (IAP) proteins. The activity of the IAPs is regulated by endogenous IAP antagonists such as SMAC (also termed DIABLO). Antagonism of IAP proteins by SMAC occurs via binding of the N-terminal tetrapeptide (AVPI) of SMAC to selected BIR domains of the IAPs. Small molecule compounds that mimic the AVPI motif of SMAC have been designed to overcome IAP-mediated apoptosis resistance of cancer cells. Here, we report the preclinical characterization of birinapant (TL32711), a bivalent SMAC-mimetic compound currently in clinical trials for the treatment of cancer. Birinapant bound to the BIR3 domains of cIAP1, cIAP2, XIAP, and the BIR domain of ML-IAP in vitro and induced the autoubiquitylation and proteasomal degradation of cIAP1 and cIAP2 in intact cells, which resulted in formation of a RIPK1:caspase-8 complex, caspase-8 activation, and induction of tumor cell death. Birinapant preferentially targeted the TRAF2-associated cIAP1 and cIAP2 with subsequent inhibition of TNF-induced NF-κB activation. The activity of a variety of chemotherapeutic cancer drugs was potentiated by birinapant both in a TNF-dependent or TNF-independent manner. Tumor growth in multiple primary patient-derived xenotransplant models was inhibited by birinapant at well-tolerated doses. These results support the therapeutic combination of birinapant with multiple chemotherapies, in particular, those therapies that can induce TNF secretion.
Highlights
Apoptosis or programmed cell death is a genetically encoded process that results in the clearance of damaged cells without inducing an inflammatory response and can be initiated by either intrinsic or extrinsic cellular cues
Other reagents included AbuRPFK(5Fam)-NH2 peptide (Biomer Technologies), anti-cIAP1, -cIAP2, and -ML-inhibitor of apoptosis (IAP) antibodies were from R&D, Antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) and -PARP antibodies were from Santa Cruz Biotechnology, anti-XIAP antibody (Stressgen), anti-inhibitor of NF-kB a (IkBa) antibody was from Cell Signaling, anti-receptor-interacting protein kinase 1 (RIPK1) antibody was from BD, anti–caspase-8 and TNF receptor–associated factor 2 (TRAF2) antibodies were from both Santa Cruz Biotechnology and Cell Signaling
The direct interaction of birinapant with native XIAP, cIAP1, cIAP2, and ML-IAP proteins was confirmed by using a pulldown assay with biotin-labeled birinapant (Supplementary Fig. S1) and the total cell lysate from either IGROV-1 or SKMEL-28 tumor cells; pretreatment of IGROV-1 cells with TNF induced the expression of cIAP2, which was detected in this assay
Summary
Apoptosis or programmed cell death is a genetically encoded process that results in the clearance of damaged cells without inducing an inflammatory response and can be initiated by either intrinsic or extrinsic cellular cues. Apoptotic signaling culminates in the activation of a family of cysteine proteases with aspartate specificity. Resistance to apoptosis is a fundamental property of cancer [1]. The acquisition of genetic lesions, which dysregulate the execution of apoptosis, is known to cooperate with other cellular defects such as oncogene activation leading to tumor initiation and progression [1, 2]. The emergence of drug resistance has been attributed, in part, to defects in apoptosis [3, 4]. The development of drugs aimed at regulation of the apoptotic signaling of cancer cells represents a new paradigm in cancer treatment and several of these agents have entered clinical trials
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