Birinapant, A Bivalent Smac-Mimetic, Promotes Efficient Cellular Iap E3 Ligase Activity And Formation Of A Pro-Apoptotic Ripk1:Caspase-8 Complex While Monovalent Iap Inhibitors Are Less Efficient - Implications For Therapeutic Utility

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The second mitochondria-derived activator of caspases (SMAC) is thought to exert its pro-apoptotic activity as a homodimeric protein. Both monovalent and bivalent peptidomimetics of the SMAC tetrapeptide are being developed for cancer therapy. Birinapant/TL32711 is a bivalent SMAC-mimetic that targets the inhibitor of apoptosis (IAP) proteins whose gene abnormalities have been implicated in various cancers. Owing to structural differences between bivalent SMAC-mimetics and monovalent IAP-inhibitors, we sought to compare and contrast the biochemical activity of birinapant with several monovalent IAP-inhibitors including a monovalent- version of birinapant/TL32711, MV711. Previous studies have shown that both bivalent and monovalent agents promote auto-ubiquitylation and subsequent degradation of cIAP1 and cIAP2, which triggers tumor necrosis factor receptor (TNFR)-mediated cell death in certain tumor cell lines. However, birinapant showed substantial differences from IAP-inhibitors in degrading TRAF2-associated cIAP1 and cIAP2. Here we show that MV711 was less efficient at degrading cIAP1 by a factor of 7-fold (16 vs. 118 nM, birinapant vs. MV711, respectively) and inhibiting TNF-mediated NF-κB activation by 220-fold (9 vs. 1985 nM, respectively). In addition, a linker-lengthened variant of birinapant was less able to inhibit NF-κB activation by 71-fold (9 vs. 642 nM, respectively). We also studied the effect of birinapant or IAP-inhibitor treatment on SKOV-3, MDA-MB-231 and EVSA-T cancer cell lines in vitro. Comparable cIAP1 BIR3 domain binding constants and IC50 values for the degradation of cIAP1 and cIAP2 (ΔcIAP1/2) were observed for these two classes of agents, and both birinapant and IAP-inhibitors showed dose-dependent induction of cell death. However, despite such comparable potencies, the IAP-inhibitors did not completely kill SKOV-3 or MDA-MB-231 cells even with concentrations >100-times their ΔcIAP1/2 IC50 values. Birinapant revealed the highest suppression of cancer cell growth in the cell lines tested, even after the agent was removed, whereas the cell lines treated with the IAP-inhibitors showed rapid restoration of cell proliferation within 24 h following removal of the agents. These results suggested that monovalent IAP-inhibitors require maintenance of high steady state levels of drug to effectively suppress tumor growth in vivo. In agreement with their inability to induce cell death, IAP-inhibitors were less efficient in stimulating the formation of a RIPK1:caspase-8 complex when compared to birinapant in EVSA-T or SKOV-3 cells. These observations may be partly attributed to the reduced ability of IAP-inhibitors to degrade TRAF2-associated cIAP1 which serves a central role in the activation of NF-κB via TNFR. Citation Format: Yasuhiro Mitsuuchi, Christopher A. Benetatos, Thomas Haimowitz, Yijun Deng, Angeline C. Mufalli, Martin E. Seipel, Jennifer M. Burns, Gurpreet S. Kapoor, C. Glenn Begley, Stephen M. Condon. Birinapant, a bivalent SMAC-mimetic, promotes efficient cellular IAP E3 ligase activity and formation of a pro-apoptotic RIPK1:caspase-8 complex while monovalent IAP inhibitors are less efficient - implications for therapeutic utility. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1806. doi:10.1158/1538-7445.AM2014-1806