Abstract 2278: The SMAC-mimetic birinapant regulates autocrine TNF production by caspase-8:RIPK1 complex via p38MAPK pathway
Authors: Kapoor Gurpreet Singh , Benetatos Christopher A. , Mitsuuchi Yasuhiro , Neiman Eric M. , Yu Guangyao , Mckinlay Mark A. , Burns Jennifer , Silke John , Condon Stephen M. , Chunduru Srinivas K. 
Published: May 2015

Abstract
Birinapant (TL32711), a SMAC-mimetic currently in clinical trials, antagonizes multiple members of the inhibitor of apoptosis (IAP) protein family to promote apoptosis in cancer cells through the formation of a caspase-8:RIPK1 complex and induction of autocrine tumor necrosis factor (TNF). Evidently, birinapant-resistant cells do not produce TNF upon drug treatment. However, the mechanism by which birinapant-sensitive cells produce TNF is not clear. Owing to the fact that mitogen-activated protein kinases (MAPKs) regulate a variety of cellular processes, we explored the role of MAPKs in birinapant-induced TNF production. Western blot analyses on a panel of birinapant-sensitive (SK-OV-3, EVSA-T, TOV-21G: all with an IC50 &lt; 200nM) and birinapant-resistant (IGROV-1, HCT116, WIDR & SK-MEL-28: all with an IC50 &gt; 10μM) cell lines showed that birinapant induced rapid and robust phosphorylation of p38MAPK in sensitive cells, which corresponded to increased TNF production as assessed by ELISA. However, resistant cells were not affected at multiple time points. The p38MAPK inhibitor, LY2228820, reduced the levels of birinapant-induced TNF (2-fold with 5nM birinapant after 24h) in birinapant-sensitive cancer cells (SK-OV-3), whereas inhibitors of Erk1/2 (PD98059) and JNK (SP600125) had no effect on TNF production. Interestingly, RNAi-mediated inhibition of p38α (MAPK14) isoform significantly inhibited birinapant-induced TNF production (∼3-fold with 5nM birinapant after 24h) in SK-OV-3 cells while inhibition of p38β (MAPK11) isoform upregulated TNF production (∼3-fold), suggesting that there may be a balance between p38MAPK isoforms to regulate TNF production. Pharmacological and RNAi-mediated inhibition of RIPK1 and caspase-8 activities in SK-OV-3 cells resulted in reduced birinapant-induced p38MAPK phosphorylation and TNF production. Furthermore, commercially available pooled siRNAs targeting different isoforms of cFLIP, a catalytically-inactive homologue of caspase-8, upregulated p38MAPK phosphorylation (∼4-fold) and TNF production (∼8 to 26-fold) in birinapant-resistant cancer cell lines (T24, HT1376 & IGROV-1), and conferred differential sensitivity to the drug treatment (T24-IC50: 7nM; HT1376-IC50: &gt;10nM; IGROV-1-IC50: &gt;1μM). Experiments involving cFLIP long (cFLIP-L) and short (cFLIP-S) isoform-specific siRNAs showed that inhibition of cFLIP-L, but not cFLIP-S, in T24 & HT1376 cells induced p38MAPK phosphorylation and TNF production, and conferred sensitivity to birinapant. However, the combined inhibition of cFLIP-L and cFLIP-S in IGROV-1 cells was required to induce p38MAPK phosphorylation, TNF production and apoptosis. Collectively, these observations indicate that birinapant-induced autocrine TNF production is triggered by the activation of the caspase-8/RIPK1/p38MAPK axis, and that cFLIP isoforms may confer a context-dependent negative regulatory effect.
Citation Format: Gurpreet Singh Kapoor, Christopher A. Benetatos, Yasuhiro Mitsuuchi, Eric M. Neiman, Guangyao Yu, Mark A. Mckinlay, Jennifer Burns, John Silke, Stephen M. Condon, Srinivas K. Chunduru. The SMAC-mimetic birinapant regulates autocrine TNF production by caspase-8:RIPK1 complex via p38MAPK pathway. [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 2278. doi:10.1158/1538-7445.AM2014-2278