Abstract 3974: Combination of birinapant and TRAILR2 agonist antibody enhances cell death in HPV-positive head and neck squamous cell carcinomas
Authors: An Yi , Sun Lillian , Derakhshan Adeeb , Carlson Sophie , Chen Zhong , Waes Carter Van 
Published: August 2018

Abstract
Head and neck squamous cell carcinomas (HNSCC) induced by human papilloma virus (HPV) have increased recently in the US. Analysis of HNSCC TCGA datasets provide evidence for distinct alterations in expression of NF-κB components and death pathways in HPV(+) HNSCC. Previously, we have found that Birinapant, a novel SMAC mimetic that inhibits inhibitor of apoptosis proteins (IAPs), exhibits single agent activity and sensitizes a subset of HPV(-) HNSCC cell lines to death agonists like TNF-α and TRAIL. Birinapant exhibited anti-tumor effects in vivo for HNSCC with FADD amplification. However, Birinapant alone exhibited minimal activity against HPV(+) tumor in vivo. To explore how to enhance the sensitivity of HPV(+) cells to Birinapant, we treated a panel of cell lines with Birinapant alone, and in combination with TRAIL or TNF-α in vitro. Birinapant sensitized HPV(+) cell lines UPCI-SCC-90 and UM-SCC-47 to TRAIL or TNF-α. The IC50 for Birinapant was under 50nM when combined with TRAIL or TNF-α for UPCI-SCC-90 and UM-SCC-47 cell lines. To explore the therapeutic potential of enhancing TRAILR mediated death signaling in HPV(+) HNSCC cells, we investigated the effects of an agonistic polyclonal TRAILR2 antibody. Anti-TRAILR2, TRAIL, or TNF-α alone showed little or no inhibitory effect on proliferation of UPCI-SCC-90 and UM-SCC-47 cells in vitro. However, a combination of Birinapant and TRAILR2 antibody, and triple combination of Birinapant, TRAIL, and TRAILR2 antibody showed additive or synergistic effects to inhibit cell density in a dose dependent manner. Combination of Birinapant with TRAIL, TRAILR2 antibody, or triple combination led to cell death and subG0 DNA fragmentation, as demonstrated by propidium iodide staining and flow cytometry. To further analyze the mechanism of cell death, we treated cells with the inhibitor of pan-caspase (ZVAD), caspase-8 (ZIETD), or RIPK1 (necrostatin) inhibitors. In both UPCI-SCC 90 and UM-SCC 47 cells, caspase inhibition (ZVAD and ZIETD) completely abolished the effects of Birinapant treatment in combination with TRAIL, TRAILR2 antibody, and triple combination, whereas RIPK1 inhibition (necrostatin) did not reverse the effects significantly. Our data suggest that Birinapant and TRAIL receptor mediated cell death is predominantly caspase 8 dependent. Taken together, our study indicates that TRAIL or TRAILR2 agonist antibody enhances Birinipant anti-tumor activity, and triple combination exhibits the strongest synergistic effects. The triple combination of targeting death pathway by TRAIL and TRAILR2 antibody with Birinapant could be a potent strategy to treat aggressive subset of HPV(+) HNSCC, and warrants the testing in future clinical trials. (Supported by NIDCD intramural projects ZIA-DC-000016, 74).
Citation Format: Yi An, Lillian Sun, Adeeb Derakhshan, Sophie Carlson, Zhong Chen, Carter Van Waes. Combination of birinapant and TRAILR2 agonist antibody enhances cell death in HPV-positive head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3974.