Abstract 5175: Birinapant enhances death agonist antibody against TRAILR2 anti-tumor activity in HPV-positive head and neck squamous cell carcinomas
Authors: Yi An, Jun W. Jeon, Lillian Sun, Adeeb Derakhshan, Jianhong Chen, Hui Cheng, Xinping Yang, Christopher Silvin, Carter Van Waes, Zhong Chen
Journal: Cancer Research (2020)

Abstract Human papilloma virus positive (HPV+) head and neck squamous cell carcinomas (HNSCC) exhibit a better prognosis and response to therapies than HPV(-) cancers. Analysis of HNSCC TCGA dataset provides evidence for distinct alterations in expression of components of the NF-κB and cell death pathways in HPV(+/-) HNSCC. Previously, we have found that birinapant, a novel SMAC mimetic that inhibits inhibitor of apoptosis proteins (IAPs), sensitizes a subset of HPV(-) HNSCC cell lines to death agonists like TNF-α and TRAIL. In our current study, we have observed that birinapant also sensitizes several HPV(+) cell lines to TNF-α and TRAIL in vitro. The IC50 for birinapant was under 50nM with TRAIL and TNF-α for the HPV (+) UPCI-SCC-90 and UM-SCC-47 cell lines. As TRAIL is known as a selective cancer cell death inducer, we explored its potential for enhancing death signaling in HPV(+) HNSCC cells via the effects of an agonistic polyclonal TRAILR2 antibody. Flow cytometry analysis confirmed the presence of TRAILR2 expression on the cell surface of the two HPV(+) cell lines. Treatment of cells with the antibody or TRAIL alone showed little or no inhibitory effect on growth of either cell lines. However, treatment with birinapant plus antibody, and especially the triple combination of birinapant, TRAIL, and antibody showed additive or synergistic effects to inhibit cell proliferation in a dose dependent manner. The Fixed Apoptotic Necrotic test using anti-phosphatidylserine and Zombie fixable dye showed that late apoptosis was the predominant mode of cell death for all combination groups for both cell lines at 48 hours post treatment. The proportion of cells undergoing apoptosis/necrosis was highest in the triple combination group in both cell lines, followed by birinapant and TRAIL combination for UM-SCC-47, or birinapant and antibody combination for UPCI-SCC-90, which was consistent with the relative percentage of sub-G0 DNA determined by flow cytometry analysis using propidium iodide staining. Enhanced apoptosis/necrosis with the triple combination was observed earlier than with single or dual treatment (12 versus 24 hours post treatment). Specific cell death mechanism was analyzed using pan-caspase (ZVAD), caspase-8 (ZIETD), and RIPK1 (necrostatin) inhibitors. In both UPCI-SCC-90 and UM-SCC-47 cells, caspase inhibition (ZVAD and ZIETD) completely reversed the effects of the double or triple combination treatments, whereas necrostatin did not. This suggests that the TRAILR2 agonist antibody mediated cell death in combination with birinapant is predominantly caspase-8 dependent. These results indicate that the TNF-α, TRAIL, and TRAILR2 agonist antibody sensitized birinapant anti-tumor activity, and triple combination exhibited synergistic effects. Supported by NIDCD projects ZIA-DC-000016, -73, -74, and the Medical Research Scholars Program. Citation Format: Yi An, Jun W. Jeon, Lillian Sun, Adeeb Derakhshan, Jianhong Chen, Hui Cheng, Xinping Yang, Christopher Silvin, Carter Van Waes, Zhong Chen. Birinapant enhances death agonist antibody against TRAILR2 anti-tumor activity in HPV-positive head and neck squamous cell carcinomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5175.