Abstract C144: LCL161, a SMAC mimetic, induces preferential radiosensitization in human papillomavirus negative head and neck squamous cell carcinoma through induction of apoptosis

Abstract

Abstract Background and purpose: Evasion of apoptosis contributes to radioresistance of head and neck squamous cell carcinoma (HNSCC), calling for novel strategies to overcome apoptotic resistance. Second mitochondria-derived activator of caspase (SMAC) - mimetics are a new class of targeted drugs that specifically induce apoptotic cell death and block pro-survival signalling by antagonizing selected members of the inhibitor of apoptosis protein (IAP) family. The present study was designed to investigate the radiosensitizing effect of a SMAC mimetic, LCL161, in HNSCC and the underlying mechanisms for radiosensitization. Material and methods: We examined the correlation between mRNA expression of apoptotic pathway associated molecules and HPV status by using The Cancer Genome Atlas Database (TCGA) in which 279 HNSCC tumors are included. = Protein expression was investigated in 6 HNSCC cell lines (3 HPV[+], 3 HPV[-]), normal oral epithelial cells (NOE) by immunoblotting, and in primary HNSCC tumors by immunohistochemistry (IHC). Clonogenic survival and Alamar Blue assays were carried out in order to explore the potential of LCL161 as a radiosensitizer in HNSCC cell lines. Cell cycle analysis and Annexin-V assays were performed to investigate mechanisms of radiation-induced tumor cell death. Human tumor xenografts were generated to explore the radiosensitization effect of LCL161 on HPV[-] tumors in vivo. Results: TCGA database analysis indicated significantly higher mRNA expression of cIAP1 and RELA, lower expression of CASP3, CASP9, and BCL2 in HPV[-] compared with HPV[+] HNSCC tumors. Consistent with these findings, immunoblotting confirmed that that protein expression of cIAP1 and p65 encoded by RELA were elevated in HPV[-] HNSCC cells.LCL161 displayed minimal single-agent cytotoxicity with IC50 values between 32μM – 95μM in 6 HNSCC cells. LCL161 is a potent inhibitor of cIAPs, and caused downregulation of cIAP1 at nanomolar concentrations in less than 1 hour, while it showed no obvious effect on NFκB, Stat3, Erk, or Akt activation. Interestingly, we found that LCL161 could induce radiosensitization only in HPV[-] HNSCC cell lines, but not in HPV[+] HNSCC cells. LCL161 mediated radiosensitisation was associated with enhanced activation of caspases-3, -7, -8, -9, and PARP. Blockage of caspase activation via a pan-caspase inhibitor, z-VAD-fmk, largely abrogated LCL161 radiosensitization. Conclusion: These results suggest that LCL161, a SMAC-mimetic, significantly radiosensitizes a subset of HNSCC tumors, via a mechanism involving caspase activation and apoptosis induction. This agent holds promise for future clinical development as a novel radiosensitizer in the treatment HNSCC HPV[-] tumors. Citation Format: Linlin Yang, Bhavna Kumar, Mitchell Romito, Theodoros N. Teknosa, Amab Chakravarti, Terence M. Williams. LCL161, a SMAC mimetic, induces preferential radiosensitization in human papillomavirus negative head and neck squamous cell carcinoma through induction of apoptosis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C144.