Abstract 3045: The SMAC-Mimetic LCL161 exhibits strong synergy with radiation in vivo in head and neck squamous cell carcinoma by promoting tumor cell apoptosis
Authors: Yang Linlin , Kumar Bhavna , Li Tianyun , Romito Mitchell , Teknosa Theodoros N. , Chakravarti Amab , Williams Terence M. 
Published: July 2016

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) - mimetic is a new class of targeted drugs that specifically induce apoptotic cell death and block pro-survival signaling 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: Extent of radiation enhancement of 6 HNSCC cell lines (3 HPV[+], 3 HPV[-]) was assessed by in vitro clonogenic assay with appropriate target inhibition verified by immunoblotting. Combination effect of LCL161 and ionizing radiation was evaluated by cell cycle analysis, Annexin-V assay, and immunoblotting. Correlation between apoptosis associated molecules and HPV status was examined by analyzing mRNA expression in The Cancer Genome Atlas Database (TCGA) in which 279 HNSCC tumors are included, and screening protein expression in 6 HNSCC cell lines via immunoblotting. Human tumor xenografts in nude mice were generated and treated with LCL161, radiation, or the combination. Target inhibition in vivo was confirmed by xenografts lysate immunoblotting, and tissue section immunomhistochemistry.

Results: LCL161 displayed minimal single-agent cytotoxicity with IC50 values ranging 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. Interestingly, we found that LCL161 could induce radiosensitization only in HPV[-] HNSCC cell lines, but not in HPV[+] HNSCC cells.TCGA database analysis indicated significantly higher mRNA expression of cIAP1 in HPV[-] compared with HPV[+] HNSCC tumors. Consistent with this finding, immunoblotting confirmed that that protein expression of cIAP1 was elevated 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, attenuated LCL161 radiosensitization. Finally, LCL161 monotherapy had minimal activity in attenuating tumor growth in HNSCCxenografts. However, the combination of LCL161 and radiation synergistically reduced xenograft tumor volume, with pharmacodynamic inhibition of cIAP1 and caspase activation confirmed in vivo.

Conclusion:Taken together, these results suggest that LCL161, a SMAC-mimetic, significantly radiosensitizes a subset of HNSCC tumors, via a mechanism involving caspase activation and apoptosis induction both in vitro and in vivo. These data provide support for the combination of LCL161 and radiation in the treatment of HNSCC.

Citation Format: Linlin Yang, Bhavna Kumar, Tianyun Li, Mitchell Romito, Theodoros N. Teknosa, Amab Chakravarti, Terence M. Williams. The SMAC-Mimetic LCL161 exhibits strong synergy with radiation in vivo in head and neck squamous cell carcinoma by promoting tumor cell apoptosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3045.