Abstract 3046: Combining Chk1/2 inhibition with cetuximab and irradiation enhances in vitro and in vivo cytotoxicity of head and neck cancer models
Authors: Zeng Ling , Beggs Reena , Cooper Tiffiny , Yang. Eddy Shih-Hsin 
Published: July 2016

Abstract
Background:
Epidermal growth factor receptor (EGFR) overexpression is present in 80-100% of head and neck cancers, and targeting EGFR with cetuximab has improved outcomes when combined with radiotherapy (XRT). However, over half of these patients still succumb to this disease, necessitating novel therapies. Checkpoint kinase 1 and 2 (Chk1/2) are serine/threonine kinases that prevent cell cycle progression and serve as critical regulators of the DNA-damage response. LY2606368 monomesylate monohydrate (CHKi), a potent inhibitor of Chk1/2, exhibits single agent activity in tumors of squamous cell histology, including head and neck. Because of the role of Chk1/2 in DNA damage response, we hypothesized that CHKi may enhance the cytotoxicity induced by cetuximab and XRT in head and neck cancer.
Methods:
The HPV-negative UM-SCC1 and HPV-positive UM-SCC47 head and neck cancer cells were used. Doses of drug compounds are as follows: CHKi (LY2606368, Eli Lilly) 1nM; Cetuximab 0.25μg/mL. Cell proliferation was assessed using a Beckman Coulter Counter. Clonogenic survival was measured with colony formation assays. Apoptosis was analyzed using Annexin V-FITC staining and western blot analysis for cleaved and full length caspase-3 and 9. DNA damage and checkpoint signaling was investigated via western blot analysis for phospho- and total Chk1, Chk2, ATM, ATR, and γ-H2AX. Animal studies were performed using orthotopic tongue injection of UM-SCC1-Luc or heterotopic flank injection of UM-SCC47 cells in athymic nude mice, which were then subjected to 3 cycles of once-weekly CHKi (4mg/kg), cetuximab (0.1mg), and 2Gy XRT.
Results:
The addition of CHKi to cetuximab and XRT resulted in the greatest reduction of cell proliferation and increased cytotoxicity in both HPV positive (HPV+) UM-SCC47 and HPV negative (HPV-) UM-SCC1 cancer cells in vitro. Triple combination treatment effectively reduced cell growth by ∼89% in UM-SCC1 and ∼63% in UM-SCC47 at 96hrs compared to untreated cells. A robust decrease in clonogenic survival in both cell lines was also observed (approximately 90% at 2Gy). Furthermore, this combination abrogated the DNA damage checkpoints induced by cetuximab and XRT, resulting in persistent DNA damage and activation of apoptosis. Specifically, we observed CHKi-mediated downregulation of total and phospho- Chk1, Chk2, ATR, and ATM, with concomitant increases in cleaved caspase-3 and caspase-9. Importantly, combining CHKi, cetuximab, and XRT led to a significant tumor growth delay in mice bearing orthotopic or heterotopic head and neck tumor xenografts compared to control mice. Additionally, no significant weight loss or toxicity was observed in the treatment.
Conclusions:
CHKi enhanced the in vitro and in vivo cytotoxicity of cetuximab and XRT against human head and neck tumor models. A clinical trial to test this treatment for head and neck cancer patients is underway (NCT02555644).
Citation Format: Ling Zeng, Reena Beggs, Tiffiny Cooper, Eddy Shih-Hsin Yang. Combining Chk1/2 inhibition with cetuximab and irradiation enhances in vitro and in vivo cytotoxicity of head and neck cancer models. [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 3046.