Abstract 981: Wee-1 kinase inhibitor AZD-1775 radiosensitizes esophageal cancer through targeting G2 checkpoint activation
Authors: Yang Linlin , Pettit Cory , Hu Andrew , Li Tianyun , Williams Terence M. 
Published: August 2018

Background: Esophageal cancer is a deadly cancer that has a 5-year survival rate of 18%, which has remained unchanged for decades. Due to a high incidence of p53 mutation, esophageal cancer cells often lack a functional G1-S checkpoint, which increases reliance upon the G2-M checkpoint for adaptation to stress and DNA repair, making G2-M checkpoint inhibition a target for novel therapeutic development. AZD-1775, a potent inhibitor against the critical G2-M checkpoint protein Wee-1, is currently in clinical trials across a number of tumor types. The purpose of this study was to determine the capacity of AZD-1775 to abrogate the radiation-induced G2 checkpoint arrest and modulate radiosensitivity in esophageal cancer cell (ECC) line models. Materials and Methods: AlamarBlue assay and clonogenic assays were carried out in 4 ECC cell lines (squamous cells: Flo-1, KYSE-30; adenocarcinoma cells: OE-33, SK-4) to calculate IC50 and potential of AZD-1775 as a radiosensitizer in ECC cell lines, respectively. The effects of AZD-1775 on radiation induced checkpoint response of ECC cells were determined in vitro by analyzing cell cycle via flow cytometry, detecting alterations on cell cycle markers through immunoblotting. We also determined AZD-1775 function on radiation caused DNA damage response through mitotic catastrophe analysis by immunofluorescence staining, and p-H2A.x expression analysis by immunoblotting. The role of AZD-1775 in fractionated radiation in comparison to single fraction radiation was also investigated through colony formation assay. Eventually, human esophageal cancer cell xenografts were generated to explore the radiosensitization effect of AZD-1775 on ECC tumors in vivo. Results: AlamarBlue assay showed that the IC50 concentrations of AZD-1775 on ECC cells are between 300 - 600 nM, and exposure to 100 nM of AZD-1775 didn't significantly increase cytotoxicity. However, 100 nM of AZD-1775 was sufficient to effectively sensitize ECC cells to radiation therapy in clonogenic assays in 3 of 4 cell lines. Study of mechanisms underlying AZD-1775-mediated radiosensitization revealed that AZD-1775 significantly abrogates radiation induced G2 checkpoint arrest, and attenuates radiation induced phosphorylation of CDC2. Additionally, AZD-1775 altered expression of various mediators of the cell cycle, consistent with its functions of abrogating G2 checkpoint.AZD-1775 prevented effective repair of DNA damage after radiation, and eventually led to cell death, as supported by increased mitotic catastrophe and increased and persistent p-H2A.x expression after radiation.Conclusion: These results show that inhibition of WEE-1by AZD-1775 has the potential to be an effective strategy for radiation sensitization in esophageal cancer cells.

Citation Format: Linlin Yang, Cory Pettit, Andrew Hu, Tianyun Li, Terence M. Williams. Wee-1 kinase inhibitor AZD-1775 radiosensitizes esophageal cancer through targeting G2 checkpoint activation [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 981.