Synergy between AZD1775 and DNA Damaging Agents in Treatment of Esophageal Adenocarcinoma

Background: Incidence of esophageal adenocarcinoma (EAC) has increased considerably in the U.S. and Western Europe relative to esophageal squamous cell carcinoma. EAC carries a five-year survival rate of 17.9% despite aggressive treatment with radiation and multi-drug chemotherapy. These treatments are highly toxic and significantly affect patient quality of life, necessitating the development of new treatment options to improve patient care. T53 is the most frequently inactivated gene in EAC. TP53 functions as a key regulator of the cell cycle. Loss of TP53 function in tumor cells results in dependence on the G2/M checkpoint for DNA repair. The Wee1 kinase is one of several proteins responsible for activating G2 arrest in response to DNA damage. Functional Wee1 is necessary for cells, particularly TP53 inactivated tumor cells, to repair DNA and survive. AZD1775, a recently developed small molecule inhibitor of Wee1, prevents Wee1 induction of G2 arrest in response to DNA damage, potentiating apoptosis. The combination of AZD1775 with DNA damaging agents has been investigated in several malignancies; these studies support that the combination is more efficient at eliciting cell death than DNA damaging agents alone in tumors with TP53 mutations. Based on the prevalence of TP53 mutation in EAC, we hypothesized that AZD1775 in combination with DNA damaging agents would have efficacy in EAC. Methods: Four EAC cell lines of known p53 mutational status were treated with the DNA damaging agents cisplatin, docetaxel, and radiation with AZD1775 either omitted or included. Following treatment, cells were assayed for viability as well as markers of apoptosis and Wee1 inhibition via standard immunoblotting. Results: Western blot analysis revealed that AZD1775, in combination with cisplatin or radiation, results in increased DNA damage and apoptosis compared to cisplatin or radiation alone. AZD1775 also functionally inhibited Wee1, shown by reduced phosphorylation of cdc2, a target of Wee1. Treatment with cisplatin/AZD1775 resulted in reduced cell viability compared to cisplatin alone, AZD1775 alone, and the cisplatin/docetaxel combination. Conclusion: Our findings support that the synergy between AZD1775 and DNA damaging agents has therapeutic effects in EAC. This work suggests that integration of AZD1775 into the treatment modalities for EAC may improve patient outcomes and quality of life by allowing clinicians to reduce or alter the schedule of doses of toxic DNA damaging agents.