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Wee1 Kinase Inhibitor AZD1775 Effectively Sensitizes Esophageal Cancer to Radiotherapy

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Wee1 Kinase Inhibitor AZD1775 Effectively Sensitizes Esophageal Cancer to Radiotherapy Author Manuscript Published OnlineFirst on March 27, 2020; DOI: 10.1158/1078-0432.CCR-19-3373 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Wee1 Kinase Inhibitor AZD1775 Effectively Sensitizes Esophageal Cancer to Radiotherapy Linlin Yang1, Changxian Shen1, Cory Pettit1, Tianyun Li1, Andrew Hu1, Eric Miller1, Junran Zhang1, Steven H. Lin2, Terence M. Williams1, * 1The Ohio State University Medical Center, Arthur G. James Comprehensive Cancer Center and Richard J. Solove Research Institute, Columbus, Ohio. 2The University of Texas MD Anderson Cancer Center, Houston, Texas. *Corresponding Author: Terence M. Williams, Department of Radiation Oncology, The Ohio State University, 460 W. 12th Avenue, BRT/Room 492, Columbus, OH 43210-1280. Phone: (614) 293-3244. Fax: 614-293-4044. E-mail: [email protected] Running title: Targeting Wee1 for radiosensitization of esophageal cancer Key words: Wee1, AZD1775, G2 checkpoint, mitotic catastrophe, esophageal cancer Conflicts of Interest: The authors report no potential conflicts of interest. Financial Disclosure Statements: All authors have no competing financial interests to disclose. Funding Support: This work was supported by the following grants: The Ohio State University Comprehensive Cancer Center (OSU-CCC), National Institutes of Health (P30 CA016058 and R01 CA198128), and National Center for Advancing Translational Sciences (KL2TR001068). 1 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 27, 2020; DOI: 10.1158/1078-0432.CCR-19-3373 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT Purpose: Esophageal cancer (ESCA) is a deadly malignancy with a 5-year survival rate of only 5-20%, which has remained unchanged for decades. ESCA possesses a high frequency of TP53 mutations leading to dysfunctional G1 cell cycle checkpoint, which likely makes ESCA cells highly reliant upon G2/M checkpoint for adaptation to DNA replication stress and DNA damage after radiation. We aim to explore whether targeting Wee1 kinase to abolish G2/M checkpoint sensitizes ESCA cells to radiotherapy. Experimental Design: Cell viability was assessed by cytotoxicity and colony forming assays, cell cycle distribution was analyzed by flow cytometry, and mitotic catastrophe was assessed by immunofluorescence staining. Human ESCA xenografts were generated to explore the radiosensitizing effect of AZD1775 in vivo. Results: The IC50 concentrations of AZD1775 on ESCA cell lines were between 300 - 600 nM. AZD1775 (100 nM) as monotherapy did not alter the viability of ESCA cells, but significantly radiosensitized ESCA cells. AZD1775 significantly abrogated radiation-induced G2/M phase arrest and attenuation of p-CDK1-Y15. Moreover, AZD1775 increased radiation-induced mitotic catastrophe, which was accompanied by increased H2AX levels, and subsequently reduced survival after radiation. Importantly, AZD1775 in combination with radiotherapy resulted in marked tumor regression of ESCA tumor xenografts. Conclusions: Abrogation of G2/M checkpoint by targeting Wee1 kinase with AZD1775 sensitizes ESCA cells to radiotherapy in vitro and in mouse xenografts. Our findings suggest that inhibition of Wee1 by AZD1775 is an effective strategy for radiosensitization in esophageal cancer and warrants clinical testing. 2 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 27, 2020; DOI: 10.1158/1078-0432.CCR-19-3373 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. TRANSLATIONAL RELEVANCE Stage II/III esophageal cancers are commonly treated with radiation or chemoradiation, with or without surgery. However, esophageal cancer has very poor prognosis, and preclinical studies have shown esophageal cancer cells are resistant to radiation. The majority of both esophageal adenocarcinoma and squamous cell carcinomas harbor mutations in TP53, an important tumor suppressor gene that also functions to promote cell cycle arrest in G1/S after DNA damage from radiation. In this preclinical study, we target the G2/M cell cycle checkpoint with AZD1775, a Wee1 kinase inhibitor, in combination with radiation in order to enhance therapeutic efficacy. We find that in TP53-mutated cells lacking an effective G1/S checkpoint, AZD1775 markedly radiosensitizes esophageal cancer cells to radiation both in cell culture assays and animal studies. Our results justify a clinical trial to determine the safety and efficacy of combining AZD1775 and radiation in patients with esophageal cancer. 3 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 27, 2020; DOI: 10.1158/1078-0432.CCR-19-3373 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. INTRODUCTION Esophageal cancer is the 6th leading cause of cancer-related death and affects more than 450,000 people worldwide (1). Standard-of-care therapy for localized esophageal cancer is radiotherapy and chemotherapy followed by surgery, but recurrence rates remain high. Moreover, approximately of 50% patients diagnosed with esophageal cancer present with unresectable or metastatic disease (2). In the past decade, although great advances have been made for the prevention and control of many cancers such as lung cancer and breast cancer, the overall 5-year survival rate of esophageal cancer patients remains below 20% and the incidence is increasing rapidly worldwide (1,3,4). Therefore, there is an urgent need to develop novel effective therapies for the management of esophageal cancer (2,5). Proper cell proliferation and accurate genetic material duplication depends on the tight and fine coordination of the cell cycle surveillance systems including G0/G1, S, G2 and M cell cycle checkpoints (6). Cell cycle progression is controlled by cyclin-dependent kinases (CDKs), which are regulated by cell growth and mitogenic signals. In response to ever-changing intracellular and extracellular genotoxic insults, cells activate DNA damage, replication and mitotic checkpoints, which function to inhibit the activity of CDKs and halt cell cycle progression in order to provide time to repair DNA damage and fix chromatin disruption (7). The fine coupling of cell cycle and DNA damage checkpoints ensures genome integrity and cell survival (7). Aberrant activation of CDKs and hence uncontrolled cell cycle progression is a hallmark of cancer cells (8). Many human cancers have deficits in G1/S checkpoint due to mutations in the p53 signaling axis including mutations of TP53, CDKN2A, and RB (9). Treatment of these cells with radiation induces a G2/M arrest, allowing time for DNA repair, thus leading to a higher level of dependence of these cancer cells on G2/M checkpoint for survival. In these cases, genetic abrogation of the G2/M checkpoint may allow entry of cells into mitosis with incompletely-repaired damaged DNA, ultimately leading to mitotic catastrophe and cell death (10). It has therefore been proposed that small molecules targeting G2/M checkpoint are promising cancer therapy agents either as monotherapy or in combination with radiotherapy and chemotherapy (5,11-13). Wee1 kinase is essential for scheduled cell division through inhibitory phosphorylation of CDK1 and CDK2 at the conserved tyrosine15 residue (14). Particularly, Wee1-mediated 4 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 27, 2020; DOI: 10.1158/1078-0432.CCR-19-3373 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. phosphorylation and inhibition of CDK1 plays a critical role in G2/M checkpoint under normal cell growth and in response to DNA damage or replication stress (15). DNA damage or replication stress activates ATM/CHK2 and ATR/CHK1 signaling cascades to maintain genome stability as well as cell viability (13). Activation of CHK1 by ATR in response to various types of DNA lesions phosphorylates and stimulates Wee1 activation to suppress CDK1 activity thereby preventing entry into mitosis (15). Forced cell cycle progression in the setting of DNA damage perpetuates DNA and chromatin damage, and leads to cell death because of irreparable genetic lesions (11). Interestingly, Wee1 expression is upregulated in many cancers and associated with the survival of cancer patients (16-18). Given the pivotal role for Wee1 in the regulation of CDK1 activity, targeting Wee1 has been proposed for the sensitization of cancer cells to radiotherapy and chemotherapy (11,19-21). Large-scale genomic studies have found that esophageal cancer has an extremely high frequency of TP53 mutations, ranging from 44% to 93% (22,23). Recently, The Cancer Genome Atlas (TCGA) demonstrated that TP53 mutations were the single most common significantly mutated gene in ESCA, occurring in ~71% and ~91% of esophageal adenocarcinoma and esophageal squamous cell carcinoma, respectively (24). Therefore, esophageal cancer cells may depend on G2/M checkpoint for survival and may be very sensitive to G2/M checkpoint abrogation by Wee1 inhibition. AZD1775 is a novel small molecule
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