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.

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).

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.

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.

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

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 inhibitor that disrupts G2/M checkpoint by directly inhibiting Wee1 kinase (25). Previous studies have demonstrated that the sensitivity of AZD1775 depends on p53 functional loss in various types of cancers including non-small cell lung cancer (26-29). However, it has also been reported that Wee1 inhibition could radiosensitize carcinoma cells without TP53 mutations (30). In addition to inhibiting G2/M checkpoint, AZD1775 has been shown to induce DNA replication stress via nucleotide exhaustion (31,32), and to reduce homologous recombination repair (33). Although Wee1 is not the core component of the replication stress response pathway, activation of Wee1 by CHK1 induces CDK1 and CDK2 to halt cell cycle progression in response to DNA damage (34). Thus, targeting Wee1 may force cells to enter mitosis in the presence of incomplete DNA replication, which might exacerbate replication stress and development of lethal DNA damage (35,36). AZD1775 has been tested preclinically in many types of cancers, and has been shown to radiosensitize and chemosensitize certain cancers, including pancreatic, breast, prostate, lung, and glioblastoma cancers (20,21).

However, the effects of AZD1775 on esophageal cancer (a disease with an extraordinary high rate of TP53 mutations) as monotherapy or in combination with other therapeutics remains to be determined. In this study, we investigated the anti-neoplastic properties of AZD1775 in combination with radiation in esophageal cancer. We hypothesized that TP53-deficient cells are sensitive to a G2/M checkpoint inhibitor and as such, combining Wee1 inhibition and radiation should target TP53-deficient ESCA cells. We found that abrogation of G2/M checkpoint by targeting Wee1 kinase with AZD1775 markedly sensitizes esophageal cancer cells to radiotherapy in vitro and in mouse xenografts. Our findings suggest that AZD1775 in combination with radiotherapy may improve the therapeutic outcome of esophageal cancer patients.

MATERIALS AND METHODS Antibodies, chemicals, and cell culture OE33, SK4, FLO1, KYSE30, and AGS cell lines were maintained at 37oC in 5% CO2 in DMEM medium supplemented with 10% fetal bovine serum (Sigma), 1% penicillin/streptomycin (Life Technologies, Grand Island, NY). The detailed cell line information is listed in Supplementary Table S1. AZD1775 was obtained under a material transfer agreement from NCI-CTEP through AstraZeneca (Cambridge, UK) and was dissolved in DMSO (Sigma) and added to medium with a final concentration of no more than 0.1% DMSO. Total CDK1, phospho-CDK1 (Tyr15), phospho-Wee1 (Ser642), phospho-H2AX (S139), phospho-histone H3 (S10), cyclin A2, cyclin B1, cyclin E1, cyclin E2 and GAPDH primary antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-rabbit and anti-mouse secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE).

AlamarBlue® assay and IC50 determination AlamarBlue® assay was performed according to manufacturer’s instructions (Roche, Basel, Switzerland). Briefly, cells were seeded in 96-well plates in 6 replicates at a density of 2,000 cells per well in 100 µL medium. The next day, the cells were treated with AZD1775 at various concentrations. After 72 hours, alamarBlue® reagent was added and incubated at 37°C for 4 hours, and absorbance was measured at 490 nm. Half maximal inhibitory concentration (IC50)

was determined using the nonlinear four-parameter regression function in GraphPad Prism (La Jolla, CA).

Immunoblotting Immunoblotting was performed as described previously (37). Briefly, cell lysates were prepared using RIPA buffer (ThermoFisher, Waltham MA) supplemented with 1x protease inhibitors (Complete, Roche, Indianapolis, IN) and phosphatase inhibitors (PhosSTOP, Roche) followed by protein quantification with the Dc protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of protein were loaded and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Membranes were incubated in 5% bovine serum albumin (BSA) in Tris buffered saline with 0.1% Tween-20 (TBST) blocking buffer for 1 hour at room temperature. Primary antibodies with dilution of 1:200-1000 were allowed to bind overnight at 4̊C, or for 2 hours at room temperature. After washing in TBST, the membranes were incubated with immunofluorescent secondary antibodies at a 1:5000 dilution for 1 hour at room temperature. Membranes were washed with TBST and allowed to air dry prior to imaging via LI-COR Odyssey® CLx Imaging System (ThermoFisher).

Radiation clonogenic assay Radiation clonogenic assays were performed essentially as previously described (38). In brief, single cells seeded in 60 mm or 100 mm tissue culture plates were incubated with DMSO or AZD1775 for 3 hours and then irradiated with various doses (0-8 Gy). Radiation was performed with 160kV, 25mA at a dose rate of approximately 113cGy/min using a RS-2000 biological irradiator (RadSource, GA), and cells were fixed seven to ten days after seeding. The number of colonies containing at least 50 cells was counted using a dissecting microscope (Leica Microsystems, Inc. Buffalo Grove, IL) and dose enhancement ratio (DER) was calculated as previously reported (39).

Immunofluorescence for mitotic catastrophe Cells on coverslips were fixed with 2% paraformaldehyde, permeabilized with 1% Triton X-100, and blocked with 3% BSA in phosphate buffered saline (PBS). Briefly, for paraffin-embedded

tissue sections from tumor xenografts, sections were cut onto slides and deparaffinized in xylene and rehydrated through a graded alcohol series. Then, slides were washed in distilled water. Cells on coverslips or tissue sections were stained with anti-tubulin antibody (Cell Signaling Technology), washed, and incubated with a fluorophore-conjugated secondary antibody (Biotium, Hayward, CA). Following nucleus counterstaining with DAPI, the slides were examined on a Zeiss fluorescence microscope. For each experiment, mitotic catastrophe was determined in at least 100 cells.

Flow cytometry Cells were seeded into 6-well plates at a density of 200,000 cells per well in 2 mL medium for 16 hours. The cells were treated with AZD1775 for 3 hrs, followed by ionizing radiation (IR) and then cultured for 24 hours. Cells were fixed in 70% ethanol at -20°C and stained with DNA staining solution containing propidium iodide and RNaseA (Sigma-Aldrich) overnight. All data were acquired on LSRII cytometry (BD Biosciences) and each sample was assessed using a collection of 10000 events, followed by analysis using FlowJo software (FlowJo, Ashland, OR).

In vivo studies Animal studies were conducted in accordance with an approved protocol adhering to the IACUC policies and procedures at The Ohio State University. Six to eight-week-old male athymic nude mice (Taconic Farms Inc., NY) were caged in groups of five or less, and fed with a diet of animal chow and water ad libitum. OE33 and FLO1 cells were injected subcutaneously into the flanks of each mouse at 2 × 106 and 2.5 × 106 cells per injection, respectively. Treatment regimens were started once tumors reached ~150mm3 in size, 2-4 weeks post-injection. AZD1775 powder was suspended in 0.9% sodium chloride containing 5% dextrose. AZD1775 was administered orally using a sterile 18G gavage needle at 50 mg/kg b.i.d. for 5 consecutive days. Using a custom shielding apparatus to block non-targeted areas, 4 Gy of radiation was administered directly to tumors once daily for 5 consecutive days. For combination treatment, mice were treated with radiation 2-3 hours after the first daily dosing of AZD1775. To obtain a tumor growth curve, perpendicular diameter measurements of each tumor were measured every 2-5 days from the first day of injection with digital calipers, and volumes were calculated using

the formula (L × W × W)/2. Two mice from each group were sacrificed after three days treatment, to isolate tumor xenografts for immunoblotting and immunofluorescence staining.

Data analysis Data were analyzed similarly as previously described (38), and are presented as the mean ± standard error of the mean (s.e.m.).

RESULTS Wee1 is a potential therapeutic target in human ESCA The sensitization of many cancers to radiotherapy and chemotherapy by targeting Wee1 and the high mutation rate of TP53 suggests that Wee1 inhibition is a potential therapeutic strategy for ESCA. To this end, we first analyzed the TCGA and GTEx databases with GEPIA (http://gepia.cancer-pku.cn/) for WEE1 and CDK1 mRNA expression in ESCA. Significant elevation of CDK1 mRNA expression was found in ESCA tumors compared to normal tissues, suggesting increased cell proliferation or mechanisms to promote transition through G2/M phases (Fig. 1A). Interestingly, there was also higher WEE1 mRNA expression in ESCA tumors than that in normal tissues, although not statistically significant. Interestingly, MCM10, CCNE1, CCNE2, FBXO5, and CLSPN have been shown to be biomarkers predictive for responsiveness to AZD1775 (40). Similarly, analysis of TCGA and GTE databases revealed that these 5 genes were significantly overexpressed in ESCA tumor versus normal tissues (Fig. 1B). Further analysis of the Oncomine database (http://oncomine.org/) confirmed overexpression of these genes in human ESCA tumors relative to normal tissues (Supplementary Fig. S1). These findings of CDK1 upregulation and AZD1775 responsiveness gene signature suggested targeting Wee1 with AZD1775 may sensitize ESCA to radiotherapy.

Wee1 kinase inhibition with AZD1775 sensitizes ESCA cells to radiation To evaluate the potential radiation-sensitizing efficacy of Wee1 inhibition by AZD1775 in ESCA, we initially investigated the independent cytotoxic effect of AZD1775 on ESCA cell lines. All four cell lines used in our study have TP53 mutation, including esophageal adenocarcinoma cell lines (OE33, SK4, and FLO1) and an esophageal squamous cell carcinoma

cell line (KYSE30). The cytotoxic effect of AZD1775 in ESCA cell lines was assessed by alamarBlue® assay. We found that the IC50 values of AZD1775 in these ESCA cell lines ranged from 252 to 624 nM (Fig. 2A). To explore the potential of AZD1775 as a radiosensitizer for ESCA cancer, FLO-1 and OE33 cells were treated with increasing doses of radiation in the presence or absence of 100 nM AZD1775, followed by clonogenic (colony-forming) assay. At 100 nM of AZD1775 alone, we noted minimal cytotoxicity of the drug (Supplementary Fig. S2). In combination with radiation however, AZD1775 could effectively sensitize ESCA cells to radiation treatment, with dose enhancement ratios (DER) up to 3.14 in SK4 cells, 1.46 in OE33 cells, 1.34 in FLO1 cells, and 1.23 in KYSE cells (Fig. 2B).

Wee1 kinase inhibition abrogates radiation-induced G2/M phase cell cycle arrest Wee1 kinase plays a key role in promoting G2/M cell cycle arrest after DNA damage (e.g. by ionizing radiation) to allow time for cells to undergo DNA repair, by inactivating CDK1. We explored whether the radiosensitizing effects of AZD1775 are associated with abrogation of radiation-induced G2/M cell cycle arrest in asynchronously growing cells by flow cytometry assay. In both FLO1 and OE33 cells, AZD1775 treatment (100 nM) decreased while 4 Gy radiation alone increased cells in G2/M phase. However, pre-treatment of cells with AZD1775 at 3 hrs before radiation significantly reduced the accumulation of G2/M phase cells after radiation (Fig. 3A). Activation of Wee1 kinase prevents cells from entering into mitosis through phosphorylating and subsequent inactivation of CDK1. Immunoblotting analyses showed AZD1775 inhibited Wee1 and CDK1 phosphorylation in a time-dependent manner, with maximal changes noted 24 hrs after treatment (Fig. 3B). Of note, radiation alone mildly increased the phosphorylation of both Wee1 and CDK1 at 24 hrs after radiation. Exposing cells to AZD1775 before radiation attenuated radiation-induced Wee1 and CDK1 phosphorylation, most notable at 24 hrs after radiation. Interestingly, AZD1775 alone induced H2AX expression and enhanced radiation-mediated increase of H2AX particularly after 24 hrs of treatment. Most studies have shown that the therapeutic effects AZD1775 is related to the abrogation of the G2 checkpoint and/or unscheduled mitotic entry. However, emerging evidence suggest that Wee1 inhibition suppresses DNA damage repair and induces replication stress (32,33), both of which leads to phosphorylation of H2AX. These results suggest that AZD1775 inhibition of Wee1

abrogates radiation induced G2/M cell cycle checkpoint arrest by promoting CDK1 activity and increasing replication stress, thereby potentiating radiation-mediated DNA damage in ESCA cells.

Wee1 inhibitor enhances radiation-induced mitotic cell death Premature entrance into mitosis with unrepaired DNA lesions (particularly DSBs) leads to lethal consequences in cells. The abrogation of G2/M phase cell cycle arrest and enhancement of DNA damage by AZD1775 in ESCA cells treated with radiation suggests that AZD1775 can promote irradiated ESCA cells to prematurely enter into cell mitosis before completion of DNA repair. To test this hypothesis, FLO1 and OE33 cells were cultured on cover slides, and treated with vehicle, AZD1775, 4Gy radiation, or the combination of AZD1775 for 3 hrs followed by 4Gy. After 72 hrs, the cover slides were collected, and stained with DAPI and tubulin by immunofluorescence. Mitotic cell death was determined by the number of cells demonstrating mitotic catastrophe (multinuclear cells with more than 2 nuclear lobes, or cells with several micronuclei) (Fig. 4A). In comparison to vehicle control, AZD1775 alone did not induce mitotic catastrophe. Conversely, 4 Gy radiation treatment resulted in accumulation of cells experiencing mitotic catastrophe, which was significantly enhanced by AZD1775 in both cell lines (Fig. 4B and 4C). Thus, the combination of AZD1775 with radiation resulted in a significantly higher incidence of mitotic catastrophe than radiation treatment alone.

Wee1 inhibition attenuates DNA damage repair during fractionated radiation We found that AZD1775 attenuates radiation-induced G2/M phase arrest, enhances radiation mediated DNA damage, causes premature entrance into mitosis, and finally leads to mitotic cell death. To further support our observations with more clinically relevant doses of radiation (i.e. 2 Gy per day), we extended our study to investigate the capacity of AZD1775 for radiosensitization using standard fractionated radiation doses (i.e. 2 Gy per fraction) using colony formation assays. In the single fraction ionizing radiation experiments, FLO1 and OE33 cells were pre-treated with 100 nM AZD1775 or vehicle control for 3 hrs, followed by treatment with a single radiation dose of 4 Gy. In the fractionated ionizing radiation experiment, the cells were pre-treated with 100 nM AZD1775 or vehicle control for 3 hrs, followed by treatment with

2 Gy, which was repeated 24 hrs later (Fig. 5A). Twenty-four hours post radiation, the cells were cultured in fresh medium without AZD1775/vehicle for 10 additional days before colony fixation and staining. Cell recovery rate was calculated by dividing the percentage of colonies formed following fractionated radiation (2 Gy x 2 fractions) by the percentage of colonies formed following a single fraction of 4 Gy. As expected, 2 Gy x 2 led to a higher surviving fraction compared to a single 4Gy dose of radiation, likely due to sublethal DNA repair occurring between fractions, leading to a survival enhancement rate of 1.37 and 1.84 for FLO1 and OE33 cells, respectively. Similar to our previous data with single fraction radiation in Fig. 2B, exposure of FLO1 or OE33 cells to AZD1775 before and during fractionated radiation still effectively radiosensitized ESCA cells and abrogated cell recovery (Fig. 5B). These results indicate a comparable enhancement of cell death by AZD1775 in ESCA cells when combining AZD1775 with either fractionated or single fraction radiation. These findings have important clinical implications as patients with ESCA are treated with fractionated radiation to typical doses of 1.8-2.0 Gy per day.

Wee1 inhibition markedly radiosensitizes ESCA cells in mouse tumor xenografts To determine if Wee1 inhibition could effectively radiosensitize ESCA cells in vivo, we further explored the combined treatment of AZD1775 and radiation in vivo using nude mice xenografts with FLO1 and OE33 cells. When tumors reached 100-150 mm3, the mice were randomized to groups of treatment with vehicle, AZD1775 alone, 4 Gy radiation alone, or the combination of AZD1775 + 4Gy (mice were treated with AZD1775 2 hrs before radiation). The treatments lasted for 5 consecutive days during Days 1-5 (Fig. 6A). AZD1775 was delivered by oral gavage with a dose of 50 mg/kg, twice a day as previously described (29). AZD1775 monotherapy and radiation alone resulted in partial tumor growth delay. However, AZD1775 in combination with radiation treatment led to remarkable and sustained tumor regression of both FLO1 and OE33 xenografts (Fig. 6B and 6C). In order to investigate whether the treatment combination is indeed inducing tumor cell death through mitotic catastrophe, we performed mitotic catastrophe assay in tumors derived from mouse xenografts. Consistent with our in vitro data, the combination of AZD1775 and IR significantly increased mitotic catastrophe in tumor xenografts compare to IR treatment alone (Fig. 6D). Moreover, the majority of FLO1 and OE33 tumors showed no

evidence of tumor recurrence after treatment with AZD1775 in combination with radiation (survival curves shown in Supplementary Fig. S3). In terms of toxicity, mice tolerated the treatment well, and no mice died early from treatment toxicity. Mice who received IR treatment did lose weight during the first week, but fully recovered within two weeks (Supplementary Fig. S4). We further performed immunoblotting of the tumor lysates 2 hrs after Day 3 treatment in each group to assess pharmacodynamics effects of AZD1775. AZD1775 reduced the phosphorylation of Wee1 and CDK1 as well as the protein levels of cyclin A2, B1, E1 and E2, while increasing levels of phospho-histone H3 (a marker of mitotic cells) (Fig. 6E). Taken together, these findings indicate AZD1775 is promoting G2/M phase cell cycle progression. Radiation increased the phosphorylation of Wee1 and CDK1, as well as the protein levels of cyclin A2, B1, E1 and E2, which were all prevented by Wee1 inhibition. These results demonstrate that potent inhibition of Wee1 leads to promotion of cells through mitosis despite DNA damage from radiation, thus leading to marked radiosensitization by AZD1775 in vivo.

DISCUSSION Radiation therapy induces DNA damage, resulting in activation of apoptotic pathways or inducing post-mitotic death due to unrepaired DNA damage. Following DNA damage, cells rely on cell cycle checkpoints to provide time for DNA repair prior to cell division. Esophageal cancer cells often lack a functional G1 checkpoint due to a high frequency of TP53 mutations. Based on TCGA data, up to 91% of squamous cell carcinoma and 71% of adenocarcinoma esophageal cancers possess a TP53 mutation making them heavily dependent on the G2/M checkpoint to survive DNA damage and replication stress. In the present study, we demonstrated that a potent Wee1 kinase inhibitor AZD1775 sensitized ESCA cells to radiation therapy in in vitro cell cultures and mouse tumor xenografts. In addition, AZD1775 treatment led to a comparable enhancement of cytotoxicity in ESCA cells treated with either fractionated radiation or single dose radiation. Mechanistically, AZD1775 attenuated radiation-induced G2/M phase arrest, which was accompanied by enhanced radiation-induced mitotic catastrophe and DNA damage. Our findings suggest that Wee1 kinase specific inhibitor AZD1775 is an effective radiosensitizer for esophageal cancer.

Wee1 is a tyrosine kinase and is activated following DNA damage. Wee1 kinase is a critical regulator of the G2/M checkpoint and thus genomic stability by mediating inhibitory phosphorylation of CDK1, resulting in cell cycle arrest and permitting DNA repair prior to proceeding with mitosis (41,42). It has been shown that inhibiting Wee1 results in replication stress, loss of genomic integrity, nucleotide shortage, and subsequent double-strand DNA breaks (43). It has been proposed that targeting Wee1 kinase is a promising strategy for the radiosensitization and chemosensitization of cancer cells with a defective G1 cell cycle checkpoint (21). Accordingly, several Wee1 kinase small molecule inhibitors, including AZD1775, have been developed (27). Previous studies have shown that AZD1775 is a potent and selective small molecule inhibitor of Wee1 kinase and has been shown to sensitize tumor cells to both chemotherapy and radiation (27,28,33). In the present study, we found that both CDK1 and WEE1 are overexpressed in ESCA, as well as genes associated with an AZD1775 responsiveness gene signature (40). Moreover, we demonstrated that AZD1775 potently inhibited the phosphorylation of both Wee1 and CDK1 in the absence or presence of radiation. Consistent with the role for Wee1 in G2/M checkpoint regulation, AZD1775 prevented IR-induced G2/M phase cell cycle arrest, which was accompanied with enhanced mitotic catastrophe and γH2AX, indicative of enhanced cell death and DNA damage. Interestingly, we noted AZD1775 caused reductions in E, A, and B-type cyclins. This observation is consistent with our knowledge of the temporal expression of cyclins during the cell cycle. Specifically, E-type cyclins are upregulated during the G1/S phase transition and then fall down, while A-type cyclins are upregulated in the G2 phase and then fall down prior to and during entry of calls into mitosis. Lastly, B-type cyclins are upregulated at the G2/M transition then fall down sharply upon entry to mitosis. Taken together, AZD1775 potently inhibits Wee1 in ESCA cells, thereby promoting entry from S and G2 through M phase, and thus preventing Wee1 from protecting ESCA cells from the effects of radiotherapy. Esophageal cancer remains a global problem and is the 8th most common cancer worldwide (1). Globally, while esophageal squamous cell carcinoma remains the predominant histology in Asia, Africa, and South America, the incidence of esophageal adenocarcinoma has surpassed that of squamous cell carcinoma in North America, Australia, and Europe. Current predictions are that by 2030, up to 1 in 100 men will be diagnosed with esophageal adenocarcinoma during their

lifetime in the Netherlands and the United Kingdom (44). In the U.S., the incidence of cancers of the esophagus and gastroesophageal junction has increased dramatically in recent decades, largely driven by the rising incidence of adenocarcinoma (45). Nearly 40-50% of patients present with either unresectable disease or evidence of distant metastasis (45). For patients with resectable disease, the current standard of care includes administering chemotherapy, most commonly carboplatin and paclitaxel, concurrently with radiation therapy followed by surgical resection (46,47). Overall, approximately 30% of patients with esophageal cancer will demonstrate a pathologic complete response (pCR) at the time of surgery (47-49) with pCR rates closer to 43-49% for squamous cell cancer (47,50) and 16-25% for adenocarcinoma (47,51,52). A pCR is associated with an improvement in overall survival (48,49). Patients with a pCR, arguably, may be able to avoid esophagectomy without compromising survival (53). Esophagectomy is associated with impaired quality of life and remains a morbid operation with substantial mortality (54-58). With current pCR rates at or below 50%, improvements in therapy, including the potential addition of radiosensitizing agents, may help to make organ preservation a reality in esophageal cancer. Currently, even with the most aggressive approach of trimodality therapy, approximately 50% of patients will experience a locoregional and/or distant recurrence within 5 years of treatment completion (46). Therefore, there is an urgent need to develop novel strategies that will improve clinical outcomes of patients diagnosed with localized esophageal cancer. Strategies aimed at improving local control, including modalities such as radiation therapy, will likely have an impact on improving survival. Our results showed that AZD1775 in combination with radiotherapy resulted in virtually complete tumor regression of ESCA tumor xenografts. Therefore, our study suggests that AZD1775 inhibition of Wee1 is an effective strategy for radiation sensitization in ESCA cells. Interestingly, we observed that the DER of SK4 was much higher than that of the other three cell lines (Fig. 2B). All the four ESCA cell lines used in this study have TP53 mutations. However, besides TP53 mutation, SK4 cells have additional KRAS and PIK3CA mutation (https://portals.broadinstitute.org/ccle). KRAS and PIK3CA mutations drive tumorigenesis via multiple mechanisms, one of which is to induce DNA replication stress leading to genomic instability (59,60). As mentioned, Wee1 has an important role in replication stress response (35). Thus, ESCA cells with KRAS and PIK3CA mutations might may have

become more dependent on Wee1 kinase for survival due to increased replication stress. It will be important to determine whether ESCA with KRAS and/or PIK3CA mutations are hypersensitive to Wee1 inhibitors in future preclinical and clinical studies. In addition, it will be critical to determine whether TP53 mutant status confers increased sensitivity to AZD1775 and radiation. In our preliminary studies, we found that the combination of AZD1775 and radiation did not radiosensitize TP53 intact AGS adenocarcinoma cells (Supplementary Fig. S5). Finally, further studies are needed to assess whether AZD1755 sensitizes ESCA cells to DNA-damaging chemotherapies such as platinum-based drugs or other chemotherapeutics which promote replication stress. Based on our results, we feel that our data support a clinical trial testing the combination of Wee1 kinase inhibitor and radiation for ESCA. It would be interesting to also explore combining Wee1 inhibitor with chemoradiation, or replacing a chemotherapy drug commonly used in ESCA with a Wee1 kinase inhibitor. Standard chemotherapy regimens used in combination with radiation for ESCA include paclitaxel and carboplatin, or 5FU and oxaliplatin. Recently, a phase II trial of AZD1775 in combination with gemcitabine and radiation was published for pancreatic cancer, showing tolerable safety profile of the combination and significant efficacy compared to historical controls (61). This trial was based on preclinical data combining Wee1 inhibitor with gemcitabine and radiation. Thus, additional preclinical studies may be needed to establish the optimal combination of Wee1 inhibitor with certain chemotherapy drugs and radiation for the treatment of esophageal cancer. In summary, our results demonstrated a potent inhibitory role for Wee1 kinase inhibitor AZD1775 in cell cycle checkpoints in response to IR-induced DNA double strand breaks; importantly, AZD1775 enhanced IR–mediated cell death in vitro and maintained the suppression of ESCA mouse xenografts by radiotherapy in vivo. There are several phase I and II studies which have been performed using AZD1775 both as monotherapy and in combination with multiple chemotherapy regimens with preliminary data showing that combination therapy appears to be reasonably tolerated (62-65). Due to the high incidence of TP53 mutations and the dependence on the Wee1-mediated G2/M checkpoint to survive DNA damage, esophageal cancer is a logical site to consider the addition of a Wee1 kinase inhibitor to standard

neoadjuvant therapy. We believe our study warrants a phase I trial testing AZD1775 in combination with radiation or chemoradiation for esophageal cancer.

Authors' Contributions Conception and design: T.M. Williams, L. Yang, C. Shen Development of methodology: L. Yang, C. Shen, A. Hu, C. Pettit, T.M. Williams Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Yang, E. Miller, J. Zhang, S.H. Lin, T.M. Williams Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L. Yang, C. Shen, E. Miller, J. Zhang, S.H. Lin, T.M. Williams Writing, review, and/or revision of the manuscript: L. Yang, C. Shen, T.M. Williams Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T.M. Williams Study supervision: T.M. Williams

ACKNOWLEDGEMENTS This data was presented in part at the American Association of Cancer Research (AACR) annual meeting 2018 and Radiation Research Society annual meeting 2019. Research reported in this publication was supported by The Ohio State University Comprehensive Cancer Center (OSU- CCC) and the National Institutes of Health under grant number P30 CA016058, as well as RSG- 17-221-01-TBG (to T.M.W.), award number grant KL2TR001068 from the National Center for Advancing Translational Sciences (to T.M.W.), and NIH grant R01 CA198128 (to T.M.W.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

REFERENCES

1. Lagergren, J., Smyth, E., Cunningham, D., and Lagergren, P. (2017) Oesophageal cancer. Lancet 390, 2383-2396 2. van Rossum, P. S. N., Mohammad, N. H., Vleggaar, F. P., and van Hillegersberg, R. (2018) Treatment for unresectable or metastatic oesophageal cancer: current evidence and trends. Nat Rev Gastroenterol Hepatol 15, 235-249

3. Pennathur, A., Gibson, M. K., Jobe, B. A., and Luketich, J. D. (2013) Oesophageal carcinoma. Lancet 381, 400-412 4. Siegel, R. L., Miller, K. D., and Jemal, A. (2018) Cancer statistics, 2018. CA Cancer J Clin 68, 7-30 5. Smyth, E. C., Lagergren, J., Fitzgerald, R. C., Lordick, F., Shah, M. A., Lagergren, P., and Cunningham, D. (2017) Oesophageal cancer. Nat Rev Dis Primers 3, 17048 6. Otto, T., and Sicinski, P. (2017) Cell cycle proteins as promising targets in cancer therapy. Nat Rev Cancer 17, 93-115 7. Jeggo, P. A., Pearl, L. H., and Carr, A. M. (2016) DNA repair, genome stability and cancer: a historical perspective. Nat Rev Cancer 16, 35-42 8. Hanahan, D., and Weinberg, R. A. (2011) Hallmarks of cancer: the next generation. Cell 144, 646- 674 9. Ciccia, A., and Elledge, S. J. (2010) The DNA damage response: making it safe to play with knives. Mol Cell 40, 179-204 10. Castedo, M., Perfettini, J. L., Roumier, T., Andreau, K., Medema, R., and Kroemer, G. (2004) Cell death by mitotic catastrophe: a molecular definition. Oncogene 23, 2825-2837 11. Rundle, S., Bradbury, A., Drew, Y., and Curtin, N. J. (2017) Targeting the ATR-CHK1 Axis in Cancer Therapy. Cancers (Basel) 9 12. Goldstein, M., and Kastan, M. B. (2015) The DNA damage response: implications for tumor responses to radiation and chemotherapy. Annu Rev Med 66, 129-143 13. Blackford, A. N., and Jackson, S. P. (2017) ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response. Mol Cell 66, 801-817 14. Parker, L. L., and Piwnica-Worms, H. (1992) Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase. Science 257, 1955-1957 15. Bartek, J., and Lukas, J. (2003) Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell 3, 421-429 16. Masaki, T., Shiratori, Y., Rengifo, W., Igarashi, K., Yamagata, M., Kurokohchi, K., Uchida, N., Miyauchi, Y., Yoshiji, H., Watanabe, S., Omata, M., and Kuriyama, S. (2003) Cyclins and cyclin- dependent kinases: comparative study of hepatocellular carcinoma versus cirrhosis. Hepatology 37, 534-543 17. Iorns, E., Lord, C. J., Grigoriadis, A., McDonald, S., Fenwick, K., Mackay, A., Mein, C. A., Natrajan, R., Savage, K., Tamber, N., Reis-Filho, J. S., Turner, N. C., and Ashworth, A. (2009) Integrated functional, gene expression and genomic analysis for the identification of cancer targets. PLoS One 4, e5120 18. Mir, S. E., De Witt Hamer, P. C., Krawczyk, P. M., Balaj, L., Claes, A., Niers, J. M., et al. (2010) In silico analysis of kinase expression identifies WEE1 as a gatekeeper against mitotic catastrophe in glioblastoma. Cancer Cell 18, 244-257 19. Benada, J., and Macurek, L. (2015) Targeting the Checkpoint to Kill Cancer Cells. Biomolecules 5, 1912-1937 20. Do, K., Doroshow, J. H., and Kummar, S. (2013) Wee1 kinase as a target for cancer therapy. Cell Cycle 12, 3159-3164 21. Matheson, C. J., Backos, D. S., and Reigan, P. (2016) Targeting WEE1 Kinase in Cancer. Trends Pharmacol Sci 37, 872-881 22. Song, Y., Li, L., Ou, Y., Gao, Z., Li, E., Li, X., et al. (2014) Identification of genomic alterations in oesophageal squamous cell cancer. Nature 509, 91-95 23. Dulak, A. M., Stojanov, P., Peng, S., Lawrence, M. S., Fox, C., Stewart, C., et al. (2013) Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity. Nat Genet 45, 478-486 18

24. Kim, J., Bowlby, R., Mungall, A. J., Robertson, A. G., Odze, R. D., Cherniack, A. D., et al. (2017) Integrated genomic characterization of oesophageal carcinoma. Nature 541, 169-+ 25. Leijen, S., Beijnen, J. H., and Schellens, J. H. (2010) Abrogation of the G2 checkpoint by inhibition of Wee-1 kinase results in sensitization of p53-deficient tumor cells to DNA-damaging agents. Curr Clin Pharmacol 5, 186-191 26. Ku, B. M., Bae, Y. H., Koh, J., Sun, J. M., Lee, S. H., Ahn, J. S., Park, K., and Ahn, M. J. (2017) Mutational status of TP53 defines the efficacy of Wee1 inhibitor AZD1775 in KRAS-mutant non- small cell lung cancer. Oncotarget 8, 67526-67537 27. Hirai, H., Iwasawa, Y., Okada, M., Arai, T., Nishibata, T., Kobayashi, M., Kimura, T., Kaneko, N., Ohtani, J., Yamanaka, K., Itadani, H., Takahashi-Suzuki, I., Fukasawa, K., Oki, H., Nambu, T., Jiang, J., Sakai, T., Arakawa, H., Sakamoto, T., Sagara, T., Yoshizumi, T., Mizuarai, S., and Kotani, H. (2009) Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensitizes p53-deficient tumor cells to DNA-damaging agents. Mol Cancer Ther 8, 2992-3000 28. Bridges, K. A., Hirai, H., Buser, C. A., Brooks, C., Liu, H., Buchholz, T. A., Molkentine, J. M., Mason, K. A., and Meyn, R. E. (2011) MK-1775, a novel Wee1 kinase inhibitor, radiosensitizes p53- defective human tumor cells. Clin Cancer Res 17, 5638-5648 29. Rajeshkumar, N. V., De Oliveira, E., Ottenhof, N., Watters, J., Brooks, D., Demuth, T., Shumway, S. D., Mizuarai, S., Hirai, H., Maitra, A., and Hidalgo, M. (2011) MK-1775, a potent Wee1 inhibitor, synergizes with gemcitabine to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts. Clin Cancer Res 17, 2799-2806 30. Cuneo, K. C., Morgan, M. A., Davis, M. A., Parcels, L. A., Parcels, J., Karnak, D., Ryan, C., Liu, N., Maybaum, J., and Lawrence, T. S. (2016) Wee1 Kinase Inhibitor AZD1775 Radiosensitizes Hepatocellular Carcinoma Regardless of TP53 Mutational Status Through Induction of Replication Stress. Int J Radiat Oncol Biol Phys 95, 782-790 31. Parsels, L. A., Karnak, D., Parsels, J. D., Zhang, Q., Velez-Padilla, J., Reichert, Z. R., Wahl, D. R., Maybaum, J., O'Connor, M. J., Lawrence, T. S., and Morgan, M. A. (2018) PARP1 Trapping and DNA Replication Stress Enhance Radiosensitization with Combined WEE1 and PARP Inhibitors. Mol Cancer Res 16, 222-232 32. Pfister, S. X., Markkanen, E., Jiang, Y., Sarkar, S., Woodcock, M., Orlando, G., Mavrommati, I., Pai, C. C., Zalmas, L. P., Drobnitzky, N., Dianov, G. L., Verrill, C., Macaulay, V. M., Ying, S., La Thangue, N. B., D'Angiolella, V., Ryan, A. J., and Humphrey, T. C. (2015) Inhibiting WEE1 Selectively Kills Histone H3K36me3-Deficient Cancers by dNTP Starvation. Cancer Cell 28, 557-568 33. Kausar, T., Schreiber, J. S., Karnak, D., Parsels, L. A., Parsels, J. D., Davis, M. A., Zhao, L., Maybaum, J., Lawrence, T. S., and Morgan, M. A. (2015) Sensitization of Pancreatic Cancers to Gemcitabine Chemoradiation by WEE1 Kinase Inhibition Depends on Homologous Recombination Repair. Neoplasia 17, 757-766 34. Nam, A. R., Park, J. E., Bang, J. H., Jin, M. H., Bang, Y. J., and Oh, D. Y. (2018) DNA damage response (DDR)-targeting strategy by targeting WEE1 and or ATM/ATR works in biliary tract cancer. Cancer Res 78 35. Geenen, J. J. J., and Schellens, J. H. M. (2017) Molecular Pathways: Targeting the Protein Kinase Wee1 in Cancer. Clinical Cancer Research 23, 4540-4544 36. Matheson, C. J., Backos, D. S., and Reigan, P. (2016) Targeting WEE1 Kinase in Cancer. Trends Pharmacol Sci 37, 872-881 37. Williams, T. M., Flecha, A. R., Keller, P., Ram, A., Karnak, D., Galban, S., Galban, C. J., Ross, B. D., Lawrence, T. S., Rehemtulla, A., and Sebolt-Leopold, J. (2012) Cotargeting MAPK and PI3K signaling with concurrent radiotherapy as a strategy for the treatment of pancreatic cancer. Mol Cancer Ther 11, 1193-1202 19

38. Estrada-Bernal, A., Chatterjee, M., Haque, S. J., Yang, L., Morgan, M. A., Kotian, S., Morrell, D., Chakravarti, A., and Williams, T. M. (2015) MEK inhibitor GSK1120212-mediated radiosensitization of pancreatic cancer cells involves inhibition of DNA double-strand break repair pathways. Cell Cycle 14, 3713-3724 39. Fertil, B., Dertinger, H., Courdi, A., and Malaise, E. P. (1984) Mean inactivation dose: a useful concept for intercomparison of human cell survival curves. Radiat Res 99, 73-84 40. Mizuarai, S., Yamanaka, K., Itadani, H., Arai, T., Nishibata, T., Hirai, H., and Kotani, H. (2009) Discovery of gene expression-based pharmacodynamic biomarker for a p53 context-specific anti-tumor drug Wee1 inhibitor. Mol Cancer 8, 34 41. Dominguez-Kelly, R., Martin, Y., Koundrioukoff, S., Tanenbaum, M. E., Smits, V. A. J., Medema, R. H., Debatisse, M., and Freire, R. (2011) Wee1 controls genomic stability during replication by regulating the Mus81-Eme1 endonuclease. J Cell Biol 194, 567-579 42. Watanabe, N., Broome, M., and Hunter, T. (1995) Regulation of the Human Wee1hu Cdk Tyrosine 15-Kinase during the Cell-Cycle. Embo J 14, 1878-1891 43. Beck, H., Nahse-Kumpf, V., Larsen, M. S., O'Hanlon, K. A., Patzke, S., Holmberg, C., Mejlvang, J., Groth, A., Nielsen, O., Syljuasen, R. G., and Sorensen, C. S. (2012) Cyclin-dependent kinase suppression by WEE1 kinase protects the genome through control of replication initiation and nucleotide consumption. Molecular and cellular biology 32, 4226-4236 44. Arnold, M., Laversanne, M., Brown, L. M., Devesa, S. S., and Bray, F. (2017) Predicting the Future Burden of Esophageal Cancer by Histological Subtype: International Trends in Incidence up to 2030. Am J Gastroenterol 112, 1247-1255 45. Thrift, A. P. (2016) The epidemic of oesophageal carcinoma: Where are we now? Cancer Epidemiol 41, 88-95 46. Shapiro, J., van Lanschot, J. J. B., Hulshof, M., van Hagen, P., van Berge Henegouwen, M. I., Wijnhoven, B. P. L., et al. (2015) Neoadjuvant chemoradiotherapy plus surgery versus surgery alone for oesophageal or junctional cancer (CROSS): long-term results of a randomised controlled trial. The Lancet. Oncology 16, 1090-1098 47. van Hagen, P., Hulshof, M. C., van Lanschot, J. J., Steyerberg, E. W., van Berge Henegouwen, M. I., Wijnhoven, B. P., et al. (2012) Preoperative chemoradiotherapy for esophageal or junctional cancer. The New England journal of medicine 366, 2074-2084 48. Alnaji, R. M., Du, W., Gabriel, E., Singla, S., Attwood, K., Nava, H., Malhotra, U., Hochwald, S. N., and Kukar, M. (2016) Pathologic Complete Response Is an Independent Predictor of Improved Survival Following Neoadjuvant Chemoradiation for Esophageal Adenocarcinoma. Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract 20, 1541-1546 49. Donahue, J. M., Nichols, F. C., Li, Z., Schomas, D. A., Allen, M. S., Cassivi, S. D., Jatoi, A., Miller, R. C., Wigle, D. A., Shen, K. R., and Deschamps, C. (2009) Complete pathologic response after neoadjuvant chemoradiotherapy for esophageal cancer is associated with enhanced survival. The Annals of thoracic surgery 87, 392-398; discussion 398-399 50. Yang, H., Liu, H., Chen, Y., Zhu, C., Fang, W., Yu, Z., et al. (2018) Neoadjuvant Chemoradiotherapy Followed by Surgery Versus Surgery Alone for Locally Advanced Squamous Cell Carcinoma of the Esophagus (NEOCRTEC5010): A Phase III Multicenter, Randomized, Open-Label Clinical Trial. Journal of clinical oncology : official journal of the American Society of Clinical Oncology, JCO2018791483 51. Stahl, M., Walz, M. K., Stuschke, M., Lehmann, N., Meyer, H. J., Riera-Knorrenschild, J., Langer, P., Engenhart-Cabillic, R., Bitzer, M., Konigsrainer, A., Budach, W., and Wilke, H. (2009) Phase III comparison of preoperative chemotherapy compared with chemoradiotherapy in patients with 20

locally advanced adenocarcinoma of the esophagogastric junction. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 27, 851-856 52. Walsh, T. N., Noonan, N., Hollywood, D., Kelly, A., Keeling, N., and Hennessy, T. P. (1996) A comparison of multimodal therapy and surgery for esophageal adenocarcinoma. The New England journal of medicine 335, 462-467 53. Borggreve, A. S., Mook, S., Verheij, M., Mul, V. E. M., Bergman, J. J., Bartels-Rutten, A., et al. (2018) Preoperative image-guided identification of response to neoadjuvant chemoradiotherapy in esophageal cancer (PRIDE): a multicenter observational study. BMC cancer 18, 1006 54. Busweiler, L. A., Wijnhoven, B. P., van Berge Henegouwen, M. I., Henneman, D., van Grieken, N. C., Wouters, M. W., van Hillegersberg, R., van Sandick, J. W., and Dutch Upper Gastrointestinal Cancer Audit, G. (2016) Early outcomes from the Dutch Upper Gastrointestinal Cancer Audit. The British journal of surgery 103, 1855-1863 55. Djarv, T., Lagergren, J., Blazeby, J. M., and Lagergren, P. (2008) Long-term health-related quality of life following surgery for oesophageal cancer. The British journal of surgery 95, 1121-1126 56. Kassis, E. S., Kosinski, A. S., Ross, P., Jr., Koppes, K. E., Donahue, J. M., and Daniel, V. C. (2013) Predictors of anastomotic leak after esophagectomy: an analysis of the society of thoracic surgeons general thoracic database. The Annals of thoracic surgery 96, 1919-1926 57. Mc Cormack, O., Zaborowski, A., King, S., Healy, L., Daly, C., O'Farrell, N., Donohoe, C. L., Ravi, N., and Reynolds, J. V. (2014) New-onset atrial fibrillation post-surgery for esophageal and junctional cancer: incidence, management, and impact on short- and long-term outcomes. Annals of surgery 260, 772-778; discussion 778 58. Schandl, A., Lagergren, J., Johar, A., and Lagergren, P. (2016) Health-related quality of life 10 years after oesophageal cancer surgery. European journal of cancer 69, 43-50 59. Forment, J. V., and O'Connor, M. J. (2018) Targeting the replication stress response in cancer. Pharmacol Therapeut 188, 155-167 60. Macheret, M., and Halazonetis, T. D. (2015) DNA Replication Stress as a Hallmark of Cancer. Annu Rev Pathol-Mech 10, 425-448 61. Cuneo, K. C., Morgan, M. A., Sahai, V., Schipper, M. J., Parsels, L. A., Parsels, J. D., Devasia, T., Al- Hawaray, M., Cho, C. S., Nathan, H., Maybaum, J., Zalupski, M. M., and Lawrence, T. S. (2019) Dose Escalation Trial of the Wee1 Inhibitor Adavosertib (AZD1775) in Combination With Gemcitabine and Radiation for Patients With Locally Advanced Pancreatic Cancer. Journal of Clinical Oncology 37, 2643-+ 62. Do, K., Wilsker, D., Ji, J., Zlott, J., Freshwater, T., Kinders, R. J., Collins, J., Chen, A. P., Doroshow, J. H., and Kummar, S. (2015) Phase I Study of Single-Agent AZD1775 (MK-1775), a Wee1 Kinase Inhibitor, in Patients With Refractory Solid Tumors. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 33, 3409-3415 63. Leijen, S., van Geel, R. M., Pavlick, A. C., Tibes, R., Rosen, L., Razak, A. R., Lam, R., Demuth, T., Rose, S., Lee, M. A., Freshwater, T., Shumway, S., Liang, L. W., Oza, A. M., Schellens, J. H., and Shapiro, G. I. (2016) Phase I Study Evaluating WEE1 Inhibitor AZD1775 As Monotherapy and in Combination With Gemcitabine, Cisplatin, or Carboplatin in Patients With Advanced Solid Tumors. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 34, 4371-4380 64. Leijen, S., van Geel, R. M., Sonke, G. S., de Jong, D., Rosenberg, E. H., Marchetti, S., Pluim, D., van Werkhoven, E., Rose, S., Lee, M. A., Freshwater, T., Beijnen, J. H., and Schellens, J. H. (2016) Phase II Study of WEE1 Inhibitor AZD1775 Plus Carboplatin in Patients With TP53-Mutated Ovarian Cancer Refractory or Resistant to First-Line Therapy Within 3 Months. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 34, 4354-4361 21

65. Mendez, E., Rodriguez, C. P., Kao, M. C., Raju, S., Diab, A., Harbison, R. A., Konnick, E. Q., Mugundu, G. M., Santana-Davila, R., Martins, R., Futran, N. D., and Chow, L. Q. M. (2018) A Phase I Clinical Trial of AZD1775 in Combination with Neoadjuvant Weekly Docetaxel and Cisplatin before Definitive Therapy in Head and Neck Squamous Cell Carcinoma. Clin Cancer Res 24, 2740-2748

FIGURE LEGENDS Figure 1. CDK1 and genes associated with Wee1 inhibitor sensitivity are overexpressed in ESCA. Relative mRNA expression levels of CDK1 and WEE1 (A) or genes associated with sensitivity to Wee1 inhibitor (B) in ESCA (esophageal carcinoma) tumor (T) versus normal (N) tissues. Box plots were derived from Gene Expression Profiling Interactive Analysis (GEPIA) based on TCGA and GTEx databases. Red and black boxes represent the relative mRNA expression levels of the genes in the tumor and normal samples, respectively. The y-axis represents the relative mRNA expression levels of the genes in terms of log2 (TPM+1). Tumor samples=182; normal samples=286, *p<0.05. TPM= transcripts per million. Figure 2. AZD1775 effectively sensitizes esophageal cancer cells to radiation. (A) IC50s of AZD1775 in esophageal cancer cells. OE33, SK4, FLO1 and KYSE cells were treated with different concentrations of AZD1775 for 72 hr. The cell viability was assessed by alamarBlue assay, and IC50 values were calculated. (B) Cells were cultured in media containing 100 nM AZD1775 at 3 hr prior to radiation with 0 (no IR), 2, 4, 6 and 8 Gy doses, followed by radiation clonogenic survival assay. Each dose was prepared in triplicate per experiment, and no less than 2 experiments were performed per cell line. Dose enhancement ratios (DER) at 2 Gy were compared between vehicle and AZD1755. *p<0.05, **p<0.01.

Figure 3. AZD1775 inhibition of Wee1 abrogates radiation-induced G2/M cell cycle arrest. (A) FLO1 and OE33 cells were cultured in media containing 100 nM AZD1775 (AZD-100) at 3 hrs prior to radiation with 0 (ctr) or 4 Gy doses. 24 hrs after 4 Gy or sham radiation, cells were prepared for flow cytometry analysis of cell cycle distribution. Each dose was prepared in triplicate per experiment, and no less than 2 experiments were performed per cell line. Note AZD1775 significantly reduced G2//M phase fractions after 4 Gy radiation. **p<0.001. (B) FLO1 cells were cultured in media containing 100 nM AZD1775 (AZD-100) for 3 hr prior to

radiation with 0 or 4 Gy doses. At the indicated time points following radiation (1, 4, 16, and 24 hrs), the cells were lysed and subjected to immunoblotting with GAPDH as loading control.

Figure 4. AZD1775 enhanced radiation-induced mitotic catastrophe in ESCA cells. (A) FLO1 and OE33 cells were treated with 4 Gy radiation, 100 nM AZD1775 alone, or in combination (100 nM AZD1775 added 3 hrs prior to 4Gy radiation). At 72 hrs post radiation, the cells were subjected to immunofluorescence staining of tubulin and DAPI to show the signs of mitotic catastrophe (micro- and multinucleated cells as shown by red arrows). Representative images of mitotic catastrophe in OE33 cells are shown. The graphs shows percentage of mitotic catastrophe cells in 100 counted FLO1 (B) and OE33 (C) cells. **p<0.001.

Figure 5. AZD1775 effectively radiosensitizes ESCA cells during fractionated radiation. (A) Schema of two radiation treatment schedules for clonogenic assay. (B) AZD1775 sensitized FLO1 and OE33 cells to radiation whether cells were treated with fractionated radiation (2 Gy x 2) or single fraction radiation (4 Gy). IR, ionizing radiation. w/o, without. AZD= AZD1775 (100 nM). *p<0.05

Figure 6. Wee1 inhibition effectively radiosensitizes ESCA cells in xenograft tumor models. Mice were injected with 2x106 FLO1 or OE33 cells, and randomized to start treatment once tumors reached 100-200 mm3. AZD1775 was administered via oral gavage at 50 mg/kg BID for 5 days (control group received vehicle at same intervals), 2 hrs prior to radiation when radiation was given. Radiation was administered at 4 Gy daily for 5 consecutive days. Tumor size was calculated by measuring length and width via calipers. (A) Schema of in vivo experimental plan using tumor xenografts in mice. (B) Growth curves of FLO1 xenograft tumors. (C) Growth curves of OE33 xenograft tumors. (D, E) FLO1 tumor xenografts were isolated from mice on Day 3 (2 hrs after treatment completed) and subjected to mitotic catastrophe assay (D) or immunoblotting analysis (E) of the indicated proteins with GAPDH as loading control. HH3= histone H3. Ctr= control (vehicle). IR= ionizing radiation. *p<0.05.

Wee1 Kinase Inhibitor AZD1775 Effectively Sensitizes Esophageal Cancer to Radiotherapy

Linlin Yang, Changxian Shen, Cory J Pettit, et al.

Clin Cancer Res Published OnlineFirst March 27, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-3373

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2020/03/27/1078-0432.CCR-19-3373.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2020/03/27/1078-0432.CCR-19-3373. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.