Wee1 Kinase Inhibitor AZD1775 Effectively Sensitizes Esophageal Cancer to Radiotherapy Linlin Yang1, Changxian Shen1, Cory J

Published OnlineFirst March 27, 2020; DOI: 10.1158/1078-0432.CCR-19-3373

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

Wee1 Kinase Inhibitor AZD1775 Effectively Sensitizes Esophageal Cancer to Radiotherapy Linlin Yang1, Changxian Shen1, Cory J. Pettit1, Tianyun Li1, Andrew J. Hu1, Eric D. Miller1, Junran Zhang1, Steven H. Lin2, and Terence M. Williams1

ABSTRACT ◥ Purpose: Esophageal cancer is a deadly malignancy with a 5-year (100 nmol/L) as monotherapy did not alter the viability of survival rate of only 5% to 20%, which has remained unchanged for esophageal cancer cells, but significantly radiosensitized esoph- decades. Esophageal cancer possesses a high frequency of TP53 ageal cancer cells. AZD1775 significantly abrogated radiation- mutations leading to dysfunctional G1 cell-cycle checkpoint, which induced G2–M phase arrest and attenuation of p-CDK1-Y15. – likely makes esophageal cancer cells highly reliant upon G2 M Moreover, AZD1775 increased radiation-induced mitotic checkpoint for adaptation to DNA replication stress and DNA catastrophe, which was accompanied by increased gH2AX damage after radiation. We aim to explore whether targeting Wee1 levels, and subsequently reduced survival after radiation. – kinase to abolish G2 M checkpoint sensitizes esophageal cancer Importantly, AZD1775 in combination with radiotherapy cells to radiotherapy. resulted in marked tumor regression of esophageal cancer tumor Experimental Design: Cell viability was assessed by cytotoxicity xenografts. – and colony-forming assays, cell-cycle distribution was analyzed by Conclusions: Abrogation of G2 M checkpoint by targeting flow cytometry, and mitotic catastrophe was assessed by immuno- Wee1 kinase with AZD1775 sensitizes esophageal cancer cells to fluorescence staining. Human esophageal cancer xenografts were radiotherapy in vitro and in mouse xenografts. Our findings generated to explore the radiosensitizing effect of AZD1775 in vivo. suggest that inhibition of Wee1 by AZD1775 is an effective Results: The IC50 concentrations of AZD1775 on esophageal strategy for radiosensitization in esophageal cancer and warrants cancer cell lines were between 300 and 600 nmol/L. AZD1775 clinical testing.

Introduction response to ever-changing intracellular and extracellular genotoxic insults, cells activate DNA damage, replication, and mitotic check- Esophageal cancer is the sixth leading cause of cancer-related death points, which function to inhibit the activity of CDKs and halt cell- and affects more than 450,000 people worldwide (1). Standard-of-care cycle progression to provide time to repair DNA damage and fix therapy for localized esophageal cancer is radiotherapy and chemo- chromatin disruption (7). The fine coupling of cell cycle and DNA therapy followed by surgery, but recurrence rates remain high. More- damage checkpoints ensures genome integrity and cell survival (7). over, approximately of 50% patients diagnosed with esophageal cancer Aberrant activation of CDKs and hence uncontrolled cell-cycle pro- present with unresectable or metastatic disease (2). In the past decade, gression is a hallmark of cancer cells (8). Many human cancers have although great advances have been made for the prevention and deficits in G –S checkpoint due to mutations in the p53 signaling axis control of many cancers such as lung cancer and breast cancer, the 1 including mutations of TP53, CDKN2A, and RB (9). Treatment of overall 5-year survival rate of patients with esophageal cancer remains these cells with radiation induces a G –M arrest, allowing time for below 20% and the incidence is increasing rapidly worldwide (1, 3, 4). 2 DNA repair, thus leading to a higher level of dependence of these Therefore, there is an urgent need to develop novel effective therapies cancer cells on G –M checkpoint for survival. In these cases, genetic for the management of esophageal cancer (2, 5). 2 abrogation of the G –M checkpoint may allow entry of cells into Proper cell proliferation and accurate genetic material duplication 2 mitosis with incompletely-repaired damaged DNA, ultimately leading depends on the tight and fine coordination of the cell-cycle surveillance to mitotic catastrophe and cell death (10). It has therefore been systems including G –G ,S,G, and M cell-cycle checkpoints (6). 0 1 2 proposed that small molecules targeting G –M checkpoint are prom- Cell-cycle progression is controlled by cyclin-dependent kinases 2 ising cancer therapy agents either as monotherapy or in combination (CDK), which are regulated by cell growth and mitogenic signals. In 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 1The Ohio State University Medical Center, Arthur G. James Comprehensive 2 residue (14). Particularly, Wee1-mediated phosphorylation and inhi- Cancer Center and Richard J. Solove Research Institute, Columbus, Ohio. The – University of Texas MD Anderson Cancer Center, Houston, Texas. bition of CDK1 plays a critical role in G2 M checkpoint under normal cell growth and in response to DNA damage or replication stress (15). Note: Supplementary data for this article are available at Clinical Cancer DNA damage or replication stress activates ATM/CHK2 and Research Online (http://clincancerres.aacrjournals.org/). ATR/CHK1 signaling cascades to maintain genome stability and cell Corresponding Author: Terence M. Williams, Ohio State University Wexner viability (13). Activation of CHK1 by ATR in response to various types Medical Center, 460 W. 12th Avenue, Room 492, Columbus, OH 43235. of DNA lesions phosphorylates and stimulates Wee1 activation to Phone: 614-366-2621; Fax: 614-293-4044; E-mail: [email protected] suppress CDK1 activity thereby preventing entry into mitosis (15). Forced cell-cycle progression in the setting of DNA damage perpe- Clin Cancer Res 2020;XX:XX–XX tuates DNA and chromatin damage, and leads to cell death because doi: 10.1158/1078-0432.CCR-19-3373 of irreparable genetic lesions (11). Interestingly, Wee1 expression is 2020 American Association for Cancer Research. upregulated in many cancers and associated with the survival of

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xenografts. Our findings suggest that AZD1775 in combination with Translational Relevance radiotherapy may improve the therapeutic outcome of patients with Stage II/III esophageal cancers are commonly treated with esophageal cancer. 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 Materials and Methods majority of both esophageal adenocarcinoma and squamous cell Antibodies, chemicals, and cell culture carcinomas harbor mutations in TP53, an important tumor sup- OE33, SK4, FLO1, KYSE30, and AGS cell lines were maintained at – pressor gene that also functions to promote cell-cycle arrest in G1 S 37 Cin5%CO2 in DMEM medium supplemented with 10% FBS after DNA damage from radiation. In this preclinical study, we (Sigma), 1% penicillin/streptomycin (Life Technologies). The detailed target the G2–M cell-cycle checkpoint with AZD1775, a Wee1 cell line information is listed in Supplementary Table S1. AZD1775 was kinase inhibitor, in combination with radiation to enhance ther- obtained under a material transfer agreement from NCI-CTEP apeutic efficacy. We find that in TP53-mutated cells lacking an through AstraZeneca and was dissolved in DMSO (Sigma) and added – effective G1 S checkpoint, AZD1775 markedly radiosensitizes to medium with a final concentration of no more than 0.1% DMSO. esophageal cancer cells to radiation both in cell culture assays and Total CDK1, phospho-CDK1 (Tyr15), phospho-Wee1 (Ser642), phos- animal studies. Our results justify a clinical trial to determine the pho-H2AX (S139), phospho-histone H3 (S10), cyclin A2, cyclin B1, safety and efficacy of combining AZD1775 and radiation in cyclin E1, cyclin E2, and GAPDH primary antibodies were purchased patients with esophageal cancer. from Cell Signaling Technology. Anti-rabbit and anti-mouse secondary antibodies were purchased from LI-COR Biosciences.

AlamarBlue assay and IC50 determination patients with cancer (16–18). Given the pivotal role for Wee1 in AlamarBlue assay was performed according to manufacturer's instruc- the regulation of CDK1 activity, targeting Wee1 has been proposed tions (Roche). Briefly, cells were seeded in 96-well plates in six replicates at for the sensitization of cancer cells to radiotherapy and a density of 2,000 cells per well in 100 mL medium. The next day, the cells chemotherapy (11, 19–21). Large-scale genomic studies have found were treated with AZD1775 at various concentrations. After 72 hours, that esophageal cancer has an extremely high frequency of TP53 alamarBlue reagent was added and incubated at 37C for 4 hours, and mutations, ranging from 44% to 93% (22, 23). Recently, The Cancer absorbance was measured at 490 nm. IC50 was determined using the Genome Atlas (TCGA) demonstrated that TP53 mutations were the nonlinear four-parameter regression function in GraphPad Prism. single most common significantly mutated gene in ESCA, occurring in 71% and 91% of esophageal adenocarcinoma and esophageal Immunoblotting squamous cell carcinoma, respectively (24). Therefore, esophageal Immunoblotting was performed as described previously (36). Brief- – cancer cells may depend on G2 M checkpoint for survival and may ly, cell lysates were prepared using RIPA buffer (Thermo Fisher – fi be very sensitive to G2 M checkpoint abrogation by Wee1 inhibition. Scienti c) supplemented with 1 protease inhibitors (Complete; – AZD1775 is a novel small molecule inhibitor that disrupts G2 M Roche) and phosphatase inhibitors (PhosSTOP; Roche) followed by checkpoint by directly inhibiting Wee1 kinase (25). Previous studies protein quantification with the Dc Protein Assay Kit (Bio-Rad). Equal have demonstrated that the sensitivity of AZD1775 depends on p53 amounts of protein were loaded and resolved by SDS-PAGE and functional loss in various types of cancers including non–small cell transferred onto nitrocellulose membranes. Membranes were incu- lung cancer (26–29). However, it has also been reported that Wee1 bated in 5% BSA in Tris-buffered saline with 0.1% Tween-20 (TBST) inhibition could radiosensitize carcinoma cells without TP53 muta- blocking buffer for 1 hour at room temperature. Primary antibodies – tions (30). In addition to inhibiting G2 M checkpoint, AZD1775 has with dilution of 1:200 to 1,000 were allowed to bind overnight at 4 C, been shown to induce DNA replication stress via nucleotide exhaus- or for 2 hours at room temperature. After washing in TBST, the tion (31, 32), and to reduce homologous recombination repair (33). membranes were incubated with immunofluorescent secondary anti- Although Wee1 is not the core component of the replication stress bodies at a 1:5,000 dilution for 1 hour at room temperature. Mem- response pathway, activation of Wee1 by CHK1 induces CDK1 and branes were washed with TBST and allowed to air dry prior to imaging CDK2 to halt cell-cycle progression in response to DNA damage (34). via LI-COR Odyssey CLx Imaging System (Thermo Fisher Scientific). Thus, targeting Wee1 may force cells to enter mitosis in the presence of incomplete DNA replication, which might exacerbate replication Radiation clonogenic assay stress and development of lethal DNA damage (21, 35). AZD1775 Radiation clonogenic assays were performed essentially as described has been tested preclinically in many types of cancers, and has been previously (37). In brief, single cells seeded in 60 or 100 mm tissue shown to radiosensitize and chemosensitize certain cancers, including culture plates were incubated with DMSO or AZD1775 for 3 hours and pancreatic, breast, prostate, lung, and glioblastoma cancers (20, 21). then irradiated with various doses (0–8 Gy). Radiation was performed However, the effects of AZD1775 on esophageal cancer (a disease with with 160 kV, 25 mA at a dose rate of approximately 113 cGy/min using an extraordinary high rate of TP53 mutations) as monotherapy or in a RS-2000 biological irradiator (RadSource), and cells were fixed 7 to combination with other therapeutics remains to be determined. 10 days after seeding. The number of colonies containing at least 50 In this study, we investigated the anti-neoplastic properties of cells was counted using a dissecting microscope (Leica Microsystems, AZD1775 in combination with radiation in esophageal cancer. We Inc.) and dose–enhancement ratio (DER) was calculated as reported TP53 fi – hypothesized that -de cient cells are sensitive to a G2 M check- previously (38). point inhibitor and as such, combining Wee1 inhibition and radiation should target TP53-deficient ESCA cells. We found that abrogation of Immunofluorescence for mitotic catastrophe – fi G2 M checkpoint by targeting Wee1 kinase with AZD1775 markedly Cells on coverslips were xed with 2% paraformaldehyde, permea- sensitizes esophageal cancer cells to radiotherapy in vitro and in mouse bilized with 1% Triton X-100, and blocked with 3% BSA in PBS. Briefly,

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for paraffin-embedded tissue sections from tumor xenografts, sections also higher WEE1 mRNA expression in esophageal cancer tumors than were cut onto slides and deparaffinized in xylene and rehydrated that in normal tissues, although not statistically significant. Interest- through a graded alcohol series. Then, slides were washed in distilled ingly, MCM10, CCNE1, CCNE2, FBXO5, and CLSPN have been shown water. Cells on coverslips or tissue sections were stained with anti- to be biomarkers predictive for responsiveness to AZD1775 (39). tubulin antibody (Cell Signaling Technology), washed, and incubated Similarly, analysis of TCGA and GTE databases revealed that these with a fluorophore-conjugated secondary antibody (Biotium). Fol- five genes were significantly overexpressed in esophageal cancer tumor lowing nucleus counterstaining with DAPI, the slides were examined versus normal tissues (Fig. 1B). Further analysis of the Oncomine on a Zeiss fluorescence microscope. For each experiment, mitotic database (http://oncomine.org/) confirmed overexpression of these catastrophe was determined in at least 100 cells. genes in human ESCA tumors relative to normal tissues (Supplemen- tary Fig. S1). These findings of CDK1 upregulation and AZD1775 Flow cytometry responsiveness gene signature suggested targeting Wee1 with Cells were seeded into six-well plates at a density of 200,000 cells per AZD1775 may sensitize esophageal cancer to radiotherapy. well in 2 mL medium for 16 hours. The cells were treated with AZD1775 for 3 hours, followed by ionizing radiation (IR) and then Wee1 kinase inhibition with AZD1775 sensitizes esophageal cultured for 24 hours. Cells were fixed in 70% ethanol at 20C and cancer cells to radiation stained with DNA staining solution containing propidium iodide and To evaluate the potential radiation-sensitizing efficacy of Wee1 RNaseA (Sigma-Aldrich) overnight. All data were acquired on LSRII inhibition by AZD1775 in ESCA, we initially investigated the inde- cytometry (BD Biosciences) and each sample was assessed using a pendent cytotoxic effect of AZD1775 on esophageal cancer cell lines. collection of 10,000 events, followed by analysis using FlowJo software All four cell lines used in our study have TP53 mutation, including (FlowJo). esophageal adenocarcinoma cell lines (OE33, SK4, and FLO1) and an esophageal squamous cell carcinoma cell line (KYSE30). The cytotoxic In vivo studies effect of AZD1775 in esophageal cancer cell lines was assessed by Animal studies were conducted in accordance with an approved alamarBlue assay. We found that the IC50 values of AZD1775 in these protocol adhering to the IACUC policies and procedures at The Ohio esophageal cancer cell lines ranged from 252 to 624 nmol/L (Fig. 2A). State University. Six- to eight-week-old male athymic nude mice To explore the potential of AZD1775 as a radiosensitizer for esoph- (Taconic Farms Inc.) were caged in groups of five or less, and fed ageal cancer, FLO-1 and OE33 cells were treated with increasing doses with a diet of animal chow and water ad libitum. OE33 and FLO1 cells of radiation in the presence or absence of 100 nmol/L AZD1775, were injected subcutaneously into the flanks of each mouse at 2 106 followed by clonogenic (colony-forming) assay. At 100 nmol/L of and 2.5 106 cells per injection, respectively. Treatment regimens AZD1775 alone, we noted minimal cytotoxicity of the drug (Supple- were started once tumors reached 150 mm3 in size, 2 to 4 weeks mentary Fig. S2). In combination with radiation however, AZD1775 postinjection. AZD1775 powder was suspended in 0.9% sodium could effectively sensitize esophageal cancer cells to radiation treat- chloride containing 5% dextrose. AZD1775 was administered orally ment, with DER up to 3.14 in SK4 cells, 1.46 in OE33 cells, 1.34 in FLO1 using a sterile 18G gavage needle at 50 mg/kg twice a day for 5 cells, and 1.23 in KYSE cells (Fig. 2B). consecutive days. Using a custom shielding apparatus to block non- targeted areas, 4 Gy of radiation was administered directly to tumors Wee1 kinase inhibition abrogates radiation-induced G2–M once daily for 5 consecutive days. For combination treatment, mice phase cell-cycle arrest fi – were treated with radiation 2 to 3 hours after the rst daily dosing of Wee1 kinase plays a key role in promoting G2 M cell-cycle arrest AZD1775. To obtain a tumor growth curve, perpendicular diameter after DNA damage (e.g., by ionizing radiation) to allow time for cells to measurements of each tumor were measured every 2 to 5 days from the undergo DNA repair, by inactivating CDK1. We explored whether the first day of injection with digital calipers, and volumes were calculated radiosensitizing effects of AZD1775 are associated with abrogation of using the formula (L W W)/2. Two mice from each group were radiation-induced G2–M cell-cycle arrest in asynchronously growing sacrificed after three days treatment, to isolate tumor xenografts for cells by flow cytometry assay. In both FLO1 and OE33 cells, AZD1775 immunoblotting and immunofluorescence staining. treatment (100 nmol/L) decreased whereas 4 Gy radiation alone – increased cells in G2 M phase. However, pretreatment of cells with Data analysis AZD1775 at 3 hours before radiation significantly reduced the accu- – Data were analyzed similarly as described previously (37), and are mulation of G2 M phase cells after radiation (Fig. 3A). Activation of presented as the mean SEM. Wee1 kinase prevents cells from entering into mitosis through phos- phorylating and subsequent inactivation of CDK1. Immunoblotting analyses showed AZD1775 inhibited Wee1 and CDK1 phosphoryla- Results tion in a time-dependent manner, with maximal changes noted Wee1 is a potential therapeutic target in human esophageal 24 hours after treatment (Fig. 3B). Of note, radiation alone mildly cancer increased the phosphorylation of both Wee1 and CDK1 at 24 hours The sensitization of many cancers to radiotherapy and chemother- after radiation. Exposing cells to AZD1775 before radiation attenuated apy by targeting Wee1 and the high mutation rate of TP53 suggests that radiation-induced Wee1 and CDK1 phosphorylation, most notable at Wee1 inhibition is a potential therapeutic strategy for ESCA. To this 24 hours after radiation. Interestingly, AZD1775 alone induced end, we first analyzed the TCGA and GTEx databases with GEPIA gH2AX expression and enhanced radiation-mediated increase of (http://gepia.cancer-pku.cn/) for WEE1 and CDK1 mRNA expression gH2AX particularly after 24 hours of treatment. Most studies have in esophageal cancer. Significant elevation of CDK1 mRNA expression shown that the therapeutic effects AZD1775 is related to the abro- was found in esophageal cancer tumors compared with normal tissues, gation of the G2 checkpoint and/or unscheduled mitotic entry. How- suggesting increased cell proliferation or mechanisms to promote ever, emerging evidence suggest that Wee1 inhibition suppresses DNA – transition through G2 M phases (Fig. 1A). Interestingly, there was damage repair and induces replication stress (32, 33), both of which

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A CDK1 WEE1 86 6 4 4 2 2 0 0

Esophageal cancer Esophageal cancer [num (T) = 182; num (N) = 286] [num (T) = 182; num (N) = 286] B MCM10 CCNE1 CCNE2 FBXO5 CLSPN 6 54321 86 6 6 23 23 24 24 145 145 0 0 0 0 0

Esophageal cancer Esophageal cancer Esophageal cancer Esophageal cancer Esophageal cancer [num (T) = 182; num (N) = 286] [num (T) = 182; num (N) = 286] [num (T) = 182; num (N) = 286] [num (T) = 182; num (N) = 286] [num (T) = 182; num (N) = 286]

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 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 (TPMþ1). Tumor samples ¼ 182; normal samples ¼ 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 hours. The cell viability was assessed by alamarBlue assay, and IC50 values were calculated. B, Cells were cultured in media containing 100 nmol/L AZD1775 at 3 hours 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 two experiments were performed per cell line. DER at 2 Gy were compared between vehicle and AZD1755 (, P < 0.05; , P < 0.01).

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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 nmol/L AZD1775 (AZD-100) at 3 hours prior to radiation with 0 (ctr) or 4 Gy doses. Twenty-four hours 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 two 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 nmol/L AZD1775 (AZD-100) for 3 hours prior to radiation with 0 or 4 Gy doses. At the indicated time points following radiation (1, 4, 16, and 24 hours), the cells were lysed and subjected to immunoblotting with GAPDH as loading control.

leads to phosphorylation of H2AX. These results suggest that Wee1 inhibition attenuates DNA damage repair during – AZD1775 inhibition of Wee1 abrogates radiation induced G2 M fractionated radiation – cell-cycle checkpoint arrest by promoting CDK1 activity and increas- We found that AZD1775 attenuates radiation-induced G2 M phase ing replication stress, thereby potentiating radiation-mediated DNA arrest, enhances radiation mediated DNA damage, causes premature damage in esophageal cancer cells. entrance into mitosis, and finally leads to mitotic cell death. To further support our observations with more clinically relevant doses of Wee1 inhibitor enhances radiation-induced mitotic cell death radiation (i.e., 2 Gy per day), we extended our study to investigate Premature entrance into mitosis with unrepaired DNA lesions the capacity of AZD1775 for radiosensitization using standard frac- (particularly DSBs) leads to lethal consequences in cells. The abro- tionated radiation doses (i.e., 2 Gy per fraction) using colony formation gation of G2–M phase cell-cycle arrest and enhancement of DNA assays. In the single fraction ionizing radiation experiments, FLO1 and damage by AZD1775 in ESCA cells treated with radiation suggests that OE33 cells were pretreated with 100 nmol/L AZD1775 or vehicle AZD1775 can promote irradiated ESCA cells to prematurely enter into control for 3 hours, followed by treatment with a single radiation dose cell mitosis before completion of DNA repair. To test this hypothesis, of 4 Gy. In the fractionated ionizing radiation experiment, the cells FLO1 and OE33 cells were cultured on cover slides, and treated with were pretreated with 100 nmol/L AZD1775 or vehicle control for vehicle, AZD1775, 4 Gy radiation, or the combination of AZD1775 for 3 hours, followed by treatment with 2 Gy, which was repeated 24 hours 3 hours followed by 4 Gy. After 72 hours, the cover slides were later (Fig. 5A). Twenty-four hours postradiation, the cells were collected, and stained with DAPI and tubulin by immunofluorescence. cultured in fresh medium without AZD1775/vehicle for 10 additional Mitotic cell death was determined by the number of cells demonstrat- days before colony fixation and staining. Cell recovery rate was ing mitotic catastrophe (multinuclear cells with more than two nuclear calculated by dividing the percentage of colonies formed following lobes, or cells with several micronuclei; Fig. 4A). In comparison to fractionated radiation (2 Gy 2 fractions) by the percentage of vehicle control, AZD1775 alone did not induce mitotic catastrophe. colonies formed following a single fraction of 4 Gy. As expected, 2 Conversely, 4 Gy radiation treatment resulted in accumulation of cells Gy 2 led to a higher surviving fraction compared with a single 4 Gy experiencing mitotic catastrophe, which was significantly enhanced by dose of radiation, likely due to sublethal DNA repair occurring AZD1775 in both cell lines (Fig. 4B and C). Thus, the combination of between fractions, leading to a survival enhancement rate of 1.37 and AZD1775 with radiation resulted in a significantly higher incidence of 1.84 for FLO1 and OE33 cells, respectively. Similar to our previous data mitotic catastrophe than radiation treatment alone. with single fraction radiation in Fig. 2B, exposure of FLO1 or OE33

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Figure 4. AZD1775 enhanced radiation-induced mitotic catastrophe in ESCA cells. A, FLO1 and OE33 cells were treated with 4 Gy radiation, 100 nmol/L AZD1775 alone, or in combination (100 nmol/L AZD1775 added 3 hours prior to 4 Gy radiation). At 72 hours postradiation, 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 show percentage of mitotic catastrophe cells in 100 counted FLO1 (B) and OE33 (C) cells (, P < 0.001).

cells to AZD1775 before and during fractionated radiation still effec- tent with our in vitro data, the combination of AZD1775 and IR tively radiosensitized esophageal cancer cells and abrogated cell recov- significantly increased mitotic catastrophe in tumor xenografts com- ery (Fig. 5B). These results indicate a comparable enhancement of cell pare to IR treatment alone (Fig. 6D). Moreover, the majority of FLO1 death by AZD1775 in esophageal cancer cells when combining and OE33 tumors showed no evidence of tumor recurrence after AZD1775 with either fractionated or single fraction radiation. These treatment with AZD1775 in combination with radiation (survival findings have important clinical implications as patients with esoph- curves shown in Supplementary Fig. S3). In terms of toxicity, mice ageal cancer are treated with fractionated radiation to typical doses of tolerated the treatment well, and no mice died early from treatment 1.8 to 2.0 Gy/day. toxicity. Mice who received IR treatment did lose weight during the first week, but fully recovered within 2 weeks (Supplementary Fig. S4). Wee1 inhibition markedly radiosensitizes esophageal cancer We further performed immunoblotting of the tumor lysates 2 hours cells in mouse tumor xenografts after Day 3 treatment in each group to assess pharmacodynamics To determine if Wee1 inhibition could effectively radiosensitize effects of AZD1775. AZD1775 reduced the phosphorylation of ESCA cells in vivo, we further explored the combined treatment of Wee1 and CDK1 as well as the protein levels of cyclin A2, B1, AZD1775 and radiation in vivo using nude mice xenografts with FLO1 E1, and E2, while increasing levels of phospho-histone H3 (a marker and OE33 cells. When tumors reached 100 to 150 mm3, the mice were of mitotic cells; Fig. 6E). Taken together, these findings indicate – randomized to groups of treatment with vehicle, AZD1775 alone, 4 Gy AZD1775 is promoting G2 M phase cell-cycle progression. Radi- radiation alone, or the combination of AZD1775 þ 4 Gy (mice were ation increased the phosphorylation of Wee1 and CDK1, as well as treated with AZD1775 2 hours before radiation). The treatments lasted the protein levels of cyclin A2, B1, E1, and E2, which were all for 5 consecutive days during Days 1–5(Fig. 6A). AZD1775 was prevented by Wee1 inhibition. These results demonstrate that delivered by oral gavage with a dose of 50 mg/kg, twice a day as potent inhibition of Wee1 leads to promotion of cells through previously described (29). AZD1775 monotherapy and radiation alone mitosis despite DNA damage from radiation, thus leading to resulted in partial tumor growth delay. However, AZD1775 in com- marked radiosensitization by AZD1775 in vivo. bination with radiation treatment led to remarkable and sustained tumor regression of both FLO1 and OE33 xenografts (Fig. 6B and C). To investigate whether the treatment combination is indeed inducing Discussion tumor cell death through mitotic catastrophe, we performed mitotic Radiotherapy induces DNA damage, resulting in activation of catastrophe assay in tumors derived from mouse xenografts. Consis- apoptotic pathways or inducing postmitotic death due to unrepaired

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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 2) or single fraction radiation (4 Gy). IR, ionizing radiation; w/o, without; AZD, AZD1775 (100 nmol/L; , P < 0.05).

DNA damage. Following DNA damage, cells rely on cell-cycle check- shortage, and subsequent double-strand DNA breaks (42). It has been points to provide time for DNA repair prior to cell division. Esophageal proposed that targeting Wee1 kinase is a promising strategy for the cancer cells often lack a functional G1 checkpoint due to a high radiosensitization and chemosensitization of cancer cells with a defec- TP53 frequency of mutations. On the basis of TCGA data, up to tive G1 cell-cycle checkpoint (21). Accordingly, several Wee1 kinase 91% of squamous cell carcinoma and 71% of adenocarcinoma esoph- small molecule inhibitors, including AZD1775, have been devel- ageal cancers possess a TP53 mutation making them heavily dependent oped (27). Previous studies have shown that AZD1775 is a potent on the G2–M checkpoint to survive DNA damage and replication and selective small molecule inhibitor of Wee1 kinase and has been stress. In this study, we demonstrated that a potent Wee1 kinase shown to sensitize tumor cells to both chemotherapy and radia- inhibitor AZD1775 sensitized ESCA cells to radiation therapy in tion (27, 28, 33). In this study, we found that both CDK1 and WEE1 in vitro cell cultures and mouse tumor xenografts. In addition, are overexpressed in ESCA, as well as genes associated with an AZD1775 treatment led to a comparable enhancement of cytotoxicity AZD1775 responsiveness gene signature (39). Moreover, we demon- in esophageal cancer cells treated with either fractionated radiation or strated that AZD1775 potently inhibited the phosphorylation of both single dose radiation. Mechanistically, AZD1775 attenuated radiation- Wee1 and CDK1 in the absence or presence of radiation. Consistent – – induced G2 M phase arrest, which was accompanied by enhanced with the role for Wee1 in G2 M checkpoint regulation, AZD1775 fi – radiation-induced mitotic catastrophe and DNA damage. Our ndings prevented IR-induced G2 M phase cell-cycle arrest, which was accom- suggest that Wee1 kinase specific inhibitor AZD1775 is an effective panied with enhanced mitotic catastrophe and gH2AX, indicative of radiosensitizer for esophageal cancer. enhanced cell death and DNA damage. Interestingly, we noted Wee1 is a tyrosine kinase and is activated following DNA damage. AZD1775 caused reductions in E, A, and B-type cyclins. This obser- – Wee1 kinase is a critical regulator of the G2 M checkpoint and thus vation is consistent with our knowledge of the temporal expression of genomic stability by mediating inhibitory phosphorylation of CDK1, cyclins during the cell cycle. Specifically, E-type cyclins are upregulated – resulting in cell-cycle arrest and permitting DNA repair prior to during the G1 S phase transition and then fall down, whereas A-type proceeding with mitosis (40, 41). It has been shown that inhibiting cyclins are upregulated in the G2 phase and then fall down prior to and Wee1 results in replication stress, loss of genomic integrity, nucleotide during entry of calls into mitosis. Finally, B-type cyclins are

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A -Day 14 Day 1 Day 5 Day 52–90 D FLO1 70 60 50 Tumor cell injection 5 days treatment Study end 20 B FLO1 cells Vehicle 10 400.00% AZD1775 ALONE Vehicle + IR 0

300.00% % Cell with mitotic catastrophe AZD1775 + IR Ctr IR

AZD1775 200.00% AZD1775 + IR

100.00% E Vehicle AZD1775 4 Gy 4 Gy + AZD1775 % Volume change % Volume 0.00% 122 12121 15 10 151924295485 p-WEE (S642) −100.00% Days p-CDK1 (Y15)

C 1400.00% Cyclin A2 OE33 cells 1200.00% Vehicle Cyclin B1 1000.00% AZD1775 ALONE Vehicle + IR 800.00% Cyclin E1 AZD1775 + IR 600.00% Cyclin E2 400.00% % Volume change % Volume 200.00% p-HH3 (S10)

0.00% GAPDH 15 10 151924293338 43 47 52 Days

Figure 6. Wee1 inhibition effectively radiosensitizes esophageal carcinoma cells in xenograft tumor models. Mice were injected with 2 106 FLO1 or OE33 cells, and randomized to start treatment once tumors reached 100 to 200 mm3. AZD1775 was administered via oral gavage at 50 mg/kg twice a day for 5 days (control group received vehicle at same intervals), 2 hours 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 and E, FLO1 tumor xenografts were isolated from mice on Day 3 (2 hours 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; , P < 0.05).

upregulated at the G2–M transition then fall down sharply upon entry tion (45, 46). Overall, approximately 30% of patients with esophageal to mitosis. Taken together, AZD1775 potently inhibits Wee1 in cancer will demonstrate a pathologic complete response (pCR) at the esophageal cancer cells, thereby promoting entry from S and G2 time of surgery (46–48) with pCR rates closer to 43% to 49% for through M phase, and thus preventing Wee1 from protecting esoph- squamous cell cancer (46, 49) and 16% to 25% for adenocarcino- ageal cancer cells from the effects of radiotherapy. ma (46, 50, 51). A pCR is associated with an improvement in overall Esophageal cancer remains a global problem and is the eighth most survival (47, 48). Patients with a pCR, arguably, may be able to avoid common cancer worldwide (1). Globally, although esophageal squa- esophagectomy without compromising survival (52). Esophagectomy mous cell carcinoma remains the predominant histology in Asia, is associated with impaired quality of life and remains a morbid Africa, and South America, the incidence of esophageal adenocarci- operation with substantial mortality (53–57). With current pCR rates noma has surpassed that of squamous cell carcinoma in North at or below 50%, improvements in therapy, including the potential America, Australia, and Europe. Current predictions are that by addition of radiosensitizing agents, may help to make organ preser- 2030, up to 1 in 100 men will be diagnosed with esophageal adeno- vation a reality in esophageal cancer. carcinoma during their lifetime in the Netherlands and the United Currently, even with the most aggressive approach of trimodality Kingdom (43). In the United States, the incidence of cancers of the therapy, approximately 50% of patients will experience a locoregional esophagus and gastroesophageal junction has increased dramatically and/or distant recurrence within 5 years of treatment completion (45). in recent decades, largely driven by the rising incidence of adenocar- Therefore, there is an urgent need to develop novel strategies that will cinoma (44). Nearly 40% to 50% of patients present with either improve clinical outcomes of patients diagnosed with localized esoph- unresectable disease or evidence of distant metastasis (44). For patients ageal cancer. Strategies aimed at improving local control, including with resectable disease, the current standard of care includes admin- modalities such as radiation therapy, will likely have an impact on istering chemotherapy, most commonly carboplatin and paclitaxel, improving survival. Our results showed that AZD1775 in combination concurrently with radiation therapy followed by surgical resec- with radiotherapy resulted in virtually complete tumor regression of

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esophageal cancer tumor xenografts. Therefore, our study suggests with multiple chemotherapy regimens with preliminary data showing that AZD1775 inhibition of Wee1 is an effective strategy for radiation that combination therapy appears to be reasonably tolerated (61–64). sensitization in esophageal cancer cells. Interestingly, we observed that Because of the high incidence of TP53 mutations and the dependence – the DER of SK4 was much higher than that of the other three cell lines on the Wee1-mediated G2 M checkpoint to survive DNA damage, (Fig. 2B). All the four esophageal cancer cell lines used in this study esophageal cancer is a logical site to consider the addition of a Wee1 have TP53 mutations. However, besides TP53 mutation, SK4 cells have kinase inhibitor to standard neoadjuvant therapy. We believe our additional KRAS and PIK3CA mutation (https://portals.broadinstitute. study warrants a phase I trial testing AZD1775 in combination with org/ccle). KRAS and PIK3CA mutations drive tumorigenesis via radiation or chemoradiation for esophageal cancer. multiple mechanisms, one of which is to induce DNA replication stress leading to genomic instability (58, 59). As mentioned, Wee1 has Disclosure of Potential Conflicts of Interest an important role in replication stress response (35). Thus, esophageal E.D. Miller reports receiving other remuneration from a CTEP-approved career cancer cells with KRAS and PIK3CA mutations might may have development LOI combining AZD1775 with radiation therapy for inoperable or become more dependent on Wee1 kinase for survival due to increased metastatic esophageal cancer. S.H. Lin reports receiving commercial research grants from Beyond Spring Pharmaceuticals, Genentech, and Hitachi Chemical Diagnostic, replication stress. It will be important to determine whether esophageal speakers bureau honoraria from AstraZeneca and Varian Medical Systems, holds KRAS PIK3CA cancer with and/or mutations are hypersensitive to ownership interest (including patents) in STCube Pharmaceuticals, and is an advisory Wee1 inhibitors in future preclinical and clinical studies. In addition, it board member/unpaid consultant for AstraZeneca and Beyond Spring Pharmaceu- will be critical to determine whether TP53 mutant status confers ticals. No potential conflicts of interest were disclosed by the other authors. increased sensitivity to AZD1775 and radiation. In our preliminary studies, we found that the combination of AZD1775 and radiation did Authors’ Contributions not radiosensitize TP53 intact AGS adenocarcinoma cells (Supple- Conception and design: L. Yang, C. Shen, J. Zhang, T.M. Williams mentary Fig. S5). Finally, further studies are needed to assess whether Development of methodology: L. Yang, C. Shen, S.H. Lin, T.M. Williams Acquisition of data (provided animals, acquired and managed patients, provided AZD1755 sensitizes esophageal cancer cells to DNA-damaging che- facilities, etc.): L. Yang, C.J. Pettit, T. Li, A.J. Hu, T.M. Williams motherapies such as platinum-based drugs or other chemotherapeu- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, tics which promote replication stress. computational analysis): L. Yang, C. Shen, C.J. Pettit, T. Li, E.D. Miller, S.H. Lin, On the basis of our results, we feel that our data support a clinical T.M. Williams trial testing the combination of Wee1 kinase inhibitor and radiation for Writing, review, and/or revision of the manuscript: L. Yang, C. Shen, C.J. Pettit, esophageal cancer. It would be interesting to also explore combining E.D. Miller, S.H. Lin, T.M. Williams Administrative, technical, or material support (i.e., reporting or organizing data, Wee1 inhibitor with chemoradiation, or replacing a chemotherapy constructing databases): J. Zhang drug commonly used in esophageal cancer with a Wee1 kinase inhibitor. Standard chemotherapy regimens used in combination with Acknowledgments radiation for esophageal cancer include paclitaxel and carboplatin, or This work was supported by the following grants: The Ohio State University 5FU and oxaliplatin. Recently, a phase II trial of AZD1775 in com- Comprehensive Cancer Center (OSU-CCC), NIH (P30 CA016058 and R01 bination with gemcitabine and radiation was published for pancreatic CA198128) and National Center for Advancing Translational Sciences cancer, showing tolerable safety profile of the combination and (KL2TR001068). These data were presented in part at the American Association of significant efficacy compared with historical controls (60). This trial Cancer Research (AACR) annual meeting 2018 and Radiation Research Society annual meeting 2019. Research reported in this publication was supported by The was based on preclinical data combining Wee1 inhibitor with gemci- Ohio State University Comprehensive Cancer Center (OSU-CCC) and the National tabine and radiation. Thus, additional preclinical studies may be Institutes of Health under grant number P30 CA016058, as well as RSG-17-221-01- needed to establish the optimal combination of Wee1 inhibitor with TBG (to T.M. Williams), award number grant KL2TR001068 from the National certain chemotherapy drugs and radiation for the treatment of esoph- Center for Advancing Translational Sciences (to T.M. Williams), and NIH grant R01 ageal cancer. CA198128 (to T.M. Williams). The content is solely the responsibility of the authors fi In summary, our results demonstrated a potent inhibitory role for and does not necessarily represent the of cial views of the NIH. Wee1 kinase inhibitor AZD1775 in cell-cycle checkpoints in response The costs of publication of this article were defrayed in part by the payment of page to IR-induced DNA double-strand breaks; importantly, AZD1775 charges. This article must therefore be hereby marked advertisement in accordance – in vitro enhanced IR mediated cell death and maintained the sup- with 18 U.S.C. Section 1734 solely to indicate this fact. pression of esophageal cancer mouse xenografts by radiotherapy in vivo. There are several phase I and II studies which have been Received October 14, 2019; revised February 20, 2020; accepted March 24, 2020; performed using AZD1775 both as monotherapy and in combination published first March 27, 2020.

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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.

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