Purpose:

Esophageal cancer is a deadly malignancy with a 5-year survival rate of only 5% to 20%, which has remained unchanged for decades. Esophageal cancer possesses a high frequency of TP53 mutations leading to dysfunctional G1 cell-cycle checkpoint, which likely makes esophageal cancer 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 esophageal cancer 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 esophageal cancer xenografts were generated to explore the radiosensitizing effect of AZD1775 in vivo.

Results:

The IC50 concentrations of AZD1775 on esophageal cancer cell lines were between 300 and 600 nmol/L. AZD1775 (100 nmol/L) as monotherapy did not alter the viability of esophageal cancer cells, but significantly radiosensitized esophageal cancer 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 γH2AX levels, and subsequently reduced survival after radiation. Importantly, AZD1775 in combination with radiotherapy resulted in marked tumor regression of esophageal cancer tumor xenografts.

Conclusions:

Abrogation of G2–M checkpoint by targeting Wee1 kinase with AZD1775 sensitizes esophageal cancer 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 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.

Esophageal cancer is the sixth 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 patients with esophageal cancer 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 (CDK), 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 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 and 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 patients with cancer (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 (21, 35). 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 patients with esophageal cancer.

Antibodies, chemicals, and cell culture

OE33, SK4, FLO1, KYSE30, and AGS cell lines were maintained at 37°C in 5% CO2 in DMEM medium supplemented with 10% FBS (Sigma), 1% penicillin/streptomycin (Life Technologies). The detailed cell line information is listed in Supplementary Table S1. AZD1775 was obtained under a material transfer agreement from NCI-CTEP through AstraZeneca 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. Anti-rabbit and anti-mouse secondary antibodies were purchased from LI-COR Biosciences.

AlamarBlue assay and IC50 determination

AlamarBlue assay was performed according to manufacturer's instructions (Roche). Briefly, cells were seeded in 96-well plates in six 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. IC50 was determined using the nonlinear four-parameter regression function in GraphPad Prism.

Immunoblotting

Immunoblotting was performed as described previously (36). Briefly, cell lysates were prepared using RIPA buffer (Thermo Fisher Scientific) supplemented with 1× protease inhibitors (Complete; Roche) and phosphatase inhibitors (PhosSTOP; Roche) followed by protein quantification with the Dc Protein Assay Kit (Bio-Rad). Equal amounts of protein were loaded and resolved by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were incubated in 5% 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 to 1,000 were allowed to bind overnight at 4°C, or for 2 hours at room temperature. After washing in TBST, the membranes were incubated with immunofluorescent secondary antibodies at a 1:5,000 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 (Thermo Fisher Scientific).

Radiation clonogenic assay

Radiation clonogenic assays were performed essentially as described previously (37). In brief, single cells seeded in 60 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 160 kV, 25 mA at a dose rate of approximately 113 cGy/min using a RS-2000 biological irradiator (RadSource), and cells were fixed 7 to 10 days after seeding. The number of colonies containing at least 50 cells was counted using a dissecting microscope (Leica Microsystems, Inc.) and dose–enhancement ratio (DER) was calculated as reported previously (38).

Immunofluorescence for mitotic catastrophe

Cells on coverslips were fixed with 2% paraformaldehyde, permeabilized with 1% Triton X-100, and blocked with 3% BSA in 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). 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 six-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 hours, 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 10,000 events, followed by analysis using FlowJo software (FlowJo).

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.) 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 ∼150 mm3 in size, 2 to 4 weeks postinjection. 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 twice a day for 5 consecutive days. Using a custom shielding apparatus to block nontargeted 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 to 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 to 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 described previously (37), and are presented as the mean ± SEM.

Wee1 is a potential therapeutic target in human esophageal cancer

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 esophageal cancer. Significant elevation of CDK1 mRNA expression was found in esophageal cancer tumors compared with 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 esophageal cancer 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 (39). Similarly, analysis of TCGA and GTE databases revealed that these five genes were significantly overexpressed in esophageal cancer 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 esophageal cancer to radiotherapy.

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 (TPM+1). Tumor samples = 182; normal samples = 286; *, P < 0.05. TPM, transcripts per million.

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 (TPM+1). Tumor samples = 182; normal samples = 286; *, P < 0.05. TPM, transcripts per million.

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Wee1 kinase inhibition with AZD1775 sensitizes esophageal cancer 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 esophageal cancer 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 esophageal cancer cell lines was assessed by alamarBlue assay. We found that the IC50 values of AZD1775 in these esophageal cancer cell lines ranged from 252 to 624 nmol/L (Fig. 2A). To explore the potential of AZD1775 as a radiosensitizer for esophageal cancer, FLO-1 and OE33 cells were treated with increasing doses of radiation in the presence or absence of 100 nmol/L AZD1775, followed by clonogenic (colony-forming) assay. At 100 nmol/L of AZD1775 alone, we noted minimal cytotoxicity of the drug (Supplementary Fig. S2). In combination with radiation however, AZD1775 could effectively sensitize esophageal cancer cells to radiation treatment, with 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).

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).

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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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 nmol/L) decreased whereas 4 Gy radiation alone increased cells in G2–M phase. However, pretreatment of cells with AZD1775 at 3 hours 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 hours after treatment (Fig. 3B). Of note, radiation alone mildly increased the phosphorylation of both Wee1 and CDK1 at 24 hours after radiation. Exposing cells to AZD1775 before radiation attenuated radiation-induced Wee1 and CDK1 phosphorylation, most notable at 24 hours after radiation. Interestingly, AZD1775 alone induced γH2AX expression and enhanced radiation-mediated increase of γH2AX particularly after 24 hours 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 esophageal cancer cells.

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.

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.

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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, 4 Gy radiation, or the combination of AZD1775 for 3 hours followed by 4 Gy. After 72 hours, 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 two 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 C). Thus, the combination of AZD1775 with radiation resulted in a significantly higher incidence of mitotic catastrophe than radiation treatment alone.

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).

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).

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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 pretreated with 100 nmol/L AZD1775 or vehicle control for 3 hours, followed by treatment with a single radiation dose of 4 Gy. In the fractionated ionizing radiation experiment, the cells were pretreated with 100 nmol/L AZD1775 or vehicle control for 3 hours, followed by treatment with 2 Gy, which was repeated 24 hours later (Fig. 5A). Twenty-four hours postradiation, 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 × 2 fractions) by the percentage of colonies formed following a single fraction of 4 Gy. As expected, 2 Gy × 2 led to a higher surviving fraction compared with a single 4 Gy 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 esophageal cancer cells and abrogated cell recovery (Fig. 5B). These results indicate a comparable enhancement of cell death by AZD1775 in esophageal cancer cells when combining AZD1775 with either fractionated or single fraction radiation. These findings have important clinical implications as patients with esophageal cancer are treated with fractionated radiation to typical doses of 1.8 to 2.0 Gy/day.

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).

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).

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Wee1 inhibition markedly radiosensitizes esophageal cancer 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 to 150 mm3, the mice were randomized to groups of treatment with vehicle, AZD1775 alone, 4 Gy radiation alone, or the combination of AZD1775 + 4 Gy (mice were treated with AZD1775 2 hours 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 C). 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 2 weeks (Supplementary Fig. S4). We further performed immunoblotting of the tumor lysates 2 hours 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.

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).

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).

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Radiotherapy induces DNA damage, resulting in activation of apoptotic pathways or inducing postmitotic 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. On the basis of 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 this 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 esophageal cancer 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 (40, 41). It has been shown that inhibiting Wee1 results in replication stress, loss of genomic integrity, nucleotide shortage, and subsequent double-strand DNA breaks (42). 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 this study, we found that both CDK1 and WEE1 are overexpressed in ESCA, as well as genes associated with an AZD1775 responsiveness gene signature (39). 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, whereas A-type cyclins are upregulated in the G2 phase and then fall down prior to and during entry of calls into mitosis. Finally, 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 esophageal cancer cells, thereby promoting entry from S and G2 through M phase, and thus preventing Wee1 from protecting esophageal cancer cells from the effects of radiotherapy.

Esophageal cancer remains a global problem and is the eighth most common cancer worldwide (1). Globally, although 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 (43). In the United States, the incidence of cancers of the esophagus and gastroesophageal junction has increased dramatically in recent decades, largely driven by the rising incidence of adenocarcinoma (44). Nearly 40% to 50% of patients present with either unresectable disease or evidence of distant metastasis (44). 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 (45, 46). Overall, approximately 30% of patients with esophageal cancer will demonstrate a pathologic complete response (pCR) at the time of surgery (46–48) with pCR rates closer to 43% to 49% for squamous cell cancer (46, 49) and 16% to 25% for adenocarcinoma (46, 50, 51). A pCR is associated with an improvement in overall survival (47, 48). Patients with a pCR, arguably, may be able to avoid esophagectomy without compromising survival (52). Esophagectomy is associated with impaired quality of life and remains a morbid operation with substantial mortality (53–57). 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 (45). 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 esophageal cancer tumor xenografts. Therefore, our study suggests that AZD1775 inhibition of Wee1 is an effective strategy for radiation sensitization in esophageal cancer 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 esophageal cancer 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 (58, 59). As mentioned, Wee1 has an important role in replication stress response (35). Thus, esophageal cancer 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 esophageal cancer 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 esophageal cancer cells to DNA-damaging chemotherapies such as platinum-based drugs or other chemotherapeutics which promote replication stress.

On the basis of our results, we feel that our data support a clinical trial testing the combination of Wee1 kinase inhibitor and radiation for esophageal cancer. It would be interesting to also explore combining Wee1 inhibitor with chemoradiation, or replacing a chemotherapy drug commonly used in esophageal cancer with a Wee1 kinase inhibitor. Standard chemotherapy regimens used in combination with radiation for esophageal cancer 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 with historical controls (60). 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 esophageal cancer 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 (61–64). Because of 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.

E.D. Miller reports receiving other remuneration from a CTEP-approved career development LOI combining AZD1775 with radiation therapy for inoperable or metastatic esophageal cancer. S.H. Lin reports receiving commercial research grants from Beyond Spring Pharmaceuticals, Genentech, and Hitachi Chemical Diagnostic, speakers bureau honoraria from AstraZeneca and Varian Medical Systems, holds ownership interest (including patents) in STCube Pharmaceuticals, and is an advisory board member/unpaid consultant for AstraZeneca and Beyond Spring Pharmaceuticals. No potential conflicts of interest were disclosed by the other authors.

Conception and design: L. Yang, C. Shen, J. Zhang, T.M. Williams

Development of methodology: L. Yang, C. Shen, S.H. Lin, T.M. Williams

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Yang, C.J. Pettit, T. Li, A.J. Hu, T.M. Williams

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L. Yang, C. Shen, C.J. Pettit, T. Li, E.D. Miller, S.H. Lin, T.M. Williams

Writing, review, and/or revision of the manuscript: L. Yang, C. Shen, C.J. Pettit, E.D. Miller, S.H. Lin, T.M. Williams

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Zhang

This work was supported by the following grants: The Ohio State University Comprehensive Cancer Center (OSU-CCC), NIH (P30 CA016058 and R01 CA198128) and National Center for Advancing Translational Sciences (KL2TR001068). These data were 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. Williams), award number grant KL2TR001068 from the National Center for Advancing Translational Sciences (to T.M. Williams), and NIH grant R01 CA198128 (to T.M. Williams). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Lagergren
J
,
Smyth
E
,
Cunningham
D
,
Lagergren
P
. 
Oesophageal cancer
.
Lancet
2017
;
390
:
2383
96
.
2.
van Rossum
PSN
,
Mohammad
NH
,
Vleggaar
FP
,
van Hillegersberg
R
. 
Treatment for unresectable or metastatic oesophageal cancer: current evidence and trends
.
Nat Rev Gastroenterol Hepatol
2018
;
15
:
235
49
.
3.
Pennathur
A
,
Gibson
MK
,
Jobe
BA
,
Luketich
JD
. 
Oesophageal carcinoma
.
Lancet
2013
;
381
:
400
12
.
4.
Siegel
RL
,
Miller
KD
,
Jemal
A
. 
Cancer statistics, 2018
.
CA Cancer J Clin
2018
;
68
:
7
30
.
5.
Smyth
EC
,
Lagergren
J
,
Fitzgerald
RC
,
Lordick
F
,
Shah
MA
,
Lagergren
P
, et al
Oesophageal cancer
.
Nat Rev Dis Primers
2017
;
3
:
17048
.
6.
Otto
T
,
Sicinski
P
. 
Cell cycle proteins as promising targets in cancer therapy
.
Nat Rev Cancer
2017
;
17
:
93
115
.
7.
Jeggo
PA
,
Pearl
LH
,
Carr
AM
. 
DNA repair, genome stability and cancer: a historical perspective
.
Nat Rev Cancer
2016
;
16
:
35
42
.
8.
Hanahan
D
,
Weinberg
RA
. 
Hallmarks of cancer: the next generation
.
Cell
2011
;
144
:
646
74
.
9.
Ciccia
A
,
Elledge
SJ
. 
The DNA damage response: making it safe to play with knives
.
Mol Cell
2010
;
40
:
179
204
.
10.
Castedo
M
,
Perfettini
JL
,
Roumier
T
,
Andreau
K
,
Medema
R
,
Kroemer
G
. 
Cell death by mitotic catastrophe: a molecular definition
.
Oncogene
2004
;
23
:
2825
37
.
11.
Rundle
S
,
Bradbury
A
,
Drew
Y
,
Curtin
NJ
. 
Targeting the ATR-CHK1 axis in cancer therapy
.
Cancers
2017
;
9
.
doi: 10.3390/cancers9050041
.
12.
Goldstein
M
,
Kastan
MB
. 
The DNA damage response: implications for tumor responses to radiation and chemotherapy
.
Annu Rev Med
2015
;
66
:
129
43
.
13.
Blackford
AN
,
Jackson
SP
. 
ATM, ATR, and DNA-PK: the trinity at the heart of the DNA damage response
.
Mol Cell
2017
;
66
:
801
17
.
14.
Parker
LL
,
Piwnica-Worms
H
. 
Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase
.
Science
1992
;
257
:
1955
7
.
15.
Bartek
J
,
Lukas
J
. 
Chk1 and Chk2 kinases in checkpoint control and cancer
.
Cancer Cell
2003
;
3
:
421
9
.
16.
Masaki
T
,
Shiratori
Y
,
Rengifo
W
,
Igarashi
K
,
Yamagata
M
,
Kurokohchi
K
, et al
Cyclins and cyclin-dependent kinases: comparative study of hepatocellular carcinoma versus cirrhosis
.
Hepatology
2003
;
37
:
534
43
.
17.
Iorns
E
,
Lord
CJ
,
Grigoriadis
A
,
McDonald
S
,
Fenwick
K
,
Mackay
A
, et al
Integrated functional, gene expression and genomic analysis for the identification of cancer targets
.
PLoS One
2009
;
4
:
e5120
.
18.
Mir
SE
,
De Witt Hamer
PC
,
Krawczyk
PM
,
Balaj
L
,
Claes
A
,
Niers
JM
, et al
In silico analysis of kinase expression identifies WEE1 as a gatekeeper against mitotic catastrophe in glioblastoma
.
Cancer Cell
2010
;
18
:
244
57
.
19.
Benada
J
,
Macurek
L
. 
Targeting the checkpoint to kill cancer cells
.
Biomolecules
2015
;
5
:
1912
37
.
20.
Do
K
,
Doroshow
JH
,
Kummar
S
. 
Wee1 kinase as a target for cancer therapy
.
Cell Cycle
2013
;
12
:
3159
64
.
21.
Matheson
CJ
,
Backos
DS
,
Reigan
P
. 
Targeting WEE1 kinase in cancer
.
Trends Pharmacol Sci
2016
;
37
:
872
81
.
22.
Song
Y
,
Li
L
,
Ou
Y
,
Gao
Z
,
Li
E
,
Li
X
, et al
Identification of genomic alterations in oesophageal squamous cell cancer
.
Nature
2014
;
509
:
91
5
.
23.
Dulak
AM
,
Stojanov
P
,
Peng
S
,
Lawrence
MS
,
Fox
C
,
Stewart
C
, et al
Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity
.
Nat Genet
2013
;
45
:
478
86
.
24.
Kim
J
,
Bowlby
R
,
Mungall
AJ
,
Robertson
AG
,
Odze
RD
,
Cherniack
AD
, et al
Integrated genomic characterization of oesophageal carcinoma
.
Nature
2017
;
541
:
169
.
25.
Leijen
S
,
Beijnen
JH
,
Schellens
JH
. 
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
2010
;
5
:
186
91
.
26.
Ku
BM
,
Bae
YH
,
Koh
J
,
Sun
JM
,
Lee
SH
,
Ahn
JS
, et al
Mutational status of TP53 defines the efficacy of Wee1 inhibitor AZD1775 in KRAS-mutant non-small cell lung cancer
.
Oncotarget
2017
;
8
:
67526
37
.
27.
Hirai
H
,
Iwasawa
Y
,
Okada
M
,
Arai
T
,
Nishibata
T
,
Kobayashi
M
, et al
Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensitizes p53-deficient tumor cells to DNA-damaging agents
.
Mol Cancer Ther
2009
;
8
:
2992
3000
.
28.
Bridges
KA
,
Hirai
H
,
Buser
CA
,
Brooks
C
,
Liu
H
,
Buchholz
TA
, et al
MK-1775, a novel Wee1 kinase inhibitor, radiosensitizes p53-defective human tumor cells
.
Clin Cancer Res
2011
;
17
:
5638
48
.
29.
Rajeshkumar
NV
,
De Oliveira
E
,
Ottenhof
N
,
Watters
J
,
Brooks
D
,
Demuth
T
, et al
MK-1775, a potent Wee1 inhibitor, synergizes with gemcitabine to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts
.
Clin Cancer Res
2011
;
17
:
2799
806
.
30.
Cuneo
KC
,
Morgan
MA
,
Davis
MA
,
Parcels
LA
,
Parcels
J
,
Karnak
D
, et al
Wee1 kinase inhibitor AZD1775 radiosensitizes hepatocellular carcinoma regardless of TP53 mutational status through induction of replication stress
.
Int J Radiat Oncol Biol Phys
2016
;
95
:
782
90
.
31.
Parsels
LA
,
Karnak
D
,
Parsels
JD
,
Zhang
Q
,
Velez-Padilla
J
,
Reichert
ZR
, et al
PARP1 trapping and DNA replication stress enhance radiosensitization with combined WEE1 and PARP inhibitors
.
Mol Cancer Res
2018
;
16
:
222
32
.
32.
Pfister
SX
,
Markkanen
E
,
Jiang
Y
,
Sarkar
S
,
Woodcock
M
,
Orlando
G
, et al
Inhibiting WEE1 selectively kills histone H3K36me3-deficient cancers by dNTP starvation
.
Cancer Cell
2015
;
28
:
557
68
.
33.
Kausar
T
,
Schreiber
JS
,
Karnak
D
,
Parsels
LA
,
Parsels
JD
,
Davis
MA
, et al
Sensitization of pancreatic cancers to gemcitabine chemoradiation by WEE1 kinase inhibition depends on homologous recombination repair
.
Neoplasia
2015
;
17
:
757
66
.
34.
Nam
AR
,
Park
JE
,
Bang
JH
,
Jin
MH
,
Bang
YJ
,
Oh
DY
. 
DNA damage response (DDR)-targeting strategy by targeting WEE1 and or ATM/ATR works in biliary tract cancer [abstract]
.
In: Proceedings of the American Association for Cancer Research Annual Meeting 2018
; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 
2018
;
78
(13 Suppl):Abstract nr 323.
35.
Geenen
JJJ
,
Schellens
JHM
. 
Molecular pathways: targeting the protein kinase Wee1 in cancer
.
Clin Cancer Res
2017
;
23
:
4540
4
.
36.
Williams
TM
,
Flecha
AR
,
Keller
P
,
Ram
A
,
Karnak
D
,
Galban
S
, et al
Cotargeting MAPK and PI3K signaling with concurrent radiotherapy as a strategy for the treatment of pancreatic cancer
.
Mol Cancer Ther
2012
;
11
:
1193
202
.
37.
Estrada-Bernal
A
,
Chatterjee
M
,
Haque
SJ
,
Yang
L
,
Morgan
MA
,
Kotian
S
, et al
MEK inhibitor GSK1120212-mediated radiosensitization of pancreatic cancer cells involves inhibition of DNA double-strand break repair pathways
.
Cell Cycle
2015
;
14
:
3713
24
.
38.
Fertil
B
,
Dertinger
H
,
Courdi
A
,
Malaise
EP
. 
Mean inactivation dose: a useful concept for intercomparison of human cell survival curves
.
Radiat Res
1984
;
99
:
73
84
.
39.
Mizuarai
S
,
Yamanaka
K
,
Itadani
H
,
Arai
T
,
Nishibata
T
,
Hirai
H
, et al
Discovery of gene expression-based pharmacodynamic biomarker for a p53 context-specific anti-tumor drug Wee1 inhibitor
.
Mol Cancer
2009
;
8
:
34
.
40.
Dominguez-Kelly
R
,
Martin
Y
,
Koundrioukoff
S
,
Tanenbaum
ME
,
Smits
VAJ
,
Medema
RH
, et al
Wee1 controls genomic stability during replication by regulating the Mus81-Eme1 endonuclease
.
J Cell Biol
2011
;
194
:
567
79
.
41.
Watanabe
N
,
Broome
M
,
Hunter
T
. 
Regulation of the human Wee1hu Cdk tyrosine 15-kinase during the cell-cycle
.
EMBO J
1995
;
14
:
1878
91
.
42.
Beck
H
,
Nahse-Kumpf
V
,
Larsen
MS
,
O'Hanlon
KA
,
Patzke
S
,
Holmberg
C
, et al
Cyclin-dependent kinase suppression by WEE1 kinase protects the genome through control of replication initiation and nucleotide consumption
.
Mol Cell Biol
2012
;
32
:
4226
36
.
43.
Arnold
M
,
Laversanne
M
,
Brown
LM
,
Devesa
SS
,
Bray
F
. 
Predicting the future burden of esophageal cancer by histological subtype: international trends in incidence up to 2030
.
Am J Gastroenterol
2017
;
112
:
1247
55
.
44.
Thrift
AP
. 
The epidemic of oesophageal carcinoma: where are we now?
Cancer Epidemiol
2016
;
41
:
88
95
.
45.
Shapiro
J
,
van Lanschot
JJB
,
Hulshof
M
,
van Hagen
P
,
van Berge Henegouwen
MI
,
Wijnhoven
BPL
, et al
Neoadjuvant chemoradiotherapy plus surgery versus surgery alone for oesophageal or junctional cancer (CROSS): long-term results of a randomised controlled trial
.
Lancet Oncol
2015
;
16
:
1090
8
.
46.
van Hagen
P
,
Hulshof
MC
,
van Lanschot
JJ
,
Steyerberg
EW
,
van Berge Henegouwen
MI
,
Wijnhoven
BP
, et al
Preoperative chemoradiotherapy for esophageal or junctional cancer
.
N Engl J Med
2012
;
366
:
2074
84
.
47.
Alnaji
RM
,
Du
W
,
Gabriel
E
,
Singla
S
,
Attwood
K
,
Nava
H
, et al
Pathologic complete response is an independent predictor of improved survival following neoadjuvant chemoradiation for esophageal adenocarcinoma
.
J Gastrointest Surg
2016
;
20
:
1541
6
.
48.
Donahue
JM
,
Nichols
FC
,
Li
Z
,
Schomas
DA
,
Allen
MS
,
Cassivi
SD
, et al
Complete pathologic response after neoadjuvant chemoradiotherapy for esophageal cancer is associated with enhanced survival
.
Ann Thorac Surg
2009
;
87
:
392
8
.
49.
Yang
H
,
Liu
H
,
Chen
Y
,
Zhu
C
,
Fang
W
,
Yu
Z
, et al
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
.
J Clin Oncol
2018
;
36
:
2796
803
.
50.
Stahl
M
,
Walz
MK
,
Stuschke
M
,
Lehmann
N
,
Meyer
HJ
,
Riera-Knorrenschild
J
, et al
Phase III comparison of preoperative chemotherapy compared with chemoradiotherapy in patients with locally advanced adenocarcinoma of the esophagogastric junction
.
J Clin Oncol
2009
;
27
:
851
6
.
51.
Walsh
TN
,
Noonan
N
,
Hollywood
D
,
Kelly
A
,
Keeling
N
,
Hennessy
TP
. 
A comparison of multimodal therapy and surgery for esophageal adenocarcinoma
.
N Engl J Med
1996
;
335
:
462
7
.
52.
Borggreve
AS
,
Mook
S
,
Verheij
M
,
Mul
VEM
,
Bergman
JJ
,
Bartels-Rutten
A
, et al
Preoperative image-guided identification of response to neoadjuvant chemoradiotherapy in esophageal cancer (PRIDE): a multicenter observational study
.
BMC Cancer
2018
;
18
:
1006
.
53.
Busweiler
LA
,
Wijnhoven
BP
,
van Berge Henegouwen
MI
,
Henneman
D
,
van Grieken
NC
,
Wouters
MW
, et al
Early outcomes from the Dutch Upper Gastrointestinal Cancer Audit
.
Br J Surg
2016
;
103
:
1855
63
.
54.
Djarv
T
,
Lagergren
J
,
Blazeby
JM
,
Lagergren
P
. 
Long-term health-related quality of life following surgery for oesophageal cancer
.
Br J Surg
2008
;
95
:
1121
6
.
55.
Kassis
ES
,
Kosinski
AS
,
Ross
P
 Jr
,
Koppes
KE
,
Donahue
JM
,
Daniel
VC
. 
Predictors of anastomotic leak after esophagectomy: an analysis of the Society of Thoracic Surgeons general thoracic database
.
Ann Thorac Surg
2013
;
96
:
1919
26
.
56.
Mc Cormack
O
,
Zaborowski
A
,
King
S
,
Healy
L
,
Daly
C
,
O'Farrell
N
, et al
New-onset atrial fibrillation post-surgery for esophageal and junctional cancer: incidence, management, and impact on short- and long-term outcomes
.
Ann Surg
2014
;
260
:
772
8
.
57.
Schandl
A
,
Lagergren
J
,
Johar
A
,
Lagergren
P
. 
Health-related quality of life 10 years after oesophageal cancer surgery
.
Eur J Cancer
2016
;
69
:
43
50
.
58.
Forment
JV
,
O'Connor
MJ
. 
Targeting the replication stress response in cancer
.
Pharmacol Therapeut
2018
;
188
:
155
67
.
59.
Macheret
M
,
Halazonetis
TD
. 
DNA replication stress as a hallmark of cancer
.
Annu Rev Pathol
2015
;
10
:
425
48
.
60.
Cuneo
KC
,
Morgan
MA
,
Sahai
V
,
Schipper
MJ
,
Parsels
LA
,
Parsels
JD
, et al
Dose escalation trial of the Wee1 inhibitor adavosertib (AZD1775) in combination with gemcitabine and radiation for patients with locally advanced pancreatic cancer
.
J Clin Oncol
2019
;
37
:
2643
50
.
61.
Do
K
,
Wilsker
D
,
Ji
J
,
Zlott
J
,
Freshwater
T
,
Kinders
RJ
, et al
Phase I study of single-agent AZD1775 (MK-1775), a Wee1 kinase inhibitor, in patients with refractory solid tumors
.
J Clin Oncol
2015
;
33
:
3409
15
.
62.
Leijen
S
,
van Geel
RM
,
Pavlick
AC
,
Tibes
R
,
Rosen
L
,
Razak
AR
, et al
Phase I study evaluating WEE1 inhibitor AZD1775 as monotherapy and in combination with gemcitabine, cisplatin, or carboplatin in patients with advanced solid tumors
.
J Clin Oncol
2016
;
34
:
4371
80
.
63.
Leijen
S
,
van Geel
RM
,
Sonke
GS
,
de Jong
D
,
Rosenberg
EH
,
Marchetti
S
, et al
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
.
J Clin Oncol
2016
;
34
:
4354
61
.
64.
Mendez
E
,
Rodriguez
CP
,
Kao
MC
,
Raju
S
,
Diab
A
,
Harbison
RA
, et al
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
2018
;
24
:
2740
8
.

Supplementary data