MOLECULAR CANCER THERAPEUTICS | CANCER BIOLOGYAND TRANSLATIONAL STUDIES

Synthetic Lethality with Trifluridine/Tipiracil andCheckpoint Kinase 1 Inhibitor for Esophageal SquamousCell Carcinoma A C

Shinya Ohashi1, Osamu Kikuchi1,2, Yukie Nakai1, Tomomi Ida1, Tomoki Saito1, Yuki Kondo1,Yoshihiro Yamamoto1, Yosuke Mitani1, Trang H. Nguyen Vu1, Keita Fukuyama1, Hiroshi Tsukihara3,Norihiko Suzuki3, and Manabu Muto1

ABSTRACT◥

Esophageal squamous cell carcinoma (ESCC) is a diseasecharacterized by a high mutation rate of the TP53 gene, whichplays pivotal roles in the DNA damage response (DDR) and isregulated by checkpoint kinase (CHK) 2. CHK1 is another keyDDR-related protein, and its selective inhibition is suggested tobe particularly sensitive to TP53-mutated cancers, because a lossof both pathways (CHK1 and/or CHK2–p53) is lethal due to theserious impairment of DDR. Such a therapeutic strategy istermed synthetic lethality. Here, we propose a novel therapeuticstrategy based on synthetic lethality combining trifluridine/tipir-acil and prexasertib (CHK1 inhibitor) as a treatment for ESCC.Trifluridine is a key component of the antitumor drug combi-nation with trifluridine/tipiracil (an inhibitor of trifluridinedegradation), also known as TAS-102. In this study, we demon-strate that trifluridine increases CHK1 phosphorylation in ESCC

cells combined with a reduction of the S-phase ratio as well as theinduction of ssDNA damage. Because CHK1 phosphorylation isconsidered to be induced as DDR for trifluridine-mediated DNAdamage, we examined the effects of CHK1 inhibition on triflur-idine treatment. Consequently, CHK1 inhibition by short hairpinRNA or treatment with the CHK1 inhibitor, prexasertib, marked-ly enhanced trifluridine-mediated DNA damage, represented byan increase of gH2AX expression. Moreover, the combination oftrifluridine/tipiracil and CHK1 inhibition significantly sup-pressed tumor growth of ESCC-derived xenograft tumors. Fur-thermore, the combination of trifluridine and prexasertibenhanced radiosensitivity both in vitro and in vivo. Thus, thecombination of trifluridine/tipiracil and a CHK1 inhibitor exhi-bits effective antitumor effects, suggesting a novel therapeuticstrategy for ESCC.

IntroductionEsophageal squamous cell carcinoma (ESCC) is the major his-

tologic type of esophageal cancers (1), which are the seventh leadingcause of cancer-related mortality and the eighth most commoncancer worldwide (2). Despite recent progress in therapeutics, theprognosis of patients with ESCC remains poor (3–5). Specifically,the 5-year survival rates of patients with ESCC (stages IIB–IVB)receiving chemoradiotherapy or chemotherapy are 19.1% and 5.3%,respectively (5). Therefore, the development of novel chemother-apeutic strategies is required to improve the outcomes of patientswith ESCC.

Recently, a novel therapeutic strategy targeting the DNA damageresponse (DDR) was reported (6, 7). DDR is a critical mechanismthat maintains genome stability (8), and it is coordinated by twodistinct kinase signaling cascades: ataxia telangiectasia Rad3-related(ATR)-checkpoint kinase 1 (CHK1; ATR–CHK1) and ataxia tel-

angiectasia mutated (ATM)-checkpoint kinase 2 (CHK2; ATM–CHK2) pathways (9, 10). These pathways regulate specific cell-cyclecheckpoints: the ATR–CHK1 pathway regulates the S-phase andG2-phase checkpoint (7, 11), whereas the ATM–CHK2 pathwayregulates the G1-phase checkpoint (7).

The tumor suppressor protein p53 (encoded by the TP53 gene) isregulated by the ATM–CHK2 pathway (12), and, therefore, p53plays a pivotal role in DDR (13). TP53 is frequently mutated incancers (14) and is defective in >50% of human tumors (13).Accordingly, ATM-CHK2-p53–mediated DDR during the G1-phase checkpoint is compromised in many malignant p53-defectivetumor cells (15) and, alternatively, those tumor cells are dependenton the ATR-CHK1–mediated S and G2–M-phase checkpoints forDNA damage repair (15). Thus, ATR-CHK1–mediated DDR issuggested to play an important role in the survival of p53-defectivecancer cells.

The selective inhibition of CHK1 has been predicted to be partic-ularly sensitive in those p53-defective cells because the loss of bothpathways (CHK1 and/or CHK2–p53 pathway) is lethal (13, 16).Such a therapeutic strategy has been termed synthetic lethali-ty (13, 16, 17). Indeed, treatment for p53-defective tumors withCHK1 inhibitors is potentiated by combining it with other anti-cancer drugs that induce DNA damage (17). Because the TP53mutation frequency in ESCC is as high as approximately 79.8%–93% (18–22), we consider that ESCC treatment with CHK1 inhi-bitors may effectively potentiate the antitumor effect by promotingsynthetic lethality.

TAS-102 (C10H11F3N2O5�C9H11ClN4O2�HCl: CAS Number:733030-01-8) is an orally administered combination drug of a tri-fluridine (C10H11F3N2O5) as a thymidine-based nucleic acid analog,and a tipiracil hydrochloride (C9H11ClN4O2�HCl; ref. 23) as a

1Department of Therapeutic Oncology, Graduate School of Medicine, KyotoUniversity, Sakyo-ku, Kyoto, Japan. 2Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts. 3Translational Research Labo-ratory, Taiho Pharmaceutical Co., Ltd., Kawauchi-cho, Tokushima, Japan.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Shinya Ohashi, Kyoto University, 54, Kawahara-cho,Shogoin, Sakyo-ku, Kyoto 6068507, Japan. Phone:þ81-75-751-4770; Fax:þ81-75-751-3519; E-mail: ohashish@kuhp.kyoto-u.ac.jp

Mol Cancer Ther 2020;19:1–10

doi: 10.1158/1535-7163.MCT-19-0918

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thymidine phosphorylate inhibitor (TPI). TPI prevents the degrada-tion of trifluridine, allowing the maintenance of adequate plasmatrifluridine levels (24). Trifluridine is the active cytotoxic compo-nent of TAS-102 (trifluridine/TPI); its triphosphorylated form isincorporated into the DNA, leading to antitumor effects (25).Trifluridine/TPI is approved for metastatic colorectal cancers pre-viously treated with chemotherapy such as fluoropyrimidine, oxa-liplatin, and irinotecan (26). It has also been applied for metastaticgastric cancers that have progressed after treatment with otherchemotherapy regimens (27).

In this study, we propose a novel therapy combining trifluridine/TPI and a CHK1 inhibitor for ESCC treatment. We found thattrifluridine markedly increased CHK1 phosphorylation in ESCC cells.CHK1 phosphorylation is considered to be induced by trifluridine-mediated DNA damage, and the combination of trifluridine andCHK1 inhibition resulted in the induction of severe DNA damage aswell as antitumor effects, indicating the efficient induction of syntheticlethality. Furthermore, we show that the combination therapy oftrifluridine and a CHK1 inhibitor enhances the radiosensitivity ofESCC cells.

Materials and MethodsCells and reagents

We used human ESCC cell lines TE-1, TE-10, and TE-11, whichwere obtained from the Riken BioResource Center. TE-11R cells are5-fluorouracil–resistant ESCC cells established with TE-11 cells, asdescribed previously (28). We used TE-11R cells in this study becausethey generate tumors in BALB/cAJcl-nu/nu mice, while other cells(TE-1, TE-10, and TE-11) did not result in xenograft tumors in ourprevious study (28). These cells were cultured in RPMI1640 medium(Life Technologies Corp.), supplemented with 10% FBS (Life Tech-nologies Corp.), 100 mg/mL of streptomycin, and 100 units/mL ofpenicillin (Life Technologies Corp.). All cells were cultured at 37�C in a5% CO2 incubator.

Chemical compoundsPrexasertib (C18H19N7O2: CAS Number: 1234015-52-1) was

purchased as the CHK1 inhibitor from Selleck Chemicals. Triflur-idine was purchased from Tokyo Chemical Industry, Co., Ltd.Trifluridine/TPI (TAS-102) was provided by Taiho PharmaceuticalCo., Ltd.

EdU flow cytometry assayA Click-iT EdU Flow Cytometry Assay Kit (Thermo Fisher Scien-

tific, Corp.) was used to assess the effects of trifluridine on the cell cycle.After the cells were cultured in the presence or absence of trifluridine,they were incubated with Click-iT EdU, harvested, and treated accord-ing to the manufacturer's instructions. Cells were analyzed by flowcytometry (LSRFortessa Flow Cytometer; BD Biosciences), and thedata were analyzed using BD FACSDiva Software (BD Biosciences).The percentages of cells in the S-phase were determined; cells in aproliferating population (S-phase) show high fluorescence intensity,whereas cells in nonproliferating populations show low fluorescenceintensity.

Cell viability assayCell viability was determined using the WST-1 Assay (Roche

Applied Science) according to the manufacturer's instructions. Cells(1–5� 103 cells/well) were seeded in 96-well plates and exposed to the

indicated concentrations of trifluridine with or without prexasertib(10 nmol/L) for 72 hours. All data were obtainedwith aMultiwell PlateReader (Infinite 200 Pro, Tecan Group Ltd.) at wavelengths of 450 and630 nm.

ImmunofluorescenceTrifluridine-mediated ssDNA damage was detected using

immunofluorescence. Cell pellets were fixed in methanol, andheated in formamide, according to the manufacturer's instructions(Enzo Life Sciences, Inc.). After the heating, they were blockedwith 1% nonfat dry milk in PBS for 15 minutes at room temper-ature. Then, they were incubated with mouse anti-single-strandedDNA mAb (F7-26; 10 mg/mL: Enzo Life Sciences, Inc.) for 15minutes at room temperature followed by a fluorescein-conjugatedgoat anti-mouse IgM (Sigma, catalog no. F-9259) for 15 minutes atroom temperature. Nuclei were counterstained by 40,6-diamidino-2-phenylindole using VECTASHIELD Mounting Medium (VectorLaboratories, Inc.). Stained objects were examined with an EVOSFL Cell Imaging System (Thermo Fisher Scientific, Corp.).

RNA isolation, cDNA synthesis, and real-time reversetranscriptase-PCR

RNeasy Midi Kit (Qiagen, Inc.) was used to isolate total RNA fromcultured cells. RNA was treated with DNase I (Invitrogen). cDNAsynthesis was carried out with the SuperScript First-Strand SynthesisSystem (Invitrogen) using 3.3 mg of total RNA as a template. ThecDNA synthesis reactions without reverse transcriptase yielded noamplicons in the PCRs. Real-time reverse transcriptase-PCR (RT-PCR) was performed with the LightCycler 480 Instrument II Real-Time PCR System (Roche Diagnostics Ltd.). The relative expression ofeach mRNA was normalized to b-Actin as an internal control. Theprimers used in this study were as follows: CHK1: forward 50-CGGT-ATAATAATCGTGAGCG-30 ; reverse 50-TTCCAAGGGTTGAGG-TATGT-30; and ACTB: forward 50-TTGTTACAGGAAGTCCCT-TGCC-30; reverse: 50-ATGCTATCACCTCCCCTGTGTG-30.

Western blottingWhole-cell lysates were prepared as follows. Cells were washed

twice with ice-cold PBS and lysed with a RIPA Buffer (NacalaiTesque). After 30 minutes on ice, the cell lysates were centrifuged at14,000 rpm at 4�C for 30 minutes. Protein concentrations weredetermined by BCA Protein Assay (Pierce Biotechnology). Protein(10–15 mg) was heat-denatured in a sample buffer solution withreducing reagent (6�) for SDS-PAGE (Nacalai Tesque) at 70�C for10 minutes. The protein samples were separated on Mini-PROTEAN TGX Gels (Bio-Rad Laboratories, Inc.) and transferredto a polyvinylidene difluoride membrane (Trans-Blot Turbo Trans-fer Pack, Bio-Rad Laboratories, Inc.). The membrane was blocked in5% nonfat milk and 1% BSA, pH 5.2, Fraction V (Wako PureChemical Industries) in TBS-T (10 mmol/L Tris, 150 mmol/L NaCl,pH 8.0, and 0.1% Tween 20) for 1 hour at room temperature.Membranes were probed with the primary antibody diluted in 5%nonfat milk and 1% BSA in TBS-T overnight at 4�C, washed threetimes in TBS-T, incubated with secondary antibody in 5% nonfatmilk and 1% BSA in TBS-T for 1 hour at room temperature, and,finally, washed three times in TBS-T. The signal was visualized withan enhanced chemiluminescence solution (SuperSignal West FemtoMaximum Sensitivity Substrate, Thermo Fisher Scientific or Immo-bilon Western Chemiluminescent HRP Substrate, Merck Millipore)and exposed to a ChemiDoc Touch Imaging System (Bio-Rad

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Laboratories, Inc.). Densitometric analyses of Western blot bandswere performed using Image Lab Software (Bio-Rad Laboratories,Inc.). Data were calibrated with b-Actin as a loading control inarbitrary units.

Primary antibodies and the titers used in this study were asfollows: mouse monoclonal anti-CHK1 antibody (2G1D5, #2360,Cell Signaling Technology, Inc.; 1:1,000), rabbit monoclonal anti-phospho-CHK1 (Ser 345) antibody (133D3, #2348, Cell SignalingTechnology, Inc.; 1:1,000), rabbit polyclonal anti-phospho-CHK1(Ser 296) antibody (#2349, Cell Signaling Technology, Inc.; 1:1,000),rabbit monoclonal anti-phospho-Histone H2A.X (Ser 139) anti-body (20E3, #9718, Cell Signaling Technology, Inc.; 1:1,000),and rabbit monoclonal anti-b-Actin antibody (HRP conjugate;13E5, #5125, Cell Signaling Technology, Inc.; 1:3,000). The sec-ondary antibodies and titers were anti-rabbit IgG, HRP-linkedwhole Ab donkey (NA-934, GE Healthcare; 1:2,000) and anti-mouse IgG, HRP-linked whole Ab sheep (NA-931, GE Healthcare;1:2,000).

Short hairpin RNAThe following lentiviral vectors were used in this study: GIPZ

nonsilencing Lentiviral shRNA control (control for CHK1 knock-down), V3LHS_637957 (shCHK-1), and V3LHS_637959 (shCHK1-2). They were transfected into HEK293T cells to produce viralparticles using the Trans-Lentiviral shRNA Packaging Kit (GEHealthcare Dharmacon, Inc.). TE-11R cells were incubated withRPMI1640 medium containing these viral particles and 10 mg/mLhexamethrine bromide, and then centrifuged at 1,800 rpm, at 32�Cfor 1 hour. After centrifugation, the medium was replaced withfresh RPMI1640 medium containing viral particles and 10 mg/mLhexamethrine bromide, and centrifuged, again, using the sameconditions. After centrifugation, the medium was changed tonormal RPMI1640. Puromycin (1.5 mg/mL) was used for cellselection.

In vivo experimentsESCC cell–derived xenograft tumors and/or ESCC patient–

derived xenograft tumors were utilized to assess the therapeuticeffects of trifluridine/TPI and/or prexasertib in vivo. Moreover, wealso examined the synergistic therapeutic effects of trifluridine/TPIand prexasertib with radiotherapy. All animal experiments con-formed to the relevant regulatory standards and were approved bythe Institutional Animal Care and Use Committee of Kyoto Uni-versity (Kyoto, Japan, Med Kyo 18284, 18285) and the EthicsCommittee of Kyoto University (Kyoto, Japan, G0770-2) or Insti-tutional Animal Care and Use Committee of Taiho PharmaceuticalCo. Ltd (18PB02, 18PB13).

To establish that xenograft tumors derived from ESCC cells, TE-11R cells (5 � 106 cells) were suspended in 50% Matrigel (BDBiosciences), followed by subcutaneous implantation in the leftflank of 6-week-old BALB/cAJcl-nu/nu mice (CLEA Japan, Inc.). Toestablish patient-derived xenograft (PDX) tumors, biopsy speci-mens taken from human ESCC tissues were placed in a subcuta-neous pocket created by a 5-mm incision in the left flank of 6-week-old hairless SCID male (Crlj: SHO-PrkdcscidHrhr) mice (CharlesRiver Laboratories Japan, Inc.), which was then closed by suturing.Those xenografted tumors were used for the following experimentswhen they reached a volume of about 150–300 mm3. The mice wererandomly assigned to groups (n ¼ 5 or 6, each) and they weretreated with trifluridine/TPI (200 mg/kg/day, orally) and/or pre-

xasertib (20 mg/kg/day, subcutaneously). 0.5% hydroxypropylmethylcellulose (10 mL/kg) and/or 20% captisol (CyDex Pharma-ceuticals, Inc.) was used as a control for trifluridine/TPI andprexasertib, respectively.

Radiation treatment [RT, 4 grays (Gy)] was conducted in asingle fraction to tumors, as follows. Mice were positioned in amodified 50 mL conical plastic tube to allow irradiation of thetumor area while keeping the rest of the body outside the RT fieldusing a collimator. The tumors were locally irradiated with 4 Gy of137Cs g-rays using a Gammacell 40 Exactor (MDS NordionInternational).

Tumor growthwas evaluated every 2 or 3 days until 10–14 days afterfinal treatment. The tumor diameters were measured with a caliper,and the tumor volume (mm3) was calculated using the followingformula: (length) � (width)2 � 0.5.

Histologic and IHC stainingTissue samples were fixed in 4% paraformaldehyde phosphate

buffer solution (Wako Pure Chemical Industries, Ltd.) overnightat 4�C, embedded in paraffin, and cut into 4-mm sections forstandard hematoxylin and eosin (H&E) staining and IHC. Incu-bation and washing procedures were carried out at room tem-perature. Deparaffinization and antigen retrieval by incubation inprotease solution (Nichirei Biosciences) were carried out for5 minutes. The glass slides were washed in PBS (2 times, 2 minuteseach) and mounted with 3% BSA in PBS for 30 minutes.The primary antibody, a rabbit monoclonal anti-g-H2AX (20E3,#9718, Cell Signaling Technology, Inc.) at 1:100 dilution wassubsequently applied for 60 minutes and followed by PBS washes(3 times, 5 minutes each). Slides were incubated with a secondaryantibody solution in a Histofine Simple Stain MAX PO (R)Kit (Nichirei Biosciences) for 30 minutes and followed by PBSwashes (3 times, 5 minutes each). A coloring reaction was carriedout with diaminobenzidine, and nuclei were counterstained withhematoxylin.

Immunostained tissues were assessed using a BIOREVO BZ-9000Microscope (Keyence), for gH2AX staining. Positive cells were scoredby counting at least 300 cells per high-power field under lightmicroscopy.

Clonogenic survival assayTE-11 cells were plated at 0.1–1.5 � 103 cells per well in 6-well

plates and incubated for 24 hours. Trifluridine (4 mmol/L) and/orprexasertib (3 nmol/L) was added to the plates and incubated for 10minutes, and then the plates were irradiated with 8, 6, 4, 2, or 0 Gy.The plates including medium with trifluridine and/or prexasertibwere incubated for 24 days, and then the medium was changed tothe normal medium, and the plates were incubated for 9 days. Cellswere fixed with 20% glutaraldehyde, stained with 0.05% crystalviolet, and counted. Plating efficiency (PE: number of coloniesformed/number of cells seeded) was determined and used tocalculate toxicity. Assays were performed in triplicate, and inde-pendent experiments were carried out three times. Radiation dosemodification factors (DMF) were calculated by taking the ratios ofradiation doses at the 10% survival level (90% toxic irradiationdose; DMF ¼ 90% toxic control irradiation dose/each 90% toxicirradiation dose in the trifluridine and/or prexasertib treatmentgroups; ref. 29). The 90% toxic irradiation dose was calculated usingXLfit software. DMF values greater than 1.0 indicate enhancedradiosensitivity (29).

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Statistical analysisData are presented as the means � SD of triplicate experiments,

unless otherwise stated. The two-tailed Student t test was selected fordata analysis of two groups. The interaction between trifluridine/TPIand prexasertib treatments for TE-11R–derived xenograft tumors aswell as ESCC PDX tumors, or trifluridine/tipiracil þ prexasertibtreatment and RT for TE-11R–derived tumors was assessed usingtwo-way ANOVA. When significant interactions were noted, analysisofmore than two groupswas conductedwithTukeyHonest SignificantDifference test. A P < 0.05 was considered significant. All statisticalanalyses were performed using SPSS 21 for Windows (SPSS Inc., IBMCorp.).

ResultsEffects of trifluridine on cell proliferation and CHK1phosphorylation (Serine 317 and Serine 345) in ESCC cells

To examine the effects of trifluridine on ESCC cells, we assessedthe proliferation of ESCC cells after treatment with trifluridine.When we treated ESCC cells (TE-1, TE-10, and TE-11) withtrifluridine, the ratio of the S-phase was sharply reduced(Fig. 1A). Moreover, ssDNA damage was induced by treatmentwith trifluridine (Fig. 1B). Next, we examined whether trifluridineinfluenced the phosphorylation of CHK1 because the cell cycle inthe S-phase is regulated by CHK1 (11) and induced by ssDNAdamage (30, 31). As shown in Fig. 1C, trifluridine increased CHK1

Figure 1.

Effects of trifluridine (FTD) on ESCC cell proliferation, ssDNAdamage induction, and CHK1 phosphorylation. A, Cell cyclesof TE-1, TE-10, and TE-11 cells treated with or without triflur-idine for 24 hours, analyzed by EdU assay. The experimentswere conducted in triplicate, and representative data areshown. B, ssDNA induction in TE-11 cells treated with triflur-idine (100 mmol/L) for 24 hours, analyzed by immunofluo-rescence. Note the presence of ssDNA-positive cells (arrow)in trifluridine-treated cells. The images of untreated or staur-osporine-treated cells are presented as a negative or positivecontrol, respectively. Scale bar, 20 mm. C, Phosphorylated(Serine 317 and Serine 345) and total CHK1 protein levels inESCC cells (TE-1, TE-10, and TE-11) treated with the indicatedconcentrations of trifluridine for 30 minutes, determined byWestern blotting, are shown. b-Actin served as a loadingcontrol.

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phosphorylation (Serine 317 and Serine 345) dose dependently inall ESCC cells.

Effects of CHK1 inhibitors on trifluridine-mediated cytotoxicityand DNA damage in ESCC cells

Next, we examined the effects of the CHK1 inhibitor on trifluridine-mediated cytotoxicity and DNA damage. The inhibitory effect onCHK1 was evaluated by expression levels of CHK1 phosphorylation(Serine 296) because CHK1 is autophosphorylated at S296 followingphosphorylation of S317 and S345 (32), and S296 phosphorylation isinvolved in subsequent DDR (33). As shown, prexasertib, a CHK1inhibitor, successfully suppressed CHK1 phosphorylation (S296)in a dose-dependent manner (Fig. 2A). Next, we examined thecytotoxic effects of trifluridine and/or prexasertib on ESCC cells

(Fig. 2B and C). As shown in Fig. 2B, prexasertib enhancedtrifluridine-mediated cytotoxicity, and the IC50 value for trifluridinein ESCC cells was markedly reduced. In addition, combinationtreatment with trifluridine and prexasertib strongly inducedgH2AX compared with the treatment with trifluridine or prexa-sertib alone (Fig. 2D).

Effects of CHK1 knockdown on trifluridine-mediated DNAdamage and/or tumor growth

We then examined the effects of CHK1 knockdown on trifluridine-mediated DNA damage and/or tumor growth. We created CHK1-knockdown ESCC cells (TE-11R cells) using two types of short hairpinRNAs (shRNA). As shown in Fig. 3A and B, CHK1 expression levelswere reduced by both shRNAs at mRNA as well as protein levels.

Figure 2.

Effects of prexasertib on cytotoxicity and DNA damage intrifluridine (FTD)-treatedESCC cells.A,Phosphorylated (Ser-ine 296) and total CHK1 protein levels in TE-11R cells treatedwith the indicated concentrations of prexasertib for 30 min-utes, determined usingWestern blotting. b-Actin served as aloading control. B, Enhancement of trifluridine-mediatedcytotoxicity by coculturewith prexasertib. Cell growth curvesand IC50 values of trifluridine in ESCC cells (TE-1, TE-10, andTE-11) treated with trifluridine alone or the combination oftrifluridine and prexasertib (10 nmol/L) for 72 hours areshown. C, Cell survival curve in TE-1, TE-10, and TE-11 cellstreated with prexasertib alone for 72 hours. IC50 values ofprexasertib in each cell are shown. D, DNA damage, repre-sented by gH2AX expression levels in ESCC cells (TE-1, TE-10,and TE-11) treated with trifluridine in the presence or absenceof prexasertib (TE-1, 10 nmol/L; TE-10, 1 nmol/L; and TE-11,10 nmol/L) for 24 or 48 hours (TE-1, 48 hours; TE-10 and TE-11,24 hours) are shown. b-Actin served as a loading control.

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Induction of trifluridine-mediated DNA damage was increased byCHK1 knockdown (Fig. 3C). When xenograft tumors derived fromCHK1-knockdown or control TE-11R cells were treated with triflur-idine/TPI, the tumor growth inhibitory effect of trifluridine/TPItreatment in CHK1-knockdown cells was significantly stronger thanin control cells (mean tumor growth inhibitory rate due to trifluridine/TPI treatment in each group: scramble control: 46% � 0%, shCHK1#1 group: 16% � 12%, and shCHK1 #2 group: 12% � 4%; Fig. 3Dand E).

Antitumor effects of trifluridine/TPI and CHK1 inhibitors onTE-11R–derived xenograft and/or ESCC PDX tumors

Furthermore, we examined whether the combination therapy oftrifluridine/TPI and aCHK1 inhibitor exhibits actual antitumor effectsin ESCC cell (TE-11R)-derived xenograft and/or ESCC PDX tumors.As shown in Fig. 4A, the tumor growth rates (on day 28) in the groupsreceiving trifluridine/TPI monotherapy, prexasertib monotherapy,and/or combination therapy with trifluridine/TPI and prexasertibcompared with the control group were 60.3, 67.7, and 25.9%,

Figure 3.

Antitumor effects of CHK1 knockdown on trifluridine (FTD)-treated ESCC cells. A, CHK1 mRNA levels in CHK1-knockdownTE-11R cells: RT-PCR showed efficient CHK1 knockdown usingshRNA in TE-11R cells. Relative CHK1 mRNA levels of each celltype relative to that of control cells are indicated. b-Actinserved as an internal control (�� ,P<0.01, vs. scramble control;n¼ 3).B,CHK1 protein levels in CHK1-knockdownTE-11R cells:Western blotting showed efficient CHK1 knockdown usingshRNA in TE-11R cells. b-Actin served as a loading control.C, Increased expression level of gH2AX in CHK1-knockdownTE-11R cells treated with trifluridine for 24 hours. b-Actinserved as a loading control. D, Enhancement of trifluridine-mediated antitumor effects by CHK1 inhibition in TE-11R cells.TE-11R cells with (shCHK1#1 or shCHK1#2) or without CHK1knockdown (scramble) were injected into BALB/c Jcl-nu/numice, and xenograft tumors were treated with trifluridine/tipiracil (200mg/kg, orally) for 3weeks (on day 1–5, 8–12, and15–19). Tumor growth was evaluated every 2 to 3 days untilday 29. Time courses of tumor volumes in each group areshown, n ¼ 5. E, Tumor growth inhibition rate in trifluridine/TPI-treated xenograft tumors derived from TE-11R cells withor without CHK1 knockdown. The tumor growth inhibitionrate is calculated by comparing the tumor volume of eachgroup treatedwith trifluridine/TPI on day 29with the averagetumor volume of each untreated group (�� , P < 0.01; n ¼ 5).

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respectively. Two-way ANOVA analysis revealed that trifluridine/TPItreatment or prexasertib treatment significantly suppressed tumorgrowth of TE-11R–derived xenograft tumors, compared with controlgroups for TE-11R–derived xenograft tumors (P < 0.05, trifluridine/TPI treatment vs. control; P < 0.05, prexasertib treatment vs. control),without significant interaction between the trifluridine/TPI treatmentand the prexasertib treatment (P¼ 0.9027). Here, we assessed gH2AXexpression in tumors treated with trifluridine/TPI and/or prexasertib.Consequently, the combination treatment of trifluridine/TPI andprexasertib showed a significant induction of gH2AX in the xenograft

tumors compared with the trifluridine/TPI monotherapy, prexasertibmonotherapy, or vehicle control (Fig. 4B and C).

In ESCC PDX tumors, the tumor growth rates (on day 22) in themonotherapy group with trifluridine/TPI or prexasertib and/orcombination therapy with trifluridine/TPI and prexasertib groupcompared with the control group were 41� 10, 37� 12, and 10� 5,respectively. Two-way ANOVA analysis revealed that there was asignificant positive interaction between trifluridine/TPI treatmentand/or prexasertib treatment for ESCC PDX tumors (P ¼0.000156), so a multiple comparison of all groups was conducted.As a result, we found that the tumor growth rate in each treatmentgroup was significantly suppressed compared with the controlgroup (P < 0.01, trifluridine/tipiracil alone vs. control; P < 0.01,prexasertib alone vs. control; and P < 0.01, trifluridine/TPI andprexasertib vs. control). Moreover, tumor growth in the groupreceiving combination therapy with trifluridine/TPI and prexasertibwas also significantly suppressed compared with the group receivingmonotherapy with trifluridine/TPI or prexasertib (P < 0.01, tri-fluridine/TPI and prexasertib vs. trifluridine/TPI alone or prexa-sertib alone; Fig. 5A).

No mice died and apparent ill effects were not observed in theseexperiments. No significant weight loss was observed in groups on day29 of the experiment using TE-11R–derived xenograft tumors (Sup-plementary Fig. S1) or day 22 of the experiment using ESCC PDXtumors (Fig. 5B).

Figure 4.

Antitumor effects of trifluridine (FTD)/TPI and/or prexasertib on ESCC cell–derived xenograft tumors. A, Time course of tumor volumes in TE-11R–derivedxenograft tumors treated with trifluridine/TPI and/or prexasertib. TE-11R cell–derived xenograft tumors were treated with trifluridine/TPI (200mg/kg, orally)and/or prexasertib (20 mg/kg, subcutaneously) for 3 weeks (on days 1–5, 8–12,and 15–19 for trifluridine/TPI, on days 1–3, 8–10, and 15–17 for prexasertib). Tumorgrowth was evaluated every 2–3 days until day 29. n ¼ 6. B, H&E images ofxenografted tumor tissues in each treatment group on day 29, and IHC stainingimages for gH2AX. Scale bar, 50 mm. C, A gH2AX positively stained nuclei rateobserved in six random fields (�� , P < 0.01 between the indicated groups).

Figure 5.

Effects of treatment with trifluridine (FTD)/TPI and/or prexasertib on ESCC PDXtumors. A, The time course of tumor volumes in ESCC PDX tumors treated withtrifluridine/TPI and/or prexasertib is shown. ESCCPDX tumorswere treatedwithtrifluridine/TPI (200 mg/kg, orally) and/or prexasertib (20 mg/kg, subcutane-ously) for 2 weeks (on days 1–5 and 8–12 for trifluridine/TPI and on days 1–3 and8–10 for prexasertib). Tumor growth was evaluated every 2–3 days until day 22.�� , P < 0.01 versus vehicle control; #, P < 0.01, trifluridine/TPI þ prexasertibversus trifluridine/TPI alone; $, P < 0.01, trifluridine/TPI þ prexasertib versusprexasertib alone; n ¼ 7 in each group. B, The time course of body weightchanges in mice treated with trifluridine/TPI and/or prexasertib is shown. n¼ 7in each group. Animalweights onday22were not significantly different betweenthe treatment groups.

Synthetic Lethality for Esophageal Squamous Cell Carcinoma

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Radiosensitizing effects of trifluridine and CHK1 inhibitor inin vitro and in vivo experimental models

To assess the radiosensitizing effect of trifluridine and/or prexa-sertib in vitro, we conducted a clonogenic assay. The average PE in eachtreatment group with or without irradiation (0, 2, 4, 6, and 8 Gy) isshown in Fig. 6A. To determine DMF, we calculated the irradiationdoses required to suppress the colony formation rate to 90% (90% toxicirradiation dose). The irradiation doses required to suppress the colonyformation rate to 90%were 9.67Gy in the control group, 6.74 Gy in thetrifluridine group, 9.04 Gy in the prexasertib group, and 3.92 Gy in the

trifluridine þ prexasertib group. Accordingly, DMFs in the mono-therapy group with trifluridine or prexasertib and/or the combinationtherapy of trifluridine and prexasertib were 1.4, 1.1, and 2.5, respec-tively. Because DMF values greater than 1.0 indicate an enhancementof radiosensitivity, a radiosensitizing effect of trifluridine/TPI orprexasertib alone or in combination was observed in ESCC cells(Fig. 6A).

We also examined the antitumor effects of the combination oftrifluridine/TPI and prexasertib in the presence or absence of RT onTE-11R–derived xenograft tumors. As shown in Fig. 6B, the tumorgrowth rates (on day 22) in the groups receiving chemotherapyalone (trifluridine/TPI and prexasertib), RT alone, and/or combi-nation therapy of trifluridine/TPI, prexasertib, and RT comparedwith the control group were 51% � 7.5%, 47% � 10%, and 19% �11%, respectively. Two-way ANOVA analysis revealed a significantpositive interaction between RT and/or trifluridine/TPI þ prexa-sertib treatment (P ¼ 0.0182), so that we conducted a multiplecomparison of all groups. The tumor growth rate in each treatmentgroup was significantly suppressed compared with the controlgroup [P < 0.01, chemotherapy (trifluridine/TPI and prexasertib)alone vs. control; P < 0.01, RT alone vs. control; and P < 0.01,trifluridine/TPI, prexasertib, and RT vs. control]. Moreover, tumorgrowth in the group receiving combination chemoradiotherapy oftrifluridine/TPI, prexasertib, and RT was also significantly sup-pressed compared with that in the chemotherapy alone group[trifluridine/TPI and prexasertib, P < 0.01, trifluridine/TPI, pre-xasertib, and RT vs. chemotherapy (trifluridine/TPI and prexaser-tib) alone] or RT alone (P < 0.01, trifluridine/TPI, prexasertib, andRT vs. RT alone; Fig. 6B). No mice died and apparent ill effects werenot observed in this experiment, and no significant body weight losswas observed on day 22 in groups (Fig. 6C).

DiscussionIn this study, we showed that trifluridine markedly increased

CHK1 phosphorylation (S317 and S345) in ESCC cells. BecauseCHK1 is phosphorylated at S317 and S345 by ATR (34, 35), which isactivated by DNA damage (i.e., ssDNA break; refs. 36, 37), CHK1phosphorylation is suggested to be caused by trifluridine-mediatedDNA damage. Consequently, CHK1 phosphorylation leads to theactivation of CHK1 signaling through autophosphorylation ofCHK1 at Serine 296 (32) as well as subsequent induction ofDDR (33, 38). Our data suggest that trifluridine treatment reducesthe relative number of cells in the S-phase via DDR inductionthrough CHK1 signal activation.

This is the first reported preclinical study that CHK1 inhibitionenhances trifluridine/TPI-mediated antitumor effects in ESCCcells. Because CHK1 is considered to be activated to repair triflur-idine-induced DNA damage, we examined the effects of CHK1inhibition on trifluridine-mediated DNA damage and/or its anti-tumor effects. As a result, the combination of trifluridine and CHK1inhibition via CHK1 knockdown or a CHK1 inhibitor, prexasertib,resulted in potent cytotoxic effects as well as antitumor effects withmarked DNA damage. Our data suggest that CHK1 inhibitionsuppresses the repair of trifluridine-induced DNA damage and thatthe accumulation of DNA damage has antitumor effects. Therefore,we believe that increasing trifluridine-mediated cell death by sup-pressing physiologic DDR activation via CHK1 inhibition isreasonable.

In this study, trifluridine was considered to work as a trigger toinduce DNA damage. Consequently, trifluridine-mediated DNA

Figure 6.

Radiosensitizing effect of trifluridine (FTD) and prexasertib in ESCC cells.A, Radiosensitizing effect of trifluridine and prexasertib in ESCC cells in vitro.The average PE in each treatment group with or without irradiation (0, 2, 4, 6,and 8 Gy) was evaluated. DMFs (the control radiation dose at 10% survivaldivided by the drug-treated radiation dose at 10% survival) are shown.B, Radiosensitizing effect of trifluridine/TPI and prexasertib in ESCC cellsin vivo. The time course of tumor volumes in TE-11R–derived xenograftedtumors treated with trifluridine/TPI and prexasertib in the presence orabsence of radiation therapy is shown. TE-11R cell–derived xenograft tumorswere treated with trifluridine/TPI (200 mg/kg, orally) and/or prexasertib(10 mg/kg, subcutaneously) for 2 weeks (on days 1, 3, 5, 8, 10, and 12 fortrifluridine/TPI, and on days 1, 3, 5, 8, 10, and 12 for prexasertib). Thetumors were irradiated (4 Gy in a single fraction) on day 4. Tumor growthwas evaluated every 2–3 days until day 22. �� , P < 0.01 versus vehicle control;#, P < 0.01, trifluridine/TPI þ prexasertib þ radiation versus trifluridine/TPI þprexasertib; $, P < 0.01, trifluridine/TPI þ prexasertib þ radiation versusradiation alone; n ¼ 5 in each group. C, Body weight changes in eachtreatment group. n ¼ 5 in each group. Animal weights on day 22 were notsignificantly different between the treatment groups.

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damage is not successfully repaired when TP53-mutated tumor cellsare treated with trifluridine and CHK1 inhibitors. Thus, syntheticlethality is caused by the impairment of DDR due to the inhibition ofboth ATR–CHK1 and the ATM–CHK2–p53 pathway (17). In ESCC,the TP53 mutation frequency is as high as approximately 79.8%–93% (18–22, 39), whereas otherDDR-related genes such asATR,ATM,CHEK1, and CHEK2 are not frequently mutated (refs. 18, 39–41;Supplementary Fig. S2A and S2B). Therefore, inhibition of theseDDR-related proteins is considered to be reasonable in most cases of ESCCwith TP53 mutations.

In this study, we examined the TP53 mutation status in ESCCcells (TE-1, TE-10, and TE-11) by TP53 target sequence, andconfirmed TP53 mutation presence in all cells as follows: TE-1:V272M, TE-10: C242Y, and TE-11: R110L. Because C242Y andR110L show a loss of p53 function (42, 43), TE-10, TE-11, andTE-11R cells are p53-defective cells. TP53 mutations (V272M) inTE-1 cells were described as variants of uncertain significance in theNCBI ClinVar variation report (44); however, a recent report oncomprehensive libraries of TP53 mutants generated by mutagenesisusing Integrated TilEs (MITE) showed that TP53V272M mutationcaused resistance to the MDM inhibitor, Nutlin-3, and suscepti-bility to etoposide, which indicates loss of function (45). Thus,combination therapy with trifluridine and CHK1 inhibitor is con-sidered to be effective for TP53-mutant tumors. However, someTP53 missense variants have transcriptional activity (45), so thatTP53-mutant tumors do not effectively equate to TP53 loss offunction. Therefore, a one-size-fits-all approach to TP53 might belimited in some cases.

Prexasertib is a small-molecule checkpoint inhibitor, mainlyactive against CHK1, with minor activity against CHK2 (46). Inthis study, we confirmed that prexasertib markedly inhibits CHK1phosphorylation but only weakly inhibits CHK2 phosphorylation,as shown in Supplementary Fig. S3. According to the data, wesuggest that combination therapy with trifluridine and a CHK1inhibitor might be effective even in TP53 wild-type cancers. Indeed,DNA damage (gH2AX induction) was also induced by the combi-nation treatment of trifluridine and a CHK1 inhibitor (AZD7762)in TP53 wild-type lung cancer cells (A549 cells) in our in vitroexperiments (Supplementary Fig. S4).

In this study, antitumor effect on combination treatments withtrifluridine/TPI and prexasertib were stronger than that on eithertreatment alone. Here, we have shown a significant positive interaction(synergistic effects) between trifluridine/TPI and prexasertib treat-ment in the experiments with ESCC PDX tumors, not with TE-11R–derived xenograft tumors (additive rather than synergistic). Thesediscrepancies might be due to the different treatment durations(2 weeks for ESCC PDX tumors, and 3 weeks for TE-11R–derivedxenograft tumors) and/or the variation in tumor size. However, thebenefits of combinational therapies are thus apparent even in theabsence of demonstrated synergism, promising as a new treatmentstrategy.

By combining both drugs as well as monotherapy with each agentin in vitro experiments, we demonstrated that trifluridine andprexasertib potentiated radiosensitivity. Moreover, we showed thatthe combination treatment of trifluridine/TPI and prexasertibenhanced radiosensitivity in in vivo experiments. This is the firstreported study on the radiosensitizing effect of prexasertib and/ortrifluridine. Ionizing radiation causes DNA damage by activatingG2-phase checkpoint signaling through CHK1 (47), and so CHK1

inhibitors are considered potential therapeutic targets in combina-tion with radiation. Indeed, the radiosensitizing effects of CHK1inhibitors such as AZD7762 and MK8776, or a combination ofCHK1 inhibitors and other anticancer agents has been shown inseveral studies (48–50). Therefore, we suggest that triple treatmentwith trifluridine, prexasertib, and radiation therapy might be anovel, powerful therapeutic strategy for ESCC.

As a limitation, in vivo studies regarding the radiosensitivity onreceiving trifluridine or prexasertib monotherapy was not conductedin this study, and so further examination is necessary. In addition,neither trifluridine/TPI nor prexasertib has yet been applied in ESCCtreatment; therefore, it is difficult to propose their clinical usefulness atthe current time for patients with ESCC. We suggest that a phase Istudy should be carefully designed.

In conclusion, the combination of trifluridine and CHK1 inhi-bition demonstrates actual antitumor effects in ESCC cells viasynthetic lethality, suggesting a novel therapeutic strategy forESCC. Combination therapy with trifluridine/TPI and a CHK1inhibitor may offer a potential therapeutic benefit for patients withESCC.

Disclosure of Potential Conflicts of InterestS. Ohashi reports receiving other commercial research support from Taiho.

H. Tsukihara is a researcher (paid consultant) at Taiho Pharmaceutical Co., Ltd.N. Suzuki is a researcher (paid consultant) at Taiho Pharmaceutical Co., Ltd.M.Mutoreports receiving other commercial research support from Taiho. No potentialconflicts of interest were disclosed by the other authors.

Authors’ ContributionsConception and design: S. Ohashi, O. Kikuchi, N. Suzuki, M. MutoDevelopment of methodology: S. Ohashi, O. Kikuchi, H. Tsukihara, N. Suzuki,M. MutoAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): O. Kikuchi, Y. Nakai, T. Ida, T. Saito, Y. Kondo, Y. Mitani,T.H. Nguyen Vu, H. Tsukihara, N. Suzuki, M. MutoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Ohashi, O. Kikuchi, K. Fukuyama, H. Tsukihara,N. Suzuki, M. MutoWriting, review, and/or revision of the manuscript: S. Ohashi, O. Kikuchi,N. Suzuki, M. MutoAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): Y. Nakai, T. Ida, T. Saito, Y. Kondo, Y. Yamamoto,Y. MitaniStudy supervision: M. Muto

AcknowledgmentsThe authors are grateful for the technical assistance from the Center for

Anatomical, Pathological and Forensic Medical Researches and the MedicalResearch Support Center, Kyoto University Graduate School of Medicine(Kyoto, Japan). This study was also conducted through the Joint Usage/ResearchCenter Program of the Radiation Biology Center, Kyoto University (Kyoto,Japan). This work was financially supported by Grant-in-Aid for ScientificResearch (19K08369) and the Gastrointestinal Cancer Project funded byNakayama Cancer Research Institute (all to S. Ohashi). This study was alsofinancially supported in part by Taiho Pharmaceutical Co., Ltd (to S. Ohashiand M. Muto).

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

Received September 24, 2019; revised December 19, 2019; accepted April 2, 2020;published first May 5, 2020.

Synthetic Lethality for Esophageal Squamous Cell Carcinoma

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CarcinomaKinase 1 Inhibitor for Esophageal Squamous Cell Synthetic Lethality with Trifluridine/Tipiracil and Checkpoint

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