chk1 inhibition radiosensitizes head and neck cancers to … · 2 arrest in the p53-deﬁcient...

Small Molecule Therapeutics

CHK1 Inhibition Radiosensitizes Head andNeck Cancers to Paclitaxel-BasedChemoradiotherapyHolly E. Barker1, Radhika Patel1, Martin McLaughlin1, Ulrike Schick1,2, Shane Zaidi1,3,Christopher M. Nutting3, Katie L. Newbold3, Shreerang Bhide1,3, and Kevin J. Harrington1,3

Abstract

Head and neck squamous cell carcinoma (HNSCC) is a leadingcause of cancer-related deaths, with increasingly more casesarising due to high-risk human papillomavirus (HPV) infection.Cisplatin-based chemoradiotherapy is a standard-of-care forlocally advanced head and neck cancer but is frequently ineffec-tive. Research into enhancing radiation responses as a means ofimproving treatment outcomes represents a high priority. Here,we evaluated a CHK1 inhibitor (CCT244747) as a radiosensitizerand investigated whether a mechanistically rational triple com-bination of radiation/paclitaxel/CHK1 inhibitor deliveredaccording to an optimized schedulewould provide added benefit.CCT244747 abrogated radiation-induced G2 arrest in the p53-deficient HNSCC cell lines HN4 and HN5, causing cells to entermitosis with unrepaired DNA damage. The addition of paclitaxelfurther increased cell kill and significantly reduced tumor growth

in an HN5 xenograft model. Importantly, a lower dose of pacli-taxel could be used when CCT244747 was included, thereforepotentially limiting toxicity. Triple therapy reduced the expressionof several markers of radioresistance. Moreover, the more radio-resistant HN5 cell line exhibited greater radiation-mediatedCHK1 activation and was more sensitive to triple therapy thanHN4 cells. We analyzed CHK1 expression in a panel of head andneck tumors and observed that primary tumors from HPVþ

patients, who went on to recur postradiotherapy, exhibited sig-nificantly stronger expression of total and activated CHK1. CHK1may serve as a biomarker for identifying tumors likely to recurand, therefore, patients who may benefit from concomitanttreatment with a CHK1 inhibitor and paclitaxel during radio-therapy. Clinical translation of this strategy is under develop-ment. Mol Cancer Ther; 15(9); 2042–54. �2016 AACR.

IntroductionHead and neck squamous cell carcinoma (HNSCC) is the sixth

leading cancer worldwide (1), with smoking, alcohol consump-tion, and high-risk human papillomavirus (HPV) infection beingthe most common risk factors (2–4). Radiotherapy is a standard-of-care treatment for patients with HNSCC. Tumors of the samesize and stage, however, may respond variably to radiation andnew approaches to overcoming radioresistance and preventingtumor recurrence are continually being sought. Ionizing radiationinduces a DNA damage response (DDR) that involves the coop-eration of a complex network of proteins, which play roles in cell-cycle checkpoints and DNA repair (5). In normal cells, radiation-induced DNA damage predominantly induces a G1–S cell-cyclearrest as a result of p53 activation. This G1–S checkpoint is

typically lost in cancer cells most often due to p53 loss, mutation,or inactivation (i.e., following HPV infection), or disruption ofp53-regulated processes (6). These cells, therefore, dependheavilyon the S or G2–M cell-cycle checkpoints to repair DNA damagefollowing ionizing radiation, a dependency that can be exploitedtherapeutically (7).

Ataxia telangiectasia and Rad3-related (ATR) and Ataxia telan-giectasia mutated (ATM) are the initial DDR kinases activated bythe sensors of DNA damage and, in turn, activate the downstreamcheckpoint effector kinases CHK1 and CHK2 (8). One conse-quence of CHK1 activation is inhibition of the phosphataseCDC25C and subsequent arrest of cells in the G2–M phase ofthe cell cycle. CHK1 activity has also been shown to play a role intheG1–S, intra-S, andmitotic spindle checkpoints, indicating thatCHK1 is a fundamental component of the DDR and represents anideal target for therapeutic intervention (9). We, and others, haveshown that CHK1 inhibitors can enhance radiation cytotoxicity incancer cells (reviewed in ref. 7). In the presence of CHK1 inhibi-tors, cells will attempt to undergo mitosis despite harboringradiation-induced DNA damage. This can lead to chromosomalmissegregation or loss of chromatid fragments, resulting in abnor-mal cell division and cell death in a process called mitoticcatastrophe (10).

Patients with locally advanced HNSCC typically receive cis-platin-based chemoradiotherapy (CCRT), but this is frequentlyineffective and associated with severe acute and late toxicities(11). Therefore, alternative approaches to CCRT for HNSCC needto be developed. Taxanes are potent radiosensitizers and havebeen shown to be tolerable and active in combination with

1Targeted Therapy Team, Division of Cancer Biology, The Institute ofCancer Research, Chester Beatty Laboratories, London, United King-dom. 2Radiation Oncology Unit, University Hospital, Brest, France.3Head and Neck Unit, The Royal Marsden NHS Foundation Trust,London and Surrey, United Kingdom.

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

Corresponding Author: Holly E. Barker, The Institute of Cancer Research,Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UnitedKingdom. Phone: 020-7153-5150; Fax: 020-7352-3299; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0998

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 15(9) September 20162042

on February 15, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst July 15, 2016; DOI: 10.1158/1535-7163.MCT-15-0998

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radiotherapy in patients with HNSCC (12). Because they disruptnormal mitotic progression, they represent attractive agents tocombine with radiation and radiosensitizers that target the G2–Mphase of the cell cycle (e.g., CHK1 inhibitors).

CCT244747 is a selective and orally bioavailable CHK1 inhib-itor, which has previously been shown to enhance the antitumoractivity of several genotoxic agents (13). Here, we investigatedwhether including paclitaxel provided added benefit toCCT244747 and radiation combination therapy and defined theschedule-dependence of the combined therapy. We also investi-gated possible biomarkers of radiation resistance and assessed thepotential for this triple combination therapy in overcomingradiation resistance in head and neck cancer patients.

Materials and MethodsCell lines

HN4 and HN5 humanHNSCC cell lines (LICR-LON-HN4 andLICR-LON-HN5) were kindly provided by Sue Eccles (The Insti-tute of Cancer Research, Sutton, United Kingdom) and wereauthenticated by short tandem repeat analysis using the PuregeneCell (Qiagen) and GenePrint 10 (Promega) kits. Results werecompared with STR profiles published online at http://web.expasy.org/cellosaurus, matching for the followingmarkers: ame-logenin, CSF1PO, D16S539, D5S818, D7S820, TH01, TPOX, andvWA. SCC090 cells were obtained commercially from DMSZwithin the last 6 months. Cells were grown in DMEM supple-mented with 5% FCS, 1% glutamine, and 0.5% penicillin/strep-tomycin. Cells were maintained at 37�C and 5% CO2 in ahumidified incubator and routinely tested for mycoplasma.

Drug preparation and irradiationThe CHK1 inhibitor CCT244747 was manufactured at the ICR.

CCT244747 was dissolved in DMSO for in vitro experiments and in10%DMSO, 5% Tween-80, 20% PEG400, and 65%H2O for in vivoexperiments. Paclitaxel was obtained from the Royal MarsdenHospital (London, United Kingdom). Irradiation was carried outusing an AGO HS MP1 X-ray machine (AGO X-ray Ltd.) at 250 kV.

MTT assaysCells were plated in 96-well plates, treated with increasing

concentrations of CCT244747 or paclitaxel, and cell viabilityassessed 72 (IC50 and synergy assays) or 144 (long-term tripletherapy assays) hours later using the MTT [3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma] assay accord-ing to manufacturer's instructions. Absorbance was measured at550 nm on a SpectraMax M5 plate reader (Molecular Devices).

Flow cytometric analysis of cell cycleCells were treated with IC50 concentrations of CCT244747,

paclitaxel, and/or 4 Gy radiation. Cells were trypsinized and fixedin 80% ethanol. For cell-cycle analysis, cells were washed andresuspended in propidium iodide (PI) solution (20 mg/mL PI and100 mg/mL RNase A; Sigma-Aldrich), incubated at 37�C for 30minutes and analyzed on a BD LSRII Flow Cytometer (BDBiosciences). To quantify the mitotic population, cells wereincubated with phospho-Ser10 histone H3 antibody conjugatedto Alexa Fluor 647 (Cell Signaling Technology).

Western blottingLysates were prepared in RIPA buffer (50 mmol/L Tris; pH 7.5,

150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1%

SDS) supplemented with a complete mini protease inhibitorcocktail tablet (Roche), 200 mmol/L sodium fluoride, and200mmol/L sodium vanadate. Proteins were separated on Crite-rion TGX precast gels (Bio-Rad). Gels were transferred onto Trans-Blot Turbo Midi PVDF membranes using the Trans-Blot TurboTransfer system (Bio-Rad). Membranes were probed with thefollowing antibodies: phospho-S345 CHK1, CHK1, phospho-Thr68 CHK2, CHK2, phospho-Ser1981 ATM, ATM, phospho-S139 histone H2AX (g-H2AX), Cyclin A2, Cyclin B1, MCL1,Caspase-3 (Cell Signaling Technology), phospho-Ser10 histoneH3 (Millipore), PARP (Santa Cruz Biotechnology), or b-actin(Abcam).

Clonogenic assaysFor radiosensitization studies, cells were treated with DMSO

(vehicle control) orCCT244747 and irradiatedwith 1, 2, or 4Gy 1hour after drug or vehicle exposure. CCT244747 was washed offafter 48 hours. After 10 to 14 days, colonies were fixed and stainedwith 5%gluteraldehyde, 0.5%crystal violet solution and counted.Surviving fractions (SF) were calculated by normalizing forCCT244747 treatment. For triple combination studies, cells weretreated with DMSO, CCT244747, paclitaxel, and/or 2 Gy radia-tion according to schedule 1 or schedule 2.

Synergy assaysCells were treated with 0.25�, 0.5�, 1�, 2�, and 4� IC50

concentrations of CCT244747 or paclitaxel or both, and cellviability assessed 72 hours later using theMTT assay. Cell viabilitywas calculated by comparing to vehicle-treated cells. Synergy wasdetermined using the Bliss Independence Model, defined by theequationEexp¼ExþEy� (ExEy),whereEexp is the expected effect ifthe two drugs are additive and Ex and Ey are effects of theindividual drugs (14). The equation DE ¼ Eobs � Eexp is thenused to ascertain synergy. If DE and the 95% CI values are allgreater than 0, the two drugs demonstrate synergy.

In vivo experimentsHN5 cells were injected subcutaneously in female 6- to 8-week-

old NOD scid gamma mice (NSG, JAX Mice). Once tumors hadreached approximately 100 mm3, animals were divided intoeight treatment groups (8–12 mice per group). Treatment wasgiven three times on alternate days. Mice received CCT244747(125 mg/kg) by oral gavage, paclitaxel (5 mg/kg) by intraperito-neal injection, and/or 2 Gy radiotherapy according to schedule 2.Tumor volumewas obtainedusing the formula: volume¼width�length � depth � 0.524 mm3. Mice were culled when tumorsreached a maximum diameter of 15 mm in one dimension. Allanimal studieswere conducted in accordancewithNational CancerResearch Institute (NCRI) guidelines (15). All animal research wasreviewed and approved by the Institute of Cancer Research EthicsCommittee. These experimentswereperformedunder the authoritygiven by UK Home Office Project License PPL 70/7947.

Immunocytochemistry for DNA damage and nuclearmorphology

Cells were treated with CCT244747, paclitaxel, and/or 4 Gyradiation according to schedule 2. Cells were fixed with 4%paraformaldehyde, blocked with blocking buffer (1% BSA, 2%FCS), and incubated with rabbit anti-g-H2AX (Cell SignalingTechnology) and mouse anti-a-tubulin (Sigma-Aldrich) anti-bodies. Anti-rabbit 488 and anti-mouse 633 Alexa Fluor

CHK1 Inhibition Chemoradiosensitizes HNSCC Cells

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secondary antibodies were used (Invitrogen, MolecularProbes). Nuclei were counterstained with 40,6-diamidino-2-phenylindole dihydrochloride (DAPI; Invitrogen, MolecularProbes). Cells were imaged using a LSM 710 inverse laserscanning microscope (Zeiss) and captured with a LSM T-PMTdetector (Zeiss). At least 275 cells from five fields of view andthree independent experiments were counted. Cells with �5g-H2AX foci/nucleus were scored as positive.

Long-term growth assaysCells were treated with CCT244747, paclitaxel, and/or 4 Gy

radiation according to schedule 2. Cells were trypsinized at 96,168, and 336 hours and counted. Before trypsinization at 336hours, cells were imaged using an Eclipse TS100 microscope(Nikon) and images captured using a Digital Sight DS-L1 camera(Nikon).

Apoptosis arrayCells were treated with vehicle or triple therapy according to

schedule 2 and lysed after 48 hours in RIPA buffer. Membranesfrom a Human Apoptosis Array Kit (R&D Systems) were incu-bated with prepared cell lysates and developed as per manufac-turer's instruction. Dot intensity was calculated using ImageJsoftware (NIH).

Patient samples and IHCThe PREDICTR-HNC study was approved by the Coventry

Research Ethics Committee (reference number 10/H1210/9).Patient samples were collected at the Royal Marsden Hospital(London, United Kingdom). All tumors were of oropharynxorigin. Biopsies were taken before patients received varyingdoses of radiotherapy with or without neoadjuvant chemother-apy. See Supplementary Table S1 for full tumor and therapydetails. IHC was carried out on sections using the EnVision FLEXSystem (Dako) and antibodies specific for phospho-S345 CHK1(Thermo Fisher Scientific), CHK1, phospho-Ser1981 ATM(Abcam), Survivin (Dako), and Ki67 (Leica Biosystems). Sec-tions were scanned using an Ariol SL50 slide scanner and BX61microscope (Olympus) and images captured using a U-CMAD3camera (Olympus).

Statistical analysisData were analyzed using the Student t test and considered

significant when the P value was <0.05. All statistical tests weretwo-sided. Bar graphs represent the mean and SE across at leastthree independent experimental repeats. Statistical significancerepresentations: �, P < 0.05; ��, P < 0.01; ��, P < 0.001; and ��,P < 0.0001.

ResultsCHK1 inhibition radiosensitizes HN4 and HN5 cells

To test the effect of inhibiting CHK1 on radiation-mediated G2

arrest, cells were treated with the CHK1 inhibitor, CCT244747,1 hour before irradiation and then collected at indicated timepoints. Mitotic cells were identified by phospho-Ser10 histoneH3 (p-HH3) staining and flow cytometry. IC50 concentrationswere calculated for CCT244747 (1 mmol/L for HN4 cells and0.4 mmol/L for HN5 cells) and were used in subsequent experi-ments unless otherwise stated (Supplementary Fig. S1A). Asexpected, radiation reduced the percentage of cells entering mito-sis, suggesting that cells were arrested at the G2 phase of the cellcycle (Fig. 1A). CCT244747 administration significantly abro-gated the radiation-mediated reduction of mitotic HN4 andHN5 cells at 4 and 8 hours after 4 Gy (Fig. 1A). We, and others,have previously shown that phospho-S345 CHK1 is the mostconsistent pharmacodynamic biomarker of CHK1 inhibition(16, 17). Western blot analysis confirmed CCT244747 drug-on-target activity as demonstrated by increased p-CHK1 expression(Fig. 1B). p-HH3 expression was reduced in cells followingirradiation as before, whereas cells pretreated with CCT244747had higher levels of p-HH3 expression at 4 and 8 hours afterirradiation (Fig. 1B). Continued cell cycling after irradiation,due to CCT244747 treatment, resulted in the maintenance ofDNA double-strand breaks (DSB) as shown by increased phos-phorylation of histone H2AX at Ser139 (g-H2AX) expression,most notably at 12, 24, and 48 hours after irradiation in bothcell lines. Analysis of the sub-G1 population indicated thatCCT244747 pretreatment significantly increased apoptosis at48 hours in both cell lines, although levels of apoptosis overallwere very low in HN5 cells (Supplementary Fig. S1B). Impor-tantly, CCT244747 pretreatment led to significant radiosensi-tization of both HN4 and HN5 cells in clonogenic assays (Fig.1C and Supplementary Fig. S1C).

CHK1 inhibition reduces paclitaxel-mediated mitotic arrest,but the combination is not synergistic in HN4 and HN5 cells

To test the effect of combining CCT244747 with paclitaxel onthemitotic population, cells were treatedwithCCT2447471hourbefore paclitaxel treatment and then collected at indicated timepoints for quantification of p-HH3–expressing cells by flowcytometry. IC50 concentrations were determined for paclitaxelusing MTT assays (8 nmol/L for HN4 cells and 4 nmol/L for HN5cells) and were used in subsequent experiments unless otherwisestated (Supplementary Fig. S1A). Paclitaxel treatment increasedthe percentage of HN4 and HN5 cells in mitosis up to 24 hours(Fig. 1D). When cells were pretreated with CCT244747

Figure 1.The CHK1 inhibitor CCT244747 overcomes radiation-mediated G2 arrest and reduces paclitaxel-mediated mitotic arrest in HN4 and HN5 cells. A, quantification ofmitotic cells following 4 Gy of radiation (RT) with or without CCT244747 (CCT) pretreatment. Cells were fixed at indicated time points, stained with phosho-S10Histone H3 (p-HH3), and analyzed by flow cytometry. Data expressed as fold changes (0 hour ¼ 1). B, Western blot analysis of CCT drug on target activity(accumulation of phospho-S345 CHK1), mitotic cells (expression of p-HH3), and sustained DNA DSBs (expression of g-H2AX) after 4 Gy of radiation with orwithout CCT pretreatment. Expression of b-actin provided a loading control. C, analysis of CCT244747 mediated radiosensitization using clonogenic assays.Cells were treated with 0.2 or 0.7 mmol/L CCT 1 hour before exposure to indicated doses of radiation and data normalized for drug effect. D, quantificationof mitotic cells following exposure to paclitaxel (PTX) with or without CCT pretreatment, as described in A. E, Western blot analysis of CCT drug on targetactivity, mitotic cells, and sustained DNA DSBs after exposure to PTX with or without CCT pretreatment. Expression of b-actin provided a loading control.F, quantification of cell viability following CCT or PTX monotherapy, or combined treatment, at the indicated ratios of IC50 doses for CCT and PTX. Cellviability was quantified using MTT assays and calculated as a fraction of control cells. G, analysis of synergy using the Bliss Independence Model. The differencein observed and expected effects (DE¼ Eobs � Eexp) indicates synergy when DE and the 95% CI values are all greater than 0. � , P < 0.05; �� , P < 0.01; �� , P < 0.001;and �� , P < 0.0001.






accumulation in mitosis was initially unaffected; however, after12 hours, the mitotic population was significantly reduced inboth cell lines (P ¼ 0.01 and 0.014 for HN4 and HN5 cells,respectively; Fig. 1D). Western blot analysis confirmedCCT244747 drug-on-target activity as demonstrated by increasedp-CHK1 expression (Fig. 1E). p-HH3 expression was reducedslightly after 12 hours in cells pretreated with CCT244747 com-pared with those treated with paclitaxel alone; however, a p-HH3reduction was most pronounced at 24 hours. Although theWestern blotting andflowcytometry results vary slightly (possiblydue to the sensitivities of the different antibodies used), bothmethods indicate that CHK1 inhibition enables cells to prema-turely escape a paclitaxel-induced mitotic arrest. Moreover,CCT244747 pretreatment results in increased DNA DSBs, dem-onstrated by increased expression of g-H2AX at 24 hours in bothcells lines (Fig. 1E). Analysis of the sub-G1 population indicatedthat CCT244747 pretreatment did not increase apoptosis in HN4or HN5 cells (Supplementary Fig. S1D). MTT assays were carriedout to determine whether CCT244747 and paclitaxel acted syn-ergistically. HN4 andHN5 cells were treated with varying ratios ofIC50 concentrations of CCT244747 and paclitaxel independentlyor in combination and cell viability calculated relative to vehicle(DMSO) treated cells (Fig. 1F). Bliss analysis of the MTT resultsindicated that CCT244747 and paclitaxel only behaved synergis-tically in HN5 cells at a concentration of 0.5 � IC50 (Fig. 1G).Clonogenic assays showed a significant decrease in cell survivalwhen cells were treated with CCT244747 and paclitaxel doubletherapy compared with monotherapy (Supplementary Fig. S1E).

A schedule for CCT244747, paclitaxel, and radiation tripletherapy synergistically kills HN4 and HN5 cells

We hypothesized that cells prematurely entering mitosis withradiation-induced DNA damage, due to pretreatment with theCHK1 inhibitor, could be subsequently exposed to paclitaxelresulting in greater cell kill. Flow cytometry results suggestedthat treatment with paclitaxel 4 to 8 hours after CCT244747/radiation double therapy would affect the maximum numberof damaged cells entering mitosis (Fig. 1A). In contrast, flowcytometry revealed that irradiation of cells 4 to 8 hours afterCCT244747/paclitaxel double therapy would target the greatestnumber of cells arrested in mitosis, the most radiosensitivephase of the cell cycle (refs. 18–20; see Fig. 1D). Therefore, wetested two schedules where cells were treated with CCT244747and then exposed to radiation before paclitaxel (Schedule 1) orvice versa (Schedule 2) over the same 6-hour therapeuticwindow (Fig. 2A). The short-termmaintenance of DNA damagewas determined by Western blotting for g-H2AX expression.Triple therapy according to Schedule 2 resulted in a greaterg-H2AX signal at 24 hours in HN4 and HN5 cells comparedwith schedule 1 (Fig. 2B). The concurrent g-H2AX and p-HH3expression suggests that mitosis is occurring despite unrepairedDNA DSBs being present. Therefore, cells are at significant riskof undergoing mitotic catastrophe. In long-term clonogenicassays, HN4 and HN5 cells were dosed with 0.5 mmol/LCCT244747, 3 nmol/L paclitaxel, and 2 Gy of radiation. TheSF of cells treated with triple therapy was significantly lowerthan the SF of cells treated with CCT244747/radiation doubletherapy only in HN5 cells when using schedule 2 (P ¼0.031; Fig. 2C). Normalizing SF values for CCT244747 andpaclitaxel treatment again demonstrated a significant decreasein SF after triple therapy compared with CCT244747/radiation

double therapy in HN5 cells when schedule 2 was used (P ¼0.001; Fig. 2D). Clonogenic assays carried out with lowerconcentrations of 0.2 mmol/L CCT244747 and 1 nmol/L pac-litaxel with 2 Gy of radiation resulted in a significantly lower SFfollowing triple therapy only when compared with paclitaxel/radiation double therapy in HN5 cells using schedule 2 (P ¼0.0002; Supplementary Fig. S2A). Normalizing for drug effect, alower SF after triple therapy was only seen in HN5 cells usingschedule 2 but this difference was not significant (Supplemen-tary Fig. S2B). We used the Bliss Independence Model todetermine whether paclitaxel synergized with CCT244747/radiation double therapy. Synergy was only observed for sched-ule 2 at the high drug concentrations in HN5 cells (Fig. 2E).Taking into account all of the results obtained for the two tripletherapy schedules, schedule 2 was selected for further analysis.

Triple therapy reduces the growth rate of HN5 xenograftsTo assess the efficacy of the triple therapy in vivo, HN5 cells were

implanted subcutaneously in NSG mice and treated according toschedule 2 (Fig. 3A). Relatively lowdoses of all agentswere used toensure effects of the combination therapies would be apparentand to reduce toxicity in the combination groups. Radiotherapywas the only single agent to show a significant reduction in tumorgrowth compared with controls (P ¼ 0.0343). Triple therapysignificantly decreased tumor growth compared with all doubletherapy treatment groups (P < 0.0001 when comparing withCCT244747 double combinations and P ¼ 0.0008 when com-paring with the paclitaxel/radiotherapy combination; Fig. 3B).HN5 tumors appeared to be more sensitive to radiation in vivothan might have been expected from in vitro data, possibly due tothe fractionated nature of the in vivo treatment regimen. Interest-ingly, combining CCT244747 or paclitaxel with radiation did notcause a significant reduction in tumor growth above what wasseen for tumors treated with radiation alone. Three tumors fromeach group were collected 2 hours after the final treatment forpharmacodynamic analysis. Western blotting of lysates preparedfrom these tumors demonstrated that, in most cases, CCT244747activity could be detected by expression of p-CHK1. Furthermore,CCT244747 caused an increase in the mitotic population withinthe tumors as assessed by p-HH3 staining (Fig. 3C).

Cells treated with CCT244747, paclitaxel, and radiation tripletherapy undergo mitotic catastrophe

To assess the appearance of the nuclei in cells after triple therapyusing schedule 2, treated cells were fixed at 24 and 48 hours andanalyzed by confocalmicroscopy. Cells were stainedwith g-H2AXto visualize DNA DSBs, and DAPI and a-tubulin to visualizenuclei and microtubules, respectively. Both HN4 and HN5 cellsexhibited increased g-H2AX foci after radiation, pan-H2AX stain-ing (whole nuclear g-H2AX staining) after CCT244747 treatment[as has been previously reported (21)], and abnormal nuclei afterpaclitaxel treatment (Fig. 4A and Supplementary Fig. S3A). Cellswere quantified according to 6 categories: normal nuclear appear-ance, g-H2AX foci, pan-H2AX staining, abnormal nuclear mor-phology (micronuclei and/or multinuclear), abnormal nuclearmorphology with g-H2AX foci, and abnormal nuclear morphol-ogy with pan-H2AX staining (Fig. 4B). Forty-eight hours aftertriple therapy, very few of the remaining cells had normal nuclearappearance. The most common characteristic in triple-treatedcells was abnormal nuclear morphology, which was observed in44.3% of HN4 cells and 67.5% of HN5 cells. HN4 cells exhibited

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Figure 2.

Scheduling of CCT244747, paclitaxel, and radiation triple therapy. A, schematic of the two schedules chosen for further analysis. B, Western blot analysis ofCCT244747 (CCT) drug on target (accumulation of p-CHK1), mitotic cells (expression of p-HH3), and sustained DNA DSBs (expression of g-H2AX) 24 hoursafter triple therapy according to schedule 1 or 2. Expression of b-actin provided a loading control. C, quantification of cell survival after triple therapy usingclonogenic assays. Cells were treated with vehicle, 0.5 mmol/L CCT, 3 nmol/L paclitaxel (PTX), and/or 2 Gy radiation (RT) according to schedule 1 or 2. SFs werecalculated by comparing treated wells with control wells. D, quantification of the SF when normalizing for drug effects. E, analysis of synergy using the BlissIndependence Model. The effect of triple therapy is compared with the effects of CCT and chemoradiotherapy (PTXþRT) independently. � , P < 0.05; �� , P < 0.01;�� , P < 0.001; and �� , P < 0.0001.






significantlymoremicronuclei following triple therapy comparedwithHN5 cells (P¼ 0.001) andHN5 cells were significantlymorelikely to be multinuclear following triple therapy compared withHN4 cells (P¼ 0.044; Fig. 4C), indicating mitosis was aberrant inboth cell lines. Pan-H2AX staining occurred in cells exposed toCCT244747 with 43.0% and 51.9% of HN4 and HN5 triple-treated cells exhibiting pan-H2AX staining 48 hours after tripletherapy, respectively. Pan-H2AX staining has previously beenlinked to stalled replication (S-phase arrest), as well as being anindicator of inappropriate mitotic entry of cells harboring unre-paired DNA damage from replication stress or checkpoint inhi-bition (22, 23). We have previously observed byWestern blottingthatmitosis is occurring after triple therapy despite the presence ofunresolved DNA DSBs (Fig. 2B). In addition, flow cytometryrevealed that there was a 2.2- and 2.7-fold increase in the S-phasepopulation of HN5 cells 24 and 48 hours after triple therapy,respectively (Supplementary Fig. S3B). Both of these findingscould account for the increased pan-H2AX staining observed.

Triple therapy induces apoptosis and reduces clonogenicity insurviving cells

Pan-H2AX staining may also be an early indicator of cellsdestined for apoptosis (24). To assess whether HN4 and HN5cells were undergoing apoptosis, sub-G1 analysis and Westernblotting were carried out 48 hours after triple therapy. Flowcytometry revealed significantly more HN4 cells (19.9 � 2.8%,compared with 6.1 � 2.1%HN5 cells) were undergoing the latestages of apoptosis (as assessed by the percentage of cells insub-G1) 48 hours after triple therapy (P ¼ 0.016; Fig. 5A).Western blotting confirmed that HN4 cells were dying byapoptosis by virtue of decreased expression of MCL1 and theappearance of PARP and caspase-3 cleavage in lysates harvestedfrom treated cells 48 hours after triple therapy (Fig. 5B).Decreased MCL1 expression could not be detected by Westernblotting in HN5 cell lysates 48 hours after triple therapy, andonly faint bands representing PARP and caspase-3 could bedetected (Fig. 5B).

Figure 3.

Triple therapy significantly delaysHN5xenograft tumor growth. A, schematicof in vivo triple therapy schedule usedfor treatment of mice-harboring HN5xenografts. B, tumor volume of HN5xenografts after indicated therapies.Tumors were measured biweekly inthree dimensions. Significance is onlyindicated between groups that differby a single agent (n¼ 8–12 per group).C, Western blot analysis ofCCT244747 (CCT) drug on targetactivity (accumulation of p-CHK1) anddrug effect [CCT, paclitaxel (PTX), andradiation (RT)] on the mitoticpopulation (expression of p-HH3).Expression of b-actin provided aloading control. � , P <0.05; �� , P <0.01;�� , P < 0.001; and �� , P < 0.0001.

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To assess whether HN5 cells were recovering at later time-points following triple therapy, as very few appeared to beundergoing apoptosis at 48 hours, long-term growth assayswere carried out. Neither HN4 nor HN5 cells appeared torecover up to 2 weeks following treatment (Fig. 5C) and manyof the cells that remained appeared large and flat, or small and

apoptotic (Fig. 5D). Assays for senescence and autophagy werecarried out with negative results (data not shown). The expres-sion of apoptotic markers and cell-cycle proteins was assessedat later time points (96 and 120 hours) following triple ther-apy. Western blotting revealed that HN5 cells appeared toundergo delayed apoptosis because PARP and caspase-3

Figure 4.

HN4 and HN5 cells have abnormal nuclear appearance after triple therapy. A, confocal analysis of HN4 and HN5 cells treated with CCT244747 (CCT) 1 hourbefore treatment with paclitaxel (PTX) and 6 hours before 4 Gy of radiation (RT). Cells were fixed and stainedwith anti-g-H2AX antibody (green) to detect sustainedDNA DSBs. Cells were costained with anti-a-tubulin antibody (red) and counterstained with DAPI (blue) to distinguish nuclear morphology. Representativeimages from one experimental repeat of cells fixed and stained at 48 hours are shown. Scale bar, 12.5 mm. B, quantification of confocal data for HN4 and HN5 cellsat 24 and 48 hours following triple therapy as described in A. C, quantification of cells with micronuclei and multinuclear cells 48 hours after triple therapy asdescribed in A. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; and �� , P < 0.0001.






Figure 5.

HN5 cells exhibit delayed apoptosis and loss of clonogenicity after triple therapy. A, quantification of cells in the sub-G1 population following indicated treatmentsaccording to schedule 2. Cells were fixed at 24 and 48 hours, stained with PI and analyzed by flow cytometry. B, Western blot analysis of cell lysates48 hours after indicated treatments according to schedule 2. Induction of apoptosis was analyzed by blotting for MCL1, PARP, and caspase-3. Expression of b-actinprovided a loading control. C, long-term growth assays of treated cells. Drugs were washed off after 48 hours and cells counted at indicated time points. D,representative images of cells remaining at the end of the long-term growth assays described in A. Scale bar, 100 mm. E, Western blot analysis of HN4and HN5 cell lysates 96 and 120 hours after indicated treatments. Apoptosis was analyzed by blotting for MCL1, PARP, and caspase-3. Cell-cycle progressionwas analyzed by blotting for Cyclin A2 and Cyclin B1. Expression of b-actin provided a loading control. � , P < 0.05; ��, P < 0.01; �� , P < 0.001; and �� , P < 0.0001.

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cleavage could be easily detected by 120 hours, despite constantexpression of MCL1 (Fig. 5E). HN4 cells continued to displayreduced expression of MCL1 following triple therapy, as well assustained PARP and caspase-3 cleavage. Interestingly, CHK1appeared to be reactivated 96 and 120 hours after tripletherapy, more so in HN5 cells (demonstrated by p-CHK1expression, as CCT244747 was washed off at 48 hours). Con-sequently, the expression of Cyclin A2 was reduced, mostprominently at 120 hours, whereas Cyclin B1 expressionremained constant (Fig. 5E), suggesting cells arrested inS-phase. These results imply that reactivation of CHK1 occursin triple therapy–treated cells after drug wash-off, due to thepresence of persistent DNA damage. When damage is too great,cells undergo apoptosis at this later stage, whereas survivingcells appear to express reduced levels of Cyclin A2 and lose theirclonogenicity.

Triple therapy reduces the expression of markers of radiationresistance

To further study changes in protein expression in HN4 andHN5 cells following triple therapy, we used an apoptosis array.Cells were treated with triple therapy according to schedule 2 andlysates collected after 48 hours were used to probe an apoptosisarray. The fold change in expression was calculated by comparingintensity of blots probed with lysate from treated cells with blotsprobed with lysates from vehicle-treated cells and converted toLog2 values (Supplementary Fig. S4A). The expression of a num-ber of proteins was decreased by at least half (�1 on Log2 scale) inat least one cell line (Fig. 6A). The expression of cleaved caspase-3was increased 3.1-fold and 1.6-fold in HN4 and HN5 cells,respectively, correlating with our previous Western blots (Fig.5B). The expression of Claspin, a CHK1 adaptor protein, whichundergoes proteasomal degradation during apoptosis, wasdecreased 9.0- and 4.0-fold in HN4 and HN5 cells, respectively,further confirming treatment efficacy. In addition to these find-ings, the expression of a number of proteins previously implicatedin radiation resistance was reduced following triple therapy.Expression of the inhibitor of apoptosis proteins Survivin andXIAPwas reduced 4.0- and 2.1-fold, respectively, inHN4 cells and2.9- and 3.1-fold, respectively, in HN5 cells. We confirmed byWestern blotting that downregulation of Survivin and XIAPpersisted at least 120 hours after triple therapy (Fig. 6B).

CHK1 expression and activity asmarkers of radiation resistanceDuring this study, we noted that, despite HN5 cells being

more resistant to radiation than HN4 cells, they appeared to bemore sensitive to triple therapy. To investigate the DDR path-ways activated in HN4 and HN5 cells in response to radiation,cells were treated with 10 Gy and collected at specific time-points for analysis. DDR pathway activation was analyzed inprepared lysates by Western blotting for phospho-S1981 ATM(p-ATM), p-CHK1, and phospho-T68 CHK2 (p-CHK2). p-HH3expression confirmed that radiation-mediated G2 arrestoccurred within 2 hours and was maintained at least until 6hours (Fig. 6C). HN4 cells exhibited strong activation of CHK2(p-CHK2 expression), whereas HN5 cells had strong CHK1activation (p-CHK1 expression; Fig. 6C). To determine whetherCHK1 expression levels and activity could be a marker ofresistance to radiotherapy, we analyzed a panel of HPVþ andHPV� head and neck tumors by IHC. Interestingly, high expres-sion of total nuclear CHK1 protein and increased nuclear

p-CHK1 expression significantly correlated with HPVþ tumorsthat recurred following radiotherapy (P ¼ 0.005, Fig. 6D and Eand Supplementary Fig. S5). We noted that high cytoplasmicexpression of CHK1 and p-CHK1 also significantly correlatedwith recurring HPVþ tumors (P¼ 0.005 and 0.016, respectively;Supplementary Figs. S4B and S5). We also analyzed the expres-sion of p-ATM, Ki67, and Survivin in these tumors. Highexpression of p-ATM was associated with recurring HPVþ

tumors and low expression of Survivin was associated withnonrecurring HPVþ tumors; however, neither association wassignificant (P ¼ 0.078 and 0.053, respectively; SupplementaryFigs. S4C and S5). No correlation was observed between Ki67expression levels and tumor recurrence.

Finally, we confirmed these findings in the SCC090 HNSCCcell line, which was isolated from a recurring HPVþ tumor.These cells were highly resistant to radiation in clonogenicassays (Supplementary Fig. S6A) and exhibited strong activa-tion of CHK1 after exposure to 10 Gy radiation (SupplementaryFig. S6B). In accordance with these findings, SCC090 cells werevery sensitive to CCT244747 (IC50 ¼ 0.2 mmol/L; Supplemen-tary Fig. S6C) and, consequently, triple therapy was highlyeffective in clonogenic and long-term MTT assays (Supplemen-tary Fig. S6D and S6E).

DiscussionDetailed in vitro studies have been conducted to assess the

efficacy of a range of CHK1 inhibitors in radiosensitizing cancercells that rely on theG2–Mcheckpoint forDNAdamage repair (7).However, CHK1 inhibitors have not yet been combined withradiotherapy in clinical trials, possibly due to inadequate selec-tivity and/or unexpected toxicities observed with earlier com-pounds (25, 26). The CHK1 inhibitor CCT244747 is a highlyselective, potent, orally active ATP-competitive CHK1 inhibitorpreviously shown to enhance the activity of several genotoxicagents, including ionizing radiation (13). The activity ofCCT244747 has not previously been investigated in HNSCC celllines. Here we confirmed that CCT244747 could overcome radi-ation-mediated G2 arrest in the p53-deficient HN4 andHN5 cells,causing cells to enter mitosis with unrepaired DNA damage. Thisled to significant radiosensitization of both HN4 and HN5 cellsin vitro.

We hypothesized that CCT244747 pretreated p53-deficientcells enteringmitosis harboring radiation-mediatedDNAdamagecould be further damaged with an antimitotic, such as paclitaxel.Indeed, greater cell kill was observed for triple therapy using thisschedule (schedule 1). However, exploiting the ability of pacli-taxel to arrest cells inmitosis, themost radiosensitive phase of thecell cycle, before exposing them to ionizing radiation proved to bemore effective (schedule 2). These findings reinforce the criticalimportance of optimizing scheduling when combining differenttypes of anticancer agents. Importantly, including CCT244747 inour triple therapy enabled a much lower dose of paclitaxel to beused, therefore potentially limiting toxicity. We hypothesize thattumors not harboring p53 mutations would also be sensitized totriple therapy as many still rely on the G2 checkpoint for efficientDNA damage repair. However, normal tissue would be relativelyprotected due to harboring an intact G1 checkpoint, thus wewould see an improvement in the therapeutic index whenCCT244747 is present as part of a combination regimen. Finally,administration of the triple therapy according to schedule 2






significantly reduced the growth rate ofHN5 xenografts comparedwith all double combination therapies.

Cells receiving triple therapy exhibited many characteristics ofmitotic catastrophe. Themost common feature of cells exposed totriple therapy was the presence of nuclear abnormalities; cellsoften harbored micronuclei and/or were multinucleated. HN4cells displayed more micronuclei, which could signal for them tobe eliminated by apoptosis (27). Indeed, markers of apoptosis(namely an increased sub-G1 population, as well as caspase 3 andPARP cleavage) were increased in HN4 cells 48 hours after triple

therapy. HN5 cells, however, were more likely to be multinucle-ated following triple therapy. Cell-cycle arrest was thought to bethe primary response to multinucleation in HN5 cells, as the S-phase populationmore than doubled after triple therapy. S-phasearrest has also previously been associated with CHK1 activationanddecreased expression of CyclinA2 (28).Wehypothesized thatreactivation of CHK1 (after drug wash-off) due to persisting DNAdamage could lead to cell-cycle arrest at later time points. If cellsare unable to repair DNA damage at this later stage, they mayundergo delayed apoptosis. Indeed, markers of apoptosis could

Figure 6.

Triple therapy downregulates markersof radiation resistance and high CHK1expression and activity correlate withrecurring HPVþ head and neck tumors.A, analysis of protein expression in HN4and HN5 cells 48 hours after tripletherapy according to schedule 2 usingan apoptosis array. Proteins withgreatest fold change (compared withuntreated controls) shown. B, Westernblot analysis of cell lysates 120 hoursafter indicated treatments according toschedule 2. Findings from the apoptosisarray described in Awere confirmed byblotting for Survivin and XIAP.Expression of b-actin provided aloading control. C, Western blotanalysis of HN4 and HN5 cells followingexposure to 10 Gy of radiation (RT).Checkpoint activity was determined byblotting for p-ATM, p-CHK1, and p-CHK2. The mitotic population was alsoanalyzed by blotting for p-HH3.Expression of b-actin provided aloading control. D, quantification ofCHK1 nuclear staining by IHC in HPVþ

and HPV� HNSCC samples obtainedfrom patients in the PredictR-HNC trial.E, quantification of p-CHK1 nuclearstaining in patient samples as describedin D. �� , P < 0.01.


Barker et al.




be detected 96 and 120 hours after triple therapy. Surviving cellsappeared to have lost their clonogenicity and, as such, expresseddecreased levels of the S-phase regulator Cyclin A2.

Finally, we found that triple therapy reduced the expression ofseveral markers of radiation resistance, including Survivin andXIAP, in both HN4 and HN5 cells. High expression of theseproteins has previously been linked to radiation resistance andworse prognosis (29–32). During this study, it was noted thatdespite HN5 cells being highly radioresistant they were moresensitive to CCT244747 monotherapy, as well as CCT244747combined therapies, than HN4 cells. Certainly, HN5 cells exhib-ited greater activation of CHK1 following irradiation comparedwith HN4 cells. This correlates with findings from two recentstudies carried out in non–small cell lung cancer where CHK1levels and activity indicated sensitivity to CHK1-targeted therapy(33, 34). Likewise, wehypothesized that highCHK1activity couldbe a marker of radiation resistance and may serve as a biomarkerfor tumors likely to relapse and, thus, identify the patients whowould benefit the most from triple therapy. Analysis of a smallpanel of HPVþ and HPV� head and neck tumors revealed therewas a significant correlation between high total CHK1 expressionin the nucleus or cytoplasm with recurrence after radiotherapy inHPVþ tumors. High p-CHK1 expression in the nucleus or cyto-plasm also significantly correlated with recurring HPVþ tumors.Although this is a small sample set, high CHK1 expression and/oractivity has previously been correlated with therapy-resistantcancer cells (35, 36) and associated with high-grade, high-risktumors often resistant to therapy and with a poor prognosis(36–40). Confirmation of our findings in a larger head and neckcancer dataset will be required.

We propose that cells with high CHK1 proficiency, which resistthe first round of treatment with chemotherapy or radiotherapy,will become dominant in a tumor and drive recurrence andrelapse. Not only is it possible that CHK1 expression and activitywill help identify those tumors associated with a worse prognosis,but also CHK1 is likely to continue being an important target forcancer treatment. The triple therapy we propose here; adminis-tration of a CHK1 inhibitor 1 hour prior to paclitaxel treatmentand followed 6 hours later with radiotherapy, holds great promisefor treating all tumors reliant on aG2 checkpoint forDNAdamagerepair but also for overcoming therapy resistance and hopefullypreventing recurrence. The schedule thatwe have defined is highlycompatible with current clinical practice in HNSCC. Biomarker-

driven clinical studies in patients with locally advanced HNSCCrepresent a rapid and rational route to translating this strategy andoffer a means of mitigating the severe toxicity associated withexisting CCRT.

Disclosure of Potential Conflicts of InterestIntellectual property from the research collaboration with Sareum Ltd. on

Chk1 inhibitors was licensed from The Institute of Cancer Research to SareumLtd. The Institute of Cancer Research has benefited from this and requires itsemployees to declare this potential conflict of interest. This applies toH.E. Barker, R. Patel, M. McLaughlin, S. Zaidi, C. Nutting, K. Newbold, S. Bhideand K.J. Harrington. In addition, K. Newbold has received honoraria forspeakers bureau and advisory boards for Eisai and Astra-Zeneca. U. Schickdeclared no potential conflicts of interest.

Authors' ContributionsConception and design: H.E. Barker, S. Zaidi, K.J. HarringtonDevelopment of methodology: H.E. Barker, M. McLaughlin, S. Zaidi,C.M. Nutting, K.J. HarringtonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H.E. Barker, U. Schick, K.L. NewboldAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H.E. Barker, C.M. Nutting, K.J. HarringtonWriting, review, and/or revision of the manuscript: H.E. Barker, R. Patel,S. Zaidi, C.M. Nutting, K.L. Newbold, S. Bhide, K.J. HarringtonAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H.E. BarkerStudy supervision: S. Zaidi, K.J. Harrington

AcknowledgmentsWe would like to thank Ian Collins, Michelle Garrett and Thomas Matthews

for providing the CCT244747 compound; and Frances Daley, Anne Lowe,Margaret Smith and Jemma Shead in the Breakthrough Histopathology Facilityat the Royal Marsden Hospital for carrying out the staining of the patientsamples from the PredictR-HNC study.

Grant SupportThis work was supported by grants from the Cancer Research UK Pro-

gramme Grants C46/A10588 and C7224/A13407 (all authors), the NIHRRoyal Marsden/Institute of Cancer Research Biomedical Research Centre (allauthors), the Oracle Cancer Trust (to H.E. Barker) and Rosetrees Trust (to K.J.Harrington).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 23, 2015; revised June 6, 2016; accepted June 10, 2016;published OnlineFirst July 15, 2016.

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Barker et al.




2016;15:2042-2054. Published OnlineFirst July 15, 2016.Mol Cancer Ther Holly E. Barker, Radhika Patel, Martin McLaughlin, et al. Paclitaxel-Based ChemoradiotherapyCHK1 Inhibition Radiosensitizes Head and Neck Cancers to

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