the checkpoint kinase 1 inhibitor prexasertib induces regression … · julie stewart, jennifer...

Cancer Therapy: Preclinical

The Checkpoint Kinase 1 Inhibitor PrexasertibInduces Regression of Preclinical Models ofHuman NeuroblastomaCaitlin D. Lowery, Alle B. VanWye, Michele Dowless,Wayne Blosser, Beverly L. Falcon,Julie Stewart, Jennifer Stephens, Richard P. Beckmann, Aimee Bence Lin, andLouis F. Stancato

Abstract

Purpose: Checkpoint kinase 1 (CHK1) is a key regulator ofthe DNA damage response and a mediator of replication stressthrough modulation of replication fork licensing and activa-tion of S and G2–M cell-cycle checkpoints. We evaluatedprexasertib (LY2606368), a small-molecule CHK1 inhibitorcurrently in clinical testing, in multiple preclinical models ofpediatric cancer. Following an initial assessment of prexasertibactivity, this study focused on the preclinical models ofneuroblastoma.

Experimental Design:We evaluated the antiproliferative activ-ity of prexasertib in a panel of cancer cell lines; neuroblastoma celllines were among the most sensitive. Subsequent Westernblot and immunofluorescence analyses measured DNA damageand DNA repair protein activation. Prexasertib was investigatedin several cell line–derived xenograft mouse models ofneuroblastoma.

Results: Within 24 hours, single-agent prexasertib promotedgH2AX–positive double-strandDNAbreaks andphosphorylationof DNA damage sensors ATM and DNA–PKcs, leading to neuro-blastoma cell death. KnockdownofCHK1and/orCHK2by siRNAverified that the double-strand DNA breaks and cell death elicitedby prexasertib were due to specific CHK1 inhibition. Neuroblas-toma xenografts rapidly regressed following prexasertib admin-istration, independent of starting tumor volume. Decreased Ki67and increased immunostaining of endothelial and pericyte mar-kers were observed in xenografts after only 6 days of exposure toprexasertib, potentially indicating a swift reduction in tumorvolume and/or a direct effect on tumor vasculature.

Conclusions: Overall, these data demonstrate that prexasertibis a specific inhibitorofCHK1 inneuroblastomaand leads toDNAdamage and cell death in preclinical models of this devastatingpediatric malignancy. Clin Cancer Res; 23(15); 4354–63. �2017 AACR.

IntroductionNeuroblastoma is derived from neural crest precursor cells of

the peripheral sympathetic nervous system, typically developingin the adrenal medulla or paraspinal ganglia (1, 2). These tumorscomprise 5% of all childhoodmalignancies and 10% of pediatricpatient deaths, emphasizing the critical need for novel therapies(1). Clinical behavior of the disease ranges from localized tumorswhich can be cured with surgical excision to invasive and/ormetastatic disease which is often refractory to aggressive multi-modal treatment regimens (3). Patient risk is stratifiedon thebasisof age at diagnosis; tumor characteristics such as stage, grade, andhistology; DNA ploidy; and status of MYCN genomic amplifica-tion (4). Nearly half of high-risk neuroblastoma patients experi-ence a relapse, which is usually fatal; those who survive are oftenfaced with damaging complications from intensive therapeutic

interventions composed of surgery, chemotherapy, and radiation(1, 3). Therefore, it is essential to evaluate targeted agents to offerthese patients more efficacious treatment options.

The serine/threonine kinase checkpoint kinase 1 (CHK1) iscritical for replication initiation through licensing of replicationforks; furthermore, CHK1 regulates DNA damage response andrepair mechanisms following stalled replication forks or single-strand DNA breaks through its modulation of the S-phase andG2–M cell-cycle checkpoints (5–7). Loss of CHK1 activity due topharmacologic inhibition or knockdown of total protein by RNAinterference in specific tumor types impairs the DNA damageresponse and the ability to mitigate replication stress, leading tostalled forks, double-strand DNA breaks, and eventual cell deathvia replication catastrophe (8). Similarly, neuroblastoma cellviability was reduced upon depletion or pharmacologic inhibi-tion of CHK1 and, interestingly, sensitivity to CHK1 inhibitionwas not influenced by p53 status or baseline level of DNAdamage (9).

Prexasertib (LY2606368) is a CHK1 small-molecule inhibitorcurrently in clinical evaluation as both a single agent and incombination with targeted agents or chemotherapy in adultpatients with solid tumors (10). Furthermore, clinical develop-ment of the molecule includes a phase I trial in pediatric solidtumors (NCT02808650). A previous preclinical study reportedthe efficacy of prexasertib in promoting extensive DNA damage inadult carcinoma cell lines and xenograftmousemodels, which led

Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Louis F. Stancato, Oncology Discovery, Lilly CorporateCenter, Indianapolis, IN 46285. Phone: 317-655-6910; Fax: 317-276-1414; E-mail:[email protected].

doi: 10.1158/1078-0432.CCR-16-2876

�2017 American Association for Cancer Research.

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to cell death due to replication catastrophe (8). Here, we dem-onstrate that single-agent prexasertib induced tumor regression inmultiple preclinical mouse models of neuroblastoma.

Materials and MethodsCell culture conditions

Human neuroblastoma cell lines IMR-32 and SH-SY5Y, pan-creatic cell line PANC-1, and primary neonatal epidermal mela-nocytes were purchased from ATCC. KELLY, MHH-NB-11, andNBL-S were obtained from The German Collection of Microor-ganisms and Cell Cultures (DSMZ). Cell culture conditions aredetailed in Supplementary Table S1. All cells were maintained at37�C and 5% CO2 in tissue-culture–treated flasks.

Test compoundPreclinical studies use 2940930, which is the mesylate mono-

hydrate salt of LY2606368 (prexasertib), and will be henceforthreferred to as prexasertib. Prexasertib (LY2606368, Eli Lilly andCompany) was dissolved in DMSO at a stock concentration of 10mmol/L for in vitro use and prepared in 20% Captisol for in vivoexperiments.

Cell proliferation assayProfiling of prexasertib in a panel of more than 300 cancer cell

lines was performed as previously reported in ref. 11. For com-parison with standard of care (SOC), pediatric cancer cell lineswere plated in 96-well microtitre plates and treated with prexa-sertib, doxorubicin, cisplatin, or gemcitabine across a range ofconcentrations. Cell proliferationwas assayed after twodoublingsby CellTiter Glo Luminescent Cell Viability Assay (Promega,catalog no. G7571). For in-depth evaluation of pediatric neuro-blastoma cell lines, PANC-1, and primary melanocytes, cell pro-liferation was assayed after 72 hours. Luminescence was normal-ized to the average of the DMSO control for each individual cellline and plotted as the percent of control over the log concen-tration. EC50 values were calculated from triplicate experimentsusing GraphPad Prism 6 (GraphPad Software, Inc., Version 7.00).

Western blot analysisCells were lysed in 1% SDS (Fisher BioReagents, catalog no.

BP2436-200) supplemented with 1x HALT protease and phos-phatase inhibitor (Thermo Fisher Scientific, catalog no. 78440);

lysates were briefly sonicated and boiled at 95�C. Protein wasquantified using the DC Protein Assay (Bio-Rad, catalog no.5000116). Whole-cell lysates (30–50 mg of protein per well) wereelectrophoresed on 4%–20% Tris-Glycine gels (Novex, ThermoFisher Scientific) and transferred using a semi-dry method tonitrocellulose (Bio-Rad, catalog no. 170-4159). Membranes wereblocked in 5% milk diluted in 1� Tris-buffered Saline þ 0.1%Tween-20 (1� TBST), probed with primary antibodies diluted in5% BSA in 1� TBST, and incubated with horseradish peroxidase–conjugated secondary antibodies diluted in 5%milk. Protein wasvisualized using SuperSignal West Femto ChemiluminescentSubstrate (Thermo Fisher Scientific, catalog no. 34095) andimaged with a Bio-Rad ChemiDoc XRS. Antibodies and condi-tions are listed in Supplementary Table S2.

ImmunofluorescenceHigh-content cell imaging and subsequent analysis were con-

ducted as described previously (12, 13). Briefly, neuroblastomacells, PANC-1, or primary melanocytes were seeded in clear-bottom black 96-well plates coated with poly-D-lysine. Aftertransfection with siRNA and/or treatment with prexasertib, cellswere fixed in 3.7% formaldehyde (Sigma, catalog no. F-1268) or1� PREFER (Anatech Ltd, catalog no. 414) in D-PBS, permeabi-lized with 0.1% Triton X-100 in D-PBS, and blocked with 1% BSAin D-PBS. Cells were incubated with primary antibodies over-night, followed by three washes and incubation with secondaryantibodies for 1 hour at room temperature. Antibody details areshown in Supplementary Table S2. DNA was stained withHoescht 33342. TUNEL was performed using the In Situ CellDeath Detection Kit, Fluorescein (Sigma Aldrich, catalog no.11684795910). Cells were imaged using a CellInsight NXT plat-form and analyzed by the TargetActivation V.4 Bioapplication(Thermo Fisher Scientific). Percent responders (percent positivefor desired marker) were gated on the basis of the DMSO-treatedgroup for each cell line.

RNAi-mediated knockdownIndividual and pooled siRNA against CHK1 and nontargeting

siRNA were purchased from Dharmacon (Supplementary TableS3). Neuroblastoma and control cells were reverse-transfectedwith siRNA using the Lipofectamine-RNAiMax according to themanufacturer's protocol (Thermo Fisher Scientific, catalog no.13778-075). Degree of knockdown was evaluated 72 hoursposttransfection by Western blot analysis. For experiments eval-uating the effects of prexasertib treatment on CHK1 knockdowncell lines, treatment commenced 48 hours posttransfection for anadditional 24 hours.

In vivo evaluation of prexasertibIn vivo studies were approved by the Eli Lilly and Company

Animal Care and Use Committee. To evaluate the effects ofprexasertib on neuroblastoma xenograft growth, cells were har-vested during log phase growth and resuspended in Hank bal-anced salt solution (HBSS). Suspended cells were diluted 1:1 withBD Matrigel Matrix (catalog no. 356234) and 5 � 106 cells (0.2-mL cell suspension) were injected subcutaneously into the rightflank of female CB-17 SCID beige mice. Tumors were monitoredbeginning 7 days after injection. When tumor volumes averaged200 or 500 mm3, mice were randomized into treatment groups(n¼ 6/group) based on tumor volume and bodyweight. Animalswere given vehicle (20% Captisol in water, pH 4) or 10 mg/kg

Translational Relevance

Prexasertib is a small-molecule inhibitor of checkpointkinase 1 (CHK1), currently in clinical evaluation in adultcancer patients. A previous study identified CHK1 as a ther-apeutic target in neuroblastoma, a devastating pediatricmalig-nancy generally treated with an intensive and often ineffectualmultimodal regimen. Here we demonstrate that prexasertib isa potent inhibitor of CHK1 in multiple preclinical models ofneuroblastoma, resulting in DNA damage and tumor celldeath; furthermore, tumor regression was observed in twoxenograft mouse models following prexasertib treatment.These data suggest that neuroblastoma is sensitive to prexa-sertib-mediated CHK1 inhibition and further supports assess-ment of prexasertib in pediatric patients in an ongoing clinicaltrial (NCT02808650).

Prexasertib Induces Regression of Neuroblastoma Models

www.aacrjournals.org Clin Cancer Res; 23(15) August 1, 2017 4355




prexasertib by subcutaneous injection twice daily for 3 days,followed by 4 days of no treatment for a total of 4 weeks[(twice daily� 3 days, rest� 4days)� 4weeks)], unless otherwiseindicated in the figure legend. For combination studies, two studyarms were added and animals were given 5 mg/kg doxorubicinintravenously with or without 10 mg/kg prexasertib [(twice daily� 3 days, rest � 4 days )� 4 weeks].

To evaluate the effects of prexasertib on tumor health andangiogenesis, cells were injected and tumors monitored asdescribed above. Animals were sacrificed 6 days after the begin-ning of treatment and tumors were promptly excised and fixed in10% neutral buffered formalin. Multiplexed immunohistochem-ical analysis was performed on vehicle- and prexasertib-treatedtumors as described previously (14). TUNELwas performed usingthe Roche kit (catalog no. 12156792910) and antibodies aredescribed in Supplementary Table S2.

ResultsPediatric cancer cell lines are highly sensitive to prexasertib

Prexasertib was evaluated across a panel of well-characterized,commercially available cancer cell lines encompassing a widespectrumof adult andpediatricmalignancies. The EC50s of severalpediatric tumor types, including neuroblastoma, fell below theaverage plasma concentration [Cavg at 24 hours postinfusion(46.9 ng/mL)] reported in a phase I trial in adult carcinomapatients following the dosing schedule of 105mg/m2 via infusionon Day 1 every 14 days (Fig. 1; Supplementary Table S4; ref. 10).When compared with a series of SOC in vitro, single-agent pre-xasertib wasmore potently antiproliferative in 19 pediatric cancer

cell lines (Table 1). Five neuroblastoma cell lines were selected fordetailed characterization with respect to CHK1 inhibition in thistumor type. Primary neonatal melanocytes share the neural crestlineage with neuroblastoma and are derived from infant foreskin,providing an age-appropriate normal cell control (15). The adultpancreatic cancer cell line PANC-1, previously reported to have anEC50 of prexasertib above 1 mmol/L, served as an intrinsicallyprexasertib-resistant cell line (8). Consistentwith the results of thecancer cell line sensitivity panel, additional cellular assays con-firmed the antiproliferative activity of prexasertib in the lownanomolar range (Fig. 2A).In addition, prexasertib induced apo-ptosis, as evidenced by increased activation of capases 3 and 7,within 24 hours of treatment in the majority of neuroblastomalines evaluated (Supplementary Fig. S1).

Neuroblastoma cell death is observed following prexasertib-induced DNA damage in vitro

Upon replication stress and/or DNA damage, CHK1 is directlyphosphorylated on serines 317 and 345 (S317 and S345) by theDNA damage sensing kinase ataxia telangiectasia and Rad3-relat-ed (ATR) in anATM-independent or -dependentmanner, depend-ing on the nature of the genotoxic stress (16–19). Phosphoryla-tion at both S317 and S345 is required for autophosphorylationof CHK1 at serine 296 (S296), resulting in full activation of thekinase (20, 21). As increased transcriptional activity can causereplication stress, neuroblastoma cell lines with high MYC levelsdue to gene amplification or increased expression were selectedfor further analysis. Endogenous levels of C-MYC and N-MYCprotein were confirmed via Western blot analysis of KELLY,

Figure 1.

Cancer cell sensitivity profile of prexasertib. Prexasertib was evaluated for efficacy in a panel of more than 300 adult and pediatric cancer cell lines. AverageEC50 values for proliferation for a subset of these lines after prexasertib treatment is displayed here and are grouped by histology. Several pediatric tumor types,including neuroblastoma, exhibited sensitivity to prexasertib treatment over two cell doublings, with EC50 values within the clinically achievable range based on theaverage plasma concentration 24 hours postinfusion (Cavg, 24) reported in the phase I trial in adult patients with solid tumors (Hong 2016). NS, not specified.

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NBL-S, and SH-SY5Y lysates (Fig. 2A). Baseline phosphorylationof CHK1 at S296has been reported inKELLY cells (9); in addition,endogenous CHK1 S296 was detected in NBL-S and SH-SY5Y aswell as PANC-1 (Fig. 2B). Prexasertib treatment over 24 or 48hours reducedphosphorylation atCHK1S296 (Fig. 2B) and led to

an accrual of gH2AX-positive double-strand DNA breaks in neu-roblastoma cell lines, primary melanocytes, and PANC-1 cells(Fig. 3; Supplementary Table S5). In response to CHK1 inhibitionand the resulting double-strand breaks, activated DNA damagesensors ataxia telangiectasia mutated (ATM; phosphorylated at

Table 1. EC50 (mmol/L) comparison of prexasertib and SOC in pediatric cancer cell lines

Cell line Cancer type Prexasertib Doxorubicin Cisplatin Gemcitabine

MOLT-4 <0.001 0.026 1.121 0.014MOLT-3 Acute lymphocytic leukemia <0.001 0.006 0.227 0.003CCRF-CEM <0.001 0.036 1.443 0.017RD-ES

Ewing sarcoma0.003 0.267 2.944 0.070

SK-NM-C <0.001 0.074 0.214 0.010DAOY

Medulloblastoma<0.001 0.072 1.170 0.035

D238 <0.001 0.138 2.407 0.007KELLY <0.001 0.030 1.660 0.002TGW Neuroblastoma 0.001 0.190 3.499 0.029IMR-32 <0.001 0.010 0.005 0.004SH-SY5Y <0.001 0.038 0.420 0.034SJSA1 0.001 >0.200 13.260 >0.200HOS Osteosarcoma <0.001 0.040 6.774 >0.200SAOS-2 0.001 0.043 1.445 0.002Y79 Retinoblastoma 0.001 0.028 1.383 0.001A204 Rhabdoid <0.001 0.027 2.400 0.003TE 381.T 0.001 0.026 2.040 0.003SJCRH30 Rhabdomyosarcoma <0.001 0.007 1.384 0.001RD <0.001 0.013 0.781 0.002

Figure 2.

Prexasertib reduces neuroblastomacell proliferation and inhibits CHK1autophosphorylation. A, Neuroblastomacell lines, PANC-1, and primarymelanocytes were assayed forproliferation after 72 hours of prexasertibtreatment and EC50 values werecalculated. Experiments were repeatedin triplicate, and error bars representSEM.B,After 24 or 48 hours of treatmentwith 50 nmol/L prexasertib, cells werelysed, and the indicated total andphosphorylated proteins were assessedby Western blot analysis.






serine 1981) and DNA protein kinase-catalytic subunit (DNA-PKcs; phosphorylated at serine 2056) localized to the nucleus inKELLY and NBL-S posttreatment (Supplementary Fig. S2A; Sup-

plementary Table S5). Activation of the DNA damage responsewas confirmed by concomitant increases in phosphorylation ofCHK2 at threonine 68 andCHK1 at S345,which are target sites for

Figure 3.

Prexasertib-induced double-strand DNAbreaks leads to neuroblastoma cell death invitro. KELLY, NBL-S, PANC-1, and primarymelanocytes were incubated with DMSO or50 nmol/L prexasertib for 24 hours andsubsequently fixed. Cells wereimmunostained for gH2AX (green) andcleaved PARP (red). DNA was stained withHoescht 33342 (blue). Representative singlechannel and composite images taken with a20� objective using the appropriate filtersare shown. Experiments were repeated atleast twice.

Lowery et al.





ATM and ATR (Fig. 2B). Furthermore, both total and phosphor-ylated replication protein A 32/2 (RPA32/2), necessary for stabi-lizing stalled replication forks and coating single-strand DNA,were elevated within 24 hours of prexasertib treatment (Supple-mentary Fig. S3). Interestingly, although prexasertib led to dou-ble-strand DNA breaks in all of the cell lines, apoptosis wasobserved in neuroblastoma cells, but not PANC-1 or primarymelanocytes, as measured by increased cleaved PARP, cleavedcaspase-3, and TUNEL (Figs. 2B and 3; Supplementary Fig. S2B;Supplementary Table S5). This dichotomous outcome was notlinked to the expression of cyclin-dependent kinase 2 (CDK2) orthe phosphatase CDC25A, two proteins previously shown to benecessary for prexasertib efficacy (Supplementary Fig. S3.)

Anti-neuroblastoma effects of prexasertib are linked specificallyto CHK1 inhibition

While prexasertib preferentially binds to and inhibits the activ-ity of CHK1, prexasertib also inhibits checkpoint kinase 2 (CHK2)with an IC50 of <10 nmol/L in vitro, while inhibition of CHK2autophosphorylationwas achievedwith an IC50 of <31 nmol/L incellular assays (8). To further validate CHK1 as the functionaltarget of prexasertib in neuroblastoma, CHK1 and CHK2 weretransiently knocked down using siRNA in KELLY, which was theneuroblastoma cell line most sensitive to prexasertib and theintrinsically resistant cell line PANC-1, either individually or incombination, and then treated with DMSO or prexasertib (Fig. 4;Supplementary Fig. S4). Depletion of CHK1, but not CHK2,increased gH2AX and cleaved PARP in the absence of prexasertibin KELLY cells (Fig. 4A). Furthermore, knockdown of CHK2 didnot alter the antiproliferative response of KELLY cells to prexa-sertib treatment, while siCHK1 drastically reduced proliferationrelative to the siNT control (Fig. 4B). Prexasertib treatment ofKELLY cells with siCHK1 did not alter the observable effects onPARP cleavage or gH2AX levels. As expected due to its observedintrinsic resistance to prexasertib, PANC-1 cell proliferation wasnot affected by CHK1 or CHK2 knockdown.

Pharmacologic inhibition of CHK1 results in regression ofneuroblastoma xenografts

As prexasertib reduced neuroblastoma cell viability in vitro,prexasertib was evaluated in vivo using IMR-32 or KELLY subcu-taneous xenograft mouse models. To examine the effects ofprexasertib in the context of primary lesion size, treatment wasinitiated when tumors reached an average volume of 200 or

500 mm3. Importantly, clinically relevant plasma concentrationsof prexasertib were achieved in these mouse models using the 3days on twice daily treatment, 4 days rest dosing schedule (8, 10).

Durable complete regressions were observed in prexasertib-treated animals harboring KELLY xenografts, regardless of initialtumor size (Fig. 5A). Similarly, IMR-32 xenografts initially andcompletely regressed upon administration of prexasertib. How-ever, unlike KELLY xenografts which did not regrow after the endof treatment, IMR-32 tumor regrowth was observed 4 weeks aftertreatment cessation (Fig. 5C). The reemergent tumors retainedsome sensitivity to prexasertib as evidenced by an ensuing partialregression; however, complete regression was not achieved fol-lowing rechallenge with drug. No significant changes in bodyweight were observed during the treatment period (Supplemen-tary Fig. S5).

Combination with SOC does not enhance tumor growthinhibition

First-generation CHK1 inhibitors have been used as chemopo-tentiators, enhancing the genotoxic stress triggered by DNA-dam-aging chemotherapies via abrogation of cell-cycle checkpoints(22). Recent studies have demonstrated that single agent CHK1inhibition in the absence of exogenous DNA damage is sufficientto cause tumor cell death (8, 23). While single-agent prexasertibwas sufficient to induce KELLY xenograft regression in mousemodels, this was not the case in IMR-32 and the C-MYC–drivenneuroblastoma model SH-SY5Y, suggesting combination treat-ment may yield synergistic antitumor effects. To evaluate thecombinatorial effects of prexasertib plus SOC, IMR-32 and SH-SY5Y xenograft models were treated with doxorubicin, which hasbeen used in multimodal pediatric neuroblastoma treatmentregimens for more than 40 years (24–26), with/without prexa-sertib (Supplementary Fig. S6). No superior effects on tumorgrowth inhibition were observed with combination treatmentwhen comparedwith the single-agent prexasertib arms, indicatingthat using this dose and schedule doxorubicin treatment neitherenhances prexasertib efficacy nor prevents tumor recurrence inthese preclinical neuroblastoma models.

Prexasertib alters endothelial andpericytemarker expression inneuroblastoma xenografts

Because of the rapid regressionof tumors upon initial treatmentwith prexasertib, two additional study arms were sacrificed 6 daysafter dosing commenced. These tumors were subjected to

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Figure 4.

CHK1 is the functional target ofprexasertib in neuroblastoma. CHK1 andCHK2 were knocked down individuallyand in combination with siRNA for 72hours, then treated with 50 nmol/Lprexasertib for an additional 24 hours.A,Whole-cell lysates were analyzed byWestern blot analysis for expressionand/or phosphorylation of theindicated proteins. B, Effects of siRNAagainst CHK1 or CHK2 with or withoutadditional prexasertib treatment on cellproliferation were evaluated byCellTiter Glo in KELLY (top) and PANC-1(bottom). Error bars represent SEMfrom technical triplicates.






multiplexed IHC to assess tumor health and angiogenesis (Fig. 5BandD; Supplementary Fig. S7). Cell proliferation, asmeasured byKi67, significantly decreased with prexasertib treatment in bothtumor models, although there was more Ki67 after treatment inIMR-32 cells (Fig. 5D). The number of TUNEL–positive tumorscells did not change within the first 6 days of treatment. Surpris-ingly, increased coincident expression of vascular markers MECA-32, CD31, and smooth muscle actin (SMA) was detected inprexasertib-treated xenografts compared with the control, indi-cating an enrichment of pericyte-covered vessels in both models(Supplementary Fig. S7). Furthermore, significantly fewer smallvessels were observed in prexasertib-treated IMR-32 xenografts,while an increase in the number of large vessels was noted(Supplementary Fig. S7C).

Prexasertib does not affect established endothelial cords in vivoThe increase in vascular markers and large vessels combined

with fewer smaller vessels following prexasertib treatment ofneuroblastoma xenografts suggested that prexasertib may inhibitthe formation of new vessels while leaving the established vas-culature relatively unaffected. Therefore, we utilized an in vitroendothelial cord formation assay to model the potential activityof prexasertib on the vascular endothelium. Treatment with

increasing concentrations of prexasertib strongly reducedVEGF-driven cord formation, as measured by total tube area,back to basal levels but did not significantly alter establishedcords (Supplementary Fig. S8). In addition, we concluded that theprexasertib-dependent decrease in cord formation was not due totreatment effects on the feeder layer. The ability of neuroblastomacell lines to support cord formation was also investigated in atumor-driven cord formation assay; the total tube area of neuro-blastoma-driven cords was comparable with VEGF treatment(Supplementary Fig. S9A and S9B). Furthermore, IMR-32, KELLY,and SH-SY5Y cells were found to produce VEGF, VEGF-C, andVEGF-D in coculture conditions, suggesting that these neuroblas-toma cell lines could promote neovascularization in vitro and invivo (Supplementary Fig. S9C).

DiscussionCHK1 is a master regulator of replication fork licensing and of

cell-cycle checkpoints in response to genotoxic stress and DNAdamage. Inhibition of its activity by either therapeutic agents orRNA interference leads to excessive replication origin firing, expos-ing single-strand DNA to endonucleases and resulting in double-strand DNA breaks. In addition, abrogation of the intra-S-phase

Figure 5.

Neuroblastoma xenografts rapidly regress during prexasertib treatment. KELLY (A) or IMR-32 (C) xenograft models were treated subcutaneously with vehicleor 10 mg/kg prexasertib twice daily following a 3 day on, 4 day off dosing schedule for 4 weeks (n ¼ 5 for all arms). KELLY (B) and IMR-32 (D) xenograftswere harvested after 6 days of vehicle or prexasertib treatment and subjected to an immunofluorescence-based tumor health panel. Cell proliferation, celldeath, and vessel-positive area were measured by Ki67, TUNEL, and MECA-32 immunostaining, respectively. Average percent positive area (¼ 100 � marker þphantoms/total Hoescht þ phantoms) for each group (KELLY: n ¼ 5/group; IMR-32: n ¼ 6/group) � SEM is displayed below representative images taken ata 10X magnification. Vehicle: *; prexasertib starting at 200 mm3 average tumor volume: &; prexasertib starting at 500 mm3 average tumor volume: ~.Thin and thick dashed lines represent dosing period for 200 mm3 and 500 mm3 starting tumor volume, respectively. � , P < 0.05; �� , P < 0.0001.

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and G2–M cell-cycle checkpoints allows for unbridled progres-sion through the cell cycle regardless of the integrity of thegenome. Prexasertib (LY2606368), a second-generation small-molecule inhibitor of CHK1, promotes cell death through rep-lication catastrophe in solid tumor models, leading to clinicalevaluation of the drug in adult cancer patients (8, 10). In thisstudy, we report that prexasertib is potently antiproliferative inmodels of pediatric cancer, especially neuroblastoma. Further-more, prexasertib induces regression of neuroblastoma xeno-grafts in mouse models. High-risk neuroblastoma is a devastat-ing pediatric malignancy responsible for approximately 10% ofpediatric cancer deaths annually and leaves survivors with debil-itating long-term side effects following severe combinatorialtherapeutic regimens.

Previously, an RNAi screen identified CHK1 as a therapeutictarget in neuroblastoma cell lines. Furthermore, recent studieshave shown that treatment with CHK1 inhibitors, either alone orin combination with systemic or targeted agents, was sufficient tokill neuroblastoma cells (9, 27). As a single agent, prexasertib wasmore potent in pediatric cancer cell lines than several current SOCagents. Although efficacy was not directly compared with othertargeted agents, prexasertib treatment reduced cell proliferation inpediatric cell lines at low nanomolar concentrations, approxi-mately 10- to 100-fold less than EC50 values reported for first-generation CHK1/2 inhibitors in neuroblastoma models (9, 27,28). Prexasertib can also inhibit CHK2, though it is unlikely thatblockade of CHK2 kinase activity contributed to the rapid celldeath observed following drug treatment. CHK2 is a potentialtumor suppressor gene in adult tumors due to its role in theDNA damage response and regulation of p53; however, onco-genic alterations or mutations of CHEK2 are rare in neuroblas-toma and other pediatric tumor types, suggesting that CHK2activity is not essential for neuroblastoma tumorigenesis (29).Indeed, knockdown of CHK2 in KELLY cells did not increaseDNA double-strand DNA breaks, induce the DNA damageresponse, or cause cell death, while depletion of CHK1 reca-pitulated the effects of prexasertib treatment, validating thatCHK1 is the functional target of prexasertib in neuroblastoma.Importantly, treatment of CHK1-depleted cells with prexasertibdid not enhance PARP cleavage or gH2AX levels, indicating thatoff-target effects of prexasertib resulting in DNA damage andcell death are unlikely and furthering the notion that theefficacy of prexasertib in neuroblastoma is primarily a resultof CHK1 inhibition.

The tumor suppressor p53 is a key mediator of the G1 cell-cycle checkpoint in response to DNA damage (30, 31). In p53-deficient tumors, cells are more reliant on the intra-S and G2–Mcheckpoints regulated by CHK1 to maintain a level of genomicstability necessary for continued proliferation and survival. Inprimary neuroblastoma, TP53mutations are rare (32); however,the p53 pathway has been shown to be repressed due to directinhibition by the ubiquitin ligase MDM2 and/or loss of keynodes within the pathway which could prevent activation of theG1 checkpoint (33, 34). In addition, mutant TP53 and/or inac-tivation of the p53 pathway have been identified in some celllines derived from recurrent tumors (35–37). All neuroblastomacell lines evaluated in this study have intact wild-type p53(33, 38, 39) and yet are highly sensitive to prexasertib treatment.Although other small molecule inhibitors of CHK1 have beenshown to be particularly effective in combination with SOC inp53-deficient tumors due to more permissive S-phase entry of

cells with damaged DNA (28, 40, 41), previous data along withthis study suggests that p53 status does not influence prexasertibefficacy as a monotherapy (8).

In addition to its well-characterized role in enforcing the DNA-damage response through monitoring the intra-S and G2–Mphase cell-cycle checkpoints, CHK1 is also responsible for regu-lating replication fork licensing tominimize replication stress andmaintain genome integrity. Therefore, it is possible that inhibi-tion of CHK1 may lead to DNA damage and cell death due toreplication catastrophe in some tumor types where replicationstress is inherently high due to elevated rates of cellular prolifer-ation or expression of specific oncogenes. For example, augment-ed replication origin firing and increased replication stress havebeen reported in mammalian cells with increased expression ofthe MYC family of transcription factors (42). Genomic amplifi-cation ofMYCN is observed in approximately 20% of neuroblas-toma cases and denotes poor prognosis (4). MYC family expres-sion is prevalent in established neuroblastoma cell lines, with theamplicon present in KELLY, IMR-32, and MHH-NB-11, and highC-MYC was expression detected in SH-SY5Y, which may contrib-ute to the sensitivity of neuroblastoma to CHK1 inhibition.However, prexasertib has not yet been evaluated in neuroblasto-ma cell lines with endogenously low levels of N-MYC and C-MYCexpression with or without the MYCN amplicon to fully under-stand the contribution of MYC activity to prexasertib sensitivity.Interestingly, a reduction in N-MYC or C-MYC protein wasobserved in neuroblastoma and PANC-1 cells following 48-hourtreatment with prexasertib (Fig. 2B), suggesting that inhibition ofCHK1 activity may potentially influence MYC protein expressionor stability.

An unexpected finding of our study is the effect of prexasertibon xenograft vasculature, whereby drug treatment resulted in aninflux of vascular markers and an increase in large vessels, whilethe number of smaller vessels was diminished. In vitro investiga-tions revealed that neuroblastoma cell lines can support endo-thelial cord formation, most likely due to the production andsecretion of VEGF ligands. Furthermore, although prexasertibtreatment blocked new cord formation, it was unable to alterestablished cords, suggesting that prexasertib blocks neovascular-ization in vivo. Therefore, it is possible that the observed increasein the number of large vessels is a consequence of rapidly regres-sing xenografts, resulting in the same number of mature vesselswithin a smaller tumor area. Our data indicate that single-agentprexasertib, a potent CHK1 inhibitor, promotes DNA damageleading to tumor cell death and xenograft regression in preclinicalmodels of neuroblastoma. Inhibitors of CHK1 have traditionallybeen used in combination with SOC, to enhance the genotoxiceffects of these chemotherapeutic agents (5, 41, 43). However,these data add to the growing body of literature that indicates thatCHK1 inhibitors also have activity as a single agent (5, 10, 44, 45).In this study, cotreatment with doxorubicin did not enhance theefficacy of prexasertib nor prevent tumor regrowth in two neu-roblastoma mouse models. However, combination with doxo-rubicin in a preclinical model of alveolar rhabdomyosarcoma, apediatric malignancy driven by a fusion transcription factor, wassufficient to block tumor regrowth and acquired resistance toprexasertib (46). Therefore, efficacious combination of prexaser-tib with chemotherapeutics may be tumor-type dependent. Selec-tion and validation of different genotoxic therapies as combina-tion partners as well as alterations in dosing schedule (e.g., CHK1inhibition following administration of SOC) may improve the






degree or duration of response in neuroblastoma. In addition,prexasertib efficacy may be enhanced through combination withother targeted agents, such as an inhibitor against WEE1, whichhas shown synergistic drug interactions with first-generationCHK1 inhibitors in preclinical neuroblastoma models (27). Inpreliminary studies conducted by our group, WEE1 phosphory-lation decreased with prexasertib treatment in a time-dependentmanner in KELLY cells, while treatment did not affect WEE1phosphorylation in prexasertib-resistant PANC-1 cells (data notshown). Further investigation into potential biomarkers of pre-xasertib sensitivity as well as effective combination therapies toimprove efficacy and prevent tumor recurrence in preclinicalmodels of neuroblastoma are warranted.

Disclosure of Potential Conflicts of InterestR.P. Beckmann and A.B. Lin hold ownership interest (including patents) in

Eli Lilly and Company. No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: C.D. Lowery, M. Dowless, J. Stephens, R.P. Beckmann,L.F. StancatoDevelopment of methodology: A.B. VanWye, J. Stewart, L.F. Stancato

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C.D. Lowery, M. Dowless, W. Blosser, B.L. Falcon,J. Stewart, J. StephensAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C.D. Lowery, A.B. VanWye, M. Dowless, B.L. Falcon,J. Stewart, J. Stephens, A.B. Lin, L.F. StancatoWriting, review, and/or revision of themanuscript:C.D. Lowery, A.B VanWye,M. Dowless, B.L. Falcon, J. Stephens, R.P. Beckmann, A.B. Lin, L.F. StancatoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.D. Lowery, W. BlosserStudy supervision: L.F. Stancato

AcknowledgmentsThe authors would like to thank Dr. Sean Buchanan and Dr. YueWebster for

their assistance with the Cancer Cell Sensitivity Profile database.

Grant SupportThis study was funded by Eli Lilly and Company, Lilly Corporate Center.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 15, 2016; revised December 13, 2016; acceptedMarch 2,2017; published OnlineFirst March 7, 2017.

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2017;23:4354-4363. Published OnlineFirst March 7, 2017.Clin Cancer Res Caitlin D. Lowery, Alle B. VanWye, Michele Dowless, et al. of Preclinical Models of Human NeuroblastomaThe Checkpoint Kinase 1 Inhibitor Prexasertib Induces Regression

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