LY2606368 Causes Replication Catastrophe and Antitumor ......catastrophe. Changes in the ratio of...

Small Molecule Therapeutics

LY2606368 Causes Replication Catastrophe andAntitumor Effects through CHK1-DependentMechanismsConstance King1, H. Bruce Diaz1, Samuel McNeely1, Darlene Barnard1, Jack Dempsey1,Wayne Blosser1, Richard Beckmann1, David Barda2, and Mark S. Marshall1

Abstract

CHK1 is a multifunctional protein kinase integral to both thecellular response to DNA damage and control of the number ofactive replication forks. CHK1 inhibitors are currently underinvestigation as chemopotentiating agents due to CHK1's role inestablishing DNA damage checkpoints in the cell cycle. Here, wedescribe the characterization of a novel CHK1 inhibitor,LY2606368, which as a single agent causes double-stranded DNAbreakage while simultaneously removing the protection of theDNAdamage checkpoints. The action of LY2606368 is dependentupon inhibition of CHK1 and the corresponding increase inCDC25A activation of CDK2, which increases the number ofreplication forks while reducing their stability. Treatment of cellswith LY2606368 results in the rapid appearance of TUNEL andpH2AX-positive double-stranded DNA breaks in the S-phase cell

population. Loss of the CHK1-dependent DNA damage check-points permits cells with damaged DNA to proceed into earlymitosis and die. The majority of treated mitotic nuclei consist ofextensively fragmented chromosomes. Inhibition of apoptosis bythe caspase inhibitor Z-VAD-FMK had no effect on chromosomefragmentation, indicating that LY2606368 causes replicationcatastrophe. Changes in the ratio of RPA2 to phosphorylatedH2AX following LY2606368 treatment further support replica-tion catastrophe as the mechanism of DNA damage. LY2606368shows similar activity in xenograft tumormodels, which results insignificant tumor growth inhibition. LY2606368 is a potentrepresentative of a novel class of drugs for the treatment of cancerthat acts through replication catastrophe. Mol Cancer Ther; 14(9);2004–13. �2015 AACR.

IntroductionTraditional chemotherapeutics that induce DNA damage are

the most widely used class of anticancer drugs today and willcontinue to be so into the foreseeable future (1). Lacking the strictcontrol of cell division inherent in normal cells, cancerous cellmasses are in general more sensitive to agents targeting DNAintegrity due to an increased fraction of actively replicating cells.Normal replicating cells are somewhat protected from DNAdamage resulting from chemotherapy due to functional cell-cyclecheckpoints and DNA repair processes. Paradoxically, although arapidly growing tumor mass may be sensitive to DNA-damagingchemotherapy, individual cancer cells are relatively tolerant ofgenomic damage because of resistance to cell senescence andapoptosis (2). Depending upon the functionality of each of theDNA repair pathways in a tumor, certain types ofDNAdamage areless well tolerated than others. Many types of DNA-damaging

therapeutics have been developed that utilize widely differingmechanisms of action (3). These differences in mechanism makeit possible to find a therapy that is effective against cancers that areresistant to other types of DNA-damaging agents. Chemothera-peutic drugs are classified by mechanism, chemical structure, orsimilarity to other agents. On the basis of these criteria, there arefour main groups of DNA-damaging chemotherapeutics: alkylat-ing agents, antimetabolites, topoisomerase inhibitors, and anti-tumor antibiotics. Most chemotherapeutics also have nonspecificcytotoxic effects that can contribute to patient toxicity (3).

A recent strategy to improve the action ofDNA-damaging drugsin cancer treatment is toprevent the activationof theDNAdamagecheckpoints in tumor cells during treatment with chemotherapy(4). Upon sensing DNA damage, normal cells stop progressionthrough the cell cycle at specific points in theG1, S, andG2 phases.Thepurpose of these checkpoints is toprovide the cellwith time torepair breaks and chemical damage to DNA and to determine thefinal fate of the cell. Mutated in the majority of cancers, the p53protein is the primary regulator of the G1 and G2–M checkpointsand controls the decision tree leading to cell survival, death, orsenescence (5). Although loss of p53 function occurs in mosthuman tumors, sufficient pathway redundancy exists tomaintainfunctional intra-S and G2–M checkpoints. One key regulator ofthese two checkpoints is the checkpoint kinase 1 (CHK1; ref. 6). Inthe presence of DNA damage, CHK1 is activated through phos-phorylation by the ataxia-telangiectasia andRAD3-related protein(ATR) leading to the phosphorylation of the CDC25 phospha-tases leading to the degradation of CDC25A and the nuclearexclusion of CDC25B and CDC25C. CDC25A is a key regulatorof CDK2 and DNA replication. CHK1-mediated loss of CDC25A

1Oncology Discovery Research, Lilly Research Laboratories, Lilly Cor-porateCenter, Eli Lilly andCompany, Indianapolis, Indiana. 2ChemistryDiscovery Research, Lilly Research Laboratories, Lilly Corporate Cen-ter, Eli Lilly and Company, Indianapolis, Indiana.

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

Corresponding Author: Mark S. Marshall, Oncology Discovery Research, LillyResearch Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indiana-polis, IN 46285. Phone: 317-433-2506; Fax: 317-276-6510; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-14-1037

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

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activity suspends CDK2 in an inactive phosphorylated stateblocking initiation of DNA replication origins. Cytosolic seques-tration of CDC25B and CDC25C prevents the activation of CDK1resulting in cell-cycle arrest at the G2–M boundary. A number ofCHK1 inhibitors have been developed and entered the clinic ascheckpoint inhibitors to increase the efficacy of chemotherapy inpatients with p53-mutant cancer (7, 8).

One unexpected consequence of CHK1 inhibition is the gen-eration of double-stranded DNA breaks (DSB; ref. 9). WithoutCHK1 to regulate CDC25A during a normal cell cycle, CDK2activity is increased leading to unscheduled DNA replicationinitiating at both normal and dormant replication origins (10).Simultaneous activation of such an excess of replication originsresults in the slowing and stalling of replication forks, apparentlydue to an insufficient number of replication proteins (11, 12). In acascade effect, loss of CHK1 activity may also lead to fork insta-bility and collapse. A recent study using inhibitors of ATR andCHK1 reported that the abundance of ssDNA during replicationstress exhausts the available pool of RPA increasing the likelihoodthat unprotected ssDNA will be cleaved by endonucleases (13).Stalled forks are believed to be repaired primarily, but not exclu-sively, by homologous recombination (14, 15). The resultingDNA structure intermediates generated during homologousrecombination repair (HRR; i.e., Holliday junctions) are cleavedby endonucleases such as MUS81/EME1 yielding a double-stranded break. However, inhibition of CHK1 function alsoprevents localization of RAD51 to the invading repair strandduringHRR,maintaining the accumulated breaks in the collapsedforks (16). Disaster continues to escalate for the CHK1 inhibitedcell as the loss of the intra-S checkpoint permits the cell tocontinue up to the G2–M checkpoint with broken DNA (7).Ultimately, the cell enters mitosis with fragmented chromosomesresulting in cell death. Cell death caused by failure of the ATR/CHK1 axis during replication stress has been described as repli-cation catastrophe (13).

Although a number of chemotherapeutics yield DSB, CHK1inhibitors are unique in that not only do they causeDNAdamage,but also abrogate critical DNA damage checkpoints and hamperHRR. One such inhibitor is LY2606368. It is currently in clinicaldevelopment as a single agent and in combination with bothcytotoxic and targeted agents. In this report, we describe thebiochemical and biologic properties of LY2606368. LY2606368causes replication catastrophe in vitro and in vivo and is unique inits mechanism of action from all of the major classes of DNA-damaging agents.

Materials and MethodsCell culture and antibodies

HeLa cervical cancer cells (lot 2619582obtained in 2003),NCI-H460 non–small cell lung cancer cells (lot 1613811 obtained in2001), PANC-1 pancreas cancer cells (lot 1077384 obtained in2000), HT-29 colon cancer cells (lot 2463682 obtained in 2003),and HCT 116 colon cancer cells (lot 1562770 obtained in 2002)were from ATCC. Calu-6 non–small cell lung cancer cells and U-2OS osteosarcoma cells were obtained from ATCC before 2003.Upon receipt from ATCC, each cell line was revived, expanded(two to eight passages) and frozen working stocks prepared. Celllines were maintained as recommended by ATCC and passagednomore than ten times following revival fromworking stock. Thefrozen working stock of each cell line was authenticated in

December 2014 by IDEXX-Radil using STR-based DNA profilingand multiplex PCR. The genetic profiles for the samples wereidentical to the genetic profiles reported for these cell lines.

The following antibodies against corresponding proteins andphosphoproteins were purchased and used according to themanufacturer's instructions. Abbreviations used are "p" for phos-pho protein followed by the amino acid letter code and positionof phosphorylation. Antibodies against CDK2 (#05-596), pH2AX(S139) (#05-636), and pH3 (S10) (#06-570) were from Milli-pore. pCHK1 (S296) (#2349) and pCHK1 (S345) (#2341) anti-bodies were from Cell Signaling Technology. Other antibodieswere to CHK1 (Stressgen, #KAM-CC111), GAPDH (Fitzgerald,#10R-G109a), and RPA32/RPA2 (Abcam, #61184), pRPA32 (S4/S8; Bethyl Labs, #A300-245A). CDC25A (#SC-7389) and donkeyanti-goatHRP (SC-2020)were fromSanta Cruz Technology. Goatanti-mouse-Alexa-555 (#A21422), donkey anti-rabbit-Alexa-555(#A31572), and goat anti-mouse-Alexa-488 (#A11001) werefrom Invitrogen. Goat anti-mouse-Dylight 550 (#84540) wasfrom Thermo Scientific, donkey anti-rabbit HRP (NA934V) andsheep anti-mouse HRP (NA9310V) were from Amersham.

Cell proliferation assayCell proliferation assays were performed as previously

described using aCell Titer-96 AQkit (Promega; ref. 17). AbsoluteIC50 values were calculated in Microsoft EXCEL using an XLFitsoftware add-in (ID Business Solutions).

Test compoundsDoxorubicin (Sigma) was prepared as a 10 mmol/L stock in

H2O. Phorbol 12-myristate 13-acetate (Sigma)was prepared as a10 mmol/L stock in ethanol. SN38 (Tocris Bioscience), Z-VAD-FMK (R&D Systems), BI-D1870 (Symansis), U0126 (Alexis), andstaurosporine (Eli Lilly & Co.) were prepared as separate 10mmol/L stocks in DMSO. Hydroxyurea (HU; Calbiochem) wasprepared as a 1 mol/L stock in H2O. LY2606368 (Eli Lilly & Co.)was prepared as a 10mmol/L stock inDMSO for in vitrouse and in20% Captisol (CyDex Inc), pH4, for in vivo use.

High content cell imagingHigh content cell imaging and analysis were performed using

the Thermo Scientific Cell Insight NXT platform and softwaresuite as described (18). Cells (2,500–5,000 per well) were platedin 96-well poly D-lysine–coated black clear bottom plates (BDBiocoat). Following appropriate experiment time, the cells wereformaldehyde fixed, permeabilized, then blocked with BSA, andstained according tofigure legends.Hoechst 33342was purchasedfromMolecular Probes. TUNEL assay was performed using the insitu cell death detection kit purchased from Roche Diagnostics.

Chromosome spreadsHeLa cells were plated onto T25 flasks and allowed to recover

for 24 hours. LY2606368 was then added to give final con-centrations of 33 or 100 nmol/L. In some experiments,20mmol/L Z-VAD-FMK was included during the drug treatment.Cells were treated for 12 hours, and during the last 2 hours,colchicinewas added to 1mg/mL. Fixationof nuclei formetaphasespreads was done following the method of Bayani and Squire(19). Chromosome spreads were done with a modification of themethod of Deng and colleagues (20). A 12-mL volume of cellsuspension in 3:1methanol/ acetic acidfixativewas dropped from

LY2606368 Induces Replication Catastrophe

www.aacrjournals.org Mol Cancer Ther; 14(9) September 2015 2005




a height of 3 cm onto dry glass slides or coverslips. The slides werethen heated for 45 seconds on a 43�C metal block, before beingremoved to allow drying to complete at room temperature.Coverslips were mounted on slides with Vectashield Hard Setmounting medium with DAPI (Vector laboratories, Inc.). Slideswere examined with a Leica DMR fluorescence microscope andimages were captured using a SPOT RT3 Slider camera.

siRNA knockdownTransfection of U-2 OS cells with siRNAs followed the Invitro-

gen Lipofectamine RNAiMAX (cat. 13778-075) reverse-transfec-tionprotocol. Cellswere platedwith the transfectionmixtures andtreated with LY2606368 or DMSO 48 hours later. The siRNA usedas a control was the ON-TARGETplus non-targeting pool fromThermo Scientific (cat. D-001810-10-20), whereas theCDK2 (cat.L-003236-00-0005); and CDC25A (cat. SC-29254) targeted siR-NAswere obtained fromDharmacon/ThermoScientific andSantaCruz Biotechnology, respectively. The final concentration ofsiRNA used for each transfection was 20 nmol/L.

Flow cytometryCellswere harvested and thenfixed in ice-cold 70%ethanol and

stored at �20�C. The fixed cells were recovered by centrifugationand thenwashed in PBS. The final cell pellets were resuspended in500mLof 0.1%TritonX-100/propidium iodide solution andwereincubated for 30minutes at room temperature. The samples wereanalyzed on a Beckman Coulter FC500 flow cytometer and cell-cycle profiles were generated using ModFit LT software (VeritySoftware).

Immunoblot analysisThe cells were lysed either in RIPA or Cell Extraction Buffer

(Invitrogen) supplemented with phosphatase inhibitors (Sig-ma) and protease inhibitors (Roche Diagnostics) using ice-coldsonication. The protein concentration of each lysate was firstdetermined using the Pierce BCA Protein Assay Kit (ThermoScientific) followed by dilution with 4 � Laemmli SampleBuffer (21) and heating at 95�C. Proteins were separated usingSDS-PAGE Criterion gels (Biorad), transferred onto Immobi-lon-P (Millipore) membranes. Primary antibodies were incu-bated with the membranes overnight at 4�C and secondaryantibodies coupled to HRP were incubated with the mem-branes for 2 hours at room temperature. Membrane-boundantibody complexes were detected with Pierce Supersignal WestPico or Femto Chemiluminescent Substrate (Thermo Scientif-ic). Immunoblot band intensity was determined using a LAS-4000 imaging system (FUJIFILM Corp) and quantified usingTotalLab gel analysis software (Nonlinear Dynamics LTD).Only the brightness and contrast of each image were adjustedfor optimal printing.

In vivo biochemistry and tumor growth inhibitionFemale CD-1 nu-/numice (26–28 g) from Charles River Labs

were used for this study. Tumor growth was initiated by subcu-taneous injection of 1�106Calu-6 cells in a 1:1mixture of serum-free growth medium and Matrigel (BD Bioscience) in the rearflank of each subject animal. When tumor volumes reachedapproximately 150 mm3 in size, the animals were randomizedby tumor size and body weight, and placed into their respectivetreatment groups. Vehicle consisting of 20%Captisol (CyDex Inc)

pH4 or LY2606368 was administered by subcutaneous injectionin a volume of 200 mL. Four, eight, 12, 24, and 48 hours after drugadministration, blood for plasma drug exposure was extracted viacardiac puncture and assayed on a Sciex API 4000 LC/MS-MSsystem. The xenograft tissue was promptly removed and preparedas previously described (17). Lysates were analyzed by immuno-blot analysis for protein phosphorylation levels. Group means,SEs and P values were calculated using Kronos (22).

To measure xenograft tumor growth inhibition, tumors wereimplanted, established, and the animals randomized as above.Eight animals were used in each treatment group. Vehicle alone orLY2606368 was administered BIDx3, followed by 4 days of restand repeated for an additional two cycles. Tumor size and bodyweight were recorded biweekly and compared between vehicle-and drug-treated groups (23).

ResultsProperties of LY2606368

The properties of LY2606368 have been previously reported atscientific conferences but not yet in a peer-reviewed journal. Thechemical structure of LY2606368 consists of a cyanopyrazinegroup linked to by an amine to a pyrazole core as shown (Fig. 1).The pyrazole core is further substitutedwith a 2,6 dialkoxy phenylgroupwhere one of the alkoxy groups bears a pendant amine. Thegeneral characteristics of the compound are briefly summarizedhere in Table 1 with supporting data to be found in the supple-mentary data section. LY2606368 is an ATP-competitive proteinkinase inhibitor with a Ki of 0.9 nmol/L against purified CHK1(Supplementary Fig. S1). In cell assays measuring CHK1 activitythrough autophosphorylation of serine 296 induced by doxoru-bicin or gemcitabine, LY2606368 had an EC50 of

autophosphorylation with an IC50 of less than 31 nmol/L (Sup-plementary Fig. S3). However, 100 nmol/L LY2606368 did notinhibit PMA-stimulated RSK but instead weakly stimulated phos-phorylation of S6 on serines 235/236 (Supplementary Fig. S4).LY2606368 was broadly antiproliferative with IC50 values typi-cally

chromosomes from the LY2606368-treated cells were shreddedinto hundreds to thousands of single chromosome fragments.Cotreatment of cells with LY2606368 and with the apoptosisinhibitor Z-VAD-FMK had no effect upon the number of mitoticnuclei with fragmented chromosomes (Supplementary Table S4).

Another hallmark of replication catastrophe caused by loss ofATR/CHK1 origin control is depletion of the pool of RPA proteinavailable to protect ssDNA at replication forks (13). This is due tothe excess of open replication forks with exposed ssDNA and canbe measured by high-content image analysis of the coaccumula-tion of RPA2 protein and phosphorylated H2AX (S139) on thechromatin of each cell. When RPA2 becomes limited duringreplication catastrophe,H2AX (S139) phosphorylation continuesto associate with new DSBs. This is characterized by a time-

dependent increase in pH2AX (S139) staining intensity in cellswithout a corresponding increase in chromatin-associated RPA2staining.Graphing theRPA2andpH2AX (S139) staining intensityof each individual nucleus results in a diagnostic upturn inpH2AX(S139) intensity as a function of time when replication catastro-phe is occurring. In order to get a confirmation of whether or notLY2606368 was causing DNA fragmentation through replicationcatastrophe, HeLa cells were treated with either vehicle orLY2606368 over a time span of 0.5 hours to 9 hours. Cells werefixed at the various time points, stained with antibodies specificfor RPA2 and pH2AX (S139) and analyzed by high-contentimagining. RPA2 chromatin localization in S-phase nuclei wasdetected by one hour and increased to a maximum intensity by 3hours, indicating replication stress in the LY2606368-treated cells.

DNA TUNEL pH2AX Merged

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Figure 2.Exposure to LY2606368 results inDNA damage during S-phase. HeLacells were treatedwith 0.4%DMSO, 33nmol/L LY2606368, 100 nmol/LLY2606368, or 100 nmol/LLY2606368 plus 20 mmol/L Z-VAD-FMK for 7 hours. Following fixation,the plate was stained for DNA(Hoechst 33342) and DSB usingTUNEL and an antibody specific forpH2AX (S139). A, representative visualfields of DMSO or LY2606368-treatedcells were photographed with a �10objective using the appropriate filterto capture staining as labeled abovethe figure. B, the relative intensity foreach stain was measured on a singlecell basis as described in Materials andMethods. DNA content (top row) isshown by plotting DNA intensities(x-axis) verses a sliding average forthe number of cells staining at thatintensity (y-axis). pH2AX (S139;middle row) or TUNEL (bottom row)staining intensity was plotted (y-axis)versus the relative DNA content(x-axis) for each cell. The red bar alongthe x-axis designates cells in S-phase.

King et al.

Mol Cancer Ther; 14(9) September 2015 Molecular Cancer Therapeutics2008




H2AX phosphorylation was detected by 3 hours (Fig. 5A). From 6hours on, deflection in each subsequent plot shows how indi-vidual cells continued to phosphorylate H2AX (S139) without anincrease in the colocalization of additional RPA2 (red boxes).While not as pronounced as that reported for an ATR inhibitorplus hydroxyurea (HU), the upturned shape of the plot of thenuclear ratio of RPA2 to pH2AX (S139) following LY2606368treatment is a characteristic fingerprint of cells undergoing repli-cation catastrophe. The time lag between RPA2 loading andH2AX(S139) phosphorylation suggests that ssDNA breakage is notimmediate as has been reported previously for ATR inhibitionduring replication stress (13). Cells were also treated with HU, aDNA-damaging agent that causes extensive stalling of replicationforks and endonuclease-dependent DSB (25, 26). HU-treatedcells were similar to LY2606368 in that replication stress was first

noted by RPA2 association with chromatin by one hour and wasmaximal by 3hours (Fig. 5B).However, HUwas less efficient thanLY2606368 at causing replication catastrophe andDSBs based onlower levels of pH2AX (S139) phosphorylation (red boxes). Thegreatest degree of replication catastrophe was observedwhen cellswere treated together with HU and LY2606368 (Fig. 5C).

LY2606368 causes DNA damage and growth inhibition intumor xenografts

To determine whether LY2606368 also caused DNA damage inin vivo models of cancer, mice bearing Calu-6 tumor xenograftswere dosed with either vehicle or a single subcutaneous dose of15mg/kg LY2606368. Tumors were removed 4 hours after dosingand at increasing intervals of time out to 48 hours. Lysateswere prepared from the tumors, which were then probed by

Figure 4.LY2606368 causes chromosomalfragmentation. HeLa cells weretreated with 0.5% DMSO as a diluentcontrol or 33 nmol/L LY2606368 for 12hours. Cells were harvested andprometaphase chromosome spreadsprepared as described inMaterials andMethods. Chromosomes were stainedwith DAPI and photographed under a�63 objective. Representativespreads for the most commonmorphologies for each condition areshown in the top row. Inset boxesshow regions magnified and shown inthe bottom row.

Figure 3.The DNA damage effects of LY2606368 aredependent upon CDC25A andCDK2. U-2 OS cellswere transfected with siRNA targeting CDC25Aand CDK2 and a scrambled sequence siRNA asa control. At 48 hours after transfection, cellswere treated with 4 nmol/L LY2606368. A andC, after 6 hours LY treatment, cells wereharvested and the relative levels of CDC25A,CDK2, pH2AX (S139), and GAPDH weredetermined by immunoblot analysis. B and D,24 hours following LY2606368 addition,samples were harvested and analyzed by flowcytometry to determine the effects of siRNAsand compound on DNA content.






immunoblotting with antibodies specific for CHK1 autopho-sphorylation and DNA damage (Fig. 6A). Basal CHK1 (S296)autophosphorylation was reduced 3- to 3.5-fold between 4 hoursand 12 hours following dosing with activity restored by 24 hours.CHK1 inhibition tracked with drug exposure in the blood, withmeasured plasma exposures of 7 ng/mL at 12 hours and 3 ng/mLby 24 hours (Fig. 6B). This was in close agreement with a previousexperiment providing an EC50 exposure of 8 ng/mL for in vivoCHK1 inhibition by LY2606368. Phosphorylation of both H2AX(S139) and RPA2(S4/S8) was also detectable at 4 hours afterdosing of LY2606368, showing the rapid occurrence of DNAdamage (Fig. 6A). The intensity of phosphorylation of these twoDDR markers gradually increased out to 24 hours after dosingeven while CHK1 activity returned. DNA damage was still detect-able by 48 hours. The persistence of these markers even 24 hoursafter treatment indicates that the type of damage induced byLY2606368 is not readily repaired and is consistent with observa-tions in tissue culture models.

The accumulation of DNA damage in LY2606368-treatedtumors was also demonstrated to result in clear antitumoractivity. Animals bearing Calu-6 xenograft tumors were dosed

twice daily (BID) for 3 days with 1, 3.3, or 10 mg/kg ofLY2606368 followed by 4 days of rest for three cycles. Tumorgrowth inhibition was determined by tumor volume measure-ment performed twice a week until the end of the study on day64. As shown in Fig. 6C, all three doses of LY2606368 causedstatistically significant tumor growth inhibition (up to 72.3%).LY2606368 was well tolerated in this experiment with animalweight loss not exceeding 3% in any of the treatment groups.Furthermore, tumor regrowth of the highest dose group wasslow during the 28-day recovery period, indicating a durableresponse to LY2606368.

DiscussionThere is a growing realization that a potent inhibitor of

CHK1 might not only potentiate traditional drugs, but alsoact as a stand-alone antitumor agent (7, 27, 28). It is welldocumented that chemically induced DNA damage activatesCHK1-dependent S and G2–M checkpoints to aid DNA repair.Abrogation of these checkpoints by a CHK1 inhibitor dramat-ically increases the sensitivity of cancer cells to traditional DNA-

Figure 5.LY2606368 induces replication stressand depletes the pool of availableRPA2 for binding to DNA. HeLa cellswere treated with 0.4% DMSO, 100nmol/L LY2606368 (A), 2,000 nmol/Lhydroxyurea (B), or with bothLY2606368 and hydroxyurea (C).Plateswere fixedat various timepointsfrom 0.5 to 9 hours followingcompound addition and stained forDNA (Hoechst 33342), RPA2, andpH2AX (S139). The relative intensityfor each stain was measured on asingle cell basis using the ThermoScientific Cell Insight NXT. S-phasecells were determined by measuringDNA content and used for furtheranalysis. RPA2 staining intensity (x-axis) was plotted versus pH2AX (S139)staining intensity for each conditionand time point. The 0.5-hour DMSOcontrol is representative of all DMSOtime points and is illustrated as astarting control condition. Regionswith increased ratios of pH2AX (S139)to RPA2 are boxed in red and indicatethe depletion of RPA2 associated withreplication catastrophe.

King et al.





damaging agents (8). The key role that CHK1 plays in the rapidactivation of the S and G2–M checkpoints is due to the tightintegration of CHK1 into normal DNA replication and DNAdamage repair. During replication stress, CHK1 suppressesinitiation of new replication origins through downregulationof CDK2 and association with the Treslin protein at origins

(29). Loss or chemical inhibition of CHK1 kinase activityduring an unperturbed replication cycle results in significantlyincreased replication origin activity and increased exposure ofssDNA to cleavage by endonucleases (30).

Recent work with an ATR inhibitor has provided evidence thatRPA protein is the rate-limiting step for fork stabilization duringreplication stress (13). Inhibition of ATRduring severe replicationstress results in massive replication fork firing, exposing ssDNA,which absorbs the limited pool of available RPA, leaving manyreplication forks unprotected and subject to endonucleolyticcleavage. Similar results were also obtained with UCN-01, amoderately selective CHK1 inhibitor (13). Without the ATR/CHK1-dependent DNA damage checkpoints, the cell will contin-ue to progress through the cell-cycle programdespite DNAbreaks.The persistence of DSBmay be a consequence of the inhibition ofCHK1 phosphorylation of RAD51 and a resulting loss of HRR.Cell death brought about by this combination of replicationfailure, DSB generation, and loss of DNA damage checkpointshas been called replication catastrophe (13). A number of CHK1inhibitors have entered clinic trials, primarily in combinationwithDNA-damaging agents (7, 8). LY2606368was developed as asecond-generation CHK1 inhibitor to have increased potency invivo and is being assessed clinically both as a single agent(NCT01115790) as well as in combination with cytotoxic andtargeted agents (NCT02124148). Not simply a DNA damagecheckpoint drug, LY2606368 appears to cause replication catas-trophe in the absence of exogenous replication stress, through theinhibition of both the replication control and the DNA damageresponse activities of CHK1.

In this study, we have confirmed that LY2606368 kills cancercells through a CHK1-dependent mechanism. The potency ofLY2606368 is sufficiently high that complete inhibition of CHK1is achieved in cells at low compound concentrations. Treatmentwith LY2606368 phenocopies genetic models of CHK1 loss andacts in part by removing canonical CHK1 suppression of repli-cation origin activation and DNA damage checkpoint arrest in Sand G2–M-phases. This activity results in rapid saturation of RPAbinding to chromatin, presumably regions of ssDNA, followed bythe appearance of DSBs and chromosome fragments. Dysregula-tion of CHK1-dependent replication and checkpoint control byLY2606368 is an extremely efficient method of shredding chro-mosomes in cancer cells.

Although many clinical agents cause DNA DSB, they differsignificantly in how the breaks are generated. Differences in thecell-cycle timing of the break as well as the structure of the freeends of the break determine which pathway(s) will be used torepair the damage (31). This is one reason that different tumortypes show tendencies to respond better to one particular classof DNA-damaging agent. Tumors generally possess inactivatingmutations in genes involved in one or more DNA repair path-ways resulting in resistance to some classes of agents andsensitivity to others (32–34). Because of the lack of deepunderstanding of predicting drug sensitivity, the discovery ofnovel agents helps to increase the number of responsive indica-tions. The mechanism of action of LY2606368 is uniquelydifferent from traditional DNA-damaging agents. Not only didCHK1 Inhibition by LY2606368 induce DSBs by replicationcatastrophe, it also effectively abrogated the S and G2–M DNAdamage checkpoints, reduced HRR, and led to cell death inmitosis. Although LY2606368 is also a potent inhibitor ofCHK2, we have no evidence that inhibition of CHK2

Figure 6.LY2606368 causes DNA damage and growth inhibition in tumor xenografts.CD-1 nu/nu mice bearing Calu-6 tumor xenografts were dosed with eithervehicle or a single subcutaneous dose of 15 mg/kg of LY2606368. A, tumorswere removed at 4, 8, 12, 24, and 48 hours after dosing and protein lysatesprepared, standardized for protein concentration, and used forimmunoblotting. The DNA damage response was determined bymeasuring the relative quantity of pCHK1(S296), pRPA2(S4/S8), and pH2AX(S139) in each sample using specific antibody reagents. B, the plasmaconcentration of LY2606368 was determined for each animal from bloodharvested at the time of tumor removal. C, on day 20 after implant,Calu-6 xenograft tumor-bearing CD-1 nu/numice were administered 1, 3.3, or10 mg/kg LY2606368 subcutaneously, twice daily for 3 days, followed by4 days of rest (BIDx3, rest 4 days) for three cycles. Dose groups are asindicated in the figure. Tumor response was determined by tumorvolume measurement performed twice a week during the course of thestudy. � , P

contributes to the known biologic effects of LY2606368. CHK2is activated by DSBs through ATM and has been implicated inthe p53-dependent G1 DNA damage checkpoint, chromosomestability, and surveillance of DNA damage in female meiosisand mitotic exit (35, 36). Mutations in the CHEK2 geneincrease susceptibility to cancer, likely due to impairment of theG1 checkpoint (35). Comparison of selective CHK1 and dualCHK1/CHK2 inhibitors in vitro shows no differences in abilityto cause DNA damage and kill cells (17).

Finally, CHK1 is required for RAD51 localization to DSBs topromote HRR. The lack of a strong HRR response may be thereason that LY2606368 induces DNA damage lasting manyhours. Persistent levels of DNA damage were observed intumors treated with efficacy doses of LY2606368 linking theantitumor activity of the molecule with replication catastropheas a result of CHK1 inhibition. LY2606368 efficiently causedthe hallmarks of replication catastrophe in vitro; depletion ofRPA2 pools followed by massive DSB and chromosome frag-mentation (13). Significantly, this occurred in the absence ofadditional chemically induced replication stress. In theabsence of the CHK1-dependent DNA damage checkpoints,cells entered into mitosis with highly fragmented chromo-somes followed by cell death. LY2606368 is a potent CHK1inhibitor that helps to define a new class of drug capable ofkilling cancerous cells through RPA-dependent replicationcatastrophe.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C. King, S. McNeely, R. Beckmann, D. Barda,M.S. MarshallDevelopment of methodology: C. King, H.B. Diaz, S. McNeely, D. Barnard,W. Blosser, M.S. MarshallAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. King, H.B. Diaz, S. McNeely, D. Barnard,J. Dempsey, W. Blosser, M.S. MarshallAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. King, H.B. Diaz, S. McNeely, D. Barnard,W. Blosser, D. Barda, M.S. MarshallWriting, review, and/or revision of the manuscript: C. King, H.B. Diaz,D. Barnard, R. Beckmann, D. Barda, M.S. MarshallAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. King, D. Barnard, M.S. MarshallStudy supervision: R. Beckmann, M.S. Marshall

AcknowledgmentsThe authors acknowledge the essential contributions of the CHK1 biology

and medicinal chemistry team members formerly located at Icos Pharmaceu-ticals: Phyllis Goldman, Erik Christenson, Darcey Clark, Jeff Dantzler, FrankDiaz, Heather Douanpanya, Francine Farouz, Ryan Holcomb, Angela Judkins,Adam Kashishian, Ed Kesicki, Kim McCaw, Harch Ooi, Vanessa Rada, FuqiangRuan, Alex Rudolf, Frank Stappenbeck, Janelle Taylor, Gene Thorsett, JenTreiberg,Margaret Weidner, and SteveWhite. They also thankMichele Dowless,Karen Cox, Lisa Kays, and Bonita Jones for superb technical support; and EricWestin, Aimee Bence Lin, and Robert Ilaria for insightful intellectual input.

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 4, 2014; revised June 9, 2015; accepted June 27, 2015;published OnlineFirst July 3, 2015.

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2015;14:2004-2013. Published OnlineFirst July 3, 2015.Mol Cancer Ther Constance King, H. Bruce Diaz, Samuel McNeely, et al. through CHK1-Dependent MechanismsLY2606368 Causes Replication Catastrophe and Antitumor Effects

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