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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
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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-/nu- mice (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
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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
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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
DM
SO
LY26
0636
8LY
2606
368
33 n
mo
l/L10
0 n
mo
l/L
DMSO LY2606368 LY2606368 LY2606368100 nmol/L 100 nmol/L + ZVAD33
nmol/L
DNA content
TU
NE
Lp
H2A
X (
S13
9)C
ell n
um
ber
A
B
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
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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.
LY2606368 Induces Replication Catastrophe
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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.
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Therapeutics2010
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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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LY2606368 Induces Replication Catastrophe
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CHK1-Dependent MechanismsLY2606368 Causes Replication Catastrophe
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