Cytosine-based nucleoside analogs are selectively lethal to DNA mismatch repair-deficient tumour cells by enhancing levels of intracellular oxidative stress.

2 MB·English

by  Hewish, M

FULL PAPER
British Journal of Cancer (2013) 108, 983–992 | doi: 10.1038/bjc.2013.3
Keywords: cytarabine; mismatch repair; MLH1; MSH2; synthetic lethality; cancer
Cytosine-based nucleoside analogs are
selectively lethal to DNA mismatch repair-
deficient tumour cells by enhancing levels
of intracellular oxidative stress
M Hewish1,2,3, S A Martin1,2,4, R Elliott1, D Cunningham3, C J Lord*,1 and A Ashworth*,1,2
1TheBreakthroughBreastCancerResearchCentre,TheInstituteofCancerResearch,FulhamRoad,London,SW36JB,UK;2CRUK
GeneFunctionLaboratory,TheInstituteofCancerResearch,FulhamRoad,LondonSW36JB,UKand3DepartmentofMedicine,
Royal MarsdenHospital NHS Trust, London andSurrey, UK
Background:DNAmismatchrepairdeficiencyispresentinasignificantproportionofanumberofsolidtumoursandisassociated
withdistinct clinical behaviour.
Methods:Toidentifythetherapeuticagentsthatmightshowselectivityformismatchrepair-deficienttumourcells,wescreeneda
pairofisogenicMLH1-deficientandMLH1-proficienttumourcelllineswithalibraryofclinicallyuseddrugs.Totestthegenerality
of hitsinthe screen,selective agents wereretested incells deficient intheMSH2mismatch repairgene.
Results:Weidentifiedcytarabineandotherrelatedcytosine-basednucleosideanaloguesasbeingselectivelytoxictoMLH1and
MSH2-deficient tumour cells. The selective cytotoxicity we observed was likely caused by increased levels of cellular oxidative
stress, asit couldbe abrogated byantioxidants.
Conclusion:Weproposethatcytarabine-basedchemotherapyregimensmayrepresentatumour-selectivetreatmentstrategyfor
mismatch repair-deficient cancers.
Mismatchrepairdeficiency(dMMR)isrecognisedasanimportant et al, 2012). Given the widespread occurrence of dMMR and the
cause of mutagenesis and driver of oncogenesis in human cancer availability of robust biomarkers to determine the presence of
(Jiricny, 2006; Hewish et al, 2010). Mismatch repair deficiency is dMMRinhumantissue(Hewishetal,2010),wesoughttoidentify
manifest in a small proportion of solid tumours that occur in new therapeutic strategies for this subgroup of cancers.
association with the inheritance of a pathogenic mutation in an TheclassicalMMRpathwayprimarilyexiststorepairbase–base
MMR gene (Lynch syndrome) (Lynch et al, 2008), but is also mismatches and short insertion–deletion loop mismatches that
frequently involved in sporadic tumorigenesis. Fifteen per cent of form during DNA replication (Jiricny, 2006). Canonical MMR
all colorectal cancers (CRC) exhibit dMMR, with the majority involves two protein containing heterodimers, MutS and MutL.
occurringasaconsequenceofhypermethylationoftheMLH1gene The MSH2-containing heterodimer (MutS) senses the mismatch
promoter (Hewish et al, 2010; Tejpar et al, 2010). More recently, and triggers the recruitment and activation of the MLH1-
with access to high-throughput sequencing techniques, sporadi- containing heterodimer (MutL) that in turn coordinates other
cally occurring mutations in MMR genes have been reported in proteins, which sequentially excise, resynthesise, and religate the
cancers not classically associated with Lynch syndrome (Maeng repairedDNAatthemismatchedsite(Jiricny,2006;Hewishetal,
*Correspondence:DrCJLord;E-mail:[email protected];E-mail:[email protected]
4Present address: Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London,
EC1M6BQ,UK
Received27July2012;revised16November2012;accepted16December2012;publishedonline29January2013
&2013CancerResearchUK.Allrightsreserved0007–0920/13
www.bjcancer.com|DOI:10.1038/bjc.2013.3 983
BRITISHJOURNAL OF CANCER dMMRcancer cellsare sensitivetocytarabine
2010). In the absence of functional MMR, for example, caused by Cytarabine (Ara-C), azacytidine, cyclocytidine, and nucleoside
MSH2orMLH1mutation,thisprocessbecomesdysregulatedand triphosphates (Sigma Aldrich, Dorset, UK) were dissolved in
canleadtoa‘mutatorphenotype’,withtherapidaccumulationof phosphate-buffered saline (PBS). N-acetyl-l-cysteine (Sigma) was
thousandsofmutationsthroughoutthegenome,includingthosein dissolved in media containing HEPES (4-(2-hydroxyethyl)-1-
microsatellite repeat regions. The majority of dMMR tumours, piperazineethanesulfonic acid) buffer, adjusting pH using 1M
including over 90% of those deficient in the commonest MMR NaOH. 3H-cytarabine was obtained from Moravek Biochemicals
defects, MLH1 and MSH2, exhibit microsatellite instability (MSI) (Brea, CA, USA). Drugs were stored at (cid:2)201C.
(Hewishetal,2010).InadditiontotheirroleintherepairofDNA
Drugscreeningandvalidation. Thecontentsofthecommercially
lesions during DNA replication, it is increasingly appreciated that
available US Drugs library (http://www.msdiscovery.com) were
MMR proteins also play important roles in other processes,
dissolved in DMSO and stored at (cid:2)201C. For all cell viability
including the repair of oxidatively damaged DNA (Macpherson
experiments,cellswereseededin96or384-wellplatesandexposed
et al, 2005). These additional functions are thought to be
to serial dilutions of drug or DMSO/PBS control 24h later. Cell
responsible for the altered responses of dMMR cancer cells to
viability was assessed at the end of the experiment using the
cytotoxicchemotherapy,includingtherelativeresistanceofdMMR
CellTiter-Gloluminescentassay(PromegaUK,Southampton,UK)
CRC cells to 5-fluorouracil (Hewish et al, 2010).
according to the manufacturer’s instructions with an automated
MMR genes function as classical tumour suppressor genes, with
plate reader. Luminescence readings for wells exposed to control
loss of function of both alleles being generally required to cause the
werecomparedwithwellsexposedtotestdruginordertocalculate
dMMRphenotype(Hewishetal,2010).Astherapeuticrestorationof
a surviving fraction (SF). Data were analysed using GraphPad
normalgenefunctioniscomplex,syntheticlethalstrategieshavebeen
Prism (GraphPad Software Inc., La Jolla, CA, USA) and survival
proposedasameansoftargetingcancersdrivenbytumoursuppressor
curves determined using the Four Parameter Logistic Regression
gene dysfunction(Ashworth etal, 2011). Synthetic lethality describes
Model. For clonogenic assays, cells were seeded in six-well plates
therelationshipbetweentwogenesorpathways,wherelossoffunction
and exposed to serial dilutions of drug of DMSO/PBS control.
of one gene or pathway in a cell is compatible with cellularviability,
Drug or media was replaced twice a week till the end of the
whereaslossofbothisnot.Inpart,thepromiseofsyntheticlethality
experiment (10–14 days). Colonies were washed in PBS, fixed in
has been borne out by the successful translation of the sensitivity of
methanol, stained using crystal violet solution, washed and
tumoursdeficientinBRCA1orBRCA2totreatmentwithpoly(ADP-
colonies counted using an automated colony counter (Oxford
ribose)polymerase(PARP)inhibitors(Tuttetal,2010;Ashworthetal,
Optronics, Oxford, UK), and SFs calculated.
2011).SyntheticlethalapproachestodMMRhavealsobeenproposed,
includinginhibitionofPINK1asbeingselectivelycytotoxictoMMR- Short interfering RNA transfections. Cells were reverse trans-
deficient cells (Martin et al, 2011) and exposure to methotrexate as fected with short interfering RNA (siRNA) duplexes targeting
beingselectivelycytotoxictoMSH2-deficientcells(Martinetal,2009). MLH1, POLA, POLA2, PLK1 or non-targeting control
Here,wedescribeacomprehensivedrugscreentoidentifyadditional (Supplementary Methods). Twenty-four hours after transfection,
synthetic lethal targets for dMMR and in doing so highlight the cells were trypsinised, counted, and reseeded in 96-well plate
potential for cytosine-based nucleoside analogues as a potential format,exposedtodrugorcontrol24hlater,andviabilityassessed.
therapeuticstrategy.
Proteinanalysis. Whole-cellextractswerepreparedbylysingcells
in NP-40 lysis buffer with protease inhibitors (Complete mini
protease inhibitor cocktail tablets, Roche, Welwyn Garden City,
MATERIALS AND METHODS UK). Lysates were electrophoresed on NuPAGE Tris-acetate pre-
castgels(Invitrogen,LifeTechnologies),andimmunoblottedwith
Cell lines, culture conditions, and reagents. HCT116 and thefollowingantibodies:anti-PARP(SantaCruz,Sc-8007,1:1000,
HCT116þChr3 were a gift from Dr A Clark, National Institute of Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MLH1
Environmental Health Sciences, Research Triangle Park, NC, USA. (Calbiochem PC56 ab-2, 1:1000, Calbiochem, Nottingham, UK),
BVEC F7bro and BVEC E2 are HCT116 derivatives and were and anti-Phospho (Ser139) H2AX (Upstate 05–636, 1:1000).
donated by Professor Bert Vogelstein, Johns Hopkins University of Immunoblotting for anti-b-actin-HRP (Santa Cruz Sc-1616,
Medicine, Baltimore, MD, USA. LoVo (and LoVoþChr2 cells, 1:250) was used as a loading control. This was followed by
created by DrMinoru Koi) weredonated by ProfessorRickBoland, incubation with anti-IgG-horseradish peroxidase and chemilumi-
Baylor University Medical Centre, Dallas, TX, USA and Dr nescent detection (Supersignal West Pico chemiluminescent
Christopher Gasche, Medical University of Vienna, Austria. substrate) (Pierce, Thermofisher Scientific, Rockwell, IL, USA).
A2780cp70 a2 and A2780cp70 e1 cells were donated by Professor
Immunofluorescence. Cellsgrownoncoverslipswerewashedand
Robert Brown, Institute of Cancer Research, Surrey, UK. SW48,
fixedwith4%paraformaldehyde.Cellswerepermeabilisedin0.2%
CACO2, HCT-15, RKO, HRT-18, and SW480 were obtained from
(v/v) Triton X-100 in PBS for 30min, washed, blocked and
theATCC,andHT29,LS174T,COLO205andCOLO-741fromthe
incubated with anti-Phospho (Ser139) H2AX antibody (Cell
ECACC.Cellswereculturedingrowthmediasupplementedwith10%
Signaling 9718S, 1:1000, Cell Signaling, New England Biolabs,
Fetal bovine serum (Gibco, Life Technologies, Paisley, UK), 2mM Hitchin, UK), followed by an Alexa Fluor 555nm (Molecular
L-glutamine (Gibco), 50Uml(cid:2)1 penicillin and 50mgml(cid:2)1 in a 5% Probes, Invitrogen, Life Technologies)-conjugated secondary anti-
CO2 environment at 371C. HCT116 and derivatives, and HRT-18 body diluted 1:1000. Coverslips were washed in DAPI (40,6-
weremaintainedinMcCoy’s5Amedia(Gibco),withtheadditionof
diamidino-2-phenylindole) diluted 1:10000 in PBS (358nm
the selection pressure of 400mgml(cid:2)1 geneticin to HCT116þChr3.
excitation, Molecular Probes) to visualise cell nuclei, placed on
LoVoweremaintainedinIscove’smodifiedDulbecco’smedium,with
Vectashieldmountingmedium(Vectorlabs,Peterborough,UK)on
the addition of 700mgml(cid:2)1 geneticin to LoVoþChr2. A2780cp70,
a microscope slide, and viewed on a Leica TCS-SP2 confocal
RKO, COLO205 and COLO-741 were maintained in Roswell Park
microscope (Leica Microsystems, Milton Keynes, UK).
Memorial Institute (RPMI) media (Gibco), with the addition of
200mgml(cid:2)1 hygromycin B to A2780cp70 a2 and A2780cp70 e1. Analysis of cytarabine incorporation into DNA. Cells were
LS174T, HCT-15, SW48, SW480, CACO2, and HT29 were treatedwith50nMcytarabinemixedwith20mCiof3H-cytarabine.
maintained in Dulbecco’s modified Eagle’s medium (Gibco). Cells DNAfromcytarabine-treatedcellsandcontrolswaspurifiedusing
wereexpandedfortwopassagesandthencryopreserved. the QIAamp DNA Blood Mini Kit (Qiagen, Manchester, UK).
984 www.bjcancer.com|DOI:10.1038/bjc.2013.3
dMMRcancercells aresensitive tocytarabine BRITISH JOURNALOF CANCER
Cells seeded
day 1
Controls
Drug treated days 2
HCT116 HCT116+Chr3 and 4
Assay readout:
cellular viability day 6
(Cell Titer-Glo
luminescent assay)
Hit selection
2.0 US drugs primary screen
3 DMSO
onChr 1.5 Drugs
g fractiT116+ 10..05
vinHC 0.0
Log survi2CT116 vs –––011...505 DesmfeeCrsoyyxtlaaartmaebininee Eth5i0n0yl oestradiol Menadione 1000
H
–2.0
Cytarabine – short term assay Cytarabine – clonogenic assay
1.25 1.25
HCT116 HCT116
on 1.00 HCT116+Chr3 on 1.00 HCT116+Chr3
cti cti
g fra 0.75 g fra 0.75
vin 0.50 vin 0.50
vi vi
ur 0.25 ur 0.25
S S
0.00 0.00
0 10–9 10–8 10–7 10–6 10–5 0 10–10 10–9 10–8 10–7 10–6
Concentration (M) Concentration (M)
Figure1.PrimaryscreenofMLH1-deficientandproficientcancercellsidentifiescytarabineasMLH1-deficientselective.(A)TheMLH1-deficient
CRCcelllineHCT116andtheMLH1-proficientcomparatorHCT116þChr3werescreenedinparallel.Cellswereplatedonday1and
exposedtodrugorcontrolcontinuouslyfromday2.Viabilitywasassessedusingaluminescentassayonday6.Datawereanalysedtoobtain
MLH1-deficientselectivehits.(B)Scatterplotoftheprimaryscreen.Log SFforHCT116wascomparedwiththelog SFforHCT116þChr3and
2 2
plottedagainstscreenposition.(C,D)Survivalcurvesfromassaysoftheeffectsofcytarabineon(C)cellviabilityinHCT116þ/(cid:2)Chr3cells
exposedcontinuouslytodrugand(D)clonogenicsurvivalinHCT116þ/(cid:2)Chr3cellsexposedto24hofcytarabine.Assayswereperformedin
triplicateorquadruplicate.Errorbarsrepresentthestandarderrorofthemean(s.e.m.).
DNAsampleswerequantifiedandcountsperminutemeasuredon Analysis of reactive oxygen species. Reactive oxygen species
a2500TRliquidscintillationcounterPackard.Theincorporationof (ROS)andreactivenitrogenspecies(RNS)incellularsamplesand
3H-cytarabine in each sample was calculated from the activity of culture supernatant samples were assessed using the OxiSelect In
3H-cytarabine recorded expressed as DPM/l (disintegrations per VitroROS/RNSAssayKit(CellBiolabs,Inc,SanDiego,CA,USA),
minute), compared with total DNA in the sample analysed. accordingtothemanufacturer’sinstructions.Cellsandsupernatant
samples were homogenised and particulate matter removed.
Analysis of 8-oxo-7,8-dihydro-20-deoxyguanosine (8-oxodG). Samples were assayed for the presence of fluorescent dichlorodi-
Genomic, nuclear, and mitochondrial DNA were extracted from hydrofluorescein (DCF), produced on the oxidation of DCFH-
cell lines as previously described (Martin et al, 2011). Following DioOxyQ(DCFDiOxyQ),andcomparedagainstastandardcurve.
quantification, DNA samples were standardised, digested with Fluorescence wasquantified usinganautomatedplatereader, and
nuclease P1 (Sigma), treated with calf intestinal phosphatase and normalised to cell number.
denatured.8-oxo-7,8-dihydro-20-deoxyguanosine (8-oxodG)levels
were quantified using an 8-oxodG ELISA assay in 96-well plate
format (Cayman Chemicals, Ann Arbor, MI, USA) following RESULTS
the manufacturer’s instructions. Standard curves were calculated
from serial dilutions of 8-oxodG standard to calculate reaction Isogenic MMR-deficient andproficient drug screens. To model
efficiency. Absorbance was quantified at 405nm using an the effects of MLH1 deficiency in vitro, we used the human CRC
automated plate reader. cell line HCT116 and its isogenic comparator HCT116þChr3.
www.bjcancer.com|DOI:10.1038/bjc.2013.3 985
BRITISHJOURNAL OF CANCER dMMRcancer cellsare sensitivetocytarabine
Cytarabine Cytarabine
1.25 BVEC F7 bro 1.25
BVEC E2 Lovo
Surviving fraction 1000....07520505 Surviving fraction 1000....07520505 Lovo+Chr2
0.00 0.00
0 10–9 10–8 10–7 10–6 10–5 0 10–9 10–8 10–7 10–6
Concentration (M) Concentration (M)
106
P=0.0388* MSI P=0.0474*
1.0 )M 104 MSS
(cid:2)e ()M 0.8 (cid:2)bine ( 102
IC50 cytarabin 0000....6420 MSI MSS IC50 cytara 111000––024 GP5dC2BBe1HCT-116SW620SW48LS-513RKOCW-2LoVoLS-411NCOLO-741HCC2998HT55NCI-H747HT-29RCM-1SW1116NCI-H630SW1463HCT-15SW948KM12SNU-C2BCaR-1SW1417SW837O-320-HSRT84LS-123COLO-678LS-1034
Microsatellite status L
O
C
Colorectal cancer cell line
i Cytarabine ii
A2780cp70 a2 siCON
1.25 A2780cp70 e1 siCON
A2780cp70 e1 siMLH1*1
A2780cp70 e1 siMLH1*2
1 2
1.00 A2780cp70 e2 siMLH1*1 N N H1* H1*
O O L L
n C C M M
actio 0.75 a2 si e1 si e1 si e1 si
ng fr MLH1
urvivi 0.50
S
(cid:3)-Actin
0.25
0.00
0 10–10 10–9 10–8 10–7
Concentration (M)
Figure2.MultiplemodelsofMMRdeficiencyareselectivelysensitivetocytarabine.(A)CellviabilityassayofBVECF7broandE2cellsexposed
tocytarabinefor24h(Pp0.0001usingANOVA).(B)CellviabilityassayofLoVoandLoVoþChr2cells,followingcontinuousexposureto
cytarabine.(C)BoxandwhiskerplotofcytarabineIC50valuesobtainedfromtheSangerInstitutedataset,stratifiedbyMSIstatus.(P¼0.0388
usingtwo-tailedt-test).(D)WaterfallplotofcytarabineIC50valuesinCRCcelllinesobtainedfromthesamedataset,stratifiedbyMSIstatus
(P¼0.0474usingtwo-tailedt-test).(E(i))SurvivalcurvesfromcellviabilityassaysofA2780cp70a2ande1cellstransfectedwithnon-targeting
controlsiRNAorsiRNAtargetingMLH1,andcontinuouslyexposedtovariousconcentrationsofcytarabine,vscontrol,48haftertransfection.
Assayswereperformedintriplicateorquadruplicate.Errorbarsrepresentthes.e.m.(E(ii))ImmunoblotofMLH1expressionincelllysatesobtained
fromthisexperiment,withb-actinasloadingcontrol.
HCT116isMLH1-deficientbecauseofthepresenceofhomozygous transformed average readings from control wells with drug-
mutationsinexon9oftheMLH1gene,whileHCT116þChr3has treated wells on that plate, to obtain a log SF. The log SFs for
2 2
beenrenderedMLH1-proficientduetothestabletransferofacopy each cell line were compared with select MLH1-deficient selective
of chromosome 3 (and a wild-type MLH1 gene) using microcell drugs.Thescreenwasperformedtwiceandresultscombinedinthe
fusion (Koi et al, 1994). To identify potential drugs that selectively final analysis.
target MLH1-deficient cells, we screened both cell lines with the A scatter plot comparing the log ratio of SFs obtained in the
2
contents of the commercially available US Drug Collection, MLH1-deficient and MLH1-proficient cell lines is shown in
containing1040 drugsthathaveentered clinicalassessment. Figure 1B. Drugs demonstrating MLH1-deficient selectivity are
Thedruglibrarycontentswerearrayedin96-wellplateformat, listed in Supplementary Table 1. Among drugs identified as being
with control wells in each plate. Cell solutions were dispensed MLH1-deficient selective were menadione, ethinyl estradiol, and
using an automated liquid handling device, and cells exposed to desferrioxamine, which have been associated previously with
drug (at a concentration of 10mM) or control (0.1% DMSO) in modulation of cellular oxidative stress (for example (Laux and
growth media for 5 days, 24h after seeding (Figure 1A). Cell Nel, 2001)), consistent with our previous findings (Martin et al,
viability was estimated at the end of the experiment using the 2009, 2011). While menadione has been previously validated as
luciferase-based CellTiter-Glo assay Promega, which quantifies being MSH2-deficient selective (Martin et al, 2009), it has been
ATP.Theeffectofeachcompoundwasassessedbycomparinglog- withdrawn from clinical use. Nevertheless, the identification of
986 www.bjcancer.com|DOI:10.1038/bjc.2013.3
dMMRcancercells aresensitive tocytarabine BRITISH JOURNALOF CANCER
HCT116 HCT116+Chr3 Treatment
– + – – + – Ara-C 50nM
– – + – – + Ara-C 100nM
Total PARP
Cleaved
MLH1
(cid:3)-Actin
(cid:4)-H2AX
HCT116 HCT116+Chr3
Cytarabine-treated cells
P(cid:2)0.001
(cid:4)n H2AX-mpared withontrols 543 Control
Fold change iositive cells countreated c210 e 10(cid:2)m
p HCT116 CT116+Chr3 Cytarabin
H
Figure3.TheexcesscytotoxicityofcytarabineinMMR-deficientcellsisassociatedanincreaseinmarkersofapoptosisandDNAdamage.
(A)Immunoblotassessingtheeffectsofcytarabinetreatmentonproteinlevels.LysateswereobtainedfromHCT116þ/(cid:2)Chr3cellsexposedto
cytarabine(ara-C),orcontrol,for72h.LysateswereimmunoblottedandprobedforexpressionofPARP,MLH1,gH2AX,andb-actinasaloading
control.(B)BargraphdemonstratingpercentagechangeingH2AX-positivecellsbyconfocalmicroscopyfollowingtreatmentwith50nMcytarabine
for24h,comparedwithcontrols.Cellswerefixed24hfollowingcytarabineexposure.X5focipercellwasdefinedasapositivecell.Atleast
100cellswerecountedinaminimumofsevenrepresentativefields.(C)Mergedimagesofcontrol(PBS-treated)HCT116andHCT116þChr3cells
(toppanels)andcytarabine-treatedcells(bottompanels).CellswerestainedforgH2AX(red)andDAPI(blue).
menadione demonstrated that we could identify MMR selective effect of cytarabine in other isogenic MLH1 and MSH2-deficient
agents from this screen. cell line pairs. We used the CRC cell lines, BVEC F7bro (MLH1-
deficient) and BVEC E2 (MLH1-proficient) (Weiss et al, 2007),
Cytarabine is selective for MLH1-deficient cells. We noted that as well as LoVo (MSH2-deficient) and LoVoþChr2 (MSH2
the nucleoside analogue, cytarabine was selective for MLH1- proficient) (Watanabe et al, 2000). We found that cytarabine was
deficientHCT116cellsinthescreenwithasignificantdifferencein selective forMMR-deficient celllinesinbothsystems(Figures2A
SFs (SF at 10mM cytarabine for HCT116¼0.0891 vs HCT116þ and B), suggesting that this phenotype was applicable to multiple
Chr3SF¼0.3289;log ratio (cid:2)1.134)(SupplementaryTable1).As models.
2
screensarepronetoasignificantfalse-positiverate,weassessedthe To determine the generality of our observations in a non-
effects of cytarabine using short-term viability assays (with isogenic setting, we interrogated the publicly available drug
identical conditions to the original screen) (Figure 1C), and sensitivity data published by the Wellcome Trust Sanger Institute
clonogenic assays, the gold standard of cellular viability (Brown (http://www.cancerrxgene.org/translation/Drug). We observed a
andAttardi,2005),wherecellswereexposedtocytarabinefor24h trend towards sensitivity to cytarabine in the initial data sets,
(Figure 1D). We observed that cytarabine was MLH1-deficient stratified by mutations in MSH2 vs wild-type (IC50 0.067mM vs
selectiveinbothassays,andthatthemagnitudeofMLH1-deficient 0.788mM) and mutations in MSH6 vs wild-type (IC50 0.002mM vs
selective cytotoxicity was increased with increased length of drug 0.788mM), with no clear association present with mutations in
exposure (Supplementary Figure 1), suggesting that the observed MLH1 vs wild-type (0.682mM vs 0.780mM). Given that in the
differential phenotype was perhaps due to a cumulative effect. clinical setting the majority of MLH1 loss results from epigenetic
A synthetic lethal interaction that is relatively unaffected by modifications rather that the presence of mutations, we then
other genetic changes has been termed a ‘hard’ synthetic lethality stratified the IC50 data for cytarabine by MSI status, as MSI is
(Ashworth et al, 2011). To determine whether our observations present in the majority of dMMR tumours (Supplementary
were generally applicable to models of dMMR, we assessed the Table2).Weobservedastatisticallysignificantdifferencebetween
www.bjcancer.com|DOI:10.1038/bjc.2013.3 987
BRITISHJOURNAL OF CANCER dMMRcancer cellsare sensitivetocytarabine
25 P=0.385 Cytarabine
1.25 HCT116
bine 20 on 1.00 HHCCTT111166++dCChTr3P
3DPM/I H cytara(cid:2)per g DNA 11505 Surviving fracti 111...752505 HCT116+Chr3+dCTP
0 1.00
HCT116 CT116+Chr3 0 10–9Con1c0e–n8tratio1n0 (–M7) 10–6
H
Cytarabine Cytarabine
1.25 HCT116 1.25 HCT116
1.00 HHCCTT111166++CdThTr3P on 1.00 HHCCTT111166++dCUhTr3P
Surviving 11..7550 HCT116+Chr3+dTTP urviving fracti 11..7550 HCT116+Chr3+dUTP
1.25 S 1.25
1.00 1.00
10–9 10–8 10–7 10–6 10–9 10–8 10–7 10–6
Concentration (M) Concentration (M)
0.5
Proportional survival
n 0.4 increase of dCTP in
g fractio 0.3 HCT116
urvivin 0.2 PsduCrorTvpPivo aritnli oinncarlease of
1- S 0.1 HCT116+Chr3
0.0
–9 –8 –7 –6
Concentration (M)
Figure4.CytarabineisnotincorporatedintoDNAmoreinMLH1-deficientcellscomparedwithMLH1-proficientcells.(A)Bargraphof
incorporationof3H-cytarabineintoHCT116andHCT116þChr3celllines.Cellsweretreatedwithcytarabinemixedwith3H-cytarabinefor72h.
GenomicDNAwasextractedandquantified.Levelsof3H-cytarabinepermgofDNAareshown(P¼0.3845(two-tailedt-test)).Theexperimentwas
performedinduplicate.(B–D)Survivalcurvesofcytarabineexposureinthepresenceorabsenceof10mMdCTP,dTTP,ordUTP.Cellularviability
wasquantifiedafter72husingtheCellTiter-Gloassay,andSFcalculated.(E)TheincrementalchangeinSFwhenHCT116orHCT116þChr3cells
wereexposedtocytarabine,withtheadditionofdCTP,whencomparedwithcytarabineexposurealone(calculatedfromB).
cancer cells exhibiting MSI and those exhibiting microsatellite A2780cp70e1(MLH1-proficient)ovariancancercells(Plumbetal,
stability(P¼0.0388)(Figure2C).GiventherelevanceofdMMRto 2006), and performed the same experiment (Figure 2E(i)). While
CRCinparticular,weassessedthedataarisingfromCRCcelllines non-targeting siRNA did not ostensibly alter the response to
alone(waterfallplotinFigure2D),andalsoobservedastatistically cytarabine in either clone, MLH1 silencing in the previously
significant difference in cytarabine sensitivity dependent on MSI MLH1-proficient e1 line (immunoblot in Figure 2E(ii)) did result
status across this smaller panel of cell lines (P¼0.0474). Taken incytarabinesensitivity.Takentogether,theseresultsdemonstrate
together, these data suggest that thepresence of dMMR/MSI may thatcytarabinesensitivitycouldbeinducedbyMLH1silencingand
be one important modifier of sensitivity to cytarabine, although that mutations secondary to MMR deficiency were unlikely to
multiple other genetic and epigenetic factors are also important explain the effects observed.
particularly given the multiple mechanisms of action of this drug
Other cytosine-based nucleoside analogues exhibit MMR-
(discussed below).
deficient selectivity. To begin to address the basis of the
GiventhatMMRdeficiencycausesamutatorphenotype,itwas
cytarabine selectivity, we performed cell viability assays to assess
still possible that genetic drift between isogenic clones could
whether other cytosine-based nucleoside analogues might exhibit
explainthephenotypeweobserved.Wedirectlyaddressed thisby
the same MLH1-deficient selective phenotype. Both cyclocytidine
inducing MLH1 deficiency in two isogenic systems using an
and azacytidine caused an MLH1 selective effect in isogenic CRC
orthogonal,siRNAapproach.WetransfectedtheMLH1-proficient
cells (Supplementary Figures 3a and b).
cell line used in the screen, HCT116þChr3, with non-targeting
siRNA(siCON)orsiRNAtargetingMLH1,andobservedtheeffect The cytarabine response of MLH1-deficient cancer cells is
on cytarabine response (Supplementary Figure 2). We noticed a associated with increased apoptosis and a DNA damage
partialsensitization tocytarabineonMLH1suppression. Notably, response. As cytarabine is known to have multiple mechanisms
others have reported that adequate levels of MLH1 for MMR ofaction,manyofwhicharenotwellelucidated(Kufeetal,1985;
function in HCT116þChr3 may persist even after loss of Grant, 1998), we next assessed the modalities of cell death and
expression of MLH1 by western blot (Chauhan et al, 2000), We, DNAdamageassociatedwithexposuretocytarabineinthesecells.
therefore, selected the MLH1 isogenic system with the highest Cytarabine treatment has been associated with the induction of
siRNA transfection rate, the A2780cp70 a2 (MLH1-deficient) and apoptosis in multiple models of cancer (Backway et al, 1997). To
988 www.bjcancer.com|DOI:10.1038/bjc.2013.3
dMMRcancercells aresensitive tocytarabine BRITISH JOURNALOF CANCER
2.5 HCT116+50nM Ara-C replicationforks,interferingwithchainelongationandactingasa
G HCT116+100nM Ara-C chain terminator. Cytarabine exposure has not been associated
od 2.0 HCT116+Chr3+50nM Ara-C with potentially lethal double-strand DNA breaks (DSBs) in the
ge in 8-oxmpared1.5 HCT116+Chr3+100nM Ara-C mWsuarern,ontgehareteroeffomsroaem,rkdeeerctyerotomftoinrxeeipcdliactghaeteinotansbi(lGfiotyrraknotf,stc1ay9ltl9ian8rga;/bPDinoSmeBmtofioeirrn,md2au0tci0eo6n)a,.
hanco1.0 histone H2AX phosphorylation (gH2AX) (Bonner et al, 2008).
c
d 0.5 H2AXisspecificallyphosphorylatedonaC-terminalserineresidue
ol
F inresponsetoDSBsandothertypesofDNAdamage,includingthe
0.0 stalling of DNA replication forks (Bonner et al, 2008). gH2AX
24 48 72
levels were induced by cytarabine, and were significantly more
Hours after start of treatment
elevated in MLH1-deficient HCT116 cells than MLH1-proficient
HCT116þChr3 cells. The effect of cytarabine on gH2AX was
1.25 Cytarabine
HCT116 concentration dependent and could be elicited at nanomolar
HCT116+Chr3 concentrations,previouslyobservedtobeMLH1-deficientselective
1.00 HCT116+NAC
(Figures 3A and 1C). As H2AX can also be phosphorylated in
n HCT116+Chr3+NAC
ng fractio 0.75 ftrooesrpmDoSantBsioefnotroumsiaantpgioopnimtoamsnisdu,ntowhfeelufoaorlrsemoscaeatnisocsene,ssowefdhsictnahullceisldearrreelapgtlHiivcea2ltAyioXnspfefooccirfukicss
vivi 0.50 (Bonner et al, 2008). Cytarabine exposure additionally caused a
Sur significant elevation in the percentage of gH2AX-positive cells in
0.25 MLH1-deficientcellscomparedwithMLH1proficient(Figures3B
and C and Supplementary Figure 4). Collectively, these data
suggestedtheMMR-deficientselectivityassociatedwithcytarabine
0.00
0 10–10 10–9 10–8 10–7 10–6 was associated with induction of apoptosis and activation of the
Concentration (M) DNA damage response.
Investigation of the potential mechanism of MMR selectivity.
0.5 Proportional survival
increase of NAC in Cytarabine is initially converted to a monophosphate form, and
on 0.4 HCT116 then to a diphosphate and triphosphate (cytarabine triphosphate;
cti Proportional survival ara-CTP), followed by incorporation into DNA. The degree of
a
ng fr 0.3 iHnCcrTe1a1s6e+ oCf hNrA3C in ara-CTP incorporation correlates with the degree of resulting
vivi 0.2 cytotoxicity (Grant, 1998). As we had observed induction of the
ur DNA damage response, we next assessed whether the MMR-
1-S 0.1 deficient selective effect of cytarabine could be explained by
differential incorporation into DNA. We determined the relative
0.0
incorporation of 3H-cytarabine into DNA in MLH1-deficient and
0 10–10 10–9 10–8 10–7 10–6
proficient cells, and did not observe a significantly differing rate
Concentration (M)
between the two models (Figure 4A). We also assessed the effects
Figure5.CytarabinetreatmentinMMR-deficientcellsisassociated ofexogenousnucleosides(Figure4B–D).Althoughthepresenceof
withanincreaseinoxidativeDNAdamage,andcanbereversedby dCTP did ameliorate the inhibitory effects of cytarabine, it did so
antioxidants.(A)ResultsofanELISAassaytoquantifylevelsof8-oxodG in equal measure in both cell lines (Figure 4E). Taken together,
inHCT116andHCT116þChr3followingtreatmentwithcytarabine. these data suggested that the increased sensitivity of MLH1-
Cellswereplatedandexposedtocytarabinecontinuously24hlater. deficient cell lines could not be explained by the differential
DNAwasextractedfromwhole-celllysatesafter24,48,and72h,and incorporation of cytarabine into DNA.
quantified;assayloadingwasnormalisedtototalDNAconcentration. Cytarabine and other nucleoside analogues are also known to
Datafromonerepresentativeexperiment,performedinduplicateand inhibit the DNA polymerase POLA, with a lesser inhibition of
analysedaccordingtothemanufacturer’sprotocol.8-oxodGwas POLB, albeit weakly at micromolar concentrations (Grant, 1998;
quantifiedagainstastandardcurve.(B)Clonogenicassayoftheeffects Wills et al, 2000). In view of these data, and data from our
ofNAConcytotoxicityobservedwithcytarabinetreatment.HCT116 laboratorysuggestingsyntheticlethalrelationshipsbetweenMLH1
andHCT116þChr3cellswereexposedto10mMNAC,andthen and POLG, and MSH2 and POLB (Martin et al, 2010), we tested
cytarabineorcontrolfor24h.Cellsweremaintainedingrowthmedia theMLH1selectiveeffectsofinhibitionofPOLAoritsregulatory
untiltheendoftheexperiment(10–12days).(C)Proportional subunit POLA2 using siRNA in HCT116 and HCT116þChr3
incrementinSFwiththeadditionofNACtocytarabineinHCT116and cells. We did not observe a significant difference in response
HCT116þChr3cells(calculatedfromB). (SupplementaryFigure5). Theabsence ofMLH1-POLA synthetic
lethality coupled with the concentrations at which this phenotype
wasobserved,suggestedthattheMMRselectiveeffectofcytarabine
assess whether apoptosis was induced by cytarabine in these
waslesslikelytobeexplainedbyaneffectofcytarabineonPOLA.
dMMRmodels,weextractedcelllysatesfromMLH1-deficientand
proficientcellsexposedtocytarabine/ara-cfor72h,andquantified The MLH1-deficient selective effect of cytarabine can be
levels of cleaved PARP, produced as a consequence of caspase abrogated by antioxidants and is associated with increased
activation by immunoblotting. A significant concentration-depen- oxidatively damaged DNA. Our previous studies (Martin et al,
dent increase in PARP cleavage was observed in MLH1-deficient 2009,2010,2011),togetherwiththoseofothers(Macphersonetal,
cells when compared with MLH1-proficient comparators at 2005),havehighlightedthedMMRselectivepotentialofdrugsand
nanomolar concentrations (Figure 3A). Cytarabine is known to othercellularperturbationsthatinduceintracellularoxidativestress.
act as an inhibitor of DNA synthesis, competing with dCTP One hypothesis is that these observations can be explained by the
(Grant, 1998). Following conversion to its triphosphate form and role of MMR in the repair of oxidatively damaged DNA, and a
incorporation into DNA, cytarabine triggers stalling of DNA relative failure of these processes in MMR-deficient tumour cells
www.bjcancer.com|DOI:10.1038/bjc.2013.3 989
BRITISHJOURNAL OF CANCER dMMRcancer cellsare sensitivetocytarabine
HCT116
HCT116 + 50nM cytarabine
HCT116 + 100nM cytarabine
HCT116+Chr3
HCT116+Chr3 + 50nM cytarabine
HCT116+Chr3 + 100nM cytarabine
i 2 h ii 24 h
2.0 2.5
S P=NS S P(cid:2)0.05
S/RN 1.5 P=NS S/RN 2.0 P(cid:2)0.05
e ROpared 1.0 e ROpared 1.5
hangcom 0.5 hangcom 1.0
c c 0.5
d d
ol ol
F 0.0 F 0.0
iii 48 h iv 72 h
1.5 P(cid:2)0.05 8
NS P=NS NS P(cid:2)0.001
R R
OS/d 1.0 OS/d 6
e Rpare e Rpare 4
gm gm
hanco 0.5 hanco P=NS
c c 2
d d
ol ol
F F
0.0 0
i 2 h ii 24 h
1.5 1.5 P(cid:2)0.001
NS P=NS P=NS NS P(cid:2)0.01
R R
S/ S/
Od 1.0 Od 1.0
e Rpare e Rpare
gm gm
hanco 0.5 hanco 0.5
c c
d d
ol ol
F F
0.0 0.0
iii 48 h iv 72 h
2.0 4 P(cid:2)0.001
NS P(cid:2)0.001 NS
S/R 1.5 P=NS S/R 3
Od Od
e Rpare 1.0 e Rpare 2 P=NS
gm gm
chanco 0.5 chanco 1
d d
ol ol
F F
0.0 0
Figure6.CytarabinetreatmentinMMR-deficientcellsisassociatedwithanincreaseinROSinadose-dependentmanner.QuantificationofROS
inHCT116andHCT116þChr3cellsexposedtocytarabineorcontrolfordifferentdurations.Samplesweretakenfromhomogenisedcells(A)and
growthmedia(B).Theresultingfluorescentsignalwasquantifiedusinga96-wellplatereader.Assayswereperformedintriplicate.Errorbars
representthes.e.m.Statisticalvalueswereobtainedusingatwo-tailedt-test.
(Martin et al, 2010). As it has previously been demonstrated et al, 2010), we examined 8-oxodG levels in mitochondrial DNA,
that cellular exposure to cytarabine was associated with and observed a particularly marked increase in the levels of
increased oxidative stress (Geller et al, 2001), and that oxidative mitochondrial 8-oxodG in MLH1-deficient cells when compared
stress can result in DNA damage, with excess levels potentially with nuclear DNA (Supplementary Figure 6).
resulting in formation of gH2AX and cytotoxicity (Trachootham If cytarabine caused MLH1 selective effects via an oxidative
et al, 2009), we reasoned that oxidative stress and oxidatively stress-mediated mechanism, then we reasoned that then antiox-
damaged DNA might be responsible for the MMR-deficient idant compounds might modify the impact of cytarabine in
selectivityofcytarabine. MLH1-deficient cells. We exposed HCT116 and HCT116þChr3
We exposed the MLH1-deficient and proficient cells to cells to media containing 10mM N-acetyl-L-cysteineNAC, a
cytarabine, and measured levels of 8-oxodG in DNA, a well- scavenger of ROS (Raj et al, 2011), and cytarabine. We found
validated marker ofoxidatively damaged DNA(Evans et al,2010; that NAC almost totally abrogated the excess cytotoxicity of
Cadet et al, 2012) using an ELISA assay, as previously described cytarabineobservedinMLH1-deficientcells(Figure5B).Exposure
(Martinetal,2010).Cytarabineexposureresultedinanincreasein to NAC reduced the difference in the size of the therapeutic
levelsof8-oxodGinMLH1-deficientcellsafter72h,whichwasnot window observed between the cell lines, suggesting that a pro-
observed in MLH1-proficient cells (Figure 5A). Given the role of oxidant mechanism might be important in mediating MLH1
MLH1 in the repair of mitochondrial oxidative damage (Martin selectivity (Figure 5C).
990 www.bjcancer.com|DOI:10.1038/bjc.2013.3
dMMRcancercells aresensitive tocytarabine BRITISH JOURNALOF CANCER
The MLH1-deficient selective effect of cytarabine is associated model exhibited increased sensitivity to cytarabine, along with a
with presence of increased levels of ROS and collapse of the leukaemia cell line in which expression was inhibited using short
mitochondrial membrane potential. As cytarabine has been hairpinRNA.Takahashietal(2005)reportedthatMMR-deficient
associated with the production of ROS (Geller et al, 2001), we cells were sensitised to DNA polymerase reaction inhibitors,
investigatedwhethercytarabineinducedROSattheconcentrations includingcytarabine.However,inviewofthefactthatweobserved
eliciting MMR selectivity over a time course. We observed nodifferentialselectivitywhenwespecificallyinhibitedPOLA,and
modestlyincreasedlevelsofROSinMLH1-deficientcellsexposed thatweobservedsensitization atonehundredthoftheconcentra-
tocytarabineafter2hoftreatment.Reactiveoxygenspecieslevels tionthatisrequiredforsignificantinhibitionofDNApolymerases
becamefurtherelevatedovertime,inadose-dependentmanner,in (Furth and Cohen, 1968; Grant, 1998), a causal relationship
both cells and later in culture supernatant samples (Figure 6A appearslesslikelyfromourdata.Ourdatainfourisogenicmodels
andB).Notably,whilstsomeinitialincreaseinROSwasobserved together with analysis of publicly available data sets assessing
in MLH1-proficient cells (Figure 6A(i) and (ii)), this was not multiple non-isogenic models demonstrates that MMR selectivity
observed at later time points (Figure 6A(iii) and (iv)). of cytarabine in epithelial and haematological cancer cells is a
Finally, given previous data suggesting that the apoptosis relatively robust effect, and provides greater impetus that
associated with cytarabine treatment occurred following collapse cytarabine should be assessed clinically in patients with MMR-
of the mitochondrial membrane potential (DCm) (Backway et al, deficient malignancies.
1997), we quantified the reduction of DCm with Mitotracker red In order to take forward these observations into the clinical
(Molecular Probes, Invitrogen, Life Technologies) after cytarabine setting, robust biomarkers are required to ensure that the target
treatment over a time course, along with Annexin V and DAPI effectisachievedinvivo;inthiscase,thatoxidativelydamagedDNA
stainingtofurthercharacterisecellpopulationsforcellularviability isgenerated.The8-oxodGELISAassayhasbeeninwidespreaduse
andapoptosis.Mitotrackerredisacellpermeabledyeselectivefor in research outside the field of oncology in the investigation of
mitochondria, withmitochondrial accumulation only taking place oxidative stress in Parkinson’s disease and diabetes. The efforts of
inthepresenceofanadequatemitochondrialmembranepotential. ESCULA(EuropeanStandardCommitteeofUrinary(DNA)Lesion
We observed an increase in the proportion of Mitotracker red- Analysis) among others (Evans et al, 2010) will be essential to the
negative cells (both Annexin V negative cells, and Annexin V furtherdevelopment ofreliableassaysfor clinical use.
positive,apoptoticcells),dependentonlengthofexposuretodrug Intheclinic,low-dosecytarabinetreatmentresultsinplasmadrug
and concentration, and greater in the MLH1-deficient cells levelsof42-64nmoll(cid:2)1(Kufeetal,1985).Thishasbeenreportedas
(SupplementaryFigures7a–d).Takentogether,ourdatasuggested sufficient to result in many of the cytarabine-associated phenotypes,
that cytarabine caused increased cytotoxicity in MLH1-deficient including the delayed replication of human leukaemic cells in vitro,
CRCcellsduetoitsabilitytogenerateinstabilityoftheDCmand and oxidative stress. Provided that these in vitro results can be
generate ROS. Firstly, this could lead directly to apoptosis, as replicated in vivo, a clinical trial of low-dose cytarabine, or a
excess levels of ROS were not controlled and gradually accumu- cytarabine-based combination, in the dMMR subset of epithelial
lated, with ROS levels presumably reaching a critical threshold cancersmostlikelytorespondtoitrepresentsanintriguingpossibility.
becauseofhighpre-existing endogenouslevelsinMLH1-deficient
cells (Trachootham et al, 2009). Secondly, this could lead to
suboptimal repair of oxidatively damaged DNA. The presence of ACKNOWLEDGEMENTS
higher levels of oxidatively damaged DNA likely leads to an
increasednumberofstalledreplicationforks,collapsedreplication We acknowledge National Health Service funding to the National
forks,andultimatelythepotentialfortheformationoflethalDSBs Institute for Health Research Royal Marsden Hospital/Institute of
and an apoptotic response (Supplementary Figure 8). Cancer Research Biomedical Research Centre. Madeleine Hewish
was in receipt ofa ClinicalResearch Training Fellowship from the
Medical Research Council. This work was also funded by a
programme grant from Cancer Research UK (C347/A8363) (Alan
DISCUSSION
Ashworth)andannualgrantsfromBreakthroughBreastCancer(BC
08/09,BC09/10(AlanAshworth)andCTR-Q3Y-Y1(ClareIsacke)).
The findings described here demonstrate the potential value of
comprehensive drug screens in re-purposing established drugs for
the treatment of particular molecular subsets of cancer. We CONFLICT OFINTEREST
demonstrated through a screen of isogenic MLH1-deficient and
proficientcancercelllinesthatcytosine-basednucleosideanalogues
ProfessorAshworthandDrLordareco-investigatorsonpatentsheld
were selectively cytotoxic to MMR-deficient cells, most likely as a
with Kudos-Astra Zeneca for the development of PARP inhibitors
resultoftheirabilitytoaltertheDCmandgenerateROS.Ourdata
and may also benefit through the Institute of Cancer Research
suggest that treatment of CRC cells with low concentrations of
‘RewardstoInventors’scheme.ProfessorAshworth,DrLord,andDr
cytarabineresultsinearlyproductionofROSanddestabilisationof
Martin have a patent pending on the use of methotrexate in
themitochondrialmembranepotential.IntheabsenceofMLH1or
mismatch repair-deficient cancers. ProfessorAshworth has received
MSH2,apoptosismayresultbothfromuncontrolledROSlevelsdue
honorariafromRoche,MerckSerono,andClovisOncology,andisa
to an inadequate or overwhelmed antioxidant response, and also
member of the scientific advisory board of GSK. The remaining
fromaninabilitytorepairoxidativelydamagedDNAleadingtoan
authorsdeclarenoconflictofinterest.
increaseinpotentiallylethalDSBformationandapoptosis.Notably,
othershavereportedthattheabilityofcytarabinetocauseapoptosis
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MartinSA,HewishM,SimsD,LordCJ,AshworthA(2011)Parallelhigh-
throughputRNAinterferencescreensidentifyPINK1asapotential ment. After 12 months the work will become freely available and
therapeutictargetforthetreatmentofDNAmismatchrepair-deficient the license terms will switch to a Creative Commons Attribution-
cancers.CancerRes71:1836–1848. NonCommercial-Share Alike 3.0 Unported License.
Supplementary Information accompanies this paper on British Journal of Cancer website (http://www.nature.com/bjc)
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