rst4444kkkcom

REVIEWCheckpointcontrolandcancerRHMedema1,3andLMacurek21DepartmentofMedicalOncology,UniversityMedicalCenter,Utrecht,TheNetherlandsand2DepartmentofGenomeIntegrity,InstituteofMolecularGenetics,AcademyofSciencesoftheCzechRepublic,Prague,CzechRepublicDNA-damagingtherapiesrepresentthemostfrequentlyusednon-surgicalanticancerstrategiesinthetreatmentofhumantumors.
Thesetherapiescankilltumorcells,butatthesametimetheycanbeparticularlydamagingandmutagenictohealthytissues.
TheefcacyofDNA-damagingtreatmentscanbeimprovediftumorcelldeathisselectivelyenhanced,andtherecentapplicationofpoly-(ADP-ribose)polymeraseinhibitorsinBRCA1/2-decienttumorsisasuccessfulexampleofthis.
DNAdamageisknowntotriggercell-cyclearrestthroughactivationofDNA-damagecheckpoints.
Thisarrestcanbereversedoncethedamagehasbeenrepaired,butirreparabledamagecanpromoteapoptosisorsenescence.
Alterna-tively,cellscanreenterthecellcyclebeforerepairhasbeencompleted,givingrisetomutations.
InthisreviewwediscussthemechanismsinvolvedintheactivationandinactivationofDNA-damagecheckpoints,andhowthetransitionfromarrestandcell-cyclere-entryiscontrolled.
Inaddition,wediscussrecentattemptstotargetthecheckpointinanticancerstrategies.
Oncogene(2012)31,2601–2613;doi:10.
1038/onc.
2011.
451;publishedonline3October2011Keywords:DNAdamage;checkpoint;cancer;check-pointrecoveryIntroductionTomaintaingenomeintegrity,cellsneedtoadequatelyrespondtovariousmodesofgenotoxicstress.
ThisisachievedbyactivationofevolutionarilyconservedDNA-damageresponse(DDR)pathwaysthatabrogatecell-cycleprogressionwhenthegenomeisdamagedandstimulateDNArepair.
DependingontheextentofDNAdamage,cellseithermanagetorepairalllesionsandre-enterthecellcycle(checkpointrecovery),ortheyareeliminatedbyprogrammedcelldeath(apoptosis).
Alternatively,cellscanremainpermanentlyarrestedafteraDNA-damaginginsult(senescence).
ActivationofthecomponentsofDDRpathwaythatallowsefcientDNArepairhasbeenrecentlyreviewedextensivelyelsewhere(Bekker-JensenandMailand,2010;CicciaandElledge,2010;PoloandJackson,2011).
Thisreviewwillfocusonthemechanismsthatactivatecheckpointsaftergenotoxicstressandsilencingofcheckpointsduringtherecoveryprocessthatcanoccuraftersuccessfulrepairofthedamage.
ActivationofDNA-damagecheckpointsIngeneral,activationofDNA-damagecheckpointsisenabledbyrecognitionofDNAdamagebysensors,followedbyanorderedactivationofupstreamandeffectorkinases,thelatterofwhichcandirectlytargetthemajorcell-cyclecontrolmachinery(Figure1a).
DNAdamagecaninduceacell-cyclearrestintheG1,intra-SorG2phaseofthecellcycle.
Dependingonthephaseofthecellcyclewhendamageoccurs,andthemodeofDNAdamage,cellscanactivatedistinctpathwaysinresponsetogenotoxicstress.
TheprimaryresponsetoDNAdamageisaccom-plishedbyupstreamkinasesofthephosphatidylinositol-3-OHkinase-relatedfamily,whichincludesATM(ataxiateleangiectasiamutatedkinase),ATR(ATM-andRad3-relatedkinase)andDNA-PK(DNA-depen-dentproteinkinase).
WhereasATMandDNA-PKareactivatedbydouble-strandbreaks(DSBs),ATRre-spondstoabroaderspectrumofDNA-damaginglesionsthatgeneratesingle-strandedDNA(includingstalledreplicationforkscausedbybulkybaseadductsorUVphotoproducts).
Thissimplisticview,however,isinfactmorecomplexbecauseDSBsareprocessedduringtheS/G2phasesofthecellcyclebytheendonucleaseCtIPandgeneratesingle-strandedDNAlesionsleadingtoasecondaryactivationoftheATR(Jazayerietal.
,2006;Sartorietal.
,2007).
Allphosphatidylinositol-3-OHkinasesrespondingtoDNAdamageshowsimilarsubstratespecicities,andrecentphosphoproteomicscreensrevealasubstantialoverlapoftheirsubstrates(Matsuokaetal.
,2007;Bennetzenetal.
,2010;Bensimonetal.
,2010).
ThebestexampleisprobablythehistonevariantH2AX,whichisrapidlyphosphorylatedatSer-139(producinggH2AX)byATM/ATRorDNA-PKinthechromatinankingthedamagesite.
Ontheotherhand,thesekinasesalsohavesomeexclusivesubstrates,whicharenotsharedbytheothermembers.
ThusATMphosphorylatescheckpointkinase-2(Chk2)atThr-68,whereasATRphosphorylatesChk1attworesidues,Ser-317andSer-345(BartekandLukas,2003).
Ingeneral,Received10July2011;revised16August2011;accepted29August2011;publishedonline3October2011Correspondence:DrLMacurek,DepartmentofGenomeIntegrity,InstituteofMolecularGenetics,AcademyofSciencesoftheCzechRepublic,Videnska1083,Prague414200,CzechRepublic.
E-mail:libor.
macurek@img.
cas.
cz3Currentaddress:DivisionofCellBiology,NetherlandsCancerInstitute,Amsterdam,TheNetherlandsOncogene(2012)31,2601–2613&2012MacmillanPublishersLimitedAllrightsreserved0950-9232/12www.
nature.
com/oncphosphorylationofChk2andChk1leadstotheiractivationandfurthertransmissionofthecheckpointsignal(seebelow).
Activationoftheupstreamcheckpointkinasesre-quirestherecognitionoftheDNAlesion.
IncaseofDSBs,thisisachievedbytheMre11–Rad50–Nbs1complexthatdirectlybindstotheexposedendsoftheDNA,recruitsATMandinitiatesitsactivation(Falcketal.
,2005).
ActiveATMgeneratesgH2AXatthesurroundingchromatin,whichisrecognizedbyphosphopeptide-bindingBRCTdomainsthatarepresentinmanyproteinsinvolvedintheDDR,andservesasadockingsiteforalargeadaptorprotein,MDC1(Stuckietal.
,2005).
Recruitmentofmultipleadditionalcompo-nentsofthecheckpointthenfollowsinanorderlymanner,includingRNF8,RNF168,HERC2,53BP1andBRCA1(reviewedelsewhereBekker-JensenandMai-land,2010).
Ingeneral,recruitmentoftheseproteinstothechromatinisessentialforefcientDNArepairandmayalsohavesomeroleinfurtheramplicationofthecheckpointsignaling(thatis,byrecruitingmoreATM)(Leeetal.
,2010;Shibataetal.
,2010).
ATMisrapidlyautophosphorylatedatSer-1981,whichwasoriginallyproposedtoinducethedissociationofaninactivehomodimerintoactivemonomers(BakkenistandKastan,2003).
However,micelackingthecorrespondingATMautophosphorylationsitesarestillabletofullyactivatethecheckpointafterexposuretoionizingradiation(IR),stronglysuggestingthatATMautopho-sphorylationisnotanessentialstepforitsactivation(butausefulmarkerofATMactivity)(Pellegrinietal.
,2006).
Instead,DNAdamage-inducedacetylationofATMatLys-3016bytheacetyltransferaseTip60wasrecentlyproposedtoactivatetheATM(Sunetal.
,2005,2007).
Inthismodel,Tip60isrecruitedtoDSBsbyadirectinteractionwiththeMre11–Rad50–Nbs1complexanditsenzymaticactivitytowardATMisincreasedafterbindingofitschromodomaintotrimethylatedhistoneH3(H3K9me3)(Sunetal.
,2009;Chailleuxetal.
,2010).
Bycontrast,single-strandedDNAlesions(suchasthestalledreplicationforksorresectedDSBs)arerapidlycoatedbyreplicationprotein-Acomplexes,whichfurtherrecruitandactivateATRinastablecomplexwithitscofactorATRIP(ZouandElledge,2003).
Inparallel,replicationprotein-AbindstoRad17andrecruitsaring-shapedtrimericcomplex,Rad9–Hus1–Rad1(9-1-1),tothesiteofdamage.
This9-1-1complexisthenphosphorylatedbyATRandcanbindtotheBRCTdomainsofanadaptorprotein,TopBP1,whichfurtherbooststheactivityofATR(CimprichandCortez,2008).
RHINO,arecentlyidentied9-1-1-andTopBP1-interactingprotein,isrequiredforefcientactivationofATR(Cotta-Ramusinoetal.
,2011).
ActiveATRhasseveralhundredsofsubstrates,includingChk1,whichisessentialforinductionofthecheckpointresponse.
Importantly,Chk1needstoformacomplexwithamediatorprotein,claspin,tobeefcientlyactivatedbyATR(ChiniandChen,2003;Kumagaietal.
,2004).
Uponphosphorylation,Chk1isreleasedfromthechromatinandcanfreelydiffusethroughthenucleustophosphorylatemultiplesubstratestoestablishthecheckpointresponse(seebelow)(Smitsetal.
,2006).
Checkpointkinases—effectorsofthecheckpointProliferatingcellsrepeatedlypassthroughtheG1(growthphase),S(replicationofDNA)andG2phasescyclinCdkATMp53Chk2DSBp21Cdc25A/B/CWee1Nek11Chk1ClaspinATRssDNARPA9-1-1ATRIPTopBP1RHINOCtIP(SorG2phase)BaxNoxaAPOPTOSISMRNH2AXMDC1IRorchemotherapyUVorreplicationstressp38MK2CHECKPOINTARRESTGADD45CHECKPOINTMAINTENANCE&SURVIVALMdm2Plk1Aurora-AWip1ATMp53p21Chk1ClaspinATRMdm2MdmXH2AXCdc25A/B/CWee1GTSE1hBoracyclinBCdk1CHECKPOINTRECOVERYFigure1(a)Checkpointactivation.
DNAdamageisrecognizedbyvarioussensorandadaptorproteins,whichleadstoactivationofATMandATRkinasesandallowsestablishingofaDNA-damagecheckpoint.
ThemajorcomponentsactivatedduringthisresponseareChk1andp53(green).
Chk1regulatescdc25A/B/CandWee1,whichleadstoinhibitoryphosphorylationofCdk(yellowdots),preventsitsactivation(red)andcausescell-cyclearrest.
p53isstabilizedbymultipleposttranslationalmodications(dots),andbyincreasingthetranscriptionofaCdkinhibitor,p21,andrepressingthetranscriptionofcyclin-B,itcontributestoinhibitionofcyclin–Cdk.
Inaddition,cellsactivatetheChk2andp38/MK2pathwaysthatareinvolvedinapoptosisandcheckpointmaintenance,respectively.
(b)RecoveryfromtheG2checkpoint.
AfterDNArepair,multipleproteinsarede-phosphorylatedbyWip1phospha-tase,whichleadstosilencingofthecheckpointsignalingandinactivationofp53.
Inparallel,Plk1kinasetargetsclaspinandWee1fordegradation,andactivatesCdc25A/B/C,whichallowsactivationofcyclin-B–Cdk1andcheckpointrecovery.
CheckpointcontrolandcancerRHMedemaandLMacurek2602Oncogeneofthecellcycle,followedbynucleardivision(mitosis)andcellulardivision(cytokinesis).
Transitionsbetweenthedifferentphasesofthecellcyclearefacilitatedbyevolutionarilyconservedcyclin-dependentkinases(Cdks),whichactincomplexwithvariouscyclins(mainlyCdk2/cyclin-EinG1/SandCdk1/cyclin-BinG2/Mtransition).
ActivityofaCdkisnegativelyregulatedbyphosphorylationofThr-14andTyr15byWee1andMyt1kinases.
Conversely,Cdksareactivatedbyde-phosphorylationoftheseresiduesbytheCdc25familyofphosphatases.
Thecell-cyclearrestinducedinresponsetoDNAdamageisatleastinpartestablishedthroughinhibitionofCdc25activity.
HumancellsexpressthreedistinctisoformsofCdc25,Cdc25A,BandC,allofwhichcandephosphorylateandactivateCdks.
AfterDNAdamage,Cdc25Aisrapidlydegraded,whichleadstoincreasedinhibitoryphosphorylationatTyr-15ofCdk2orCdk1,andcausesaG1orG2checkpointarrest,respectively(Mailandetal.
,2000).
DestructionofCdc25AisexecutedbybTrCP–SCF-dependentubiquitinationandproteasomaldegradation,andisinducedbyCdc25AphosphorylationatSer-76mainlybyChk1(Jinetal.
,2003,2008).
Chk1alsoautophosphorylatesatSer-296,whichgeneratesamotifrecognizedby14-3-3gthatmediatestheinteractionofChk1withCdc25A(Kasaharaetal.
,2010).
Inparallel,Chk1alsoactivatesNek11(Neverinmitosisgene-A-relatedkinase-11),whichfurtherphosphorylatesCdc25AatSer-82andenablesitsrecognitionbythebTrCP–SCF(SKP/Cullin/F-boxprotein)complex(Melixetianetal.
,2009).
TheATM–Chk2pathwaywasoriginallyalsosuggestedtobeinvolvedinthedegradationofCdc25A(Falcketal.
,2001);howeverthendingthatIR-induceddegradationofCdc25AinHCT116-Chk2/cellsoccurswiththesamekineticsasinwt-HCT116cellsindicatesthatthecontributionofChk2todegradationofCdc25Aiseitherfunctionallyredundantoroccursinacelltype-specicmanner(Jinetal.
,2008).
Inaddition,Cdc25BandCdc25Carephosphory-latedbyChk1/2atSer-323andSer-216,respectively,andarefunctionallyinactivatedbybindingto14-3-3proteins(Pengetal.
,1997;Sanchezetal.
,1997;ForrestandGabrielli,2001).
Nonetheless,micedecientinCdc25BandCdc25CarestillabletofullyactivatetheDNA-damagecheckpoints,indicatingthatmereregulationofCdc25Aissufcienttoestablishthecheckpoint(Fergu-sonetal.
,2005).
ThisobservationisinlinewiththecurrentviewoffunctionalredundancyinthemolecularmachinerythatcontrolstheG2/Mtransition(Lindqvistetal.
,2009b).
InadditiontoitseffectonCdc25A,Chk1canalsophosphorylateandactivateWee1kinase,whichcandirectlyincreasetheinhibitorymodicationofCdk2andCdk1(O'Connelletal.
,1997).
Importantly,theroleofChk1incheckpointcontrolisevolutionarilyconservedfromyeasttohumansandhumanChk1operatesbythesamemolecularmechan-ismsasitsyeasthomolog(Sanchezetal.
,1997).
Chk2(homologoustoRad53andCds1inyeast)isstructurallydistinctfromChk1,butbothkinasessharesimilarsubstratespecicityandmaythereforehaveoverlappingsubstrates.
DatafromChk2-knockoutmiceandalsofromHCT116-Chk2/humancoloncancercellsindicatethat,incontrasttoyeast,Chk2isfunctionallyredundantincheckpointactivationinhighereukaryotes(Jacketal.
,2002;Jinetal.
,2008).
Instead,Chk2seemstoadoptauniqueroleintheregulationoftheapoptoticpathwaysinducedbyDNAdamage,asChk2/micearemoreresistanttoIRcomparedwiththecontrolanimals(Hiraoetal.
,2002;Takaietal.
,2002).
IRstimulatesATMtoactivateChk2byphosphorylationatThr-68,whichinturnleadstostabilizationofthetumorsuppressorp53andpromotesthetranscriptionofseveralpro-apoptoticgenessuchasBax,PumaandNoxa(Ahnetal.
,2000;Hiraoetal.
,2000,2002;Takaietal.
,2002).
Phosphorylationofp53atSer-20wasoriginallyproposedtoberesponsiblefortheChk2-dependentactivationofp53;however,thismodicationwasretainedinChk2/cellsaswellasinChk2-depletedcells,suggestingthatadditionalmechanismsmayexistbywhichChk2modulatesapoptoticresponses(Ahnetal.
,2003;Jallepallietal.
,2003).
Indeed,Chk2hasalsobeenreportedtoregulateapoptosisaftertreatmentwithetoposideinanp53-independentmannerthroughphosphorylationofE2F1(Stevensetal.
,2003).
Chk2seemstobespecicallyinvolvedinregulatingapoptoticresponsestoagentscausingDSBsasUV-inducedapoptosisisnotaffectedinChk2/cells(Hiraoetal.
,2000).
ThisisingoodagreementwithChk2beingspecicallyactivatedbyATMbutnotATR.
Strikingly,thefunctionalredundancyofChk2forcheckpointactivationislostwhencellslosep53(Jiangetal.
,2009).
Incontrasttop53-procientcells,depletionofChk2inp53-decientcellsinterfereswiththeIR-inducedcheckpointarrest(Jiangetal.
,2009).
Reasonsforrewiringofcheckpoint-activatingpathwaysinp53-decientcellsarecurrentlyunclear;however,aninter-estingpossibilityisthat,bylosingp53,highereukaryoticcellssimplyregainsimilarmolecularpathwaysthatactinyeastandthatweresuppressedduringevolution.
AnothermechanismimplicatedinG1checkpointinductionisrapiddegradationofcyclin-D1afterexposuretoIR(AgamiandBernards,2000).
Degrada-tionofcyclin-D1isbelievedtoreleasetheCdkinhibitorp21fromCdk2/cyclin-D1complexes,whichinturnallowsp21toinhibitCdk2/cyclin-EandpreventtheG1/Stransition.
Degradationofcyclin-D1wasoriginallyreportedtodependonadestructionmotifwithincyclin-D1andubiquitinationbytheAPCE3ligasecomplex(AgamiandBernards,2000).
However,recentevidencesuggeststhatafterDNAdamage,cyclin-D1isubiqui-tinatedbytheSCFE3ligasecomplexandthisdependsbothonphosphorylationofcyclin-D1atThr-286byglycogensynthasekinase-3bandadirectphosphoryla-tionoftheF-boxproteinFBXO31byATM(Santraetal.
,2009).
Thus,itispossiblethattheprecisemechanismbywhichcellsdegradecyclin-D1afterDNAdamageiscelltype-dependent.
Moreover,italsoappearsthatDNAdamagespecicallyinducesthedegradationofcyclin-D1(butnotcyclinsD2andD3)indicatingthattheroleofthispathwayintheDDRinG1mightdependontheexpressionproleofD-typecyclinsinvarioustissues.
CheckpointcontrolandcancerRHMedemaandLMacurek2603OncogeneFinally,p38anditsdownstreameffectorMAPKAPkinase-2(MK2)haverecentlybeenimplicatedintheDDRinG2afterexposuretoUV(Bulavinetal.
,2001;Mankeetal.
,2005).
Thecontributionofp38totheDDRwasattributedtothephosphorylationofcdc25Bandcdc25CbyMK2(Mankeetal.
,2005).
Moreover,rapidandtransientactivationofthep38/MK2pathwaywasalsoreportedafterIR,ortreatmentwithdoxor-ubicinoralkylatingagentssuchasmethylmethanesul-fonate(MMS)(Mikhailovetal.
,2004;Ramanetal.
,2007;Lafargaetal.
,2009;Phongetal.
,2010).
Activationofthep38/MK2branchofthecheckpointhasbeenreportedtoinvolveATM/ATR-dependentactivationofThousandandoneaminoacidproteinkinases(TAO)thatbelongtotheMAPKKKfamily.
TAO-dependentphosphorylationofMEK3andMEK6thaninducestheactivityofp38(Ramanetal.
,2007).
Inaddition,thep38pathwaymightbeinvolvedintheinitiationoftheG1checkpointbyarapidstabilizationofp21mRNAcausedthroughphosphorylationofthemRNA-bindingproteinHuRbyp38atThr-118(Lafargaetal.
,2009).
Despiteallthis,theroleofthep38pathwayincheckpointactivationhasrecentlybeenchallengedwhenp38pathwaywassuggestedtohaveanessentialroleinsurvivalafterDNAdamagethroughinductionofmultiplesurvivalgenes(Phongetal.
,2010).
Inaddition,p38/MK2seemstobeinvolvedincheck-pointmaintenance(seebelow).
Thus,thepreciseroleoftheATM/ATR–p38–MK2pathwayinDDRsremainsincompletelyunderstoodanditispossiblethatitlargelydependsonthemodeandamplitudeofDNAdamage.
CheckpointmaintenanceAnappropriatecheckpointresponsetogenotoxicstressneedstobefastenoughtopreventtransitiontothenextphaseinthecellcyclewithdamagedDNA,butdurableenoughtoallowtimeforefcientDNArepair.
Recentworkhasmadeitclearthatinductionandmaintenanceofthecheckpointsaregovernedbydistinctmolecularmechanisms.
Asdiscussedabove,thefastcheckpointinductionlargelydependsonthetransmissionofthesignalthroughphosphorylationofmultiplesubstratesbyATM/ATRandChk1/2kinases,affectingtheiractivity,proteinstabilityorboth.
Conversely,pathwaysthatdependonchangesinthetranscriptionoftargetgenesactconsiderablyslowerandaremainlyinvolvedincheckpointmaintenance.
Thebest-studiedexampleofcheckpointmaintenanceisthecontributionofthetumorsuppressorp53anditstranscriptionaltargetp21.
p53isextensivelymodiedposttranslationally,especiallyintheN-terminaltransactivationdomain(includingphos-phorylationofSer-15andSer-20byATM/ATR/DNA-PKandChk1/2,respectively)andintheC-terminalregu-latorydomain(includingacetylationofLys-382andLys-320byCBP/p300andPCAF,respectively),whichtogetherreducetheafnityofp53foritsnegativeregulatorMdm2.
Thisallowsstabilizationandtetra-merizationofp53;promotesassociationofp53withtranscriptionalcoactivators(orco-repressors);andbindingofp53tothepromotersofitsmultipletargetgenes(forarecentreviewonp53posttranslationalmodicationsseeDaiandGu(2010)).
Oneofthese,thecyclin-dependentkinaseinhibitorp21,bindstoandinhibitsthecyclin-E/Cdk2andcyclin-A/Cdk2com-plexes,andhasamajorroleinthecontrolofG1arrest.
Similarly,p21alsocontributestomaintenanceoftheG2checkpoint(Bunzetal.
,1998).
Inaddition,p53canalsocontroltheG2checkpointindependentlyofp21throughtranscriptionalrepressionofmitoticinducers,includingcyclin-B,cdc25Bandpolo-likekinase-1(Plk1)(Imbrianoetal.
,2005;McKenzieetal.
,2010;Dalvaietal.
,2011).
Interestingly,recentdataindicatethatthedynamicsofp53'sresponsetoIRdoesnotoccurinalinearmannerbutinsteadappearsinmultiplerepeatingpulseswithxedamplitudeandduration(Lahavetal.
,2004;Batcheloretal.
,2008).
Theseoscillationshadlongremainedmaskedowingtoaveragingacrossapopulationofcells,buttheirobservationbecamepossiblewithrecentadvancesinsingle-cellimagingtechniques(Batcheloretal.
,2009).
Theoscillatorybehaviorofp53isinducedbyactivationofp53byATMatthetimeDNAdamageispresent,whichsubsequentlyresultsinthetranscriptionalactivationofMdm2andWip1(seebelow),bothnegativeregulatorsofp53,whichestablishnegativefeedbackloops(Batcheloretal.
,2009)(Figure2).
InresponsetoDSBs,cellsstabilizep53,generatingtherstpulse,whichisthenfollowedbypulsesofasimilarintensityrepeatingin4-to7-hintervalsuntiltheDNAisfullyrepaired.
Strikingly,spontaneouspulsesofp53expressionofcomparableintensitycanalsooccurinnormalcyclingcells;however,posttranslationalmodicationskeepp53inactiveintheabsenceofsustaineddamage(Loeweretal.
,2010).
Bycontrast,UVirradiationcausesaninductionofp53,butdoesnotinduceoscillationsasseenafterIR.
Instead,UVinducesasinglep53pulsetheamplitudeofwhichincreaseswiththedoseofUV(Batcheloretal.
,2011).
TheroleofIR-inducedp53pulsesindeterminingcellfatestillneedstobeaddressedfully;however,itopensupanexcitingpossibilitythat,byrecurringphasesofstrongcheck-pointoutputwithweakcheckpointoutput,cellscansensetheactualstrengthoftheDNAdamageandcanpreventinappropriateinitiationofapoptoticcelldeath(Batcheloretal.
,2009).
Importantly,theDDRinG1isprimarilydependentonp53.
Asaconsequence,cancercellsthathavelostp53failtoestablishaG1arrestinresponsetoDNAdamage,butarrestinG2instead,aresponsethatremainsrelativelyintactalsoincancercells(KuntzandO'Connell,2009).
Thisimpliesthat,apartfromp53,additionalmechanismsmustexisttopreventp53-decientcellsfromentryintomitosiswithdamagedDNA.
Survivalofp53-decientcellsafterDNAdamagewasrecentlyreportedtodependonthep38/MK2pathway(Reinhardtetal.
,2007).
WhereastheChk1pathwaywasfoundtobeimportanttoestablishtheG2checkpoint,thep38/MK2pathwaywasshowntobecriticalforlong-termmaintenanceoftheG2checkpoint(Reinhardtetal.
,2010).
Strikingly,Chk1andMK2kinasessharethesameoptimalphosphorylationmotifCheckpointcontrolandcancerRHMedemaandLMacurek2604Oncogene(Arg-X-X-pSer/Thr)andthusmayhaveoverlappingsubstrates(Mankeetal.
,2005).
However,thisdoesnotappeartobetheentirestory,asbothkinasesshowadistinctsubcellulardistributionaftertreatmentwithdoxorubicin.
WhereasChk1remainsmainlynuclearafterDNAdamage,activatedMK2rapidlyredistributestothecytoplasm,whereitphosphorylatesdistinctsubstrates(Reinhardtetal.
,2010).
ByphosphorylatinganRNA-bindingproteinhnRNPA0andaribonucleasePARN,activatedMK2inducestheaccumulationofgrowtharrestandDNA-damage-inducibleprotein-a(GADD45a)mRNA,leadingtoincreasedproteinlevelsofGADD45a(Reinhardtetal.
,2010).
Inturn,GADD45awassuggestedtoactinapositivefeedbackloopandtoinducep38/MK2-dependentphosphoryla-tionofcdc25B/C,bindingof14-3-3proteinandcytosolicsequestrationofcdc25B/C(Reinhardtetal.
,2010).
Thisndingisinlinewiththeobservationthatthepro-survivalactivityofGADD45ainhematopoieticcellsexposedtoUVdependsonthep38pathway(Guptaetal.
,2006).
However,GADD45awasalsoreportedtodirectlyassociatewithandinhibitcyclin-B/Cdk1(Wangetal.
,1999;Zhanetal.
,1999),andthusitispossiblethatGADD45apreventsprematuremitoticentrythroughmultipleindependentmechanisms.
Inaddition,thep38pathwaywasalsoreportedtoregulatecheckpointmaintenancethroughstabilizationofp27Kip,whichcanfurthersuppressanyresidualCdkactivityincasetheDNAdamagepersists(Cuadradoetal.
,2009;Liontosetal.
,2010).
AscheckpointsneedtobemaintaineduntiltheDNAdamageisfullyrepaired,DNA-repairpathwaysarelikelytobefunctionallyconnectedwithcheckpointsignaling.
Indeed,shortinterferingRNAscreensforDNArepairfactorsinvolvedincheckpointregulationrevealedthattwohomologousrecombination-repairproteins,BRCA2andPALB2,arerequiredforG2checkpointmaintenance(Cotta-Ramusinoetal.
,2011;Menzeletal.
,2011).
ItispossiblethatbyusingDNA-repairfactors,cellscoordinatecheckpointsignalingwithongoingDNArepair;however,theexactmechanismbywhichBRCA2andPALB2communicatewiththecheckpointstillremainstobeexplored.
SilencingofcheckpointsbyphosphatasesandcheckpointrecoveryAftersuccessfulDNArepair,cellsre-enterthecellcycleinaprocesscalledcheckpointrecovery.
Owingtotheinactivationanddegradationofamultitudeofcell-cycle-regulatoryproteins,whichoccursinresponsetoDNAdamage,cellsthatarearrestedinresponsetoDNAdamagearebiochemicallydifferentfromunda-magedcellsinthesamephaseofthecellcycle.
Thishastheconsequencethatthecell-cyclemachineryoperatesdifferentlyduringrecoveryascomparedwithcontrolofthecellcycleinunperturbedcells.
Currently,thebestunderstoodmechanismsthatpromoterecoveryaremechanismsresponsibleforcheckpointrecoveryfromtheG2checkpoint(Figure1b).
WhereasthereisaconsiderabledegreeoffunctionalredundancyinmechanismsregulatingunperturbedG2/Mprogres-sion,certainpathwaysbecomeessentialwhencellsrecoverfromDNAdamage(Lindqvistetal.
,2009b).
Inparticular,thisistrueforPlk1(anditsyeasthomologcdc5)depletionorinhibitionofwhichdoesnotaffectnormalmitoticentrybutcompletelyblockscheckpointrecovery(Toczyskietal.
,1997;vanVugtetal.
,2004;Lenartetal.
,2007).
Plk1containsaconservedthreonineresidue(Thr-210)withintheT-loop,whichhastobephosphorylatedtofullyactivatePlk1(Jangetal.
,2002).
InlateG2,Aurora-Akinase,togetherwithitscofactor,hBora,phosphorylatesPlk1atThr-210,resultinginactivationPlk1(Macureketal.
,2008;Sekietal.
,2008).
InresponsetoDNAdamage,activityofPlk1iskeptlowthroughinhibitionofThr-210phosphorylation(Smitstimep53levelsIRrepairATMp53Wip1Mdm2IRFigure2Dynamicsofthep53responseinducedbyDNAdamage.
Inunstressedcells,basallevelsofp53arekeptlowbyMdm2-dependentdegradation.
InresponsetoIR-induceddamage,ATMisactivatedandphosphorylatesp53andMdm2bothresultinginstabilizationofp53.
Activep53stimulatesthetranscriptionofMdm2andWip1mRNA,whichleadstoadelayedincreasedexpressionoftheseproteins.
Inturn,Wip1de-phosphorylatesp53,ATMandMdm2,generatingnegativefeedbackloopsthatleadtodestabilizationofp53.
Thisresponseresultsinregularoscillationsofp53levelsuntiltheDNAdamageisrepaired.
Spontaneouspulsesofp53transcriptioncanbegeneratedbytransientdamagethatoccursduringthecell-cyclephaseassociatedwithintrinsicDNAdamage.
Thegreenlinesindicateactivation,redlinesinactivation.
CheckpointcontrolandcancerRHMedemaandLMacurek2605Oncogeneetal.
,2000;TsvetkovandStern,2005).
Plk1activationcanpromotemitoticentryinanunperturbedcellcycle,butfollowingaDNA-damaginginsult,cellscometocompletelyrelyonPlk1tore-enterthemitoticcycle(vanVugtetal.
,2004).
Topromoterecovery,Plk1targetsclaspinandWee1forbTrCP–SCF-dependentdegrada-tion(Watanabeetal.
,2004;Mailandetal.
,2006;Mamelyetal.
,2006;Peschiarolietal.
,2006),promotesnucleartranslocationofCdc25B/C(Toyoshima-Mor-imotoetal.
,2002;Lobjoisetal.
,2009)anddirectlyinhibitsChk2(vanVugtetal.
,2010).
Asaresult,Plk1effectivelypreventsfurtheractivationofbothChk1/2andcontributestoactivationofcyclin-B/cdk1com-plexes,stimulatingcheckpointsilencingandrecoveryatmultiplelevels(vanVugtetal.
,2004).
Inaddition,Plk1phosphorylatesG2-andS-phase-expressedprotein-1,whichactsasanegativeregulatorofp53andthusPlk1activitycontributestosuppressionofp53duringcheckpointrecovery(Liuetal.
,2010).
Plk1activityisessentialbutnotsufcientforcheckpointrecovery,indicatingthatadditionalcontrolmechanismsexist.
Amongthem,phosphatases,whichcounteractthemultitudeofproteinphosphorylationsimplementedbythecheckpointmachinery,arelikelycandidatestobeinvolvedinsilencingthecheckpointandpromotingcheckpointrecovery(FreemanandMonteiro,2010).
Dataanalysisfromlargephosphopro-teomicscreensindicatesthatphosphatasesmayhaveamoreactiveroleintheregulationofDDRsthanoriginallyanticipated(Bensimonetal.
,2010).
Sofartheonlywell-characterizedphosphatasethathasbeendescribedtoactivelyparticipatetoestablishthecell-cyclearrestisPP2Aincomplexwithitsregulatorysubunit,B56g.
Thus,B56gisstabilizedinanATM-dependentmannerandactivatesp53throughde-phosphorylationofpThr-55(Lietal.
,2007;Shouseetal.
,2011).
Ontheotherhand,themajorcellularphosphatasePP2AhasalsobeenshowntolimitbasalphosphorylationofATM–pSer-1981,Chk2–pThr-68,Chk1–pSer-317andgH2AX(Goodarzietal.
,2004;Chowdhuryetal.
,2005;Leung-Pinedaetal.
,2006;Carlessietal.
,2010;Freemanetal.
,2010).
BykeepingarelativelyhighbasalactivityofPP2A,cellscanthereforepreventinadequateactivationofthecheckpointundernormalconditions.
DNAdamage(perhapswhenexceedingacertainthreshold)rapidlydisruptstheinteractionofPP2AwithATM,Chk1andChk2,allowingfullactivationofthecheckpoint(Goodarzietal.
,2004;Carlessietal.
,2010;Freemanetal.
,2010).
Similarly,PP1phosphataseincomplexwithitschroma-tin-targetingsubunit,Repo/Man,wasrecentlydemon-stratedtoinhibittheactivityofATMunderunstressedconditions,thusdeterminingthethresholdforactivationoftheDNA-damagecheckpoint(Pengetal.
,2010).
ItispossiblethataftersuccessfulDNArepair,there-establishedinteractionofPP2Aand/orPP1withvariouscomponentsoftheDDRpathwayhelpstorevertproteinphosphorylationbacktotheoriginallevel,contributingtothesilencingofthecheckpoint.
However,itremainsuncleartowhatextentcellsactivelyregulatethesephosphatasesduringtheDDR.
Moreisknownaboutthecontributionofphospha-tasestocheckpointsilencingandrecovery.
RecentdatafromseveralgroupsindicatethataPP2Cdphosphatase(PPM1D,hereafterreferredtoasWip1)hasacentralroleinterminationofthecheckpointsignalingandincheckpointrecovery(Luetal.
,2008;LeGuezennecandBulavin,2010).
SeverallinesofevidencesuggestthatthefunctionofWip1istightlylinkedwithregulationoftheDDR.
First,depletionofWip1byRNAinterferenceresultsinprolongedcheckpointactivationwhereasoverexpressionofWip1causesefcientcheckpointoverride,indicatingthatWip1hasthepotentialtoregulatecheckpointsignaling(Luetal.
,2005;Lindqvistetal.
,2009a).
Second,thesubstratespecicityoftheWip1seemstocorrespondwiththemotifsphosphory-latedduringtheDDR,namelypSQ/pTQmotifsthatareselectivelyphosphorylatedbyATM/ATRandthepTxpYmotifinp38thatisalsophosphorylatedinresponsetoDNAdamage(Yamaguchietal.
,2007).
Byrecognizingsuchmotifs,Wip1couldefcientlytargetallofthesubstratesofATM/ATR,andthisishasthusfarbeenconrmedforATM–pSer-1981,Chk1–pSer-317,Chk2–pThr-68,p53–pSer-15,Mdm2–pSer-395andgH2AX(Takekawaetal.
,2000;Fujimotoetal.
,2005;Luetal.
,2005,2007;Shreerametal.
,2006a;Chaetal.
,2010;Macureketal.
,2010;Moonetal.
,2010).
Third,PPM1Disatargetgeneofp53andexpressionofWip1isincreasedaftergenotoxicstress(Fiscellaetal.
,1997).
Likethis,cellscancyclewithlowlevelsofWip1(limitingtheriskofanundesiredde-phosphorylationofanyphosphoproteininvolvedinnormalcellprogres-sion),whereasDNAdamagereleasesanegativefeed-backloopinwhichexpressionofWip1willlimittheamplitudeofthecheckpointtopreventanirreversiblearrest.
Fourth,prematuretranslationofWip1mRNAisblockedbymiR-16,whichisalsoinducedbygenotoxicstress(Zhangetal.
,2010).
ThismechanismassuresthatearlyafterDNAdamageWip1levelsarekeptlowtoallowefcientcheckpointinductionandDNArepair.
Fifth,Wip1isanuclearproteinandisenrichedatthechromatin,whereitcangetinclosecontactwiththeproteinsoftheDDRpathway(Macureketal.
,2010).
Finally,althoughWip1isnotpresentinyeast,itappearsthatloweukaryotesusethesamefamilyofPP2Cphosphatases(namelyPtc2andPtc3)toregulatecheckpointrecovery,indicatingpartialconservationinevolution(Leroyetal.
,2003).
AmongmanysubstratesofWip1,p53seemstohaveaspecialroleincheckpointrecovery.
WehavenotedthatevencompleteinhibitionofChk1/2,ATM/ATRandp38isnotsufcienttopromoterecoveryinWip1-depletedcells(Lindqvistetal.
,2009a).
Ontheotherhand,cellslackingbothWip1andp53recoverednormally,indicatingthatp53isthecrucialtargetofWip1tocontrolrecovery.
Thisalsoimpliesthatde-phosphorylationofallotherWip1substratesislessrelevantforefcientrecovery.
Whatismore,wecouldshowthatWip1isrequiredthroughoutthecheckpointresponse,notjustatthestagethatthecheckpointisdenitivelyterminated(Lindqvistetal.
,2009a).
ThismeansthattheactivityofWip1isrequiredthroughoutCheckpointcontrolandcancerRHMedemaandLMacurek2606OncogenetheDNA-damagecheckpointtopreventexcessivep53activationandisessentialtoretaincheckpointrecoverycompetence(Lindqvistetal.
,2009a).
Interestingly,Wip1canregulatep53bymultiplemechanisms.
Apartfromdirectde-phosphorylationofpSer15onp53,Wip1hasbeenshowntoactivatetheubiquitinE3ligasemdm2,whichtargetsp53forproteasomaldegradation,andactivateMdmX,whichdirectlyinhibitsthetranscrip-tionalactivityofp53(Luetal.
,2005,2007;Zhangetal.
,2009).
Whichoneofthesemechanismsisthemostphysiologicallyrelevantiscurrentlyunclear;however,counteractingp53'sfunctionseemstobethemajorroleforWip1topreventanirreversiblecell-cyclearrest.
Inaddition,thetwonegativefeedbackloopsinwhichWip1inactivatesp53andATMarerequiredtoestablishthedynamicbehaviorofthep53responsethatfollowsafterDSBs(seeabove).
Importantly,Wip1isoverexpressed(usuallybecauseofamplicationofthe17q23locus)inmultiplehumancancers,includingmedulloblastomas,neuroblastomas,breast,ovarianandgastriccarcinomas(Bulavinetal.
,2002;Lietal.
,2002;Saito-Oharaetal.
,2003;Castellinoetal.
,2008;Tanetal.
,2011).
Moreover,overexpressionofWip1islinkedtopoorprognosisinlungadenocarci-noma(Satohetal.
,2011).
HighexpressionlevelsofWip1areusuallyfoundintumorsthatretainwild-typep53,whereasoverexpressionofWip1israreintumorscarryingmutationsorlackingp53(Bulavinetal.
,2002).
Strikingly,Wip1-knockoutmiceareviableandareresistanttooncogene-inducedaswellasspontaneouscancerdevelopment(Bulavinetal.
,2004;Harrisonetal.
,2004;Belovaetal.
,2005;Nannengaetal.
,2006;Shreerametal.
,2006b),suggestingthatinhibitionofWip1activitymaypotentiallybebenecialincancertherapy.
Duringprolongedcheckpointactivation,cellsneedtomaintainexpressionofcriticalcell-cycleregulatorsaboveaminimalleveltoremaincompetentforeventualcheckpointrecoveryaftersuccessfulrepair.
ThishasbeenshowntobethemostcrucialfunctionofWip1duringacheckpointresponseinG2.
WhencellsdepletedofWip1aretreatedwithDNA-damagingagents,expressionofcyclin-B(aswellasanumberofothercell-cycle-regulatoryproteins)decreasesbelowtheminimallevelrequiredforrecovery(Lindqvistetal.
,2009a).
Theexcessivereductionincyclin-Bexpressionisduetodisproportionateactivationofp53,whichoccursincellslackingWip1(Lindqvistetal.
,2009a).
Whenexpressionofcyclin-Breducesbelowacriticalthreshold,cellslosethecompetencetorecover.
ThisdemonstratestheimportanceofcontinuedexpressionoftheG2clusterofcell-cycle-regulatedgenesduringaDNA-damage-inducedarrestinG2.
Indeed,itwasdemonstratedrecentlythatminimalbasalCdkactivityandtransactivationbythetranscriptionalfactorFoxM1isneededtosustaincyclin-Bexpressionatalevelthatcanfacilitaterecoveryoncethecheckpointissilenced(Alvarez-Fernandezetal.
,2010).
TargetingcheckpointcomponentsincancertherapyInductionofcelldeathbyexcessiveDNAdamagerepresentsthegeneralprincipleofvariouscancertherapies,suchasradiotherapyandalargeproportionofchemotherapeutictherapies.
Thistreatmenttargetsgeneticallyinstablecancercells(thatarepronetocelldeathcausedbygenotoxicstress)butalsohitsnormaltissues,especiallythosewithhighproliferativerates(suchasepitheliainthegastrointestinaltract,hairfolliclesandbonemarrow).
UndesiredtargetingofhealthytissuesbyDNA-damagingagentsrepresentsamajorproblemintheclinicsandlimitsefciencyincuringcancer.
AlargesubsetofcancertypesisincapabletoestablishtheG1checkpoint,mostoftenbecauseofmutationorlossofthep53tumorsuppressor.
SurvivalofthesecellsafteraDNA-damagingtreatmentdependsonactivationoftheG2checkpointbytheATR/Chk1,p38/MK2andATM/Chk2pathways(Wangetal.
,1996;Zhaoetal.
,2002;Reinhardtetal.
,2007;Jiangetal.
,2009).
Asaconsequence,cellsdecientofp53aremorevulnerabletoinactivationoftheG2checkpoint.
Thisndingledtoanovelstrategyofchemosensitization,whichcombinestreatmentwithaDNA-damagingagentwithcheckpointinhibitor(s),thuspreventingcell-cyclearrestandpro-motingcelldeaththroughmitoticcatastrophe(ZhouandBartek,2004)(Figure3).
PreviousstudiesshowedthatdisruptionoftheChk1pathway,eitherbyshortinterferingRNAorbytreatmentwiththeChk1inhibitorUCN-01,abrogatesG2checkpointactivationinducedbyvariousDNA-damagingagents(includingIR,5-uorouracil,cisplatinandcampotecin),andleadstoprematuremitoticentryandcelldeath(Zhaoetal.
,2002;Xiaoetal.
,2003).
Subsequently,UCN-01wasusedinphase-Itrialsincombinationwithcisplatinoririnotecan(Laraetal.
,2005;Fracassoetal.
,2010;Healthycellsp53mutatedcancercellsp53wtcancercellsIRATM/Chk2/p53ATR/Chk1CheckpointarrestIRATM/Chk2/p53ATR/Chk1IR/Chk1iATM/Chk2/p53ATR/Chk1MitoticcatastropheIR/Wip1iorIR/NutlinATM/Chk2/p53TreatmentApoptosisActivatedpathwayOutcomeCheckpointarrestATR/Chk1Figure3Checkpointregulatorsandcancertherapy.
Healthycellswithintactcheckpointcomponentsareabletofullyactivatethecheckpointaftergenotoxicstress.
Tumorcellslackingfunctionalp53showaweakG1checkpointbutarestillabletoarrestinG2thankstotheATR/Chk1pathway.
ExposureofthesecellstoDNAdamagetogetherwithinhibitionofChk1preventscheckpointactivationandleadstocelldeathbymitoticcatastrophe.
Tumorcellswithwild-typep53activatethecheckpointwhenexposedtoDNAdamage;however,stimulationofp53bytreatmentwithnutlin-3orbyinhibitionofWip1phosphatasemayleadtoextensivep53responseandapoptoticcelldeath.
CheckpointcontrolandcancerRHMedemaandLMacurek2607OncogeneMaetal.
,2011).
However,lowspecicityofUCN-01forChk1,aswellasunfavorablepharmacokineticproles,preventedfurtheruseofUCN-01intheclinic.
AnewgenerationofATP-competitiveinhibitorsofChk1showshigherselectivitytowardChk1/2thanUCN-01(Maetal.
,2011).
Forexample,AZD7762iscomparablyefcienttowardChk1andChk2(IC505–10nM),whereasthePF477736andSCH900776com-poundsselectivelyinhibitChk1(IC500.
49and3nM,respectively).
PreclinicaltestingshowedthatthesenovelChk1inhibitorsefcientlyabrogatetheG2checkpointandsensitizep53-decientcellstovariousDNA-dama-gingagents(Maetal.
,2011),anditwillbeinterestingtoseehowtheseinhibitorswillperforminongoingclinicaltrials.
AnimportantaspectthatneedstobeconsideredwhenusingChk1inhibitorsintheclinicsistheroleoftheChk1outsidethecheckpointcontrol.
EssentialfunctionsofChk1attheorganismallevelarewell-documentedbytheearlyembryoniclethalityofChk1-knockoutmiceaswellasbyseveredefectsinthemammaryepitheliaintheconditionalChk1haploinsuf-ciencymousemodel(Liuetal.
,2000;Takaietal.
,2000;Lametal.
,2004).
Chk1appearstobeessentialformaintenanceofgenomeintegritybymonitoringreplica-tionforksduringnormalS-phaseprogression(Takaietal.
,2000;Syljuasenetal.
,2005;Maya-Mendozaetal.
,2007).
ThusinhibitionofChk1togetherwithtreatmentsthatcausestallingofreplicationforksmayleadtoirreversiblereplicationforkcollapse(Maya-Mendozaetal.
,2007).
Inaddition,recentreportssuggestthatChk1mayalsocontributetotheregulationofmultipleaspectsofunperturbedmitoticprogressionandcelldivision(Krameretal.
,2004;Zachosetal.
,2007;Wilskeretal.
,2008;Peddibhotlaetal.
,2009).
InthisrespectitmightbebenecialtopharmacologicallytargetdownstreameffectorsofChk1specicallyactinginthecheckpointpathway.
ApromisingexampleistheWee1kinase,whichactsdownstreamfromChk1andregulatestheG2/Mtransition.
MK1775,asmall-moleculeinhi-bitorofWee1,hasrecentlybeendevelopedandshowedchemosensitizationofp53-decienttumorcellsandxenograftstogemcitabine,cisplatinand5-uorouracil,basicallymimickingtheeffectsofChk1inhibition(Hiraietal.
,2009,2010;Rajeshkumaretal.
,2011).
Similarly,thePD0166285compound,whichalsoinhibitsWee1,showedradiosensitizationofvariouscancercelllines(Wangetal.
,2001).
Asdiscussedabove,efcientcheckpointactivationincellsthatlackp53requiresalsoactivityofChk2,andithasbeensuggestedthatinhibitionoftheATM/Chk2pathwaymightsensitizep53-decienttumorstoDNA-damagingtreatmentsortopoly-(ADP-ribose)polymeraseinhibitors(Jiangetal.
,2009;Andersonetal.
,2011).
TheuseofChk2inhibitorsmightalsobringadditionalbenetstosurroundinghealthytissuesexpressingintactp53,asthiscouldpreventanapoptoticresponsewhenChk2isinhibited(ZhouandBartek,2004).
Bycontrast,usingChk2inhibitorsontumors,whichretainwild-typep53,woulddramaticallyincreasetheirresistancetoirradiation,leadingtounfavorableclinicaloutcomes.
Thustransla-tionofthistreatmentstrategyintotheclinicwillrequiretheestablishmentofreliablepredictivebiomarkers(suchasthestatusofthep53andATMpathways)andarguablywillbelimitedtosituationswheretumortissuesamplesareavailable(Jiangetal.
,2009).
Theobserva-tionthatsurvivalofp53-decientcellsexposedtogenotoxicstresscriticallydependsonthep38/MK2pathway(Reinhardtetal.
,2007)opensanintriguingpossibilitythatpatientsreceivingchemotherapycouldpotentiallybenetfromadjuvanttherapywithp38inhibitors;howeverthisnovelconceptawaitsexperi-mentaltesting.
Importantly,chemosensitizationseemstoworkef-cientlyonlyincellswithadecientp53pathway(ZhouandBartek,2004;Maetal.
,2011).
Thus,althoughthisstrategycouldbeusefulincancerswithmutatedp53,thereisaneedtodevelopotherstrategiessuitableforcancersthatretainwild-typep53.
Nutlin-3awastherstidentiedsmall-moleculeantagonistofMDM2thatspecicallyblocksinteractionbetweenMDM2andp53,leadingtostabilizationofp53andinducingapoptosisorcellularsenescence(Vassilevetal.
,2004).
Strikingly,Nutlin-3ahasastrongantitumoractivityintumorcellsthatretainwild-typep53,andpromisingpreclinicalresultsforNutlin-3awerereportedwithvarioustumortypes(Secchieroetal.
,2011;ShenandMaki,2011).
SimilarlytoNutlin-3a,MI-219blockstheMDM2–p53interaction,butitshowshigherafnitytoMDM2andexcellentpharmacokineticproperties(Shangaryetal.
,2008).
Anotherpotentialpharmacologicaltargetinp53-positivetumorsisWip1,whichactsinsilencingoftheG2checkpointprimarilythroughblockingtheactivityofp53(Lindqvistetal.
,2009a).
DepletionofWip1byantisenseoligonucleotidesorbyRNAinterferencedecreasedviabilityincelllinesderivedfromneuroblas-toma,glioma,breastadenocarcinomaandovarianclearcellcarcinoma,indicatingthatasubsetofcancerpatientssufferingfromp53-positivetumorscouldbenetfromblockingtheactivityofWip1(Saito-Oharaetal.
,2003;Rayteretal.
,2008;Tanetal.
,2009;Wangetal.
,2011).
Indeed,PPM1D-knockoutmiceareviableandareprotectedfromtumordevelopment,indicatingthatlossofWip1activityiswell-toleratedandmayblocktumorgrowth(Bulavinetal.
,2004;Harrisonetal.
,2004).
However,developmentofspecicWip1inhibitorsrepresentsamajorchallengeowingtotherelativelyshallowgrooveintheactivesiteandthehighhomologywithotherPP2Cfamilyphosphatases(Harrisonetal.
,2004).
CyclicphosphopeptidesthatmimicthebindingofasubstratetotheactivesiteofWip1wereshowntoinhibittheactivityofWip1invitro;howevertheirefciencyininhibitingWip1incellsstillrequiresfurthertesting(Yamaguchietal.
,2006).
ChemicallibraryscreeningyieldedacompoundthatinhibitedWip1activityinvitroandalsoincreasedthephosphorylationofp38,JNKandextracellularsignal-regulatedkinaseintransformedmouseembryonicbro-blasts(Belovaetal.
,2005).
However,asthiscompoundaffectedthephosphorylationstatusofallmitogen-activatedproteinkinases,concernsremainaboutitsspecicitytowardWip1andfurthermodicationmaybenecessarytogenerateamoreselectiveinhibitorofWip1CheckpointcontrolandcancerRHMedemaandLMacurek2608Oncogene(Belovaetal.
,2005).
Anothercell-permeable,small-moleculeinhibitorofWip1(CCT007093,IC508.
4mM)hasrecentlybeenreportedtodecreasetheviabilityoftheMCF7,KPL1andMCF3Btumorcelllines(Rayteretal.
,2008).
Interestingly,thiscytotoxiceffectofWip1inhibitionseemstobespecicfortumorsthatover-expressWip1,whereascellswithnormalWip1levelstoleratethelossofWip1activity(Rayteretal.
,2008).
ThecelldeathinducedbyCCT007093wasdependentonp38,whichisawell-describedsubstrateofWip1,andmimickedtheeffectofWip1RNAinterference,indicat-ingthattheobservedphenotypeindeedresultsfromspecicinhibitionofWip1.
ItwillbeinterestingtoseehowthispromisingWip1inhibitorwillperformonabroaderspectrumoftumorsandinxenografttumormodels.
ConcludingremarksOverthelastdecades,wehavegainedalotofinsightintothemechanismsthatacttocoordinatethecellularresponsetoDNAdamage.
IthasbecomeclearthatvariouspathwayscanactinparalleltopromoteDNArepairandseveralothersacttogethertoinhibitcell-cycleprogression.
WehavealsolearnedthatdifferentcomponentsoftheoverallDDRcanbelostincancercells,renderingthemmoredependentontheremainingintactpathwaystopromoterepairandarrestthecellcycle.
Thishasalreadyledtothedevelopmentoftailoredtherapiesthatexploitthecancer-specicDDRdefect(s).
ThesepromisingapproachescallforamoreadvancedmolecularunderstandingoftheDDRaswellasthedevelopmentofsmall-moleculeinhibitorstargetingvariouscomponentsoftheDNA-damagecheckpoint,whichcanopenadditionalavenuesforpersonalizedcancertreatment.
ConictofinterestTheauthorsdeclarenoconictofinterest.
AcknowledgementsRHMwasfundedbytheNetherlandsGenomicsInitiativeoftheNetherlandsOrganizationforScienticResearchandbytheDutchCancerSociety(GrantUU2009-4478).
LMwassupportedbytheGrantAgencyoftheCzechRepublic(P301/10/1525andP305/10/P420).
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