自噬的发展趋势

2022-06-15 来源：知库网

Oncogene(2016)35,1–11

REVIEW

Emergingstrategiestoeffectivelytargetautophagyincancer

VWRebeccaandRKAmaravadi

Autophagyservesadichotomousroleincancerandrecentadvanceshavehelpeddelineatetheappropriatesettingswhereinhibitingorpromotingautophagymayconfertherapeuticefﬁcacyinpatients.Ourevolvingunderstandingofthemolecularmachineryresponsibleforthetightlycontrolledregulationofthishomeostaticmechanismhasbeguntobearfruitinthewayofautophagy-orientedclinicaltrialsandpromisingleadcompoundstomodulateautophagyfortherapeuticbeneﬁt.Inthismanuscriptwereviewtherecentpreclinicalandclinicaltherapeuticstrategiesthatinvolveautophagymodulationincancer.Oncogene(2016)35,1–11;doi:10.1038/onc.2015.99;publishedonline20April2015INTRODUCTION

Theimpactautophagyhasonhumanhealthanddiseasearefarandwide,withreportsdemonstratingimportantfunctionsinbacterial1andviralinfections,2suppressionofinﬂammation,3adaptiveimmuneresponses4andimmunosurveillance,5neurodegeneration,6heartdisease7andcancer.8Aberrantauto-phagicactivityisanemerginghallmarkofcancer,9servingacriticalfunctioninthepathogenesis,survivalandresponsetotherapyinagrowingnumberofcancers.Ingeneral,autophagyprovidesthemeansbywhichcellsmitigatemetabolicandtherapeuticstresses,removewasteandmanagetoxicbyproductsofanabolismandcatabolism,suchasreactiveoxygenspecies.10Theroleautophagyservesspeciﬁcallyincancerhasbeencontroversial,withsomereportsindicatingautophagysuppressestumordevelopment,whereasotherreportsprovidingevidencethatautophagypromotesthegrowthofestablishedtumors.11Theoverarchingquestioniswhetherornotautophagycanbeeffectivelymodulatedtoimpaircancerinitiationorprogression.Recentadvancesinthefundamentalunderstandingofthecontext-dependentconsequencesofautophagydefectsinthesettingofactivatedoncogeneswilllikelypavethewayfornewstrategiestoeitherinduceorimpairautophagytherapeutically.Meanwhile,theﬁrstdeliberateattempttomodulateautophagytherapeuticallyhasbeenaccomplishedthroughthepublicationoftheﬁrstsevenclinicaltrialsinvolvinghydroxychloroquine(HCQ)incancerpatients.12–18Lessonslearnedfromtheseclinicaltrialshaveraisednewquestionsthatcanbeansweredinthelaboratory.Finally,adeeperunderstandingofhowautophagyisregulatedatthegenetic,epigeneticandposttranslationallevel,andhowautop-hagycanregulateitselfandberegulatedbydrugs,extracellularcomponentsandmetabolites,maypointtonewtherapeutictargetsthatcandirectlyorindirectlymodulateautophagy.Herewediscussthelatestdevelopmentsintheﬁeld’sunderstandingofautophagyincancerandnovelstrategiestoeffectivelymodulateautophagicactivity.

AUTOPHAGYFORMANDFUNCTION

Thedissectionoftheautophagypathwaywasﬁrstdescribedinyeast19whereitclearlyservesasanintracellular,self-preservationmechanismprovidinginternalnutrientstocellsintimesofstress.20Althoughautophagyisevolutionarilyconservedacrossorganisms,itsroleinmulticellularorganismsismorenuancedthanitisinyeast.Recentevidenceindicatesautophagicﬂuxisnotonlydependentontheexpressionofthecanonicalautophagymachinery,butthroughgenetic,epigenetic,metabolic,post-translationalandextracellularregulationofthismachinery.Thiscomplexregulationofautophagymayenableitsmultiplerolesincancer.Autophagicﬂuxoccursatabasalrateinalleukaryoticcellstomaintainequilibriumthroughtherecyclingofnonessentialcomponentswithinthecell.8Underchallengingconditionssuchasnutrientdeprivation,21hypoxia22ortargetedtherapy,23autophagicﬂuxcanbeincreasedviamultiplestimulitoelicithomeostaticregulationovercriticalmetabolicbuildingblocksincludingaminoacids,nucleicacidsandmonosaccharidesnecessaryforcellsurvival(Figure1).Multipleformsofautophagyexistinmammaliancells,eachwithwell-characterizedmechan-ismsthatdifferinthewaymaterialdestinedfordegradationissequesteredandtransportedtothelysosome(micro,chaperonemediatedandmacroautophagy).24Macroautophagyrepresentsthemostmultifunctionalandbest-describedformofautophagy,comprisingacomplex,tightlyregulatedprocesswheredouble-membraneautophagicvesicles(termedautophagosomes)aregenerated.Autophagosomesfunctionbysequesteringdamagedormisfoldedproteins,engulﬁngmitochondria(termedmito-phagy)andinternalizingendoplasmicreticulum(ER;amongstothercytoplasmiccomponents)throughtheaidofcargoadaptorproteinsbeforeultimatelyfusingtothelysosomefordegradationandrecyclingofinternalcontentstosustaincellularviability.25,26Autophagycanbecharacterizedascanonicalornon-canonical,dependinguponthemolecularmachineryinvolvedinthebiogenesisofautophagosomes.Canonicalautophagyisregulatedbyanumberofautophagy-related(ATG)proteinsandnon-ATGproteins(suchasclassIIIphosphatidylinositol3-kinase(PI3KIII),p150and(activatingmoleculeinBeclin-1-regulatedautophagy)Ambra1)thatchoreographtheinitiation,elongation,maturationandfusionstagesofthepathway.27Non-canonicalautophagyisnotaswellunderstood,whereautophagosomescanbecreatedindependentlyofAtg5orAtg7.28Recently,ferritinclustershavebeenreportedtoaccumulateatthesiteofautophagosome

TheDepartmentofMedicineandAbramsonCancerCenter;UniversityofPennsylvaniaSchoolofMedicine,Philadelphia,PAUSA.Correspondence:DrRKAmaravadi,TheDepartmentofMedicineandAbramsonCancerCenter,UniversityofPennsylvaniaSchoolofMedicine,16PennTower,3400SpruceStreet,Philadelphia,PA19063,USA.E-mail:ravi.amaravadi@uphs.upenn.edu

Received31December2014;revised18February2015;accepted18February2015;publishedonline20April2015

AutophagyinhibitortherapyVWRebeccaandRKAmaravadi

extracellularintracellularNutrient /EnergySensorsAMPKmTORNucleationMaturationCargoAdaptorsPost-TranslationalbaPhosphorylationAcetylationUbiquitinationBeclin 1Vps34ATG14Lp150LC3ATG4ATG5ATG7ATG12ATG16P62NixNBreMetabolicAmino AcidsAmmoniaGlucoseInitiationULK1/2ATG101mATG13FIP200Autophagy LysosomaldegradationLipidsRecyclingTranscriptionalcAutophagy genesFoxk proteinsTFEBp53ATF4XBP1AcMeTfd••••EpigeneticHistoneMethylationDemethylationAcetylation DeacetylationNucleusPotentially druggable targetsFigure1.Autophagyregulatorsandpointsofintervention.(a)Autophagyoccursthroughamultistepprocessthatincludesfourcontrolpoints:initiation,nucleation,maturation,andlysosomalfusionanddegradationofautophagosomecontents.Successfulautophagyresultsintherecyclingofnutrientsintothecytoplasm.(b–e)Autophagyisregulatedonmultiplelevelswithfourmajorclassesofregulationincludingposttranslational,transcriptional,epigeneticandmetabolicregulation.Potentialdruggabletargetsaredepicted(redstar)withapromisetobettermodulateautophagythanstrategiescurrentlybeingimplored.

formationalongwithp62incellslackingAtg5,possiblysheddinginsightregardingnon-canonicalautophagosomebiogenesisdynamics.29Theclassicalandperhapsbest-characterizedenviron-mental-mediatedregulationofcanonicalautophagyoccursviathegrowthfactor/receptortyrosinekinase/phosphoinositide3-kinase(PI3K)/proteinkinaseB(alsoknownasAKT)/mechanistictargetofrapamycincomplex1(mTORC1)signalingaxis,whichdirectlycontrolsautophagicactivitythroughthephosphorylationandinhibitionofUnc-51-likekinase1(ULK1),partoftheﬁrstproteincomplexinvolvedinautophagicvesicleformation.30Underconditionsinwhichgrowthfactorsandnutrientssuchasaminoacidsarerichintheextracellularspace,thePI3K/AKT/mTORC1pathwayishighlyactiveandmTORC1inhibitsULK1throughthephosphorylationatitsserine-757residue.31However,whengrowthfactorsbecomelimited,mTORC1becomesinactiveandcannolongerrepressthecomplexconsistingofULK1,focaladhesionkinasefamily-interactingprotein200kDa,ATG13andATG101,whichfavorstheinitiationofautophagy(theﬁrstphaseofautophagy).32AMP-activatedproteinkinase,inresponsetoeitherglucosestarvationoramino-aciddeprivation,canalsoregulateULK1activityviaﬁne-tuningofthephosphorylationstatusofULK1.33Onceactivated,ULK1formsacomplexwithBeclin-1viaassistancefromTRIM5α,actingasaproteinplatform,leadingtothephosphorylationandactivationofBeclin-1.34Onceactive,Beclin-1activatestheclassIIIPI3Kvacuolarsortingprotein34(Vps34),acomponentnecessarybothforendocyticsortingandintheabilityofcellstorespondtoﬂuctuationsinnutrientssuchasaminoacidsandinsulin.Vps34activityhasalsobeendemon-stratedtonotbeinhibitedbytheTORC1inhibitorrapamycin,suggestingthatVps34canalsofunctionupstreamofmTOR,servingasavehicleformTORtomonitorthelevelsofawidernetofcriticalnutrientsforcellsurvival.35FollowingVps34activation,autophagycytoplasmicmachineryisrecruitedontothephospho-lipidmembranesderivedfromvarioussourcesincludingthe

Oncogene(2016)1–11

endoplasmicreticulum,36plasmamembrane,37mitochondria38andGolgiapparatus.39Thesecondphaseofautophagy(nuclea-tion)marksthebeginningofautophagosomeformationwiththenucleationofmembranesbyBeclin-Vps34andeitherATG14L,Rubicon,Ambra,amongotherproteins.Thethirdphase(elonga-tionandmaturation)allowsforthematurationofautophago-somesandrequiresaubiquitinligase-likeATG5-ATG12-ATG16Lcomplex(formedwiththeaidofATG7andATG10).40ATG4canalsocontributetotheelongationphase,andhasrecentlybeenimplicatedasabiomarkerandpotentialtherapeutictargetforchronicmyeloidleukemiastem/progenitorcells(Figure1).41Theubiquitin-likeproteinLC3/Atg8issubsequentlyconjugatedtothelipidphosphatidylethanolamineonthesurfaceofautophagosomemembranes.Onceintegratedinthelipidbilayer,LC3interactswithadaptorproteins(autophagyreceptors)suchasp62,Nbr1,TRIM5αandNIX,whichrecruitcargofromthecytoplasmandpromoteautophagosomeclosure.34,42ProteomicnetworkanalysisincellsundergoingautophagyrevealhighconnectivitybetweenLC3/Atg8andupstreamautophagycomponentssuchasULK1,Vps34andATG2A,suggestingthatLC3/Atg8mayserveamoresigniﬁcantroleinregulatingautophagosomeformationthanwaspreviouslyappreciated.43Onceautophagosomeshaveengulfedcargoandclosed,theyareultimatelytrafﬁckedandfusedtolysosomesformingautophagolysosomes.ThisfusionallowsforthepH-dependentdegradationofcytosoliccargoviahydrolaseslocatedwithintheacidicenvironmentoftheautophagolysosome.44Lysosomalpermeasessuchasspinsterpermitthereleaseofdegradationproductsrangingfromsugars,aminoacidsandnucleicacidsintothecytosolforreusebythecell45(Figure1).Ourgrowingunderstandingofhowautophagyisregulatedhasshedlightonthepotentialnoveldruggablecomponentsforautophagyinhibition,whichwillbediscussedlater(seeFigure1andbelow).

AutophagyinhibitortherapyVWRebeccaandRKAmaravadi

MOUSEMODELSADDRESSTHEROLEOFAUTOPHAGYINTUMORINITIATIONANDMAINTENANCE

AmajorbreakthroughinunderstandingtheroleofautophagyintumorigenesiswasmadewhenspontaneouslungandlivertumorswerefoundtoariseinBeclin-1+/−mice.46MonoallelicdeletionofthehumanhomologofBeclin-1(BECN1)wasinitiallyreportedtooccurin40–75%ofcasesofhumansporadicovarian,breastandprostatecancer.47TakentogethertheseresultsestablishedBECN1astheﬁrstautophagy-associatedtumorsuppressorgene.47,48However,theproximityofBECN1totheovarianandbreasttumorsuppressorgeneBRCA1onchromosome17q21hasdecreasedthecertaintyofBeclin-1’sroleasabonaﬁdetumorsuppressorgene.ArecentreportdemonstratedBeclin-1allelelosstobearareevent,assessedinhumanprostate,breastandovariantumorsequencingdatafromTheCancerGenomeAtlasandotherdatabases,exceptinthesettingoflossofneighboringgeneBRCA1.49Further,alargerpanelofcancerswasanalyzedwithnoevidenceforBECN1mutationorloss,leavingthefunctionofBECN1asatumorsuppressorinhumancancerunclear.AddingmorecomplexitytotheroleBeclin-1servesinmalignancyisareportshowingBeclin-1toshareregulationwithp53atthelevelofproteasomaldegradationinanubiquitin-dependentmanner;thereforesug-gestingthatthespontaneousmalignancyinBeclin-1+/−experimentalsystemsmaybeduetolowerp53levels.50Alongasimilarvein,Beclin-1andtheantiapoptoticBcl-2familymembermyeloidcellleukemia(Mcl-1)proteinarebothstabilizedbybindingtothedeubiquitinaseUSP9X(ubiquitin-speciﬁcpeptidase9X-linked),andnegativelymodulatetheexpressionofeachotherthroughcompetitivedisplacementofUSP9X.51Beclin-1expres-sionlevelswerediscoveredtodecreaseinpatient-derivedmelanomatissuesasMcl-1levelsincreasedinasigniﬁcantinterdependentmanner,independentofautophagy.51ThoughBeclin-1hasrecentlybeendemonstratedtohavearoleintheresponseoflungcancertoepidermalgrowthfactorreceptorinhibition,52furtherexperimentalvalidationisneededtodeter-minethepracticalconsequencesofBECN1heterozygosityinhumantumorsandtodelineatewhethertheobservationsinvolvingBeclin-1areindeeddependentontheroleautophagyservesineachoftheseexperimentalsystems,orratherduetotheconfoundingimplicationsBRCA1,p53andMcl-1eachprovideoncancercellviabilityanddiseaseprogression.

BeyondBeclin-1,mousemodelswithmosaicdeletionofAtg5andliver-speciﬁcdeletionofAtg7alsoresultedinagreaterincidenceofspontaneousliveradenomas;however,thetumorswerebenignsuggestingautophagymaybenecessaryfortheprogressionbeyondthebenignstate.53DeletionofFip200alsopreventedthedevelopmentofbreastcancer.54,55Numerousmousemodelshavedemonstratedautophagytoserveacriticalcapacityindiseaseprogressioninestablishedoncogene-driventumors,whereinhibitionofautophagyresultsinareductionin

tumorvolumeinestablishedtumors.InamousexenograftmodelutilizingimmortalizedbabymousekidneyepithelialcelllinesengineeredtoexpressconstitutiveactivityofRAS(H-rasV12)whilealsopossessingdefectsinapoptoticmachinery(Bax/Bak-deﬁ-cient),autophagywasfoundtosupportsurvivalofcancercellsundergoingmetabolicstressandwaslocalizedtothepoorlyvascularized,hypoxiccoresoftumors.56Further,celllinesengineeredwithconstitutiveactivityofAKT(myr-AKT)alongwithapoptoticdefectsdisplayedhighlevelsofnecrosis,mechanisti-callyduetothecoordinateinhibitionofapoptosis(viaBax/Bakdeﬁciency)andautophagy(inhibitedbyAKTactivity).

Althoughthesedatawerecritical,whatweresorelyneededweregeneticallyengineeredmousemodelsofoncogene-drivencancerswithandwithoutdefectsinautophagygenes.Thesemodelshaveemergedrecently(Table1)andrevealathemewherethemajorityofmicewithdefectsinkeyautophagymachinerydisplayacceleratedthedevelopmentofbenigntumors,however,autophagyappearstobeessentialfortheprogressionofbenigntumorstoamoremalignantstate.Onceatumorisestablished,autophagyhasbeenclearlydemonstratedtoalsohavearoleinpromotingthesurvivalofexistingtumorcellswithinthetumormicroenvironment.57TwomodelsofspontaneousKras-drivenlungcancer,onewithtumorcelldeletionofAtg758andonewithtumorcelldeletionofAtg5,59exploredtheimportanceofautophagyinthecontextofRasoncogenes(Table1).IntheKrasG12D/Atg7ﬂ/ﬂmodel,thedeletionofAtg7resultedinasigniﬁcantreductionintumorburdenandanincreaseintumorlipidaccumulation;however,nodifferenceintheoverallsurvivalcouldbenotedduetoanincreaseindeathbyinﬂammationinmicewithAtg7-deﬁcienttumors.58IntheKrasG12D/Atg5ﬂ/ﬂmodel,thedeletionofAtg5resultedinincreasedtumorinitiation;however,tumorcellsexhibiteddecreasedmitochondrialbioenergetics,andthedeletionofAtg5alsoenhancedsurvivalofmice.59Eachofthesemousemodelsrevealedautophagytobenecessaryforcancercellproliferationandprogressionoflungtumorsfromadenomastocarcinomas.TheseﬁndingsstrengthentheconceptthatRas-drivencancersrelyonautophagyforsustainedmetabolismandgrowth.AmousemodelwithCre-activatableBRAF(BrafV600E)drivenlungcancer,withandwithouttheconditionalknockoutofAtg7wasgeneratedtodeterminetheroleofautophagyinBRAF-drivenlungcancers.AutophagywasrequiredforthegrowthofestablishedBrafV600E-drivenlungcancersviathepreservationofmitochondrialfunctionandthesupplyofmetabolicsubstratescriticalforsustainedtumorigenesis.60Atg7-deﬁcientmiceexperiencedincreasedearlytumorigenesisinanoxidativestress-dependentmannercomparedwithmicewithintactAtg7;however,asintheKras-drivenlungcancermodel,Atg7deletionconvertedBrafV600E-drivenadenomastotumorsthathadthehistologicalappearanceofbenignoncocytomasratherthancarcinomas.60Table1.Mousemodelstestingtheeffectsoftumor-speciﬁcautophagydeﬁciencyincancerCancertypeAtgknockoutTumorinitiationaDecreasedNDIncreasedIncreasedIncreasedIncreasedIncreasedIncreasedTumorprogressionaDecreasedDecreasedDecreasedDecreasedDecreasedIncreasedDecreasedDecreasedMousesurvivalfollowingReferencespost-tumorautophagyinhibitionIncreasedNodifferenceIncreasedIncreasedNDDecreasedDecreasedIncreasedWeietal.55Guoetal.58Raoetal.59Stroheckeretal.60Karsli-Uzunbasetal.63Rosenfeldtetal.64Rosenfeldtetal.64Iacobuzio-Donhueetal.68GenotypeMMTV-PyMTBreastFIP200lox-stop-lox-KrasG12D;Tp53ﬂox/ﬂoxLungAtg7lox-stop-lox-KrasG12DLungAtg5V600Eﬂox/ﬂoxBraf;Tp53LungAtg7frt-stop-frt-KrasG12D;Tp53frt/frtLungAtg7lox-stop-lox-KrasG12D;Tp53ﬂox/ﬂox;Pdx-crePancreasAtg5orAtg7lox-stop-lox-KrasG12D;Pdx-crePancreasAtg5orAtg7lox-stop-lox-KrasG12D;Tp53ﬂox/+;Pdx-crePancreasAtg5Abbreviation:ND,notdetermined.aFollowinggeneticautophagyinhibition©2016MacmillanPublishersLimitedOncogene(2016)1–11

AutophagyinhibitortherapyVWRebeccaandRKAmaravadi

Inmousemodelsofpancreaticcancer,autophagywasdiscoveredtobevitalandessentialfortumorigenicgrowthofpancreaticcancersdenovo.61Pancreaticductaladenocarcinoma(PDAC)celllinesandprimarytumorpossessconstitutivelyactivatedautophagy(asseenbyGFP-LC3punctaandcleavedLC3-AIHC(LC3-II))andauniquedependenceuponautophagy.Importantly,thegenetic(suppressionofATG5expressionbyshRNAs)orchemicalinhibition(chloroquine)ofautophagyleadstorobusttumorregressionandprolongedsurvivalinpancreaticcancerxenograftsandgeneticmousemodels.61KRASmutationsareoneoftheknowndriversinPDAC,andarecentreportleveraginganinduciblemousemodelofmutatedKras(KrasG12D)inap53Lox/WTbackgroundshedfurtherlightontheroleautophagyservesinpancreaticcancer.Inatemporalandpancreas-speciﬁcmanner,theauthorsablatedKRASactivity,whichresultedinpancreatictumorregressionwithin2–3weeksfollowedbyrelapseafewmonthsthereafter.ThecancercellssurvivingKRASablationwerestudiedwithtranscriptomeanalysisandgenesetenrichmentanalysisrevealingasigniﬁcantenrich-mentofgenesinvolvedinlysosomalactivity,mitochondrialelectrontransportchainandautophagy,amongothercellularprocesses.62Althoughthegeneticallyengineeredmousemodelsdescribedabovewereincrediblyusefulinsheddinglightontheeffectsofautophagydefectsonthetumorigenesisofoncogene-drivencancer,theydidnoteffectivelymodelthetherapeuticablationofautophagy.Withcancertherapy,drugswilltypicallyimpactthepathwaythroughoutthebodyandareoftenadministeredonlyafterthetumorbecomesapparent(stageIV)orinahigh-risk(stageIII)setting.Althoughtumorxenograftsaddressthistosomedegree,thosemodelsareartiﬁcialbecausemicelackimmunesystemsﬂengineeredanksofandthemousemice.thetumorTomodeladdressistypicallyofanallgrownoutofcontextintheinducibleoftheseKrasconcerns,-drivenalunggeneticallycancerwasgeneratedwhereAtg7couldbesystemicallydeletedinaconditionalmanner.WhensystemicAtg7deletionwasengagedinadultmice,miceinitiallywereasymptomatic,buteventuallydiedofneurodegenerationatroughly3months.63However,whenAtg7wassystemicallyablatedinmicebeforetheinductionofKras-drivenlungcancer,therateoflungnodulesappearedtoincrease,butthenodulesfailedtoprogresstocancerbeforethemicesuccumbedtotheeffectsofsystemicAtg7depletion.WhenAtg7wassystemicallydeletedinmiceafterKras-driventumorswereallowedtoform,massivetumorregressionandapoptosiswasobservedbeforethetoxicityofAtg7depletiononnormaltissuewasevident.Theseobservationsarevaluableastheyrevealthatchronicautophagyinhibitionmayyieldtoxicities,supportingtheexplorationofoptimaltreatmentregimensthatminimizeexpo-suretoautophagyinhibitorswhilestillmaximizingtheantitumorbeneﬁtconferredfromautophagyinhibition.

Ingeneral,mousemodelsshowthatautophagyiscriticalinthetransitionfrompremalignanttomalignant,however,autophagypromotesgrowthofestablishedtumors.Theserecentresultspartiallyreconcilethedichotomyofautophagyintumorigenesis,andsupportarolefortheinhibitionofautophagyasatherapeuticstrategyincertainadvancedcancers.Therewasanexceptionreported,whereamodelofpancreas-speciﬁcKras-mutant,Trp53−/−tumorswastreatedwithautophagyinhibitionwitheithergeneticablationofAtg5orAtg7,orchemicallywithHCQ,resultinginthepromotionoftumorigenesis(Table1).64Fromboththestrategies,autophagyinhibitionwasfoundtoacceleratetheformationofPDACinmiceduetoenhancedglucoseuptakeandenrichmentofanabolicpathways.65Awrinkleinthismodelisitsuseofanembryonicpancreas-speciﬁchomozygousdeletionofTrp53inthecontextofKrasmutation,whichresultsinadvancedcancersinearlydevelopment.Innature,p53ismostfrequentlyfoundasmissensemutationsinKras-mutantpancreaticcancers.66TheheterozygousexpressionofmutantTrp53inthecontextof

Oncogene(2016)1–11

oncogenicKrasispostulatedtogiverisetoprecancerouslesionscalledpancreaticintraepithelialneoplasias,withthesubsequentlossofheterozygosityofthewild-typeTP53alleledrivingtheprogressionfrom64pancreaticintraepithelialneoplasiastoPDAC.65Thus,themodelutilizinghomozygousdeletionofTrp53didnotfullyrecapitulatethestep-wiseprogressionofpancreascancerasisfoundinhumans.Toaddressthisimportantissue,apancreas-speciﬁcKras-mutantTrp53+/−mousemodelwasgeneratedthatexperienceslossofheterozygosityofthewild-typeTrp53alleleduringPDACprogression,thereforemirroringthestep-wisedevelopmentofhumanpancreascancer.67WithinthismodelwithTrp53(lossofheterozygosity),autophagyinhibitionviaablationofAtg5orwithHCQwasfoundtoincreasetheoverallsurvivalinamousepreclinicaltrialleveragingcohortsofgeneticallycharacterized,patient-derivedxenografts.Trp53statuswasnotfoundtocorrelatewiththeresponseintumorcelllinesorpatient-derivedxenograftmodels,andalthoughautophagyinhibitioninthepancreasleadtoanincreaseintumorinitiation,fewofthesepremalignantlesionscoulddevelopintoinvasivetumorsandthemicetreatedwithautophagyinhibitionlivedlongeroverall.67Theseﬁndingsareoftheupmostimportance,asconclusionsdrawnfromtheTrp53modelthatdidnotrecapitulatehumanpancreascancerdevelopment64leadtoprematurerecommendationsthatpatientswithTrp53mutationsshouldnotreceivetreatmentwithHCQ.68DuetothehighproﬁleoftheJournalinwhichthisopinionpiecewaspublished,itispossiblethatpatientswhomayhavebeneﬁtedfromclinicaltrialsutilizingHCQmayhavebeendirectedtoothertherapiesbytheirphysicians.

Insightfromthesestudieswillalsohelpdesigntherapyregimens,whereexposuretoautophagyinhibitorswillbestrategicallytimedtoallowforoptimaltherapeuticbeneﬁtintheabsenceofpotentialhazardsfromthechronicinhibitionofautophagy.Itappearsthatinmostcasesautophagydefectsleadtoacceleratedtumorinitiation,butimpairedtumormaintenance.Itisforthesereasonswhymucheffortindevelopingtherapeuticstargetingautophagyisfocusedonadvancedcancerswhereconcernsaboutdevelopingsecondarybenigntumorswillbelessproblematiciftheadvancedcancerthatisputtingthepatient’slifeimmediatelyatriskcanbehaltedorregressed.Adeeperunderstandingofhowautophagyisregulatedonmultiplelevelscouldunraveltheswitchthatturnsautophagyfromatumorsuppressortoatumorpromoter.

CANCERIMMUNOGENICTHERAPYCANPRODUCEAUTOPHAGIC/

AUTOPHAGY

CELLDEATH:THEARGUMENTTOINDUCEObservationsthattherapy-inducedautophagycanhavearoleintumorcellcytotoxicityhavebeenreported;however,theycommonlydependuponpre-existingdefectiveapoptoticmachin-eryinorderfortheautophagiccelldeathtomanifest.Bcl-2homology3mimeticssuchasgossypolhavebeendemonstratedtoelicitautophagiccelldeathinapoptosis-deﬁcientmalignantgliomaandprostatecancer,bywayofdisruptingphysicalinteractionsbetweenBcl-2familymembersandBeclin-1.69Autophagiccelldeathreferstocelldeaththatisaccompaniedbyextensivecytoplasmicvacuolization,oftencorrelatedtoincreasedautophagicﬂux.70Theuseofthetermautophagiccelldeathiscontroversial,assinceitsconceptionthephraseiscommonlymisusedtosuggestthatautophagyactivelycontri-butestocelldeath.Althoughautophagyfrequentlyoccursconcurrentlywithregulatedcelldeath,autophagyisdirectlyresponsibleforthedeathoftumorcellsinonlyafewcases.71Todate,therehavebeennodeliberateattemptstoinduceautophagyspeciﬁcallyinacancermodel.Autophagyappearstoberesponsibleforthedeathofsomecancercellswithdefectiveapoptoticmachinery,suchasinhibitedcaspase-8,inanATG7and

Beclin-1-dependentmannerinvitro.72Anotherstudyreportedre-expressionof(ARHI)aplasiaRashomologIinhumanovariancancercelllinesresultedinautophagiccelldeathinvitro.73However,invivoautophagyenabledthesecellstoremaindormantinthecontextofARHIre-expression,withchloroquinetreatmentmarkedlyreducingtheregrowthofxenografts.SimilarresultswerealsoobservedinvitrowhencellswereculturedwithfactorsfoundinvivosuchasIGF-I,M-CSFandIL-8,suggestingautophagyservesaprotectiverolewhenexperimentalconditionsrecapitulatethosefoundwithinthetumormicroenvironment.Arecentconsensusstatementoncelldeathnomenclaturewarnedaboutthefactthatregulatedcelldeathmechanismsfrequentlyinteractwitheachotheranditmaybethatinmanycasespersistentautophagycanactivateotherformsofcelldeaththatareactuallyresponsibleforthedeaththatensues.71Theremayexistmultiplecheckpointsthatlimitautophagiccelldeathfromoccurringinvivo,suchasgrowthfactoravailabilityandfunctionalapoptoticmachinery.

Interestingly,autophagyhasalsobeenreportedtoservearoleintherecruitmentofimmunesystemeffectors.Chemotherapyinautophagy-competentcancersrecruiteddendriticcellsandTlymphocytestothetumorbedinanATP-dependentfashion.74InhibitingautophagysuppressedthereleaseofATPandattenuatedtherecruitmentofimmunecells.Similarresultswereobservedinmelanomawherechemotherapy75orradiotherapy76eachledtoanincreaseinmannose-6-phosphatereceptoronthetumorcellsurface,makingtumorcellsmoresusceptibletolysisbycytotoxicTcells,inanautophagy-dependentmanner.Theimplicationstheseﬁndingsholdinregardtotheclinicalutilizationofautophagyinhibitorsmovingforwardremaintobedetermined.Apotentialcombinationofanimmunecheckpointinhibitor,suchasanti-PD-1antibody,77withanautophagyinhibitorcanbeenvisionedtoensurepotentialsecondaryeffectsontheimmuneresponsetocancercellsdonotblunttheantitumoreffectofautophagyinhibition.

CANCERAUTOPHAGY:THERAPYTHECANARGUMENTPRODUCETOCYTOPROTECTIVEINHIBITAUTOPHAGY

Autophagywasconvincinglyshownasakeysurvivalmechanisminapoptosis-defectivetransformedcellssubjectedtogrowthfactorwithdrawal.Cellsthatsurvivedgrowthfactorwithdrawalorothermodesofstarvationcouldbekilledwhenautophagywasinhibitedwitheither3-methyladenineorCQ,andtheautophagicphenotypewasreversibleoncegrowthfactorswerereplenished.21UtilizingaMyc-inducedmodeloflymphoma,theroleofautophagyinthesurvivaloftumorcellsinvivowasdemonstratedwheretreatmentwitheitherCQorATG5shRNAsenhancedtheabilityofalkylatingdrugtherapytoinducetumorcelldeath.78Sincethen,amultitudeofpapershavebeenpublisheddemonstrating11utilityincombiningautophagyinhibitorswithcancertherapy.Inadditiontoautophagyservingacriticalroleintumorigenesis,manycancerdrugshavebeenreportedtoinduceautophagythatcanbecytoprotective.TraditionalcytotoxicchemotherapeuticsandtargetedtherapiesinduceautophagythroughanumberofsignalingpathwaysincludingtheDNAdamageresponse,mTORandAMP-activatedproteinkinasesignaling,theERstressresponseandothers.11Inhibitionofautophagywithchloroquineinpreclinicalmodelsimprovestheresponseoftumorcellstoalkylatingagents,suggestingthatautophagypromotessurvival.79Anotherreportobservedcyto-protectiveautophagytoserveacriticalresistance23mechanismtoBRAFinhibitioninBRAF-mutantmelanoma.Thisﬁndingwasofparticularinterest,astheroleautophagyhasinresistancetotargetedtherapiesthattargetPI3K/AKT/mTORsignalinghavebeenwellstudied;80,81however,thefunctionofautophagyinthecontextofMAPKpathwayinhibitionhasnotbeenwellcharacterized.Mechanistically,BRAFinhibitionleadstoaphysical

AutophagyinhibitortherapyVWRebeccaandRKAmaravadi

interactionbetweenmutantBRAFandGRP78,amasterregulatorofERstressactivity,whichresultsinthedownstreamactivationoftheERstresspathwayeffectorPERK.PERKactivationresultsinaninductionofcytoprotectiveautophagy.BRAFinhibitor-inducedautophagywasobservedatahighrateintumorsobtainedatthetimeofprogressiononBRAFinhibitortherapy.23TargetingautophagywithHCQconcurrentlywithBRAFinhibitortherapyresultedinsigniﬁcanttumorregressioninmousexenograftsstudies.ThisﬁndingwasreproducedininvitroandinvivostudiesinpediatricgliomasthatharborBRAFV600Emutations,andtheadditionofHCQtoaBRAFinhibitorovercametheresistancetoBRAFinhibitioninapatientwithpediatricglioma.82Manyotherexamplesexistsupportingtheconceptofcombiningchemo-therapyortargetedtherapywithachloroquinederivative,providingrationaleforlaunchingcancerclinicaltrialsinvolvingHCQ.CLINICALTRIALSOFHCQ,THEFIRSTAUTOPHAGYINHIBITORTheseminaldiscoveriesoftheserecentmousemodelsandpreclinicalﬁinvestigationsdovetailnicelywith(Tablerstset2).ofSixHCQphaseclinicalI/IItrialstrialswereinpatientsperformedwiththeinadvancedpublishinghumanpatientscancersofthediagnosed17withglioblastomamultiforme,16relapsed/refractorymyeloma–15andmelanomainadditiontootheradvancedtumors.13OneadditionalclinicaltrialwaspublishedwhereinpetdogsdiagnosedwithspontaneouslyoccurringlymphomawerealsotreatedwithHCQ-basedcombinationtherapies.12Eachtrialinvolvedacombinationtherapy–86thathadpreclinicalstudiestojustifyclinicaltranslation.78,83Themajorﬁndingfromthesetrialsisthat,basedonelectronmicroscopy-basedpharmacody-namicassays,autophagycanbemodulatedtherapeuticallywithchloroquinederivatives.Remarkably,acrossallofthetrialso10%ofpatientshadseverenon-hematologicaltoxicity.Speciﬁcally,therewasnoevidenceofextensivemetabolictoxicity,liverinjuryorneurologicimpairmentinthesetrialsdespitesomeevidencethatchronicmodulationofautophagywasachievedinpatients,asseenbytheaccumulationofautophagicvesiclesinperipheralbloodmononuclearcellsandtumorcells.Whencombinedwithradiationtherapyandconcurrentandadjuvanttemozolomide,HCQproduceddose-limitingmyelosuppressionatdosesabove600mgHCQ.Atthesedosesonlyasubsetofpatientshadevidenceofautophagymodulationdetectableintheirperipheralbloodmononuclearcells,whichmaybeonereasontherewasnosigniﬁcantimprovementintheoverallsurvivalcomparedwiththehistoricalcontrolsoftemozolomideandradiationalone.16Signiﬁcanttherapy-associatedincreasesinAVsandLC3-IIwereobservedinperipheralbloodmononuclearcellsinaconcentration-dependentmanner,demonstratingHCQcouldmodulateautophagyinvivo.CombinedtreatmentwiththeproteasomeinhibitorbortezomibandHCQresultedinagreaterperturbationoftumorcellautophagycomparedwithperipheralbloodmononuclearcellautophagy,arguingthatHCQmayselectivelyaccumulateintumorcells.17SimilarresultswereobservedinthephaseItrialofvorinostatandHCQ13andinthecaninelymphomatrialusingdoxorubicinwithHCQ.12AlthoughthesephaseIstudieswerenotpoweredtodetermineefﬁcacy,responseratesinunselectedpatientpopulationsweregenerallylow.However,therewereanumberofstrikingresponsesandprolongedstablediseaseobservedinpatientswithmelanoma,renalcellcarcinoma,coloncancerandmyeloma,thatsuggestthataspeciﬁcsubsetofcancersmaybesusceptibletoregimenscontainingchloroquine-basedautophagyinhibitors.Criticaltothefuturesuccessofautophagy-orientedclinicaltrialsarebiomarkersthatmayaidinpatientselection.Currentbiomarkerstoassessautophagymodulationinclinicaltrialsconsistofmonitoringtheaccumulationofautophagicvesiclesinperipheralbloodmono-nuclearcellsandtumorcellsbyelectronmicroscopy,aswellascheckingforchangesinLC3lipidationbywesternblottingand

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AutophagyinhibitortherapyVWRebeccaandRKAmaravadi

CydMtoiolBvibP;dtclheaatrtleiawraheciplisnrloiltleeclpcl;,lesarCioweytiidrBtmsvQpiettMlauocaPtCirnH;baeeodlltiredaotutﬁnoctaitiramitnﬁlNlsuuileeneIpotcnw,;ersmfmeoeyssbQeochtneriilCnuctncvasaaoicptscHtauiCeosecoilisraTneEbisfllaeaaolconpd-aPrduvoiiqvmhclotia%-iranognirm0hiiNUuCHleu0giScST1HehtN20054709343222,ICgNmgk;x0/aieltD0gnpaT8mepmnMta5.ooo2hcisge1punsmi,eghmcr0iDtyypt0aTalmp6fMe4aDu3d/pTsgg3,aMomrleeeeef/TennndmsisDdoLy0oooa0prTatDM08NNNG06eSMrGegrICatNci,t)ssIirmCneatnuNethi,gnkceroartencecilreiemd5lznSbie,nu1MMﬁoPtorndaFR(f,,,/TOsnnnAa–reBTCnnnnoalnmtAaoai;SPePePeSCDesnoaadmmolelooocbnna,raalellaleenommetral,,,asssmrrromooohicxammpiammteauuumamsotttye,lamrlDeedddciiigTsoiylllnilDGMooooaMSSSDP;eeeesgggalayymeaatgeymommacaahawdsaaadRdpehAeOCAodttaNoAATDtuePDrNmNHAnPDDoy.thsQipChar,HCg+AoDmnHoto;QientCaQnoiiiHdusaqsigrQCHQon/CCQrmeCoeidH+HQvHlliehnom+d+CsH+coyrtvinobnxiimuilioom+tcosiairoszmllbdploooazotszruyirmeoiorhT,s,EtetoxQrPltmmniroCaoeeoHCQ;lIcIbttvdi/HleInIIIIIII:csisltneeeeeeeCsssssssnreaaaaaaaoaeighhhhhhhtl.A2PPPPPPPaciuvenelrolabibnraTT1234567boAmOncogene(2016)1–11

totalLC3proteinbyimmunohistochemistry.Interestingly,arecentstudyproﬁledthesecretedfactorsuniquetotumorcellswithhighlevelsofautophagyrelativetothosewithlowlevelsofautophagy,suggestingthemeasurementoftheseautophagy-associatedsecretedproteinsinplasmamayserveassurrogatesforintratumoralautophagylevels.87AnadditionalphaseIItrialwasrecentlypublishedwherepatientswithpreviouslytreatedmetastaticpancreaticcancerwereadministeredHCQasasingleagent.18AlthoughHCQmono-therapydidnotdemonstratesigniﬁcanttherapeuticefﬁcacy,high-doseHCQwaswelltolerated.HCQhasalsobeendemonstratedtosynergizewithchemotherapeuticsandtargetedagents,whichmayexplainthelackofefﬁcacyasasingleagent.TherearenumerousongoingtrialsutilizingHCQincombinationtherapies,asummaryofwhichcanbefoundinTable3.MorepotentinhibitorsofautophagypossessinggreaterinvivoactivityrelativetowhatiscurrentlyachievablebyHCQareurgentlyneeded.InhibitorssuchasLys05(seebelow)havebeendevelopedandareinthestepsofoptimizationforclinicaluse,whichshouldresultinanincreaseindetectableautophagyinhibitioninpatientsandanincreaseinclinicalbeneﬁt.Adeﬁnitivetestoftherolethatautophagyservesinthesettingofanticancertherapyforpatientsawaitsrando-mizedstudiesofHCQandthenewgenerationofautophagyinhibitorswhereautophagycanbemorerobustlyinhibitedinvivo.Insightgainedfromrecentpreclinicalandclinicalstudiesidentifypotentialside-effectsfromautophagyinhibitioninvivoincludingmyelosuppression,lymphopeniaandPanethcelldysfunction,acharacteristicresemblingtheintestinalphenotypeofhumanswithgeneticdefectsinATG16L1.88OngoingtrialsutilizingHCQincombinationtherapywillexpandourknowledgeregardingthepropercontextwhereautophagyinhibitionmayelicitthegreatestclinicalactivity(Table3).

OTHERINHIBITORSAGENTSFORBEINGCLINICALDEVELOPEDTRIALS

ASAUTOPHAGYOurunderstandingoftheautophagicpathwayanditsimportanceincancerhasincreasedexponentiallywithinthelastdecade,providingnewpromisingmoleculartargetsforcancertherapy.DruggableautophagytargetsincludeBeclin-1,ULK1,ATG4,ATG7andrecentlyVps34(Figure1).Todate,nokinaseinhibitorsagainstULK1haveenteredclinicaltrials,however,apeptidehasbeendescribedthatmayhaveutilityinmodulatingautophagy.High-throughputscreeningeffortstoidentifynovelautophagyinhibitorsresultedinthedevelopmentofSAR405,alow-molecularmasskinaseinhibitorofVps34.SAR405wasrecentlydescribedtopossessauniquebindingmodeandmolecularinteractionwithintheATP-bindingcleftofhumanVps34.89InhibitionofVps34withSAR405ledtosigniﬁcantimpairmentoflysosomalfunctionandcouldpreventtheautophagyinducedbystarvationconditionsortheinhibitionofmTORwitheverolimus.ThisstudyrevealedsynergybetweenSAR405andeverolimusinrenalcellcarcinomastudies.AnotherstudyutilizingtheselectiveVps34inhibitorPIK-III,demonstratedPIK-IIIpotentlyinhibitedtheformationofmCherry-positiveautolysosomes(incellsexpressingthemCherry-GFP-LC3reporter),andpreventedtheclearanceofmitochondriainacarbonylcyanidem-chlorophenylhydrazone-inducedmitophagymodel.90TheseﬁndingsrevealVps34tohaveapivotalroleintheinitiationofautophagyanddegradationofsubstrates,andencouragefurtherstudiestoestablishwhetherVps34inhibitorsshouldbeexploredinfutureclinicaltrials.

AlthoughitisclearthatHCQexertspartofitseffectsthroughitsactiononautophagy,chloroquinederivativeslikelyharmcancercellsbyengagingothertargets.ThisobservationisreverberatedwitharecentreportdemonstratingtheefﬁcacyofCQinvivorelieduponitsabilitytonormalizetumorvesselstructureandincreaseperfusion,consequentlyreducinghypoxia,cancercellinvasionandmetastasis,irrespectiveofautophagyinhibition.91Inaddition,

AutophagyinhibitortherapyVWRebeccaandRKAmaravadi

Table3.PathwayPI3KPI3KPI3KMAPKMAPKMAPKEpigeneticImmuneOtherOtherOtherTherapiesundergoingcombinatorialtestingwithHCQincancerTargetAKTmTORmTORBRAFDualMEK/BRAFPan-Raf/Pan-RTKHDACInterleukin-2EGFREGFRNucleosideClinicalagentsMK2206Sirolimus(rapamycin)Everolimus(RAD001)Vemurafenib(PLX4032)Trametinib(GSK1120212)dabrafenib(GSK2118436)SorafenibVorinostatAldesleukin(IL-2)GeﬁtinibErlotinibGemcitabineCompanyMerckPﬁzerNovartisRoche/PlexxiconGlaxoSmithKlineGlaxoSmithKlineBayerMerckPrometheusAstraZenecaGenentech/AstellasEliLillyTrialIDNCT01480154NCT01842594NCT01510119NCT01897116NCT02257424NCT01634893NCT01023737NCT01550367NCT00809237NCT01026844NCT01506973Abbreviations:EGFR,epidermalgrowthfactorreceptor;IL-2,interleukin2;mTOR,mechanistictargetofrapamycincomplex;PI3K,phosphoinositide3-kinase;RTK,receptortyrosinekinase.clinicaltrialsindicatethathighdosesofHCQproduceonlymodestautophagymodulationinsurrogatetissues.Effortstoidentifymorepotentautophagyinhibitorshavecommenced.Theexistenceofnon-canonicalautophagybringsupthepossibilitythatanytherapeuticstrategypoisedatmodulatingacanonicalautophagyproteincanbecircumventedbyanincreaseinthefunctionofnon-canonicalautophagy;however,bothcanonicalandnon-canonicalautophagyultimatelyrelyonthelysosomeforﬁnaldegradation,providingapotentiallyidealtarget,whichiscurrentlybeinginvestigated.Lys05,anoveldimericderivativeofchloroquinewasshowntohavesigniﬁcantinvivoactivitybothasasingleagent88andincombinationwithaBRAFinhibitor.23EffortsareunderwaytooptimizeLys05forclinicaltrials.VATG-027,apotentautophagyinhibitoridentiﬁedthroughahigh-throughputscreenofanti-malarialcompoundswasfoundtohaveactivityinmelanomacells.92Theinterestingobservationwasmadethattheabilitytoinhibitautophagywasseparatefromthecytoxicityproﬁlesofthecompoundstested.

CELLINTRINSICREGULATIONOFAUTOPHAGYPOINTSTONEWTHERAPEUTICTARGETS

Recentworkhasincreasedourunderstandingofthecellintrinsicregulationofautophagyincancercells,andbydoingsomaypointthewaytowardbettertherapeutictargets.Oncogeneandtumorsuppressor-dependentgeneregulationhasbeeninvesti-gatedleveragingthemousemodelsmentionedabove,whichpossessKrasmutationsandp53deletionstounderstandhoweachmayregulateautophagy.Theseexperimentsmaynotrecapitulatethehumanconditionwhereoncogenesandtumorsuppressorgenesaremutatedinthecontextofinnumerableothergeneticandepigeneticalterationsincancerthatmayconvertasignalthatsuppressesautophagyintoonethatpromotesit.93,94Addingtothecomplexityofpredictingautophagyregulationbystudyingrecurrentsomaticmutationsassociatedwithcancer,itisincreas-inglyevidentthatbesidesgeneticregulationofautophagy,transcriptional,epigeneticandposttranslationalregulationofautophagyhasamajorimpactontheeventualroleofautophagywithinagivencancercell.

TranscriptionalregulationofautophagyhasbeendemonstratedthroughFoxkproteins(Foxk1andFoxk2)actingastranscriptionalrepressorsofautophagygenes.95Mechanistically,mTORpromotesthetranscriptionalactivityofFoxk1innutrient-richconditions,resultingintheco-localizationofFoxk1withSin3Aatthepromotersof79knownautophagy-associatedgenes.Interestingly,ablationofFoxk1withsiRNAresultedintheupregulationofcriticalcomponentsoftheUlk1andVps34machinery,reinforcingthenegativeimpactonautophagyservedbyFoxk1transcriptional

activity.95AutophagyhasbeenlinkedtolysosomalbiogenesisthroughobservationsthatstarvationactivatesatranscriptionalprogramlargelycoordinatedbythetranscriptionfactorEB(TFEB),whichresultsintheupregulationofautophagyandlysosomalgenestoenablethecelltosurvive.96TFEB,whenoverexpressed,signiﬁcantlyincreasesthenumberofautophagosomesincells,andwasfoundtoberegulatedthroughthephosphorylationofitsserine142residuebyERK2,belongingtotheMAPKpathway.P53hasalsobeenshowntohavearoleinthetranscriptionofautophagygenes,whichcomplimentthemousemodelsdescribedinvestigatingtherolemutantp53mayserveonthesensitivitytoautophagy-basedtherapy.Globalgenomicproﬁlinginmouseembryoﬁbroblastsrevealedp53totranscriptionallyregulateamultitudeofautophagygenes,whereinresponsetoDNAdamage,aninductionofautophagyreliedonp53transcrip-tionalactivity.97Itisworthnotingthatautophagyhasbeendemonstratedtostilloccurintheabsenceoffunctionalp53,suggestingthatp53doesnotsolelyregulateautophagybutratherhasapartinthehighlyorchestratedsymphonythatisautophagy.63ERstressalsoresultsintheupregulationofautophagyviaactivatingtranscriptionfactor4increasingULK1mRNAandproteinexpressionincellsundergoingsevereERstress,98andhasrecentlyrepresentedasigniﬁcantresistancemechanisminmelanomacellstreatedwithBRAFinhibitortherapy.23Althoughtranscriptionfactorsarenottraditionallythoughtofasdruggabletargets,effortsareunderwaytodevelopstrategiestoactivateorimpairthetranscriptionalactivityofp53,TFEBandFOXOproteins(Figure1).

Epigenetically,theacetylationstatusofhistoneH4lysine16(H4K16)wasfoundtoregulatelifeordeathdecisionsinautophagiccells,whereaninductionofautophagyresultsinadecreaseofH4K16acetylation(H4K16ac)andultimatelyadecreaseintheexpressionofATGgenesonagenome-widelevel.99AntagonizingthereductioninH4K16acuponautophagyinductionresultsinanincreaseinautophagiccelldeath.99Anothercheckpointisrepresentedbythenutrientsreleasedfromautophagicdegradationsuchasaminoacids,whichstimulatetheRagulatorcomplex,andresultintheactivationofmTORC1andnegativefeedbackonautophagicactivitytomaintainhomeostasis.100Themetaboliteacetyl-coenzymeA,recentlyreportedtofunctionasasuppressorofcytoprotectiveautophagyinagingcells,alsooccursmechanisticallythroughhyper-acetylationofhistoneH3leadingtotranscriptionaldownregula-tionofanumberofautophagygenes.101Methylationalsohasaroleinautophagyregulation,withagenome-widemethylationanalysisrevealinghyper-methylationoftheULK2gene,resultingintheinhibitionofautophagyinglioblastomacells.102EpigeneticagentssuchasHDACs85anddemethylatingagents103have

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AutophagyinhibitortherapyVWRebeccaandRKAmaravadi

alreadyﬁmodulators.

ndingsbeencouldshownguidetotheirmodulatedevelopmentautophagy,furtherandastheseautophagynewPosttranslationally,theautophagicmachineryisregulatedatmultiplelevelsincludingphosphorylation,acetylationandubiqui-tination.Thephosphorylationstatusofmultiplekeyplayersintheautophagypathwayhassigniﬁcantrolesintheregulationofautophagy.Whenphosphorylated,mTORC1isactiveandresultsintheinhibitionofautophagythroughthedirectphosphorylationofULK1bymTORC1.LC3canalsobephosphorylatedbyPKAandPKC,resultingintheinabilityforLC3tobecomelipidated,anessentialstepneededforLC3incorporationwithintheautopha-gosomebilayer.104Lysineacetylationhasaninhibitoryrole,whereunderconditionsofnutrientstarvation,lossofacetylationresultsinaninductionofautophagy.105Silencingofacetyl-coenzymeAsynthetase,leadingtoadecreaseintheoverallacetylationofcytoplasmicproteins,hasalsobeenreportedtoresultinenhancedautophagyinDrosophilabrains.106Ubiquitinationalsohelpsregulateautophagy,withanemergingrolefortheE3ubiquitinligasesNedd4,107Parkin108andTRIM13109intheinitiationofautophagy,mitochondrialhomeostasisandinsubstratespeciﬁcityforautophagicdegradation.110Metabolicregulationofautophagyoccursthroughtheabilityofupstreamautophagy-regulatingeffectorstosensetheintracellularlevels10ofammonia,aminoacids,growthfactors,glucoseandlipids(Figure1).Ammoniaiscreatedviaamino-acidcatabolismandinducesautophagybywayofactivatingAMP-activatedproteinkinaseandleadingtotheERstressresponse.111Adropinamino-acidlevelsissensedbyafewdifferentmechanisms,whichinclude(1)sensingoftheresultingaccumulationofunchargedtRNAspeciesbyGCN2,112(2)lysosomalsensingthatrecruitsmTORC1tothelysosomalsurface,113(3)sensingofintracellularacetyl-CoAstoresthatarenegativelyimpactedbylowlevelsofvariousaminoacids114and(4)sensingthedepletionofthemetabolicintermediate115α-ketoglutarate,anotherresultoflowamino-acidlevels.Alloftheseamino-acidsensingmechanismsresultinaninductionofautophagytoincreasetheintracellulardegradationofnonessentialcomponentsinanattempttoincreasethepoolofavailableaminoacidstocontinuemetabo-lism.Understandingtheseepigenetic,posttranslationalandmetabolicregulatorycircuitsmayhelpdeﬁnetheautophagicswitchthatappearstooccurinthetransitionfromtumorsuppressortotumorpromoter.Thedevelopmentofsmall-moleculeinhibitorsthattargetcancermetabolismwillcertainlyhaveanimpactonautophagy,andperhapsinsomecasesthesedrugscanberepositionedorreconsideredasautophagymodulatorsiffurtherresearchindicatesthatautophagyisresponsibleforthemainchangesobservedwiththeseinhibitors.NEWROLESFORTHEFUNCTIONALEFFECTSOFAUTOPHAGYAutophagyfunctionallyprotectscellsbywayofdegradingintracellularcomponents,whichwouldhaveotherwiseledtothelossofcellularﬁtness,whilealsosimultaneouslycateringtotheever-changingmetabolitedemandsofthecellwithfreshlydigestedbuildingblocksforsurvival.Althoughitisclearthatthedegradationthroughautophagyofproteinsubstrateshasaroleincellularsurvival,thespeciﬁcityofthisprocessisunknown.Globalproteomeanalysiscomparingcellswithintact-autophagy/−versuscellswithdefectiveautophagy(Atg5+/+andAtg5−)revealedthatautophagypreferentiallydegradesproteinsthataretoxicornonessentialforsurvivalunderstressfulconditions,seemingtospareproteinsinvolvedin116themaintenanceoffunctionalautophagyandstresssurvival.Interestingly,proteinsfoundtoincreaseinresponsetoautophagyinductionwereinvolvedinvesicle-mediatedtrafﬁckingandlysosomalproteindegradation,potentiallyprovidinganewsuiteoftherapeutictargetsthatmayaugmentsstrategiesofinhibitingautophagy.Anotherreport

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identiﬁedaspeciﬁcproteinturnovermechanismwhereautop-hagywasresponsibleforthedegradationoftheinhibitoryp53isoformΔ133p53αthroughinteractionofthechaperone-associatedE3ubiquitinligaseSTUB1.117AutophagywasalsofoundtohaveakeyroleinthedegradationofdamagednuclearDNAincellsdeﬁcientofDnase2a.118DNAaccumulatedinautophagy-deﬁcientcells,whichresultedinSting-mediatedinﬂammation.Autophagicactivitycangovernthesecretoryproﬁleofcancercells,wherehighautophagyisassociatedwithmelanomametastasis,andserumfrommetastaticmelanomapatientswithhightumorautophagylevelscontainasecretorysignaturefoundtocorrelatewithcellsdisplayinghighautophagicactivity.87Theseﬁndingsareofimmenseimportanceastheyprovideapotentialavenuetoassessautophagicactivityoftumorswithinpatientsfromserumsamplesaswellasthepotentialtoprovideameanstostratifypotentialrespondersinfutureautophagy-basedtherapyregimens.Autophagyalsofunctionallyinhibitsapoptosisthroughindirectinhibitionofp53-upregulatedmodulatorofapoptosis,whichdemonstrateshowautophagycandeterminecellfate.119Inaddition,autophagyhasbeendemon-stratedtodirectlyimpactproliferationbywayofAMBRApromotingthedephosphorylationofc-MycSer62,resultingintheproteasomal120degradationofc-Mycandadecreaseintherateofcelldivision.Finally,autophagyhasafunctionalroleintheimmunogenicclearanceofcancercells.Immunogeniccelldeath(ICD)reliesinpartonthereleaseofATPfromdyingcells121andautophagyhasbeenfoundtobecriticalintheICD-associatedsecretionofATP.122Mechanistically,ATPwasfoundtoreleaseinamannerdependentuponthelysosomalproteinLAMP1andtheopeningofPANX1(pannexin1)channels.ImplicationsonwhateffectutilizationofautophagyinhibitorsmayconferuponICDremainstobedetermined.However,futurecombina-tionregimenscanbeenvisionedwhereanautophagyinhibitoralongwithanimmune-enhancingtherapycanprovidethebestofbothworldsofinhibitingcytoprotectiveautophagywhileconcurrentlylaunchinganeffectiveimmuneresponseagainstthetumorcells.

Insummary,aﬁrstseriesofhurdles,includingexperimentsinxenograftsandgeneticallyengineeredmousemodels,followedbytheﬁrstseriesofHCQtrialshavebeenovercomedemonstratingtheapplicationofautophagyinhibitorsinpatientswithadvancedcancerscouldbedonesafely,andhasresultedinencouragingantitumorresultsinselectedpatients.Thestageisnowsetforthetestingofmorepotentandspeciﬁcinhibitorsoftheautophagicmachinery.Whilethisisbeingdoneintheclinic,translatingknowledgeabouttheregulationofautophagyanditsfullspectrumoffunctionsinmulticellularorganismswillpermitthedevelopmentofnewstrategiesforautophagymodulationincancer.

CONFLICTOFINTEREST

Theauthorsdeclarenoconﬂictofinterest.

ACKNOWLEDGEMENTS

ThisworkwassupportedbyR01CA169134(RKA)fromtheNationalInstitutesofHealth.

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