Discrete roles and bifurcation of PTEN signaling and mTORC1-mediated anabolic metabolism ...

20 Pages·2017·4.39 MB·English

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GENETICS Copyright©2018
TheAuthors,some
Discrete roles and bifurcation of PTEN signaling and
rightsreserved;
exclusivelicensee
mTORC1-mediated anabolic metabolism underlie
AmericanAssociation
– fortheAdvancement
IL-7 driven B lymphopoiesis
ofScience.Noclaimto
originalU.S.Government
Hu Zeng,1* Mei Yu,2 Haiyan Tan,3,4 Yuxin Li,3,4 Wei Su,1,5 Hao Shi,1 Yogesh Dhungana,1 Cliff Guy,1 Works.Distributed
Geoffrey Neale,6 Caryn Cloer,1 Junmin Peng,3,4† Demin Wang,2† Hongbo Chi1† underaCreative
CommonsAttribution
NonCommercial
Interleukin-7 (IL-7) drives early B lymphopoiesis, but the underlying molecular circuits remain poorly under- License4.0(CCBY-NC).
stood, especially how Stat5 (signal transducer and activator of transcription 5)–dependent and Stat5-
independentpathwayscontributetothisprocess.Combiningtranscriptomeandproteomeanalysesandmouse
geneticmodels,weshowthatIL-7promotesanabolicmetabolismandbiosyntheticprogramsinpro-Bcells.IL-
7–mediatedactivationofmTORC1(mechanistictargetofrapamycincomplex1)supportedcellproliferationand
metabolism in a Stat5-independent, Myc-dependent manner but was largely dispensable for cell survival or
Rag1andRag2geneexpression.mTORC1wasalsorequiredforMyc-drivenlymphomagenesis.PI3K(phospha-
D
tidylinositol 3-kinase) and mTORC1 had discrete effects on Stat5 signaling and independentlycontrolledBcell o
w
development.PI3KwasactivelysuppressedbyPTEN(phosphataseandtensinhomolog)inpro-Bcellstoensureprop- n
lo
er IL-7R expression, Stat5 activation, heavy chain rearrangement, and cell survival, suggesting the unexpected bi- a
d
furcation of the classical PI3K-mTOR signaling. Together, our integrative analyses establish IL-7R–mTORC1–Myc ed
andPTEN-mediatedPI3KsuppressionasdiscretesignalingaxesdrivingBcelldevelopment,withdifferentialeffects fro
onIL-7R–Stat5signaling. m
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INTRODUCTION BindingofIL-7toIL-7RinitiatesphosphorylationofJanuskinase ://ad
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Blymphopoiesisisahighlyordereddevelopmentalprocesscharacter- 1(Jak1)andJak3,whichrecruitandactivatethetranscriptionfactor a
n
izedbythesequentialrearrangementsofimmunoglobulinheavy(IgH) Stat5 (signal transducer and activator of transcription 5). IL-7R– ce
s
andlightchain(IgL)locithataccompanydifferentiationoflymphoid mediated proliferation and survival of pro-B cells are critically .s
c
precursorstopro-Bcells,pre-Bcells,andimmatureBcellsinthebone dependentontheJak1/3-Stat5signalingaxis(10–13).Ectopicexpres- ie
n
marrow (BM). The stepwise progression of B cell development is sionofconstitutivelyactiveStat5considerably,althoughincompletely, ce
dependentuponanetworkoftranscriptionfactorsandextrinsicsignals rescuesBcelldevelopmentaldefectsinIL-7Ra–deficientmice(14), ma
g
(1),especiallyinterleukin-7(IL-7)(2).IL-7isproducedbyBMstromal suggestingtheinvolvementofotherpathwaysdownstreamofIL-7R. .o
cellsandsignalsthroughIL-7receptor(IL-7R;composedoftheIL-7Ra Publishedstudiesindicatethatphosphatidylinositol3-kinase(PI3K) rg
chainandacommongcchain)expressedonpro-Bandpre-Bcellsto signalingmediatesIL-7Rsignalinginpre-Bcells(15),althoughfor on/
promotetheirproliferation,survival,anddifferentiation(3).Becauseof pro-Bcelldevelopment,deletionofp110aandp110d(16),pharmaco- No
v
itspotenttrophiceffectonlymphocytes,IL-7therapyhasbeendevel- logicalinhibitionofPI3K,ordeficiencyofp85adoesnotexertastrong e
m
opedforclinicalapplication.Inparticular,infusionofIL-7inducesa effect(17).Thus,thenatureandfunctionofStat5-independentpathways b
e
markedexpansionofBcellprecursorsinhumanpatients(4,5),suggest- inIL-7Rsignaling,andtheextenttowhichPI3Kcontributestoearly r 2
8
ingastrongtrophiceffectofIL-7inadulthumanBcellprecursors.Yet, B cell development, remain elusive. Furthermore, PI3K has been , 2
thelackoffullunderstandingoftheIL-7–mediatedsignalingnetwork traditionallyassociatedwiththeactivationofthemechanistictarget 0
1
8
hindersitsrationalutilizationinclinics(6).DespiteitsimportanceinB ofrapamycin(mTOR)(18,19).DespiterecentgeneticstudiesofmTOR
lymphopoiesis,howIL-7influencespro-Bcelldevelopment,especially signalinginBcelldevelopmentandfunction(20,21),theupstreamand
aboutthemechanismslinkingIL-7Ractivationtotranscriptionaland downstream regulators of mTOR and the relationship with PI3K
translationalevents,hasnotbeenexaminedinasystematicmanner. signalinginBlymphopoiesisarenotwellunderstood.Moreover,how
Furthermore,althoughIL-7hasbeenlinkedtolymphocytemetabolism mTOR,PI3K,andStat5signalingareintegratedislargelyunknownin
(7–9),thedetailedprocessesandmolecularregulatorsareunclear. lymphocytedevelopment.
To understand the proteome landscape and signaling networks
mediatedbyIL-7signaling,weperformedtemporalproteomicprofiling
and network analysis of IL-7–stimulated pro-B cells using multi-
1DepartmentofImmunology,St.JudeChildren’sResearchHospital,Memphis,TN
plexedtandem mass tag(TMT)andtwo-dimensional(2D)liquid
38105,USA.2BloodResearchInstitute,BloodCenterofWisconsin,Milwaukee,WI
53226, USA. 3Departments of Structural Biology and Developmental Neuro- chromatography–tandemmassspectrometry(LC/LC-MS/MS)(22,23).
biology,St.JudeChildren’sResearchHospital,Memphis,TN38105,USA.4St.Jude Ourproteomicprofiling,togetherwithtranscriptomeanalysis,revealed
ProteomicsFacility,St.JudeChildren’sResearchHospital,Memphis,TN38105, enrichmentofanabolicmetabolismandactivationofmTORandMyc
UScSiAen.c5IentCeegnrateter,dMBeiommpehdisic,aTlNSc3ie8n1c6e3s,UPrSoAg.r6aHma,rtUwneivlleCrseitnyteorffToernBnieosisnefoermHeaatlitchs in IL-7–stimulated pro-B cells. Furthermore, using hCd2-iCre–
andBiotechnology,St.JudeChildren’sResearchHospital,Memphis,TN38105,USA. mediatedtargetedmutagenesis(24),wegeneticallydefinedtheroles
*Presentaddress:DivisionofRheumatology,DepartmentofMedicine,andDe- and mechanisms of mTOR and its two complexes, mTORC1 and
partmentofImmunology,MayoClinic,Rochester,MN55905,USA.
†Correspondingauthor.Email:[email protected](H.C.);demin.wang@bcw. mTORC2,aswellasMycandPI3Ksignaling,inearlyBcelldevelop-
edu(D.W.);[email protected](J.P.) ment.OurresultsshowedthatmTORC1,butnotmTORC2,iscritical
Zengetal.,Sci.Adv.2018;4:eaar5701 31January2018 1of19
SCIENCE ADVANCES | RESEARCH ARTICLE
forBlymphopoiesis,bylinkingIL-7Rsignalingtopro-Bcellmetabo- (FDR)<0.01,withatleast1.5foldchange(FC);dataS1A].Weapplied
lism.TheseeffectsareseparatedfromPI3K-Aktandareindependentof weightedgenecorrelationnetworkanalysis(WGCNA)(32)oftheseDE
Stat5signalingandcellsurvival,butmTORC1activityisrequiredfor genestoidentifyRNAclusters(RCs)ofhighlycorrelatedgenes,which
IL-7–inducedMycproteintranslation.Further,mTORC1andMyc maysharesimilarbiologicalfunctionsormechanismsofregulation
formafeed-forwardcircuitryinlymphomagenesis,andRaptorde- (33).Weidentifiednineclustersthatcontainedatleast10DEgenes
ficiencycompletelyblockstumordevelopmentinamodelofMyc- (Fig.1Bandfig.S1G).Thelargestcluster,RC7(865genes),showeda
inducedBcelllymphoma(25,26).Unexpectedly,wefoundthatactive gradualup-regulationfromfractionAtofractionC′cellsandthen
suppressionofPI3KactivitybyPTEN(phosphataseandtensinhomo- down-regulation(Fig.1B),apatternsimilartometabolicchangesand
log)isrequiredforproperBcelldevelopment.UncontrolledPI3Kdis- IL-7Raexpressiondescribedabove.PathwayanalysisrevealedthatRC7
ruptspro-Bcelldevelopment,associatedwithimpairedIL-7R–Stat5 wasenrichedwithmTORC1signaling,Myctargets,metabolicpathways
signaling,reducedlineagetranscriptionfactorexpression,diminished (includingglycolysis,oxidativephosphorylation,andone-carbonme-
IgHchainrearrangement,andgreatlyelevatedapoptosis.Together, tabolism),andcellcycleregulators(Fig.1C,fig.S1H,anddataS1B),
our results uncover two signaling axes, namely, PTEN-mediated furthersupportingthedynamicregulationofmetabolicactivitiesinear-
PI3KsuppressionandIL-7R–mTORC1–Myc,crucialforearlyBcell lyBcelldevelopment.
developmentwithdiscrete mechanismsand effectsonIL-7R–Stat5 Tocomprehensivelyexaminethedirectrolesandtemporaleffects
signalingandimmunoglobulinrearrangement,andpointtotheun- ofIL-7ontheproteomelandscapeandmetabolicprogramsinpro-
expectedbifurcationoftheconventionalPI3K-mTORsignalingaxisin Bcells,weappliedmultiplexedTMTandLC/LC-MS/MSapproaches
IL-7–drivenBlymphopoiesis. (22,23,34)toquantifythewholeproteomeoffreshlyisolated(0hour) D
orIL-7–stimulated(1,4,and16hours)pro-Bcells(fig.S1I).Wequan- ow
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tified7733uniqueproteins.Thenullcomparisonsoftheexperimental lo
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RESULTS replicatesshowedrandomdistribution(fig.S1J,left),whereascompar- de
d
IpLr-o7-Baccteivllastes mTOR, Myc, and anabolic metabolism in icsaonntsdoifffethreentcweosbtiemtweepeoninfrtess(h1l6yihsooulartsedvearnsudsa0cthivoautre)dsphroow-Bedcesillgsnbifui-t from
Cellmetabolismiscloselyassociatedwithlymphocyteactivationand consistencybetweenreplicates(fig.S1J,right).Clusteringanalysisof h
fsutonocdtio(n27,)b.uTtoitsinrvoelsetiignatiemmmeutanbeodliecvreelogpumlateinontrdeumrainingsBpoceolrllydeuvnedloepr-- DWEepidroetnetiinfsie(dFi8g5.14DD)Efuprtrhoetreivnesritfiheadttshiegnreipfircoadnutlcyibcihliatynogfedtheberetwsueletsn. ttp://ad
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ment,weexaminedmitochondriarespirationandglycolysisinfreshly anytwotimepointsusingone-wayanalysisofvariance(ANOVA; a
n
isolatedBMBcellsubsets(thegatingstrategyisshowninfig.S1A)by 10%FDR,zscoreofatleast2;Fig.1DanddataS2A).Wethenap- ce
s
measuringoxygenconsumptionrate(OCR)andextracellularacidifica- pliedWGCNAfortheseDEproteinsandidentifiedfiveclustersof .s
tionrate(ECAR),respectively.Bcellprecursors[Lin−B220+CD43+IgM−, coexpressed proteins, named as whole protein clusters WPC1 to cie
n
includingfractionA,B,C,andC′cells(28)]hadthehighestOCR, WPC5.WPC1,thelargestcluster, showed late down-regulation at ce
relativetoB220+CD25+pre-Bcells,immatureBcells(B220+IgM+), 16 hours of IL-7 stimulation, whereas WPC2, the second largest ma
and circulating mature B cells (B220hiIgM+; Fig. 1A). ECAR was cluster,exhibitedlateup-regulationat16hours.Inaddition,WPC3 g.o
modestinallcellsubsetsexamined,butBcellprecursorsstillcon- toWPC5showedup-regulationat4hoursbuthaddistinctpatternsof org/
tained the highest level (fig. S1B). We next examined additional expressionat16hours,namely,continuedup-regulation(amplification), n
N
metabolicparameters,includingtheexpressionofthenutrientreceptor unchangedexpression(plateau),ormarkeddown-regulation(atten- o
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transferrin(CD71),whichcorrelateswithcellmetabolicrate(29)(fig. uation),respectively(Fig.1E). e
m
S1C),proliferationvia5-ethynyl-2′-deoxyuridine(EdU)incorpora- PathwayenrichmentanalysisrevealedthatWPC1containedpro- b
e
tionassay(fig.S1D),andglucoseuptake,asindicatedbystainingwith teinsinvolvedincytoskeletalregulation,includingactin-bindingpro- r 2
8
theglucoseanalog2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)- teinsandmultipleintegrinmolecules,suggestingapotentialeffectof , 2
2-deoxyglucose (2-NBDG; fig. S1E). We found that developing IL-7oncytoskeletalreorganizationinpro-Bcells(Fig.1Fanddata 01
8
BcellsgraduallyincreasedCD71expression,proliferation,andglu- S2B).NegativeregulatorsofthecellcyclewerealsoidentifiedinWPC1,
coseuptakeduringtheirdevelopmentalprogressionfromfraction including CDKN2C and CDKN2D. BCL6(B cell lymphoma6),a
A[Lin−B220+CD43+IgM−BP-1−CD24−,accordingtotheHardyclassi- transcriptionalregulatorsuppressedbyIL-7signalingandinduced
fication(30)]tofractionB(Lin−B220+CD43+IgM−BP-1−CD24+)and bypre-BCR(Bcellreceptor)(35),wasalsoidentifiedinWPC1(data
then fraction C and C′ (Lin−B220+CD43+IgM−BP-1+CD24+) stage S2B).WPC2andWPC3containedthemajorityofproteinsup-regulated
(seefig.S1Aforgating),whereaspre-Bcellsdown-regulatedallthese byIL-7.InadditiontothemodestlyenrichedJak-StatandIL-2–Stat5
parametersrelativetofractionC/C′cells(fig.S1,CtoE).Accompany- pathways,mTORC1signalingandcholesterolhomeostasispathways
ingthedynamicchangesofmetabolicactivities,theexpressionofIL- weremoresignificantlyenrichedinWPC2andWPC3,includingsome
7Ra wasactivelyregulatedduring B celldevelopment.Specifically, ofthekeyenzymesinlipidbiosynthesissuchasHMGCS1,PMVK,
IL-7RaexpressionwasmarkedlyincreasedonfractionBandfrac- MVD,SQLE,IDI1,ACSL3,andFASN(dataS2B).Enzymesinvolved
tionC/C′cellscomparedtofractionAcells,followedbysequential inaminoacidmetabolism,suchasBCAT1,SLC7A5,andASNS,were
down-regulationinlaterdevelopmentalstages(fig.S1F),suggesting alsoidentifiedinWPC2andWPC3(dataS2B).Furthermore,Myctar-
coordinatedregulationofIL-7Rexpressionandmetabolicprograms getsandorganelleformationpathwayswereenrichedinWPC2(Fig.1F
duringBcelldevelopment. anddataS2B). The heat mapsofmTORC1signalingcomponents
FurtherbioinformaticsanalysisoftranscriptomeofdevelopingBcell (Fig.1Gandfig.S1K)andMyctargets(Fig.1H)inWPC2andWPC3
subsetsintheImmunologicalGenomeProject(ImmGen)(31)revealed demonstratedapronouncedup-regulationofthesepathwaysbyIL-7at
2499genesthathadsignificantlyalteredexpressionbetweenanytwo the16-hourtimepoint,indicatingarelativelylateactivationkinetics.
subsets,thatis,differentiallyexpressed(DE)genes[falsediscoveryrate WPC4 and WPC5 showed enrichment of the E2F target pathway
Zengetal.,Sci.Adv.2018;4:eaar5701 31January2018 2of19
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Fig.1. RegulationofmetabolicactivityindevelopingBcellsandIL-7–mediatedproteomelandscapeinpro-Bcells.(A)OCRofLin−B220+CD43+IgM−cells,CD25+
pre-Bcells(Lin−B220+CD43−IgM−CD25+),immatureBcells(B220+IgM+),andcirculatingmatureBcells(B220hiIgM+)measuredusingaSeahorseXF24analyzer.(B)RC7
frommicroarraydataofcommonlymphoidprogenitors(CLPs)andeachdevelopingBcellsubsetfromImmGen(geneexpressionrelativetoCLP).Thecentralrectangle
representsthefirsttothirdquartile,withnotchintheplotcorrespondingtothemedianexpressionofallgenesinthesample.Endofthewhiskersrepresentsminimum
andmaximumlevelofexpressionofthegenesinthecluster.(C)FunctionalannotationsofRCsbyGeneOntology(GO),KyotoEncyclopediaofGenesandGenomes
(KEGG),andHallmarkdatabases(FDR<0.2).(D)ClusteranalysisofDEproteinsintemporalproteomicprofilingofpro-Bcellsstimulatedwith0,1,4,and16hoursofIL-7.
(E)FiveWPCsduringpro-BcellactivationweredefinedbyWGCNA.Eachlineindicatestherelativeabundanceofeachproteinandiscolor-codedbythecluster
membershipdeterminedbycorrelationanalysisbetweenindividualproteinandtheconsensusoftheWPC(seethecolorbar).(F)FunctionalannotationsofWPCs
byGO,KEGG,andHallmarkdatabases(FDR<0.2).(GandH)IL-7–mediatedtemporalexpressionofmTORC1signaling(G)andMyctargets(H).(I)Fourinterconnected
modulesoforganellesandribosomecomponentswerederivedfromWPC2.Thenumberandnamesofproteinsandtherepresentativefunctionaltermforeach
moduleareshown.Seealsofig.S1anddataS1andS2.
Zengetal.,Sci.Adv.2018;4:eaar5701 31January2018 3of19
SCIENCE ADVANCES | RESEARCH ARTICLE
and sialyltransferase activity (36), respectively, indicating dynamic B cells (fig. S2C), including follicular (CD21intCD23hi; fig. S2D) and
effectsofIL-7oncellcycleprogressionandproteinglycosylationin marginalzone(CD21hiCD23low)Bcellsinthespleen(fig.S2E).There-
pro-Bcells.Finally,despitecloseconnectionbetweenPI3KandmTOR fore,RaptorandmTORkinasearerequiredforfractionC/C′topre-
signalingpathways(19),thePI3K-Aktpathwaywasnotidentifiedin B cell transition, and their deficiencies lead to disrupted pro-B cell
anyoftheWPCs(dataS2B).Thus,inresponsetoIL-7stimulation, development,agreatlydiminishedpre-Bcellcompartment,andde-
pro-Bcellslikelyundergochangesintheirmetabolicfeaturesassociated velopmentofprofound lymphopenia ofperipheral B cells.
withtheactivationofmTORC1andMycpathways. Incontrast,BMofCd2icreRictorfl/flmicecontainedasmall(P>0.05)
We further superimposed proteins within each WPC onto the increaseofB220+CD43+IgM−Bcellprecursors(Fig.2E)andamodest
protein-proteininteraction(PPI)networktoidentifyputativefunc- elevationofB220+CD25+pre-Bcells(Fig.2G).Theseobservationsare
tional modules altered during temporal IL-7 stimulation on the consistentwitharecentreportusingMx1-Cre–mediatedsystemicde-
basis of the assumption that coexpressed genes/proteins are more letionofRictor(41).Withinthepro-Bcellcompartment,Rictordefi-
likelytosharecommonmolecularfunctions(33,34).Weidentified ciencydidnotsubstantiallyalterthedistributionofpro-Bcellsubsets
39modules,withsizesrangingfrom2to35proteins(dataS2C).The (Fig. 2F).Despiteincreasedpre-Bcells, Cd2icreRictorfl/flmicehada
largest module (WPC1.M5) was composed of molecules involved in normalimmatureBcellcompartment(Fig.2H),indicatingthatthe
celladhesion,migration,andcytoskeleton,includingmultipleintegrin modestperturbationduringearlierBcelldevelopmentdidnotaffect
familymembers(dataS2C).InWPC2,wefoundfourinterconnected immatureBcellgeneration.However,Cd2icreRictorfl/flmicehadsignif-
modules (WPC2.M1 to WPC2.M4) with 86 combined components icantlyreducedcirculatingmatureBcellsintheBM(fig.S2B)and
that were enriched for intracellular organelle or ribosome compo- diminishednumbersofsplenicBcells,includingfollicularandmarginal D
o
nents (Fig. 1I), suggesting that IL-7 may coordinate up-regulation of zoneBcells(fig.S2,CtoE).ThesedataindicatethatmTORC2ac- w
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organelle biogenesis. Collectively, our integrative network analyses tivityisnotessentialforearlyBcelldevelopmentbutcontributesto lo
a
predict that IL-7 likely regulates cell adhesion and migration and peripheralBcellmaturation,consistentwitharecentreportusingpan- de
d
concomitantly activates mTORC1 and Myc signaling, biosynthetic hematopoieticandacutedeletionsystems(21).Combineddeletionof fro
metabolism, and organelle biogenesis in pro-B cells. bothRaptorandRictorledtodevelopmentalphenotypesthatwerelarge- m
lysimilartodeletionofRaptoralone(Fig.2,EtoH,andfig.S2,AtoE)or h
mprTeO-BRCce1l,lbtruatnnsiottiomnTORC2, activity is critical for pro-B to mopTmOeRnt(aFlidge.f2e,ctAsdtoueDt)o.FRuarptthoerrmdeofircei,entocyexwaemreinceelwl-hinettrhinersiBc,cweellgdeenveerl-- ttp://ad
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WenextinvestigatedthefunctionalimportanceofmTORsignalinginB atedmixedBMchimerasthatcontained50%ofWTcongeniccellsand a
n
celldevelopment.WecrossedmicebearingconditionalloxP-flanked foundthatmTORC1-dependentBcelldevelopmentwascell-intrinsic ce
s
Mtor(Mtorfl/fl)allelewithhCd2-iCre(Cd2icre)transgenicmice,inwhich (fig.S2,FandG).Collectively,mTORC1activityplaysapredominant, .s
c
anoptimizedvariantofCrerecombinase(iCre)isexpressedundera intrinsic role in pro-B celldevelopment and in the pro-B to pre-B ie
n
humanCD2promoterandlocuscontrolregion.Thistransgenedrives transition.Incontrast,mTORC2hasarelativelysmallsuppressiveeffect ce
m
iCreexpressioninlymphocytelineages(24),leadingtoefficientrecom- onearlyBcellgenerationbutcontributestoperipheralBcellmaturation. a
g
binationinalldevelopingBcellsincludingfractionAcells(37).Previous .o
studieshaveshownthatahypomorphicMtorallelewithconstitutive mTORC1 mediates IL-7R signaling in a Stat5-independent org/
butpartiallossofmTORactivitypartlyblockspro-Btopre-Btransition but Myc-dependent manner n
and reduces pre-B cells, but Cd19-Cre–driven [which initiates WenextexploredthemechanismsunderlyingmTORC1-mediated No
incompletetargetgenedeletionatthepro-B/pre-Bcellstage(38)]dele- early B cell development. Raptor-deficient fraction C/C′ cells, but ve
m
tionofMtordoesnotaffectBcelldevelopmentintheBM(39,40).In not fraction A or fraction B cells, had reduced CD71 expression b
e
contrast,weobservedanaccumulationofB220+CD43+IgM−Bcellpre- (Fig.3A)andEdUincorporation(Fig.3B).However,Raptordeficiency r 2
8
cursors(Fig.2A)intheabsenceofmTOR,whichwasattributedto didnotaffectcellapoptosis,asindicatedbyactivecaspase-3staining , 2
increased fraction C/C′ cells with abnormally high expression of (Fig.3C).SimilarphenotypeswereobservedinCd2icreMtorfl/flmice 01
BP-1(Fig.2B).Inaddition,therewerefewCD25+pre-Bcells(Fig.2C) (fig.S3,AandB),indicatingthatmTORC1impingesuponaspecific 8
andimmatureandcirculatingmatureBcellsintheBM(Fig.2D)of setofphysiologicalactivities.
Cd2icreMtorfl/flmice.Thus,themTORkinaseisessentialforearlyBcell BecauseIL-7isthemajorgrowthfactordrivingearlyBcellpro-
development. liferation,wetestedinvitroIL-7–inducedproliferativeresponseof
mTORsignalingconsistsofmTORC1andmTORC2complexes. BMcellsfromWTandCd2icreRptorfl/flmice.WTBMcellsprolifer-
ToinvestigatetheextenttowhichmTORC1andmTORC2contrib- atedinresponsetoIL-7stimulation,butRaptor-deficientBMcells
ute to early B cell development, we generated Cd2icreRptorfl/fl and showed no population expansion (Fig. 3D). To circumvent the
Cd2icreRictorfl/flmice,inwhichRaptorandRictorweredeletedinde- potentialcomplicationfromthealteredcompositionofdeveloping
veloping B cells. Flow cytometry analysis of BM cells revealed that Bcells,wepurifiedlineage-negativehematopoieticprogenitorcells
Cd2icreRptorfl/fl mice hada significantreduction oftotal B220+ cells fromWTandCd2icreRptorfl/flmiceand,ascontrols,thosefromRag-1−/−
(fig.S2A)andanincreasednumberofB220+CD43+IgM−Bcellpre- andJak3−/−miceandculturedthemwithOP9stromalcellsinthe
cursors(Fig.2E).SimilartoCd2icreMtorfl/flmice,Cd2icreRptorfl/flmice presenceofIL-7.ThenormalanddefectiveBcelldifferentiationfrom
had normal numbers of fraction A and fraction B cells but signifi- Rag-1−/−andJak3−/−progenitorcells,respectively,indicatedthatBcell
cantlyincreasednumbersoffractionC/C′cellswithunusuallyhigh differentiationisindependentofpre-BCRbutisdependentuponIL-7R
expression of BP-1 (Fig. 2F). Strikingly, B220+CD25+ pre-B cells signaling in this experimental system (fig. S3C). Raptor-deficient
(Fig.2G),immatureBcells(Fig.2H),andcirculatingmatureBcells progenitorcells,similartoJak3-deficientcells,failedtodifferentiateinto
(Fig.2Handfig.S2B)wereallseverelydiminishedintheabsenceof Bcells(fig.S3C),indicatingthatIL-7Rsignalingisseverelyimpairedin
Raptor.Consequently,Cd2icreRptorfl/flmiceweredevoidofperipheral Raptor-deficientprogenitorcells.
Zengetal.,Sci.Adv.2018;4:eaar5701 31January2018 4of19
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Fig.2. mTORC1,butnotmTORC2,isrequiredforpro-Bcelldevelopmentandpro-Btopre-Bcelltransition.(AtoD)RepresentativeflowcytometryplotsofBMcells
fromwild-type(WT)andCd2icreMtorfl/flmice.(A)ExpressionofB220andCD43onBMlymphocytes,withthepercentagesofB220+CD43+IgM−cellsindicated(afterfurther
gatingIgM+cellsoutofB220+CD43+cells;seefig.S1Aforgatingscheme).Right:Numberofdesignatedpopulations.(B)ExpressionofBP-1andCD24onB220+CD43+IgM−
cells,withthepercentagesoffractionA(CD24−BP-1−),fractionB(CD24+BP-1−),andfractionC/C′(CD24+BP-1+)cellsindicated.Right:Numberofindicatedsubsetcells.
(C)ExpressionofB220andCD25onBMlymphocytes,withthepercentageofB220+CD25+pre-Bcellsindicated.Right:NumberofB220+CD25+pre-Bcells.(D)Expres-
sionofB220andIgMonBMlymphocytes,withthepercentagesofpro-B/pre-Bcells(B220+IgM−),immatureBcells(B220+IgM+),andcirculatingmatureBcells
(B220hiIgM+)indicated.Right:NumberofimmatureandmatureBcells.(EtoH)RepresentativeflowcytometryplotsofBMcellsofWT,Cd2icreRptorfl/fl,Cd2icreRictorfl/fl,and
Cd2icreRptorfl/flRictorfl/flmiceandthenumberofindicatedpopulations.(E)ExpressionofB220andCD43onBMlymphocytes,withthepercentageofB220+CD43+IgM−cells
indicated.(F)Hardyclassificationofpro-BcellsubsetsintheB220+CD43+IgM−gate[from(E)],withthepercentagesoffractionA(CD24−BP-1−),fractionB(CD24+BP-1−),and
fractionC/C′(CD24+BP-1+)cellsindicated.(G)ExpressionofB220andCD25onBMlymphocytes,withthepercentageofB220+CD25+pre-Bcellsindicated.(H)Expressionof
B220andIgMonBMlymphocytes,withthepercentagesofpro-B/pre-Bcells(B220+IgM−),immatureBcells(B220+IgM+;statisticsareontheright),andcirculating
matureBcells(B220hiIgM+)indicated.NS,notsignificant.*P<0.05,**P<0.01,***P<0.001,****P<0.0001(one-wayANOVAwithTukey’stest).Resultsrepresentatleast
fourindependentexperiments.Dataaremeans±SEM.Numbersindicatepercentageofcellsingates.Seealsofig.S2.
Zengetal.,Sci.Adv.2018;4:eaar5701 31January2018 5of19
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The failure of Raptor-deficient BM cells to respond to IL-7 ToaccuratelymeasureMycproteinexpressiononasingle-celllevel,
prompted us to examine the IL-7R signaling machinery. Whereas wecrossedWTandCd2icreRptorfl/flmicewithMycEGFPmice,whichex-
WTmicesubstantiallyup-regulatedIL-7Raexpressioninfraction pressanN-terminalfusionenhancedgreenfluorescentprotein(EGFP)–
BandC/C′cellsrelativetofractionAcells,Raptor-deficientfraction MycproteinthatfaithfullymarksendogenousMycexpression(49).
BandC/C′cellsshowedonlymodestup-regulationofIL-7Raexpression In WT cells, EGFP-Myc expression progressively increased from
(fig. S3D). However, Rictor-deficient, Raptor/Rictor-deficient, and fractionAtofractionC′cells,withthehighestexpressioninthemore
mTOR-deficientBcellprecursorsubsetsmaintainedlargelynormal maturefractionC′cells,consistentwithapreviousstudy(Fig.3Jand
IL-7Raexpression(fig.S3,DandE).ExceptforthedifferentialIL- fig.S4B)(48).TheincreasedEGFP-Mycexpressionwasmarkedlyre-
7Raexpression,Cd2icreRptorfl/flRictorfl/flandCd2icreMtorfl/flmiceshare ducedinRaptor-deficientfractionCandC′cells(Fig.3J,leftpanel,and
almostidenticalBcelldevelopmentaldefectsasCd2icreRptorfl/flmice, fig.S4B).IL-7treatmentfurtherinducedEGFP-MycexpressioninWT
asdescribedabove,therebylargelyexcludingthepossibilitythatthe fractionBandC/C′cellsbutnotinfractionAcells(Fig.3J,rightpanel,
reducedIL-7Raexpression inCd2icreRptorfl/flmiceis functionally andfig.S4B),inlinewiththeexpressionofIL-7Rinthesecellsubsets
importantfortheirBcelldevelopmentaldefects. described above. IL-7–induced EGFP-Myc expression was greatly
WethereforeexaminedIL-7–inducedStat5activationandother diminishedintheabsenceofRaptorinfractionCandC′cells(Fig.3J
signalingeventsinpro-Bcells.Raptor-deficientpro-Bcellshadlargely andfig.S4B).Further,whereasthepercentageofEGFP-Myc+cellsin
normalStat5activationinresponsetoIL-7stimulation(Fig.3E).Also, fractionBcellsfromCd2icreRptorfl/flmicewasnotsignificantlyreduced
IL-7–inducedStat5nucleartranslocationwasnotalteredbymTORC1 comparedtothatofWTcells,themeanfluorescenceintensity(MFI)of
inhibition(fig.S3F).Furtherdownstream,theIL-7R–Stat5signaling EGFP-MycinRaptor-deficientcellswassignificantlylowerthanthatin D
o
pathwaydrivestheexpressionofanumberoftranscriptionfactorsthat WTcells(fig.S4C).Thus,althoughIL-7isknowntopromoteMyc w
n
orchestrate B cell lineage differentiation; for example, TCF3 (also transcriptionindevelopingBcells(50),ourresultsidentifyanovel lo
a
knownasE2A),throughactivationofEbf1andPax5,promotesBcell mTORC1-dependent regulation of Myc protein expression down- de
development(2,10,42–44).TheexpressionofTcf3,Ebf1,andPax5in streamofIL-7signaling.Thefunctionalsignificanceofthisinteraction d fro
Raptor-deficientpro-Bcellswaslargelynormal,indicativeofcompara- wasfurtherverifiedbythedrasticallyimpairedBcelldevelopmentin m
bleStat5-dependentfunction(fig.S3G).Incontrast,IL-7inducedpo- hCd2-iCre–mediateddeletionofMycindevelopingBcells(fig.S4,D h
t4eEnBtPm1T(eOuRkCar1yoatcitcivtaratinosnla,taiosninidniictiaatteidonbyfacpthoors4pEh-obriynldatiniognporfotSe6inan1)d, tRoaFp)t.oMrdyecfdiceifeinciceync(fyigb.loSc4kEe)d,wBhcieclhldmevaeylorpemfleecnt,taintapnareta,rtliheersdtiafgfeertehnacne ttp://ad
v
whichwascompletelyabolishedinRaptor-deficientcells(Fig.3E).Sim- betweencompleteandpartialreductionofMycexpressioninthese a
n
ilarly,Raptor/RictordoubledeficiencyandmTORdeficiencydidnot models(Fig.3Jandfig.S4,BandC).Theseresultscollectivelydemon- ce
affectIL-7–inducedStat5phosphorylationbutabolishedmTORC1ac- stratethatmTORC1activityisrequiredforIL-7–inducedMycexpres- s.s
c
tivation(fig.S3,HandI).WefurthertestedtheroleofmTORC1inIL-7 sionbutnotforStat5activationinpro-Bcells. ie
n
signalingviapharmacologicalapproaches.IL-7treatmentofinvitro ce
m
culturedBcellsinducedrapidS6kinase(S6K)andS6phosphorylation, mTORC1 promotes B cell development through a
g
whichwasblockedbyrapamycin.However,rapamycintreatmentdid transcriptional and translational programs .o
notaffectIL-7–mediatedStat5activation(Fig.3F).Together,Raptor WenextdeterminedmTORC1-dependenttranscriptionalprograms. org/
actsdownstreamofIL-7RtomediatemTORC1activationbutnotStat5 Tocircumventpotentialsecondaryeffectsintroducedbychronicdefi- n
signaling,suggestingauniqueprogramorchestratedbyRaptor-mTORC1 ciencyofRaptor,wecrossedRptorfl/flmicewithRosa26-Cre-ERT2(a No
v
inBcelldevelopment. Cre-ERfusiongenewasrecombinedintotheubiquitouslyexpressed e
m
TodirectlyexaminetheroleofIL-7–mTORC1signalinginearly Rosa26locus)micetogenerateCreERRptorfl/flmice,inwhichRptor b
e
Bcelldevelopmentinvivo,weinjectedrecombinantIL-7intomice wasacutelydeletedafterinjectionoftamoxifen.Usingtheacutedeletion r 2
treatedwithorwithoutrapamycin,followedbyanalysisofpro-Bcell system,weperformedfunctionalgenomicstoidentifyIL-7–mediated 8, 2
subsetsafter2daysofIL-7treatment.ExogenousIL-7expandedboth molecularpathwaysthatarecontrolledbymTORC1.Pro-Bcellsfrom 01
fractionCandC′populationsandenhancedCD71expression(Fig.3,G tamoxifen-treatedCreERRptorfl/flandcontrol(CreERRptorfl/+)micewere 8
andH).Strikingly,treatmentofrapamycinnearlycompletelyblockedthe culturedinthepresenceorabsenceofIL-7for24hours,andRNAwas
expansionoffractionC′cellsandCD71up-regulationinducedbyIL-7 subjectedtoglobalgeneexpressionprofiling.Principalcomponents
(Fig.3,GandH).Thus,transientinhibitionofmTORC1blocksIL-7– analysisshowedthatRaptor-deficientpro-Bcellshaddistinctgeneex-
mediatedpro-Bcelldevelopmentalprogression. pressionprofilescomparedtocontrolpro-BcellsandthatIL-7hada
Recent studies establish an important role of IL-7 in B cell de- morepronouncedeffectonthegeneexpressionofcontrolpro-Bcells
velopment in adult humans (45, 46). To test whether IL-7 can ac- thanthatofRaptor-deficientcells(fig.S5A).Specifically,IL-7treatment
tivate human pro-B cells, we sorted BM pro-B cells from healthy ofcontrolpro-Bcellsresultedinatotalof1315up-regulatedand713
donors and stimulated them with IL-7. IL-7 stimulation induced down-regulatedprobes(bygreaterthan0.5log FC).Bycomparison,
2
CD71up-regulationandcellgrowth,indicatingthatIL-7mayalso IL-7treatmentofRaptor-deficientcellsresultedinonly452increased
enhance cell metabolism in human pro-B cells (Fig. 3I). and 213 decreased probes (fig.S5B). Gene setenrichment analysis
The proto-oncogene Myc is essential for B cell development (GSEA)identifiedcellcycle–relatedgenesasoneofthemostdown-
(26,47,48).GiventheconcomitantactivationofmTORC1andMyc regulatedpathwaysinRaptor-deficientcellswithorwithoutIL-7stim-
inresponsetoIL-7stimulation(Fig.1,GandH),weinvestigatedthe ulation(fig.S5C),consistentwiththehighlyreducedEdUincorporation
effectofRaptordeficiencyonMycexpressionindevelopingBcell (Fig.3B)inRaptor-deficientpro-Bcells.Furthermore,althoughIL-7
subsets.MycmRNAwaslowandcomparableinfractionAcellsfrom modestlypromotedtheexpressionofcellcyclegenesinRaptor-
WTandCd2icreRptorfl/flmicebutwaselevatedinfractionBandC/C′cells deficientpro-Bcells,possiblythroughintactStat5signaling,themag-
inRaptor-deficientmicecomparedwithWTcounterparts(fig.S4A). nitudeofactivationwasmuchsmallerthanthatincontrolpro-Bcells
Zengetal.,Sci.Adv.2018;4:eaar5701 31January2018 6of19
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Fig.3. Raptor-mTORC1isessentialtomediateIL-7signalingandMycinductionbutnotStat5activation.(A)ExpressionofCD71onfractionA,B,andC/C′cells
fromWTandCd2icreRptorfl/flmice.NumbersindicatetheMFIofCD71fromarepresentativeexperiment.(BandC)EdUincorporation(at1hourafterintravenous
injectionofEdU)(B)andactivecaspase-3staining(C)infractionA,B,andC/C′cellsfromWTandCd2icreRptorfl/flmice,withthepercentagesofEdU+andactive
caspase-3+cellsindicatedfromarepresentativeexperimentplottedinthegraphs.(D)CellularityofBMcellsafterIL-7stimulationinvitro.TotalBMcellswerecultured
inthepresenceofIL-7(5ng/ml)for1week,andcellnumberswerecountedondays0,4,and7.(E)Immunoblotanalysisofp-Stat5,p-S6,andp-4EBP1infreshlysorted
pro-BcellswithorwithoutIL-7stimulation.(F)Immunoblotanalysisofp-Stat5,p-S6K,andp-S6intheinvitroculturedBcells.Lin−BMcellswereculturedwithOP9
stromalcellswithIL-7for5days,andcellswerewashedandrestedovernightbeforetheywererestimulatedwithIL-7fortheindicatedtimepointsinthepresenceor
absenceofrapamycin.DMSO,dimethylsulfoxide.(G)ExpressionofBP-1andCD24onBMLin−B220+CD43+IgM−cellsfrommiceinjectedwithvehiclecontrol,recom-
binantIL-7(250mg/kg),rapamycin(15mg/kg),orIL-7plusrapamycin,withthepercentagesoffractionA,B,C,andC′cellsindicated.Right:NumberoffractionCandC′
cells.(H)RelativeCD71expressiononfractionCcellsfrommicetreatedin(G).TheMFIofCD71frommicetreatedwithvehiclecontrolwassetas1.(I)HumanBMpro-B
cells(CD3−CD19+CD10+CD34+CD127+)weresortedfromhealthydonors.CellswerestimulatedwithrecombinanthumanIL-7(10ng/ml)for24hours.Expressionof
CD71(left)andcellsize[measuredbyforwardlightscatter(FSC);right]wereanalyzedbyflowcytometry.NumbersinplotsindicatetheMFI.(J)FrequencyofEGFP-Myc+
cellsinfractionA,B,C,andC′cellsfromWT/MycEGFPandCd2icreRptorfl/fl/MycEGFPmiceculturedfor4hoursinmedium(left)orwithIL-7(right).(K)FiveRWPCsduring
pro-BcellactivationweredefinedbyWGCNA.Thecentralrectanglerepresentsthefirsttothirdquartile,withnotchintheplotcorrespondingtothemedianexpression
ofallproteinsinthesample.Endofthewhiskersrepresentsminimumandmaximumlevelofexpressionoftheproteinsinthecluster.(L)Functionalannotationsof
RWPCsbyHallmarkandKEGGdatabases(FDR<0.1).(MandN)Rapamycin-dependentIL-7–inducedtemporalexpressionofproteinsinmTORC1signaling(M)andMyc
targets(N).**P<0.01,***P<0.001[Mann-Whitneytest(J)andone-wayANOVAwithTukey’stest(GandH)].Resultsrepresentthree(AtoC,E,andF)ortwo(D,G,andI)
independentexperimentsorarepooledfromfour(J)ortwo(GandH)independentexperiments.Dataaremeans±SEM.Numbersindicatepercentageofcellsingates.
Seealsofigs.S3toS5anddataS3.
Zengetal.,Sci.Adv.2018;4:eaar5701 31January2018 7of19
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(fig.S5C).Inadditiontothecellcyclepathway,thecholesterolbio- Cd2icreRptorfl/fl mice, had highly reduced B cells in circulation
synthesispathwaywasalsosignificantlydown-regulatedinRaptor- (Fig. 4E). Thus, these data demonstrate that mTORC1 signaling in
deficientcells(fig.S5D). earlyBcellprogenitorsiscritical forMyc-inducedBcell lymphoma.
BecausemTORC1alsocontrolsgeneexpressionatthetranslational Mechanistically,Mycoverexpressionledtoincreasedphosphoryl-
level,asinthecaseofregulatingMycexpression(Fig.3Jandfig.S4,Ato ation of 4EBP1 but reduced phosphorylation of S6 in pro-B cells
C),weprofiledthemTORC1-dependentproteomeinIL-7–stimulated (Fig.4F).BecauseIL-7–mediatedMycup-regulationwasdependent
pro-Bcells.Theproteomeoffreshlyisolated(0hour),IL-7–stimulated onmTORC1(Fig.3G),wetestedwhetherMycoverexpressionin
(16hours),orIL-7/rapamycin–treatedpro-Bcellswasanalyzedusing Em-MycmicewasalsodependentonmTORC1.Mycproteinexpression
multiplexedTMTandLC/LC-MS/MSproteomicprofiling.Variance inCd2icreRptorfl/flEm-Mycpro-BcellswasalmostaslowasthatinWT
analysisrevealedconsistencybetweenreplicates(fig.S5E).Weiden- cells,incontrasttothemassiveMycexpressionfoundinEm-Myccells
tifiedthat455DEproteins(dataS3A)significantlychangedusingone- (Fig.4G).VariousmTORinhibitorsalsoreducedMycexpressionin
wayANOVA(10%FDR,zscoreofatleast2betweenIL-7andIL-7/ Burkitt’s lymphoma cells (fig. S6G). In contrast, Myc mRNA level
rapamycintreatmentgroups).WeappliedWGCNAfortheseproteins wassimilarbetweenEm-MycandCd2icreRptorfl/flEm-Mycpro-Bcells
andidentifiedfiveclustersofcoexpressedproteins,namedasrapamycin- (fig.S6H),suggestingthatmTORC1regulatesMycproteinexpression
dependentwholeproteinclustersRWPC1toRWPC5(Fig.3Kanddata inEm-Mycmice.Furthermore,Mycproteinstabilitywasnotaffectedby
S3B).RWPC1andRWPC2,thetwolargestclusters,showedup-regulation mTORC1inhibition(fig.S6I).TodirectlyexamineMycproteinsynthe-
uponIL-7treatmentbutpartialandmorecompletesuppressionwith sis,weusedstochasticopticalreconstructionmicroscopy(STORM)to
rapamycin,respectively.RWPC4alsoshowedrapamycin-mediated imagethenascentMycproteinbindingtopolyribosomesinIL-7– D
o
suppressiondespitethelackofstrongeffectsinducedbyIL-7,whereas stimulatedpro-Bcells(Fig.4Handfig.S6J).Wefoundasignificant w
n
RWPC3 and RWPC5 revealed rapamycin-mediated activation. reductionofMycproteinbindingtopolyribosomesinRaptor-deficient lo
a
PathwayenrichmentanalysisrevealedthatRWPC1andRWPC2were pro-Bcells(Fig.4H),indicatingthatmTORC1activityisrequiredfor de
d
enrichedwithMycandmTORC1signalingtargets,aswellasdiffer- efficientMycproteinsynthesis. fro
entorganellecomponentsandmultiplelipidbiosynthesispathways Translationinitiationisamajorstepforproteinsynthesisandan m
(Fig.3L).TheinhibitoryeffectsofrapamycinonIL-7–inducedmTORC1 important target for cancer therapies (53). Eukaryotic translation h
amnadpMs(yFciga.ct3iv,aMtioannadnNd,liapniddbfiigo.sySn5t,hFesaisnwdeGre).ilTluhsetrsaeteddatinadtheemhoena-t ienIFit4iaAt,iotnheccoampp-bleinxd4inFg(esuIFb4uFn)itiseIaF4teEr,naanrdytchoemspclaefxfocldoimngpopsreodteoinf ttp://ad
stratethatIL-7–mediatedmultiplemetabolicprogramsmaydepend eIF4G.ThebindingofeIF4EtoeIF4Giscontrolledby4EBP1,whose va
n
uponmTORC1signalingandprovideadditionalvalidationsofthe phosphorylationand degradationaremediated bymTORC1(53). ce
s
conclusionsfromFig.1aswellasourextensivebiochemicalandge- HowmTORC1andMyccoordinateeIF4Factivityispoorlydefined .s
c
neticanalyses. (54).WefoundthatMycoverexpressionledtoincreasedphospho- ie
n
rylationofeIF4EbutdidnotsignificantlyaffecteIF4Gphosphoryl- ce
m
Deficiency of mTORC1 protects mice from Myc-induced ation(Fig.4G).Incontrast,thephosphorylationofbotheIF4Eand a
B cell lymphoma eIF4GwasabolishedinCd2icreRptorfl/flandCd2icreRptorfl/flEm-Myc g.o
Mycisoneofthemoststudiedproto-oncogenes,whereasaberrantup- pro-Bcells(Fig.4G)ormarkedlysuppressedbymTORinhibition org/
regulation of mTORC1 activity is also observed in multiple types of (fig. S6G),suggestingthatmTORC1activityisrequiredforeIF4G n
tumors including B cell malignancies (51). To investigate how phosphorylationandMycoverexpression–inducedeIF4Ephosphoryl- No
v
mTORC1regulatesBcelltumorigenesisandfurtherexploretheinter- ation.Together,MycoverexpressionpromotesBcelltransformation e
m
play between mTORC1 and Myc, we bred Cd2icreRptorfl/fl with Em- through a Myc-mTORC1 feed-forward circuitry, which can be b
e
Myc transgenic mice, which develop B cell lymphoma induced by interruptedthroughmTORC1inhibition(fig.S6K). r 2
8
overexpression of Myc via the IgH enhancer (Em) (25). Strikingly, , 2
RaptordeficiencycompletelysuppressedMyc-inducedlymphomagen- Pro-B cell development requires active suppression of PI3K 01
8
esisandearlydeath(Fig.4A).LymphomasarisingfromEm-Mycmice PI3K-Aktisawell-establishedactivatorofmTORinmanycelltypes
areatvariousstagesofBcelldevelopment(52).IntheBM,B220+cells (19).However,deletionofp110aandp110d(16),orp85a(17),does
from mostpre-tumorEm-Mycmiceshowedacontinuous expression notaffectpro-Bcelldevelopment.Moreover,weobservedelevatedex-
ofCD43,withanincreasedfrequencyofB220+CD43+IgM−Bcellpre- pressionofPTEN,acrucialnegativeregulatorofPI3K,inpro-Bcells
cursors,whereasBcelldevelopmentinCd2icreRptorfl/flEm-Mycmicewas andpre-Bcellsthaninothersubsets(Fig.5A),whichcorrelatedwith
largelyarrestedatthisprecursorstage(Fig.4Bandfig.S6A).Withinthe increasedIL-7Ra expression(fig.S1F).Totestthefunctionalimpor-
B cell precursor compartment, cells from Cd2icreRptorfl/flEm-Myc mice tanceofPI3Kregulation,wedeletedPtenusingthehCd2-iCresystem.
showed aphenotype similar to that ofCd2icreRptorfl/fl mice, with high Cd2icrePtenfl/flmicecontainedanormalnumberofB220+CD43+IgM−
expression of BP-1 and an expanded fraction C/C′ cell popula- cells(Fig.5B),includingnormalnumbersoffractionAandfraction
tion, whereas B cells from Em-Myc mice were either BP-1+CD24+ B cells, but markedly reduced fraction C/C′ cells (Fig. 5C). Further,
or BP-1−CD24+ (fig. S6, B and C). Em-Myc mice had reduced im- B220+CD25+ pre-B cell percentage and number were decreasedsub-
matureandcirculatingmatureBcells,whichwasfurtherexacerbated stantiallyintheBMofCd2icrePtenfl/flmice(Fig.5D).Inaddition,BM
in Cd2icreRptorfl/flEm-Myc mice (Fig. 4C and fig. S6D). Myc overex- immatureandcirculatingmatureBcells(fig.S7A)wereallprofoundly
pressiondroveincreasedDNAsynthesis(measuredbyEdUincor- lostintheabsenceofPTEN.ThesedatashowthatdeficiencyofPTEN
poration; fig. S6E), anabolic metabolism (indicated by enhanced selectivelyblocksB cell development.
CD71 and CD98 expression), and cell growth (measured by FSC BecausePTENhasnuclearfunctionsindependentofPI3K-Akt
profile; Fig. 4D and fig. S6F), which were all largely rectified by (55,56), weusedhCd2-iCre toectopicallyexpress aconstitutively
Raptor deficiency. Consequently, Cd2icreRptorfl/flEm-Myc mice, like activeformofthePI3Kcatalyticsubunitp110a(encodedbyPik3ca)
Zengetal.,Sci.Adv.2018;4:eaar5701 31January2018 8of19
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Fig.4. LossofRaptorpreventsMyc-inducedBcellmalignancy.(A)SurvivalcurveofEm-Myc,Cd2icreRptorfl/+Em-Myc,andCd2icreRptorfl/flEm-Mycmice(P=0.0005,log- 18
ranktest).(BandC)RepresentativeflowcytometryplotsofBMcellsofWT,Em-Myc,Cd2icreRptorfl/fl,andCd2icreRptorfl/flEm-Mycmice.(B)ExpressionofB220andCD43on
BMlymphocytes,withthepercentagesofB220+CD43−andB220+CD43+cellsindicated.(C)ExpressionofB220andIgMonBMlymphocytes,withthepercentagesof
pro-B/pre-Bcells(B220+IgM−),immatureBcells(B220+IgM+),andcirculatingmatureBcells(B220hiIgM+)indicated.(D)AnalysisofCD71andCD98expressionandcell
size(viaFSC)ofpro-Bcells.NumbersindicatetheMFI.(E)FrequencyofCD19+BcellsinthebloodofWT,Em-Myc,Cd2icreRptorfl/fl,andCd2icreRptorfl/flEm-Mycmice.(F)Immunoblot
analysisofp-S6andp-4EBP1inpro-BcellsfromWT,Em-Myc,Cd2icreRptorfl/fl,andCd2icreRptorfl/flEm-Mycmice.(G)ImmunoblotanalysisofMycexpressionandphosphorylationof
eIF4GandeIF4Einpro-BcellsfromWT,Em-Myc,Cd2icreRptorfl/fl,andCd2icreRptorfl/flEm-Mycmice.(H)3DrenderingofSTORMimagingofpolyribosomes(red)andMyc(green)in
pro-BcellstreatedwithIL-7.Left:Representativeimagesofunoccupied(top)andMyc-occupiedpolyribosome(bottom).Right:ThepercentageofMyc-occupiedpolyribosomes
ineachimagedcellwasenumerated.Datawerepooledfromthreeindependentexperiments,withatotalof35cellsfromWTand45cellsfromCd2icreRptorfl/flmiceexamined.
***P<0.001,****P<0.0001[one-wayANOVAwithTukey’stest(E)andMann-Whitneytest(H)].Resultsrepresentfour(BandC),three(D,F,andH),ortwo(EandG)independent
experiments.Dataaremeans±SEM.Numbersindicatepercentageofcellsingates.Seealsofig.S6.
(57)indevelopingBcells(referredtoasPik3ca*).Bcelldevelopment PI3K and mTORC1 independently control
wasblockedatthepro-Bcellstage,withsubstantiallossoffraction B cell development
C/C′ cells in Cd2icrePik3ca* mice (fig. S7B), and few pre-B cells Theaboveobservationswereincongruentwiththeconventionalmodel
emergedfromCd2icrePik3ca*mice(fig.S7C).Therefore,uncontrolled inwhichPI3K-AktfunctionsupstreamtoactivatemTORC1(19);for
PI3Kinpro-BcellsblocksearlyBcelldevelopment,highlightingthe example,mTORC1isrequiredformediatingPTEN-deficientpheno-
importanceofactivesuppressionofPI3Kinpro-Bcells. typesinothercontextsincludinghematopoieticstemcells(58).To
Zengetal.,Sci.Adv.2018;4:eaar5701 31January2018 9of19
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Fig.5. DeletionofPTENinhibitsBcelldevelopmentindependentofmTORC1.(A)ImmunoblotanalysisofPTENinfractionAcells,pro-Bcells,pre-Bcells,and
immatureBcellswithorwithoutIL-7stimulationfor4hours.(BtoD)Left:RepresentativeflowcytometryplotsofBMcellsofWTandCd2icrePtenfl/flmice.Right:Absolute
numberofindicatedpopulations.(B)ExpressionofB220andCD43onBMlymphocytes,withthepercentageofB220+CD43+cellsindicated.Right:NumberofB220+CD43+IgM−
Bcellprecursors.(C)ExpressionofBP-1andCD24onBcellprecursors,withthepercentagesoffractionA(CD24−BP-1−),fractionB(CD24+BP-1−),andfractionC/C′(CD24+BP-1+)
cellsindicated.Right:Numberofeachsubset.(D)ExpressionofB220andCD25onBMlymphocytes,withthepercentageofB220+CD25+pre-Bcellsindicated.Right:Numberof
B220+CD25+pre-Bcells.(EtoH)RepresentativeflowcytometryplotsofBMcellsofWT,Cd2icrePtenfl/fl,Cd2icreRptorfl/fl,andCd2icrePtenfl/flRptorfl/flmice.(E)ExpressionofB220and
CD43onBMlymphocytes,withthepercentageofB220+CD43+IgM−cellsindicated.Right:NumberofB220+CD43+IgM−cells.(F)Hardyclassificationofpro-Bcellsubsetsinthe
B220+CD43+IgM−gate[from(B)],withthepercentagesoffractionA(CD24−BP-1−),fractionB(CD24+BP-1−),andfractionC/C′(CD24+BP-1+)cellsindicated.Bottom:Numbersof
fractionA,B,andC/C′cells.(G)ExpressionofB220andCD25onBMlymphocytes,withthepercentageofB220+CD25+pre-Bcellsindicated.Right:NumberofB220+CD25+pre-B
cells.(H)ExpressionofB220andIgMonBMlymphocytes,withthepercentageofpro-B/pre-Bcells(B220+IgM−),immatureBcells(B220+IgM+),andmatureBcells(B220hiIgM+)
indicated.Right:NumberofimmatureBcells.*P<0.05,**P<0.01,****P<0.0001(one-wayANOVAwithTukey’stest).Resultsrepresentatleastfourindependentexperiments.
Dataaremeans±SEM.Numbersindicatepercentageofcellsingates.Seealsofig.S7.
Zengetal.,Sci.Adv.2018;4:eaar5701 31January2018 10of19