EMBO MolecularMedicine PeerReview ProcessFile- EMM-2013-02524 © EMBO 1 Activinreceptor-like kinase5 inhibition suppresses mouse melanomabyubiquitindegradationofSmad4,thereby derepressing Eomesodermin in cytotoxic T lymphocytes Jeong-HwanYoon,SuMyungJung,SeokHeePark,MitsuyasuKato,TadashiYamashita,In-Kyu Lee,KatsukoSudo,SusumuNakae,JinSooHan,Ok-HeeKim,Byung-ChulOh,TakayukiSumida, MasahikoKuroda,Ji-HyeonJu,KyeongCheonJung,SeongHoePark,Dae-KeeKim,andMizuko Mamura Correspondingauthor:MizukoMamura, TokyoMedicalUniversity Review timeline: Submissiondate: 24 January 2013 EditorialDecision: 28 February 2013 Revisionreceived: 19 July 2013 EditorialDecision: 31 July 2013 Revisionreceived: 20 August2013 EditorialDecision: 23 August2013 Revisionreceived: 25 August2013 Accepted: 06 September2013 TransactionReport: (Note:With the exception ofthe correction oftypographicalor spelling errors thatcould be a source ofambiguity, letters and reportsare notedited.The originalformatting oflettersand referee reportsmaynotbe reflected in this compilation.) Editors: CélineCarret/ Roberto Buccione 1stEditorialDecision 28 February 2013 ThankyouforthesubmissionofyourmanuscripttoEMBO MolecularMedicine.Wehavenow heard back from thetwo refereeswhom weasked to evaluateyourmanuscript.Although the referees find the study to be of potentialinterest,they also raise a number of concerns thatneed to beaddressed in amajorrevision ofyourmanuscript. Asyouwillseefrom thereportsbelow,concernsfrom bothrefereesaremainlyofanexperimental nature,butthey do insistthatbetterquality and morecontrolled assaysshould beperformed.In addition,mechanistic insightshould be provided.Referee 2 would also like to see a better integration of the results within the accepted literature. In our view the suggested revisions would render the manuscriptmuch more compellingand interesting to a broad readership. We therefore hope that you will be prepared to undertake the recommended experimentalrevision. PleasenotethatitisEMBO MolecularMedicinepolicytoallow asingleroundofrevisiononlyand that, therefore, acceptance orrejection ofthe manuscriptwilldepend on the completenessofyour responses included in the next,finalversion of the manuscript.EMBO MolecularMedicine PeerReview ProcessFile- EMM-2013-02524 © EMBO 2 Asyouknow,EMBO MolecularMedicinehasa"scoopingprotection"policy,wherebysimilar findings thatare published by others during review or revision are not a criterion for rejection. However,Idoaskyoutogetintouchwithusafterthreemonthsifyouhavenotcompletedyour revision,to update us on the status.Please also contactus as soon as possible if similarwork is published elsewhere. I look forward to receiving your revised manuscript. ***** Reviewer'scomments***** Referee#1(CommentsonNovelty/ModelSystem): Thisisahighlyinterestingpaper,howeversometechniquesappearnotproperly controlled. These results need to be validated and consolidated. Referee#1(Remarks): In this reportinhibition of TGF-betatypeIreceptorkinaseactivity isshown to suppressmouseB16 melanomaprogressionbyinducingthedegradationofSmad4inCD8+T-cells.Consistentwith this finding,T-cellspecific deletion ofSmad4 inhibitsmelanoma progression.Furthermore,T-box transcription factor Eomesodermin was identified as specific target that is repressed by TGF-betain a Smad3/4-dependentmannerin CD8+ cells. In generalthe experiments are wellperformed and conclusions are supported by data. Specificcomments: 1.Poximity ligation (PLA)experimentsareshown in Fig.2 and 3.Moreexperimentaldetailis needed.Atpresentitsunclearhow these experiments were performed and controlled.Were no signals obtained when one antibody was added? DifferentialPSmad2 PLA data (and no effecton total level of Smad2 and Smad3) need to be consolidated with Western blot analysis. In Figure 3a:When positive signals are obtained with Smad4 antibody and ubiquitin antibody, this doesnot(necessarily)mean thatSmad4 isubiquitinated.PLA only demonstratesthattheproteins are in close proximity.Isthere a PLA signalforSmad2 and ubiquitin? 2.TheALK5inhibition induced ubiquitination of Smad4 as shown in Fig. 3B data do not look convincing.Smad4 ubiquitination laddercan notbe seen.Mostofthe Ub-Smad4havealower molecularweightthanSmad4? Itis unclear how this assay was performed.Itneeds to bedoneunderdenaturing conditions: otherwiseno distinction can bemadebetween Smad4 proteinsthathavebeen covalently modified by ubiquitin and protentsthatinteractwith Smad4 (noncovalently)thatareubiquitinated.Thisassay needsto berepeated and performed in a differentmanner. 3.Themechanism forselectiveSmad4 degradation in CD8 cellsisunclear.Theauthorscould investigate the possible involvement of Smurf. 4.IsEomeprotein differentially expressed in CD8 cellsin vivo upon Smad4 deletion in CD8+ T cellsorupon ALK5 inhibition? Referee#2(CommentsonNovelty/ModelSystem): ThemodelchosenisfinebutwouldhavebeenimprovedbyanalysisofTIL ratherthanLN cells, purification ofspecificimmunesubsetsby flow cytometry priorto geneexpression analysis,and moredetailedproofthattheanti-tumor effects are fully due to the impact on CD8 T-cells. EMBO MolecularMedicine PeerReview ProcessFile- EMM-2013-02524 © EMBO 3 Referee#2(Remarks): Themanuscript"Activinreceptor-like kinase5 inhibition suppresses mouse melanoma by ubiquitin degradation ofSmad4,thereby derepressing Eomesodermin in cytotoxic T lymphocytes" detailsa complex mechanism by which ALK5 inhibition blockselementsofTGF-b signalling leading to improved anti-tumor CTL responses. Overallthisisagoodmanuscriptworthy of publication;however,there are few items of concern generally: 1)A moreconcretedemonstration thattheeffectsoftheinhibitorson restricting tumorgrowth are fully attributable to theireffects on the CD8 T-cellcompartmentare needed.Minimally, immunocompromised (RAG-KO,Nudeetc)micebearingtumorscouldbetreatedshowingno change in tumorgrowth.An idealexperimentmightbe to antibody deplete CD8,CD4,NK1.1 cells and show in each case how much ofthe efficacy ofthe inhibitorsislost. 2)Theauthorsneed to discriminatebetween increasesin cytotoxicort-box transcription factorsat the gene expression level in the overall node vs. on a per cell basis. In a few cases they insinuate these have increased "in CD8 cells in the dLN" when they have shown thatthe numbersofCD8 cellsin the node have been increased substantially - thus we don't know if the higher Granzyme RNA (forexample)isduetomoreCD8cells,ahigherpercellexpressionofGranzyme,orboth. Theflow plotsgenerally following the RNA analysis are helpful and show changes on a per cell basis.In thefuture,theauthorsmay wish to sortpurepopulationsofCD8 cellsfrom thedLN for geneexpression analysiswhich would allow them to discriminatebetween changesin per cell expression levelvs.changesin the density ofCD8 cellsin the overallLN.We hope the authorsin the future will also consider analyzing TIL instead of dLN cells as the results can often be quite distinct. 3)Theauthorsassertthatdegradation of Smad4 leads to de-repression of Eomesodermin;however, Ichiyama and Yoshimura (2011) published thatTGF-b mediated repression ofEomeswasthrough a JNK driven,Smad-independent (at least Smad2 and 3 independent) pathway. The authors need to morethoroughly describehow theirdataintegrateswith thepublished pathway. 4)Lack ofinduction ofIFN-g in concertwith Eomesinduction isunusualgiven thatEomes (+Runx3) drives IFN-g expression moreso than itdoesGranzymeB.Ibelievethelack ofIFN-g induction seen by the authors in Figure 6 and elsewhere may be due to their activation of their T- cellsin vitro using aCD3/aCD28 beads.These beadsare the bestreagentsformeasuring proliferativechanges;however,PMA/Ionomycin stimulation isgenerally superior for studying in vitro IFN-g production. MinorSpecifics: In the abstractthe sentence starting with "Notably,progression....melanoma-bearing mice"isarun- on and unclearand should bere-worked. CallingEomes"theessentialT-box transcription factorforCTL functions" in the abstractis overstated given thatIntelkoferand Reinershowed thatEomes-/- CTLarefullyfunctional. In the introduction "heteromeric" is a notword.Perhaps heterodimeric? Itwould be nice to hear atleasta speculative explanation forwhy Alk5 inhibition leadsto Smad4 downregulation in CD8 T-cellbutnotCD4 T-cellsorB16 tumorcellsasthismechanism iscritical to the postulated effect of the inhibitors. 1stRevision - authors'response 19 July 2013 Wewouldliketo thank the editor and referees for their precious comments. We described the details oftheexperimentalproceduresand performed thesuggested experiments.Wehaveincluded the commentsby the refereesin bold,which are followed by ourresponse. Referee 1. Instructive comments and suggestions by the referee led us to elaborate on the detection of endogenousubiquitinated Smad4 in CD8+ T cells. EMBO MolecularMedicine PeerReview ProcessFile- EMM-2013-02524 © EMBO 4 1.Poximityligation (PLA)experimentsareshown in Fig.2 and 3.Moreexperimentaldetailis needed.Atpresentitsunclearhow these experimentswere performed and controlled.Were no signals obtained when one antibody was added? WeclarifiedtheprotocolofPLAbydetailedexplanationinMaterialsandMethods, Immunocytochemistry,page 20-21 in therevised version.Weperformed singlerecognition assay using DuolinkIIFluorescencekit(OLINK)withoneprimaryantibodycorrespondingtoeach protein (rabbitanti-Smad2,rabbitanti-Smad3,rabbitanti-phospho-Smad2,rabbitanti-phospho- Smad3,andrabbitanti-Smad4)andthe secondary anti-rabbitantibodies conjugated with oligonucleotides(PLA probe anti-rabbitPLUS and PLA probe anti-rabbitMINUS)to clearly detect and quantify the low level-endogenousproteinsin primary lymph node cellsfreshly isolated from the melanoma-bearing mice.Weusedtwodifferentprimaryantibodiesraisedfrom differentspecies againsteach protein,the secondary anti-rabbitantibody and the secondary anti-mouseantibody conjugated with oligonucleotides(PLA probe anti-rabbitPLUS and PLA probe anti-mouseMINUS) to detecttheinteraction oftwo proteins(rabbitanti-Smad2and mouseanti-ubiquitin,rabbitanti- Smad3andmouseanti-ubiquitin,rabbitanti-Smad4andmouseanti-ubiquitin,mouseanti-Smad2/3 and rabbitanti-Smad4).CD8 was stained with conventionalstaining method using primary anti- CD8antibodyandsecondary Alexa Fluor488 conjugated anti-ratIgG. DifferentialPSmad2PLAdata(andnoeffectontotallevelofSmad2andSmad3)needtobe consolidated with Western blotanalysis. WesternblotanalysisconfirmedthePLAdatashowingtheinhibitoryeffectofEW-7197 on phosphorylation ofSmad2/3 withoutaffecting totallevelofSmad2/3 in lymph nodesorin CD8+ T cellsin melanoma-bearing mice(Fig 2G in therevised version,2 independently pooled samples/group). In Figure 3a: When positive signals are obtained with Smad4 antibodyand ubiquitin antibody,this doesnot(necessarily)mean thatSmad4 isubiquitinated.PLA onlydemonstratesthattheproteins arein closeproximity.Istherea PLA signalforSmad2 and ubiquitin? PLA didnotdetectthesignals(closeproximity<40nm)forinteractionbetweenSmad2and ubiquitin orinteraction between Smad3 and ubiquitin (Supporting Information Fig S7 in therevised version). 2.TheALK5 inhibition induced ubiquitination ofSmad4 asshown in Fig.3B data do notlook convincing.Smad4 ubiquitination laddercannotbeseen.MostoftheUb-Smad4 havea lower molecularweightthanSmad4? Itis unclear how this assay was performed.Itneeds to be done under denaturing conditions: otherwise no distinction can be made between Smad4 proteins thathave been covalently modified by ubiquitin and protentsthatinteractwith Smad4 (non-covalently)thatare ubiquitinated.Thisassay needsto berepeated and performed in a differentmanner. Weclarified the protocolto detectlow-level endogenous ubiquitinated Smad4 in the limited amountofsamplesfrom the draining lymph node CD8+ cellsofmelanoma-bearing miceusing an UbiQapture-Q kit(EnzoLifeSciences)(Materials and Methods,page 22 in therevised version).We repeated the experimentto clearly show the Ub-Smad4withhighermolecularweightthanSmad4 (Fig 3B in the revised version).Because only the limited number of CD8+ cellscould be obtained from dLNs (<106 CD8+ cells/mouse,5-7 micewerepooled foronesample),wecould notperform otherassays.TheUbiQapturekitrelieson aproprietary peptidethatmimicsaUBA (ubiquitin associating)domain from anotherprotein bound to the resin,according to the manufacturer.In theory, it is possible that the UbiQapture matrix might pull down also proteins that interact with a ubiquitinated proteinsbound to it,astherefereepointed out,butiftheseco-pulled down proteinsare notcovalently ubiquitinated,they willnotshow up in the WB analysis. Toaddressthereferee’scomment,weperformedimmunoprecipitationwithanti-Smad4antibodyto detectubiquitinated Smad4 underdenaturing condition using primary mouseCD8+ cells(2×107 CD8+ cells/sample)stimulated in vitro in thepresenceorabsenceofEW-7197 orMG132 (Fig 3D, MaterialsandMethodsinpage22-23 in therevised version).Wedissociated non-covalentprotein interactions with 1% SDS and boiling for10 min forthis assay as following the referee’s comment. EMBO MolecularMedicine PeerReview ProcessFile- EMM-2013-02524 © EMBO 5 3.Themechanism forselectiveSmad4 degradation in CD8 cellsisunclear.Theauthorscould investigate the possible involvement of Smurf. In addition to the discussion of possible involvementof Smurf and other E3 ligases in page 12 in the first version, we performed theexperimentsto knockdown Smurf1 and/orSmurf2 by shRNA in CD8+ cellsasfollowing the referee’ssuggestion.We found thatknockdown ofSmurf1/2 alone orin combination did notaffectdown-regulation of Smad4 protein by EW-7197 (SupportingInformation FigS8intherevisedversion).We confirmed Smurf1/2 knockdown by quantitative RT-PCR. WesternblotwiththeantibodiesagainstSmurf1orSmurf2(SantaCruz)failedtodetectthe endogenousproteinsin mouse primary CD8+ T cells(datanotshown). Irrelevance of Smurf1/2 in ALK5inhibition-induced ubiquitin-mediateddegradationofSmad4wasdescribedinpage8inthe resultsection and in page 14 in the discussion section in the revised version. 4.IsEomeprotein differentiallyexpressed in CD8 cellsin vivo upon Smad4 deletion in CD8+ T cellsorupon ALK5 inhibition? In addition to the data showing the normalT cellhomeostasis in vivo upon T cell-specific Smad4 deletion orupon ALK5 inhibition in SPF environmentin Fig 4 in thefirstversion, weconfirmedthe low expression of Eomes protein in CD8+ T cellsaswellasnormalhomeostasisofimmunecellsin 16 week-old Cd4Cre;Smad4+/+, Cd4Cre;Smad4+/fl, and Cd4Cre;Smad4fl/fl miceorinC57BL/6mice treated with gastric juice or EW-7197 for8 weeksin the absence of melanoma challenge (Fig 7 and in page 10-11 in therevised version). Referee2. Wedeeplyappreciatethereviewer’sinstructivecomments.Weperformedallthesuggested experiments,which significantly improved ourmanuscript. 1)A moreconcretedemonstration thattheeffectsoftheinhibitorson restricting tumorgrowth are fully attributable to their effects on the CD8 T-cellcompartmentare needed.Minimally, immunocompromised (RAG-KO,Nudeetc)micebearingtumorscouldbetreatedshowingno change in tumorgrowth.An idealexperimentmightbe to antibody deplete CD8,CD4,NK1.1 cells and show in each casehow much oftheefficacyoftheinhibitorsislost. In addition to the discussion on CD8+ T-cellcompartmentasthe main targetofTGF-β antagonism withthepreviousreportsinthereferences(Donkoretal,2011;Goreliketal,2001;Nam etal,2008; Zhangetal,2005),weperformedtheprimarilyrecommended experiments to delete CD8+, CD4+ or NK cellsbyanti-CD8,anti-CD4,oranti-asialo GM1 antibody,respectively (deletion ofspecific cell compartmentwasconfirmed in Fig 6F and SupportingInformationFig13). As shown in Fig 6A, 6B,and 6E and page10 in therevised version,weconfirmed thattheanti-melanomaeffectofthe ALK5inhibitorwascompletelyabolishedbydeletionofCD8+ cells. EW-7197 showed significant anti-tumor efficacy in CD4+-deleted orNK-deleted mice(Fig 6C and 6D).Effectof EW-7197 on tumor growth and CD8+ T cellexpansionwasslightlyreducedinNK-deleted mice(Fig.6D and 6F). TheefficacyofEW-7197 following each antibody treatmentwascalculated asa% ofthemaximum therapeutic effect observed in the group treated with control IgG (Fig 6E). These data are consistent withthepreviousreportbyNam etal,2008,showingthattheefficacyofneutralizinganti-TGF-β antibody on a 4T1 mammary tumormodelmainly dependson CD8+ T cells. 2)Theauthorsneed to discriminate between increasesin cytotoxicort-boxtranscription factorsat the gene expression level in the overall node vs. on a per cell basis. In a few cases they insinuate these have increased "in CD8 cells in the dLN" when they have shown that the numbers of CD8cells in the node have been increased substantially - thus we don't know if the higher Granzyme RNA (for example)isdue to more CD8 cells,a higherpercellexpression ofGranzyme,orboth.The flow EMBO MolecularMedicine PeerReview ProcessFile- EMM-2013-02524 © EMBO 6 plotsgenerallyfollowing theRNA analysisarehelpful and show changes on a per cell basis. In the future, the authors may wish to sort pure populations of CD8 cells from the dLN for gene expression analysiswhich would allow them to discriminatebetween changesin percellexpression levelvs. changesin thedensityofCD8 cellsin theoverallLN. WeconfirmedthatincreasesinEomesandcytolyticmoleculesatthegeneexpressionlevelwere both in theoverallnodeand on apercellbasis.Wesorted outCD8+ cellsfrom the dLNsto repeat quantitativeRT-PCR (Fig1E,4E,and5C).GeneexpressionpatternsinsortedCD8+ dLN cellswere similarto those in the whole dLNs with 5- to 10-fold increases in the expression levels due to the enrichmentofCD8+ cells(Supporting Information Fig 3D,3E,9C,and 10D).Because ofthe limited space,we show the overlay histograms in Fig 5A and show the dotplots in Supporting Information FigS10A andB.Theseresultsobtainedfrom quantitativeRT-PCR anddotplotsshow thatboth mRNAandproteinlevelsofEomesandcytolytic molecules in CD8+ cellswere increased on a per cellbasis.Thus,increase in Eomesand cytolytic moleculesatthe mRNA expression levelin the wholedLNswasduetothechangesin both percellexpression leveland the density ofCD8+ cells (Fig 1C and 4C). WehopetheauthorsinthefuturewillalsoconsideranalyzingTILinsteadofdLNcellsastheresults can often be quite distinct. WeinvestigatedTILsasfollowingthereferee’sadvice.TILswereremarkablyincreasedin EW7197-treated or Cd4Cre;Smad4fl/fl mice,whichwerebarelyobservedinthevehicle-treated controlorwild type controlCd4Cre;Smad4+/+ mice(SupportingInformationFigS3F andS9D in the revised version, more than 1,000,000 events/sample acquired).Consistentwith the immunohistochemistry detecting themelanoma-infiltrating CD8+ T cellsonlyinEW7197-treated or Cd4Cre;Smad4fl/fl mice(Fig1H,4H,and 5D in the firstversion,Fig 1I,4I,and 5E in the revised version),CD8+ T cellswerenotdetectedinTILsisolatedfrom thevehicle-treated control or wild type control Cd4Cre;Smad4+/+ mice, whereas significant CD8+ T cellsexpressinghighlevelsof EomeswerepresentinTILsisolatedfrom EW7197-treated or Cd4Cre;Smad4fl/fl mice(Fig1H,4H, and 5D).Proportionsofthe immune cellsubsets in TILs showed no significantdifferences among the groups (Supporting Information Fig S12), although absolute numbers of TILs were different. 3)Theauthorsassertthatdegradation ofSmad4 leadsto de-repression ofEomesodermin;however, Ichiyama and Yoshimura (2011) published thatTGF-b mediated repression ofEomeswasthrough a JNK driven,Smad-independent (at least Smad2 and 3 independent) pathway.Theauthorsneed to morethoroughlydescribehowtheirdataintegrateswiththepublishedpathway. WeaddedmoredetaileddiscussiononthereportbyIchiyamaandYoshimura(2011)inpage15in the revised version:“Bycontrast,ithasbeenreported that TGF-β suppressesEomesviaSmad2/3- independent, JNK-dependentsignallingin Th17 induction (Takimoto et al, 2010; Ichiyama etal, 2011).Discrepancy between their reports and our study mightbe due to severalreasons:TGF-β signallingpathwaysto suppress Eomes mightbe differentbetween CD4+ and CD8+ T celleffector subsets,Smad4 was notinvestigated in theirreports,they used T cells from LckCreSmad2fl/flSmad3-/- (Smad2/3-DKO)orLckCreSmad2fl/flSmad3+/- (Smad2cKO/Smad3hetero) mice,so thatSmad4 alone orSmad4 and haploid expression ofSmad3 could stilltransduceTGF-β signallingto repress the Eomes gene according to ourfindings (Fig 9A and B).They speculated JNK-dependent,Smad2/3- independent pathway from the similar attenuating effect of ALK5inhibitor,SB431542andJNK inhibitor, SP600125 on Eomes repression in T cells stimulated with TCR and TGF-β.However, specificity ofALK5 inhibitors forSmad-mediatedTGF-β signallingpathway (Akhurst& Hata, 2012;Flavelletal,2010;Hawinkelsetal, 2011; Jin etal,2011)and cooperation ofSmad3 and Smad4withc-Jun/c-FostomediateTGF-β-induced transcription (Zhang et al, 1998) suggest that both Smad3/4 and JNK pathwaysareinvolved in TGF-β-induced Eomes suppression.” If TGF-β ligand antagonists,such as neutralizing anti-TGF-β antibodiesand soluble TGF-β II receptor,which block allthe intracellular TGF-β signallingpathwaysshowed thesameattenuating effectwith JNK inhibitoron Eomessuppression,whereasALK5 inhibitors,which specifically block Smadpathwayfailedtoshow suchanattenuatingeffect,onecouldconcludethatTGF-β suppresses EomesviaJNK-dependent, Smad-independent signalling. The report by Zhang et al is the important reference to show the mergence of Smad pathway and the downstream of JNK. EMBO MolecularMedicine PeerReview ProcessFile- EMM-2013-02524 © EMBO 7 4)Lackofinduction ofIFN-g in concertwith Eomesinduction isunusualgiven thatEomes (+Runx3) drives IFN-g expression more so than itdoes Granzyme B.Ibelieve the lack ofIFN-g induction seen by the authors in Figure 6 and elsewhere may be due to the activation of the T-cells in vitro using aCD3/aCD28 beads. These beads are the best reagents for measuring proliferative changes;however,PMA/Ionomycin stimulation isgenerally superiorforstudying in vitro IFN-g production. WeconfirmedthatexpressionpatternsofIFN-γ (mRNA and protein) in Smad4-/- orSmad4+/+ CD8+ T cellsstimulatedwithPMA andionomycinfor6,24,and48h(FigS15andpage11intherevised version)weresimilarto thosein CD8+ T cellsstimulatedwithanti-CD3/CD28antibodiesfor72hin vitro (Fig 7A,B,D,and E in thefirstversion,Fig 8 A,B,D,andEintherevisedversion),andthose in CD8+ T cellsfrom thedraininglymphnodesofmelanoma-bearing mice,which werere- stimulated with PMA and ionomycin for1-4 h ex vivo (Fig 6A-C inthefirstversion,Fig5A-C in the revised version). We did not show the data of CD8+ T cellsstimulatedwithPMA andionomycin for72 h,because mostof the cells were dead. AstherefereesuggestedthatRunx3cooperateswithEomesintranscriptionofIFN-γ and the cytolytic molecules,Runx3 also cooperateswith Smad3/4 to regulate human germ-line IgA genes (Pardalietal,2000;Zhang etal,2000).We discussed thatthe possible mechanism of the discrepancy in theexpression ofEomesand IFN-γ in CD8+ T cellsmightbethatthecooperationof Runx3withSmad4is required for Eomes to induce IFN-γ, but not to induce the cytolytic molecules (Discussion,page 15 in the revised version). MinorSpecifics: In the abstractthe sentence starting with "Notably,progression....melanoma-bearing mice"isa run- on and unclearand should bere-worked. Wehaveamendedthetextintheabstractbydividingthesentenceintotwosentences(thesecond and fifth sentencesin the revised abstract). CallingEomes"theessentialT-boxtranscription factorforCTL functions"in theabstractis overstated given thatIntelkoferand Reinershowed thatEomes-/- CTLarefullyfunctional. Wedeletedtheword“essential”andcorrectedthedescription“the T-box transcription factor regulating CTLfunctions”intheabstract. In the introduction "heteromeric"isa notword.Perhapsheterodimeric? R-Smad-Smad4complexescanbeheterodimersorheterotrimers.Pleaserefertothereferencessuch asMassagueetal,2005,Brownetal,2007,Hawinkelsetal,2011,whichusetheword “heteromeric” to describe R-Smad-Smad4complexes. Itwould be nice to hear atleasta speculative explanation for why Alk5 inhibition leads to Smad4 down-regulation in CD8 T-cellbutnotCD4 T-cellsorB16 tumorcellsasthismechanism iscritical to the postulated effect of the inhibitors. AmongthecandidateE3ligasestoinduceubiquitin-mediateddegradationofSmad4inCD8+ T cells,which we discussed in page 12 in the firstversion,ithasbeen reported thatIL-7 modulates TGF-β signallingviaSmurf2 activity in CD8+ T cells(Pellegrinietal,2009).Atthereferee1’s suggestion,we investigated the possible involvementofSmurf1/2.However,knockdown ofSmurf1 and/orSmurf2 by shRNA did notaffectEW-7197-induced Smad4 down-regulation in CD8+ T cells (SupportingInformationFigS8andpage7intherevisedversion).Thus far,too little is known aboutubiquitination ofSmadsin immune cellsto speculate asto the possible mechanismsofour finding morethanthediscussionsinpage14intherevisedversion. EMBO MolecularMedicine PeerReview ProcessFile- EMM-2013-02524 © EMBO 8 2nd EditorialDecision 31 July 2013 ThankyouforthesubmissionofyourrevisedmanuscripttoEMBO MolecularMedicine.Wehave now heard back from thetwo Reviewerswhom weasked to re-evaluate yourrevised manuscript. YouwillseethatwhileReviewer2isnow satisfied,Reviewer1pointstoremainingissuesthat preventusfrom considering publication atthistime. Specifically,Reviewer1hassomeimportantremainingconcernsonthePLA andubiquitination experiments. S/he disagrees on the interpretation of the PLA signal and the quality of controls for these experiments. Furthermore, this Reviewer is concerned that only very little if any effect of EW- 7197 isobserved on thesubcellulardistribution ofSmadsand that the effect of EW-7197 on ubiquitination ofSmad4 doesnotappearto bespecific.Reviewer1 also notesthatdose-dependency is not apparent from Figure 3D and would like to understand why the proteasome inhibitor does not substantially potentiate the Smad4 smear.ThisRevieweralso listsothervery important experimentalshortcomingsand requestsforclarification thatrequire youraction. Althoughwewouldnormallynotallow asecondrevision,Iam preparedinthiscase,togiveyou anotheropportunity to improve your manuscript, with the understanding that the Reviewer's concernsmustbe fully addressed with additionalexperimentaldata where appropriate and thatnext version ofthemanuscriptwillundergo athird and finalround ofreview with theReviewer. Asyouknow,EMBO MolecularMedicinehasa"scoopingprotection"policy,wherebysimilar findings thatare published by others during review orrevision are nota criterion forrejection. However,Idoaskyoutogetintouchwithusafterthreemonthsifyou havenotcompleted your revision,to update us on the status.Please also contactus as soon as possible if similar work is published elsewhere I look forward to seeing a revised form of your manuscriptas soon as possible. ***** Reviewer's comments ***** Referee#1(Remarks): Theauthorshaveimprovedtheirmanuscript.However,Iam notconvincedbythethePLA and ubiquitination experimentsand theconclusionsthataredrawn from them. Specificcomments: 1.Itisstillunclearwhethersingleprimary antibody incubationswereperformed asnegative controls.When only one primary antibody istaken along thisshould notgive significantPLA signals. 2.A PLA signalisinterpreted by authorsasinteraction.Thisisincorrect.A positive signalin PLA withantibodiesagainsttwoproteinsonlytellsusthattheproteinsareincloseproximity. 3.Figure2.TGF-beta-induced Smad2/3 phosphorylation and Smad2, Smad3 and Smad4 heteromericcomplex formation and nuclearaccumulation isinhibited by TGF-betareceptorkinase inhibitors. Why do the authors observe only very little to no effect of EW-7197 on thesubcellular distribution ofSmads? 4.In Figure3B and D EW-7197 inducesamoreintensesmearofpoly-ubiquitinated Smad4 in CD8+cells.ThesameassayshouldberepeatedwithCD4+cellsinwhichtheSmad4protein downregulation isnotobserved. 5.Figure3B,lowerpanel.Thisfigureshowsthatupon Ew-7197 treatmentalso thetotalnumberof proteinsthatgetubiquitinated increases.Thusthe effect of EW-7197 on ubiquitination ofSmad4 is notspecific.Theauthorsneed to commentupon this. 6.Figure3C.CD4+ cellsarenotindicated in thisfigure. 7.Figure3F.Thetumorcellsdo respond to TGF-betaasshown by Smad2 phosphorylation in the left panel. However, how do the authors explain why there is no effect on the subcellular distribution ofSmad4 upon TGF-betaorEW-7197 treatment? Anexpectedresultwouldhavebeen that upon TGF-betatreatmentmoreSmad4 PLA spotsaredetected in the nucleus,and thereverseEMBO MolecularMedicine PeerReview ProcessFile- EMM-2013-02524 © EMBO 9 upon EW-7197 treatment. 8.Figure3D.Thedosedependentisnotapparentfrom thisfigure:thereisno differencein Smad4 down regulation with 2.0 versusthe5.0 dose.Itisunclearwhy proteasomeinhibitordoesnot (greatly) potentiate the detection of a Smad4 smear. 9.9.Figure3E.DoesEW-7197 havean effecton Smad4 subcellulardistribution? Referee#2(Remarks): I'm very satisfied with the revisions performed by the authors.The quality and interpretability of the manuscripthasimproved significantly and Iconsiderit appropriate for publication. 2nd Revision - authors'response 20 August2013 ResponsetoReferee1 Wehavedoneourbesttorespondtothereferee’simportantremainingconcernsonthefirstrevised version.Weespecially thank therefereefortheimportantadviceson theexperimentsto detect ubiquitination ofSmad4.Wehaveincluded thecommentsby therefereein bold,which arefollowed by ourresponsein theorderofthecontentsoftheconcerns raised by the referee:PLA (1,2), ubiquitination experiments(4,5,8),subcellulardistributionsofSmads(3,7,9),and others(6). Correctionsinthesecondrevisedversionareunderlinedandmarkedwithred. 1.Itis stillunclear whether single primary antibody incubations were performed as negative controls.When only one primary antibody istaken along thisshould notgive significantPLA signals. Astherefereepointedout,wehadconfirmednobackgroundsignalswhen oneoftheprimary antibodieswasomitted in double recognitions(rabbitanti-Smad2,rabbitanti-Smad3,rabbitanti- Smad4,mouseanti-ubiquitin,mouseanti-Smad2/3,respectively,incombinationwithPLA probe anti-rabbitPLUS,PLA probe anti-mouse MINUS and ratanti-CD8antibody).Pleaserefertothe explanation in line 8-9,page22 and theimagesin theattached originaldata. 2.A PLA signalisinterpreted byauthorsasinteraction.Thisisincorrect.A positivesignalin PLA withantibodiesagainsttwo proteinsonlytellsusthattheproteinsarein closeproximity. Wehavecorrectedthedescriptionsofallthecorrespondingparts,inwhichweusedtofollowthe descriptionsby themanufacturerand thepublished papersin thepreviousversions (http://www.olink.com/products/duolink/applications/protein-interactions, http://www.olink.com/products/duolink/publications).Accordingly,weadded theexplanation in line 15-17,page7:“To confirm whetherSmad4 in closeproximity with ubiquitin by thetreatmentwith EW-7197 isubiquitinated,endogenousubiquitinated Smad4 wascaptured by UbiQapturematrices”. Wecorrectedthedescriptionsbyreplacing“interactions”with “close proximity”. Please refer to the corrected parts:line 17-18 and line22 in page6,line11,14,and 15-19 in page7 in Resultssection, line 3, page 22 in Materials and Methods section, Figure 2A-F,Figure3A inFigurelegendsinthe second revised version. 4.In Figure3B and D EW-7197 inducesa moreintensesmearofpoly-ubiquitinated Smad4 in CD8+ cells.ThesameassayshouldberepeatedwithCD4+ cellsinwhichtheSmad4proteindown- regulation is notobserved.EMBO MolecularMedicine PeerReview ProcessFile- EMM-2013-02524 © EMBO 10 WerepeatedtheexperimentsusingCD8- and CD8+ dLN cellsforFigure 3B and the experiments using CD4+ and CD8+ cellsforFigure3D.Weused two independentsamplesforeach group of EW-7197 treatment(0 and 2.5 mg/kg)forbetterverification (Figure3B).Asshown,weconfirmed that the treatment with EW-7197 induced moreintensesmearsofubiquitinated Smad4 in CD8+ cellsthan those in CD8- cells(Figure 3B)and CD4+ cells(Figure 3D).Accordingly,we added the descriptionsin Resultsectionsin line 17-18,line24-25,page7,and Figure legend 3D: “Ubiquitination ofSmad4 wasenhanced significantly in CD8+ dLN cellsby thetreatmentwith EW- 7197,whereasitwasnotaltered in CD8- dLN cells(Fig 3B)”,“EW-7197 induced ubiquitination of Smad4accompaniedwithproteindownregulationinactivatedCD8+ T cells,butnotCD4+ T cellsin a dose dependentmanner(Fig 3D)”,and “IP-WesternblotshowsendogenousubiquitinatedSmad4 in CD4+ and CD8+ cellsstimulated with anti-CD3/CD28with/withoutEW-7197 and/orMG-132 for 3 days”. 5.Figure3B,lowerpanel.Thisfigureshowsthatupon Ew-7197 treatmentalso thetotalnumberof proteinsthatgetubiquitinated increases.ThustheeffectofEW-7197 on ubiquitination of Smad4 is notspecific.Theauthorsneed to commentupon this. Werepeatedtheexperimentstoblotwithanti-Ubantibody.Weusedtwoindependentsamplesfor each group ofEW-7197 treatment(0 and 2.5 mg/kg)asexplained aboveand confirmed no significantdifferences in the totalubiquitinated proteins among the samples.We paid close attention to the lysate concentration (8×106 cellsfrom 10 mice/sample,in page 23,Western blotting and in vivo ubiquitination assay in Materialsand Methodssection, 5×106 cellsfrom 5-7 mice/samplewere used in thepreviousversion)theexposuretimefortherepeated experiments. 8.Figure3D.Thedosedependentisnotapparentfrom thisfigure:thereisno differencein Smad4 down regulation with 2.0 versusthe 5.0 dose.Itisunclearwhyproteasomeinhibitordoesnot (greatly) potentiate the detection ofa Smad4 smear. Itwas presumably due to the relatively smallnumbers of the primary T cells used for immunoprecipitation in the first revised version (107 cells in 1 mlofIP buffer/sample).We repeated the IP experiments with more T cells (5×107 cellsin 1 mlofIP buffer/sample)to provide the clearer results with a more intense Smad4 smear upon MG132 treatment(upper panel).Repeated Western blotswith anti-Smad4antibodyshowedthedose-dependentSmad4 down-regulation (middle panel). Pleasealsorefertotheoriginalgelsattachedtothefirstrevisedversionshowingthedose- dependency between 2.0 and 5.0 µM. Weadded the more detailed descriptionsaboutthe subcellularlocalization ofSmadsin page 8 and the discussion on the differences of subcellular distributions of Smads in lymph node cells and in B16melanomacellsinpage14-15 in thesecond revised version. 3.Figure2.TGF-beta-induced Smad2/3 phosphorylation and Smad2, Smad3 and Smad4 heteromericcomplexformation and nuclearaccumulation isinhibited byTGF-beta receptorkinase inhibitors. Why do the authors observe only very little to no effect of EW-7197 on thesubcellular distribution ofSmads? Thiscommentisonthe subcellular distribution of Smad2 and Smad3 in the draining lymph node cellsofmelanoma-bearing mice. Astherefereepointedout,mostofC-terminally unphosphorylated Smad2 and Smad3 proteins were stilllocated in the nucleiofdraining lymph node cells when Smad4 was down-regulated by the oral treatment with EW-7197.Lymphocytesareactivated with intensivestimulithrough T/B cell receptors in combination with co-stimulatory molecules, and/or cytokine receptors, which activate the signallingpathwaysthrough serine/threoninekinases,such asMAPKsand PKC.Although these kinasesphosphorylatethelinkerregionsorMH1 domainsofR-Smads,verylittleisknownabout the signallingnetworksbetween theseserine/threoninekinasesand Smadsin lymphocytes.We added the discussion on the nuclearlocalization ofC-terminally unphosphorylated R-SmadsintheEMBO MolecularMedicine PeerReview ProcessFile- EMM-2013-02524 © EMBO 11 draining lymph nodecellsofmelanoma-bearing micetreated with EW-7197 in page14-15 with the references (Chang etal,2011;Heldin etal,2012;Matsuzaki,2013). 7.Figure3F.Thetumorcellsdo respond to TGF-beta asshown bySmad2 phosphorylation in the left panel. However, how do the authors explain why there is no effect on the subcellular distribution ofSmad4 upon TGF-beta orEW-7197 treatment? Anexpectedresultwouldhavebeen that upon TGF-beta treatmentmoreSmad4 PLA spotsaredetected in thenucleus,and thereverse upon EW-7197 treatment. Thiscommentisonthe subcellular distribution of Smad4 in B16 cells in culture. WeloweredtheintensityofgreenfluorescenceinB16cellstransfectedwithGFPtoshowthe clearersubcellulardistribution ofSmad4 red PLA dots.We also quantified the expression ofSmad4 in the cytoplasms and nucleiusing BlobFindersoftware,which proved theexpected tendency:more Smad4PLA dotsinthenucleiuponTGF-β treatment(the expression ratio of nucleus to cytoplasm: 3.48) and more Smad4 PLA dots in the cytoplasms upon EW-7197 treatment(the expression ratio of nucleusto cytoplasm:0.36).Please refer to Figure legend 3F. 9.Figure3E.DoesEW-7197 havean effecton Smad4 subcellulardistribution? Thiscommentisonthe subcellular distribution of Smad4 in B16 melanomas in vivo. Wequantifiedtheexpression ofSmad4 protein in thecytoplasmsand nucleidetected by HRP/DAB methodusingImageJsoftware(explainedinHistology,MaterialsandMethodsinpage21).EW- 7197 inhibited thenucleartranslocation ofSmad4 in B16 melanomasasshown in theimages and the graph in Figure 3E (the expression ratio of nucleus to cytoplasm: 0.32) in the second revised version.Pleasereferto Figurelegend 3E. 6.Figure3C.CD4+ cellsarenotindicated in thisfigure. Pleaseseetheupperblotslabelled “CD4+ cells”. 3rd EditorialDecision 23 August2013 ThankyouforthesubmissionofyourrevisedmanuscripttoEMBO MolecularMedicine.Wehave now received theenclosed reportfrom therefereewho wasasked to re-assessit.Asyou willsee this reviewer is now supportive and I am pleased to inform you thatwewillbeabletoacceptyour manuscriptpendingthefollowingfinalamendments: -Pleasecommentonthereferee'sconcern,includethisinthemaintextofthemanuscript,and correctFigure 3B legend. - PleaseindicateinFig. 2G thatthedatapresented doesnotcome from a single blotbutisassembled on figureby adding averticalblack barcrossing theblot. -Itappears thatSupporting Information Figure 14 is also an assembled figure made of severalpanels puttogether,pleasevisually clarify and ideally,provide the originalgels. Pleasesubmityourrevisedmanuscriptwithintwoweeks.Ilookforwardtoseeingarevisedform of yourmanuscriptassoon aspossible. ***** Reviewer'scomments***** Referee#1(Remarks): EMBO MolecularMedicine PeerReview ProcessFile- EMM-2013-02524 © EMBO 12 Theauthorshave improved the manuscript,and answered most of my concerns. CommentsonFig.3B: Upperpanel:Smad4isindicatedontheleftside;isthisSmad4modifiedbyubiquitin(non ubiquitinated Smad4 monomershould notbedetected with theprocedurethatisused)? Is this band dueto non-specific sticking ofSmad4 oris this non-modifiedSmad4pulleddownwhenincomplex withubiquitinatedSmad4andgetsseparatedanddetecteduponWesternblotanalysis? LowerpanelFig.3B isnotexplainedinFig.3B legend. 3rd Revision - authors'response 25 August2013 ResponsetoReferee1 Wedeeplyappreciateallthepreciouscommentsandinstructionsbytherefereeduringthewhole processofreview.Wehaveincluded thecommentsby therefereein bold,which arefollowed by ourresponse.Wethank therefereeagain fortheprecioustimeand consideration. CommentsonFig.3B: Upperpanel:Smad4isindicatedontheleftside;isthisSmad4modifiedbyubiquitin(non ubiquitinated Smad4 monomershould notbedetected with the procedure that is used)? Is this band dueto non-specific sticking ofSmad4 or is this non-modifiedSmad4pulleddownwhenincomplex withubiquitinatedSmad4andgetsseparatedanddetecteduponWesternblotanalysis? Astherefereepointedout,this method is not supposed to detect non-ubiquitinated proteins.Wehad meanttoindicatethemolecularweightofSmad4(70kD),butnotnon-ubiquitinated Smad4 in the previousversion. WehavedeletedtheindicationofSmad4(pointingabitlowerthanthelowestband in theprevious version)in thefigureto avoid misunderstandings.Instead,wehaveadded theinformation of molecularweightofSmad4inthefigurelegend. LowerpanelFig.3B isnotexplainedinFig.3B legend. Wethanktherefereeforcarefulreading and instructions.Explanation for the lower panelhas been included in the figure legend.