EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 1 OncogenicRolesofPRL-3 in FLT3-ITD Induced Acute MyeloidLeukemia Jung Eun Park,Hiu Fung Yuen,Jianbiao Zhou,AbdulQaderO.Al-Aidaroos, Ke Guo, Peter J. Valk,ShuDongZhang,WeeJooChng,ChengWilliam Hong,KenMillsandQiZeng Corresponding author:QiZeng, Institute ofMolecular and CellBiology Review timeline: Submissiondate: 19 October2012 EditorialDecision: 25 January 2013 Revisionreceived: 26 April2013 EditorialDecision: 11 June 2013 Revisionreceived: 18 June 2013 Accepted: 20 June 2013 TransactionReport: (Note:With the exception ofthe correction oftypographicalor spelling errors thatcould be a source ofambiguity, letters and reports are not edited. The originalformatting oflettersand referee reportsmaynotbe reflected in this compilation.) Editor:RobertBuccione 1stEditorialDecision 25 January 2013 ThankyouforthesubmissionofyourmanuscripttoEMBO MolecularMedicine.Weareverysorry that it has taken so long to get back to you on your manuscript.In this case we experienced unusual difficultiesin securing threewilling and appropriatereviewers. YouwillseethatallthreeReviewersaregenerallysupportiveofyouworkbutraisesignificant issues that question the conclusiveness of the results and note severaltechnicalissues that preventing usfrom considering publication atthistime.Iwillnotdwellinto much detail,asthe evaluationsare detailed and self-explanatory.Iwould like,however,to highlighta few main points. Reviewer1is quite concerned about the results obtained using the PRL3 mAb in the AML mouse model,andsuggestsanumberofcrucialcontrolstovalidatetheexperiment.S/henotesadditional issues that require your action. Reviewer2alsofeelsthatsomeconclusionsareinvalid dueto experimentalflaws.Again,without going into detailIwould mention his/herperplexitieson theconclusion drawn from the immunoprecipitation experiments depicted in Fig. 2. Additionally, Reviewer 2 suggests a number of crucialexperiments required to show that PRL-3 mediatesup regulation ofc-Jun through JNK/ERK. ThisRevieweralsolistsothercriticalpointsthatrequireyourintervention. Reviewer3feelsthatnon-quantitativePCR measurementsarenotacceptable.Iagreeand would suggestyou provide quantitative PCR data.S/he also has significantreservations on the description oftheexperimentsdescribed in Fig.5 and mentionsanumberofotherissuesthatyourshould act upon,including imrpoved figurelegends.EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 2 Whilepublication ofthepapercannotbeconsidered atthisstage,wewould beprepared to consider a suitably revised submission,with the understanding thatthe Reviewers'concernsmustbe fully addressed with additionalexperimentaldata where appropriate and thatacceptance ofthe manuscriptwillentailasecondroundofreview. Sincetherequiredrevisioninthiscaseappearstorequireasignificantamountoftime,additional workandexperimentationandmightbetechnicallychallenging,Iwouldthereforeunderstand if you chose to ratherseek publication elsewhere atthisstage.Should you do so,we would welcome a messagetothiseffect. PleasenotethatitisEMBO MolecularMedicinepolicytoallow asingleroundofrevisiononlyand that, therefore, acceptance orrejection of the manuscriptwilldepend on the completeness of your responses included in the next,finalversion of the manuscript. Asyouknow,EMBO MolecularMedicinehasa"scoopingprotection"policy,wherebysimilar findings thatare published by othersduring review orrevision arenotacriterion forrejection. However,Idoaskyoutogetintouchwithusafterthreemonthsifyouhavenotcompletedyour revision,to update us on the status.Please also contactus as soon as possible if similar work is published elsewhere. ***** Reviewer'scomments***** Referee#1(CommentsonNovelty/ModelSystem): Technicalqualityishigh,albeitthedatainFig.6arecounterintuitiveandrequiremanyadditional controls.Medicalimpactismedium.Whereasidentification of PRL3 as prognostic marker is convincing (high medicalimpact),the data using PRL3-specific mAbs to targetAML cells in nude miceisnotconvincing(lowimpact). Referee#1(Remarks): Parketal.investigatedtheroleofPRL3inAML inrelation to theFLT3-ITD mutation.PRL3 expression isupregulated in a significantproportion ofFLT3-ITD positive AML patients and cell lines, which is dependent on FLT3 and Src, but not JAK activity. STAT5 is activated concomitantly and PRL3 promoteractivity is regulated by STAT5. PRL3 expression in turn is correlated with Jun/AP1 activation and enhanced proliferation.Injection ofPRL-3 mAb in micewith engrafted TF- ITD tumors led to a reduction in TF-ITD tumors.Finally,PRL3 expression was identified as a novel prognosticmarkerindependentofotherclinicalparameters. ThisisaninterestingpaperthatrevealsthesignalingpathwaythatresultsinelevatedPRL3 expression in FLT3-ITD-positivetumorcellsand itdescribestheidentification ofPRL3 asa prognosticmarkerforAML. Points 1.Fig.4.Overexpression ofPRL3 leadsto activation ofAP1 in aMEK and JNK dependentmanner. Itis notevidenthow PRL3 would mediate this effect.This should be discussed. 2.Theresultsusing PRL3 mAb in thenudeTF1-ITD mousemodelforAMLarepuzzling.Howcan antibodiesdirected againstan intracellularprotein affectgrowth ofthe targetcellsin the tumor.The authorsapparently have published thisunconventionalantibody therapy before (discussion,p.14). Thesedatawarrantrigorouscontrols,astheseresultsarenotonlyunconventional,butalso counterintuitive.DoesPRL3 mAb treatmentonly affectTF1-ITD cells or other tumors as well? For instance, are FLT3-ITD-independent AML cells that do not express elevated levelsofPRL3 also affected by PRL3 mAb?Whatisthe underlying working mechanism ofthe mAb treatment? EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 3 Referee#2(CommentsonNovelty/ModelSystem): Summaryofthemajorfindings: In this study the authors analyze the correlation between the overexpression ofPRL-3,apreviously identified metastasis-associated tyrosine phosphatase,and FLT3-ITD in AML.By statistical analysisofAML patientdata setsthe authorsshow a link between PRL-3 overexpression and FLT3- ITD mutation. ExperimentswithAML celllines show thatthe constitutive activation ofFLT3 enhances PRL-3 expression through the Src-STAT5signalingpathwayandthattheup-regulation of PRL-3 induces the expression of c-Jun.However,the study does notprovide convincing evidence thatPRL-3 acts through aMAPK cascade.Moreexperimentsareneeded to provethispoint. TheauthorsalsoanalyzeeffectsofPRL-3 on cellproliferation and apoptosisand show thatPRL-3 promotescellproliferation in MTS assayswhilereducing thenumberofcellsin apoptotic fraction in FACS analysis. TheinvivoroleofPRL-3 forFLT3-ITD-driven tumorformation wasanalyzed in amouseleukemia modelbyshowingthattreatmentwithasmonoclonalPRL-3 antibody hasanti-tumor effects. The PRL-3 mAb reducesspleen and liversize in mice injected with AML cells comparable to the treatment with FLT3 mAb and reduces AML cell infiltration into the bone marrow in xenograft models.TheauthorsthusdemonstrateapotentialbenefitofPRL-3 immunotherapy. Additionally,statisticalevidenceisprovided showing acorrelation ofPRL-3 expression with shorter survivalin AML patients and revealing PRL-3 asan independentprognosticsmarker. Overall,thepresentstudyfollowsuponfindingsofapreviouspublication(Zhouetal.2011),which firstshowed the correlation between FLT3-ITD mutation and PRL-3 overexpression.Zhou etal. also show thattreatmentofMOLM14 cellswith a PRL-3 inhibitorincreasesapoptosiswhich the presentstudy also showsusing FACS analysisin figure5B.Thepresentstudy presentsa significant amountofnew data.However,the study hassome majorflaws.The presented data wasnot convincing in severalcasesand some ofthe conclusionsdrawn were invalid.The below listed experimentsshould be repeated oraltered assuggested.Especially the study ofthe in vivo functions ofPRL-3 and thepotentialuseofaPRL-3 mAb asatreatmentforFLT3-ITD positive patients are of interest from a medical point of view and would merit re-submission ofthe manuscript. Comments: Majorissues: 1. TheregulationofPRL-3 by FLT-3 analyzed in figure2A isnotconvincing in theMOLM-14 cells, asthe down-regulation of PRL3 is hardly visible in the western blotanalysis.This experiment should be repeated in orderto prove thatFLT3 activity regulates PRL-3 expression. 2. 2.1. In figure 2D the authors analyze the interaction between Src and FLT3 by immunoprecipitation studies in TFI-ITD cells and state thatSrc an FLT3 do notinteract.However,there seems to be some residualSrc binding attimepoint0,butthishard to tellastheblotsin thefigureareofpoorEMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 4 quality.Negativecontrolsforco-immunoprecipitation experiments and western blots showing protein levelsin cellularlysatesshould beincluded in thefigures. 2.2 Theconclusion drawn from the co-immuniprecipitation experiment in figure 2D is not valid. If as claimed Src bridged the interaction between FLT3 and STAT5 then an IP:FLT3 should stillpull down Srcand STAT5 from cellularlysates.Indirectinteractionscan beseen in IPs from cellular lysates. Theauthorsshouldconductpull-down experimentsusing purified recombinantproteinsin orderto analyze whetherthe interaction between FLT3 and STAT5 isdirectorindirect. 3. In figure 4 the authors make a good case of showing that the expression of PRL-3 induces expression ofc-jun.However,they state that PRL-3 actsthrough aJNK/ERK signaling cascade. Theydonotprovideanyproveforthishypothesis.Theyshow thatinhibitorsoftheMAPK JNK and MEKaswellassiRNAtargeting JNK and ERK resultin adecreasein c-Jun phosphorylation.This is to be expected as both JNK and MEK/ERK have been shown to be upstream of c-Jun.The relationship with PRL-3 remainsobscureasexperimentsmerely show thatc-Jun phosphorylation is mediated by ERK and JNK,which isno new finding. In order to show thatPRL-3 mediatesup-regulation of c-Jun through JNK/ERK the authors should conductthe following experiments: - AMLcelllinesshouldbedepletedofPRL-3 by siRNA and itshould beshown that phosphorylation ofJNK/ERK isreduced,eitherby western blotting orby an in vitro kinaseassay. - Over-expression ofPRL-3 in PRL-3 negativecellline(i.e.TFIcells)should induce phosphorylation ofJNK and ERK 4. A morespecificJNK inhibitorinshould be used in figure 4D in orderto prove thatthe growth advantage ofPRL-3 expressing cellsrelieson activation oftheJNK pathway ascurcumin effects multiplesignalingpathways Theconclusiondrawnfrom thisexperimentisnotvalid.TheauthorssuggestthatPRL-3 isup- stream ofc-Jun.However,The experiments presented in 4A and B already show this.The only new resultis thatcurcumin does notaffectPRL-3 levels. Minorissues: 1. Westernblotanalysisofphospho-FLT3provingthattheFLT3inhibitors used in figure 2 are either missingorofpoorquality.ExpressionlevelsofFLT3incellularlysatesshowahighvariabilityat the analyzed time points. 2. A c-fos western blotshould be included in figure 4b. 3.EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 5 In figure 1 a PRL-3 band clearly visible in FLT3-ITD negative firstsample.Thus,3 outof 12 (25%) FLT3-ITD negative patients appear to show PRL-3 expression.TheFLT3 band on farrightof FLT3-ITD positive sample appears very faint.In order to strengthen the correlation between FLT3- ITD mutationandPRL-3 expression,an equalnumberofFLT3-ITD positive and negative AML patientsamplesshould beanalyzed. 4 TheeffectsofPRL-3 overexpression ordepletion on AML cellsappearsmarginalin theFACS analysisin figure 5B.The study would benefitfrom a differentapoptosisassay to prove an anti- apoptotic role ofPRL-3 moreconvincingly. Referee#3(CommentsonNovelty/ModelSystem): Importantfor the reasons given below,along with suggestions for a better model(in the future;these modelsare notsufficiently developed yet,in my opinion. Referee#3(Remarks): Summary:ThetreatmentofAML (withtheexceptionofAPL)hasnotchangedmuchinthepast40 years,certainly notsincethisreviewerlastcared foran AML patientin 1984.Therefore, it is critical that we develop novel therapies, and PRL3 is certainly an interesting candidate. Here the authors demonstratean association between FLT3-ITD and PRL3,investigate the mechanism connecting the two, and demonstrate therapeutic efficacy of PRL3antibodies.Thefindingsareimportantinthat wedesperatelyneednew therapeuticoptionsinAML. Suggestionsforimprovementofthemanuscript: Thereareanumberof"minor"butstillimportantcorrectionsthatneedtobeaddressed: - Fig.1A.Idonotunderstand why anyonedoesnon-quantitativePCR anymore.IftheRNA or samples are available,please repeatin a quantitative fashion.Ifnotavailable,do itnexttime! - ThedatasupportingtheroleofSTAT5ismuchstrongerthanthatofSRC;theauthorsshould soften theirstatements in the results and discussion. - Fig.3.Itwouldbedesirable(butnotabsolutelyrequired)toaddaSTAT5antibodysupershiftto the EMSA, and a ChIP assay demonstrating STAT5 binding in cells. The streptavidin-agarose bead assay isa nice addendum to the EMSA. - Throughthemanuscript,theauthorsrefertoproliferation(referencestoPulikkanandRangatia page8,theirown Fig.4C,etc).Ibelievethey areusing thisterm incorrectly,likemostoftheworld. If I understand Fig.4C correctly,they are measuring cellnumber,notproliferation.Please correct throughout the text (and please point out to colleagues that assays of cell number are NOT assays of cellproliferation!). - Again,inFig.5,theauthorshavecompletely confused theirassays,mixing up cellnumber, proliferation,and apoptosis.They need to beVERY precisein measuring cellnumber,proliferation, and apoptosisand be very carefulin how they state the resultsand discussion.Itistotally mixed up here,butitcan allbe easily fixed by being more precise in the labeling and language used.They need to restrain theirinterpretation oftherelativeeffectsofproliferation and apoptosison cell numberunlessthey perform moreassaysspecificforproliferation and apoptosis. EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 6 - Fig.6.Itwouldbemuchmoredesirabletoperform theseexperimentsinabettermodelofFLT3- ITD AML than intravenous injection of transformed celllines,butthe published models using FLT3-ITD alone (either retroviralor knock-in) are not robust. Newer models combining FLT3-ITD withothermutationslookmorepromising,anditwillbeofgreatinteresttotestthesemodelsinthe future with PRL3 antibody by itself and combined with FLT3-ITD inhibitors. - Fig.7.Ineverunderstand the value ofinvestigating the prognostic value unlessthe therapy is related to the gene of interest.Almostallthese patients are treated with anthracycline/AraC,so unlessresponseto thesedrugsisrelated to PRL3,Ido notknow how to interpretthedata.When PRL3antibodyisusedclinically,itwouldmakemoresense.Thisisnotnaturalhistoryofthe disease.Buteveryoneelsein theworld otherthan myselfseemsto think thesefindingshavevalue, so itis OK to leave itin the manuscript.Maybe Ijustdo notthink right. - Perhapsitisduetotheidioticwordrestrictionsimposedbyjournals,butIthinkthefiguresneedto bedescribed better: Fig.1B:?microarraystudies? Fig.1C and1D:?RNA?Protein?Whatarewelookingat? FINALLY,Please do NOT eversend me a manuscriptto read with tiny font!!!The easieritis to read a manuscript,the more the reviewer willlike it! 1stRevision - authors'response 26 April2013 Referee#1(CommentsonNovelty/ModelSystem): Technicalqualityishigh, albeit the data in Fig. 6 are counterintuitive and require many additional controls. Medical impact is medium. Whereas identification of PRL3 as prognostic marker is convincing (high medicalimpact),the data using PRL3-specific mAbs to targetAML cells in nude miceisnotconvincing(lowimpact). Referee#1(Remarks): Park et al. investigated the role of PRL3 in AML in relation to the FLT3-ITD mutation. PRL3 expression isupregulated in a significantproportion ofFLT3-ITD positive AML patients and cell lines, which is dependent on FLT3 and Src, but not JAK activity. STAT5 is activated concomitantly and PRL3 promoter activity is regulated by STAT5. PRL3 expression in turn is correlated with Jun/AP1 activation and enhanced proliferation.Injection ofPRL-3 mAb in micewith engrafted TF- ITD tumors led to a reduction in TF-ITD tumors.Finally,PRL3 expression was identified as a novel prognosticmarkerindependentofotherclinicalparameters. This is an interesting paper that reveals the signaling pathway that results in elevated PRL3 expression in FLT3-ITD-positive tumor cells and it describes the identification of PRL3 as a prognosticmarkerforAML. EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 7 Weappreciatetheimportant inputs made by the reviewer. Thecomplimentfrom thisreviewerof ‘Whereas identification of PRL3 as prognostic marker is convincing (high medical impact)’ is indeed very encouraging to our endeavor on this revision. Points 1.Fig.4.Overexpression ofPRL3 leadsto activation ofAP1 in a MEK and JNK dependentmanner. Itis notevidenthow PRL3 would mediatethiseffect.Thisshould bediscussed. Wethankandagreewiththereviewerforthisinsightfulcomment.Toaddressthis,weperformed additionalexperiments.Firstly,PRL-3 wasoverexpressed in aPRL-3 negativecell line-TF-1 (Fig 1C, lane 1) and western blot showed activations of ERK, JNK, and c-Jun. Up-regulation of the phosphorylation ofERK and JNK,concomitantwith up-regulation of phosphorylationof-c-Jun were demonstrated (new Fig 4C,b).Secondly,PRL-3 wasdepleted in two PRL-3 positiveAML celllines (MOLM-14 and TF1-ITD). Depletion of PRL-3 caused down-regulation of the ERK and JNK phosphorylation,accompanied with down-regulation of phosho-c-Jun (new Fig.4C,a).Thirdly,we used specificMEK inhibitor(U0126)orJNK inhibitor(SP600125)to examinetheconsequenceof ERK or JNK inhibitions in TF-1 cells overexpressing PRL-3 (TF1-PRL-3). We showed that suppression ofcellgrowth aftereach inhibitortreatment(new Fig.4D). It has been reported that phosphatase can activate phosphorylation-mediated signallingpathway through dephosphorylation of inhibitory protein or inhibitory residues (Jaumot & Hancock, 2001; Oryetal,2003).Thus,weexaminedseveralupstream regulatorsofMEK andJNK pathwaytofind out the possible link with PRL-3. However, our trial experiments on Ras-Raf family and Akt, upstream regulatorsofMEK-ERK (Dhillonetal,2007), did not show any observable reduction on phosphorylation level after overexpression of PRL-3. Since PRL-3 has identified as metastatic protein phosphatase in various cancers, there are several attempts to identify direct substrates of PRL-3 and severalPRL-3 interacting proteinswerereported by massspectrometry approach (Ewing et al, 2007), but only very few proteins were proved as interacting molecule of PRL-3 with experimentalevidence.And,consistentwith ourfindings,severalreportspresented thatPRL-3 can activate ERK through regulation ofupstream activatorssuch asRho family GTPase(Fiordalisietal, 2006; Mingetal,2009), integrin β1 (Pengetal,2009), or Src (Liangetal,2007) butunderlying mechanism hasnotfullyaddressedyet. Wehavedescribed thesein discussion part,page18,line5- 8. At this moment, our attempt is not enough to address reviewer’s comment, but we are actively working to answer the reviewer’s comment, “how PRL3 would mediate this effect (MAPK activation)”. We appreciate reviewer’sinsightfulscientific comments. 2.Theresultsusing PRL3 mAb in thenudeTF1-ITD mouse modelfor AML are puzzling.How can antibodies directed against an intracellularprotein affect growth of the targetcells in the tumor. The authors apparently have published this unconventional antibody therapy before (discussion,EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 8 p.14).Thesedata warrantrigorouscontrols,astheseresultsarenotonlyunconventional,butalso counterintuitive. Weapologizethattheseimportantpointswerenotclearly explained in the textand we fully agree with the reviewer that rigorous controls are important since this is an unconventional antibody therapy. In our previous studies, we did use several controls: 1. control antibodies for sham- treatment, 2. cell lines either PRL-3 expressing or PRL-3 non-expressing to show therapeutic response is depending on PRL-3 expression,and 3.controlmouse strains(immunodeficientmice: nude, scid, muMT; immunocompetent mice: C57BL6 wild type, MMTV-PymT spontaneoustumor model) to compare therapeutic efficacies and understand how lymphocytes are involved in this immunotherapy as a Proof-of-Concept. In this current study (Fig. 7, former Fig. 6), we used 3 antibodies to controleach other:1.control IgG antibody forsham-treatment, 2.FLT3antibodyto target extracellular FLT3 receptor, and 3. PRL-3 antibody to target intracellular PRL-3 phosphatase.Weused 3 cell lines: 1. TF-1 cells (non-PRL-3 expression), 2.TF1-ITD cells (both FLT3-ITD and PRL-3 expressions), and 3. TF1-ITD PRL-3 KD cells (reduced PRL-3 expression levels) to study therapeutic efficacies related to target expression levels. If the cancer cells are not expressing targetprotein (oratlow levels),cancercellswillnotrespond/be affected by the PRL-3 mAbtherapy. Antibodies are traditionally used to target extracellular (surface) proteins and have never been used to target intracellular proteins because antibodies are generally believed to be too large (~150KD) to enter cells. Indeed, many oncogenic proteins are found within the cell (such as intracellular phosphatases/kinases and transcription factors), leaving a large intracellular treasure of potential cancer-specific therapeutic targets untapped in terms ofantibody therapy orvaccination. Since 2005, we have begun to explore if intracellular oncoproteins can be targeted by antibody therapies. We have published several studies since 2008 (CancerBiolTher 2008;SciTranslMed 2011; Oncotarget2012; CancerRes2012).Ourstudieshavereceived encouraging responses from manyotherlabsforcollaborationsondifferent‘tumorspecific’intracellularoncotargets.Professor Soldano Ferrone (University of Pittsburgh) was commissioned by Sci Transl Med to write a Perspective titled ‘Hidden ImmunotherapyTargets Challenge Dogma’ to accompany our research paperin (SciTranslMed2011).Actually,since1978,asubstantialbodyofevidencesuggeststhat antibodies are able to ‘penetrate’ (most likely via endocytotic pathway) human cells to target intracellular proteins.Alarcon-SegoviaD etal.(Nature, 1978) described a human immunoglobulin G autoantibodytoribonucleoproteinsthatcouldenterviablehumanlymphocytesusingFcreceptors and react with its nuclear antigen. This naturally occurring pathogenic role of autoantibodies in autoimmune diseasesindicatesthatantibody targeting ofintracellularoncoproteinsmightpotentially cause cancercellsto self-destructaswell.Although theexactmechanism isyetto bedefined,our results so far suggest that antibody can be also used to target intracellular oncoproteins for anti- cancereffectsin mice models. EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 9 Does PRL3 mAb treatment only affect TF1-ITD cells or other tumors as well? For instance, are FLT3-ITD-independent AML cells that do not express elevated levelsofPRL3 also affected by PRL3 mAb? Manythanksfortheseexcellentquestions.Inourpreviousstudies,wedemonstrated that a variety of tumors (regardless of tumor types) expressed PRL-3 antigen was clearly inhibited by PRL-3 antibodies(SciTranslMed 2011).Conversely,iftumorsdo notexpressPRL-3 oncotarget,they will notrespond/beaffected by PRL-3 antibody therapy.Therefore,FLT3-ITD-independent AML cells that do not express elevated levels of PRL-3 willnotrespond/beaffected by PRL3 mAbbecausean antigen-antibody specific effectmustbe required in orderto achieve therapeutic efficacy. WhatistheunderlyingworkingmechanismofthemAbtreatment? Several possibilities have been proposed by a well-known immunologist Professor Soldano Ferrone(‘Perspective’ in SciTranslMed 2011) and by ourrecentreview article‘Awaiting a New Era of Cancer Immunotherapy’ (Cancer Res 2012). Three possible mechanisms are proposed as below (A-C): A, antibodies may potentially enter PRL-3–expressing cells, likely via endocytic process (Cancer Biol Ther, 2008) to target intracellular PRL-3 and neutralize its function. The interaction of antibodies with the antigens may cause destruction of the cancer cells and lead to apoptosis, a cellular phenomenon previously described for autoantibodies. B, some of the intracellular PRL-3 may be externalized and displayed on the surface of cancer cells by unconventional secretion. Bindingofantibodiestosurface-exposed intracellularproteinsmay then triggerimmune responses such as ADCC to destroy the cancercells.C, proteolytic fragments of intracellular PRL-3 may be presented by MHC class Imolecules to attractCytotoxicT cells (CTLs). It isanticipated that theEMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 10 combination ofseveralmechanismsmay be involved in achieving the final therapeutic consequence ofantibodiesagainstintracellularoncoproteins. Referee#2(CommentsonNovelty/ModelSystem): Summaryofthemajorfindings: In this study the authors analyze the correlation between the overexpression ofPRL-3,a previously identified metastasis-associated tyrosine phosphatase, and FLT3-ITD in AML. By statistical analysisofAML patientdata setstheauthorsshow a linkbetween PRL-3 overexpression and FLT3- ITD mutation. Experiments with AML cell lines show that the constitutive activation of FLT3 enhances PRL-3 expression through the Src-STAT5 signaling pathwayand thattheup-regulation ofPRL-3 induces the expression of c-Jun.However,the study doesnotprovide convincing evidence thatPRL-3 acts through a MAPK cascade. More experiments are needed to prove this point. TheauthorsalsoanalyzeeffectsofPRL-3 on cellproliferation and apoptosisand show thatPRL-3 promotescellproliferation in MTS assayswhilereducing thenumberofcellsin apoptoticfraction in FACS analysis. The in vivo role of PRL-3 forFLT3-ITD-driven tumorformation wasanalyzed in a mouseleukemia modelbyshowing that treatmentwith asmonoclonalPRL-3 antibodyhasanti- tumor effects. The PRL-3 mAb reduces spleen and liver size in mice injected with AML cells comparable to the treatment with FLT3 mAb and reduces AML cell infiltration into the bone marrow in xenograft models. The authors thus demonstrate a potential benefit of PRL-3 immunotherapy. Additionally, statistical evidence is provided showing a correlation of PRL-3 expression with shorter survival in AML patients and revealing PRL-3 as an independent prognosticsmarker. Overall,thepresentstudyfollowsuponfindingsofapreviouspublication(Zhouetal.2011),which first showed the correlation between FLT3-ITD mutation and PRL-3 overexpression. Zhou et al. also show that treatment ofMOLM14 cellswith a PRL-3 inhibitor increasesapoptosiswhich the presentstudyalso showsusing FACS analysisin figure5B.Thepresentstudypresentsasignificant amount of new data. However, the study has some major flaws. The presented data was not convincing in several cases and some of the conclusions drawn were invalid. The below listed experimentsshould be repeated or altered assuggested.Especiallythestudyofthein vivo functions ofPRL-3 and thepotentialuseofa PRL-3 mAb asa treatmentforFLT3-ITD positive patients are of interest from a medical point of view and would merit re-submission ofthe manuscript. Wearedelightedthatthereviewerforeseesthevalueofourstudyofthein vivo function of PRL-3 and the potentialuse ofa PRL-3 mAb asatreatmentforFLT3-ITD positive patients.Ourdetailed responses to these insightfulinputs are as below. EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 11 Comments: Majorissues: 1.Theregulation ofPRL-3 byFLT-3 analyzed in figure2A isnotconvincing in theMOLM-14 cells, as the down-regulation of PRL3 is hardly visible in the western blot analysis. This experiment should be repeated in order to prove thatFLT3activityregulatesPRL-3 expression. We feel sorry that our Fig. 2A was not convincing to show the downregulation of PRL-3 was affected by FLT3 inhibition. To improve this, we repeated the experiment with FLT3 inhibitors (PKC412 and CEP-701) in a dose increment manner. The results now showed that inhibitor treatment clearly reduces FLT3 phosphorylation, concomitant with a decrease in endogenous PRL-3 protein levels in MOLM-14 cells. The improved blots are presented in new Fig. 2A, c-d, which suggestthat inhibition of FLT3 activity would downregulate PRL-3 protein expression. 2.1. In figure 2D the authors analyze the interaction between Src and FLT3 by immunoprecipitation studies in TFI-ITD cells and state that Src an FLT3 do not interact. However, there seems to be some residualSrc binding attime point0,butthis hard to tellas the blots in the figure are ofpoor quality. Negative controls for co-immunoprecipitation experiments and western blots showing protein levelsin cellularlysatesshould beincluded in thefigures. We thank and agree with the reviewer’s comments on these experiments. We apologize for makingthereviewerconfusedonthephysicalinteractionbetweenSrcandFLT3.Althoughwehave repeated the co-IP experiments severaltimes respectively with FLT3, Src, or IgG antibodies using TF1-ITD total cell lysates, we could not obtain a conclusive result to reveal physical interaction between FLT3 and Src, indicating that FLT3 and Src may not directly interact in nature or could due to our technical problems in detecting FLT3 and Src bindings. Therefore,weremovedouroriginal Fig. 2D and deleted previousstatement“SrccouldbeanintermediarybetweenFLT3andSTAT5” and “interaction of both FLT3-Src and Src-STAT5 pairs was reduced upon CEP-701 mediated interaction of FLT3-ITD”. Fig. 2 was revised and results were discussed on page 8, line 2-4.We nextfollowed reviewer’skind advicesto conductpulldown assay asbelow. 2.2 Theconclusion drawn from theco-immuniprecipitation experimentin figure2D isnotvalid.If asclaimed Srcbridged theinteraction between FLT3 and STAT5 then an IP:FLT3 should stillpull down Srcand STAT5 from cellular lysates. Indirect interactionscan beseen in IPs from cellular lysates. The authors should conductpull-down experimentsusing purified recombinantproteinsin orderto analyzewhethertheinteraction between FLT3 and STAT5 isdirectorindirect. Many thanks for thesuggestion.Figure2Dwasremoved.Asadvised,weperformedGSTpull- down assay with purified recombinantGST-FLT3(571-993)protein using eitherTF1-ITD totalcell lysates or using purified recombinant proteins; Src and STAT5. Our trial result (please refer to below Figure)suggeststhat:A. GST alone could not pull down Src orSTAT5 from TF1-ITD cellEMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 12 lysate. B.GST-FLT3(571-993)could pulldown Srcand STAT5 from TF1-ITD totalcell lysates, indicating possible bindings of FLT3, Src, and STAT5. C. GST-FLT3 (571-993) could bind to recombinant protein Src much stronger than STAT5 using in vitro direct binding assay. At this stage,a conclusive resulton theirphysicalinteraction was notachieved in vivo. Therefore, we did notincorporatethisresultin thepaper.Wesincerely seek an understanding from thisreviewer. Figure. GSTpull-down assaywith GST protein aloneorGST-tagged FLT3(571-993)protein in vitro TF1-ITD totalcelllysate was used for GST pull-down assay with A.GSTproteincouldnot bind SrcorSTAT5.B. GST-tagged FLT3 (571-993)protein could bind both Src and STAT5.C. GST-tagged FLT3 (571-993) protein could bind purified recombinant Src protein stronger than STAT5. 3. In figure 4 the authors make a good case of showing that the expression of PRL-3 induces expression of c-jun. However, they state that PRL-3 acts through a JNK/ERK signaling cascade. Theydonotprovideanyproveforthishypothesis.Theyshow thatinhibitorsoftheMAPK JNK and MEKaswellassiRNAtargetingJNKandERKresultinadecreaseinc-Jun phosphorylation.This is to be expected as both JNK and MEK/ERK have been shown to be upstream of c-Jun. The relationship with PRL-3 remainsobscureasexperimentsmerelyshow thatc-Jun phosphorylation is mediatedbyERKandJNK,whichisnonewfinding. Many thanks for these important points. To address this, we performed additional experiments. Firstly, PRL-3 was overexpressed in a PRL-3 negative cell line-TF-1 (Fig. 1C, lane 1) and activations of ERK, JNK, and c-Jun were shown by western blot. Up-regulation of the phosphorylation ofERK and JNK,concomitantwith up-regulation of phosphorylationof-c-Jun were demonstrated (new Fig. 4C, b). Secondly, PRL-3 was depleted in two PRL-3 positive AML cell lines (MOLM-14 and TF1-ITD).Depletion of PRL-3 caused down-regulation of the ERK and JNK phosphorylation,accompanied with down-regulation of p-c-Jun (new Fig.4C,a).Thirdly,we used specific MEK inhibitor(U0126)orJNK inhibitor(SP600125)to examine the consequence ofERK or JNK inhibitions in TF-1 cells overexpressing PRL-3 (TF1-PRL-3) (Updated Fig. 4D). These results were incorporated and described from page 10,line 8 to page 11,line 5.EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 13 In order to show thatPRL-3 mediatesup-regulation ofc-Jun through JNK/ERK the authorsshould conductthe following experiments: - AML cell lines should be depleted of PRL-3 by siRNA and it should be shown that phosphorylation ofJNK/ERK isreduced,eitherbywestern blotting orbyan in vitro kinaseassay. Asadvisedby the reviewer, tounderstandhow PRL-3 would mediate theactivation ofAP-1 via MEK/ERK and JNK pathways, PRL-3 was depleted in TF1-ITD and MOLM-14 cell lines that highly express endogenous PRL-3 (Fig. 1C, lane 3-4) by performing siRNA knock-down experiments.Depletion ofPRL-3 caused down-regulation of the ERK and JNK phosphorylation in ‘TF1-ITD PRL-3 KD’and ‘MOLM-14 PRL-3 KD’cellswhen compared to theirrespectivemock- knock downed cell line (new Fig.4C, a).The down-regulation of ERK and JNK is accompanied withdown-regulation of phospho-c-Jun,indicating thatPRL-3 may activatec-Jun viaERK and JNK pathways. We added this result in Fig. 4C, a, and explained the results on page 10 (line 16-18). Furthermore,toconfirm ERK andJNK are importantforPRL-3 to mediateup-regulation of c-Jun, we knocked-down ERK and JNK in TF1-PRL-3 cells. As expected, depletion of ERK or JNK abolished PRL-3 mediated activation ofc-Jun (Fig.4C,c-d). - Over-expression of PRL-3 in PRL-3 negative cell line (i.e. TFI cells) should induce phosphorylation ofJNK and ERK 4. Thank you for this suggestion! We have overexpressed PRL-3 in TF-1 cell line (TF1-PRL-3), whichresultedinupregulationofthephosphorylationlevelofERK andJNK inTF1-PRL-3 cells, concomitantwith upregulation ofphospho-c-Jun (new Fig.4C,b).These results indicate thatPRL-3 activates ERK and JNK pathways to activate c-Jun/AP-1. The result was incorporated in and described on page10,line18-20. A more specific JNK inhibitor should be used in figure 4D in order to prove that the growth advantageofPRL-3 expressing cells relieson activation of the JNK pathway ascurcumin effects multiplesignalingpathwaysTheconclusiondrawn from thisexperiment isnotvalid.Theauthors suggestthatPRL-3 isup-stream ofc-Jun.However,the experimentspresented in 4A and B already show this. The only new result is that curcumin does not affect PRL-3 levels. Toaddress thegrowthadvantageofPRL-3 activesc-Jun via MEK/ERK and JNK signalling on PRL-3 overexpressing AML cells,we treated TF1-PRL-3 cellswith specificMEK/ERK inhibitor (U0126)orJNK inhibitor(SP600125)inadditiontocurcumintreatmentexperiment,andassessed cellviability ofTF1-PRL-3 cells.Asshown in new Fig.4D (a-b),both inhibitorscould respectively reduce cellnumbers by around 50% compared to untreated cells. In addition, curcumin treatment could also reduce cellgrowth in similarmanner.The cellgrowth curveswere shown in updated Fig. 4D,and resultsincluded on page11,line1-5.EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 14 Minorissues: 1. Western blot analysis of phospho-FLT3 proving that the FLT3 inhibitors used in figure 2 are either missing or of poor quality. Expression levels of FLT3 in cellular lysates show a high variability atthe analyzed time points. WehaverepeatedwesternblotanalysiswithFLT3inhibitor(PKC412andCEP-701)treatments. The improvedblotswerepresented innew Fig.2A,c-d,whichshow similarexpression levelsof total FLT3 in cellular lysates, and corresponding decrease of phospho-FLT3 upon inhibitor treatments. In addition, we repeated WB analysis and replaced our previous blots with improved ones(new Fig.2B,c-d),which show an effectof inhibitor treatmenton downstream molecules. 2.A c-fos western blot should be included in figure 4b. As suggested, new Fig. 4B (b) western blot was included, showing that depletion of PRL-3 in TF1-ITD cells reduced c-Jun butnotc-fos expression level. 3. In figure 1 a PRL-3 band clearlyvisible in FLT3-ITD negative first sample.Thus,3 outof12 (25%) FLT3-ITD negative patients appear to show PRL-3 expression.The FLT3 band on farright of FLT3-ITD positive sample appears very faint. In order to strengthen the correlation between FLT3-ITD mutation and PRL-3 expression, an equal number of FLT3-ITD positive and negative AMLpatientsamplesshouldbeanalyzed. We agree with the reviewer to include this low PRL-3 expression patient #1 asPRL-3 positive AML. We apologize that we have difficulties to collect equal number of FLT3-ITD positive and FLT3-ITD negative AML patients for this analysis.Since numbers of FLT3-ITD positive patients are lesserthan numbersofFLT3-ITD negative patients,we could only obtain these 19 samples from NationalHospitalofSingapore.PerformingqRT-PCR assuggestedbyreviewer3,andsettingthe FLT3-ITD negative patient#1 (lowestPRL-3 expression patient)asapositivereference,weshowed 3 out of 12 (25%)FLT3-ITD negative patients and 5 outof 7 (71%) FLT3-ITD positive patients expressed high PRL-3, suggesting that high PRL-3 expression is associated with FLT3-ITD mutation.Datafrom qRT-PCR waspresented inSupportingInformationFig.S1andexplained in page6,line9-11.Understanding from thisreviewerisgreatly appreciated. 4. The effects of PRL-3 overexpression or depletion on AML cells appear marginal in the FACS analysis in figure 5B. The study would benefit from a different apoptosis assay to prove an anti- apoptoticroleofPRL-3 moreconvincingly. Wethankreviewerforpointingoutthisimportantcomment.TF-1 isacytokine dependentcellline in terms of cell growth (Linetal,2007). In TF1-PRL-3 cells(TF-1 cellsoverexpressing PRL-3),theEMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 15 overexpression ofPRL-3 likely allows TF-1 cells to activatesurvivalpathwayssuch asAKT and ERK (Jin et al, 2006; Songyanget al, 1997) to allow TF1-PRL-3 cells survive in the absence of cytokines. Consistently, our results show that PRL-3 overexpression could stimulate ERK phosphorylation (Fig. 4C). To further study the anti-apoptotic effect of PRL-3, we performed Annexin-V and 7-AAD staining using TF1-GFP (as control) and TF1-PRL-3 cells, followed by FACS analysis.Weobservedmorecelldeath in TF1-GFPcells(31% apoptoticcells)thaninTF1- PRL-3 cells (6.8% apoptotic cells), supporting that PRL-3 might have a role in anti-apoptotic function in TF-1 cytokinedependentcellline.The data isnow shown in Fig.5C and described on page11,line 17-23. Notably, MOLM-14 and MV-11 are cytokine-independent cell lines. Silencing of PRL-3 did not result a substantial increase of apoptotic population as evidenced by the absence of sub-G1 cells (Fig.5B is now Fig.6.A-B,b).WhiledepletionofPRL-3 resulted in increased cellnumbersin G1 phasebutdecreased cellnumbersin S phase,suggesting thatPRL-3 hasarolein promoting G1 to S phase transition. To study PRL-3 anti-apoptotic role in these cytokine independent cell lines, AnnexinV/7-AAD staining was performed,followed by FACS analyses. We found that depletion of PRL-3 in MOLM-14 and MV4-11 cell linesdid not increaseapoptoticpopulations in ‘MOLM-14 PRL-3 KD’ and ‘MV4-11 PRL-3 KD’ cells (new Supporting information Fig. S4). These results supportthatthe role ofPRL-3 in thesecytokine-independent cell lines is primarily in promoting G1- S phasetransitionratherthananti-apoptotic function.PRL-3 knock-down mightnotrestorecytokine dependency due to the activation of other compensation pathway(s).Indeed, it has been reported that in AML, several signaling pathway such as RAS, STATs, Bcl-xL,can confercelltransforming activity of cytokine dependent cells to resist apoptosis (Mizuki et al, 2000; Nosaka et al, 1999; Spiekermannetal,2002). Referee#3(CommentsonNovelty/ModelSystem): Important for the reasons given below, along with suggestions for a better model (in the future; these models are not sufficiently developed yet, in my opinion. Weappreciatethesethoughtfulcommentsfromthereviewerandwehavefollowedhis/heradvices in preparing our revision. Ourdetailedresponsestotheseinsightfulinputsareasbelow. Referee#3(Remarks): Summary:ThetreatmentofAML (with theexception ofAPL)hasnotchanged much in thepast40 years, certainly not since this reviewer last cared for an AML patient in 1984. Therefore, it is critical thatwe develop novel therapies,and PRL3 iscertainly an interesting candidate.Here the authors demonstrate an association between FLT3-ITD and PRL3, investigate the mechanism EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 16 connecting the two, and demonstrate therapeutic efficacy of PRL3 antibodies. The findings are important in that we desperately need new therapeutic options in AML. Suggestionsforimprovementofthemanuscript: Thereareanumberof"minor"butstillimportantcorrections thatneed to be addressed: - Fig. 1A. I do not understand why anyone does non-quantitative PCR anymore. If the RNA or samples are available,please repeatin a quantitative fashion.Ifnotavailable,do itnexttime! We thank the reviewer for sharing professional experience and fully agree with that “we desperatelyneed new therapeuticoptionsin AML”.Asadvisedbythereviewer,weperformedqRT- PCR on the 19 AML patients’ bone marrows samples (Fig. 1A). Notably, patient #1 (FLT3-ITD negative) also showed low PRL-3 expression level, and we included this patient #1 as PRL-3 positive,and now showed 3 outof12 (25%)FLT3-ITD negative patients expressed PRL-3,whereas 5 out of 7 (71.4%) FLT3-ITD positive patients expressed PRL-3. This suggests that high PRL-3 expression is associated with FLT3-ITD mutations. Data from q-RT-PCR was presented in SupportingInformationFigS1and explained in page 6,line 9-11. - The data supporting the role of STAT5 is much stronger than that of SRC; the authors should soften their statements in the results and discussion. WeagreewithreviewerthatourstatementandconclusionfortheroleofSrconPRL-3 regulation should be softened since our data was not convincing. To clarify this concern, we deleted our originalstatements“Srccould bean intermediary between FLT3 and STAT5”,“CriticalroleofSrc kinase” in discussion.Wealso removed Fig.2D ‘co-Immunoprecipitation’ data which is also not convincing.We amended resultsection on page 8 (line 2-5)based on updated Fig.2. - Fig.3.Itwouldbedesirable(butnotabsolutelyrequired)toaddaSTAT5antibodysupershiftto the EMSA, and a ChIP assay demonstrating STAT5 binding in cells. The streptavidin-agarosebead assayisa niceaddendum to theEMSA. Asadvisedbythereviewer,wehadbeentryingveryhardforthisexperimentduringourrevision. Using newly purchased STAT5 antibodies (polyclonal Abs), several attempts on supershift experiments were conducted but the results were still not clear. We reasoned that either an optimized condition wasnotachieved orourantibodiesused werenotable to bind STAT5-DNA complexes.Atthisjuncture,we would like to seek thisreviewer’sunderstanding ofourdifficulties. - Throughthemanuscript, theauthors refer to proliferation (references to Pulikkan and Rangatia page8,theirown Fig.4C,etc).Ibelievetheyareusing thisterm incorrectly,likemostoftheworld. IfI understand Fig.4C correctly,they are measuring cellnumber,notproliferation.PleasecorrectEMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 17 throughout the text (and please point out to colleagues that assays of cell number are NOT assays of cellproliferation!). - Again, in Fig. 5, the authors have completely confused their assays, mixing up cell number, proliferation,and apoptosis.They need to be VERY precise in measuring cellnumber,proliferation, and apoptosisand beverycarefulin how theystatetheresultsand discussion.Itistotallymixed up here,but itcan allbeeasilyfixed bybeing moreprecisein thelabeling and languageused.They need to restrain their interpretation of the relative effects of proliferation and apoptosis on cell numberunlesstheyperform moreassaysspecificforproliferation and apoptosis. Wearegratefultothereviewerforpointingoutour misuses of ‘cellnumbers,proliferation,and apoptosis’and we feelvery sorry aboutthat.Now,we measured and presented with cellnumbersfor the effects of those inhibitors on cell growth behaviour (new Fig.4D).Cellnumbers were calculated using standard growth curve of each cell line, constructed by absorbance acquired from MTS staining corresponding to cell numbers. We also changed incorrect terms throughout the text and willkeeptheseimportantpointsinmindonourfutureworksaswell. Asadvised by thereviewer,to distinguish “cellnumber,cellproliferation,and apoptosis”in term ofcellgrowth,weperformed Annexin-V stainingtoindicateapoptoticcellsinTF1-GFPverseTF1- PRL-3 cells followed by FACS analysis. We showed more cell death in TF1-GFP cells (31% apoptotic cells)than in TF1-PRL-3 cells(6.8% apoptoticcells),suggesting thatPRL-3 playsan anti- apoptotic role in TF-1 cytokine dependentcellline.The data isshown in new Fig.5C and described in page 11, line 17-23.To study aroleofPRL-3 on apoptosisin cytokineindependentcelllines,we also performed Annexin-V stainingusingpairsof ‘MOLM-14 verseMOLM-14 PRL-3 KD’cells and ‘MV4-11 verseMV4-11 PRL-3 KD’cells.Wefound thatdepletion ofPRL-3 did notshow a increment of apoptotic population in these cytokine independent cell lines (Supporting Information. Fig.4).Sincetwocitedpapers,Pulikkan(Leukemia,2010)andRangatia(Oncogene,2003)describe the role of c-Jun in blocking myeloid differentiation,we cited these papersin the updated text,page 18,line1. - Fig.6.Itwouldbemuchmoredesirabletoperform theseexperimentsinabettermodelofFLT3- ITD AML than intravenous injection oftransformed celllines,butthe published models using FLT3- ITD alone(eitherretroviralorknock-in) are not robust. Newer models combining FLT3-ITD with other mutations look more promising, and it will be of great interest to test these models in the future with PRL3 antibody by itself and combined with FLT3-ITD inhibitors. Manythankforthisimportantinputinclinicalperspective.Asadvised,weperformedadditional animal model to see if we can improve our therapeutic effect when combined PRL-3 mAb with CEP701 inhibitor. Pre-treating mice with FLT3 inhibitor (CEP701), followed by PRL-3 antibody therapy, we found that the CEP701- PRL-3 antibody combined therapies has better therapeutic effectsthan PRL-3 antibody therapy alone. Thisisencouraging in ourinitialtrial,however,ourdataEMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 18 is not solid and it needs additionalexperimentsto reach aconclusion. At this moment, we sincerely thanks reviewer for this important suggestion from his/her clinical views (Fig. 6 is now Fig. 7). - Fig. 7. I never understand the value of investigating the prognostic value unless the therapy is related to the gene of interest. Almost all these patients are treated with anthracycline/AraC, so unlessresponseto thesedrugsisrelated to PRL3,Ido notknow how to interpret thedata.When PRL3 antibody is used clinically, it would make more sense. This is not natural history of the disease.Buteveryoneelsein theworld otherthan myselfseemsto thinkthesefindingshavevalue, so itis OK to leave itin the manuscript.Maybe Ijustdo notthink right. Weagreewiththereviewer that the prognostic value should reflect the therapy outcome related to the gene of interest. We have a couple of comments here. Firstly, patients treated with anthracycline/AraC have different outcomes therefore somegene or network mustbe influencing the response by the leukaemia cells. Additionally, the exact mechanism of action of anthracycline/AraC is not known so it may (or may not) involve PRL-3 oritsassociated networks. Secondly, the response may be related to PRL-3 directly or indirectly. Nevertheless, the survival curves do indicate that the high levels of PRL-3 can contribute a poorer survival and a less favourable response,which may be of potentialclinicaluse. In the presentstudy,we have shown thatPRL-3 overexpression hasan anti-apoptotic effectand enhances cell cycle progression. Therefore, AML patients showing high PRL-3 expression may have higher rate of cancer cell growth, aswell asmore resistant to apoptosis, than those patients withoutPRL-3 in theirAML cancercells.ThecohortanalysissuggeststhatPRL-3 overexpression contributesto poorprognosisofAML patients,and therefore,targeting orsuppressing PRL-3 may beapotentialtherapeuticapproach forPRL-3 overexpressing AML patients. The understanding of this reviewerissincerely appreciated (Fig.7 isnow Fig.8). - Perhapsitisduetotheidioticwordrestrictionsimposedbyjournals,butIthinkthefiguresneed to be described better: Manythanks!Wefeelsorryfornotpresentingclearlyinourpreviousfigurelegends.Wehavenow revised allthe Figure legends with a better writing. Fig.1B:?microarraystudies? We apologize that we confused the reviewer. Yes, Fig. 1B is microarray data from four independent AML patient cohorts; Belfast/MILE dataset (Cohort 1), GSE1159, GSE6891, and GSE15434. The data showed a strong association between FLT3-ITD mutation and high PRL-3 expression in a totalof1158 AML patients. Fig.1C and1D:?RNA?Protein?Whatarewelookingat? EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 19 We are very sorry for not being able to describe clearly in the Figure legend. Fig. 1C is the westernblotforFLT3andPRL-3 protein expression levelsin fourAML cell lines.Fig.1D isthe westernblottoshow thatdepletionofFLT3reducedPRL-3 endogenousprotein expression levels. FINALLY,PleasedoNOTeversendmeamanuscript to readwith tiny font!!!Theeasier it is to read a manuscript,the more the reviewer willlike it! We apologize for the tiny fonts used in our original Figures. To make bigger fonts wherever possible, in this revised manuscript, we have shortened the headings, and deleted unnecessary wordingsfrom theFigurestoallow largerfontsizestobeused.Wehopethereviewerwillfindthem clearernow. References Alarcon-Segovia D, Ruiz-Arguelles A, Fishbein E (1978) Antibody to nuclear ribonucleoprotein penetrateslive human mononuclear cells through Fc receptors, Nature271: 67-69 FerroneS (2011)Hiddenimmunotherapytargetschallengedogma.SciTranslMed 3:99ps38 Dhillon AS, Hagan S, Rath O, Kolch W (2007) MAP kinase signalling pathways in cancer. Oncogene26:3279-3290 EwingRM,ChuP,ElismaF,LiH,TaylorP,ClimieS,McBroom-CerajewskiL,RobinsonMD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, BukhmanYV,EthierM,ShengY,VasilescuJ,Abu-FarhaM,LambertJP,DuewelHS,Stewart,II, KuehlB,HogueK,ColwillK,GladwishK,MuskatB,KinachR,AdamsSL,MoranMF,Morin GB,TopaloglouT,FigeysD (2007)Large-scale mapping ofhuman protein-protein interactionsby massspectrometry.MolSystBiol3:89 FiordalisiJJ,KellerPJ,CoxAD (2006)PRL tyrosinephosphatasesregulaterhofamilyGTPasesto promoteinvasion and motility.Cancerresearch66:3153-3161 Jin A, Kurosu T, Tsuji K, Mizuchi D, Arai A, Fujita H, Hattori M, Minato N, Miura O (2006) BCR/ABLandIL-3 activateRap1 to stimulatetheB-Raf/MEK/ErkandAktsignalingpathwaysand to regulate proliferation, apoptosis, and adhesion. Oncogene25:4332-4340 LiangF,LiangJ,Wang WQ,Sun JP,Udho E,Zhang ZY (2007)PRL3 promotescellinvasion and proliferation by down-regulation of Csk leading to Src activation.J BiolChem 282:5413-5419 MingJ,LiuN,GuY,QiuX,WangEH(2009)PRL-3 facilitatesangiogenesisand metastasisby increasing ERK phosphorylation and up-regulating the levels and activities of Rho-A/C in lung cancer.Pathology41:118-126 MizukiM,FenskiR,HalfterH,MatsumuraI,SchmidtR,MullerC,GruningW,Kratz-AlbersK, ServeS,SteurC,BuchnerT,Kienast J, Kanakura Y, Berdel WE, Serve H (2000) Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways.Blood96:3907-3914 NosakaT,KawashimaT,MisawaK,IkutaK,MuiAL,KitamuraT(1999)STAT5 asamolecular regulator of proliferation,differentiation and apoptosis in hematopoietic cells.TheEMBO journal 18:4754-4765 PengL,XingX,LiW,QuL,MengL,LianS,JiangB,WuJ,ShouC (2009)PRL-3 promotesthe motility,invasion,andmetastasis of LoVo colon cancer cells through PRL-3-integrin beta1-ERK1/2 and-MMP2signaling.MolCancer8:110 Songyang Z, Baltimore D, Cantley LC, Kaplan DR, Franke TF (1997) Interleukin 3-dependent survivalby the Aktprotein kinase.ProceedingsoftheNational Academy of Sciences of the United StatesofAmerica 94:11345-11350 SpiekermannK,PauM,SchwabR,SchmiejaK,FranzraheS,HiddemannW (2002)Constitutive activation ofSTAT3 and STAT5 isinduced by leukemic fusion proteinswith protein tyrosine kinase activity and is sufficient for transformation of hematopoietic precursor cells. Experimental hematology30:262-271 EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 20 2nd EditorialDecision 11 June 2013 ThankyouforthesubmissionofyourrevisedmanuscripttoEMBO MolecularMedicine.Wehave now received theenclosed reportsfrom theReviewers1 and 3 (Reviewer2 wasnotavailable)that wereaskedtore-assessit.Asyou willsee,since the Reviewersare now supportive and you have also satisfactorily addressed Reviewer2'sconcerns,Iam pleased to inform you thatwe willbe able to accept your manuscript pending the following final amendments: 1)AsperourAuthorGuidelines,thedescription ofallreported datathatincludesstatisticaltesting muststatethenameofthestatisticaltestusedto generate error bars and P values, the number (n) of independent experiments underlying each data point (not replicate measures of one sample), and the actualP value foreach test(notmerely 'significant'or'P < 0.05'). 2)Please correctthe mistake in Fig. 4C as indicated by Reviewer 1 Finally,IsuggestyoutakenoteofReviewer3'sverysoundadviceinthefuture! Pleasesubmityourrevisedmanuscriptwithintwoweeks.Ilookforwardtoseeingarevisedform of yourmanuscriptassoon aspossible. ***** Reviewer'scomments***** Referee#1(Remarks): Minorpoint:Fig.4C.c-Jun should read p-c-Jun in allpanels. Referee#3(Remarks): Theauthorshavedoneagoodjobrespondingtoallofthe reviewers.I only have a few "editorial" comments: 1.Ilook forward to when PRL-3 antibodiesareused clinically in humans. 2.Iurgetheauthorsto continuein thefutureto try to understand the mechanisms of how PRL-3 works(whatarethedirecttargetsand pathways),aswellashow theantibody works.Both are importantand interesting questions. 3.Theauthorsmisunderstood my remarksaboutfonts.WhileIam pleased thatauthorsincreased thesizesofthefontsin thefigures, I was also referring to the size of the fonts used in the textof the manuscript.Assumingyoureallywantmetoreadit,whyuse10point font? Nexttime please increase the fontsize of the TEXT !(Hint: EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 21 try 12 point). Also,itwouldbeusefultolabelthefiguresaswell("Figure 1","Figure2",etc)on thesamesideofthepageasthefigure. Again,theeasieritisforthereviewertoread,thehappierwewillbe... 2nd Revision - authors'response 18 June 2013 ResponsetoEditorialcomments: 1)AsperourAuthorGuidelines,thedescription ofallreported data thatincludesstatisticaltesting muststatethe name of the statistical testused to generateerrorbarsand P values,thenumber(n)of independent experiments underlying each data point (not replicate measures of one sample), and the actualP valueforeach test(notmerely 'significant'or'P < 0.05'). Thankyouforyourguidance.WeamendedourmanuscriptaccordingtoAuthorGuidelinestoshow the name of the statistical testto generate error bars and actual P values. When actual p-valuewas low (p < 1x10-3),we used ‘p < 0.001’whereitwasapplicable.Threeindependentexperimentswere performed.Pleaseseebelow table. Amendedonmanuscript Fig.1B.Microarraydata Chi-square test In Fig.1B legend on page37 a.p = 0.001 b-d.p < 0.001 Fig.3F,a.Q-RT-PCR Student’st-test In Fig.3F legend on page39 ***p < 0.001forTF1-ITD **p = 0.011forMOLM-14 *p = 0.038forMV4-11 Fig.5C.FACS analysis Student’st-test In Fig.5C legend on page40 ***p = 0.0012EMBO MolecularMedicine PeerReview ProcessFile- EMM-2012-02183 © EMBO 22 Fig.7 7A.Student’st-test 7B.Student’st-test 7C.Kaplan-Meieranalysis In Fig.7legend on page41 *p < 0.001;**p = 0.00121 *p < 0.001 p < 0.001 Fig.8 Kaplan-Meiersurvivalanalysis In Fig.8 A. p = 0.028B. p < 0.001C. p = 0.025 Table1.Cox-regression analysis Age Univariate,p < 0.001 Multivariate,p<0.001 Cytogeneticrisk Intermediate,univariate,p < 0.001 Intermediate,multivariate,p = 0.001 Adverse,univariate,p<0.001 Adverse,multivariate,p<0.001