t-cell protein tyrosine phosphatase (tcptp) is...

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T-cellproteintyrosinephosphatase(TCPTP)isirreversiblyinhibitedby

etoposide-quinone,areactivemetaboliteofthechemotherapydrugetoposide

Qing NIAN, Jérémy BERTHELET, Wenchao ZHANG, Linh-Chi BUI, Rongxing LIU, Ximing XU,

Romain DUVAL, Saravanan GANESAN, Thibaut LEGER, Christine CHOMIENNE, Florent BUSI,

FabienGUIDEZ,Jean-MarieDUPRETandFernandoRODRIGUESLIMA

UniversitédeParis,BFA,UMR8251,CNRS,F-75013,Paris,France(QN,JB,WZ,LCB,RL,FB,JMD,

FRL);KeyLaboratoryofMarineDrugs,ChineseMinistryofEducation,SchoolofMedicineand

Pharmacy,OceanUniversityofChina,5YushanRoad,Qingdao,266003,China(XX);Université

deParis,BIGR,UMRS1134,INSERM,F-75015,Paris,France(RD);UniversitédeParis,Institutde

RechercheSaint-Louis,UMRS1131,INSERM,F-75010,Paris,France(SG,CC,FG);Universitéde

Paris, IJM, UMR 7592, CNRS, F-75013, Paris, France (TL); Service de Biologie Cellulaire,

AssistancePubliquedesHôpitauxdeParis,HôpitalSaintLouis,F-75010,Paris,France(CC)

This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on June 20, 2019 as DOI: 10.1124/mol.119.116319

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Runningtitle:EtoposidequinoneimpairsthephosphataseactivityofTCPTP

Correspondingauthor:FernandoRODRIGUESLIMA,UniversitédeParis,BFA,UMR8251,CNRS,

F-75013,Paris,France;email:[email protected]

Textpages:31

Tables:0

Figures:7

References:44

Abstract:200words

Introduction:661

Discussion:910

Abbreviations: DTT: dithiothreitol; ETOP: etoposide; EQ: etoposide ortho-quinone; IAF:

fluorescein 5-iodoacetamide; MPO: myeloperoxidase; NBT: nitroblue tetrazolium; pNPP: p-

nitrophenyl phosphate; PTPN2: Tyrosine-proteinphosphatasenon-receptor type2; TCPTP: T-

cellproteintyrosinephosphatase.


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ABSTRACTEtoposideisawidelyprescribedanticancerdrugthatis,however,associatedwithanincreased

riskofsecondaryleukaemia.Althoughthemolecularbasisunderlyingthedevelopmentofthese

leukaemias remains poorly understood, increasing evidence implicates the interaction of

etoposidemetabolites (i.e.etoposidequinone,EQ)with topoisomerase IIenzymes.However,

effectsof etoposidequinoneonother cellular targets couldalsobeatplay.We investigated

whetherTCPTP,aproteintyrosinephosphatasethatplaysakeyroleinnormalandmalignant

haematopoiesisthroughregulationofJAK/STATsignallingcouldbeatargetofEQ.

WereportherethatEQisanirreversibleinhibitorofTCPTPphosphatase(IC50=~7µM,second-

orderrate inhibitionconstantof~810M-1.min-1).Noinhibitionwasobservedwiththeparent

drug.The inhibitionbyEQwas foundtobeduetotheformationofacovalentadductat the

catalytic cysteine residue in theactive siteof TCPTP.Exposureofhumanhematopoietic cells

(HL-60and Jurkat) toEQ led to inhibitionofendogenousTCPTPandconcomitant increase in

STAT1 tyrosine phosphorylation. Our results suggest that in addition to alteration of

topoisomerase II functions, EQ could also contribute to ETOP-dependent leukaemogenesis

throughimpairmentofkeyhematopoieticsignallingenzymessuchasTCPTP.


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INTRODUCTION

Protein tyrosinephosphatases (PTPs)are important regulatorsof theactivityofnumerous

signalling pathways involved inmajor cellular processes such as cell growth, proliferation

anddifferentiation(Tonks,2006;Tiganisetal.,2007;Pikeetal.,2016).T-cellproteintyrosine

phosphatase (TCPTP) is a cytosolic tyrosine phosphatase which is ubiquitously expressed.

However,thehighestlevelsofexpressionofTCPTParefoundinhaematopoieticcellswhere

thisenzymemodulatesgrowthfactorandcytokinesignallingpathways,thuscontributingto

immune and hematopoietic cell homeostasis (Wiede et al., 2012; Pike et al., 2016). In

particular,TCPTPnegativelyregulatesJAK/STATsignallingthroughthedephosphorylationof

different tyrosinephosphorylated JAK/STATproteins suchas STAT1or JAK1 (tenHoeveet

al., 2002; Dorritie et al., 2014; Pike et al., 2016). In addition to STAT1, TCPTP also

dephophorylates STAT3 and STAT5 and negatively regulates their activation (Pike et al.,

2016).TheJAK/STATpathwayplaysacrucialroleinhaematopoiesisandaberrantactivation

of STAT signalling is involved in leukaemogenesis (Dorritie et al., 2014; Pike et al., 2016).

Interestingly, deletions or inactivating mutations of TCPTP were identified in T-cell

leukaemiaandnon-Hodgkin’s lymphomaandassociatedwithelevatedSTATsignallingand

changes ingeneexpression (Kleppeetal.,2010;Kleppeetal.,2011a;Kleppeetal.,2011b;

Pike et al., 2016). In addition, it has been reported that TCPTP is overexpressed inMYC-

driven mouse B cell lymphoma (Young et al. 2009). The importance of TCPTP in

haematopoiesis is further supported by knockout mouse models (PTPN2-/-), where the

absence of TCPTP induces severe hematopoietic defects (affecting lymphoid,myeloid and

erythroidlineages)andprogressivesystemicinflammationleadingtodeath(You-Tenetal.,

1997; Bourdeau et al., 2007; Heinonen et al., 2009; Wiede et al., 2012). These studies

highlighttheimportantroleofTCPTPinnormalandmalignanthaematopoiesis.


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Etoposide(ETOP)isawidelyusedanticancerdrugtotreatavarietyofhumanmalignancies

(Hande,1998;Baldwinetal.,2005).Themechanismsofactionproposed for itsantitumor

activity are based mainly on its interaction with topoisomerases II (Baldwin et al., 2005;

Deweeseetal.,2009).ETOPisindeedknowntoaffectthecatalyticcycleoftheseenzymes

and to stabilize topoisomerase II-bound DNA strand breaks which have the potential to

activatecelldeathpathways(Jacobetal.,2011).DespitethewideclinicaluseofETOP,this

chemotherapeutic drug is known to induce treatment-related leukaemias (Baldwin and

Osheroff, 2005; Pendleton et al., 2014). In humans, ETOP can be oxidized by cytochrome

P450(CYP3A4)andmyeloperoxidasesintoetoposidequinone(EQ)whichwasfoundtohave

aneffectontopoisomeraseIIenzymesseveraltimesstrongerthanthatoftheparentdrug

(Zhuoetal.,2004;Fan etal,2006; Jacobetal.,2011;Vlasovaetal.,2011).Although the

molecular basis for ETOP-induced leukaemogenesis is not well understood, evidence

increasingly indicates that EQ is a critical contributor to thedevelopmentof ETOP-related

secondary leukaemia, in particular through alteration of topoisomerase II-functions

(GantchevandHunting,1998;Kaganetal.,1999;Fanetal.,2006;Vlasovaetal.,2011;Smith

etal,.2014;Gibsonetal.,2016).However, interactionsofETOPreactivemetaboliteswith

other cellular proteins and macromolecules may also contribute to ETOP-dependent

leukaemogenesis(Fanetal.,2006;Rojasetal.,2009).

We show here that EQ is an irreversible inhibitor of TCPTP phosphatase activity.

Kinetic andmolecular analysesusingpurifiedhumanTCPTP indicated that the irreversible

inhibitionoftheenzymebyEQisduetotheformationofacovalentadductwithitscatalytic

cysteine. Accordingly, exposure of cultured hematopoietic cells to EQ leads to the

irreversible inhibition of the endogenous TCPTP with a concomitant increase in cellular

STAT1 phosphorylation. Interestingly, it is well known that dysregulation of the JAK/STAT


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signallingpathway is involved in leukaemogenicprocesses (Beneklietal.,2009;Dorritieet

al.,2014).Altogether,ourdatasuggestthatinadditiontothedisruptionoftopoisomeraseII

functions, EQ may also contribute to ETOP-related leukaemia through alteration of

importanthematopoieticsignalingenzymessuchasTCPTP.


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METHODS

Chemicalsandcells

Etoposide (ETOP) and etoposide quinone (EQ) were obtained from Toronto Research

Chemicals(NorthYork,Canada).Hydrogenperoxide,N-ethylmaleimide(NEM),fluorescein5-

iodoacetamide (5-IAF), myeloperoxidase (MPO), p-nitrophenyl phosphate (pNPP), sodium

orthovanadate, nitroblue tetrazolium (NBT), dimethyl sulfoxide (DMSO) were purchased

fromSigma-Aldrich(France).ETOPandEQweredilutedinDMSOatastockconcentrationof

100 mM. HL60 (human acute promyelocytic leukaemia) and Jurkat (human acute T cell

leukaemia)cellswerefromSigma-Aldrich(France).

ExpressionandpurificationofrecombinanthumanTCPTPenzyme

Human TCPTP enzyme was expressed and purified as previously described (Duval et al.,

2015),usingBL21(DE3)Escherichiacoli, transformedwithapET28aplasmidcontainingthe

cDNAofhumanPTNP2(TC45variant).Thepurifiedenzymewasreducedbyincubationwith

10mMDTTfor10minutesinicebeforebuffer-exchangewithPD-10column(GEHealthcare,

France) into 25 mM Tris-HCl, 150 mM NaCl, pH 7.5 (reaction buffer). The protein

concentrationwas determined by the Bradford reagent (Bio-Rad, France), and puritywas

assessed by SDS-PAGE and Coomassie staining. Purified recombinant human TCTPT (1

mg/ml)wasstoredat-80°Cuntiluse.

DeterminationofTCPTPactivityusingp-nitrophenylphosphate(pNPPassay)

Thephosphataseactivitywasmeasuredusingp-nitrophenylphosphate(pNPP)assubstrate

asdescribedpreviously(Montalibetetal.,2005).Typically,samplescontainingTCPTPwere

diluted10 timeswith100mMsodiumacetatebuffer (pH6,1mMDTT) containing5mM


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pNPP. The formation of the product (p-nitrophenol) was tracked by continuous

measurementoftheabsorbanceat405nmat37°Cusingathermostaticmicroplatereader

(BioTek, France) in a total volume of 250µl. The final concentration of TCPTP during the

assaywas100nM.

Determination of TCPTP activity using a tyrosine-phosphorylated STAT1 peptide (FAM-

pSTAT1)

The tyrosine phosphatase activity of TCPTP was measured by reverse phase fast liquid

chromatography (RP-UFLC)usinga fluorescein (FAM)-conjugatedpeptidederived fromthe

sequenceofhumanSTAT1(697KGTGYIKTE705wheretheY701isphosphorylated)asdescribed

previously(Duvaletal.,2015).Briefly,samplescontainingTCPTPwerediluted100-foldwith

acetatebuffer.Aliquots(100µl)were incubatedfor30minat37°C inpresenceof50µM

FAM-pSTAT1peptide (finalconcentrationofTCPTPwas10nM).Thereactionwasstopped

with 100 µl of 15% HClO4 (v/v) prior to analysis by RP-UFLC using a Shim-pack XR ODS

column(Shimadzu,France)connectedtoaProminenceShimadzuUFLCsystem.

EffectsofetoposideandetoposidequinoneonTCPTPactivity

RecombinantTCPTP (1µM)was incubatedwithETOPordifferent concentrationsofEQ in

100mMsodiumacetate,pH6for30minutesat37°C(totalvolumeof50µl).Sampleswere

diluted 10 times then assayed for residual TCPTP activity using pNPP. To obtain the IC50

value, the dose-response curves were fitted with the Hill equation,

TCPTPactivity=100/(1+10^((LogIC50-[EQ])*Hillslope)),where IC50 is thehalfmaximal inhibitory

concentrationofEQ.TheeffectsofETOPandEQontheTCPTPtyrosinephosphataseactivity


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werealsoassessedbyaRP-UFLCapproachusingatyrosinephosphorylatedSTAT1peptide

(seeabove).

EffectsofreducingagentsandbufferexchangeonTCPTPinhibitedbyEQ

TCPTP (1µM)was first incubatedwith EQ (40µM)or ETOP (100µM) in100mMsodium

acetate,pH6for30minat37°C(totalvolumeof50µl).Sampleswereeitherincubatedwith

1or10mMDTT(10minatroomtemperature)orbufferexchanged(PDSpinTrapG-25,GE

Healthcare, France) to 100 mM sodium acetate, pH 6, prior to measurement of residual

TCPTPactivityusingpNPP.

Fluorescein-conjugated iodoacetamide labelling, nitroblue tetrazolium staining and

detectionofoxidizedTCPTPcatalyticcysteine

TCPTP (1µM)was first incubatedwith ETOP (100µM)or EQ (40µM) in 100mMsodium

acetatepH6at37°Cfor30minfollowedbytheadditionof20µM5-IAF(totalvolumeof50

µl)andfurtherincubationat37°Cfor10min(inthedark).SampleswereseparatedbySDS-

PAGE and transferred onto nitrocellulose membranes. 5-IAF covalent labelling of TCPTP

cysteine residues was detected by western blot using anti-fluorescein antibodies (Sigma-

Aldrich, France, ref: # 11426346910).Membraneswere stripped and further probedwith

anti-TCPTPantibody(Sigma-Aldrich,France,ref:#SAB4200249).

The formationof covalentadductsofEQonTCPTPwasdetectedbynitroblue tetrazolium

(NBT) redox cycling staining as reported previously�Paz et al., 1991�. Briefly, TCPTP (1

µM)wasincubatedwithETOP(100µM)orEQ(40µM)in100mMsodiumacetatepH6at

37°Cfor30min(totalvolume50µl).SampleswereseparatedbySDS-PAGEandtransferred

onto nitrocellulose membranes. EQ-bound TCPTP on the membranes was stained by


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incubationwith0.6mg/mlNBTin2MpotassiumglycinatepH10for30-45mininthedarkat

roomtemperature.Next,membraneswerewashedthoroughlywithPBSandfurtherprobed

withanti-TCPTPantibody(Sigma-Aldrich,France,ref:#11426346910).

ForthedetectionofoxidizedTCPTPcatalyticcysteine,TCPTP(1µM)wastreatedwithETOP

(100µM), EQ (40µM) or H2O2 (100µM) in a total volume of 50µl. Aliquots were then

separatedbySDS-PAGEandanalysedbywesternblotwithaspecificanti-oxidizedPTPactive

site antibody (R&D system, USA, ref:# MAB 2844) as described previously (Duval et al.,

2015).

Effectsofmyeloperoxidase(MPO)-dependentbioactivationofETOPintoEQ

MyeloperoxidasewasusedtobioactivateETOPintoEQasdescribedpreviously(Kaganetal.,

1999;Fanetal.,2006;Vlasovaetal.,2011).Tothisend,MPO(5units)wasincubatedwith

ETOP(100µM)andH2O2(100µM) in100mMsodiumacetate,pH6.5for30minatroom

temperature(inatotalvolumeof1ml).Heat-inactivatedMPO(boiledfor30min)wasused

as a negative control. Catalase (300 Units/ml final concentration) was added to remove

excess H2O2. Finally, reaction mix (48 µl) was added to recombinant TCPTP (1 µM final

concentration) in 100mM sodium acetate, pH 6 and incubated for 30min at 37°C (total

volumeof50µl).After incubation,anda ten-timesdilution, the residualactivityofTCPTP

wasmeasuredusingthepNPPassay.IAFandNBTlabellinganddetectionofoxidizedTCPTP

catalyticcysteinewerealsocarriedoutonTCPTPtreatedwithMPOasdescribedabove.

KineticsofTCPTPinhibitionbyEQ

ThekineticdatawereanalysedasreportedinCopeland(2005)forirreversibleinhibitors.The

kinetic data were fitted and plotted using Qtiplot software (http://www. qtiplot.com/).


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Briefly,TCPTP(3µM)wasincubatedwithdifferentconcentrationsofEQ(pseudofirst-order

conditions) in100mMsodiumacetatepH6at37°C.Atdifferenttimepoint,aliquotswere

removedandassayedforresidualTCPTPactivityusingpNPPassay.Therateofinhibitionof

TCPTPbyEQcanberepresentedas:Ln[%residualactivity]=kobsxt(wheretistimeandkobs

istheapparentfirst-orderinhibitionrateconstant).Theapparentfirstorderinhibitionrate

constants(kobs=kinactx[EQ])canbecalculatedforeachEQconcentrationfromtheslopeof

thenatural log(Ln)ofpercentresidualactivityplottedagainsttime.Thesecond-orderrate

constant(kinact)wasdeterminedfromtheslopeofkobsplottedagainstEQconcentrations.

Effects of EQ on TCPTP in the presence of the competitive PTP inhibitor orthovanadate

(Na3VO4)

TCPTP(1µM)wasincubatedwith1mMorthovanadateinthepresenceofabsenceofEQ(40

µM)in100mMsodiumacetatepH6at37°Cfor30min.Samplesweredilutedtentimesin

100mMsodiumacetatepH6containing1mMEDTAandresidualTCPTPactivitymeasured

usingpNPPassay.

Moleculardocking

TCPTP protein structure data was obtained from RCSB PDB database (PDB_ID 1L8K). The

protein structure was prepared using the Prepwizard module in the Schrodinger suite

(SchrödingerLLC,2019,https://www.schrodinger.com).Briefly,missingsidechainsorloops

werecompleted.ThepKavaluesofresidueswerepredictedandhydrogenswereaddedat

pH7.Watermoleculeswere removed.Finally, thestructurewasoptimizedbya restrained

energyminimizationwith OPLS3 force field. The ligand (EQ)was constructed byMaestro

moduleandwasalsorefinedbyOPLS3forcefield.Covalent-dockingwascarriedoutwiththe


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catalyticresidueCys216selectedasthedockingcentre.Gridboxsizewassetto20x20x20

Å3. Michael addition reaction type was selected for the covalent docking. The final

conformationwasselected,andimageswerepreparedwithVMDprogram(Humphreyetal.,

1996).

Massspectrometryanalysis

TCPTP(1µM)wasincubatedwith100µMEQfor30minat37°Cin100mMsodiumacetate

pH 6. After reduction with DTT (10 mM), the samples were diluted 10 times in sodium

acetate buffer andunmodified thiolmoieties of cysteineswereblockedby additionof 10

mM NEM for 10 min. Samples were then incubated overnight at 37 °C with trypsin

(Promega,France)at12.5ng/μlin25mMammoniumbicarbonatepH8.0.Thesupernatant

containingpeptideswasandacidifiedwithformicacid(FA),desaltedonC18tips(PierceC18

tips, Thermo Scientific), and eluted in 10 µl 70% ACN, 0.1% FA. Desalted samples were

evaporatedusingaSpeedVacthentakenupin10µlofbufferA(bufferA:water,0.1%FA)

and 5µlwere injected on a nanoLCHPLC system (Thermo Scientific, France) coupled to a

hybrid quadrupole-Orbitrapmass spectrometer (Thermo Scientific, France). Peptideswere

loadedon a reverse phase C18µ-precolumn (C18 PepMap100, 5µm, 100A, 300µm i.d.x5

mm) and separated on a C18 column (EASY-spray C18 column, 75 µm i.d.x50 cm) at a

constantflowrateof300nl/min,witha120mingradientof2to40%bufferB(bufferB:20%

water, 80% ACN, 0.1% FA). MS analyses were performed by the Orbitrap cell with a

resolution of 120.000 (atm/z 200). MS/MS fragments were obtained by HCD activation

(collisionalenergyof28%)andacquiredintheiontrapintop-speedmodewithatotalcycle

of3seconds.ThedatabasesearchwasperformedagainsttheSwissprotdatabase(02/2017)

and the Homo sapiens taxonomy with Mascot v2.5.1 software with the following


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parameters: tryptic peptides only with up to 2 missed cleavages, variable modifications:

cysteine EQ and methionine oxidation. MS and MS/MS error tolerances were set

respectively to7ppmand0.5Da. Peptide identificationswere validatedusing a 1%False

Discovery Rate (FDR) threshold obtained by Proteome Discoverer (version 2.2, Thermo

Scientific) and the percolator algorithm. The candidate sequences modified by EQ were

manuallyinspectedfordenovosequencing.

Cellculture,TCPTPimmunoprecipitationandSTAT1phosphorylationkinetics

HL60andJurkatcellsweremaintainedinRPMI1640mediumsupplementedwith10%heat-

inactivatedfoetalbovineserumand1mML-glutamineinT-75flasks.Cells(60mlat2x106

cells/ml)werewashedwithPBSpriortoexposureto50µMEQ(orDMSO)for30minat37°C

(5%CO2)inRPMI1640medium.

For immunoprecipitation of endogenous TCPTP, treated cells were washed with PBS and

resuspended in lysis buffer (PBS, 1% Triton X-100, 1 mM sodium orthovanadate,

phosphatasecocktail inhibitor2andproteaseinhibitorscocktail(Sigma-Aldrich,France)for

20min.Celllysateswerecentrifugedat15000xgfor10minat4°Candsupernatants(whole

cellextracts)weretaken.TCPTPwasimmunoprecipitatedbyincubating1mgofwholecell

extractswith1µgofpolyclonalTCPTPantibody(Sigma-Aldrich,France,ref:#SAB4200249)

overnightat4°C.Sampleswerethenrockedforonehourat4°C inpresenceof30µlof

protein A–Agarose (Santa Cruz, Germany). The immunobeads were harvested by

centrifugation,washed3timeswithlysisbufferandsplitintotwoportions.Onepartofthe

beads was incubated with 50 µM of FAM-pSTAT1 peptide in order to measure residual

immunoprecipitated-TCPTP activity by RP-UFLC as described above. The second part of

immunobeads was incubated with non-reducing Laemmli sample buffer. Eluates were


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separated by SDS-PAGE and analysed by western blot using with an anti-TCPTP antibody

(R&DSystem,USA,ref:# MAB1930).

FortheSTAT1phosphorylationkineticanalysis,cellstreatedwithEQorDMSOwerewashed

with freshmedium and incubated for 20min inmedium supplementedwith 10 ng/ml of

human IFNg. Cells were then washed in fresh medium and aliquots (2x106 cells) were

removed at different time points,washedwith PBS and lysed. STAT1 activationwas then

assayedinlysatesbywesternblotusinganti-phospho-STAT1(phosphorylatedTyrosine701,

CellSignaling,France,ref:#9176).Membraneswerere-probedsequentiallywithanti-STAT1

(Cell Signaling, France, ref: #9167), anti-TCPTP (Sigma-Aldrich, France, ref: #SAB4200249)

and anti-b actin (Cell Signaling, France, ref: #3700) antibodies. Quantification of STAT1

phosphorylation inwesternblotswas carriedout using ImageJ software (Schneider et al.,

2012).

Statisticalanalysis

Thestudywasdesignedinsuchamannerthatthreeindependentreplicatesareconducted

for each experiment prior to statistical analysis. All data are expressed as the mean ±

standard deviation (SD). All statistical analyses were then carried out using Prism 5.03

(GraphPadSoftware,LaJolla,CA)withaP-value<0.05threasholdtoconsiderdifferencesas

statisticallysignificant.Forexperimentswheremultiplegroupswerecomparedtocontrol,a

one-wayanalysisofvariance(ANOVA)wasused.Ifstatisticalsignificancewasreached,data

werefurtheranalyzedwithDunnett’sposthoctest.Forexperimentsbetweentwogroups,a

two-tailedStudent’s t testwasused.Noexperimentwas removed from the final analysis.

For qPCR experiments (supplementary figure 3), 6 experimental pointswere used in data


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analysis,whichconsistedinaStudent’sttesttocomparetheeffectofEQvscontrolincells

inducedwithinterferongamma.


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RESULTS

EffectofetoposidequinoneonactivityofTCPTP

The effects of ETOP and EQwere assessed on the activity of recombinant human TCPTP

expressed and purified from bacteria. As a first experiment, TCPTP was incubated in the

presenceof100µMETOPor increasingconcentrationsofEQpriortothemeasurementof

theresidualenzymeactivitywiththechromogenicpNPPphosphatasesubstrate.Asshownin

Figure1A,EQinhibitsthehydrolysisofthissubstrateinadose-dependentmannerwithhalf-

maximal inhibitory concentration (IC50) values of 7.3 µM (supplementary Figure 1).

Conversely, 100 µM ETOP did not display any inhibitory effect, therefore suggesting that

inhibitionofTCPTPactivitycouldoccurwithEQbutnotwiththeparentdrug.Theseresults

were further validated with a UFLC-based enzyme assay using a more specific TCPTP

substrate consisting of a phospho-STAT1 peptide derived from the sequence of human

STAT1 (697KGTGYIKTE705 were the Y701 is phosphorylated) as described in (Duval et al.,

2015).UFLCquantificationof thedephosphorylatedSTAT1confirms thatEQbutnotETOP

inhibitsTCPTPphosphataseactivity(Figure1B).

EtoposidequinonecovalentlyreactswithTCPTPcysteineresidues

AsEQbutnotETOPinhibitsTCPTPactivity,wenexthypothesizedthatthis inhibitioncould

resultfromthegeneralchemicalreactivitypropertiesofquinonechemicalsandnotablythe

possibilitythattheyreactcovalentlywithproteins(Boltonetal.,2017;Boltonetal.,2018).

Nitrobluetetrazolium(NBT)canbeusedtostainproteinscovalentlymodifiedbyquinonesto

formquinone-proteins.Asshown inFigure2A,NBTreactiongeneratedonlyasignalwhen

TCPTPwastreatedwithEQ,confirmingthepresenceofcovalentquinoneadductsonTCPTP.


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Knowing that protein cysteinyl residues are common nucleophilic targets of quinone

electrophiles, we therefore strived to show whether TCPTP cysteinyl residues could be

modified after exposure to EQ. TCPTP was treated with EQ and then incubated with

fluorescein-conjugated iodoacetamide (IAF), a specific cysteinyl thiol reagent. Our results

(Figure 2B) show that EQ decreased IAF labelling of TCPTP, therefore indicating that EQ

reactswithcysteineresiduesofTCPTP.

Quinones chemicals can also display redox cycling properties that leads to generation of

reactive oxygen species (ROS). In addition, it is known that TCPTP can be inhibited by

reactiveoxygenspeciesthroughtheoxidationofitscatalyticcysteineresidue(Ostmanetal.,

2011).We therefore evaluatedwhether EQ could oxidize the catalytic cysteine residueof

TCPTPusinganantibody specificallydirectedagainstoxidized catalytic cysteine residueof

proteintyrosinephosphatases(Duvaletal.,2015).Figure2CshowsthatEQtreatmentdoes

notleadtoTCPTPactivesitecysteineoxidation,and,inaddition,thatthebasaloxidationof

catalyticcysteineachievedduringexperimentandobservedincontrolandETOPtreatment

isdecreasedwithEQ.ThisisagreementwiththeformationofanEQadductonthecatalytic

cysteineoftheenzyme.Therefore,thesedataaltogethersuggestalossofTCPTPactivitydue

toEQadductionofthecatalyticcysteine.

When clinically administered to patients, ETOP is biochemically metabolized to EQ by

different enzymes notably myeloperoxidase which is highly expressed in bone marrow

hematopoietic cells (Fan et al., 2006; Atwal et al., 2017). To test if EQ generated from

peroxidase/H2O2-dependentbioactivationofETOPwouldyieldsimilarTCPTP inhibition,we

usedaninvitroperoxidaseactivationsystemthatmimicsthebioactivationofETOPintoEQ

in hematopoietic cells described previously (Kagan et al., 1999; Vlasova et al., 2011). As

showninSupplementaryFigure2A, inpresenceofafunctionalperoxidase/H2O2enzymatic


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system, ETOP is capable of inhibiting TCPTP activity with concomitant quinone-adduction

(NBTlabelling)andlossofIAFcysteinelabellingofTCPTPprotein(SupplementaryFigure2B

andC). Conversely,when inactiveperoxidase is used, no inhibitionof TCPTPandquinone

adductiononcysteineresiduesisobserved(supplementaryFigure2).ThisconfirmsthatEQ

butnottheparentdrugcaninhibitTCPTPactivitythroughcovalentadduction,aresultwhich

is consistent with the electrophilic nature of the quinone moiety of EQ which enables

Michaeladditionwiththiolgroups(Fanetal.,2006;Boltonetal.,2018).

IdentificationofthecatalyticcysteineresidueofTCPTPasatargetofEQ

Orthovanadate, a transition state analogue generally used as a competitive protein

phosphotyrosinephosphataseinhibitor,wasusedtoevaluatewhetherEQinhibitioninvolves

reactionsattheactivesiteoftheenzymeasdescribedpreviously(Seineretal.,2007).Figure

3A shows that vanadate confers protection to TCPTP as the inhibition of the enzyme is

significantly slowed by the competitive inhibitor thereby providing evidence that the

reaction is active-site directed. Computational analysis using covalent docking approaches

further indicatedthatthecatalyticcysteineofTCPTP(Cysteine216)canformanadduct in

theactivesitethroughtheformationofacovalentbondbetweenthesulphuratomandthe

carbon6’ofthequinonemoiety(Figure3B).

InordertoascertainthatthecatalyticcysteineresidueofTCPTPcanbecovalentlyadducted

by EQ, we analysed recombinant protein treated with EQ using LC-MS-MS. As shown in

Figure 3C, the [M+3H]3+ molecular ion of the tryptic peptide harbouring the catalytic

cysteinedisplaysa190.9m/zincreaseaftertreatmentwithEQ,correspondingtothemass

oftheEQadduct.ThismassofEQadductisalsoconfirmedinthea162+,y172+andy203+ions

ofthefragmentedpeptide.Itiswell-knownthattyrosinephosphatasesarereadilyinhibited


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bychemicalsabletomodifytheircatalyticcysteine(Seineretal.,2007;Ostman,Frijhoffet

al.,2011).AltogethertheseexperimentsindicatethatEQimpairsTCPTPactivitythroughthe

covalentadductionofitscatalyticcysteine.

CharacterizationofreversibilityandkineticsofTCPTPinhibitionbyEQ

TCPTP inhibitionbyEQwas furtherevaluated for reversibility. To this end, TCPTPenzyme

was inhibited by EQ and subsequently subjected to a buffer exchange experiment before

measuring its residual activity.Consistentwith thedatadescribedabove,bufferexchange

could not restore TCPTP activity (Figure 4A). Depending on the chemical structure of the

quinones, reversibility of cysteine adducts through reduction has already been described

(Bolton et al., 2017; Bolton et al., 2018). We therefore examined the effect of two

concentrations of the reducing agent DTT on the reversion of TCPTP inhibition by EQ. As

showninFigure4B,evenata10mMconcentration,DTTwasnotsuccessfulatrestoringthe

activity of EQ-inhibited TCPTP, thereby confirming the stable and covalent nature of the

reactionbetweenEQandthecatalyticcysteineofTCPTP.Theseobservationsareconsistent

with previously published data on the inhibition of topoisomerases II by EQ (Jacob et al.,

2011;Smithetal.,2014;Gibsonetal.,2016).

Finally, the kinetics of TCPTP inhibition by EQ were studied under pseudo-first order

conditionswhich allowed observation of time- and dose-dependent inhibition (Figure 5A)

andpermittedthedeterminationofthesecondorderinhibitionkineticconstantkinact=~810

±73M-1.min-1derivedfromtheslopeofthegraphwithpseudo-firstorderkineticconstants

plottedagainstEQconcentration(Figure5B).Thiskineticdataalsoconfirmstheirreversible

natureoftheinhibitionofTCPTPbyEQ.


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EQaltersTCPTPactivityandenhanceSTAT1phosphorylationinHL60andJurkatcells

Taking into consideration the in vitro effects of EQ observed with recombinant TCPTP

protein,HL60andJurkatcelllinesweretreatedwithEQtostudyitseffectoncellularTCPTP.

After 30min exposure, the cells were lysed and cellular TCPTP immunoprecipitated. The

residual TCPTP activity was measured using the phospho-STAT1 peptide substrate

dephosphorylationassayusingUFLCdetection(Duvaletal.,2015)andimmunoprecipitated-

TCPTPproteincontentevaluatedbywesternblot.Figure6showsthatuponexposuretoEQ,

thecellularTCPTPactivity isdecreasedtoroughly30%and50%thatofthecontrolcells in

HL60andJurkatcellsrespectively.Thesedataareconsistentwithstudiescarriedoutonthe

inhibition of PTP1B (a tyrosine phosphatase-related to TCPTP) by naphthoquinones

(Iwamoto et al., 2007). These results indicate that EQ inhibits TCPTP in a cellular

environment, and we therefore investigated whether, under such conditions, TCPTP-

dependent intracellular signalling could be altered. Incubation of HL60 cells with IFN-g

triggersSTAT1phosphorylationsignalling.Thissignalling issustainedatahigher level (1.5-

foldfor60minutes)incellsexposedtoEQtreatmentascomparedwithuntreatedcontrols

(Figure 7A). Similar resultswere also obtainedwith Jurkat cells, withmore than two-fold

phosphorylationafter1hour(Figure7B).TofurthershowthatEQincreasesSTAT1signaling,

wemeasuredtheexpressionofthreegenes(APOL1,GBP1andIRF1)knowntoberegulated

by STAT1 (Hartman et al., 2005; Reardon andMcKay, 2007). As shown in supplementary

figure3,qPCRanalyzesindicatedthattheexpressionofAPOL1,GBP1,andIRF1isincreased

in Jurkat cells exposed to EQ. These results indicate that inhibition of TCPTP by EQ is

associatedwithincreasedSTAT1phosphorylationandhigherexpressionofSTAT1-regulated

genesinthesecellsuponstimulationwithIFN-g.


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DISCUSSION

Etoposideiscurrentlyoneofthemostcommonlyusedantitumordrugs.However,itsusein

clinicsisassociatedwithanincreasedriskofsecondaryleukaemia(Pendletonetal.,2014).

SeveralthreadsofexperimentalevidenceindicatethatEQ,areactivemetaboliteofETOP,is

involved in ETOP-related leukaemia through alteration of topoisomerase II function

(Gantchevetal.,1997;Kaganetal.,1999;Fanetal.,2006;Vlasovaetal.,2011;Jacobetal.,

2011; Smith et al., 2014; Gibson et al., 2016;). However, effects of EQ on other cellular

targetscouldalsocontributetoETOP-relatedleukaemogenesis(Haimetal.,1987;Fanetal.,

2006; Rojas et al., 2009).We investigated the possibility of protein tyrosine phosphatase

TCPTP being a target of EQ. TCPTP plays a pivotal role in normal and malignant

haematopoiesisthroughthenegativeregulationoftheJAK/STATsignallingpathway(Dorritie

etal.,2014). Interestingly,ETOPwasreportedtoactivateSTAT1signalling inHeLacells. In

addition, it hasbeen shown thatprotein tyrosinephosphatases similar to TCPTP couldbe

inhibitedbypolyaromaticquinones (Wangetal.,2004; Iwamotoetal.,2007). In linewith

this,we found in thiswork that EQ, the quinonemetabolite of ETOP,was able to inhibit

cellular TCPTP activity with subsequent increase in tyrosine phosphorylation of STAT1 in

hematopoieticcelllines.Usingfurthermolecularandkineticapproaches,weshowthatEQis

an irreversible inhibitorofTCPTP.Thesecond-order rateconstant (kinact) for this inhibition

wasfoundtobe~810M-1.min-1(Figure5B).Thisrateconstant isclosetovaluesfoundfor

naphthoquinone-dependentinhibitionofhumanprotein-tyrosinephosphatase1B(420M-1.

min-1), a tyrosine phosphatase structurally-related to TCPTP (Wang et al., 2004).

Interestingly, naphthoquinones have been shown to alter cell signalling, in particular

throughincreasedtyrosinephosphorylation(Iwamotoetal.,2007;KlotzandJacob,2014).In

addition, thekinact value for the inhibitionof TCPTPby EQ is comparable to that reported


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previously for hydrogen peroxide (600 M-1. min-1), a known endogenous regulator of

tyrosineproteinphosphatases(Seineretal.,2007;Ostmanetal.,2011).InhibitionofTCPTP

by EQwas not reversed by buffer-exchange (Figure 4A). This observation, alongwith the

time-dependentnatureofthereaction(Figure5A)indicatesthattheinhibitionofTCPTPby

EQisirreversible.Theactivityofprotein-tyrosinephosphatasessuchasTCPTPiswellknown

to rely on a reactive active site cysteine that canbemodifiedbyoxidantsor electrophilic

chemicalswith subsequent lossof activity andaltered cell signalling (Ostmanet al., 2011;

Klotzetal.,2014).Inaddition,severalquinones,suchasEQ,areMichaelacceptorsthatcan

covalentlyreactwithcysteineresiduestoformirreversibleadducts(Giorgiannietal.,2006;

Fanetal.,2006;Boltonetal.,2018).Accordingly,wefoundthat the irreversiblenatureof

theinhibitionofTCPTPbyEQisduetoformationcovalentadductsonthecatalyticcysteine

residue of the enzyme (Cysteine 216). This was confirmed by the fact that the inhibition

process of TCPTP by EQ was slowed by addition of the competitive TCPTP inhibitor

orthovanadate and by computational docking approaches. More importantly, mass

spectrometryanalysisofTCPTPinhibitedbyEQshowsthattheactivesitecysteineresidueof

the enzyme is indeed adductedby EQ (Figure 3C). Interestingly, recent experimental data

indicatethatEQpoisonstopoisomerasesIIenzymesthroughcysteineadductionmechanisms

and that this covalent modification of topoisomerases II by EQ is likely to contribute to

ETOP-relatedleukaemogenesis(Jacobetal.,2011;Smithetal.,2014).Ofnote,theactivityof

EQ against topoisomerase IIa was found to be considerably higher than that of ETOP.

Indeed,EQcaninhibitDNArelaxationat100-foldlowerlevels(1µMversus100µM)ofdrug

when compared to ETOP (Gibson et al., 2016). In their study on the inhibition of

topoisomeraseIIbbyEQandETOP,Smithetal.reportedthatwhile15µMEQinhibitedDNA

cleaveagebyTopo2b in6min lessthan15%inhibitionwasobservedwiththeparentdrug


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ETOP(Smithetal.,2014).Inaddition,EQisabletoinhibitATPhydrolysisbytopoisomerase

IIawhereasETOPcannot(Gibsonetal.,2016). Interestingly,whileEQwasfoundtoinhibit

TCPTP, the parent drug did not. This is in agreement with the electrophilic nature of EQ

which enables it to covalently reactwith the catalytic cysteine of TCPTP throughMichael

addition (Fan et al., 2006; Bolton et al., 2018). Tyrosine phosphatases closely related to

TCPTPsuchasPTP1Bhavebeenshowntobeinhibitedbynaphthoquinonesthroughactive

sitecysteinecovalentmodification(Iwamotoetal.,2007;Klotzetal.,2014).Wefoundthat

EQ was also able to inhibit the phosphatase activity of PTP1B (supplementary figure 4).

Althoughwe cannot rule out that EQmay affect STAT1 tyrosine phosphorylation through

effects onother tyrosinephosphatases, siRNA-mediated knockoutof TCPTP in Jurkat cells

and knockout mice models clearly indicate that impairment of TCPTP activity strongly

impacts STAT1 tyrosine phosphorylation (Simoncic et al., 2002; Kleppe et al., 2010).

Increasing evidence indicates that CYP/myeloperoxidase-dependentoxidative activationof

ETOP into reactivemetabolites (notably EQ) inhematopoietic cells is a key contributor to

ETOP-relatedleukaemia(Zhuoetal.,2004;Fanetal.,2006;Jacobetal.,2011;Vlasovaetal.,

2011; Smith et al., 2014; Gibson et al., 2016; Atwal et al., 2017).We show here that in

addition to disruption of topoisomerases II functions, EQ could also contribute to ETOP-

relatedleukaemiathroughinteractionswithTCPTPandsubsequentcellsignallingalteration.

ACKNOWLEDGEMENTS

Wethankthetechnicalplatform“BioProfiler-UFLC”(BFAUnit,ParisDiderotUniversity) for

provision of UFLC facilities. We are grateful to Dr. Oliver Brookes for English language

proofreadingofthismanuscript.


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AUTHORSCONTRIBUTION

Participatedinresearchdesign:QN,JB,FRL

Conductedexperiments:QN,JB,L-CB,SG,TL

Performeddataanalysis:QN,JB,WZ,L-CB,RL,RD,SG,TL,CC,FG,FB,J-MD,FRL

Wroteorcontributedtothewritingofthemanuscript:QN,JB,XX,SG,TL,FB,FRL


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FOOTNOTES

This work was supported by University Paris Diderot and CNRS. QN, WZ and RL are

supportedbyChinaScholarshipCouncil (CSC)PhDfellowships. JBwassupportedbyaPhD

fellowshipfromRégionIledeFrance(Cancéropole2015).


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LEGENDFIGURES

Figure1:EffectofETOPandEQonhumanTCPTPactivity(A) Effect of ETOPandEQonTCPTPactivitymeasuredbypNPPdephosphorylation. 1µMTCPTPwasincubatedwith100µMETOPand5to40µMEQfor30minat37°Canddiluted10-foldpriortomeasurementofresidualactivityinthepresenceof5mMpNPPasdescribedintheMethodssection.Resultsarethemeanofthreeindependentexperiments,errorbarsindicateS.D.(B) Measurements of TCPTP FAM-pSTAT1 peptide dephosphorylation activity. TCPTP wasincubatedwith100µMETOPand40µMEQfor30minat37°CandresidualPTPN2activitymeasured as described under Methods. Results are the mean of three independentexperiments, error bars indicate S.D. * p<0.05 determined using ANOVA followed byDunnett’spost-hocanalysis.Figure2:ModificationofTCPTPcysteineresiduesbyEQ(A)DetectionofHumanTCPTPadductsbyNBTstaining.HumanTCPTPwasincubatedwith40µM EQ or 100µM ETOP for 30 min. Samples were separated by SDS-PAGE andtransferredontonitrocellulosemembrane.QuinoneadductsweredetectedbyNBTstainingasdescribedinMethods.Membraneisrepresentativeof3independentexperiments.(B) 5-IAF staining of Human TCPTP to detect unmodified cysteines. Human TCPTP wasincubatedwith40µMEQor100µMETOPfor30minpriorincubationfor10minwith20µM5-IAF.SDS-PAGEandtransferredontonitrocellulosemembrane.IAFadductsweredetectedby fluorescence after SDS-PAGE as described in Methods section. Membrane isrepresentativeof3independentexperiments.(C)DetectionofTCPTPproteinoxidation.HumanTCPTPwasincubatedwith100µMETOP,40µMEQor100µMH2O2for30minprioranalysiswithwesternblotusingananti-oxidizedTCPTP active site antibody as described in Methods. Membrane is representative of 3independentexperiments.Figure3:MappingofEQadductonTCPTPproteinactivesitecysteine(A) Effectof sodiumorthovanadateonTCPTP inhibitionbyEQ. 1µMHuman recombinantTCPTPwas incubatedwith40µMEQand/or1mMorthovanadate for 30min at 37°C andresidual activity measured as described in Methods. Experiment is representative of 3independentexperiments.(B)MoleculardockingmodelofEQbound inhumanTCPTPproteinstructure.EQmoleculeadductatomcoordinateswereobtainedinsideTCPTPactivesitepocketasdescribedintheMethods section. EQ is displayedwith green sticks and is covalently bonded to the C216residue.(C) Mass spectrometry characterization of EQ adduct on the active site cysteine trypticpeptide. 1µM TCPTP protein was incubated with 100 µM EQ for 30 min at 37°C priortrypsinetreatmentandLC-MS/MSanalysisasdescribedinMethods.UpperpaneldepictsthespectrumobtainedwithcontroluntreatedTCPTPproteinwhereas lowerpanelshowsdataacquiredwithTCPTPsamplestreatedwithEQ.ThesequenceofthepeptideisdisplayedoneachpanelinadditiontothepositionoftheEQadductoncysteineinlowerpanel.


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Figure4:IrreversibilityofTCPTPinhibitionbyEQ(A)EffectofbufferexchangeonactivityofTCPTPpreincubatedwithEQ.HumanTCPTPwasincubatedwith40µMEQor100µMETOP for30min followedbybufferexchangeornotprior to residual pNPPdephosphorylation activity determination asdescribed inMethods.Resultsare themeanof three independentexperiments,errorbars indicateS.D.*p<0.05determinedusingANOVAfollowedbyDunnett’spost-hocanalysis.(B) Effect of DTT on activity of TCPTP preincubated with EQ. Human TCPTP was pre-incubatedornotwith40µMEQfor30minfollowedbythetreatmentwith1or10mMDTT.ResidualpNPPdephosphorylationactivitywasdeterminedasdescribedinMethods.Resultsare the mean of three independent experiments, error bars indicate S.D. * p<0.05determinedusingANOVAfollowedbyDunnett’spost-hocanalysis.Figure5:KineticsofTCPTPinhibitionbyEQ(A)Determinationofpseudo-firstorderinhibitionrateconstantforTCPTPinhibitionbyEQ.3µMTCPTPwas incubatedwithdifferentconcentrations (0,10,20and40µM)ofEQandassayed for residual activity at different time points as described in Methods. kobs wasdeterminedastheslopeofthelinearregressionofdataexpressedasthenaturallogarithmof the residual activity as a function of time. Results are themean of three independentexperiments,errorbarsindicateS.D.(B)Determinationof thesecondorder inhibitionrateconstant forTCPTP inhibitionbyEQ.kobs were plotted as a function of EQ concentration and the kinact was determined as theslope of the linear regression as described in Methods. Results are the mean of threeindependentexperiments,errorbarsindicateS.D.Figure6:InhibitionofTCPTPbyEQinHL60andJurkatcellsHL60(A)andJurkatcells (B)weretreatedornot (Ctrl)with50µMEQfor30minat37°C.CellswerelysedandcellularTCPTPproteinwasimmunoprecipitatedpriortoresidualactivitydeterminationandTCPTPproteincontentdeterminationbywesternblottingasdescribedinMethods.Resultsarethemeanofthreeindependentexperiments,errorbarsindicateS.D.*p<0.05determinedusingStudent'sttestasdescribedunderMethodssection.Westernblotisrepresentativeof3independentexperiments.Figure7:STAT1phosphorylationkineticanalysisinHL60andJurkatcellsinducedwithIFNγHL60(A)andJurkatcells (B)weretreatedornot (Ctrl)with50µMEQfor30minat37°C,thenwashedpriortoincubationfor20minwithIFNγ.Washedcellswerethenanalyzedatdifferenttimepointsasdescribed inMethods.Resultsarethemeanofthree independentexperiments,errorbarsindicateS.D.*p<0.05determinedusingStudent'sttestasdescribedunder Methods section. Western blot is representative of 3 independent experiments.Quantification of STAT1 phosphorylation in western blots was carried out using ImageJsoftware.


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(A

bs 4

05nm

)

Time (s)

TCPTPTCPTP + Vanadate (1 mM)TCPTP + Vanadate (1 mM) + EQ (40 μM) TCPTP + EQ (40 μM)


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A B

Figure 4

Ctrl

40 μM

EQ

100 μ

M ETOPCtrl

40 μM

EQ

100 μ

M ETOP0

20

40

60

80

100

120

% T

CPTP

Act

ivity

* *

Buffer exchange

Ctrl

1 mM D

TT

10 m

M DTT

40 μM

EQ

40 μM

EQ + 1 mM D

TT

40 μM

EQ + 10 m

M DTT

0

25

50

75

100

125

150

% T

CPTP

Act

ivity

*

* * *


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Figure 5

0 10 20 30 403.0

3.5

4.0

4.5

5.0

EQ 0 μM

EQ 10 μM

EQ 20 μM

EQ 40 μM

Time (min)

Ln (E

/E0)

0 10 20 30 40 500.00

0.01

0.02

0.03

0.04

k obs

(min

-1)

EQ (μM)


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HL60 Jurkat

TCPTP TCPTP

Figure 6

Ctrl EQ0

20

40

60

80

100

120

140

%TC

PTP

Activ

ity (p

STAT

1)

*

Ctrl EQ0

20

40

60

80

100

120

140

%TC

PTP

Activ

ity (p

STAT

1)

*


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Figure 7 Figure 7


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t-cell protein tyrosine phosphatase (tcptp) is...

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