Depletion of TRRAP Induces P53-Independent Senescence in Liver Cancer by Downregulating Mitotic Genes

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2019-06-12

Depletion of TRRAP induces p53-independent senescence in liver cancer by downregulating mitotic genes

Suet-Yan Kwan University of Massachusetts Medical School

Et al.

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Repository Citation Kwan S, Sheel A, Song C, Zhang X, Jiang T, Dang H, Cao Y, Ozata DM, Mou H, Yin H, Weng Z, Wang XW, Xue W. (2019). Depletion of TRRAP induces p53-independent senescence in liver cancer by downregulating mitotic genes. Program in Molecular Medicine Publications and Presentations. https://doi.org/10.1002/ hep.30807. Retrieved from https://escholarship.umassmed.edu/pmm_pp/104

This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in Program in Molecular Medicine Publications and Presentations by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. This article isprotected rights by All copyright. reserved. 10.1002/hep.30807 doi: betwe differences to lead may which process, not proofreading and has pagination but typesetting, copyediting, review the peer through been full undergone and publication for accepted been has article This Medical and 6 University, 5 4 Institute,9000 Cancer Bethesda,MDRockvillePike, 20892,USA 3 Medical 2 Worcester, 1 Xin Wei Wang Hien Suet down Depletionof TRRAP induces p53 Articletype : Original

Medicalresearch institute, University, Wuhan PR China Wuhan, Program Department Program RNA

Accepted National Research, Cancer for Center Carcinogenesis, Human of Laboratory Article

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Weibo Therapeutics

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YueyingCao

Bioinformatics Molecular

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This article isprotected rights by All copyright. reserved. University. su researchprogram of the Center forCancer Research, National Cancer institute. by grants ( Association.A RSG (DP2HL137167,P01HL131471 andUG3HL147367), FinancialSupport: TOP2A, domain Listof abbreviations: E 01605 † arrest KEYWORDS *These

Corresponding - pportedby the Young Thousand TalentedProgram and startup the funding from Wuhan Accepted Articlemail: - 16 ,senescence

- associatedprotein [email protected] authors Topoisomerase alphaII 093),

Z01 X.Z. and X.Z. Z.W. were partly supported by NIHgrant U24 : .S. - theLung CancerResearch Foundation,Hyundai Hope on Wheels,and ALS Hepatocellularcarcinoma, CRISPR screen, histone acetyltransferase, G2/M BC010313, Z01

contributed was supportedby F30CA232657 (NCI).

author

W.X. was W.X. supportedby grants thefrom National Institutesof Health HCC,Hepatocellular carcinoma; ; HAT,histone ; acetyltransferase; GBM, glioblastoma multiforme; :

Wen

equally - . BC010876, and

Xue,

Phone: to

368

this

Plantation

work.

774

Z01 -

455 - AmericanCancer Society (129056 BC010877

- TRRAP,

Street, 3783

H.D.and wereX.W.W. supported

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Worcester

H.Y.was -

This article isprotected rights by All copyright. reserved. secondline checkpointinhibitors and approvedadvanced HCC treatments include andis18% the major subtypeliver of cancer is hepatocellularcarcinom 700,000 deaths worldwide eachyear Liveraccountscancer for morethan 27,000 deaths the in United States and more than therapeuticstrategy for HC via activationmitotic of genes. T Conclusion: phenotype of TRRAP/KAT5 knockdown. mitotic andgene TRRAP/KAT5target, was sufficient to reca TRRAP as key TRRAP/KAT5targets in HCC,and subsequentcell cycle analyses revealed that cancer genomicsanalyses usingpatient data and RNA inhibitsHCC cell growth viap53 inductionof weHere, showthatd oncogenictransformation, buthow it functionsin cancer cellproliferat ( HCChuman cell lines developmentof new targeted therapiesHCC for characterized treatmentsand underlying the mechanisms thatdrive HCCpathogenesis remain poorly Hepatocellularcarcinoma (HCC)is aggressivean subtypeof liver cancer Abstract Accepted ArticleTRRAP) sorafenib -

andKAT5 : as essentialan gene

. Ourresults uncover a role for TRRAP/KAT5in promotingHCC cell proliferation ,

therapy Identifying andgenes pathways essential HCC for cell growth willaid the which patient extend survival by several months only, and immune the - depletedcells are arrestedat G2/M Depletionphase. of TOP2A,a Nivolumaband Pembrolizumab epletion of TRRAP ,

(3) we .

identified T C. he

forHCC clinicalneed formoreeffective argeting the TRRAP/KAT5 complex transformation/transcriptiondomain

(1,2) cell or

. itsco

The 5 The proliferation. -

m andp21 ulti . Using - factor - - yearsurvival rate forliver cancer patients kinase inhibitorkinase ,which show - independentsenescence. Integrated - ,

a kinomeCRISPR screen in se histoneacetyltransferase KAT5

TRRAPbeenhas implicatedin quencingidentified mitotic genes pitulatethe senescent HCC

s ~20%

regorafenib,lenvatinib treatments remains is ionis not established. a a (HCC) - associatedprotein potential response rates with effe few (3)

.Current

three ctive

, as a

This article isprotected rights by All copyright. reserved. established. TRRAP that TRRAPand its regulated pathways alteredare cancer. in brain Bycontrast, embryo fibroblasts Moreover,w t and uterinecancers TRRAP 10) progression transcription,DNA repair asacts a scaffold protein required forHCC transc aUsing kinomeCRISPR screen in threeHCC cell lines, we identifiedtransformation/ of targeted therapies for HCCpatients inhibitcan HCC growth amenable totargeted therapy unmetpartially because Acceptedhe TRRAPS722F hotspot mutationin melanomascan transformNIH3T3 cells Article .

- tumoriniti ription domain

alteration is m

ildtypeTRRAP isre utat and stemnessin depletion of TRRAP ating cells ed s cell

(15) andamplifi and - (11 associatedprotein (TRRAP)as the top growth. ,

andMYC - its

and determining the mechanismsinvolved, HCCisgenetically heterogeneous and 14) ,

in andreplication (17,18) functionin cancer

,and has beenimplicated in oncogenic histoneacetyltransferase (HAT) complexes

embryonicstem cells and cortical apical neural progenitors TRRAP are largelyare unknown ed quired for transformation

- can in differentcancer types,including melanomas, gastric, dependent tumor initiation in ain tumor initiation breast cancer dependent model .

Givenroleits in diversecellular processes, it ispossible

induceapoptosis and differentiation in lymphomaand ,

. a pseudokinase memberof PI3 the kinase

(8) .

cells TRRAPis , particularlyin thecontext HCC, of (1,4) also a key .

by Identifying - c rankingcandidate gene - However,the extentof myc/Hrasand E1A/Hras in rat because HCC

regulatorof cell cycle transformation. will

geneswhose depletion

aid thedevelopmentaid (5 - 7)

thatregulat

drivergenes

(11) - like family Indeed, .

i s nots (16)

e (9,

. ,

This article isprotected rights by All copyright. reserved. Accepteddaysand HepG2 cells wereselected with µg/mL 4 infectedwith lentivirus.Huh7 andHep3B cellswere selected with µg/mL 2 puromycin 3 for cells.For each cellline, duplicateswere p amplificationprotocol. Lentivirus containingthe kinome librarywas packaged using293fs (Addgene#1000000083). Thelibrary was amplifiedaccording toAddgene’s library Thehuman kinomeCRISPR pooled library w KinomeCRISPR screen service. HepG2,Hep3B and SNU FBSand 1% penicillin JunweiShi sodiumpyruvate, and 4500mg/L glucose DMEM Huh7 Articleculture Cell Materials of TRRAP/KAT5 is a potential cells. sufficientto recapitulate thesenescent phenotype observed in TRRAP survival a subsetof mitotic genes senescencein ap53 weHere, showthat

Together, and SNU475 . SNU .

in HCCpatients MG132 a

and cultured inand cultured MEM.All cell werelines grownmedia in supplementedwith 10% ndMethods

- 475 cellswerecultured in RPMI

o and chloroquinewere purchased Milliporefrom Sigma. ur

cells wereprovided byScott Dr. Lowe depletion of study -

- and p21 streptomycin andmaintaine .

Depletion of TOP2A,a mitotic andgene TRRAP/KAT5target, is thatis 475 cellswere authenticated using ATCC’s cell authentication uncover

- therapeuticstrategy HCC. for indep

TRRAP/KAT5 high

role ly en

express dent manner. for . Hep3B . and HepG2 cellswere provided by Dr. erformed.For each replicate, 2

TRRAP/KAT as a gift Johnfrom Doench and David Root supplementedwith 10 mMHEPES, 1 mM ed in HCCand is in HCCcells

d in a d in puromycin4 days. for eachFor sample, W e foundthat TRRAP/KAT5activates

37 5 . Huh7 . cellswere

in  induce C

HCCcell growth anddisruption

incubatorwith 5%CO

associated with poor s

G2/Marrest and -

and KAT5

10 culturedin 7 cellswere - depleted 2 . Huh7, This article isprotected rights by All copyright. reserved. PANTHERoverrepresentation testand annotated with the GO Ontologydatabase (released Spearman’s correlation an setsdata were obtained cBioPortalfrom genecorrelation analyses,correlation data from theHCC TCGA (n=360) and GBM(n=136) TRRAPmutations and copy groups.P highestand lowest tertiles numbers GSE1898and GSE4024 ( survival weredata obtained thefrom Human Protein Atlas and cBioPortal Atlas (version 18, listof genes thatpredict prognosisof HCC patients w BrowserXena ( ofdata TCGAHCC tumor moderated t (https://www.ncbi.nlm.nih.gov/geo/geo2r/) tumor tissue (n=220)in theGSE14520 data set w (20) Toanalyze Bioinformaticsanalysis identifiedas previously described evaluatedFastQC using (version 0.11.2)to ensure high quality. high mini (Thermokit Fisher Scientific, MA). Waltham, genomicDNA was harvested atfrom least6 Acceptedhttp://www.cbioportal.org/ Article

- and TCGAHCC data setswere used. Gene expression ofdata tumor(n=225) and non fidelityPCR master mix(Thermo Fishe - valueswere calculated using the log gene expressiongene levels between and tumor non - statisticsand adjustedusing the Benjamini http://xena.ucsc.edu/ https://www.proteinatlas.org/ s

)respectively. alysis.For analyses,GO list the of genesw weredesignated as ‘highexpression’ and ‘low expression’

and - number matched

(19) (21,2 ) and ) alterationsin HCCwere obtained cBioPortal.from For .

. 2)

The LEC The data non B .

analyzed usingthe signedWilcoxon . riefly, geneexpression data were analyzed using B FKPMvalueswere groupedin tertiles andt rScientific). riefly,P × - tumor

) 10 -

rankMantel . 6 For survivalFor analysis ere sgRNAwas amplifiedusing Phusion flash

cellsusing PureLink the GenomicDNA s - valueswere calculated

as (n=50)were obtained thefrom UCSC analyzed using GEO2R is -

obtained thefrom Human Protein Hochberg method. expressionGene Allsequencing datasets were

availableGEOwith on accession - - tumor Coxwith test GraphPadPrism. Depleted

tiss ues, t as FKPM , values and

analyzedthe using

geneswere he GSE14520

using - ranktest. The he

- This article isprotected rights by All copyright. reserved. AcceptedSanta126; (#2775;Cell Signaling Technology), Santa Signaling Technology),KAT2A (#3305 against: TRRAP (#3967 protein was loaded measuredusing BCA the assay (Thermo Fisher Scientific). For each sample, 25 ofµg phosphataseinhibitor cocktails (Thermo Fisher Scientific). The concentrationof protein was Bioproducts,Ashland Cellswerewashed twice with ice ImmunoblotAnalysis selectedwith puromycin.Sequences of sgRNAs and Huh7 Articlewerecloned intothe LentiCRISPRv2 backbone hygro (Addgene#98291). with puromycin for packagedinto lentivirus 293fs using cells.HCC cells infectedwith lentivirus wereselected tools/sgrna sgRNAswere designed using sgRNAdesignlentivirus and infection sequencing data from GSE69671 Benjamini 2018 10 days10 -

10 - cellsinfected with lentivirus wereselected with CruzBiotechnology, - 08).P - Hochberg - - CruzBiotechnology), GAPDH(MAB374; Millipore Sigma,Burlington, MA), Hsp90 design

respectively - valueswerecalculated using Fisher’s Exact testand correctedby the ,cloned into LentiCRISPRv2 the backbone (Addgene #52961)and 2 onto a - 4days. To method.

MA)supplemented with protease (

;

, , in then CellSignaling Technology,Danvers, MA), p21 (#2947 n

Dallas, TX), SDS Todetermine KAT5 binding sites, https generatedouble sgRNA - - fectedwith sgTRRAP, sgNT, sgKAT5 PAGE gel. The followingantibodies wereused probeto

cold PBSand harvested in RIPAbuffer (Boston (23) ://portals.broadinstitute.org/gpp/public/analysis TOP2A(#12286; Cell Signaling Technology),p53 (sc . ;

CellSignaling Technology),KAT5 (sc NIK(#4994; Cell Signaling Technology),LC3B

are listedare 100 and500 - Roche,Indianapolis, IN

infectedcells,sgNT and sgp21

in SupplementaryTable we analyzedChIP µg/mL hygromycin for ,

and sgKAT2A SNU - 166323 - ; 475 and

)and Cell - - 1 . ; ,

and 6 - This article isprotected rights by All copyright. reserved. protocol.RT cDNAreverse transcription (Thermokit FisherScientific) according to the manufacturer’s protocol. One microgramof RNA was usedtosynthesize cDNAusing the high DNAwas removedby on RNAwas extracted from cellsusing the RNeasy mini (Qiagen,kit Germantown,MD) and RNAand extraction RT werequantified β Huh7 SA violet.All experiments were repeatedthree times. wellplate Huh7 andSNU cellswere pla growth of cellsat day 3 and day 5 was normalizedto measurements taken one dayafter the (Promega,Madison, wasaccordingWI) performed tothe manufacturer’s protocol. The Tomeasure cel growth Cell andcolony formation assays antibody(LICOR) #F1804;Millipore Sigma) (#610419;BD Bioscience, - Acceptedgalactosidase(SA Article - β - , gal assaygal

Hep3B s . After . 11 days, cells were fixedwith 4% formalin andstained with 0.5%crystal - qPCRanaly ,and SNU ted. Each ted. repeat was averagedbetween wells. 6 colonyFor formationassays, - lgrowth, cellswere seeded onto96 as previouslydescribed 475

using the - cellswere seeded at3000 β - gal)staining was performedat pH - - 475 - . ses wereperformed using qPCR colu

Bandswere visualized San Jose,CA) OdysseyImaging system.

cellswere seeded onto mn DNasemn digestion (Qiagen)according tothe manufacturer’s

(24) , and , Flag (#2368; Cell Signaling Technologyand .

and 2000 withimmunofluorescent an secondary

- wellplate SsoFastEvaGreen supermix (Bio

- wellplate

cellsper well =

6.0 s . CellTitre .

andpositively stained cells s,

senescence

respectively - glo assayglo - capacity - associated

onto 6 onto - Rad, - This article isprotected rights by All copyright. reserved. GRCh37/ readsseq eachfrom sample werealigned using STAR (version 2.5.2b) Biosystems, Wilmington,MA) and sequenced using the Illumina NextSeq 500 system.RNA described RibosomalRNA was depletedand RNA RNA Committee protocolswere approved by UMass the MedicalInstitutionalSchool Animal Careand Use ProlabIsopro RMH3000(LabDiet cycle andwere not fasted priorto injection. Mice weremaintained on animal 22°Cfacilityat under 12 the (Ancare)and Bed UniversityMassachusetts of (UMass) Medical School. Mice wereprovided with nestlets facilities accreditedby the AmericanAssociation Laboratory for Animal Care(AALAC) at the (Allentown)ventilated cagesystems with calculatedusing the formula MA C NY Female Xenograftsin nude mice Primersequences used for qRT Hercules,CA) according to the manufacturer’s protocol

Accepted Articleellswere resuspended in PBSand mixed in a2:1 ratio with matrigel( ) ) to a ) volume final of 200

and injectedin the orright left flankwith 1x10 - sequencing and sequencing 6 - hg19human reference genome.Gene expression was

w (25)

(IACUC)and eek .

- Thelibraries were quantifiedusing the KAPA Library Quantification Kit(Kapa old - r’Nests(Andersons Lab Bedding) an NCR bioinformaticsanalysis Nu/Nu complywith allrelevant federal guidelinesand institutional policies



mice werepurchased from BioscienceTaconic (Rensselaer, L. Tumorsize was measuredby calipers and volumewas - PCR )

‐ throug hourlight/dark cycle

are listed

sequencinglibraries wereprepared previously as

1/4″Bed hout thecourse of experiment the

in SupplementaryTable

6 -

o’Cobs TRRAPstable knockdown Huh7cells.

and GAPDHwas usedas a control . Mice . wereinjected during light the .

maintainedin a pathogen

Allm (AndersonsLab Bedding)in ice were

quantifiedthrough RSEM astandard dietof Westnet Inc., Canton,Westnet

(26) housedin JAG75 2 .

againstthe Allanimal ‐ free . - .

This article isprotected rights by All copyright. reserved. indicatedotherwise. P as means Statisticalanalysis was performedusing GraphPad Prismsoftware and Statistics AllCA). data w wereanalyzed described above toidentify 2N and populations.>2N For each sample, detectedusing a mouse incubatedthen with anti an ethanol,70% permeabilized with 0.3% Triton Scientific),and incubated 30 for minutes at 37 ng/mL withBrdU a trypsinizedandwith fixed 70%ethanol, permeabilized with 0.3% Tr Deoxyuridine( cellFor cycle analysis,Huh7 Fl identifydifferentially expressed genesusing rawread counts quantifiedby htseq (v1.2.31)

Accepted Article ow cytometryanalyses

propidiumiodide (Thermo

(27)

 - standard deviation.Student’s t FITCconjugated antibody (Thermo FisherScientific). Cells were stained with

with GENCODEV19 geneannotation.

BrdU, ere using the using MACSQuantVYB Flow cytometer (MACS Miltenyi Biotec, Auburn,

analyzedby FlowJo so 10.0 ThermoFisherScientific) for 1 hourat 37 - values of < - FITCIgG. Cells were al -

γH2A.Xantibody (#9718; Cell Signaling Technology),which was and SNU

FisherScientific), 10 0.05were considered to statisticallybe significant. - 475 - testwas used todetermine P cells werepulsed with µg/mL 30 - X100and blocked in 0.8% BSA.Cells were  ftware. C.For so stained with propidium iodideas

DESeq2   H2AXanalysis, cells were fixedin

g/mLRNase A (ThermoFisher

 (1.22.0) C.Cells were then iton

(28) - atleast40,000 cells X 100 andincubated data were - valuesunless

was employedto

5 - Bromo - coun presented

- t 2´ (29) - 50 .

This article isprotected rights by All copyright. reserved. Accepted‘roleBRCA1 of DNA in damage response’and growthsignaling factor pathways correlatedwith expressionof genes thatare involved in ‘molecularmechanisms of cancer’, p We investigatedwhether TRRAPexpression iscorrelated with certain genesignatures in HCC. increase TRRAP (SupplementaryFigure 1 TRRAPmRNA expression, Analysis of the TCGAdata set Outof the 8 genes, tumorigenesis previousstudies have shown that with poorsurvival and1C, Supplementary Figure 1A). Articlewereexpressed least at 1.5 31these genes that31 geneswere significantly depleted in allthree HCC cell lines (Figure 1A). amplifiedand identifie harvested genomicDNA cellsfrom of an early passage and after 10 passages single RNAsguide (sgRNAs)per gene. HepG2(Huh7, Toidentify genes thatare requiredcell for growthHCC, in we infectedthree HCCcell lines HCC CRISPR identifies TRRAPregulatoras screen A kinome a growth ofcell in Results erformedIngenuity pathway analysis and found that TRRAPexpression ispositively

expressionishigher in tumor compared to matched normaltissue d

TRRAPexpression iscorrelated with survival poor (Figure

, (30

, 8 , genes(AURKB, BUB1B, CDC7, DTYMK, PGK1, TPR, TRRAP,VRK1) andHe

- inpatients HCC (Figure 1D 34) TRRAP d .

p3B)with a kinomeCRISPR library thattargets 763 kinases sgRNAsusing PCR C )

. - amplification and/or mutationsin the TRRAP gene isthe top depleted fold higherin HCC compared to Usingadditional published patientdata sets revealed AURKB,DTYMK, PGK1, TPR

H igh expression

Todetermine changes in sgRNA representation,w that

and d 12% HCC of

and

gene eep sequencing SupplementaryFigure 1

in allthree cell lines (Figure 1B) levels

patientsamples ha of theseof geneswere associated non , -

tumor andVRK1 ,respectively 1 D).

tissue , we , confirmedthat

We alsoWe (Figure1C), B

). promoteHCC ve increased

Of note,Of (Figure 1B, ,

. We found. We then then Among

with 8 .

and e This article isprotected rights by All copyright. reserved. sgTRRAPcells (SupplementaryFigure 2C), suggestinga degradation thatMG132 andchloroquinecould notrescue KAT2A and KAT5 protein expressionin degradation in sgTRRAPcells with MG132 and chloroquine respectively.However, we fou degradation in theabsence of TRRAP,we inhibitedproteasomal and lysosomal protein investigatewhether KAT2A and KAT5 protein becomeunstable dueto increased protein not mRNAlevel that TRRAPd localized thenucleusto in Huh7and SNU predominantlybinds KAT2Ato and KAT5 TRRAPisan adaptor protein H senescence. Collectively increase Furthermore,the molecular markers of senescence positive senescence a senescent Morphologically, in decreasedcell growth and colony formationin Hep3B To Depletionof TRRAP induces senescence HCCin cells in HCC. (Supple istone acetyltransferaseistone KAT5 is required for cellgrowth HCCin

Accepted Article understand the functionof

and SNU mentaryFigure 1D).

d in sgTRRAPd in cells compared to non , these , data suggest thatloss of TRRAPinhibit

phenotype epletion resultedin decreasedKAT5 and KAT2A expression at the protein, but ,

- the cells the became flatterand enlarged after TRRAPdepletion, reminiscentof in Huh7and SNU 475 - associated

cellsusing CRISPR (sgTRRAP) (Figure 2A). Loss of TRRAPresulted

(35) . These resultssuggest that TRRAP Indeed,loss of TRRAPin Huh7and Hep3B cells resultedin in TRRAP severalHAT complexes. Inmammalian cells, TRRAP - b eta - 475cells -

galactosidase (SA in HCC,we depleted TRRAPexpression in Huh7

- (5 475cells (Supplementary Figure 2A). - 7) (Figure 3A - target .

We thatfound We TRRAPand KAT5 co

these — ing p15,p16

cell

controls (Figure 2 - andSupplementary Figure 2B

- s

gal)staining (Figure 2 lines

cell growth byinducing ,

and p21

mayhave an (Figure2B and - independent

(35,36) Aand 2D

oncogenic role 2 — We found We C C). ) were ).

(35,36) - ). To nd .

This article isprotected rights by All copyright. reserved. cells weend, co whetherp21 is required to induce senescence in TRRAP expression of deplet Hep3Bcells are senescenceis activationoncogenes of Senescencecan be triggeredby multiple stimuli, assuch DNA damage, replicative stress, p53p21 and are dispensable senescence for inducedby loss ofTRRAP and KAT5 le endof point the study ta sgKAT5. TRRAP wenude injected mice subcutaneouslywith Huh7cells expressing sgTRRAP, sgKAT2A Toinves 2F) comparedto non Figure2E induced senescences thatdepletion ofKAT5, but notKAT2A, reduced cell growth and colony andformation, sgKAT5) KAT2Aor KAT mechanism. a Accepted rgetingcontrols. Article d . s

(Figure 4A). foundthat We

to decreasedto KAT2A and KAT5 ed tigate whethertigate depletion of TRRAP,KAT2A

TRRAPand KAT5 in Huh7and SNU ). also foundWe - depletedp21 and either TRRAPor KAT5 usingCRISP To p21,a p53

5 expression,we depletedKAT2A and KAT5using CRISPR (sgKAT2A and p53 determine p53 - - target

andKAT5 However,

activation - null,suggesting that (Figure imilar ing ,

and oxidative stress targetthatis

whether a higher - controland sgKAT2A cells(Figure 3 ly 475 cells isindependent of - KAT2Adepletion didnot have anyeffect

depleted weretumors significantly smaller

(38) to TRRAP to 3F ,

). Together,these resultssuggest that TRRAPdepletion evenin theabsence of p21, depletion of TRRAPand KAT5 .However, Huh7 and

TRRAPdepletion induce increase (Figure3

expression also importantin inducingsenescence - depleted theobserved induction ofsenescence due to

(37) p53. B and SupplementaryFigure 2D

p15 andp21 , . cells but

A keymolecular event in Sincewe observe ,

andKAT5 affect tumor growth onlyKAT5 is requiredHCC for

(Figure SNU

andKAT5 s - expression 475

senescence due toloss of 3C,3D, and Supplementary Eand Supplementary Figure

cellsare p53 R

- in Huh7 andSNU d depleted

ontumor size

thantumors in non in sgKAT5 cells i ncrease induction of

(38)

- cells. To this ). found We mutant ,

we asked in

in vivo at the

growth. the , - 475

, and

or , -

This article isprotected rights by All copyright. reserved. 5C). genes genes(BAAT, Supplementary Table relevant thatwere highlyexpressed GSE14520 data set (foldchange correlation were down candidate Next (SupplementaryFigure G cycle processes, (GO)analysis of genes thatwere down sequencing in genesthatare regulated byTRRAP to promote HCC cell growth The functionof TRRAPin transcriptionalregulation beenhas well described TRRAPand KAT5activate transcriptionof canonical inhibitedcolony formation and induced senescence (Figure4Band

Accepted Articleenes thatwere up

, we , cross referenced ourRNA - positivelycorrelated with TRRAPexpression the in TCGAHCC data set (Spearman’s regulatedin sgTRRAPcells our RNAfrom

wereinvolved in genes repressedby TRRAP(Figure5 p53 genesth  0.3), / sgTRRAP p21 pathwayis not required forsenescen ITIH1and RDH16) were liver specific,whereas thatwere

. . also performedWe analysisthis in theopposite direction to (3) including atare activated by TRRAP - were regulatedin sgTRRAPcells wereenriched nucleic in acid processing 3 , , SupplementaryFigure either 3 A). A). Huh7 cells identifyto differentially activatedand 3 overexpressed inHCC compared to non

mitosis, chromosome segregation

‘cellcycle’  2),and 4) predict poor survival in HCCpatients - sequencing resultswith published data sets - regulatedin sgTRRAPcells were enrichedcell in or B thatwere ). Using). thesecriteria, we identified22

‘mitoticcell cycle’ from the GO analysis mitotic

in HCC. s

- 3B, 3C,3B, 4 and 5 sequencing data(fold change<0.5),(2)

repressed by TRRAP(Table 1,

genes

W ce in context.this

e - looked for expressed genes. ,

and celldivision 19 , we , performedRNA ). The 3 of 22 the - tumortissue in the 4 C)

genes that , suggest TRRAP

TRRAP

(8)

identify Geneontology (Figure 5A). . To

: ing - toidentify

repressed HCC (1) when

- (Figure (Figure activated

identify thatthe - were

genes -

This article isprotected rights by All copyright. reserved. inducesenescence arrestedat G1phase Previouss TRRAPand KAT5depletion induces G2/M arrest they transcriptional and SupplementaryFigure 7A). activated genes theon expression of TRRAPtarget genes. found thatWe mRNAexpression of 5Eand Supplementary Figure 7A) by TRRAP( and thatfoundKAT5 binds to the transcriptional start sites of published ChIP whetherTRRAP target geneswere also regulated byKAT5. analyzed previouslyWe Since regulate GBM(Spearman’s correlation genesactivated by TRRAPin HCCwere initiatingcells multiforme(GBM) in anothercancer type, target We

Accepted Article confirm

focus

are overexpressedare and conferpoor survival patients.to

s TRRAP

forinhibiting HCC s Figure5Dand

ed asimilar set of ing tudies have found that

- the downregulation the of thatwere derivedhumanfrom GBMsamples

activat

- and TRRAP

sequencingdata in mouseembryonic stemcells weredownregulated in sgKAT5cells, but not in sgKAT2A cells

data data set.

KAT5 (39) ionof

(24) - we analyzed gene expression data thefrom TCGAglioblastoma SupplementaryFigure . activated

genes

Since - growth , depletedcells display a similarsenescent phenotype

but mitotic TRRAP  more 0.3,Supplementary Ta

we in both cancer types I . n summary, TRRAPand KAT5 are required

cellsundergoing oncogene genes genes for analysisfurther Toassess whether , we , investigated theeffect of KAT5 and KAT2Adepletion

genes found thatthefound majorityof TRRAPtarget genes regulate is 6 recent genesafter TRRAPdepletion required

. HCC,In these genesare positivelywithcorrelated TRRAPexpression in evidencesuggests that

6 for ).

After restrict

TRRAP ble

validat

ing 4 )

, (10) as theymight be

suggesting that TRRAP differentiation ofbrain tumor

inducedsenescence are 19/22 genes may ing .

We foundthat1 We forKAT5 bindingsites

ourRNA regulate

G2 arrestG2 can also clinicallyrelevant using

qRT activatedby - sequenc the same the for for poten - , we , asked

TRRAP PCR (Figure 5E 4

ofthe 22 tial ingdata

(Figure

since genes - (23) -

This article isprotected rights by All copyright. reserved. spanning thetranscriptional start site of TOP2A toluciferase a promoter reporter and found human TOP2Apromoter. cloned a 500 We bp ( (Figure sequencing data level(Figure confirmedWe that TRRAPand KAT5 depletionreduc and Figure to TRRAPexpression in HCCand GBM decatenation (Table 1). TOP2A onfocused depletion induce Basedour on RNA depletedcells Depletionof TOP2A is sufficient to KAT5 DNAdamage isunlikely the reason for G2/Marrestand senescence upon comparedto non  (Figure accumulateat G2/M phaseand displayed a cell cycle profile similar non to sgKAT5 cellsaccumulated at We mitosis Accepted ArticleH2AX

analyzedcell cycle profiles its depletion.

8 ,we increasedexpression marker 5 5D A).Moreover, TOP2A F

). and SupplementaryFigure 7B

adownstream targetof TRRAP reasoned s Using theChIP

during mitosis 5

i E, n sgTRRAP,sgKAT2A,

s - (23)

target senescence 6 ishighly expressed at G2/Mphaseand - B,Supplementary Figures 7A and 9A).Analysis of sequencingand bioinformatic analyses,we hypothesizedthat TRRAP

that TRRAPdepletion revealedthat ing

control - (40) sequencingdata, we

was the

was by usingBrdU and PI staining and thatfound sgTRRAPand .

We foundthat We G2/Mphase. associatedwith poorsurvival in HCC(Figurepatients 6 s down

overexpressed inHCC compared to non KAT5 bind ( SupplementaryFigure recapitulate the - and regulati patient samp ). also measuredWe DNA damage and KAT5 sgKAT5cells may ing Asexpected, - ng TOP2Aexpression 133/+367)and 1 ( kb

cause to to the predictedthe KAT5 bindingsite in the

mitotic ed TOP2Aat the mRNAand protein les — senescentphenotypeofTRRAP alterations incell cycle progression TopoisomerasealphaII (TOP2A) transcriptionalstart site

(Figure 6Aand Supplementary ,

genes.

a key and foun s

7C sgKAT2Acells did not

and regulator DNA of Toinvestigate this,w dsimilar levels was 7D -

383/+617)region published )

. . This suggests positivelycorrelated - TRRAPand target - tumortissue usingthe

of TOP2A ChIP ing

control -

e that , A

- ). . s

This article isprotected rights by All copyright. reserved. Y promoting of genesthat regulateDNA replication and chromosomalsegregation are involvedin Theonc control Investigationsinto whether these factors transcription factors specificityin recruitment chromatin of fa T involvedin mitosis, and MKI67 cycle genes senescence (Figure 6F) progression. TRRAPdepletion led tocell cycle at arrest G2/Mp weHere, demonstratethat Discussion of TRRAPand KAT5 in regulatingHCC cell growth. (SupplementaryFigure thatDNA damage found that Figure formation,induc Figure9B). Next thatoverexpression of TRRAPand KAT5 induc ranscriptionalactivation of TRRAP APand FOXM1 Accepted Article s inHCC are warranted

9C ogenic roles of G2/Mgenes in tumorigenesishave beenreported.  chromosom - (17,41 H2AXlevels in sgTOP2Acells were similar tonon E

thatare regulatedby ) ,phenotypes resembling those of tion of ,we promote chromosomalinstability, and - isnot cause the forthe observed G2/M arrest 43) andinclude genesnot ,

suchMyc as and depleted

al in . senescence 9F We We found thatt . Previousstudiesother in cell types have identified ). stability TRRAP

Together,our resul . TOP2Awith CRISPR(sgTOP2A)

TRRAP,including cyclins D1, A2, D2 andMad1, E, Mad2 , a phenotype , of aggressive cancers ,

functionsin HCC - and G2/Marrest (Figure activated genes also requiredKAT5 β hemajority of TRRAP ctorsby TRRAP. -

catenin areimportant for TRRAP

previouslyknown be to ed TOP2A promoteractivity (Supplementary ts ts suggest that TOP2Aisa key mitotictarget , TRRAPKAT5or depletion are frequentlyaltered

cell

predict

proliferationby O ther s -

- activated genesin HCCare 6B target

TRRAP hase poorprognosis

and found andsenescence - E - TRRAPtargets. dependent

and Supplementary ing

and induction of

- (45) in HCC control interacting

, indicating , a promoting

. Over reduced

. Furthermore, several Inliver cancer,

cellcycle

s in patients (8, 44) (8, - , expression

suggesting

G2/M cell colony

. we ,

This article isprotected rights by All copyright. reserved. Accepted presentIn the study, we a propose different mechanism hypoacetylation andsubsequent of histone the ac immortalizeddiploidhuman fibroblastshas been described expression mutations findinghas clinical implications HCC for treatmentas 30% ofHCC patients p53has depletion induces senescence independentof the p53 Generally,p53 been not studied. protein atlas). TRRAP mutationsare reportedin severalcancer types,but effectstheir have either GBMotheror cancers types.However, unlike in HCC, GBMand HCC. Thus, TRRAPmay regulatea similar setof genesbetween different cancer Articlesamples.However, we overlapanfound in TRRAP positive correlation between cyclin A2isalso a TRRAP partiallydue to depletion induces differentiationof brain tumor The functionof TRRAPin othercancer typesisunclear. chromosomalinstability HCC.in instabilitygene also thatfound 11 (46) .

Of note,Of our studyhas identifiedFOXM1 isoneofthe TRRAP (44)

(SupplementaryFigure 10).

and p53ismore frequentlymutated HCC in patients reduced activation isa keyevent in senescence,butwe discovered that TRRAP signature etyltransferasep300induced

of our cyclin A2

22 TRRAP (45) - activated genein HCC,although

cyclin A2and T (SupplementaryFigure .

Thus, TRRAPoverexpression may contribute to TRRAP

alterationsin DNAreplication timingand velocity fork

expressionand S/G2 progression - activated

Ap53 mRNA RRAPmRNA expression in TCGA GBMpatient - genes overlappedwith the senescence -

and p21 initi e xpressionis ating cellsderived from GBMpatients, - activated genes(14 of 22) the between 8 B /p21 -

—

independent mechanism andbased dataon thefrom Human Aprevious study found that TRRAP

TRRAPdepletion leadsto dueto globalH3 andH4 pathway (47)

weobserve did not strong a not . . In previousthis study,loss a - prognostic

activated genes. We with high (17) in HCC . We We found that chromosomal

cells TRRAP predictor . . This in (47) in . This article isprotected rights by All copyright. reserved. Accepted Conflicts of interest: Deep Sequencing Coressupport. for SontheimerE. discussions. for thankWe C. Mello, Zamore,P. Acknowledgements TRRAPand mutant TRRAPmodels to furthercharacterize role its in tumordevelopment. TRRAPconsist of only genetic kno significanceof TRRAPalterations in othercancers. Moreover, because TRRAPoverexpression and mutationsaffect its tofunction understand the extentand Articleactivatingdownstream mitotic genesin Ourresults provide importantan discovery a rolefor for senescentstrategy fortreating HCC patients. targetingthe TRRAP/KAT5complex or its downstream mitotic genes maya probe KAT5 andmitotic proteins arecurrently under development acetyltransferaseactivity of the complex mayp be thatthe crystal structure of the TRRAP/KAT5 complexwas recentlycharacterized in it yeast, mechanismidentifies the TRRAPcomplex as a potential therapeutic target in HCC.Given senescencedue to d ossible to target interaction the between TRRAPand KAT5 and disrupt histone the ownregulation of mitotic genes

The authorsdisclose conflicts.no We Y.thank Liu andWe KittlerE. in theUMass Morphologyand S. Wolfe, ckouts,it will critical be establish to constitutively

HCCcell (48) F.Sanchez .

Moreover growth , such , as - Rivera , small , molecule inhibitors targeting . TRRAP

Future work should determinehow

(49,50) , J.Zuber, , J. Shi,

TOP2A,

andits co . Taken togethe

and G2/Marrest. in vivo - factorKAT5 in

T. Fazzio, T. modelsof - r, - active

Our and

This article isprotected rights by All copyright. reserved. 2000;102:463 Cell apoptosis. and repair DNA in complex acetylase histone TIP60 the of Involvement al. 7. ATM the and Esa1p containing complex acetyltransferase H4 adaptor/histone transcription essential 6. 2001;21:6782 pre with chromatin a complexis STAGA Human al. 5. cancer. 4. 3. of cellular the origin in livercancer. Nat Rev Cancer2015;15:653 2. biomarkersof hepatocellular carcinoma. Gastroenterology2015;149:1226 1. References A.S. S.K., and advice. and reagents provided data, analyzed Z.W. and H.Y. X.W.W., H.D., data. A.S., and A.S., S.K., AuthorContributions

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HepaticOncology 2015;2:315 . - S - relatedcofactor Tra RA piig n DA damage DNA and splicing mRNA , X.Z., .,

- - 6795. 473. , - W.X. wrote the with wrote manuscript comments f W.X. Ogryzko VV Ogryzko os J Vlauv A Nut C Loe J. eei lnsae and landscape Genetic JM. Llovet JC, Nault A, Villanueva J, Rossi .. eind h study. the designed W.X.

T.J.,

Y.C. 1p.EMBO 1999;18:5108J , D.M.O , Grigoriev M Grigoriev -

319. n H.M. and -

, acetylating transcription coactivator that interactsthat coactivatortranscription acetylating Groisman R Groisman .. n A.S. and S.K. - idn f binding efre eprmns n analyzed and experiments performed - 5119.

, Wang J, Horikoshi M, Scully R, et R, ScullyM, Horikoshi J, Wang , cos n io Ml el Biol Cell Mol vivo. in actors

rom authors. all ietd h poet S.K, project. the directed - 667.

- 1239. - deconstruction 1462.

This article isprotected rights by All copyright. reserved. Tumorigenesis.Mol Cell2018. Profiling Interactome Protein 16. 1998;94:363 c the for cofactor essential an is TRRAP protein related 15. 2013;497:67 Nature carcinoma. endometrial of characterization genomic Integrated al. et H, Shen Y, Liu 14. gastricadenocarcinoma. 13. al.et Alandscape of driver mutationsin melanoma.Cell 2012;150:251 12. 446. 2011;43:442 Genet Nat melanoma. in mutated frequently as GRIN2A identifies sequencing 11. neuralprogenitor fate decisions.Cell Stem Cell 2014;14:632 cell regulates specifically acetylation histone dependent 10. self maintains differentiation ofembryonic stemcells. Stem Cells 2013;31:979 Trrap cofactor acetyltransferase Histone al. et C, 9. basedprocesses: mindthe TRRAP. Oncogene 2007;26:5358 8.

Accepted Article

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l V Hre Z Ocetain f chromatin of Orchestration Z. Herceg V, kla - 209.

Copeland TD, Cole MD. The novel ATM novel The MD. Cole TD, Copeland

- yl gn tasrpin o co to transcription gene cycle - y ad 2 ocpoen. Cell oncoproteins. E2F and Myc - 643. - 5372. - 991.

- ren - 263. wl n restricts and ewal

ntrol - - - - This article isprotected rights by All copyright. reserved. Accepted Cell p16INK4a. and p53 1997;88:593 of accumulation with associated senescence cell premature 24. embryonicstem ce co and enhancers of subset a and promoters 23. progenitorcells.Nat Med 2006;12:410 hepatocellul human of subtype prognostic novel A 22. cancer.human Nat Genet2004;36:1306 o Application 21. and patientsurvival. Gastroenterology 2012;142:957 progression carcinoma hepatocellular with associated 8p on genes of identification genomic Article20. GenomeBiol 2014;15:554. genome from genesessential identificationof robust 19. wild and mutant of accumulation lymphoma.Blood 2018;131:2789 the regulating for essential is TRRAP 18. StemCell 2010;6:37 tumor brain of regulatora as identifiesTRRAP screen RNAi 17.

ern M Ln W MCrah E Bah ,Lw S.Ocgncrs provokes ras Oncogenic SW. Lowe D, Beach ME, McCurrach AW, Lin M, Serrano II Pol active to binds complex Tip60 L. Tora M, Stierle T, Ye C, Yu S, Ravens Kaposi IS, Chu L, Libbrecht J, Heo JS, Lee SS. Thorgeirsson JK, Reddy J, Heo DF, Calvisi A, Mikaelyan IS, Chu JS, Lee I al. et R, Walker J, Ji X, Zhao Y, Chen A, Budhu EL, Long S, Roessler W Li al. et L, Wagner S, Rabe V, Dalal M, Jentzsch J, Hullein M, Slabicki A, Jethwa An al. et J, Zhang CY, Chiang J, Watson M, Lorger A, Romero S, Zhu H, Wurdak , - cmaaie ucinl eois o dniy best identify to genomics functional comparative f 602. Xu H Xu

lls.Epigenetics Chromatin 2015;8:45. , X , - 47. iao T, Cong L, Love MI, Zhang F, Irizarry RA, et al. MAGeCK enables MAGeCK al. et RA, Irizarry F, Zhang MI, Love L, Cong T, iao

- 2802. - 416.

- 1311.

- euae te c the regulates - Novak P, Calvisi DF, Mikaelyan A, et al. et A, Mikaelyan DF, Calvisi P, Novak - - 966 e912. scale CRISPR/Cas9 knockout screens.CRISPR/Cas9scaleknockout r acnm drvd rm hepatic from derived carcinoma ar

- initiating cell differen cellinitiating

- i mue oes o study to models mouse fit - y ntok n mouse in network Myc - ye 5 in p53 type tiation. Cell tiation. ntegrative This article isprotected rights by All copyright. reserved. Acceptedassociatedwith G1/S transition. Oncotarget2015;6:30130 regulators cycle cell of levels the controlling by carcinoma hepatocellularpromotes 1 kinase 34. Hippo Signaling Promoteto Cytokinesis. Gastroenterolog Reducing byCells Cancer of Proliferation Contributesto 1 Protein activating GTPase Rac of 33. promotes livercancer cell proliferation and tumorigenesis.Hep 32. stemnessand associated withprognosis. poor Oncotarget 2017;8:77734 poorly in enzymes 31. BMCCancer2010;10:461. HCC. in Overexpression B Aurora carcinoma. hepatocellular in overexpression B Aurora 30. Articlethroughputsequencing data. Bioinformatics 2015;31:166 29. RNA 28. withouta reference genome.BMC Bioinformatics 2011;12:323. 27. ultrafastuniversal RNA 26. andseq small RNA 25.

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He P He , Li J, Feng MX,LiJ, Zhang , ZhangYL,etXL, al. Overexpression ormatics2013;29:15 -- Pto faeok o ok ih high with work to framework Python a Z. EliminationPCRduplicatesZ.RNAofin ioa r sgaue ee o cancer of genes signature are cinoma

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- atology2017;65:515

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- 77751. - Seq data withdataSeq or Significance of Significance

- 528. - related

- r - This article isprotected rights by All copyright. reserved. Accepted4834. 2004;23:4824 J EMBO expression. Mad2 and Mad1 of modulation by checkpoint mitotic 43. Genet Nat 2001;29: progression. cycle cell in defects and lethality embryonic early causes Trrap 42. injury.Hepatology 2011;53:954 domain transformation/transcription 41. instabilityand personalized cancer therapy. Oncogene2015;34:4019 40. 39. ArticleRevMol CellBiol 2014;15:482 38. TrendsCell Biol 2018;28: 37. livercarcinomas. Nature 2007;445:656 murine in restoration p53 by triggered is clearance tumour and Senescence al. et C, Cardo 36. cellular during genes senescence.Cell 2003;113:703 target E2F of silencing and formation heterochromatin mediated 35.

i , unn , ur , ag Q Hre Z HT oatr ra rglts the regulates Trrap cofactor HAT Z. Herceg ZQ, Wang R, Murr C, Cuenin H, Li HullaHercegZ, GellCuenin D, LleonartM, C, JacksonS, W, ZQ.Disruption Wang of cofactor acetyltransferase histone of Loss Z. Herceg N, Dubey C, Cuenin V, Shukla Zhang S, Kopetz P, Ji Y, Sun T, Chen V,Gire Dulic V. Senescence from arrest,G2 revisited. Cell Cycle 2015;14:297 Munoz Hernandez W Xue Na 206 ia , ue S Had , aia , i A, er S, pco D, t l Rb al. et DL, Spector SA, Hearn AW, Lin M, Narita E, Heard S, Nunez M, rita - 211. - , Espin D, Serrano M. Cellular senescence: from physiology to pathology. Nat pathology. to physiology from senescence: Cellular M. Serrano D, Espin edr L Zender

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associated protein im protein associated

- 660.

. oosmrs Iapa n chromosome in IIalpha Topoisomerase W.

pairs liver regeneration after toxic after regeneration liver pairs - 4031. hnvk V Cordon V, zhanovsky

- 304.

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Dominguez C Gao Saccharomyces the of Architecture G. Cai J, Cote Z, Zhang S, Ahmad X, Wang A Prieur Inductional. et M, Knaub C, Sticht A, Geldiyev T, Lutz T, Wolf F, Pinna SME, Weiler chromosomalofsignature A SzallasiZ. LN,Harris IS,Kohane AC, EklundSL, Carter Genome Cancer wbe, address Electronic Network. Research Atlas Genome Cancer

, Bourke E Bourke , 473. enr E Besnard - Brauer C Brauer

, Scobie M Scobie , Bbe A Lmir J. 5 ad 1(N4) independent p16(INK4A) and p53 JM. Lemaitre A, Babled , Thu KL Thu ession profiles predicts clinical outcome in multiple human multiple in outcome clinical predicts profiles ession - 1048. n of a Tip60 histone acetyltransferase inhibitor. Sci Rep Sci inhibitor. acetyltransferase histone Tip60 a of n , Famme MA, Koolmeister T, Helleday T, Eriksson LA, Eriksson T, Helleday T, Koolmeister MA, Famme , , - co

Mason JM Mason eedn atrto o S of alteration dependent - firstauthorship - - - 1341 e1323. 2051 e2022. Associated Protein and Protein Associated , Blaser H Blaser

- , Bray MR, Mak TW. Targeting BrayMR,TW. Mak , 536.

- hs porsin Nat progression. phase

Forkhead Box M1 in in M1 Box Forkhead

This article isprotected rights by All copyright. reserved. 0.01 presentedas mean±SD; sgTRRAPcells were measuredby qRT s CTG the assay and normalized dayto 1. sgNT andsgTRRAP. formation andinduction se of target (sgNT)and 3 individual TRRAP sgRNAs (sgTRRAP). TRRAP Figure 2.Loss of TRRAP im P high (TCGAn=115 and LEC n=31) or low (TCGA n=118and LEC n=31) TRRAPexpression. the TCGA indicatethe geometricmean ofeach group. calculatedusing moderatedt and tumorsamples discoveryrate with HCCpatient data. proliferation. growth AFigure 1. CRISPRkinome screenidentifies TRRAP a as Figure Legends enescencewas measuredby SA -

Accepted valueswere calculated using the log Article ,

***p0.001 < . and p21 A)

(left)

Three HCC Three cell lines were identifyused to genes whose depletioninhibit HCC

Eight , , the8 candidate genesare indicated

andNIH Labora

protein expressionin Huh7

(student’s c from andidate geneswere identi B)

Cellgrowth was measuredat 1, 3 the the GSE14520 Genes Genes fromthe CRISPR screen were ranked by their false p - valueswere calculated by comparing to sgNT, 0.05*p < t - nescence ofHuh7 testand signed Wilcoxon pairscellgrowth andinduces senescence inHCC cells. test). toryExperiment of Carcinogenesis (LEC - β

- gal gal staining. - rank Mantelrank - PCRand normalized sgNTcells.to Data was (left) C)

, D) Cellswere stained with crystalviole

Hep3B and TCGA

fied byintegrating CRISPRscreen results KaplanMeier curvesHCC of patients from , Hep3B D - . ) Cox*p test. < 0.05

, C) mRNAlevels of p15, p16

andSNU - mRNAlevels of TRRAP in non , and SNU , ranktest respectively.linesBlack

(right) ,

and 5days after plating using B n

andC -

essentialgene 475 data sets - 475 cellsinfected with non )

Cell growth, colony

, cellsinfectedwith ***p <0.001. ,right , p - valueswere , )cohorts with

andp21 in for t and t

HCCcell ,

**p < - tumor

- A) This article isprotected rights by All copyright. reserved. of Figure NS formation levels knockout of Figure = values Mann nude normalized p15, days staine growth with qRT in induces Figure

not

Accepted ArticleHuh7 TRRAP/KAT5 p21.

sgNT,

PCR. p16

after - mice not significant

d Whitney in

were

of 5. 4. 3.

with A)

cells

Huh7 ,

senescence.

significant cells Huh7 and Senescence and TRRAP/KAT5 Loss

plating

Schematic B) subcutaneously sgKAT2A to

crystal calculated

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and us test

of cells (student’s

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Protein

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CTG test).

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cells

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t B)

sgNT

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test).

and generation arrest.

by assay -

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β (left) ing

sgNT, infected

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transcription

with C sgKAT5 gal. weight TRRAP

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to and KAT5

and

D) sgNT,

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blot D with

at Cell

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of Data ) cells

and

and ene

day Colony

infected

cells. KAT5 p21 *p

analysis

the growth as

KAT5 p21 were < was ontology of 30

measured (right)

and

indicated 0.05,

and

mitotic

(left) impairs formation,

levels presented

values with

was

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TRRAP/KAT5

to of depletion sgKAT5.

**p levels

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) KAT5 calculated

and mRNA

mean

Cells ***p cells qRT sizes independent

in double at C)

depletion

HCC < sgRNAs.

1, blot - of

and

Colony

PCR infected

were and ± 0.001

(right)

levels

down

SD;

and

and and

p21

using and KAT5

and ,

cell in - of

5 -

This article isprotected rights by All copyright. reserved. Representative sgTOP2A. mRNA C) Protein expression respectively p (T) correlation expression regulation Figure 0.01, SD; data of infected TSS 22 id relevant significant regulated - entified

values

Accepted ArticleHuh7

Colony TRRAP samples

p (left) of ***p - values

levels TOP2A

levels

with infected TRRAP

were

genes

< and

annotation -

formation E)

analysis activated in . Loss (right), B 0.001

in of

the

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were

Cell t of quantification

D) the calculated he

(bottom).

data senescence TOP2A in target indicated with

TOP2A

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(student’s

TCGA

of GSE14520 calculated cycle sgTRRAP p

(left -

and

genes Meier value the clusters (left) TRRAP

genes.

binding .

)

analysis . E)

and indicated SA HCC

using sgRNAs mRNA

curves using and mRNA was

(right) - t

markers

cells by p21 test).

are

(second

- C) Correlation gal data

and analysis

mode comparing

quantification

calculated

published of shown expression

are compared

sgRNAs staining levels as of

set Huh7 Huh7

KAT5

analysis

rated

TCGA measured p15,

shown left)

(n=360) at

here. of

cells

cells the

and between p16 of to by t 6 ChIP using

- induce

test HCC

here. of TRRAP

Huh7

non of BrdU B) transcriptional

(right)

TCGA

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inf TOP2A and

by the -

was

and seq Pipeline -

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patients

Data

qRT

cells targeting

and 22

p21

senescence - TRRAP

data determined activated the are HCC log

TRRAP

PCR. was PI with in with

infected

- Wilcoxon for rank

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staining. non with control (second Huh7

presented start identifying control. t the

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and

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with using

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cycle

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binds

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Spearman’s F) low 0.05, Huh7

genes

data

- and

mean

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analysis

Working

test.

TOP2A to - most

mRNA down

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cells

HCC the

sets

with the

test

< D) B)

This article isprotected rights by All copyright. reserved. expression then with cells. senescence Supplementary TRRAP samples. up A of test. samples HCC Supplementary identified Table test). comparing and mitotic model. .

AcceptedTCGA Article P regulation,

- s sgNT Black

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1. genes.

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correlation

T able

with 1

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expression GBM

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DBF4 CENPF CCNA2 BUB1B ASPM SymbolGene MKI67 MCM4 MCM2 MAD2L1 KIF4A KIF20A FOXM1 EZH2 DLGAP5 AcceptedThis article isprotected rights by All copyright. reserved. Article Table

List

of Forkheadbox M1 repressive complex2 subunit Enhancerofzeste 2 polycomb associatedDLG protein 5 DBF4zinc finger Centromereprotein F Cyclin serine/threonine kinase B BUB1 mitoticcheckpoint assembly Abnormalspindle microtubule Entrez namegene Markerofproliferation ki complexcomponent 4 Minichromosome complexcomponent 2 Minichromosomemaintenance Mitotic arrestdeficient 2 like 1 Kinesin family Kinesin familymember20A

genes A2

that

are

member4A

maintenance down

- - 67 regulated

0.41 0.46 0.38 0.45 0.41 0.23 0.38 0.38 0.42 0.46 0.39 0.35 0.42 0.46 Fold change RNA

the - seq sgTRRAP/sgNTseq

absence

4.3E 1.0E 1.9E 1.2E 5.5E 1.2E 4.5E 5.5E 2.9E 8.4E 5.7E 2.2E 2.6E 1.8E P - value ------

02 06 07 06 06 04 08 07 06 06 03 03 07 02 of

TRRAP

2.38 3.03 3.13 2.88 2.70 3.24 2.27 3.41 2.49 2.41 5.25 2.45 3.33 8.22 Fold change non GSE14520 HCC/

and tumortissue

identified

9.5E 3.2E 6.3E 4.9E 1.4E 3.8E 5.9E 2.4E 6.9E 2.6E 3.9E 1.7E 1.4E 8.3E P - value

------51 72 69 62 69 80 67 78 60 71 101 59 73 112

from

0.403 0.53 0.342 0.321 0.314 0.332 0.414 0.408 0.316 0.48 0.452 0.309 0.428 0.357 coefficient Spearman's with TRRAP TCGAHCC co our

bioinformatic

- expressed P 1.6E 5.9E 1.5E 2.3E 5.3E 5.9E 2.2E 6.1E 3.9E 7.2E 2.3E 9.8E 2.0E 1.7E - value

------analyses. 14 26 10 09 09 10 15 15 09 21 18 09 16 11

XPOT TTK TPX2 TOP2A RAD51AP1 RACGAP1 PRC1 NSD2 This article isprotected rights by All copyright. reserved.

Accepted Article

Exportintrna for protein TTK kinase factor TPX2,microtubule nucleation DNAtopoisomerase II RAD51associated protein 1 Rac gtpaseactivating protein 1 Protein domain protein 2 Nuclearreceptor binding SET

regulatorof cytokinesis 1

alpha

0.40 0.46 0.47 0.47 0.37 0.34 0.50 0.39

4.9E 3.5E 1.5E 2.0E 5.6E 5.2E 3.3E 1.2E ------09 06 08 05 08 03 04 07

5.49 2.10 2.44 3.22 3.22 8.27 2.59 4.66

1.9E 9.7E 7.3E 8.5E 2.0E 2.5E 9.2E 3.0E ------62 69 71 98 62 108 106 61

0.485 0.306 0.317 0.406 0.411 0.333 0.378 0.605

2.5E 1.4E 3.6E 8.6E 4.1E 5.1E 8.2E 2.6E ------21 08 09 15 15 10 13 35

This article isprotected rights by All copyright. reserved. Accepted Article