Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling

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1 Vol Otoer 29 doi:1.138/nture8356 Tnkyrse inhiition stilizes xin nd ntgonizes Wnt signlling Shih-Min A. Hung 1, Yuji M. Mishin 1, Shnming Liu 1, Atwood Cheung 1, rnk Stegmeier 1, Gregory A. Mihud 1, Olg Chrlt 1, Elizeth Wiellette 1, Yue Zhng 1, Stephnie Wiessner 1, Mr Hild 1, Xioying Shi 1, Christopher J. Wilson 1, Crig Miknin 1, Vi Myer 1, Aleem zl 1, Ronld Tomlinson 1, rizio Serlu 1, Wenlin Sho 1, Hong Cheng 1, Mihel Shultz 1, Christin Ru 2, Mrkus Shirle 2 {, Judith Shlegl 2, Sonj Ghidelli 2, Stephen well 1, Chris Lu 1, Dniel Curtis 1, Mr W. Kirshner 3, Christoph Lenguer 1 {, Peter M. inn 1, John A. Tllrio 1, Tewis Bouwmeester 2 {, Jeffery A. Porter 1, Andres Buer 2 { & eng Cong 1 The stility of the Wnt pthwy trnsription ftor -tenin is tightly regulted y the multi-suunit destrution omplex. Deregulted Wnt pthwy tivity hs een implited in mny ners, mking this pthwy n ttrtive trget for ntiner therpies. However, the development of trgeted Wnt pthwy inhiitors hs een hmpered y the limited numer of pthwy omponents tht re menle to smll moleule inhiition. Here, we used hemil geneti sreen to identify smll moleule,, whih seletively inhiits -tenin-medited trnsription. stimultes -tenin degrdtion y stilizing xin, the onentrtion-limiting omponent of the destrution omplex. Using quntittive hemil proteomi pproh, we disovered tht stilizes xin y inhiiting the poly-adp-riosylting enzymes tnkyrse 1 nd tnkyrse 2. Both tnkyrse isoforms intert with highly onserved domin of xin nd stimulte its degrdtion through the uiquitin-protesome pthwy. Thus, our study provides new mehnisti insights into the regultion of xin protein homeostsis nd presents new venues for trgeted Wnt pthwy therpies. The evolutionrily onserved Wnt/-tenin signl trnsdution pthwy ontrols mny iologil proesses 1. A key feture of the Wnt pthwy is the regulted proteolysis of the downstrem effetor -tenin y the -tenin destrution omplex. The prinipl onstituents of the -tenin destrution omplex re denomtous polyposis oli (APC), xin nd glyogen synthse kinse 3/ (GSK3/). In the sene of Wnt pthwy tivtion, ytosoli -tenin is onstitutively phosphorylted nd trgeted for degrdtion. On Wnt stimultion the -tenin destrution omplex dissoites, leding to the umultion of nuler -tenin nd trnsription of Wnt pthwy-responsive genes. Inpproprite tivtion of the Wnt pthwy hs een oserved in mnyners 2,3.Notly,truntingmuttionsofthetumoursuppressor APC re the most prevlent geneti ltertions in oloretl rinoms 46. The effiient ssemly of the multi-protein destrution omplex is dependent on the stedy-stte levels of its prinipl onstituents. Axin hs een reported to e the onentrtion-limiting ftor in regultingtheeffiienyofthe-tenindestrutionomplex 7,8 ndoverexpression of xin indues -tenin degrdtion in ell lines expressing trunted APC 911. Thus, it is likely tht xin protein levels need to e tightly regulted to ensure proper Wnt pthwy signlling. In ft, Wnt signllingitselfregultesthelevelofxintseverlsteps,withaxin2eing mjor trnsriptionl trget of the -tenint-ell ftor (TC) omplexndwntsignllingpromotingthedegrdtionofxin 12,13.However, the moleulr mehnisms tht regulte protein homeostsis of destrution omplex omponents nd omplex ssemly remin elusive. In this study we used hemil-geneti nd -proteomi pprohes to serh for novel modultors of the Wnt signlling pthwy. We identified low moleulr mss ompound tht n prolong the hlflife of xin nd promote -tenin degrdtion through inhiiting tnkyrse (). Our study unovers new mehnism tht ontrols xin protein stility nd Wnt pthwy signlling, nd its therpeuti exploittion holds promise for treting Wnt-pthwydependent ners. inhiits Wnt signlling y inresing xin levels ws identified s smll moleule inhiitor of the Wnt/tenin pthwy from high-throughput sreen using Wntresponsive Super-Topflsh (ST) luiferse reporter ssy in ells (ig. 1). strongly inhiited Wnt3-stimulted ST tivity in ells, ut did not ffet CRE, N-kB or TG- luiferse reporters (ig. 1). In ontrst,, lose struturl nlogue of (ig. 1), hd no effet on the Wnt3indued ST reporter (ig. 1). tretment loked Wnt3indued umultion of -tenin in ells (ig. 1), inditing tht the ompound modultes Wnt signlling upstrem of -tenin. Interestingly, lso inhiited ST tivity in SW48 ells, oloretl ner ell line hrouring trunted APC (ig. 1d). deresed -tenin undne, ut signifintly inresed -tenin phosphoryltion (S33/S37/T41) in SW48 ells (ig. 1e), inditing tht promotes the phosphoryltion-dependent degrdtion of -tenin y inresing the tivity of the destrution omplex. To explore how my inrese the tivity of the destrution omplex, we investigted whether ompound tretment lters the protein levels of known Wnt pthwy omponents. Notly, the 1 Novrtis Institutes for Biomedil Reserh, 25 Msshusetts Avenue, Cmridge, Msshusetts 2139, USA. 2 Cellzome AG, Meyerhofstrsse 1, D Heidelerg, Germny. 3 Deprtment of Systems Biology, Hrvrd Medil Shool, Boston, Msshusetts 2115, USA. {Present ddresses: Novrtis Institutes for Biomedil Reserh, CH-42 Bsel, Switzerlnd (T.B., A.B.); Novrtis Institutes for Biomedil Reserh, Cmridge, Msshusetts 2139, USA (M. S.); Snofi-Aventis, 9443 Vitry-sur-Seine, rne (C.L.) Mmilln Pulishers Limited. All rights reserved

2 NATURE Vol Otoer 29 Ativity (%) Ativity (%) HO N S N HO N N N O Log 1 [Conentrtion (µm)] d Ativity (%) Ativity (%) h Log 1 [Conentrtion (µm)] Log 1 [Conentrtion (µm)] Log 1 [Conentrtion (µm)] -ST -CRE -N-κB -TG-β Wnt3 Wnt3 SW48-ST SW48-ST-TC3VP16 protein, ut not messenger RNA, levels of xin 1 nd xin 2 were strongly inresed fter tretment (ig. 1e nd dt not shown; Supplementry ig. 1). In ddition, we noted strong inrese in xingsk3 omplex formtion, presumly euse of inresed xin protein levels (Supplementry ig. 1). Similr effets of were lso oserved in ells, nother oloretl ner ell line with trunted APC (Supplementry ig. 1, d). Importntly, short interfering RNA (sirna)-medited depletion of xin 1/2 in SW48 ells reversed the effet of on -tenin degrdtion (ig. 1f nd Supplementry ig. 1f) nd diminished the inhiitory tivity of on the ST reporter (Supplementry ig. 1e), inditing tht inhiits Wnt signlling y inresing xin 1/2 protein levels. Together, these findings support the hypothesis tht inreses the onentrtion of xingsk3 omplex, therey promoting phosphoryltion nd degrdtion of -tenin. regultes xin levels through tnkyrse inhiition To identify the ellulr effiy trget(s) through whih upregultes xin protein levels, we used three-hnnel itraq quntittive hemil proteomis pproh. This strtegy is sed on the immoiliztion of iotive nlogue of (Supplementry ig. 2) to ffinity-pture ellulr proteins from ell lystes spiked with n exess mount (2 mm) of, the intive nlogue or. Speifi inding to the immoilized ompound should e ompeted with, ut not with. Of 699 proteins quntified (Supplementry Tle 1), 18 proteins were signifintly nd speifilly ompeted-off e f p- sirna p- Atin SW48 AXIN1/2 SW48 igure 1 inhiits Wnt/-tenin signlling y inresing xin protein levels., Compound strutures of nd., (24 h) speifilly inhiits ST reporter, ut not CRE, N-kB or TG- reporter in ells (n 5 12). The orresponding reporter tivity ws normlized to. Error rs represent s.d. here nd throughout the study., (1 mm) redues Wnt3-indued stiliztion of -tenin in ells. d, (24 h) inhiits ST tivity in APC-defiient SW48 ells (n 5 12). SW48 ells expressing TC3VP16 were used s ontrol s overexpression of TC3VP16 fusion protein lrgely ypssed the requirement of -tenin on ST tivity. e, (1 mm, 16 h) dereses the undne of -tenin nd inreses the undne of xin nd phospho--tenin (p--tenin) in SW48 ells. f, The effet of on -tenin in SW48 ells is eliminted y AXIN1/ 2 sirnas. (.65%,.2s of the men) with solule (ig. 2), inluding the poly(adp-riose) polymerses, PARP2,, nd severl known sustrtes, presumly o-purified with. To estlish the ffinity of inding to the identified PARP proteins, we performed ompound ompetition experiment, nd showed tht, ut not, loks inding t.1 mm nd loks /2 inding t 1 mm (ig. 2). We further hrterized the ompound inding using Cy5-lelled nd reominnt PARP proteins. We found tht inds tightly to the tlyti (PARP) domins of nd (K d 5.99 nd.93 mm, respetively) (ig. 2). lso inds to reominnt, lthough with signifintly lower inding ffinity (K d mm). To determine whih PARP fmily memer(s) re the tul effiy trgets of, we ssessed their sirna-medited loss-of-funtion phenotypes. Co-depletion of nd phenoopied the effet of y inresing the protein levels of xin 1 nd 2, wheres omintoril /2 knokdown did not (ig. 3 nd Supplementry ig. 3, ). In ddition, ABT-888, potent nd PARP2 inhiitor 14 tht hs miniml tivity on nd Perentge ompetition versus Competitor on. (µm) PARP2 HIST1H4A H2AJ RPA3 SSRP1 SUPT16H 29 Mmilln Pulishers Limited. All rights reserved BAN1 GMDS HIST2H3D XRCC SSSCA1 EMD PARP2 XRCC6 RPA2 RPA Perentge ompetition versus mp 2, 4, 6, Protein onentrtion (nm) igure 2 Identifition of the ellulr effiy trgets of., Stter plot depiting ll proteins (n 5 398) identified nd quntified with.3 unique spetr in three-hnnel itraq quntittive hemil proteomis experiment. Proteins re plotted s funtion of the perentge ompetition with the tive ompound reltive to the vehile (), plotted on the y-xis, versus the perentge ompetition with the intive ompound reltive to, plotted on the x-xis. The lue order lines on eh xis signify the 2 s.d. from the verge ompetition vlue ross ll identified proteins with.3 spetr, whih is indited y the middle lue line. All proteins tht re speifilly ompeted with the tive ompound re highlighted in red., Immunolot nlysis for, nd PARP2 on lystes from ompound ompetition experiment., Cy5-onjugted diretly inds the PARP domin of nd with high ffinity. Rw millipolriztion (mp) dt were nlysed with one-site totl inding sturtion lgorithm. 615

3 NATURE Vol Otoer 29 d CRE (forskolin) e f ST (Wnt3 CM) lg PARP2 /2 p- Atin 1A 2A 1B 2B 1A2A 1B2B 1A/B2A/B lg Wnt3 CM 1A 2A 1A2A 1B 2B 1B2B g (M154V) h i DOX DOX DOX DOX SW48 SW48 (ig. 3i nd Supplementry ig. 3i), did not ffet the protein levels of xin nd (ig. 3j). Colletively, these results indite tht nd re the ellulr effiy trgets of. Using dditionl sirnas, we further demonstrted tht o-depletion of nd inreses -tenin phosphoryltion, dereses -tenin undne, nd inhiits the trnsription of -tenin trget genes in SW48 ells (ig. 3 nd Supplementry ig. 3, d). Notly, depletion of or lone did not inrese xin 1/2 protein levels (ig. 3), inditing tht nd funtion redundntly in regulting xin protein levels. Co-depletion of nd lso phenoopied the phrmologil effet of in nd ells (ig. 3, d nd Supplementry ig. 3e). In ddition, omined nd sirna tretment inresed xin protein levels even further (Supplementry ig. 3f). We exmined whether regultion of xin nd Wnt signlling y is evolutionrily onserved. We found tht doule-strnded RNA (dsrna) trgeting Drosophil inresed protein levels, ut not mrna levels, of exogenously expressed Drosophil xin in S2 ells (ig. 3e nd Supplementry ig. 3g), nd speifilly inhiited Wnt reporter (P,.1) (ig. 3f). We lso showed tht tretment of zerfish emryos with, ut not its intive nlogue, signifintly deresed the signl from -tenin/le1 reporter (TOPGP; green fluoresent protein) (Supplementry ig. 4) nd inhiited the mrna expression of the -tenin trget gene xin2 (Supplementry ig. 4). urthermore, onomitnt knokdown of two zerfish genes (tnks1 nd tnks1) with morpholinos speifilly deresed TOPGP expression (Supplementry ig. 4). Together, these findings indite tht the regultory funtion of in Wnt signlling is evolutionrily onserved. GST SAMPARP 1B 2B 1B2B igure 3 Tnkyrse modultes xin protein levels.,, Simultneous depletion of nd phenoopies y inresing xin protein levels nd deresing -tenin protein levels in SW48 ells. or oth nd, two independent sirnas were generted from unique trget sequenes, lelled A nd B (1A, 2A, 1B nd 2B)., Codepletion of nd inreses the protein level of xin 1 (upper pnel) nd loks Wnt3-indued -tenin umultion (lower pnel) in ells. d, Co-depletion of nd inhiits ST reporter, ut not CRE reporter, in ells (n 5 4). e, Knokdown of inreses the protein level (upper pnel), ut not the mrna level (lower pnel), of hemgglutinin-onjugted Drosophil xin (Axin33HA) in Drosophil S2 ells. dsrna ginst white ws used s ontrol (n 5 4). 616 Perentge luiferse tivity A2A 1B2B IC 5 (µm) dsrna: Axin3 HA Axin3 HA mrna expression h dsrna: PARP >18.75 >18.75 >18.75 >18.75 ABT IWR-1-endo >18.75 > IWR-1-exo >18.75 > Mmilln Pulishers Limited. All rights reserved white white j S2 Wnt signlling is required for regenertive proesses in dult zerfish, inluding fin regenertion 15,16. Inks genes re expressed in udl fin tissue, nd no trnsriptionl hnges re detetle fter injury (Supplementry ig. 4d, pnel iii)., ut not, inhiited fin regenertion (Supplementry ig. 4d, pnels i nd ii) nd Wnt-dependent tivtion of xin2 trnsription (Supplementry ig. 4d, pnel iii). This phenotype is remrkly similr to wht ws oserved for IWR-1, n xin stilizer reently identified 17, whih we hve found to lso inhiit (s desried in detil elow). The ft tht two struturlly unrelted inhiitors lok Wnt-dependent fin regenertion indites tht inhiition n ntgonize Wnt signlling in vivo. However, we did not oserve ovious erly developmentl defets ssoited with Wnt inhiition. Given tht xin umultion requires severl hours, it is possile tht the kinetis of xin stiliztion re too slow to hve n impt on erly developmentl phenotypes in this model system. Alterntively, funtion in Wnt pthwy modultion my e ell-type- or orgn-speifi (for exmple, til fin). nd modify their sustrtes through the ddition of severl ADP-riose units, referred to s poly-adp-riosyltion (PARsyltion) 18. To determine whether the PARsyltion tivity of is essentil for regulting xin protein levels, we performed sirna-resue experiments. In trnsient trnsfetion experiments, overexpression t high levels indues -tenin stiliztion in PARP-domin-independent fshion. However, this is n overexpression rteft proly medited y sequestrtion of endogenous xin y overexpressed (see Supplementry ig. 6 for detiled disussion). We therefore used lentivirl trnsdution t low multipliity of infetion for the resue experiments, in whih the Reltive luiferse tivity No lignd Lignd dsrna white white Wingless S2 ABT-888 SW48 * IWR-1-endo IWR-1-exo BMP * f, Depletion of speifilly inhiits Wnt reporter (LELu), ut not BMP reporter (BRELu), in S2 ells (*P,.1, n 5 4). g, Doxyyline (DOX)-indued expression of wild-type, ut not tlytilly intive mutnt (M154V), resues sirna-indued umultion of xin 1 in ells. h, inhiits uto- PARsyltion of. Reominnt protein used in this experiment ontins the sterile lph motif (SAM) domin nd the PARP domin. i, Compound tivity in /2 nd iohemil ssys. j, Effets of ompounds (5 mm, 24 h) on the protein levels of xin 2 nd in SW48 ells. A kground nd tht migrted right elow the nd is indited y n sterisk.

4 NATURE Vol Otoer 29 expression of is mrkedly lower thn in the trnsient trnsfetion experiments (dt not shown). Doxyyline-indued expression of wild-type, ut not the tlytilly intive (M154V) mutnt 19, resued the effet of sirnas on xin 1 protein expression (ig. 3g). Similr results were otined with (Supplementry ig. 3h). These dt indite tht the tlyti tivity of oth tnkyrses is required for the regultion of xin protein levels. Consistent with this finding, we showed tht, ut not, inhiits uto-parsyltion of in vitro (ig. 3h). In iohemil tivity ssys, strongly inhiited nd, with hlf-mximl inhiitory onentrtion vlues of.11 nd.4 mm, respetively, ut displyed muh weker effets on nd PARP2 (ig. 3i). Auto-PARsyltion of hs een reported to promote its own degrdtion through the uiquitin-protesome pthwy 2. We found tht tretment led to signifint inrese in protein levels (ig. 3j), inditing tht lso inhiits uto-parsyltion in vivo. A reent study desried two ompound series (IWR-1, -2, nd IWR-3, -4, -5) tht inrese xin protein levels nd inhiit Wnt signlling 17. It ws shown tht IWR-1-endo (lso referred to s IWR-1) inresed xin protein levels, wheres its lose nlogue IWR-1-exo hd muh weker tivity. Although the uthors suggested tht IWR-1 might stilize xin y inding to region in the roxy-terminl hlf of xin, we exmined whether IWR-1 ompounds my in ft stilize xin y inhiiting. Notly, IWR-1-endo strongly inhiited nd in iohemil ssys, with IWR-1-exo eing pproximtely tenfold less tive (ig. 3i nd Supplementry ig. 3i), onsistent with their poteny in xin stiliztion ssys (ig. 3j). In ontrst, neither of these two ompounds inhiited /2 (ig. 3i). Consistent with the tivity of IWR-1-endo in iohemil ssys, IWR-1-endo, ut not IWR-1-exo, signifintly stilized endogenous, nd xin 2 (ig. 3j nd Supplementry ig. 3j), inditing tht IWR-1-endo inhiited uto-parsyltion of in vivo. These results indite tht IWR-1-endo stilizes xin t lest in prt through inhiition. Together, our dt demonstrte tht the PARsyltion tivity of, ut not /2, is required for the regultion of xin protein levels. interts with xin to regulte xin levels We next explored how regultes xin protein levels. Using oimmunopreipittion experiments, we found tht endogenous nd ssoite with xin 2 in SW48 ells (ig. 4). In ddition, we deteted strong intertion etween xin 1/2 nd in the yest two-hyrid ssy (ig. 4 nd dt not shown). The nkyrin repet domin of ws required nd suffiient for the intertion with xin 1 (Supplementry ig. 6). Strikingly, smll mino-terminl region of xin 1 (mino ids 193), whih enompsses the most onserved streth of mino ids within xin (ig. 4), ws oth required nd suffiient to intert with (ig. 4). The speifi intertion of xin 1 with through this domin, whih we nmed tnkyrse-inding domin (TBD), ws further sustntited y glutthione S-trnsferse (GST) pull-down nd o-immunopreipittion ssys (ig. 4d, e). We next ssessed the funtionl onsequenes of disrupting the physil intertion etween xin nd. If inding etween xin nd promoted xin degrdtion, deletion of the TBD should result in the stiliztion of xin. Indeed, wheres ells expressing wild-type GPxin 1 demonstrted low sl protein levels tht were strongly inresed in response to tretment, ells expressing GPxin 1(D193) lredy exhiited high sl protein levels tht did not further respond to ompound tretment (ig. 4f nd Supplementry ig. 5). Importntly, restoring the xin 1 intertion y fusing the heterologous inding domin of either IRAP (insulin-regulted minopeptidse, lso known s LNPEP) or TR1 (telomeri repet inding ftor, lso known s TER1) to GPxin 1(D193) fully restored its response to (ig. 4f). We further hypothesized tht overexpression of the N-terminl domin of xin my ompete with endogenous xin for the inding of nd thus stilize xin. Indeed, overexpression of GPxin 1N (mino ids 187), ut not the mutnt with the AXIN2 IP: IgG WB: WB: xin 2 SW48 β-gltosidse ssy GSK3β Vetor RGS GSK3β DIX WT Humn (xin 1) ASTEDAPRPPVPGEEGELVS N88 / / Mouse ASTEDAPRPPVPGEEGELVS N215 - Xenopus GSTEDAPRPPVPGEEGELIT ΔN35 Zerfish SSTEDAPRPPVPGEEGDLVS ΔN65 Drosophil DDNECSGPRPPVPGEESRVKK ΔN88 Se urhin SGGSQLSERPPVPGEETEDRG ΔN1 ΔC15 Humn (xin 2) SSREDAPRPPVPGEEGETPP Δ GPxin d e f g 1N GPxin 1 GPxin 1 IRAP TR1 onstruts: WT Δ193 Δ193 Δ193 DOX GPxin 1(Δ193) lg IP: lg GPxin 1 WB: GP GP GP GPxin 1 GST TCL lg lg GP 1% input GST GSTxin 1N GSTxin 1N(Δ193) GSTxin 1(193) GPxin 1N (Δ193) SW48 igure 4 Tnkyrse physilly nd funtionlly interts with xin., Coimmunopreipittion of endogenous xin 2 nd in SW48 ells., Binding etween xin 1 frgments nd in the yest two-hyrid ssy (1, strong inding; 1/2, wek inding; 2, no inding). The N88 frgment retins prtil self-tivtion tivity., The TBDs of xin proteins re evolutionrily onserved. Highly onserved mino id residues re depited in red. d, The N-terminl frgment of xin 1 (mino ids 187) 29 Mmilln Pulishers Limited. All rights reserved inds to in GST pull-down ssy. e, The TBD is required for the intertion etween xin 1 nd in o-immunopreipittion ssy. f, The TBD is required for -indued xin 1 protein umultion in SW48 ells. The expression of GPxin 1 ws under the ontrol of the metllothionein promoter to ensure low trnsription. g, DOX-indued overexpression of GPxin 1N (mino ids 187) leds to umultion of endogenous xin 1 in ells. 617

5 NATURE Vol Otoer 29 deleted TBD, sustntilly inresed endogenous xin 1 protein levels while not ffeting its mrna expression (ig. 4g nd dt not shown). Together, these findings demonstrte tht the physil intertion etween xin nd, whih is medited y the evolutionrily onserved TBD, is ritil for regulting xin protein levels. Axin is degrded through PARsyltion nd uiquitintion The inrese in xin protein levels in response to tretment ould e due to modultion of trnsltion or protein stility. Consistent with the ltter possiility, tretment prolonged the hlf-life of endogenous xin 2 in SW48 ells (ig. 5). The degrdtion of xin is proly medited y the uiquitin-protesome pthwy, euse the polyuiquitintion of xin 1 inresed signifintly fter ddition of the protesome inhiitor MG132 (ig. 5). In ontrst, o-tretment of with MG132 signifintly diminished xin 1 nd xin 2 polyuiquitintion (ig. 5 nd Supplementry ig. 7), inditing tht my stilize xin y preventing its polyuiquitintion. Auto-PARsyltion of or -medited TR1 PARsyltion leds to uiquitintion nd degrdtion of or TR1, respetively 2,21. Together with our findings, this rised the possiility tht WB: xin 1 TCL (h) IP: IgG U MG132 MG132 MG132 MG132 WB: U WB: xin 1 WB: tuulin SW48 GSTxin 1(128) GSTxin 1 (128) SW48 Poly-U-xin 1 d e IP: WB: PAR WB: GP Control GP GPxin 1 GPxin 1 Wsh off with: Pre-tret with IP: xin 2 TCL WB: U WB: PAR WB: xin 2 SW48 MG132 MG132 WB: tuulin SW48 GPxin 1 GPxin 1 Poly-Uxin 2 igure 5 stilizes xin protein nd inhiits uiquitintion of xin., (1 mm) stilizes xin 2 in SW48 ells s indited y pulse-hse nlysis., Uiquitintion of xin is inhiited y (1 mm). The position of xin 1 is lelled with n rrow. Slow-migrting polyuiquitinted xin 1 onjugtes re indited., In vitro PARsyltion of GSTxin 1 (mino ids 128) y. d, (1 mm, 6 h) inhiits in vivo PARsyltion of xin 1. e, Post-trnsltionl modifition of xin 2 in ompound wsh-off experiment. SW48 ells pretreted with were wshed nd inuted with medium supplemented with the indited ompound for 1 h, nd nlysed y the immunopreipittion ssy. The position of xin 2 is indited y the rrows. IP, immunopreipittion; TCL, totl ell lystes Mmilln Pulishers Limited. All rights reserved xin degrdtion my e filitted through diret PARsyltion y. Indeed, ws le to PARsylte n xin 1 frgment (mino ids 128) ontining the TBD in vitro (ig. 5), whih ws ompletely inhiited y tretment (ig. 5). Using n ntiody tht speifilly reognizes the PAR modifition, we lso oserved tht exogenously expressed GPxin 1 ws PARsylted in ells (ig. 5d). In ddition, the PARsyltion signl ws strongly redued in the presene of (ig. 5d), inditing tht xin PARsyltion is medited y in vivo. To enhne the detetion of endogenous xin 2 uiquitintion nd PARsyltion, we pretreted ells with to inrese endogenous xin 2 levels. ws rpidly degrded within 1 h fter ws wshed off (ig. 5e). As expeted, tretment of MG132 loked xin 2 degrdtion nd strongly inresed its polyuiquitintion (ig. 5e nd Supplementry ig. 7). Tretment with nd MG132, however, ompletely loked the uiquitintion of xin 2 (ig. 5e nd Supplementry ig. 7). Interestingly, n nti-par ntiody retive signl tht o-migrted with xin 2 ws deteted when ells were treted with MG132 lone nd disppered when ells were lso treted with (ig. 5e). Together, these findings re onsistent with the hypothesis tht promotes the uiquitintion nd degrdtion of xin, whih my e medited, t lest in prt, through the diret PARsyltion of xin. inhiits growth of ner ells Beuse inhiited -tenin signlling even in APC-defiient ells, we exmined whether this ompound ould inhiit the prolifertion of APC-defiient oloretl ner ells using -tenin-dependent ells nd -tenin-independent RKO ells (Supplementry ig. 8). Under low serum growth onditions,, ut not the intive nlogue, signifintly inhiited olony formtion of ells (ig. 6). Importntly, did not ffet olony formtioninrkoells(ig.6). ws proposed to e required for the resolution of sister telomere ssoition or ssemly of ipolr spindles, nd knokdown ws reported to use strong mitoti rrest 22,23. However, using tretment or individul/omintoril / sirna knokdown, we did not oserve ny overt mitoti rrest phenotype (Supplementry ig. 8 nd dt not shown), demonstrting tht does not inhiit the prolifertion of ells through n ntimitoti funtion. If the ntiprolifertive effet of on ells ws insted medited y n inrese in xin protein levels, knokdown of AXIN1/2 my resue the growth inhiitory effets of ompound tretment. Indeed, sirna-medited depletion of xin 1 nd xin 2 proteins ompletely olished the ntiprolifertive effet of (ig. 6), inditing tht the growth inhiitory effets of in ells re due to xin-dependent inhiition of Wnt signlling. Disussion Despite the ft tht the Wnt pthwy is n ttrtive trget for ntiner therpy, there re few druggle trgets in this pthwy to generte smll moleule Wnt inhiitors. Using hemil genetis pproh, we hve disovered tnkyrses s novel trgets for Wnt inhiition nd desried novel mehnism to promote -tenin degrdtion through inhiition of tnkyrses nd stiliztion of xin (Supplementry ig. 9). This finding my pve the wy for mehnism-sed tretments of Wnt-dependent ners. Our dt indite tht tnkyrses re physiologil regultors of xin protein homeostsis nd Wnt signlling. Although the preise mehnisti detils remin unler, tnkyrses seem to promote the uiquitintion of xin, possily through diret PARsyltion of xin. Notly, -medited PARsyltion seems to lso stimulte uiquitinmedited proteolysis of TR1 nd itself 2,21. These findings indite tht oupling of these iohemil proesses my e more generl regultory mehnism, kin to the widespred ourrene of phosphodegrons tht ouple phosphoryltion to uiquitintion 24,25.

6 NATURE Vol Otoer 29 sirna AXIN1/2 sirna µm.33 µm 3.3 µm RKO µm.33 µm 3.3 µm µm 1 µm Compound (µm) Tnks1 2/2 nd Tnks2 2/2 mie re lrgely norml nd doule knokout of Tnks1/2 uses erly emryoni lethlity, inditing their redundny in mouse development 26,27. Conditionl knokout of oth Tnks1 nd Tnks2 will e importnt to understnd the role of tnkyrses in regultion of Wnt signlling in tissue homeostsis nd disese. Axin is onentrtion-limiting ftor in the -tenin degrdtion omplex 7,8. Our study further highlights the importne of xin s key regultory node in the Wnt signlling sde. We speulte tht xin my funtion more generlly s signl integrtor nd tht other proteins/pthwys my modulte Wnt pthwy tivity y impinging on this key regultory node. urther investigtion of the moleulr mehnisms tht regulte xin protein nd their potentil deregultion in disese onditions is likely to provide dditionl venues for treting Wnt-dependent ners nd other Wnt-relted diseses. METHODS SUMMARY Compound ffinity purifition nd mss spetrometry nlysis. Compound ffinity purifition, mss spetrometry nd dt nlysis were performed essentilly s previously desried 28. A derivtized, iotive nlogue LDW639 with primry mine group ws oupled to NHS (N-hydroxysuinimide)-tivted Sephrose 4 eds. ells were homogenized in lysis uffer (5 mm Tris- HCl, ph 7.5, 5% glyerol, 1.5 mm MgCl 2, 15 mm NCl, 2 mm N, 1 mm N 3 VO 4, 1 mm dithiothreitol, 5 mm lyulin A,.8% Igepl-CA63 nd protese inhiitor oktil). Cell lystes were pre-inuted with 2 mm, or for 3 min, nd inuted with LDW639-mtrix for nother 6 min. After extensive wshes, ound mteril ws eluted nd resolved y SDSpolyrylmide gel eletrophoresis. Gel lnes were ut into slies ross the full seprtion rnge nd sujeted to in-gel trypti digestion, followed y itraq lelling (Applied Biosystems). Peptides extrted from -treted smples were lelled with itraq regent 116 nd omined with extrts of ompound-treted smples lelled with itraq regents 114 Numer of olonies Numer of olonies Numer of olonies Cell Compound (µm) Cell Cell RKO µm 1 µm sirna AXIN1/2 sirna igure 6 inhiits olony formtion in n xin-dependent mnner.,, inhiits olony formtion of, ut not RKO, ells. nd RKO ells were seeded nd grown in.5% serum medium ontining the indited ompounds (n 5 3). The numer of olonies in eh well ws ounted., The effet of on olony formtion is xin-dependent. ells were trnsfeted with sirnas ginst AXIN1 nd AXIN2 or ontrol sirna, nd tested in the olony formtion ssy (n 5 3). nd 115, respetively. Liquid hromtogrphytndem mss spetrometry ws done using n Eksigent 1D1 HPLC system oupled to LTQ Oritrp mss spetrometer (Thermo-innign). Tndem mss spetr were generted using pulsed-q dissoition, enling detetion of itraq reporter ions 29. Peptide mss nd frgmenttion dt were used to query n in-house urted version of the IPI dtse using Msot (Mtrix Siene). Protein identifitions were vlidted using deoy dtse. itraq reporter ion-sed quntifition ws performed with softwre developed in-house. Reeived 7 My; epted 3 July 29. Pulished online 16 Septemer Mmilln Pulishers Limited. All rights reserved 1. Clevers, H. Wnt/-tenin signling in development nd disese. Cell 127, (26). 2. Polkis, P. The mny wys of Wnt in ner. Curr. Opin. Genet. Dev. 17, 4551 (27). 3. Brker, N. & Clevers, H. Mining the Wnt pthwy for ner therpeutis. Nture Rev. Drug Disov. 5, (26). 4. Miyki, M. et l. Chrteristis of somti muttion of the denomtous polyposis oli gene in oloretl tumors. Cner Res. 54, (1994). 5. Mori, Y. et l. Somti muttions of the APC gene in oloretl tumors: muttion luster region in the APC gene. Hum. Mol. Genet. 1, (1992). 6. Powell, S. M. et l. APC muttions our erly during oloretl tumorigenesis. Nture 359, (1992). 7. Sli, A., Lee, E., Myer, L. & Kirshner, M. W. Control of -tenin stility: reonstitution of the ytoplsmi steps of the Wnt pthwy in Xenopus egg extrts. Mol. Cell 5, (2). 8. Lee, E., Sli, A., Kruger, R., Heinrih, R. & Kirshner, M. W. The roles of APC nd xin derived from experimentl nd theoretil nlysis of the Wnt pthwy. PLoS Biol. 1, E1 (23). 9. Behrens, J. et l. untionl intertion of n xin homolog, ondutin, with -tenin, APC, nd GSK3. Siene 28, (1998). 1. Kishid, M. et l. Axin prevents Wnt-3-indued umultion of -tenin. Onogene 18, (1999). 11. Hrt, M. J., de los Sntos, R., Alert, I. N., Ruinfeld, B. & Polkis, P. Downregultion of -tenin y humn xin nd its ssoition with the APC tumor suppressor, -tenin nd GSK3. Curr. Biol. 8, (1998). 12. Leung, J. Y. et l. Ativtion of AXIN2 expression y -tenin-t ell ftor. A feedk repressor pthwy regulting Wnt signling. J. Biol. Chem. 277, (22). 13. Willert, K., Shimoto, S. & Nusse, R. Wnt-indued dephosphoryltion of xin releses -tenin from the xin omplex. Genes Dev. 13, (1999). 14. Donwho, C. K. et l. ABT-888, n orlly tive poly(adp-riose) polymerse inhiitor tht potentites DNA-dmging gents in prelinil tumor models. Clin. Cner Res. 13, (27). 15. Poss, K. D., Shen, J. & Keting, M. T. Indution of lef1 during zerfish fin regenertion. Dev. Dyn. 219, (2). 16. Stoik-Cooper, C. L. et l. Distint Wnt signling pthwys hve opposing roles in ppendge regenertion. Development 134, (27). 17. Chen, B. et l. Smll moleule-medited disruption of Wnt-dependent signling in tissue regenertion nd ner. Nture Chem. Biol. 5, 117 (29). 18. Hsio, S. J. & Smith, S. Tnkyrse funtion t telomeres, spindle poles, nd eyond. Biohimie 9, 8392 (28). 19. Sodio, J. I., Lodish, H.. & Chi, N. W. Tnkyrse-2 oligomerizes with tnkyrse-1 nd inds to oth TR1 (telomere-repet-inding ftor 1) nd IRAP (insulinresponsive minopeptidse). Biohem. J. 361, (22). 2. Yeh, T.-Y. et l. Tnkyrse reruitment to the lterl memrne in polrized epithelil ells: regultion y ellell ontt nd protein poly(adpriosyl)tion. Biohem. J. 399, (26). 21. Chng, W., Dynek, J. N. & Smith, S. TR1 is degrded y uiquitin-medited proteolysis fter relese from telomeres. Genes Dev. 17, (23). 22. Chng, P., Coughlin, M. & Mithison, T. J. Tnkyrse-1 polymeriztion of poly(adp-riose) is required for spindle struture nd funtion. Nture Cell Biol. 7, (25). 23. Dynek, J. N. & Smith, S. Resolution of sister telomere ssoition is required for progression through mitosis. Siene 34, 971 (24). 24. Winston, J. T. et l. The SC -TRCP -uiquitin ligse omplex ssoites speifilly with phosphorylted destrution motifs in IkB nd -tenin nd stimultes IkB uiquitintion in vitro. Genes Dev. 13, (1999). 25. Koepp, D. M. et l. Phosphoryltion-dependent uiquitintion of ylin E y the SC w7 uiquitin ligse. Siene 294, (21). 26. Hsio, S. J., Poitrs, M.., Cook, B. D., Liu, Y. & Smith, S. Tnkyrse 2 poly(adpriose) polymerse domin-deleted mie exhiit growth defets ut hve norml telomere length nd pping. Mol. Cell. Biol. 26, (26). 27. Ching, Y. J. et l. Tnkyrse 1 nd tnkyrse 2 re essentil ut redundnt for mouse emryoni development. PLoS ONE 3, e2639 (28). 28. Bntsheff, M. et l. Quntittive hemil proteomis revels mehnisms of tion of linil ABL kinse inhiitors. Nture Biotehnol. 25, (27). 29. Bntsheff, M. et l. Roust nd sensitive itraq quntifition on n LTQ Oritrp mss spetrometer. Mol. Cell. Proteomis 7, (28). 619

7 NATURE Vol Otoer 29 Supplementry Informtion is linked to the online version of the pper t Aknowledgements We thnk D. Ptel,. Hrinski, J. Leighton-Dvies, R. de Beumont, X. Xing, K. Ben, C. Xin, S. Zho, B. Zhng nd M. Xu for tehnil ssistne, G. Wussler, H. Urquiz nd W. Di for zerfish mintenne, nd I. Cornell Trido, S. Clever, A. Hernndez nd Y. Ben-Nerih for omments nd dvie. In ddition we re indeted to B. Kuster, J. Rik, M. Rid nd A. Sholten for ontinued support nd disussion. AuthorContriutionsS.-M.A.H., A.C.,.St., G.A.M., E.W.,V.M., S.., C.Lu,D.C.,M.W.K., C.Le., P.M.., J.A.T., T.B., J.A.P., A.B. nd.c. oneived nd designed the study. S.-M.A.H., Y.M.M., S.L., A.C.,.St., G.A.M., O.C., E.W., Y.Z., S.W., M.H., X.S., C.W., C.M., A.., R.T.,.Se., W.S., H.C., M.Sh., C.R., M.S., J.S., S.G., A.B. nd.c. designed nd implemented experiments. S.-M.A.H.,.St., A.B., Y.M.M. nd.c. wrote the pper. Author Informtion Reprints nd permissions informtion is ville t Correspondene nd requests for mterils should e ddressed to.c. (feng.ong@novrtis.om) Mmilln Pulishers Limited. All rights reserved