Selective inhibition of BET bromodomains

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1 doi:1.138/nture954 Seletive inhiition of BET romodomins Pngis Filippkopoulos 1 *, Jun Qi 2 *, Srh Piud 1 *, Yo Shen 3, Willim B. Smith 2, leg Fedorov 1, Elizeth M. Morse 2, Trey Ketes 1, Tyler T. Hikmn 4, Ildiko Felletr 1, Mrtin Philpott 1, Shongh Munro 5, Mihel R. MKeown 2,6, Yuhun Wng 7, Amnd L. Christie 8, thn West 2, Mihel J. Cmeron 4, Brin Shwrtz 4, Tom D. Heightmn 1, ihols L Thngue 5, Christopher A. Frenh 4, lf Wiest 3, Andrew L. Kung 8,9, Stefn Knpp 1,5 & Jmes E. Brdner 2,6 Epigeneti proteins re intently pursued trgets in lignd disovery. So fr, suessful efforts hve een limited to hromtin modifying enzymes, or so-lled epigeneti writers nd ersers. Potent inhiitors of histone inding modules hve not yet een desried. Here we report ell-permele smll moleule () tht inds ompetitively to etyl-lysine reognition motifs, or romodomins. High poteny nd speifiity towrds suset of humn romodomins is explined y o-rystl strutures with romodomin nd extr-terminl (BET) fmily memer BRD4, reveling exellent shpe omplementrity with the etyl-lysine inding vity. Reurrent trnslotion of BRD4 is oserved in genetilly-defined, inurle sutype of humn squmous rinom. Competitive inding y disples the BRD4 fusion onoprotein from hromtin, prompting squmous differentition nd speifi ntiprolifertive effets in BRD4-dependent ell lines nd ptient-derived xenogrft models. These dt estlish proof-of-onept for trgeting protein protein intertions of epigeneti reders, nd provide verstile hemil sffold for the development of hemil proes more rodly throughout the romodomin fmily. Gene regultion is fundmentlly governed y reversile, non-ovlent ssemly of mromoleules 1. Signl trnsdution to RA polymerse requires higher-ordered protein omplexes, sptilly regulted y ssemly ftors ple of interpreting the post-trnsltionl modifition sttes of hromtin 2. Reders of epigeneti mrks re struturlly diverse proteins eh possessing one or more evolutionrily onserved effetor modules, whih reognize ovlent modifitions of histone proteins or DA. The e--etyltion of lysine residues (K) on histone tils is ssoited with n open hromtin rhiteture nd trnsriptionl tivtion 3. Context-speifi moleulr reognition of etyl-lysine is priniplly medited y romodomins. Bromodomin-ontining proteins re of sustntil iologil interest, s omponents of trnsription ftor omplexes nd determinnts of epigeneti memory 4. There re 41 diverse humn proteins ontining totl of 57 romodomins. Despite lrge sequene vritions, ll romodomin modules shre onserved fold omprising left-hnded undle of four helies ( Z, A, B, C ), linked y diverse loop regions (ZA nd s) tht ontriute to sustrte speifiity. Co-rystl strutures with peptidi sustrtes showed tht the etyl-lysine is reognized y entrl hydrophoi vity nd is nhored y hydrogen ond with n sprgine residue present in most romodomins 5. The BET fmily (BRD2, BRD3, BRD4 nd BRDT) shres ommon domin rhiteture feturing two mino-terminl romodomins tht exhiit high levels of sequene onservtion, nd more divergent roxy-terminl reruitment domin (Supplementry Fig. 1) 6. Reent reserh hs estlished ompelling rtionle for trgeting BRD4 in ner. BRD4 remins ound to trnsriptionl strt sites of genes expressed during the M/G1 trnsition, influening mitoti progression 4. BRD4 is lso ritil meditor of trnsriptionl elongtion, funtioning to reruit the positive trnsription elongtion ftor omplex (P-TEF) 7,8. Cylin-dependent kinse-9, ore omponent of P-TEF 9 11, is vlidted trget in hroni lymphoyti leukemi 12, nd hs reently een linked to -My-dependent trnsription 13. Thus, BRD4 reruits P-TEF to mitoti hromosomes resulting in inresed expression of growth-promoting genes 14. Importntly, BRD4 hs reently een identified s omponent of reurrent t(15;19) hromosoml trnslotion in n ggressive form of humn squmous rinom 15,16. Suh trnslotions express the tndem -terminl romodomins of BRD4 s n in-frme himer with the UT (nuler protein in testis) protein, genetilly defining the so-lled UT midline rinom (MC). Funtionl studies in ptient-derived MC ell lines hve vlidted the essentil role of the BRD4 UT onoprotein in mintining the hrteristi prolifertion dvntge nd differentition lok of this uniformly ftl mlignny 17. otly, RA silening of BRD4 UT rrests prolifertion nd prompts terminl squmous differentition. These oservtions undersore the rod utility nd immedite therpeuti potentil of diret-ting inhiitor of humn romodomin proteins. A seletive nd potent inhiitor of BET fmily romodomins A mjor ollortive fous of our reserh groups onerns the development of hemil proes 18,19 nd the optimiztion of therpeuti leds for the trnsltion of smll-moleule modultors of epigeneti trgets s ner therpeutis. Motivted y the ove rtionle, we hve developed iohemil pltforms for the identifition of new inhiitors of romodomin isoforms using high-throughput sreening, s well s the nnottion of puttive lignds emerging from ollortive nd pulished reserh. In the ourse of these studies, we lerned of n 1 Deprtment of Clinil Mediine, Struturl Genomis Consortium, University of xford, ld Rod Cmpus, Roosevelt Drive, xford X3 7DQ, UK. 2 Deprtment of Medil nology, Dn-Frer Cner Institute, Hrvrd Medil Shool, 44 Binney Street, Boston, Msshusetts 2115, USA. 3 Wlther Cner Reserh Center nd Deprtment of Chemistry nd Biohemistry, University of otre Dme, otre Dme, Indin 46556, USA. 4 Deprtment of Pthology, Brighm & Women s Hospitl, Hrvrd Medil Shool, 75 Frnis Street, Boston, Msshusetts 2115, USA. 5 Deprtment of Clinil Phrmology, University of xford, ld Rod Cmpus, Roosevelt Drive, xford X3 7DQ, UK. 6 Deprtment of Mediine, Hrvrd Medil Shool, 25 Shttuk Street, Boston, Msshusetts 2115, USA. 7 Deprtment of Imging, Dn-Frer Cner Institute, Hrvrd Medil Shool, 44 Binney Street, Boston, Msshusetts 2115, USA. 8 Lurie Fmily Imging Center, Dn-Frer Cner Institute, Hrvrd Medil Shool, 44 Binney Street, Boston, Msshusetts 2115, USA. 9 Deprtment of Peditri nology, Dn-Frer Cner Institute nd Children s Hospitl, Boston, Hrvrd Medil Shool, 44 Binney Street, Boston, Msshusetts 2115, USA. *These uthors ontriuted eqully to this work. 23/3 DECEMBER 21 VL 468 ATURE Mmilln Pulishers Limited. 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2 RESEARCH ARTICLE oservtion y Mitsuishi Phrmeutils tht simple thienodizepines possessed inding tivity for BRD4 (ref. 2). Previous reserh from this group indites tht these ompounds emerged from nti-inflmmtory phenotypi studies, suh s inhiition of CD28 o-stimultion s mens of treting utoimmune diseses 21,22. A rih literture hs estlished the syntheti essiility nd fvourle phrmologil properties of this privileged lss of drug-like smll moleules 23. Indeed, the ore sffold desried ppers in FDA-pproved sustnes suh s lprzolm nd trizolm. Inferring struture tivity reltionships lso derived from moleulr modelling of ndidte lignds within the inding poket of the po rystl struture of the first romodomin of BRD4 (herefter referred to s BRD4(1); Protein Dt Bnk ode 2SS), we designed prototype lignd, (Fig. 1). is novel thieno-trizolo-1,4-dizepine, possessing n ppended, ulky t-utyl ester funtionl group t C6 in order to (1) llow for dditionl pendnt group diversity, s needed, nd (2) to mitigte inding to the entrl enzodizepine reeptor s predited y pulished struture tivity reltionships 23.Wefirstestlished high-yielding, seven-step syntheti route to ess remi (herefter referred to s ) nd derivtives (sheme 1 in Supplementry Methods). We hve lso identified route to synthesize eh enntiomer, (1)- nd (2)- (sheme 2 in Supplementry Methods). To estlish iohemil pltform for omprehensive seletivity sreening, ll humn romodomins were suloned into teril expression vetors. Testing of n verge of 15 expression onstruts per romodomin resulted in the identifition of 37 expression systems tht yielded solule protein suitle for speifiity sreening nd overed ll romodomin sufmilies (Supplementry Tle 1). Beuse the speifi sustrtes of most romodomins re unknown, generl inding ssy sed on differentil snning fluorimetry (DSF) ws implemented 24. Binding of (1)- signifintly inresed the therml stility of ll romodomins of the BET fmily (Fig. 1 nd Supplementry Tle 2) with DT m os vlues etween 4.2 uc (BRDT(1)) nd 1.1 uc (BRD4(1)). o signifint stility shifts were deteted for romodomins outside the BET fmily, inditing tht this lignd is highly seletive. In ontrst, the stereoisomer (2)- showed no signifint intertion with ny romodomin present in our pnel. Within fmily of proteins liner orreltion etween DSF DT m os vlues nd inding onstnts hs een oserved, with temperture shifts lrger thn 6 uc orresponding to ompounds with nnomolr Time (min) ( ) A B. C.1 S S ( )- 6 Cl Cl.6 A: lnk 8.7 B: ( )- BRD1 BRPF C: Molr rtio.8 CREBBP EP3 BRD2(1) BRPF3 BRD4(1) BRD3(1) BRD9 d 12 BRD7 BAZ1A BRD3(2) KIAA124 BRDT(1) ATAD2 BRDT(2) BRD2(2) 1 WDR9(2) BRD8(2) BRD8(1) BRD4(2) 8 PHIP(2) BRWD3(2) BAZ1B BAZ2B BAZ2A ZMYD11 TRIM33 MLL TAF1(1) PRKCBP1 TAF1L(1) TAF1(2) TAF1L(2) CECR2 FALZ GC5L2 PCAF WDR9(1) BRWD3(1) PHIP(1) TRIM28 TIF1 TRIM66 SP11 PB1(6) ASH1L SP14 SP1 LC93349 PB1(3) PB1(1) PB1(4) PB1(5) PB1(2) SMARCA4 SMARCA2 >9 C 7 9 C 4 7 C 1 4 C 1 C < C Figure 1 Struture nd seletivity of., Struture of the two stereoisomers. The stereoentre t C6 is indited y n sterisk., Assessment of inhiitor seletivity using differentil snning fluorimetry (DSF). Shown re verged temperture shifts (DT m os ) in degrees Celsius upon inding of 1 mm (1)-. The temperture shifts re represented y spheres s indited in the inset. Sreened romodomins re lelled nd proteins not inluded in the seletivity pnel re shown in grey. (2)- did not revel ny signifint temperture shifts to ny of the sreened romodomins., Isotherml titrtion lorimetry (ITC). Differentil power (DP) dt time ourse of rw injetion hets re shown for lnk titrtion of BRD4(1) into uffer (A), nd reverse titrtions using the intive isomer (2)- (B) nd the tive isomer (1)- (C). The inset shows normlized inding enthlpies orreted for the het of 168 ATURE VL /3 DECEMBER 21 Mmilln Pulishers Limited. All rights reserved 21 ΔP (μl s 1 ) ΔT m os ( C) e Inhiition (%) K d (nm) BRD4(1) BRD4(2) CREBBP BRD4(1) ( )- kl mol 1 of injetnt log[on.(μm)] dilution s funtion of inding site sturtion (symols s indited in the inset). Solid lines represent nonliner lest squres fit using single-site inding model. d, Therml shifts (DT m os ) show good orreltion to dissoition onstnts (K d ) determined y ITC for the BET romodomins. The dotted red line represents lest squres fit with n R of 96%. The DT m os dt represent the men 6 s.d. (n 5 3). Error for ITC dt ws sed on devitions to lest squres fit desried in. e, Competitive displement of histone peptide from humn romodomins is exhiited y using edsed proximity ssy. Alph-sreen titrtions monitoring the displement of tetr-etylted histone H4 peptide y isomers using the romodomins BRD4(1), BRD4(2) or of n etylted H3 peptide using CREBBP re shown.

3 RESEARCH dissoition onstnts (K d ) 25,26. Beuse the sensitivity of this ssy my vry etween different protein fmilies, isotherml titrtion lorimetry (ITC) ws used to determine inding onstnts preisely. Enntiomerilly pure (1)- ound with K d of out 5 nm nd 9 nm to the first nd seond romodomins of BRD4, respetively (Fig. 1 nd Supplementry Tle 3). Comprle inding to oth domins of BRD3 ws oserved, wheres the first romodomins of BRDT nd BRD2 reveled out threefold weker inding. Affinities determined y ITC nd DT os m vlues showed very good orreltion (Fig. 1d). Importntly, (1)- showed no detetle inding to romodomins tht exhiited miniml temperture shifts, suh s WDR9(2) nd CREBBP. To ssess whether (1)- inding ws ompetitive with etyllysine, we dpted luminesene proximity homogeneous ssy (lph-sreen) 27 to the BET romodomins. Binding of tetretylted histone H4 peptide to BRD4 ws strongly inhiited y (1)-, with hlf-mximum inhiitory onentrtion (IC 5 ) vlues of 77 nm nd 33 nm for the first nd seond romodomin, respetively (Fig. 1e). The IC 5 for the (2)- stereoisomer ginst BRD4(1) nd for (1)- ginst CREBBP were oth estimted to e ove 1, nm (Fig. 1e). Thus, (1)- represents potent, highly speifi nd K-ompetitive inhiitor for the BET fmily of romodomins. (1)- inds to the K inding site of BET romodomins To estlish the inding mode of we determined o-rystl strutures using remi mteril nd purified, reominnt BRD4(1) nd BRD2(2) (for dt olletion nd refinement sttistis see Supplementry Tle 4). The determined high-resolution strutures reveled tht only the (1)- enntiomer ound diretly into the K inding site (Figs 2 nd 3, ). Similr to intertions oserved in etyl-lysine omplexes 28, the trizole ring formed hydrogen ond with the evolutionrily onserved sprgine (Asn 14 in BRD4(1) nd Asn 429 in H3K14 mbrd4(1) hbrd4(1) 14 BRD2(2); Fig. 2). The inhiitor showed n extrordinry shpe omplementrity with the K inding site, oupying the entire inding poket (Fig. 2, d). In oth omplexes, lignd inding ws stilized y hydrophoi intertions with onserved BET residues in the ZA- nd BC-loop regions (Fig. 3, ). Struturl nd sequene omprison showed high onservtion of the BET K inding poket, ut reveled numer of differenes in loop regions lining the inding vity tht ould e explored for future development of isoformspeifi inhiitors (Fig. 3 ). Doking of either isomer of to BRD4(1) resulted in exellent fit of (1)- in position of perfet overlp to the rystllogrphilly determined inding mode, wheres (2)- resulted in n energetilly unfvourle onformtion with signifint distortion of the dizepine ring system due to steri lshes with residues of the ZA-loop region (Fig. 3d). To explore the dynmi fetures of BET romodomins, we rried out 2-ns moleulr dynmis simultions of BRD4(1) in the sene nd presene of (1)-. The simultions reveled little displement of the protein helies, ut the loop regions surrounding the etyl-lysine inding site showed signifint onformtionl L92 BRD4(1) L94 Y97 V87 Q85 W81 Y C136 D144 I146 M149 P82 D145 αa L383 Y386 L381 V376 K374 BRD2(2) W37 αb Y H433 C425 M438 P BRD2(1) 97 WPFRQPVDAVKLGLPDYHKIIKQPMDMGTIKRRLEYYWAASECMQDFTMFTCYIYKPTDDIVLMAQT BRD2(2) 37 WPFYKPVDASALGLHDYHDIIKHPMDLSTVKRKMERDYRDAQEFAADVRLMFSCYKYPPDHDVVAMARK BRD3(1) 57 WPFYQPVDAIKLLPDYHKIIKPMDMGTIKKRLEYYWSASECMQDFTMFTCYIYKPTDDIVLMAQA BRD3(2) 332 WPFYKPVDAEALELHDYHDIIKHPMDLSTVKRKMDGREYPDAQGFAADVRLMFSCYKYPPDHEVVAMARK BRD4(1) 81 WPFQQPVDAVKLLPDYYKIIKTPMDMGTIKKRLEYYWAQECIQDFTMFTCYIYKPGDDIVLMAEA BRD4(2) 374 WPFYKPVDVEALGLHDYCDIIKHPMDMSTIKSKLEAREYRDAQEFGADVRLMFSCYKYPPDHEVVAMARK BRDT(1) 5 WPFQRPVDAVKLQLPDYYTIIKPMDLTIKKRLEKYYAKASECIEDFTMFSCYLYKPGDDIVLMAQA BRDT(2) 293 WPFYPVDVALGLHYYDVVKPMDLGTIKEKMDQEYKDAYKFAADVRLMFMCYKYPPDHEVVTMARM d e f L94 αc V435 D434 L92 1 kt/e +1 kt/e d αa 14 αb Figure 2 Chrteriztion of BET omplexes with (1)-., Superimposition of the mouse BRD4(1) H3K14 peptide omplex 28 with the humn BRD4(1) (1)- omplex struture. The hydrogen ond formed to the onserved sprgine (14) in the peptide omplex is shown s yellow dots., 2F o 2 F mp of (1)- in omplex with BRD4(1) ontoured t 2s., Eletrostti surfe of BRD4(1) in omplex with (1)-. The lignd is shown s Corey Puling Koltun (CPK) model demonstrting the exellent shpe omplementrity with the protein etylted lysine reeptor site. d, Rion digrm of the omplex of humn BRD4(1) with (1)- in CPK representtion. The min seondry struturl elements nd the onserved tive site sprgine side hin (14) re lelled. αz αc (ns) 2 Figure 3 Binding site omprison etween - nd C-terminl romodomins in omplex with (1)-., The etyl-lysine inding poket of BRD4(1) is shown s semi-trnsprent surfe with ontt residues lelled nd depited in stik representtion. Cron toms in (1)- re oloured yellow to distinguish them from protein residues. Distinguishing surfe residues re shown in red; the fmily onserved sprgine is shown in lue., The etyl-lysine inding poket of BRD2(2) is shown in identil representtion nd orienttion s desried in., Protein sequene lignment of the humn BET su-fmily highlighting onserved (red) nd similr (yellow) residues. Mjor romodomin struturl elements re shown. The side-hin ontts with (1)- re nnotted with lk str. Contts whih differ etween the - nd C-terminl BET romodomins (red str) re highlighted. The onserved sprgine is indited y lue str. d, Models of (1)- (in yellow) nd (2)- (in green) doked into the BRD4(1) inding site. The steri lshes of the (2)- stereoisomer with Leu 92 nd Leu 94 re highlighted in red. e, Moleulr dynmis simultion demonstrting the flexiility of the ZA nd s of the BRD4(1) po-struture. Shown is the kone of BRD4(1) during 2-ns simultion s snpshots seprted y 1-ns intervls. The different strutures re distinguished y olours hnging from lue to green s indited in the inset. f, Moleulr dynmis simultion of the BRD4(1) (1)- omplex depited in 1-ns snpshots s desried in e. 2 3 / 3 D E C E M B E R 2 1 V L AT U R E Mmilln Pulishers Limited. All rights reserved

4 RESEARCH ARTICLE flexiility. Furthermore, these loops were muh more flexile in the sene (Fig. 3e) thn in the presene (Fig. 3f) of the inhiitor, inditing tht (1)- stilized the K inding vity. In ll ses, moleulr dynmis simultion energies onverged (Supplementry Fig. 2). disples BRD4 from nuler hromtin in ells To estlish whether inds romodomins ompetitively with hromtin in ellulr environment, we performed fluoresene reovery fter photolehing (FRAP) experiments. Previous reserh hs demonstrted the utility of FRAP in ssessing the pe of lterl redistriution of humn romodomins 17,29. Humn osteosrom ells (U2S) trnsfeted with GFP BRD4 show time-dependent reovery of fluoresene intensity (Fig. 4, ). In the presene of (5 nm), the oserved reovery is immedite, inditing displed nd freely diffusing nuler BRD4 (Fig. 4, ). Cellulr FRAP studies onfirmed tht the effets on the moile frtion of BRD4 re limited to the iohemilly tive (1)- stereoisomer (Supplementry Fig. 3). Hving demonstrted potent, seletive inding to BRD4 in homogeneous nd ell-sed ssys, we eme interested to explore the effets of on disese-relevnt phenotypes. Previous studies hve estlished tht the pthogeni BRD4 UT fusion protein rising from the t(15;19) trnslotion in MC inds vidly to disrete foi of etylted hromtin, onferring prolifertive dvntge nd differentition lok 17. Using FRAP, we ssessed the ility of to trget diretly the BRD4 UT onoprotein. Compred to vehile ontrol, (5 nm) mrkedly elerted time to hlf fluoresene reovery in photolehed regions of ells trnsfeted with GFP BRD4 UT (Fig. 4, d). otly, no effet ws oserved on redistriution of GFP UT (Supplementry Fig. 3). These dt re onsistent with ompetitive inding of to BRD4 in ultured ells. indues differentition nd growth rrest in MC Diret inhiition of gene produts expressed from reurrent, onogeni trnslotions is vlidted therpeuti pproh in ner 3,31. We thus endevoured to estlish the onsequenes of ompetitive inhiition of BRD4 UT in MC. A hrteristi feture of MC is the pperne of disrete nuler spekles of the BRD4 UT onoprotein y UT-direted immunohistohemistry 32. Tretment of the ptient-derived 797 MC ell line for 48 h with (5 nm) effes nuler foi, produing diffuse nuler UT stining y immunohistohemistry (Supplementry Fig. 3e). In dose- nd time-dependent mnner, provokes differentition phenotype in MC ell lines, d Reovery t 1/2 (s) f Reltive expression h Count GFP BRD4. Pre 1.7 s 5.4 s 9.1 s 6 s Time (s) e P=.2 5 BRD P=.49 S BRD4 UT S UT P= (Kertin) P<.1 P<.1 P<.1 TGM1 KRT1 KRT14 RAD21 RA 1, 5, 1, 5, 5, 1, Veh. ( )- ormlized intensity 1..5 ormlized intensity GFP BRD4 UT Time (s) t= 24 h 48 h 7 dys ( ) Figure 4 inds BRD4 ompetitively with hromtin resulting in differentition nd growth rrest of MC ells., Fluoresene reovery fter photolehing (FRAP) of GFP BRD4 demonstrtes enhned reovery in the presene of. ulei re flse-oloured in proportion to fluoresene intensity. White irles indite trget regions of photolehing.,, elertes fluoresene reovery in FRAP experiments performed with trnsfeted GFP BRD4 () nd GFP BRD4 UT (). d, Quntittive omprison of time to hlf-mximl fluoresene reovery for FRAP studies (,, Supplementry Fig. 3). Dt represent the men 6 s.d. (n 5 5), nd re nnotted with P-vlues s otined from two-tiled t-test. S, not signifint. e, Differentition of MC ells y (5 nm) is prompt nd hrterized y mrked inrese in ytokertin expression (mouse ntiytokertin lone AE1/AE3; 31, sle r is 5 mm). f, Comprtive gene i PI g Prolifertion log[on.(μm)] Per log[on.(μm)] AV (48 h) STA (48 h) 1 7 AT U R E V L / 3 D E C E M B E R 2 1 Mmilln Pulishers Limited. All rights reserved 21 Prolifertion expression studies of (1)- (red; 25 nm, 48 h) versus (2)- (grey; 25 nm, 48 h) nd vehile (lk) onfirm squmous differentition. Dt represent the men 6 s.d. (n 5 3), nd re nnotted with P-vlues s otined from two-tiled t-test. g, Growth effets of BRD4 inhiition on BRD4 UTdependent ell lines. Cells were inuted with (1)- (red irles) or (2)- (lk irles) nd monitored for prolifertion fter 72 h. (1)- uniquely ttenutes prolifertion y MC ell lines. Dt re presented s men 6 s.d. (n 5 3). Curve fit ws lulted y logistil regression. h, Flow ytometry for DA ontent in MC 797 ells. (1)- (25 nm, 48 h) indues G1 rrest ompred to (2)- (25 nm) nd vehile ontrol. Dt re presented s histogrm of nuler fluoresene intensity. i, Flow ytometri nlysis of MC 797 squmous rinom ells treted with vehile, or sturosporine (STA), s indited. AV, nnexin-v; PI, propidium iodide. 8 37

5 RESEARCH feturing ell spreding nd flttening, open hromtin nd striking spindle morphology (Fig. 4e nd Supplementry Fig. 4). Differentition is prompt (,24 h) nd hrterized y mrked hnges in ell shpe ompnied y mrkedly ugmented expression of ytokertin, hllmrk of squmous differentition (Fig. 4e). After 7 dys in ulture with sumiromolr exposures to, terminl differentition is oserved. In this mnner, phenoopies the morphologil hnges nd inresed kertin expression oserved with BRD4 UT silening y RA interferene (Supplementry Fig. 5) 17. Corroorting these studies, expression nlysis of three nonil squmous tissue genes y RT PCR identified mrked (3-fold) indution of kertin-14 y (1)- in MC 797 ells (Fig. 4f). The modest indution of kertin-1 without ffeting epiderml trnsglutminse (TGM1) my indite differentition towrds thori squmous epithelium, onsistent with the medistinl primry tumour from whih MC 797 ells derive 33. Indution of differentition with intense kertin stining is progressive over 72 h, s determined y quntittive immunohistohemistry nlysis (Supplementry Fig. 6). Supporting n on-trget mehnism-of-tion, the (2)- enntiomer is omprtively intive in MC, nd non- BRD4-dependent squmous rinom ell line (TE1) fils to exhiit differentition effets of tive (Supplementry Fig. 4). In BRD4-dependent MC ells, differentition is expetedly ompnied y growth rrest, s demonstrted y redued Ki67 stining (Supplementry Fig. 7), sustined inhiition of prolifertion (Fig. 4g; Supplementry Fig. 8) nd G1 ell-yle rrest (Fig. 4h). To understnd further the oserved G1 rrest nd to onfirm n effet of on known BRD4-dependent genes, we performed quntittive RT PCR for RAD21 nd RA (ref. 4). (1)- potently deresed expression of oth BRD4 trget genes, wheres (2)- hd no effet (Fig. 4f). Erly nd lte poptosis were ssessed with nnexin-v nd propidium iodide stining to sertin whether the ntiprolifertive effet nd irreversile differentition ws ompnied y ell deth. Indeed, indues immedite nd progressive poptosis in BRD4- dependent humn rinom ells, without triggering signifint growth rrest or ell deth in ell lines lking the BRD UT fusion (Supplementry Figs 8 nd 9). Antitumour effiy of in xenogrft models of MC To determine whether ould ttenute the growth of BRD4- dependent rinom s single gent in vivo, we developed three xenogrft models of MC in mie. First, short-term tretment studies were performed in MC 797 xenogrfts with positron-emission tomogrphy (PET) imging of 18 F-fluorodeoxygluose (FDG) uptke s primry endpoint to explore whether tivity of ould e demonstrted y non-invsive imging. Mthed ohorts of mie with estlished tumours were rndomized to tretment with (5 mg kg 21 ) or vehile, dministered y dily intrperitonel injetion. Before rndomiztion, nd fter 4 dys of therpy, mie were evluted y FDG-PET imging. A mrked redution in FDG uptke ws oserved with tretment, wheres vehile-treted mie demonstrted progressive disese (Fig. 5). Tumour-volume mesurements onfirmed redution in tumour growth with tretment (Fig. 5 nd Supplementry Fig. 1). ws well tolerted t this dose nd shedule without overt signs of toxiity or weight loss (Supplementry Fig. 1). To onfirm tht the ntineoplsti effet oserved with tretment ws ssoited with trget enggement, setioned tumour tissue ws exmined for the BRD4 UT onoprotein. As presented in Supplementry Fig. 11, tretment resulted in effement of UT nuler spekles, onsistent with ompetitive inding to nuler hromtin. Cell spreding nd inresed kertin expression onfirmed indution of squmous differentition (Fig. 5). Deresed nuler Ki67 nd inresed TUEL stining in treted nimls onfirmed n ongoing ntiprolifertive, pro-poptoti effet (Supplementry Fig. 11). To quntify the phrmodynmi iomrker of tumour kertin expression, we estlished protools for utomted immunohistohemistry imge quisition nd nlysis. Pired smples from treted nd untreted nimls were prepred nd nlysed using stndrdized protools nd ommerilly ville softwre (ImgeSope; Aperio Tehnologies), demonstrting tht indued strong (grde 31) kertin expression in MC 797 xenogrfts (Supplementry Fig. 12). In prllel with these studies, we hd osion to re for 29-yerold ptient with widely metstti BRD4 UT-positive MC rising from the medistinum. With the gol of developing more linilly relevnt disese model, we estlished short-term ultures (116 ells) using disrded linil mteril otined from pleurl fluid drining from pllitive hest tue. As presented in Supplementry Fig. 13, in vitro studies onfirmed the stereospeifi, potent effet of (1)- on ellulr viility (IC nm), growth nd ell yle progression. Four nimls engrfted with ptient-derived tumour mteril developed mesurle disese, whih ws strongly FDG-vid y PET imging (Fig. 5d). Animls were rndomly ssigned to vehile or (1)- tretment. Before tretment nd fter 4 dys of therpy, mie were evluted y PET imging. A mrked response in FDG uptke ws oserved with (1)- tretment, wheres vehile-treted nimls demonstrted progressive disese (Fig. 5e). Tumour mteril prepred for quntittive immunohistohemistry nlysis demonstrted indution of kertin expression fter (1)- tretment (Fig. 5f, g nd Supplementry Fig. 14) in this minimlly pssged MC xenogrft model. To onfirm the trnsltionl potentil of diret-ting BRD4 inhiition in MC, we further dpted the ptient-derived 116 ells to expnsion in vivo, nd performed definitive effiy studies. Mrked tumour regression nd prolonged overll survivl were oserved, fter only 18 dys of well-tolerted therpy with (1)- (Fig. 5h, i). These results were repitulted in third MC xenogrft model, using Per43 ells (Fig. 5j, k nd Supplementry Fig. 15). Together, these dt estlish in vivo proof-of-onept for trgeting BRD4 with in MC. Disussion Aross the omplex lndspe of the ner genome, reurrent hromosoml rerrngements omprise ompelling suset of ler, geneti trgets in ner. As evidened y the suessful development of firstnd seond-genertion kinse inhiitors trgeting BCR ABL in hroni myelogenous leukemi, well-hrterized proe ompounds 34,35, high-resolution rystllogrphi dt 36, trnsltionl reserh studies 37, nd informtive murine models 38, where ville, provide n optiml pltform for lignd disovery nd trget vlidtion. Herein, we provide omprle evidene supporting the BRD4 UT fusion s therpeuti trget in n inurle, genetilly-defined humn squmous rinom, using novel BRD4-direted smll-moleule inhiitor. Beyond UT-midline rinom, BET-fmily romodomins ontriute to other neoplsti nd non-neoplsti diseses. BRD4 trgets the P-TEF omplex to mitoti hromosomes, resulting in the expression of growth-promoting genes suh s -My 12,14 nd the well-estlished ner trget Auror B 39. BET fmily memers hve een reognized s essentil genes for the replition of viruses 4,41 nd in mediting inflmmtory responses 42. Thus, the vilility of (1)- will prompt informtive reserh rodly in developmentl nd disese iology. possesses mny desirle qulities of hemil proe, suh s high trget poteny in homogeneous nd ellulr ssys, well-hrterized profile of seletivity, syntheti essiility nd herein proven utility in experimentl iology 18,19. We hve lso found to exhiit few off-trget effets on ellulr reeptors nd exellent phrmokineti properties inluding 49% orl iovilility (Supplementry Figs 16 nd 17 nd Supplementry Tle 5), estlishing the plusiility of developing drug-like derivtives for therpeuti pplition. The disovery nd optimiztion of smll-moleule inhiitors of epigeneti trgets is mjor fous of urrent iomedil reserh 43. We sought to meet the hllenge of developing potent, seletive inhiitors of epigeneti reders. Here we present first, thoroughly hrterized 23/3 DECEMBER 21 VL 468 ATURE Mmilln Pulishers Limited. All rights reserved

6 RESEARCH ARTICLE 2, P =.39 Kertin Ki67 Pre Post Tumour volume (mm 3 ) 1,5 1, 5 d e 2 f Kertin Msk g 1 % ID per g 15 1 Post 5 h i j k 3, 2, 1 1 Tumour volume (mm 3 ) 2, 1, P <.1 Per ent survivl P <.1 Pre Figure 5 promotes differentition, tumour regression nd prolonged survivl in murine models of MC., PET imging of murine MC 797 xenogrfts. FDG uptke in xenogrft tumours is redued y 5 mg kg 21 tretment ompred to vehile ontrol. Arrows indite the ntomil lotion of tumour xenogrft., Tumour volume is redued in mie with estlished disese (MC 797 xenogrfts) treted dily with 5 mg kg 21 ompred to vehile ontrol. A signifint response to therpy is oserved y two-tiled t-test t 14 dys (P 5.39). Dt represent the men 6 s.d. (n 5 7)., Histopthologil nlysis of MC 797 tumours exised from nimls treted with revels indution of kertin expression (mouse ntiytokertin lone AE1/AE3, 34) nd impired prolifertion (Ki67, 34), s ompred to vehile-treted nimls (sle r is 2 mm). d, Viility of ptientderived MC 116 xenogrfts ws onfirmed y PET imging. Arrow indites the ntomil lotion of tumour xenogrft. e, Therpeuti response of primry 116 MC xenogrfts to (1)- (5 mg kg 21 dily for 4 dys) ws demonstrted y PET imging. Integrted signl enompssed within the Veh. Tumour volume (mm 3 ) 1,5 1, 5 P < Per ent survivl 5 Positivity (%) P =.1 P < tumour volume is presented s the per ent injeted dose per grm (% ID per g). f, Histopthologil nlysis of primry MC 116 tumours exised from nimls treted with (1)- revels indution of kertin expression (mouse nti-ytokertin lone AE1/AE3, 32; sle r is 2 mm), ompred to vehiletreted nimls. Quntittive nlysis of kertin indution ws performed using imge msking (f, right pnel) nd pixel positivity nlysis (g). A signifint response to therpy is oserved y two-tiled t-test (P 5.1). Dt represent the men 6 s.d. of three independent wide mirosopi fields. Comprtive imges of stined exised tumours nd quntittive msks re provided in Supplementry Fig. 14. h k,(1)- (red irles nd lines; 5 mg kg 21 dily for 18 dys) produes derese in tumour volume (h, j) nd promotes prolonged survivl (i, k) in ptient-derived 116 (h, i) nd Per43 (j, k) MC xenogrft models (n 5 1 in ll groups). A signifint response to therpy is oserved for tumour volume y two-tiled t-test (P,.1) nd for overll survivl y log-rnk test (P,.1). Blk irles nd lines, vehile. inhiitor of the BET-fmily of romodomins. The pproh outlined herein further estlishes the fesiility of rogting protein protein intertions with smll moleules, nd trgeting dditionl epigeneti reders for lignd disovery. METHDS SUMMARY The inhiitor ws synthesized in oth remi nd enntiomerilly pure formt using the syntheti route outlined in sheme 1 nd sheme 2 (Supplementry Methods) nd its struture ws fully hrterized. Humn romodomins were expressed in teri s His-tgged proteins nd were purified y nikelffinity nd gel-filtrtion hromtogrphy. Protein integrity ws ssessed y SDS PAGE nd eletro-spry mss spetrometry on n Agilent 11 Series LC/MSD TF. All rystlliztions were rried out t 4 uc using the sitting-drop vpour-diffusion method. X-ry diffrtion dt were olleted t the Swiss Light soure emline X1SA, or using Rigku FR-E genertor. Strutures were determined y moleulr replement. Isotherml titrtion lorimetry experiments were performed t 15 uc on VP-ITC titrtion mirolorimeter (MiroCl). Therml melting experiments were rried out on n Mx35p RT PCR mhine (Strtgene) using SYPR rnge s fluoresene proe. Dose-rnging smll-moleule studies of prolifertion were performed in white, 384-well pltes (Corning) in DMEM medi ontining 1% FBS. Compounds were delivered with JAUS pin-trnsfer root nd prolifertion mesurements were mde on n Envision multilel plte-reder (PerkinElmer). Murine xenogrfts were estlished y injeting MC ells in 3% Mtrigel (BD Biosienes) into the flnk of 6-week-old femle Cr nude mie (Chrles River Lortories). Tumour mesurements were ssessed y liper mesurements, nd volume ws lulted using the formul Vol L 3 W 2. All mie were humnely killed, nd tumours were fixed in 1% formlin for histopthologil exmintion. Quntittive immunohistohemistry ws performed using the Aperio Digitl Pthology Environment (Aperio Tehnologies) t the DF/HCC Core Lortory t the Brighm nd Women s Hospitl. Reeived 5 My; epted 17 Septemer 21. Pulished online 24 Septemer Ptshne, M. Binding retions: epigeneti swithes, signl trnsdution nd ner. Curr. Biol. 19, R234 R241 (29). 2. Shreier, S. L. & Bernstein, B. E. Signling network model of hromtin. Cell 111, (22). 3. Mrushige, K. Ativtion of hromtin y etyltion of histone side hins. Pro. tl Ad. Si. USA 73, (1976). 4. Dey, A., ishiym, A., Krpov, T., Mlly, J. & zto, K. Brd4 mrks selet genes on mitoti hromtin nd direts postmitoti trnsription. Mol. Biol. Cell 2, (29) AT U R E V L / 3 D E C E M B E R 2 1 Mmilln Pulishers Limited. All rights reserved 21

7 RESEARCH 5. wen, D. J. et l. The struturl sis for the reognition of etylted histone H4 y the romodomin of histone etyltrnsferse gn5p. EMB J. 19, (2). 6. Zeng, L. & Zhou, M. M. Bromodomin: n etyl-lysine inding domin. FEBS Lett. 513, (22). 7. Yng, X. J. Multisite protein modifition nd intrmoleulr signling. nogene 24, (25). 8. Yng, Z. et l. Reruitment of P-TEF for stimultion of trnsriptionl elongtion y the romodomin protein Brd4. Mol. Cell 19, (25). 9. Peng, J., Zhu, Y., Milton, J. T. & Prie, D. H. Identifition of multiple ylin suunits of humn P-TEF. Genes Dev. 12, (1998). 1. Mrshll,. F. & Prie, D. H. Purifition of P-TEF, trnsription ftor required for the trnsition into produtive elongtion. J. Biol. Chem. 27, (1995). 11. Mrshll,. F., Peng, J., Xie, Z. & Prie, D. H. Control ofra polymerseiielongtion potentil y novel roxyl-terminl domin kinse. J. Biol. Chem. 271, (1996). 12. Phelps, M. A. et l. Clinil response nd phrmokinetis from phse 1 study of n tive dosing shedule of flvopiridol in relpsed hroni lymphoyti leukemi. Blood 113, (29). 13. Rhl, P. B. et l. -My regultes trnsriptionl puse relese. Cell 141, (21). 14. Yng, Z., He,. & Zhou, Q. Brd4 reruits P-TEF to hromosomes t lte mitosis to promote G1 gene expression nd ell yle progression. Mol. Cell. Biol. 28, (28). 15. Frenh, C. A. et l. BRD4 romodomin gene rerrngement in ggressive rinom with trnslotion t(15;19). Am. J. Pthol. 159, (21). 16. Frenh, C. A. et l. BRD4-UT fusion onogene: novel mehnism in ggressive rinom. Cner Res. 63, (23). 17. Frenh, C. A. et l. BRD-UT onoproteins: fmily of losely relted nuler proteins tht lok epithelil differentition nd mintin the growth of rinom ells. nogene 27, (28). 18. Frye, S. V. The rt of the hemil proe. ture Chem. Biol. 6, (21). 19. pre, T. I. et l. A rowdsouring evlution of the IH hemil proes. ture Chem. Biol. 5, (29). 2. Miyoshi, S., oike, S., Iwt, K., Hikw, H. & Sugrh, K. Antitumor gent. Interntionl Ptent o. PCT/JP28/73864 (W/29/84693) (29). 21. Adhi, K. et l. Thienotrizolodizepine ompound nd mediinl use thereof. Interntionl Ptent o. PCT/JP26/3179 (W/26/129623) (26). 22. Sueok, H., Komtsu, H., Koyshi, H. & Ehr, S. Thienotrizolodizepine Compounds nd Mediinl Uses Thereof 1 5 (Yoshitomi Phrmeutil Industries, Ltd, 1998). 23. VonVoigtlnder, P. F. & Strw, R.. Alprzolm: Review of phrmologil, phrmokineti nd linil dt. Drug Dev. Res. 6, 1 12 (1985). 24. iesen, F. H., Berglund, H. & Veddi, M. The use of differentil snning fluorimetry to detet lignd intertions tht promote protein stility. ture Protools 2, (27). 25. Fedorov,. et l. A systemti intertion mp of vlidted kinse inhiitors with Ser/Thr kinses. Pro. tl Ad. Si. USA 14, (27). 26. Bullok, A.. et l. Struturl sis of inhiitor speifiity of the humn protoonogene provirl insertion site in moloney murine leukemi virus (PIM-1) kinse. J. Med. Chem. 48, (25). 27. Quinn, A. M. et l. A homogeneous method for investigtion of methyltiondependent protein-protein intertions in epigenetis. ulei Aids Res. 38, e11 (21). 28. Vollmuth, F., Blnkenfeldt, W. & Geyer, M. Strutures of the dul romodomins of the P-TEF-tivting protein Brd4 t tomi resolution. J. Biol. Chem. 284, (29). 29. Dey, A. et l. A romodomin protein, MCAP, ssoites with mitoti hromosomes nd ffets G 2 -to-m trnsition. Mol. Cell. Biol. 2, (2). 3. Hung, M. E. et l. Use of ll-trns retinoi id in the tretment of ute promyeloyti leukemi. Blood 72, (1988). 31. Druker, B. J. et l. Effiy nd sfety of speifi inhiitor of the BCR-ABL tyrosine kinse in hroni myeloid leukemi.. Engl. J. Med. 344, (21). 32. Hk, H. et l. Dignosis of UT midline rinom using UT-speifi monolonl ntiody. Am. J. Surg. Pthol. 33, (29). 33. Toretsky, J. A. et l. Trnslotion (11;15;19): highly speifi hromosome rerrngement ssoited with poorly differentited thymi rinom in young ptients. Am. J. Clin. nol. 26, 3 36 (23). 34. Buhdunger, E. et l. Seletive inhiition of the pltelet-derived growth ftor signl trnsdution pthwy y protein-tyrosine kinse inhiitor of the 2-phenylminopyrimidine lss. Pro. tl Ad. Si. USA 92, (1995). 35. Buhdunger, E. et l. Inhiition of the Al protein-tyrosine kinse in vitro nd in vivo y 2-phenylminopyrimidine derivtive. Cner Res. 56, 1 14 (1996). 36. Shindler, T. et l. Struturl mehnism for STI-571 inhiition of elson tyrosine kinse. Siene 289, (2). 37. Druker, B. J. et l. Effets of seletive inhiitor of the Al tyrosine kinse on the growth of Br-Al positive ells. ture Med. 2, (1996). 38. le Coutre, P. et l. In vivo erdition of humn BCR/ABL-positive leukemi ells with n ABL kinse inhiitor. J. tl Cner Inst. 91, (1999). 39. You, J. et l. Regultion of uror B expression y the romodomin protein Brd4. Mol. Cell. Biol. 29, (29). 4. You, J. et l. Kposi s srom-ssoited herpesvirus lteny-ssoited nuler ntigen interts with romodomin protein Brd4 on host mitoti hromosomes. J. Virol. 8, (26). 41. Ate, E. A., Voitenleitner, C. & Bothn, M. R. Struture of the ppillomvirus DA-tethering omplex E2:Brd4 nd peptide tht ltes HPV hromosoml ssoition. Mol. Cell 24, (26). 42. Hung, B., Yng, X. D., Zhou, M. M., zto, K. & Chen, L. F. Brd4 otivtes trnsriptionl tivtion of F-kB vi speifi inding to etylted RelA. Mol. Cell. Biol. 29, (29). 43. Cole, P. A. Chemil proes for histone-modifying enzymes. ture Chem. Biol. 4, (28). Supplementry Informtion is linked to the online version of the pper t Aknowledgements We re grteful to U. ppermnn, S. Müller, S. Slln, C. Lthn, P. Rhl, R. Young, K. Lee nd K. Shw for disussions nd shring unpulished informtion; K. Agu, S. Johnston nd L. Li for nlytil hemistry support; J. Dley for flow ytometry support; T. Bowmn, T. Cron, C. Mrvin nd S. Rodig for immunohistohemistry; nd A. Bss for shring ell lines. The Struturl Genomis Consortium is registered hrity (numer ) tht reeives funds from the Cndin Institutes for Helth Reserh, the Cndin Foundtion for Innovtion, Genome Cnd through the ntrio Genomis Institute, GlxoSmithKline, Krolinsk Institutet, the Knut nd Alie Wllenerg Foundtion, the ntrio Innovtion Trust, the ntrio Ministry for Reserh nd Innovtion, Merk & Co., In., the ovrtis Reserh Foundtion, the Swedish Ageny for Innovtion Systems, the Swedish Foundtion for Strtegi Reserh nd the Wellome Trust. This reserh ws supported y Grdute Fellowship from the Chemistry-Biohemistry-Biology Interfe Progrm t the University of otre Dme, IGMS T (to Y.S.), the DF/HCC (to C.A.F. nd J.E.B.), the tionl Institutes of Helth, the Burroughs Wellome Fund, nd the Leukemi & Lymphom Soiety (to J.E.B.). Author Contriutions P.F., J.Q., S.K. nd J.E.B. designed the study, nlysed dt nd wrote the mnusript. P.F. nd S.P. performed nd nlysed iophysil studies. J.Q. nd J.E.B. designed nd estlished the syntheti routes. Y.S. nd.w. ompleted doking nd moleulr dynmis studies..f. performed nd nlysed DSF. S.M. nd.l.t. ontriuted iohemilssys. M.R.M., M.P. ndt.d.h. performedndnlysed lph-sreen ssys. W.B.S., M.J.C. nd J.E.B. performed in vitro MC studies nd immunohistohemistry. E.M.M. performed flow ytometry studies. E.M.M. nd.w. performed prolifertion studies. T.T.H., M.J.C., C.A.F. nd J.E.B. ompleted FRAP studies. M.R.M. nd B.S. performed expression nlysis. Y.W., A.L.C. nd A.L.K. ompleted in vivo effiy studies. T.K. nd I.F. expressed nd purified proteins. S.K. nd J.E.B. supervised the reserh. Author Informtion Atomi oordintes nd struture ftors for the reported rystl strutures hve een deposited with the Protein Dt Bnk under ession odes 2SS (BRD4(1)), 3MXF (BRD4(1) (1)-) nd 3I (BRD2(2) (1)-). Reprints nd permissions informtion is ville t The uthors delre no ompeting finnil interests. Reders re welome to omment on the online version of this rtile t Correspondene nd requests for mterils should e ddressed to S.K. (stefn.knpp@sg.ox..uk) or J.E.B. (jmes_rdner@dfi.hrvrd.edu). 2 3 / 3 D E C E M B E R 2 1 V L AT U R E Mmilln Pulishers Limited. All rights reserved