© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Center, New York, New York, USA. for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston, USA. Massachusetts, Texas MD Anderson Cancer Center, Houston, Texas, USA. Durham, North Carolina, USA. Women’s Health, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, USA. Hochschule, Zurich, Switzerland. Hospital Zurich, Zurich, Switzerland. Italiana, Lugano, Switzerland. 1 and portion uncharacterized of the substrate-recognition domain ( an in occur carcinosarcoma and cancer endometrial in substitutions proteins substrate oncogenic of degradation and ubiquitination repress to fashion nant-negative domi a in act mutations these that is shown subsequently have others that transfer ubiquitin domain and interaction substrate for substrate-recognition essential the within region cleft—a substrate-binding specific the of residues acid amino the to fined cancers trial ( POZ protein (SPOP) in of 4–14% prostate and speckle-type endome adaptor ligase ubiquitin the of domain substrate-recognition the to mapping mutations point recurrent revealed have studies Genome domain. protein-coding same the within occur changes acid amino if resulting the especially implications, therapeutic have gene similar specific drug a within given mutations a recurrent to that assumed respond is it not Generally, may or whether may cancer indicate with can patient genes a cancer-related in mutations Specific with endometrial but not prostate cancer who harbor evoke opposing drug Specifically,susceptibilities. we provide a molecular rationale for the use of BET inhibitors to treat patients inhibition. pharmacologic These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain Conversely, prostate cancer–specific SPOP cancer–associated mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP–CUL3 substrates that are degraded preferentially by endometrial understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer–associated ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely studies have identified recurrent but divergent missense mutations affecting the domain of substrate-recognition the ubiquitin It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome A Garraway Levi Peter Schraml David G Mutch Craig M Bielski Anna Ulbricht Hana Janouskova on BET protein degradation and sensitivity to OpposingBET inhibitors effects of cancer-type-specific SPOP mutants nature medicine nature Received 2 March; accepted 16 June; published online 14 August 2017; Institute Institute of Oncology Research, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland. upeetr Fg 1a Fig. Supplementary Fig. Fig. 1 4 6 , , Holger Moch

7 a , , Tiziano Bernasocchi 5

) , advance online publication online advance 5 14 2– 1 , 12 ,

, , Michael E Carney 6 , , Gregory V Kryukov 2 . . In prostate cancer, , , 15 13 3 10 Centre Centre Hospitalier Vaudois,Universitaire University of Lausanne, Lausanne, Switzerland. , , Geniver El Tekle , , Peter J Wild Division Division of Gynecologic Oncology, University of Minnesota, Minneapolis, Minnesota, USA. 7 15 8– Division Division of Gynecologic Oncology, Washington University, St. Louis, Missouri, USA. ) These These authors contributed equally to this work. should Correspondence be addressed to J.P.T. ( 1 4– 5 2 Broad Broad Institute of Harvard and MIT, Cambridge, USA. Massachusetts, . In contrast, recurrent amino acid acid amino recurrent contrast, In . 6 . Given the divergent mutation mutation divergent the Given . 4 , , Francesco Bertoni SPOP SPOP 4 & Jean-Philippe P Theurillat mutations resulted in impaired degradation of BETs, promoting their resistance to 1 8 – , , Andrew Berchuck 3 12 1 5 mutations are con , , Gianluca Civenni – Department Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, USA. Massachusetts, , , Luciano Cascione 3 ,

15 , Elisa , Bellini Elisa SPOP 7 . We and and We . 1 Fig. Fig. 1 d , 2 o mutations. i , , Carlo V Catapano : 1 0 1 a - - - . . 1

0 3 9 on in those found with overlapped substantially genes expressed robustly their a well-differentiated endometrioid cancer, for experimentation because ting, we chose human Ishikawa endometrial cancer cells, derived from set experimental our in expressed being proteinswereease-relevant mutantsproteomics.spectrometry–basedmassby To ensuredisthat SPOP cancer–associated endometrial to specific landscape tination ubiqui the in changes characterized we hypothesis, this exploreTo BET protein levels Cancer-type-specific RESULTS opportunities. therapeutic distinct in resulting possibly tions, ubiquitination in comparison to prostate cancer–specific cancer with proteinaffect might associated endometrial differentially that speculated we types, tumor these in patterns vector, wild-type SPOP (SPOP-WT) or one of seven endometrial endometrial seven of one or (SPOP-WT) SPOP wild-type controlvector, overexpressed stably that cells generated we Subsequently, 4 8 , , Boris J N Winterhoff 1 , , Namrata D Udeshi / n , 1 2 m , , , Marco Losa 2 . 14

4 3 Present Present address: Molecular Oncology, Memorial Sloan Kettering Cancer 7

, , Sara Napoli 2 1 SPOP – 3 2 Faculty Faculty of Biomedical Science, Università della Svizzera mtn tmr ise ( tissues tumor -mutant 1 – 3 9 6 1 , , Matthias Peter Division Division of Gynecologic Oncology, Duke Cancer Center, Department Department of Biochemistry, TechnischeEidgenössische , 2 SPOP , , Tanya Svinkina 1 , 2 5 , Radoslav , I Radoslav Enchev , , Anna Rinaldi 8 10 mutations have opposing effects on Department Department of Obstetrics, Gynecology and 4 , , Russell R Broaddus Institute Institute of Surgical Pathology, University 11 Department Department of Pathology, University of [email protected] 6 upeetr Fg 1b Fig. Supplementary , , Steven A Carr 5 , 1

, SPOP 2 ,

s e l c i t r a 6 SPOP , mutations

11 SPOP

mutations mutations 13 , 5

Center Center ,

). muta ) 6 , 1 3  - - - - .

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. erdto, e se wih ifrnily xrse K- expressed differentially which asked we degradation, SPOP-WT within experiments ( individual SPOP-MTs were compared to those from cells ( overexpressing levels protein ubiquitination-relatedin changesfor account to ratios proteincorresponding the SPOP-WT,to normalizedtomutant were 1d Fig. acids in cell culture (SILAC)-based mass spectrometry ( (K- each case, we measured glycine-glycine remnants of M117V,ubiquitinated S80R, R121Q and D140N; SPOP-MTs) E78K, lysines( E50K, (E47K, variants SPOP mutated cancer–specific panel, each In tau-b. Kendall’s from derived to according stratified tissues cancer prostate human to according stratified tumors primary in levels ( (IHC) immunohistochemistry by BRD4 and BRD3 BRD2, for stained ( mutants SPOP ( mutants ( mutant SPOP cancer–specific endometrial an or SPOP-WT control, vector expressing stably cells Ishikawa in proteins indicated the for validation blot lines, red Dashed (SPOP-WT). SPOP wild-type versus (SPOP-MTs) mutants SPOP expressing cells cancer endometrial red in are cancer endometrial in those and blue in highlighted are cancer prostate in residues acid amino mutated Recurrently domain. substrate-recognition SPOP 1 Figure A  abundance protein with correlation inverse an showed peptides n SupplementaryTable 1

e d a AC SPO BRD4 BRD3 BRD2 AC SPOP BRD4 BRD3 BRD2 = 5). ( = 5). Because protein ubiquitination is often linked to proteasomal proteasomal to linked often is ubiquitination protein Because s e l c i t r ε TB TB -GG) after trypsin digestion and stable isotope labeling of aminodigestionstableisotopelabelingofand trypsin after -GG) P

) d 1 Endometrial cancer– and prostate cancer–associated SPOP mutants induce opposing effects on BET protein levels. ( levels. protein BET on effects opposing induce mutants SPOP cancer–associated prostate and cancer– Endometrial n Control 4

) Representative western blot for the indicated proteins in Ishikawa endometrial cancer cells expressing prostate cancer–specific SPOP SPOP cancer–specific prostate expressing cells cancer endometrial Ishikawa in proteins indicated the for blot western ) Representative Control = 3). ( = 3). Al K- All . R12

SPOP-WT SPOP-WT F125 1 K129 n Ish 22Rv 7 = 3). In In = 3). . The substrate-binding cleft is shown in green. ( green. in shown is cleft substrate-binding . The e ik

) Representative western blot for the indicated proteins in 22Rv1 prostate cancer cells stably expressing prostate cancer–associated cancer–associated prostate expressing stably cells cancer prostate 22Rv1 in proteins indicated the for blot western ) Representative SPOP-Y87C SPOP-Y87C aw ε 1 W131 D130 a F102 G vle ( values -GG

SPOP-F102C SPOP-F102C b ). K- ). –

SPOP-W131G Y8 d SPOP-W131G M1 , , 7 F133 β 17 ε -actin (ACTB) is used as a loading control. ( control. a loading as used is (ACTB) -actin -GG peptide values for cells expressingvaluesforcellspeptide -GG E5 Supplementary Fig. 2a 0 S8 n 0 1,3) dfnd s ais of ratios as defined 17,239), = f BRD4 BRD3 BRD2 E4 7 D140 n E7 SPOP-MT SupplementaryFig. 1c Negat (M indicates the number of independent experiments performed. performed. experiments independent of number the indicates 8 SPOP 117V iv Pr e ) b im mutation status. Scale bars, 20 20 bars, Scale status. mutation log protein (MTs/WT) SPOP

ary endo 2 –1 –0 0. 1. –2 –1 .5 .5 Supplementary 2 0 1 5 5 , SPOP-MT c (R121Q mutation status status mutation 0 , me W e BRD3 DE BRD2 BRD4 (endometrial cancer–associatedSPOPmutants) eak , g tr K ia ). l l ) cancer ti cancer b ) Scatterplot showing expression differences for the indicated proteins in Ishikawa Ishikawa in proteins indicated the for differences expression showing ) Scatterplot ε Supplementary Fig. 4c Fig. Supplementary Protein expressionchanges -GG -GG ). In). 2 ,0 ssue ssue 00 SPOP-W S tron f ( ) Left, representative images of primary human endometrial cancer tissues tissues cancer endometrial human primary of images representative ) Left, Supplementary Fig. 4g Fig. Supplementary NCOA3 (refs. (refs. NCOA3 and TRIM24 for reported been have mutants SPOP cancer–specific prostate by dysregulation substrate of patterns dominant-negative protein corresponding the of levels in increase an by K- in a decrease properties—showed oncogenic reported with proteins 3 (NCOA3)—all coactivator receptor (AGR-2) and nuclear homolog 2 protein gradient anterior (TRIM24), 24 partite-motif-containing ( and domain the (MATH) and homology TRAF substrates and meprin individual possi mutant to between points pointing contact substrates private SPOP ble putative and dysregula known protein in and tion ubiquitination of patterns two identified ( g Supplementary Table 1 Table Supplementary T ε -GG peptide abundance in cells expressing SPOP-MTs, followed expressing cells in abundance peptide -GG 4,00 Supplementary Figs. 2 Figs. Supplementary 6 0 µ m. ( m. BRD4 IHC BRD3 IHC BRD2 IHC 100 100 100 25% 50% 75% 25% 50% 75% 25 50 75 g NCOA 0% 0% TRIM 0% , ) Analysis of BRD2, BRD3 and BRD4 expression in primary primary in expression BRD4 and BRD3 BRD2, of ) Analysis % % d % % % % AGR2 8 ); right, corresponding quantification of protein expression expression protein of quantification corresponding right, );

advance online publication online advance Negative , R ,0 R 24 9 3 R =–0.336, =–0.328, 00 , =–0.165, 1 7 ) ). . . Weak c R AC SPOP DAXX NCOA AGR2 TRIM DE BRD4 BRD3 BRD2 correlation coefficients and and coefficients correlation

P K TB P P = 0.00 = = 0.00 = = 0.07 = 24 and and

Strong 3 and and 1 1 Control 8 Supplementary Fig. 2b Fig. Supplementary WT MT WT MT MT WT 3a SPOP-WT ). Peptides corresponding to tri to corresponding Peptides ).

± SPOP-E47K g 2 s.d. ( 2 s.d.

SPOP-E50K

BRD4 IHC BRD3 IHC BRD2 IHC Ishi Pr 100% 100% 100 25% 50% 75% 25% 50% 75% 25% 50% 75% ka im 0% 0% 0% SPOP-E78K a c % wa ary prostate ) Outer surface of the the of surface ) Outer ) Representative western western ) Representative Negative R = 0.208, =

SPOP-S80R R R nature medicine nature = 0.199, = = 0.157, =

P SPOP-M117V values were were values

cancer ti cancer Weak P 9

SPOP-R121Q = 0.11 = ,

15

P P

, = 0.02 = , = 0.03 = d 1 Strong SPOP-D140N 6 ssue ssue 5 , Fig. 1b Fig. . Similar Similar . MT WT f , h 0 5 . We ). MT WT MT WT , c - - -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. extraterminal extraterminal (BET)-motif-containing proteins, which serve as prom ( BRD4 and BRD2 BRD3, in found were level mRNA the at changes concurrent without level protein the at changes profound most the and ( protein corresponding the of robust upregulation of K- performed. experiments In control. a loading as used in shown blots western of ( ( treatment MG132 5 h of without or with HA-BRD3 and SPOP-WT expressing transiently cells Ishikawa in blot western by determined level ( beads nickel using performed was cells lysed from pulldown 8×His-Ub treatment. MG132 3 h of by followed constructs indicated the and (Ub) ubiquitin (8×His)-tagged histidine 8× express to transfected were cells SPOP-WT. by 293T degron-MT ( cells 293T transfected transiently from shown are (bottom) (WCE) extract whole-cell and (top) (IP) immunoprecipitation HA degron-MT. ( cells Ishikawa in blot western by assessed (HA-BRD3-degron-MT) degron mutated the with BRD3 HA-tagged and (HA-BRD3-WT) BRD3 wild-type ( red. in depicted is motif degron SPOP the indicated; spectrometry (K- lysines ubiquitinated the and (CTD) domain C-terminal the domain, (ET) extraterminal the BD2), and 2 (BD1 and ( 2 Figure nature medicine nature therapy cancer for targets ising f n n b a Fig. 1b Fig. d b a HA (BRD3) SPOP-WT: HA-BRD3-WT: ) Representative western blot of the indicated proteins upon knockdown knockdown upon proteins indicated the of blot western ) Representative BRD4 BRD3 BRD2 HA (BRD3 ) Schema of the BET proteins with the positions of bromodomains 1 bromodomains of positions the with proteins BET the of ) Schema = 3). ( = 3). ( = 3). HA-tagged of levels the on overexpression SPOP-WT transient of ) Effect SPOP-WT MG132 (3h): The most striking changes were found in proteins that exhibited exhibited that proteins in found were changes striking most The SPOP degron-MT: SPOP-WT: HA-BRD3- 3a 8 SPOP × VC 7 1 5 1 9 1 His-Ub: VC ), including DEK, another characterized SPOP substrate characterized DEK, another ), including with shRNA (left) or siRNA (right) in Ishikawa cells ( cells Ishikawa in (right) siRNA or (left) shRNA with HA-BRD3-Ub n L d c L , ) BET proteins are bona fide substrates of wild-type SPOP. wild-type of substrates fide bona are proteins BET c ) Interaction between SPOP-WT and BRD3-WT or HA-BRD3- or BRD3-WT and SPOP-WT between ) Interaction ) ) 1–123 and and BD In vivo In 5–147 BD 1–163 BD + – 1 + + + – – HA-BRD3-WT 1 1 Ishikaw 326–398 Supplementary Fig. 3b Fig. Supplementary + + + – – 364–436 ubiquitination of HA-BRD3-WT and HA-BRD3- and HA-BRD3-WT of ubiquitination BD 368–440 BD + – 388

a BD 2 + + + + + – + – –

293T HA-BRD3- 2 degron-MT 43 advance online publication online advance 2 b 1 544 and and + – 562–644 ε 632–714 -GG peptides coupled with downregulation 600–682 ET b + + – + + + + – ET 590 d HA (BRD3 – ET c – 66 f 661 f , , 1 are representative. Vinculin (VCL) was was (VCL) Vinculin representative. are + n Fig. 1b Fig. SPOP 1 indicates the number of independent independent of number the indicates 8 726 801 , may be increasingly ubiquitinated ubiquitinated increasingly be may , e ) 1,047–1,362 SPOP 293T SPOP-WT BRD4 BRD3 BRD2 f HA (BRD3)IP VCL HA-BRD3 n IgG CT , = 3). ( = 3). c BRD4: 27 BRD3: 24 BRD2: 287 HA (BRD3) SPOP-WT:

-WT D and and ). These bromodomain and and bromodomain These ). HA IP Ishikawa

shControl SPO ε

VC HA IP

-GG) detected by mass mass by detected -GG) HA-BRD3- Supplementary Figs. 2 Figs. Supplementary e degron-MT 8 1 P ) HA-BRD3 protein protein ) HA-BRD3 L K

shSPOP HA (BRD3) Control K- SPOP degron + – SPO SPOP ε BRD4 BRD3 BRD2 -GG -GG VC VCL Ishikawa Degron

P L MG132 (5h) (ubi + – n HA-BRD3-WT qui = 3). The The = 3). WC Ishikawa siControl ti E HA-BRD3- n)

n

degron-MT = 3). = 3). 8 304 295

siSPOP 258 . . Yet, -

on BET protein levels of those identified in endometrial cancer endometrial in identified those of levels protein BET on effect opposite the have might mutants these that ner, speculated we man dominant-negative a in substrates of ubiquitination impair to ( cells cancer prostate 22Rv1 human in RL95-2 and cells and cancer HEC-151 endometrial human in found also were changes similar and degraded by endometrial cancer–specific SPOP mutants. Notably, BET proteins are bona fide SPOP substrates and ( tissues cancer prostate human primary in noted was tion correla a positive whereas by immunohistochemistry, analyzed sues recurrent these findings, nuclear levels of BET proteins correlated inversely with In of lineage. of support cellular mutants irrespective cancer–specific tially ubiquitinated and degraded by endometrial cancer– and prostate that are differen SPOP substrates that represent BET might proteins cells epithelial prostate mouse cells epithelial prostate LHMAR human cells, cancer prostate 22Rv1 human cells, cancer endometrial Ishikawa human in levels protein BET increased Indeed, overexpression of mutants recurrent prostate cancer–specific ( HA-BRD3 ubiquitinated of levels increased treatment MG132 term in the presence or absence of the proteasome inhibitor MG132. Short- mal we degradation, cultured SPOP- and BRD3-expressing 293T cells ( BRD3 toward SPOP of KLHL21) failed to ubiquitinate BRD3 KLHL13, (KLHL9), 9 member family (kelch-like adaptors substrate ( in vitro HA-BRD3 wild-type ubiquitinated directly CUL3, and ( cells 293T in control to relative ubiquitination BRD3 decreased and E3 ubiquitin (RBX1) 1 complex ligase protein (CUL3)–RING-box cullin-3 a of binding. SPOP for necessary be to appears BRD3 within ( interaction BRD3–SPOP the disrupted variant degron BRD3 the in substitutions the whereas HA-BRD3, wild-type of immunoprecipitation after detectable was protein SPOP variant. degron the or HA-BRD3 express wild-type either ing cells in experiments immunoprecipitation performed we BRD3, to SPOP of binding direct mediated motif the whether mine ( variant degron the SPOP was not able that ment to endogenous the notion with degrade of levels BRD3 protein of to relative elevated in those controls, agree degron variant abolished the the repressive of effect SPOP in and resulted in substitutions The degradation. BRD3 mediate to SPOP-WT ( cells in Ishikawa substitutions at threonine-to-alanine the binding motif (degron-MT) First, we overexpressed HA-tagged BRD3 (HA-BRD3) harboring three it was regulated BET the most family member ( differentially ( motif SPOP-binding a is that con sequence proteins consensus BET conserved the a of tain sequences acid amino primary the esis, hypoth this with agreement In ubiquitination. promote to proteins BET with interacts directly SPOP whether determine to Wesought Supplementary Fig. 5c Supplementary Supplementary Fig. 3c Fig. Supplementary Fig. 2 Fig. Fig. 2 Fig. i. 2 Fig. Because prostate cancer–specific SPOP mutants have been found found been have mutants SPOP cancer–specific prostate Because To determine whether BRD3 ubiquitination induces its proteaso its induces ubiquitination BRD3 whether Todetermine part as BRD3 ubiquitinate could SPOP whether tested we Next, Supplementary Fig. 4 Fig. Supplementary a d , , whereas the degron variant of HA-BRD3 remained unaffected d ) and and ). Prolonged proteasomal inhibition increased HA-BRD3 HA-BRD3 increased inhibition proteasomal Prolonged ). 7 . We focused on BRD3 for experimental follow-up because because follow-up for experimental . Weon BRD3 focused SPOP Supplementary Fig. 5d Fig. Supplementary 8 2 mutations in cancer tis human endometrial primary ( 0 . Knockdown of of Knockdown . i. 1d Fig. Supplementary Fig. 5a Fig. Supplementary Fig. 2 Fig. Supplementary Fig. 5d Fig. Supplementary ). Furthermore, SPOP-WT,). Furthermore, along with RBX1 – , e e b ). ). and and Fig. 2 Fig. and and 1 9 . In aggregate, our findings suggest suggest findings our aggregate, In . upeetr Fg 3f Fig. Supplementary Supplementary Fig. 5b Fig. Supplementary c CUL3 ). Thus, the SPOP-binding motif motif SPOP-binding the Thus, ). ). Additional CUL3-dependent CUL3-dependent Additional ). in vitro increased HA-BRD3 levels levels HA-BRD3 increased ) and assessed the ability of ability the ) and assessed , , verifying the specificity ). s e l c i t r a in vivo in ). To deter ). – h ) and in in and ) Fig. 1f Fig. Fig. Fig. 1 and and 8– b 1 , 1 ). ).  g ------.

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. gate, these data are consistent with a model in which ubiquitination ubiquitination which in model a with consistent are data these gate, ( with cycloheximide levels mRNA and in impaired protein degradation after changes inhibition of protein synthesis concomitant without levels protein BET ( proteins BET endogenous of immunoprecipitation after detectable SPOP-WT of ( presence overexpression the in levels protein BET endogenous and A  degradation. proteasomal their promotes proteins BET of legend, legend, in ( by SPOP-E50K WT or HA-BRD3-degron-MT ( of HA-BRD3 ubiquitination ( constructs SPOP indicated the and HA-BRD3 overexpressing cells 293T transfected HA (top) immunoprecipitation and WCE (bottom) are shown from transiently (W131G). mutant SPOP cancer–associated prostate one and R121Q) (E50K, mutants SPOP cancer–associated endometrial SPOP-WT, with ( cells EN and Ishikawa in treatment (CHX) cycloheximide of duration specified the after proteins indicated ( treatment MG132 of h 3 without or with cells EN and Ishikawa in proteins indicated the ( s.e.m. bars, Error significant). Student’s two-tailed unpaired, by determined was significance Statistical lines. cell endometrial human EN and Ishikawa ( mutants. SPOP cancer–specific prostate and cancer– endometrial by 3 Figure a e CHX (h): c a upeetr Fg 5f Fig. Supplementary

TRIM ) Representative western blot ( blot western Representative ) SPOP-W131G: a SPOP-R121Q: SPO SPOP DAXX s e l c i t r AC BRD4 BRD3 BRD2 AC BRD4 BRD3 BRD2 – SPOP-E50K: HA (BRD3 f TB TB SPOP-WT: 24 are representative. ACTB were used as loading control. In each In each control. as loading used were ACTB representative. are P SPOP n AC

indicates the number of independent experiments performed. experiments of independent number the indicates BET proteins are differentially ubiquitinated and degraded degraded and ubiquitinated differentially are proteins BET 4 0 TB HA-BRD3-Ubn Ishikawa Ish n ) = ( 3). ik

aw EN – – + – – 293T HA-BRD3-W 1 8 a Fig. 2 Fig. – – – 0 2 e SPOP expression ) Effects of SPOP-WT and SPOP mutants on mutants SPOP and of SPOP-WT ) Effects 0. 1. n Fig. Fig. 2 + – – – – 0 5 1 5 = 3). ( 3). =

e Ishikawa and 8 4 + – – ). Moreover, Moreover, ). E n N f n.s. = ( 3). T and Supplementary Fig. 5e Fig. Supplementary c + – – – 12 ) Representative western blot of the the of blot western Representative )

b EN n ) Representative western blot of of blot western Representative ) = 4) of BET proteins and SPOP in in SPOP and proteins BET of 4) = Supplementary Fig. 5g Supplementary f n ) ) HA (BRD3) HA = 3). ( 3). = f d b In In vivo HA (BRD3) HA HA (BRD3) SPOP-E50K: MG SPO degron-MT HA-BRD3 HA-BRD3: SPOP SPO 132 (3 h) (3 132 SPO VC AC MG P n TRIM d ubiquitination of HA-BRD3- ubiquitination = 3). Western blots shown shown = Western blots 3). SPOP DAXX AC BRD4 BRD3 BRD2 P ) Interaction of HA-BRD3 HA-BRD3 of Interaction ) L TB HA-BRD3-Ub n 132: P IgG (SPOP-WT) TB knockdown increased increased knockdown 24 - : Control 293T HA-BRD3-W 293T – : + + – – t Control Ishi -test (n.s., not not (n.s., -test HA-BRD3 IP 293T His-U SPOP-WT ka + + – + – SPOP-WT WC ). SPOP was also also was ). SPOP wa

SPOP-E50K – + + + – SPOP-E50K E

, SPOP-R121Q h EN b SPOP-R121Q ). ). In aggre + + + – T

SPOP-W131G +

in in vivo SPOP-W131G

-

increased after prolonged proteasome inhibition and reduced after after reduced and inhibition proteasome prolonged after increased Supplementary Fig. 6f ( cycloheximide with synthesis of protein inhibition after proteins BET of degradation pronounced more noted we which cells, in EN in endogenous proteins BET above, to efficiently observations more bound our SPOP-R121Q with accordance in and to ( cells in Ishikawa than cells EN in inhibition proteasome short-term or depletion upon levels protein BET in increases significant more found we cells, EN in levels protein BET reduced to leads SPOP-R121Q by 6b Fig. ( mRNAs corresponding higher the of having levels despite proteins BET of levels lower exhibited cells ( SPOP-WT of levels equivalent expressing cells Ishikawa in those to cells EN in levels protein BET ( (CCLE) Encyclopedia Line that harbors a recurrent enous levels, we the identified human EN cancer endometrial cell line comparison to SPOP-WT when these mutants are expressed at endog cancer– endometrial specific of SPOP mutants properties may translate into functional reduced BET the protein levels whether in test To ubiquitination of BET proteins Cancer-type-specific SPOP mutants induce differential in the context of endometrial cancer–specific SPOP mutants are are mutants SPOP cancer–specific endometrial of context proteins BET the of in levels low with cells cancer endometrial sug that data gest These mutant. SPOP-R121Q the of context the in down to knocked had be proteins BET individual cells, in Ishikawa growth ( growth in decreased resulted of which BET the proteins, knockdown individual to susceptible were cells EN Indeed, levels. protein BET of reduction further to susceptible particularly protein become might BET levels low with cells cancer endometrial that speculated we vulnerabilities; specific create might mutants SPOP cancer–specific of of in context BET the endometrial proteins degradation enhanced and growth survival of for BET proteins tumor on presence the depend cells cancer Some SPOP Sensitivity to BET inhibitors is altered by cancer-type-specific degron. BET the and mutants SPOP between interactions by differential affected part 6j Fig. of variant degron the ( BRD3 ubiquitinate to failed SPOP-E50K Indeed, cancer– endometrial the on on SPOP degron an mutants BRD3. was intact dependent by specific mediated ubiquitination in increase ( mutants and cancer–specific prostate the with decreased and mutants SPOP cancer–specific endometrial the with increased was ( reduced 6h Fig. was F133L and W131G did whereas the interaction of the SPOP-WT prostate SPOP cancer–specific mutants than HA-BRD3 to Endometrial strongly more cells. bound R121Q) 293T and in (E50K mutants SPOP side by cancer–associated side species SPOP of different activity the analyzed we SPOP, within substitutions acid amino specific of the a were result cells Ishikawa EN and in human kinetics ( itself SPOP of turnover proteasomal indicating synthesis, protein of inhibition Supplementary Fig. 7a Fig. Supplementary To BET the different protein test and binding whether degradation upeetr Fg 6i Fig. Supplementary mutations ). These results suggest that BET protein levels are at least in in least at are levels protein BET that suggest results These ). ). In line with the notion that enhanced protein degradation degradation protein enhanced that notion the with line In ). ). In line with this observation, ubiquitination of HA-BRD3 HA-BRD3 of ubiquitination observation, this with line In ). Fig. 3 Fig. Figs. 2e 2e Figs. f ) and to reduce BRD3 protein levels ( levels protein BRD3 reduce to and ) advance online publication online advance Fig. 3 Fig. 18 and b , 2 , and and 1 g SPOP . Therefore, we sought to determine whether . whether we Therefore, to sought determine ). ). Of note, endogenous SPOP levels were also ). In contrast, to achieve a similar effect on on effect similar a achieve to contrast, In ). 3b , Supplementary Fig. 6c Fig. Supplementary Supplementary Fig. 6a Fig. Supplementary c mutation (p.R121Q) in the Cancer Cell ). We next investigated whether the the whether investigated next We ). ). Fig. 3 Fig. i. 3 Fig. a Fig. 3 Fig. ). We found that EN cancer cancer EN that Wefound ). d a and and n vivo in and and nature medicine nature Supplementary Supplementary Supplementary Supplementary Supplementary Supplementary – ). We ). compared e and and ). In addition addition In ). Fig. 3 Fig. n vitro in Fig. 3 Fig. SPOP c and e - - ,

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. of independent experiments performed. performed. experiments of independent Student’s two-tailed unpaired, mean represent Data 30 d.f.). test, ANOVA Dunnett’s post with (two-way bars compared the above ( culture in 3D semisolid SPOP-E50K expressing cells cancer urothelial human VM-CUB1 mutant and SPOP-E47K expressing cells cancer intestine ( of depletion of shRNA-mediated ( proteins BET of the constructs degron-mutant different and SPOP-E50K overexpressing stably cells by Ishikawa nM) (250 treatment of SPOP-WT.to that ( relative mutants SPOP of individual expression in protein change the represents point Each correlation). rank ( mutants SPOP cancer–specific endometrial recurrent expressing stably cells in Ishikawa spectrometry, ( (d.f.). of freedom degrees 112 test, post ( culture cell in 3D semisolid in blue) F133L; W131G, F102C, (Y87C, cancer M117V, or prostate S80R, in grey) E78K, D140N; E50K, R121Q, (E47K, cancer to endometrial specific mutants SPOP overexpressing stably 4 Figure nature medicine nature n e ) JQ1 sensitivity of: Ishikawa cells expressing SPOP-WT, EN human endometrial cancer cells expressing SPOP-R121Q, NCI-H508 human large large human NCI-H508 SPOP-R121Q, expressing cells cancer SPOP-WT, endometrial EN human expressing cells Ishikawa of: ) sensitivity JQ1 = 4). A representative immunoblot for the indicated proteins and cell lines is shown. ACTB was used as a loading control. control. as a used loading was ACTB is shown. lines cell and proteins indicated the for immunoblot = A 4). representative

Cancer-type-specific SPOP mutants alter BET inhibitor sensitivity in an opposing manner. ( in an opposing sensitivity inhibitor BET alter mutants SPOP Cancer-type-specific JQ1 (nM JQ1 (nM b a e % of growth inhibition upon JQ1 Average protein expression changes

exposure (relative to control) –3 –2 –1 % of growth inhibition upon JQ1 exposure 0 ) 100

50 (relative to control) 20 40 60 80 ) 100 0

20 40 60 80 R R R 0 advance online publication online advance

(SPOP-WT) (BRD4) = 0.4077 0.4077 = (BRD4) (B 0.9643 = (BRD2) Ish 50 RD3) = 0.9286 0.9286 = RD3) WT 100 ik JQ1 (nM aw 100 IC t a -test unless otherwise specified. * specified. otherwise unless -test ** 50 BRD2 E47K * ** (JQ1(nM)) (SPOP-R121Q) ) 150 * 150 ** ** * , ,

**

P P P P P EN BRD3 ** * E50K ** = 0.39 0.39 = 0.0028 = = 0.0067 = b * * ) Correlation of IC ) Correlation ** ** 200 * * and and ** BRD4 BRD3 BRD2

* E78K *** *** (SPOP-E47K) *** BRD4 NCI-H508 250 *** *** S80R *** ** individually on JQ1 (200 nM) sensitivity in Ishikawa cells expressing SPOP-Y87C ( SPOP-Y87C expressing cells in Ishikawa sensitivity nM) (200 on JQ1 individually

* *** (SPOP-E50K ** 50 M1 VM-CUB ** * ** (JQ1), shown in shown (JQ1), * 17V ** *** P * < 0.05, ** < 0.05, n.s *** R121Q 1 ** . n c ) * = 3). Statistical analysis was performed using two-way ANOVA Dunnett’s with two-way using performed was analysis = Statistical 3). % of growth inhibition upon JQ1 exposure *** HA-BRD2-degron-MT

(relative to control) *** D140N 60 20 40 **

HA-BRD3-degron-MT 0 P

Control * *** < 0.01, *** < 0.01, Supplementary Figure 7c Figure Supplementary HA-BRD4-degron-MT SPOP-E50K ** Y87C VM-CUB1 NCI-H508 Ish Ish ** EN ik ik * aw aw * 50 F102 a a ** P JQ1 (nM < 0.001. In each legend, legend, In each < 0.001. ** C DM 150 * W SO ± 131 s.e.m. Statistical significance was determined by determined was significance Statistical s.e.m. ) a 250 * ) Response to JQ1 treatment by Ishikawa cells cells by Ishikawa treatment to JQ1 ) Response G (150 nM (150 R F133 , with BET protein levels, as quantified by mass by mass as quantified levels, protein BET , with and and JQ 1 ** d L *

) % of growth inhibition upon JQ1 exposure P

values were calculated using Spearman Spearman using calculated were values (relative to control) 60 shControl 20 40 0

shBRD2_1 F102 Y87C E78K E50K WT ** SPOP AC BRD4 BRD3 BRD2

n shBRD2_2 * C indicates the number number the indicates ** TB

shBRD3_1 SPOP-Y87C Ish P DM **

values in values shBRD3_2 * Ishikawa ik SO ** aw shBRD4_1 Ish

EN * a c ik **

) Response to JQ1 ) Response shBRD4_2 aw (250 nM (250

NCI-H508 * s e l c i t r a n ** a JQ = ( 3).

e VM-CUB1 * 1 are indicated indicated are ) n = 3). d ) Effect ) Effect

 © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. in response to expression of SPOP mutants in Ishikawa cells cor cells concentration inhibitory Ishikawa in half-maximal the a with decrease related in mutants SPOP of expression to proteins BET response in individual of levels reduced that To found JQ1. we to end, this sensitivity to contribute levels protein BET in changes ( cells cer results were can Similar found in and endometrial HEC-151 RL95-2 ing cellular proliferation ( BET inhibitors, JQ1 and byOTX015, promoting apoptosis and reduc to both cells Ishikawa sensitized to levels) BET lower (shown protein Indeed, forced expression of endometrial cancer–specific SPOP mutants to in by BET SPOP protein corresponding mutants. differences levels influenced be might inhibitors these to cells cancer of susceptibility tumors solid for including peutics, proteins BET reported been among has overlap functional a view, this of support In particularly susceptible to further suppression of BET protein function. * specified. otherwise In performed. of validation ( ( W131G) (Y87C, cancer prostate or E50K) (E47K, cancer endometrial to specific mutants SPOP or SPOP-WT expressing stably cells Ishikawa in treatment nM) (500 JQ1 after levels expression by stratified ( tissues cancer endometrial primary human of images ( means. to correspond SPOP-WT. to Midlines BETsof compared as degradation on effect pronounced a less with mutants SPOP BETs;of gray, degradation on effect a strong with mutants SPOP Red, reads. mapped million per transcript of kilobase per reads RPKM, correction. endometrial with respectively; blue, and gray in (shown mutants SPOP cancer–specific prostate or cancer– endometrial either or SPOP-WT expressing stably cells Ishikawa of levels (bottom) protein FOSL1 and (top) ( a significant with genes of number the to corresponds Overlap treatment. JQ1 without or with W131G) (Y87C, cancer prostate versus E50K) (E47K, cancer endometrial to specific mutants SPOP expressing stably cells Ishikawa in genes expressed 5 Figure A  (IC n b e a

Control = 3). = 3). of area overlap the in genes 16 the for expression in change fold showing map ) Heat

BET inhibitors are under clinical investigation as thera BET anticancer investigation are inhibitors clinical under Strong Weak Negative s e l c i t r FOSL1 IH 50 endometrial cancer–vs.prostate 95 cancer–specific SPOPmutants Differentially expressedgenes ) during JQ1 treatment ( treatment JQ1 during ) P

Downregulation of of Downregulation values are indicated below the dose-response curves, as calculated by extra sum-of-squares sum-of-squares extra by calculated as curves, dose-response the below indicated are values 3 C Supplementary Fig. 7f Fig. Supplementary 66 SPOP 16 FOSL1 61

c FOSL1 IHC JQ1 (2 JQ1 (500nM 100 , , 20% 40% 60% 80% 3 f R 54 0% and and 0 Endometrial cancer % mutation status. status. mutation = –0.235,

and prostate and SPOP-MT 87 Strong Negative knockdown. In In knockdown. 2 µ 2 6 M) P . g 4 9 5 < 0.05, ** < 0.05, , data are represented as mean mean as represented are , data SPOP-WT ) P =0.024 33 33 Fig. Fig. 4 6 FOSL1 Weak 31– Fig. 4 Fig. HIST1H2B b a LINC00511 P FOSL1 IHC HIST1H1C 3 sensitizes Ishikawa cells to JQ1 treatment. ( treatment. JQ1 to cells Ishikawa sensitizes 100% and f S100A16 SCNN1 < 0.01, *** < 0.01, P 3 FGFBP1 20% 40% 60% 80% MYAD and and SPON 0% cancer stratified according to to according stratified cancer ). We aimed to discover whether whether discover to aimed We ). FOSL values were determined by Kendall’s tau-b. Scale bars, 80 80 bars, Scale tau-b. Kendall’s by determined were values R GLIS3 NEXN 18 H1F0

SPOP-MT =0.19, Prostate cancer b GAL Strong Negative LO NT ZYX , Supplementary Fig. 7b Supplementary ). Functionally, overexpression overexpression Functionally, ). 21 M D A S g X 1 1 4 7 3 , ACTB was used as a loading control. In each legend, legend, each In control. a loading as used was , ACTB , 23 n SPOP-WT Scaled foldchange = 3). ( = 3). P , 2 =0.823 Control –1.5 4 n 29 81 25 . We anticipated that the the that We . anticipated Weak P = 4). VCL was used as a loading control. ( control. a loading as used was VCL = 4). < 0.001. < 0.001. 0

g JQ1 (500 nM) ) Left, JQ1 dose-response curves for Ishikawa cells expressing SPOP-Y87C upon upon SPOP-Y87C expressing cells Ishikawa for curves dose-response JQ1 ) Left, 1.5 n ± = 239) stained for FOSL1; right, corresponding expression analysis on human primary tumors tumors primary human on analysis expression corresponding right, FOSL1; for stained = 239) s.e.m. Statistical significance was determined by unpaired, two-tailed Student’s Student’s two-tailed unpaired, by determined was significance Statistical s.e.m. FOSL1 mRNA fold change f JQ1 (2

SPOP-WT 0.5

0 1 µ SPOP-E47K M ) FOSL SPOP-E50K (500 nM),2 ** c VC ** – SPOP-Y87C SPOP-WT n.s. SPOP-W131G JQ SPOP-E47K FOSL1 mRNA fold change e 1 L SPOP 1.5 0.5 * SPOP-WT ) 1 SPOP-E50K 0 1 2 25 SPOP-E47K SPOP-WT SPOP-E78K , 2 SPOP-E47K h SPOP-E50K

6 SPOP-S80R mutation status. status. mutation - - - - *** . SPOP-M117V

SPOP-E50K SPOP-E78K (500 nM),4 **

SPOP-R121Q *** SPOP-Y87C ** SPOP-S80R **

expression levels of BRD2, BRD3 and BRD4 were associated with with associated sensitivity to JQ1 were in many cases ( BRD4 and BRD3 BRD2, of levels expression Decreased parallel. in levels protein BET determined we which for lines, human cell 12 different across conditions culture semisolid 3D in sensitivity JQ1 assessed we purpose, this For cancer. endometrial human of models line cell across inhibition BET pharmacologic to sensitivity predict might general in levels protein BET decreased or phenotype. this dampened SPOP-Y87C mutated of context ( bined ( individual whereas ( JQ1 to resistant more cells 22Rv1 and Ishikawa rendered SPOP-WT to comparison in mutants SPOP cancer–specific prostate of Overexpression inhibitors. BET to resistance induce contrary, the on might, the cancer in prostate of context levels protein BET increased whether investigated then We ( JQ1 to sensitization mediated of the BRD3 and BRD2, SPOP-E50K- reduced BRD4 degron variants SPOP-W131G SPOP-D140N *** JQ ** SPOP-S117V a Next, we sought to determine whether recurrent recurrent whether determine to sought we Next, 1 *** ) Venn diagram depicting the overlap of significantly differentially differentially significantly of overlap the depicting ) Venn diagram SPOP-R121Q Ishikawa

SPOP-D140N ** h FOSL1 Supplementary Fig. 7k Fig. Supplementary FOSL SPOP-Y87C * ACTB a VC

SPOP-WT *** . . ( SPOP-WT SPOP-Y87C SPOP-F102C

SPOP-E47K SPOP-F102C L d c 1 SPOP-W131G P ) ) ) ) SPOP-W131G

values were derived from unpaired unpaired from derived were values SPOP-E50K FOSL1 FOSL1 SPOP-F133L SPOP-F133L SPOP-Y87C Ishikawa advance online publication online advance SPOP-W131G µ n m. ( m. indicates the number of independent experiments experiments independent of number the indicates mRNA (normalized to cyclophilin expression) expression) cyclophilin to (normalized mRNA mRNA expression in data sets of individuals individuals of sets data in expression mRNA Fig. 4 Fig. F f P ) ) test. Right, corresponding western blot blot western corresponding Right, test. d value of <0.05 (Benjamini–Hochberg test). test). (Benjamini–Hochberg <0.05 of value FOSL1 FOSL1 expression (RPKM) g 1,000 2,000 4,000 6,000 d Relative cell viability (%) 200 400 600 800 and and Fig. 4 Fig. 100 SPOP-MT 0 20 40 60 80 mRNA (left) and FOSL1 protein (right) (right) protein FOSL1 and (left) mRNA 0 Endometrial cancer Fig. 4 Fig. ) knockdown of BET proteins in the the in proteins BET of knockdown ) n Supplementary Supplementary Fig. 8a P = 13 n.s. P Supplementary Fig. 7j Fig. Supplementary –2 = 0.0304 a < 0.0001 SPOP-WT and and

c n log (JQ1

and and = 211 –1 Supplementary Fig. 7h Fig. Supplementary Supplementary Fig. 7g Fig. Supplementary E78K E50K E47A E46K R121Q M117V D140G/N ( µ siFOSL1_3 siFOSL1_2 siFOSL1_1 siControl M)) e t 1 0 -tests with Welch’s with -tests ) Left, representative representative ) Left, nature medicine nature FOSL1

SPOP FOSL1 expression (Z score) –2 10 FOSL1 SPOP-MT 0 2 4 6 8 ACTB t – -test unless unless -test n Prostate cancer n.s. c siControl = 46 knockdown knockdown , mutations mutations ); ); however,

l siFOSL1_1 ) or com or ) SPOP-Y87 SPOP-WT

siFOSL1_2 Ishikaw n siFOSL1_3 = 510 a , C i ), ), ). ). - © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. them them from the histone acetylated tails of active sites. transcriptionally BET inhibitors bind to the bromodomains of BET proteins to displace JQ1 response Transcriptome analysis identifies ( degradation protein BET of regulation through to JQ1 directly SPOP sensitivity mutants affect protein the or levels notion supporting that the JQ1 further response, BET in changes to substantial or led NCOA3 ERG TRIM24, of DEK, through JQ1 to responses changes influence in BET protein levels. Neither indirectly knockdown nor overexpression or directly either inhibitors. BET to sensitivity with associated more broadly be 8d Fig. ( inhibition proteasomal to responsive to ularly sensitive JQ1 and low displayed BET protein that levels were encoding p.E47K and p.E50K, respectively. Both cell lines were partic harbored that Project Lines Cell (COSMIC) Cancer (VM-CUB1) cancer cell lines in the Catalogue of Somatic Mutations in endogenous locus. We identified colorectal (NCI-H508) and urothelial cancer–associated with endometrial recurrent 7f Figs. ( lines cell isogenic in generated data SPOP-R121Q mutant were sensitive to JQ1 inhibition, in line with our inhibitors BET to tibility suscep regulate that mechanisms molecular other of existence the we also found some notable exceptions to this rule, in agreement with ( SPOP-E50K. overexpressing stably cells Ishikawa from established bar, 50 Scale ( histology ( with (right) weight tumor individual ( with (right) weight 6 Figure nature medicine nature changes in we transcriptional investigated this function, Considering without ( without means. Statistical significance was determined by unpaired, two-tailed Student’s Student’s two-tailed unpaired, by determined was significance Statistical means. ( tumors of cancer-specific of

3 d 3 a Moreover, we tested whether established SPOP substrates might might substrates SPOP established whether tested we Moreover, Tumor volume (mm ) Tumor volume (mm ) 1,00 1,50 1,000 50 50 0 0 0 0 0 0 ). Thus, endometrial cancer–associated cancer–associated endometrial Thus, ). and

n Endometrial cancer–associated SPOP mutants sensitize cells to JQ1 treatment treatment JQ1 to cells sensitize mutants SPOP cancer–associated Endometrial 6 0 = 7) JQ1 treatment in xenografts established from Ishikawa cells stably overexpressing SPOP-S80R. ( SPOP-S80R. overexpressing stably cells Ishikawa from established xenografts in treatment JQ1 = 7) n n 0 = 7) (top) and quantification (bottom) of mitotic and apoptotic cells in EN and Ishikawa xenografts treated with vehicle (control) or JQ1. JQ1. or (control) vehicle with treated xenografts Ishikawa and EN in cells apoptotic and mitotic of (bottom) quantification and (top) = 7) ) for each xenograft group are shown in in shown are group xenograft each ) for 9 3 Ishikawa SPOP-E50K JQ Control 8b 3 µ Days oftreatment Days oftreatment 1 m. ( m. ). This finding led us to search for additional cell lines lines cell us led to for search additional ). finding This 1 6 SPOP 12 1 9 d n EN

) Tumor growth kinetics (left) and individual tumor weight (right) with ( with (right) weight tumor individual and (left) kinetics ) Tumor growth = 9) or without (control; (control; without or = 9) 15 1 2 advance online publication online advance 18 mutations on both BET protein levels and sensitivity to BET inhibitors (BETi). Representative images of the indicated number number indicated the of images (BETi). Representative inhibitors BET to sensitivity and levels protein BET both on mutations 5 21 JQ Control 27 2 8 24 Supplementary Fig. 9 Fig. Supplementary

1 ,

2

27 1 8 ** . Nevertheless, EN cells expressing the the expressing cells EN Nevertheless, . **

Tumor weight (mg) Tumor weight (mg) n 1,000 1,500 1,000 1,500 = 7) or without (control; (control; without or = 7) 500 500 FOSL1 Control 0 0 Control Fig. 4a Fig. JQ Control Control JQ Fig. 4 Fig. 1 1 n as a determinant of *** ** = 7) JQ1 treatment in xenografts established from EN cells. ( cells. EN from established xenografts in treatment JQ1 = 7) , JQ e JQ e SPOP and and and and SPOP 1 a 1 , , ). b , , b e mutations at the

SPOP 3 d Supplementary Supplementary Supplementary Supplementary 3

mutations may mutations Tumor volume (mm ) Tumor volume (mm ) and and 1,000 1,500 1,000 1,500 500 500 0 0 mutations mutations e n . Data in in . Data = 7) JQ1 treatment in xenografts established from Ishikawa cells. ( cells. Ishikawa from established xenografts in treatment JQ1 = 7) 0 6 0 Ishikawa SPOP-S80R JQ Control 3 e 9 3 Days oftreatment Days oftreatment ) Tumor growth kinetics (left) and individual tumor weight (right) with ( with (right) weight tumor individual and (left) kinetics ) Tumor growth 1 1 6 Ishikaw 1 9 a - - , , b 12 1 2 , , trial cancer harboring mutated mutated harboring cancer trial FOSLI protein levels were also in decreased individuals with endome ( sis analy in changes BET and protein levels the transcriptome observed the with accordance in mutants, SPOP cancer–specific, prostate to compared as cancer–specific, endometrial overexpressed that cells inhibitors target reported gene for BET proteins implicated in to sensitivity BET 2 or nM 500 (untreated, conditions all 10a Fig. altered with those expression in and the untreated conditions ( genes expressed differentially overlap the significant between a found and mutants SPOP prostate versus cancer–associated cancer– endometrial overexpressing cells in ment ( NCOA3 or TRIM24 as WT, shared of dysregulation possibly reflecting SPOP substrates such of mutants types from SPOP-both feature that discriminated second lines ( cell the across observed levels protein BET different the with of SPOP mutants ( types different the between in SPOP positioned SPOP-WT with mutants, cancer–associated prostate and cancer– endometrial the of expression to response in changes transcriptional opposing revealed mainly expression gene of analyses (MDS) scaling tidimensional ( cells Ishikawa in mutants SPOP cancer–associated prostate and cancer– response to overexpression of SPOP-WT or two recurrent endometrial a d Supplementary Fig. 3f Fig. Supplementary 15 5 and and Next, we interrogated transcriptional changes under JQ1 treat JQ1 under changes transcriptional interrogated we Next, JQ Control t 18 2 8 -test. * -test. Fig. 5 Fig. 1

Supplementary Table 2 Table Supplementary 21 1 e shown as mean tumor volume + s.e.m.; midlines represent represent midlines + s.e.m.; volume tumor mean as shown * – c in vivo in 2 c P

9 Tumor weight (mg)

). We identified 16 genes with altered expression across across expression altered with genes 16 identified We ). Tumor weight (mg) ). Notably, in human tumor tissues, tissues, tumor human in Notably, ). 1,000 1,500 1,000 1,500 . < 0.05, ** < 0.05, FOSL1 n 500 500 = 7) or without ( without or = 7) Control 0 0 Control . . ( JQ Contro Control JQ a 1 Supplementary Fig. 10a Supplementary ) Tumor growth kinetics (left) and individual tumor tumor individual and (left) kinetics ) Tumor growth mRNA and FOSLI protein levels were reduced in mRNA were reduced and protein levels FOSLI 1 n.s. l ** P JQ JQ < 0.01, *** < 0.01, Fig. 1 Fig. ). Interestingly, the MDS analysis revealed a revealed analysis MDS the Interestingly, ). 1 1 c

b Cleaved n Prostate ) Tumor growth kinetics (left) and and (left) kinetics ) Tumor growth f c

= 6) JQ1 treatment in xenografts xenografts in treatment JQ1 = 6) % of mitotic cells cancer accumulation

Control 0 1 2 3 4 5 CASP-3 H&E ). Unsupervised clustering and mul and clustering Unsupervised ). and and f SPOP ) Model showing the differential effects effects differential the showing ) Model Loss offunction Cancer-associated *** Control BE JQ1 BET proteinlevels

P BET T Supplementary Fig. 10b Fig. Supplementary < 0.001. < 0.001. , with the lowest mRNA levels levels mRNA lowest the with , SPOP EN MATH Cleaved CASP-3 µ Fig. Fig. 5a BET (% positive cells) 15 M JQ1), including including JQ1), M 10 5 Control 0 JQ

BET , *** b 1 JQ1 ). This result aligns ). well This result aligns

SPOP , b and BETi sensitivit

FOSL1 Cleaved c pointmutation s e l c i t r a Gain offunction % of mitotic cells ) Representative ) Representative CASP-3 H&E 0 1 2 3 SPOP Control MATH Supplementary Supplementary Control

BET n.s. JQ1 mRNA and and mRNA Ishikawa Ub n y degradation Endometrial = 6) or = 6) Ub Cleaved CASP-3 FOSL1 cancer s Ub

BET (% positive cells) )

Control 0 1 2 3 8 Ub JQ . n.s. 1 JQ1 , a ,  - - - - © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. and thereby alter the susceptibility of cancer cells to BET inhibitors. inhibitors. BET to cells cancer of susceptibility the alter thereby and as such genes, target of established transcription the the in levels protein BET altered The other substrates (for example, DEK) remains to elucidated. be further and proteins BET of ubiquitination and binding enhance mutations structural basis through which endometrial cancer–associated cancer–associated endometrial which through basis structural ( mechanism gain-of-function a through tion cancer–associated previously reported mutations these of ties degradation of BET proteins, in line with the proper loss-of-function cancer–associated prostate that found We oncogenic programs transcriptional driving lineage-specific in proteins these of importance critical the of because tumors solid group of proteins are under clinical investigation in hematological and this against inhibitors Small-molecule substrates. SPOP fide bona as elusive. largely remain types mutation both of implications tein relevant substrates to prostate tumorigenesis mutations associated have shown been more recently to prostabilize the same substrate-recognition domain. Although the prostate cancer– to are they even though confined tumor types, these overlap between studies ing prostate and endometrial cancers in comprehensive genome sequenc of 5–10% in found been ligase—have ubiquitin tor of a cullin–RING mutations in missense Recurrent DISCUSSION JQ1 to cells Ishikawa tized vitro in to ( JQ1 resistant treatment largely were tumors Ishikawa-cell-derived whereas apoptosis, inducing and from established Indeed, JQ1 efficiently blocked the growth of tumor xenograft models from response BET inhibitors eficial may in this setting. be identified ben a receiving patients because mutants SPOP cancer–associated endometrial of expression on focused we purpose, this For setting. an in validated be could mutants SPOP to response in JQ1 to Finally, we investigated whether our results showing altered sensitivity endometrial cancer–specific SPOP mutants JQ1 treatment blocks tumor growth in xenografts expressing transcriptional of regulation through part, in least at JQ1, to susceptibility alter mutants SPOP to response in changes level protein BET that suggest ( mutants SPOP and levels tein pro BET in changes of downstream gene this of involvement tional itself directly lowered resistance to JQ1 in this setting, indicating func ( SPOP-Y87C variant the prostate overexpressing cancer–specific cells decreased proteins ( mutants SPOP of types ent differ the between same the remained levels expression relative the treatment reduced FOSL1 expression levels in all conditions, whereas the at reported transcription, as triple occupancy of BRD2, BRD3 and BRD4 has been to SPOP mutants decrease response might and JQ1 treatment ( study our in have on the BET strongest to effects protein and JQ1 levels sensitivity to shown mutants SPOP the harbored who individuals in observed A  Fig. 4 Fig.

Our study identifies the BET proteins (BRD2, BRD3 and BRD4) BRD4) and BRD3 (BRD2, proteins BET the identifies study Our in levels protein BET in changes whether investigated we Next, s e l c i t r a data, forced expression of SPOP-E50K or SPOP-S80R sensi SPOP-S80R or SPOP-E50K of expression forced data, and and 2– 6 Supplementary Fig. 10e Fig. Supplementary FOSL1 Fig. 5d Fig. . Surprisingly, the specific genetic alterations show no no show alterations genetic specific the Surprisingly, . FOSL1 SPOP SPOP FOSL1 . , e and and -mutant EN cells by reducing cell proliferation promoter ( promoter mutations enhance BET protein degrada protein BET enhance mutations mRNA levels in JQ1-resistant Ishikawa Ishikawa JQ1-resistant in levels mRNA in vivo in Supplementary Fig. 7c Fig. Supplementary Fig. 5 Fig. Fig. 5 Fig. SPOP ( Fig. 6a Fig. f Supplementary Fig. 10d Fig. Supplementary Fig. 6d Fig. ). Knockdown of individual BET BET individual of Knockdown ). g SPOP ). Taken together, these results results Taken). these together, ). Moreover, ). —encoding a substrate recep —encoding – , c -mutant setting influence influence setting -mutant e 8 ). In accordance with the ). with In accordance SPOP ). , 9 , whereas endometrial endometrial whereas , in vivo Fig. 6 Fig. 8– mutations impair impair mutations 1 ) FOSL1 2 30– , the therapeutic , the therapeutic FOSL1 f ). The precise precise The ). 3 3 . depletion depletion (ref. (ref. 18 ) , 2 21 FOSL1 in vivo in 4 SPOP . JQ1 JQ1 . , 23 2 , 9 2 4 ), ), ------.

reported mechanisms that influence BET inhibitor sensitivity inhibitor BET influence that mechanisms reported as well cells cancer in ovarian resistance of as a acquired mechanism activity FOSL1 enhanced implicates report recent a regard, this in note Of t the in available are references, and codes accession statements of including Methods, and data availability any associated M mutations. similar of basis the on responses therapeutic tion in the setting, clinical caution may be needed when extrapolating informa of genome use cancer Given increasing the responses. drug opposing evoke protein particular a of domain same the to mapping inhibitors. BET with (prostate cancer–associated cancer–associated (endometrial may detec the specific of Thus, tion response. inhibitor BET of biomarker detectable identifies study preclinical Our ment. treat this to respond may who patients identify to need critical a is 1. claims jurisdictional in published maps and affiliations. institutional r at online available is information permissions and Reprints v The authors declare competing interests:financial details are available in the H.J. and G.E.T. contributed equally to this work. resources. J.-P.P.T., H.J. and G.E.T. interpreted the data and wrote the paper. J.-P.P.T., L.A.G., S.A.C., M.P., C.V.C., F.B. and P.J.W.samples. providedC.M.B., G.V.K.,funding and andA.R. L.C. analyzed genomic and RNA-seq data. M.E.C., A.B., B.J.N.W. and providedR.R.B. SPOP-mutant cancer endometrial prostate cancer samples with annotation for experiments and analysis. P.J.W., P.S., H.M. and E.B. provided andendometrial immune-deficient mice. M.L., H.J. and J.-P.P.T. performed immunohistochemical the data. H.J., G.C., G.E.T. and T.B. performed tumor xenograft experiments in J.-P.P.T., N.D.U., T.S., S.N., A.U. and performed experiments andR.I.E. analyzed the experiments together with H.J., G.E.T. and N.D.U. H.J., G.E.T., A.R., J.-P.P.T. originally developed the concept, elaborated further on it and designed (SWF). Zurich of University at the Humanities the and Science in Research for Foundation the and P.J.W.to grant by a part in (KLS-3384-02-2014-R) by Oncosuisse provided funded M.P. also fund was work This government Korean the from grant GRL a and (KFS-3498-08-2014) Schweiz Krebsforschungs (2013/128), IPhD SystemsX (268930), Council Research European the (310030B_160312/1), Foundation Science National Swiss The Vontobel by the Stiftung. grant a AG and Life Swiss Jubiläumsstiftung by the grant a grant, (KSL-3654-02-2015) League Cancer Swiss a grant, (PP00P3_150645) Professorship Foundation Science National J.-P.P.T. discussions. scientific Swiss by a for funded is laboratory the of members all We thank repository. also tissue the for (CHTN) Network Human Tissue Cooperative the and (OHSU) University Science & Health WeOregon the thank lines. cell KLE and EFE184 SNG-II, HEC116, HEC1B, HEC1A, AN3CA, providing for Zurich) Hospital (University Dedes K. and We Samartzis E. thank work. animal the and assistance technical for facility core animal IRB the of members We all pipeline. thank bioinformatics sequencing next-generation the with Wehelp his for Q. Zhong thank assistance. histology and handling tissue F. and with help for Prutek P. Dettwiler Storz, S. M. We Losa, Schraml, M. thank o Note: Any Supplementary Information and Source Data files are available in the C AUTH A e h e n ckn O p

r l ethods e More broadly, our results suggest a paradigm whereby mutations mutations whereby paradigm a suggest results our broadly, More BET inhibitors are development, and under currently clinical there i s r MPETING FINANCIAL INTERESTS FINANCIAL MPETING n ocoduy S & hnayn AM Tasaig eois o peiin cancer precision for genomics Translating A.M. Chinnaiyan, & S. Roychowdhury, medicine.

i i p n o e t n

a s v o O

p / e o 3 i r n wledgments R R C 4 e f s

d . Overall, . our model extends the established list Overall, of previously t i r o h e . n e x O .

Annu. Rev. Genomics Hum. Genet. Hum. Genomics Rev. Annu. p o h NTRIBUTI f a t

m p t h e l e . . Publisher’s with neutral Nature remains regard to note: Springer r advance online publication online advance SPOP

. p a p e r . mutations may be used to select patients who who patients select to used be may mutations O NS SPOP mutations) benefit from mutations) treatment benefit SPOP SPOP SPOP

15 mutation status. D.G.M., , 395–415 (2014). 395–415 , mutations as a clinically as a clinically mutations mutations) or may not not may or mutations)

h nature medicine nature t t o p : n / / l w i n w w e

. v n

a e t r u

27 s r

i e o ,

o 35

. n c n

l

o ,

i 3

n m o 6

e - - - . f /

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. nature medicine nature

ug M, eao KA, enne-otlá, . See, . Hede, B. Haendler, & S. Siegel, A., Fernández-Montalván, K.A., Gelato, M., Jung, A.C. Groner, 2 gradient anterior estrogen-regulated The B. F.Andersen, Zhao, & M.L., Salmans, Tsai,W.W. of identification Large-scale S.A. Carr, & T. Svinkina, P., Mertins, N.D., Udeshi, Establishment K. Hayashi, & H. Iwasaki, M., Kaneko, K., Kasahara, M., Nishida, C. Geng, J. An, Gan, W. C. Geng, J.P. Theurillat, M. Zhuang, C. Kandoth, S. Jones, M. Gallo, Le Blattner, M. C.E. Barbieri, agtn BT rmdmis o cne treatment. cancer (2015). for bromodomains BET Targeting cancer. biomarker. and target drug potential Res. Cancer a cancer: breast in protein (AGR2) Nature spectrometry. mass by sites ubiquitination (1985). 1103–1111 receptors. progesterone and estrogen containing cells, Ishikawa line, cell adenocarcinoma endometrial human new a of cancer. prostate in SPOP suppressor degradation. proteasome SPOP-mediated to progression. cancer prostate suppress to USA Sci. turnover. protein 3 coactivator receptor steroid regulate (SPOP) cancer. prostate SPOP-mutant in substrates ligases. ubiquitin (2009). BTB–Cul3 of architectures molecular Nature genes. remodelling (2014). chromatin in mutations frequent genes. complex ligase ubiquitin and chromatin-remodeling in mutations somatic recurrent cohorts. patient MED12 et al. et

Nat. Genet. Nat. mutations in prostate cancer. prostate in mutations 468 497 Cancer Cell Cancer et al.

et al. et et al. et 110 et al. et Truncated ERG oncoproteins from TMPRSS2-ERG fusions are resistant are fusions TMPRSS2-ERG Truncatedfrom oncoproteins ERG , 927–932 (2010). 927–932 , (2013). 67–73 , t al. et

et al. 15 SPOP promotes ubiquitination and degradation of the ERG oncoprotein et al. et t al. et , 6997–7002 (2013). 6997–7002 , t al. et t al. et Androgen receptor is the key transcriptional mediator of the tumor the of mediator transcriptional key the is receptor Androgen Prostate cancer–associated mutations in speckle-type POZ protein POZ speckle-type in mutations cancer–associated Prostate t al. et , 204 (2013). 204 , TRIM24 links a non-canonical histone signature to breast cancer. breast to signature histone non-canonical a links TRIM24 t al. et Neoplasia

SPOP eoi aaye o gneooi crioacms reveal carcinosarcomas gynaecologic of analyses Genomic Integrated genomic characterization of endometrial carcinoma. endometrial of characterization genomic Integrated

RM4 s n noei tasrpinl ciao i prostate in activator transcriptional oncogenic an is TRIM24

44

xm sqecn o sru edmtil uos identifies tumors endometrial serous of sequencing Exome 29 tutrs f PPsbtae opee: nihs into insights complexes: SPOP–substrate of Structures Exome sequencing identifies recurrent identifies sequencing Exome Ubiquitylome analysis identifies dysregulation of effector dysregulation identifies analysis Ubiquitylome advance online publication online advance , 1310–1315 (2012). 1310–1315 , , 846–858 (2016). 846–858 , mutations in prostate cancer across demographically diverse

16 , 14–20 (2014). 14–20 , Cancer Res. Cancer Nat. Genet. Nat. io Sna uik Gka Zasshi Gakkai Fujinka Sanka Nihon Mol. Cell Mol. Nat. Protoc. Nat. Mol. Cell Mol. Science

74 44 59

, 5631–5643 (2014). 5631–5643 , 346

, 685–689 (2012). 685–689 , , 917–930 (2015). 917–930 , 59

Epigenomics 8 a. Commun. Nat. , 904–916 (2015). 904–916 , , 1950–1960 (2013). 1950–1960 , , 85–89 (2014). 85–89 , o. Cell Mol.

SPOP Proc. Natl. Acad. Natl. Proc.

,

7 FOXA1 36 487–501 ,

5 39–50 , 5006 , Breast and

37 ,

32. 31. 30. 29. 28. 27. 26. 25. 24. 23. 22. 21. 20. 19. 36. 35. 34. 33.

acr eoe ta Rsac Ntok Te oeua txnm o primary of A. Kumar, taxonomy molecular The Network. Research Atlas Genome Cancer W.J.Gibson, human of Sensitivity H. Varmus, & J.E. Bradner, K., W.W.,Zejnullahu, Lockwood, Targeting H.G. Munshi, & F.D. Eckerdt, K.A., Collier, A.J., Redig, V., Sahai, S. Shu, M. Boi, P.Filippakopoulos, I.A. Asangani, Wang, C.Y. & Filippakopoulos, P. Beating the odds: BETs in disease. BET A.J. of Stonestrom, activity therapeutic the behind mechanisms The C.R. Vakoc, & J. Shi, J.E. Kwon, Blattner, M. h, X. Shi, R. Marcotte, A.M. Kurimchak, D. Robinson, castration-resistant prostate cancer. prostate castration-resistant Sci. Blood inhibition. bromodomain ligase. ubiquitin Cul3-based Chem. by ubiquitination for Daxx of adaptor an as regulationPI3K/mTORof signaling.andAR diversity among tumors from men with metastatic prostate cancer. prostate metastatic with men from tumors among diversity cancer. prostate metastasis. abdominopelvic and progression signaling proteins. epigenetic BET of inhibition targeted to lines cell adenocarcinoma lung tumors. solid in (2016). proteins bromodomain BET cancer. breast negative drugs. targeted Res. with Cancer Clin. synergizes and models tumor B-cell preclinical in pathways (2010). 1067–1073 E4558–E4566 (2016). E4558–E4566 TGF- and MYC- through resistance. and vulnerabilities, cancer. ovarian in reprogramming kinome 161 (2016). 369–378

, 1215–1228 (2015). 1215–1228 , 40

125 281 , 468–479 (2015). 468–479 , t al. et t al. et t al. et Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. , 2825–2834 (2015). 2825–2834 , , 12664–12672 (2006). 12664–12672 , et al. t al. et et al. et et al. et et al. et os f RM3 ass eitne o E boooan inhibitors bromodomain BET to resistance causes TRIM33 of Loss et al. et epne n rssac t BT rmdmi ihbtr i triple- in inhibitors bromodomain BET to resistance and Response h BT rmdmi ihbtr T05 fet pathogenetic affects OTX015 inhibitor bromodomain BET The

t al. et SPOP Cell BTB domain–containing speckle-type POZ protein (SPOP) serves (SPOP) protein POZ speckle-type domain–containing BTB usata itrniiul n lmtd nridvda genomic intraindividual limited and interindividual Substantial

et al. et t al. et The genomic landscape and evolution of endometrial carcinoma endometrial of evolution and landscape genomic The 21 Functional genomic landscape of human breast cancer drivers, cancer breast human of landscape genomic Functional Integrative clinical genomics of advanced prostate cancer. prostate advanced of genomics clinical Integrative et al. et

, 1628–1638 (2015). 1628–1638 , 163 mutation drives prostate tumorigenesis hrpui treig f E boooan rtis in proteins bromodomain BET of targeting Therapeutic Resistance to BET bromodomain inhibitors is mediated by mediated is inhibitors bromodomain BET to Resistance β ucin o BT rtis n rtri gn expression. gene erythroid in proteins BET of Functions Nature -dependent mechanisms. -dependent Selective inhibition of BET bromodomains. BET of inhibition Selective Mol. Cell Mol. , 1011–1025 (2015). 1011–1025 ,

Cell 529

54 , 413–417 (2016). 413–417 , 164 Nature

109 , 728–736 (2014). 728–736 , , 293–309 (2016). 293–309 , , 19408–19413 (2012). 19408–19413 , Cell Rep. Cell CancerCell

510 Nat. Genet. Nat. , 278–282 (2014). 278–282 , Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. Oncotarget

16

31 , 1273–1286 (2016). 1273–1286 ,

, 436–451, (2017). 48 in vivo s e l c i t r a , 848–855 (2016). 848–855 ,

7 through coordinate 53997–54009 , Trends Biochem. Nat. Med. Nat. Nature . Biol. J.

468 113

Cell 22  , , ,

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. by nanoflow-ultra-performance liquid chromatography (UPLC)–higher-energy completely using vacuum centrifugation. 50 with FAeluted and 50 with twice washed StageTips,on loaded FA. then were Peptides 50% acetonitrile (MeCN) /0.1% formic acid (FA) followed by 2 × 50 desalted using StageTips. StageTips were conditioned by washing with 50 50 × 2 with antibody the K- PBS. ice-cold with washes three by lowed IAP of ice-cold ml 1.5 fol buffer with were twice rotation.washed with Beads approximately 31 previouslydescribed as exactly pleted stituted in immunoaffinity purification (IAP) buffer, and enrichment was com to protein A beads using dimethyl pimelimidate (DMP) Technology, cat. no. 5562). Prior to enrichment, the antibody was cross-linked (K- Motif Remnant Ubiquitin PTMScan Pooled werefractions completelydried using vacuum centrifugation. ner such that every eighth fraction was combined to create eight final fractions. analysis, the remainder of each fraction was combined in a noncontiguous man that 24th was fraction every combined to create 24 final fractions. For K- 5% of was each fraction taken and combined in a noncontiguous manner such were collected across the entirety of the bRP separation. For proteome analysis, gradient and flow-rate settings previously described 5 Å, 300 mm, 250 × (9.4 umn ammonium formate (pH 10.0)/2% acetonitrile). A Zorbax 300 Extend-C18 col for replicate. Briefly, werepeptides dried reconstituted in bRP buffer A (5 mM equivalent to 30 mg of starting protein material (10 mg protein per SILAC state) described previously matography, as described as previously exactly (Waters) cartridges SPE SepPak tC18 using desalted were Peptides desalting. before centrifugation by cleared were solutions peptide the and (TFA), acid 1:50, overnight at 25 °C. Digestion reactions were quenched with trifluoroacetic ratio of using an enzyme-to-substrate trypsin with sequencing-grade digested were proteins and Tris-HCl, 8, mM pH 50 with 1:4 diluted Lysates were dark. at RT in the 10 with mM iodoacetamide ture alkylated (RT) and subsequently state. Proteins were reduced with 5 mM dithiothreitol (DTT) at room tempera SILAC per protein of amount equal combining by created were mixes SILAC Respective sample. each of concentration protein the determine to used was 20,000 at centrifugation Scientific) Fisher (Thermo assay protein (BCA) acid by A bicinchoninic min. 10 cleared was lysate The chloroacetamide. Science); 1 mM phenylmethylsulfonyl fluoride (PMSF); 50 EDTA; 2 lysis buffer containing 8 M urea; 50 mM Tris-HCl, pH 8; 150 mM NaCl; 1 mM pleted as previously described spectrometry. K- frozen. snap and harvested PBS, with twice washed were condition per cells million 100 Approximately experiments. three first the in artifacts labeling out rule to switched was line cell for this labeling The experiments. and across for within comparison line SPOP-WT cell of overexpression with line cell a included experiment Each with labeled ( experiments four into isotopically grouped and media SILAC were (MTs) mutants or (SPOP-WT) SPOP type proline with supplemented were [ and and and defi streptomycin medium penicillin, DMEM/F12 (Sigma-Aldrich), in cultured were in cient cells cancer endometrial culture. cell and labeling SILAC ONLINE METHODS nature medicine nature 13 Samples were reconstituted in 3% MeCN/0.1% FA. All samples were analyzed K- of enrichment For chro (bRP) reverse-phase pH basic by offline fractionated were Peptides  C -GG profiling and proteome analysis by liquid chromatography mass mass chromatography liquid by analysis proteome and profiling -GG 6 l l , -arginine (Arg-0) and and (Arg-0) -arginine 15 -lysine-4,4,5,5-d 3 N 7 l µ . Specifically, isogenic cell lines expressing vector control (C), wild- (C), control vector expressing lines cell isogenic . Specifically, 2 -arginine and and -arginine g/ml aprotinin (Sigma-Aldrich); 10 mg/ml leupeptin (Roche Applied ]HCl (Lys-8) (Sigma-Aldrich) for 14 d (10 doublings). All media media All doublings). (10 d 14 for (Sigma-Aldrich) (Lys-8) ]HCl Preparation of proteins for mass spectrometry analysis was com µ g of anti-K- µ 4 Ls4, r [ or (Lys-4), l of 50% MeCN/0.1% FA. Eluted peptides were dried dried were peptides FA. Eluted MeCN/0.1% 50% of l l µ -lysine and supplemented with 10% dialyzed FBS FBS dialyzed 10% with supplemented and -lysine l ε l of 0.15% trifluoroacetic acid (TFA). acid trifluoroacetic of l 0.15% Peptides were -proline to prevent the conversion of arginine to to arginine of conversion the prevent to -proline G ppie, anti-K- peptides, -GG 1 l ε 4 µ -lysine (Lys-0), (Lys-0), -lysine . Briefly, cell pellets were lysed in an ice-cold urea -GG antibody beads and incubated for 1 h at 4 °C 1 m; Agilent) was used for Agilent)separation.Using used m; was the 4 . For SILAC experiments, human Ishikawa Ishikawa human experiments, SILAC For 14 1 13 4 , 3 . Briefly, peptides were incubated with with wereBriefly,incubated . peptides 8 C . Input for each bRP separation was was separation bRP each for Input . ε 6 -GG) kit was used (Cell Signaling Signaling (Cell used was kit -GG) , 15 ε N -GG peptides were eluted from eluted were peptides -GG l 4 -arginine [ -arginine ]HCl (Arg-10) and and (Arg-10) ]HCl 1 l 4 ε , a total of 96 2-ml fractions -glutamine (Invitrogen), (Invitrogen), -glutamine Supplementary Fig. 1d Fig. Supplementary -GG antibody from the the from antibody -GG 1 4 µ . Peptides were recon M PR-619; and 1 mM 13 C 6 ]HCl (Arg-6) (Arg-6) ]HCl µ µ l of 0.1% 0.1% of l l l of 0.1% l -lysine -lysine ε g µ -GG -GG for for l l of ). ). ------

city was set to trypsin, the maximum number of missed cleavages was set to 2 to set was cleavages missed ofnumber maximum the trypsin, to set was city common laboratory contaminants was used for searching. The enzyme specifi (version 1.2.2.5) software package. The human UniProt database including 248 analysis. data spectrometry Mass SILAC state for experiment 4). for experiments 3 and 4 (6 mg per SILAC state for experiment 3 and 10 mg per A second round of bRP fractionation, K- enabled. were functions isotope-exclusion and peptide-match the and s, 20 to m used for the MS1 and MS2 scans, respectively. The isolation width was set to 2.5 tively, was used for acquisition. A maximum ion time of 10 ms and 120 ms was abundant ions. An MS1 and MS2 ion target of 3 × 10 ( scan MS1 an acquiring by operated was Exactive Q The min. 120 was proteomesample described viously pre as used were settings flow-rate and gradient the analyses, (LC–MS/MS) column heater (Pheonix ForS&T). liquid chromatography mass spectrometry 1.9- Å, tip, 50-cm length (New Objective) self-packed with 24 cm of ReproSil-Pur 120- ontoof nl/min 500 a PicoFrit column (360 system rate flow spectrometer.at a mass Samplesintoinjected the were injected were 1000 Easy-nLC an to online (Proxeon). For K- coupled Scientific) Fisher (Thermo collisional using(HCD)–MS/MS dissociation a massQ Exactive spectrometer Hs_FOSL1_3, Qiagen) or or Qiagen) Hs_FOSL1_3, nM 50 Qiagen), (Hs_SPOP_7, and 5% CO and penicillin-streptomycin 1% grown in RPMI medium (Gibco); all were supplemented with 10% FBS (Gibco), SNG-II cells and were grown in DMEM (Gibco); and HEC116 22Rv1 and EFE184 cells were AN3CA, HEC1B, HEC1A, EN, HEC-151, MEF-962, (Gibco); F12/DMEM in grownwere cells KLE and Ishikawa,RL95-2 HospitalZurich). (University Dedes K. and Samartzis E. byprovided kindly were cells KLE and EFE184 SNG-II, HEC116, 1B, HEC HEC1A, AN3CA, JCRB. from purchased were cells HEC-151 and DSMZ; from purchased were cells EN ATCC; from Sigma; RL95-2, 22Rv1, MEF-962, VM-CUB1 and NCI-H508 infection. and transfection cellsculture, Cell were purchased by −1 ( for ratios K- SILAC protein-normalized SPOP-WT, versus SPOP-MT of case the in expression protein total on effects opposing with paralleled were tides K- deregulated significantly the of which highlight and Toassess protein-normalized measurements were available for 17,239 K- accession number was used to match the protein and K- K- the normalized final data set if the corresponding protein was quantified in the proteome data. K- experiment. triple-labeled SILAC each andquantifiedidentifiedin uniquebyor morepeptidetwo per razorpeptides were they if set data analysis.Proteinsthe removedthe inincludedwere from from the “GlyGly(K)Sites” table. “proteinGroups” table. the For from obtained K- were ratios normalized data, proteome For enabled. were functions quantification peptide and acid amino filter-labeled The 1%. to set to 7, and the false discovery rate for peptide, protein and side identification was For variable modification. was set length minimumidentification, the peptide searched as variable modifications. tion, and oxidation of methionines and N-terminal acetylation of proteins were modifica fixed a as searched was cysteines of Carbamidomethylation search. ppm to6 for mainppmtoleranceset and to20 was search, the the for first the for proteome data and 4 for K- / ε For transient transfection, cells were transfected with 50 nM nM 50 with transfected were cells transfection, transient For To capture the ubiquitination changes associated with protein degradation, we K- For For K- z -GG were multiplied by their corresponding protein-level ratio and also also and ratio protein-level corresponding their by multiplied were -GG , the HCD collision energy was set to 25, the dynamic exclusion time was set µ Supplementary Supplementary Fig. 2 m C18-AQ beads). The nanoflow The C18-AQcolumnm heatedusing a was °C to 50 beads). ε ε -GG -GG data, addition of glycine-glycine to lysine was also searched as a G ad rtoe aa es rvre n cnaiat is were hits contaminant and reverse sets, data proteome and -GG R 2 . Cells . wereCells routinely tested for mycoplasma contamination. = 70,000) followed by MS/MS scans ( scans followedbyMS/MS 70,000) = ε 1 ε 4 -GG -GG and proteome samples, 4/8 -GG changes-GG proteinmeasured to their leading The levels. and the MS acquisition time used for each K- each for used time acquisition MS the and ε siControl and -GG data, normalized SILAC ratios were obtained ε -GG -GG data, the precursor mass tolerance was set l siFOSL1 -glutamate. All cells were incubated at 37 °C °C at37 wereincubated cells -glutamate.All Supplementary Supplementary Table 1 Data were processed using the MaxQuant the using processed were Data (Qiagen) using Fugene (Promega). (Promega). Fugene using (Qiagen) ε Ishikawa cells were purchased from purchased were Ishikawacells -GG -GG and MS analysis was completed ε (Hs_FOSL1_1, Hs_FOSL1_2 and and Hs_FOSL1_2 (Hs_FOSL1_1, -GG peptides were included in the the in included were peptides -GG µ m m (OD) × 75 R µ 6 = 17,500) on the 12 most 12 onthe 17,500) = l l and 1/20 and 5 × 10 ε -GG data. Quantitative, doi: ). µ m (ID), 10- m (ID), 10.1038/nm.4372 µ ε -GG -GG peptides. 4 l, respectively, ions, respec ε ε -GG pep -GG -GG and -GG siSPOP µ m m ID - - - - -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. horseradish horseradish peroxidase–conjugated antibody secondary (Promega). with TBS-T, incubation bywith followed washed was membrane The °C. 4 at overnight antibody primary the with incubation by followed buffer blocking The Scientific). membrane was incubated for 1 h in 5% nonfat milk/TBS-T dry and transferred Rad) onto (PVDF) membrane difluoride a polyvinyl (Thermo SDS–PAGE(Bio- by separated were proteins Fisher); (Thermo reagent BCA RIPA buffer (Sigma) and sonicated. Protein concentration was determined using the manufacturers. were employedantibodies All at Signaling). dilutions by suggested Cell (9661, (Asp175) caspase-3 anti-cleaved and Sigma) (9658, anti-HA Signaling), Cell anti- Signaling), Cell anti-FOSL1 (4650, anti-VCL Cruz), Signaling), Santa Cell anti-DEK (5281, (sc-271048, Signaling), anti-ERG Cell Bioscience), BD (2126, (610948, anti-NCOA3 Cruz), Santa (sc-271266, anti-TRIM24 Abcam), (ab81163, anti-SPOP Cruz), Santa anti- (sc-48772, Cruz), BRD4 Santa (sc-81202, anti-BRD3 Labs), Bethyl anti- (A302-583A, follows: BRD2 as were assays immunoprecipitation and immunoblotting in immunoprecipitation. and immunoblotting Antibodies, Signaling protocol). (J.P.T.) by H&E staining and immunohistochemistry for cleaved caspase-3 (Cell extracted, weighed and histologically analyzed by a pathologist board-certified were tumors and euthanized were mice experiment, the of end tumor. the At ( formula the using growth was recorded using digital calipers, and tumor volumes were calculated or JQ1 at 50 mg/kg bodyweight (twice per day) for the indicated duration. mice were randomized and wereTumor intraperitoneally administrated either vehicle of the dorsal flanks of nude mice. Once tumors reached approximately 100 mm each were resuspended in 200 the Swiss Veterinary Authority (no. TI-14-2014). 2 × 10 nude mice (BALB/c nu/nu, aged 4–6 weeks) according to a protocol approved model. Xenograft by microplate reader. a in measured was nm) (OD, 590 absorbance and overnight °C 37 at solution CO 5% and °C 37 at incubated were Cells (JQ1). drug or DMSO) (0.1% vehicle with treated duplicateand in 10 × 1 assay Clonogenic in methylcellulose. was measured in a microplate reader. violet was then solubilized with 10% acetic acid and absorbance (OD, 590 nm) with PBS and stained with 0.5% crystal violet solution in 25% methanol. Crystal the case of dose-response curves and 6 d for cell-growth assays cells were washedofuntreated h treatment or were96 left in forassays. After cell-growth curves dose-response to determine media in dilutions of or JQ1 OTX-015 serial with 1 × 10 assays. cell-growth and curves Dose-response Mlu1 and Scientific). Fisher Nhe1 (Thermo using pLX_TRC_307–mOrange into cloned were (ORFs) frames reading open All pCW107). 41392, plasmid as Addgene at available exchanged with mOrange or a puromycin-resistance cassette (pLX_TRC_307, cassette a and blastocidin with promoter the PGK exchanged been had moter pro CMV the which in throughout, used was vector pLX304 the of derivate (2 puromycin of presence the in selected were ( ( TRCN0000194983 and and TRCN0000013105 ( ( TRCN0000021426 BRD3 3_1 (5 POP used: were clones following the and (Sigma) vectors pLKO-1 with infected were cells experiments, knockdown stable For doi: shNCOA3_1 ′ For immunoblotting, cells were washed with PBS and subsequently in lysed -TCCAACTGCTATAAGTACAAT-3 ) and TRCN0000073344 ( 10.1038/nm.4372 ); : TRCN0000021376 ( 3 BRD2 4 and 1 × 10 cells) in methylcellulose (Methocult H4100, StemCell Technologies) StemCell H4100, (Methocult methylcellulose in cells) : TRCN00000315433 ( ) and TRCN0000365253 ( TRCN0000365253 and ) 4 cells/well) in a 96-well plate. Cells were subsequently treated All animal experiments were carried outwere animal experiments All carried in female athymic L × × shBRD4_1 W shDEK_2 2 )/2, where where )/2, shBRD3_1 shCUL-3_2 shTRIM24_2 µ 2 ), TRCN0000021427 ( TRCN0000021427 ), l l of PBS and subcutaneously injected into both for 7–14 d, colonies were stained with MTT MTT with stained were colonies d, 7–14 for ); shBRD2_1 TRIM24 ), TRCN0000021377 ( L Cells wereCells 5 (between × seeded 10 is the length and length the is ′ ); ); ); shNCOA3_2 DEK CUL-3 ); ); : TRCN0000021259 ( ), TRCN0000350530 ( SPOP NCOA3 : TRCN0000013104 ( µ Cells were seeded (between (between seeded were Cells : TRCN0000073343 : ( TRCN0000073343 g/ml). For overexpression, a overexpression, For g/ml). : TRCN0000140431 ( TRCN0000140431 : ). After infection, cells cells infection, After ). shBRD4_2 6 : TRCN0000370320 TRCN0000370320 : Ishikawa and EN cells W shBRD3_2 is the width of the the of width the is Antibodies used used Antibodies β shTRIM24_# -actin (4967, (4967, -actin ) and and ) shBRD2_2 shDEK_1 ); shCUL- shBET BRD4 3 and shS 3 ); - ) ) - : ,

overnight at 4 °C. Next, Ni-NTA beads were washed once with Buffer C, C, Buffer with once washed were beads Ni-NTA Next, °C. 4 at overnight 60 with incubated and sonicated was WCE M Na M (6 C 0.1 Buffer in guanidine–HCl, lysed were rest the and buffer RIPA in lysed were cells of amount A small Cells by centrifugation. h. collected and 3 PBS with washed then were additional an for DMSO or MG132 with treated were cells later, neddylated CUL3 and RBX1 were purified in a preassembled complex from insect cells and cloned, expressed and from purified tag affinity MBP an of instead species were cloned, expressed and purified as described previously,SPOP humanmutant and usingWild-type cells. a GSTHEK-293T transfected transiently mutant (degron-MT) constructs of HA-tagged human BRD3 were purified from vitro In immunoblotting. by detected and SDS–PAGE by separated loaded, were Samples contain imidazole. mM buffer 300 ing loading SDS 1× in boiling through processed was proteins of E Elution bound pH (25 mM Buffer 6.8). with Tris-HCl, once 20 mM imidazole; and E) Buffer of volumes C:3 Buffer of volume (1 D Buffer with twice pCW107-SPOP-WT or SPOP-MT (2 (2 SPOP-MT or (2 pCW107-SPOP-WT BRD3-degron-MT or pCW107-BRD3-WT plasmids: indicated the vivo In 015 were purchased from Selleckchem and used at the indicated concentrations. used at 10 Chemicals. presented in are immunoblots Uncropped expression. ACTB or VCL to normalized levels protein with performed was blots western the of analysis Quantitative min. 5 for °C 95 at conditions reducing under buffer sample SDS 1× of addition by 2 h, followed by two washing steps with 1% NP-40 buffer. Proteins were eluted collected using 25 IgG (sc-2025, Santa Cruz Biotechnology) at 4 °C. Subsequently, antibodies were 2 with overnight inhibitor cocktail (Complete, Roche) and sonicated; 3 mg of lysateprotease 2× NP-40) with 1% NaCl; mM was 150 Tris-HCl, 7.4; mM pH (50 incubated buffer primers were used: used: were primers method Ct ( comparative the using quantified was expression mRNA Target System. StepOnePlus Biosystems Applied an on undertaken was qPCR instructions. by the Kapa SybrFAST One-Step qRT–PCR kit according to the manufacturer’s qRT–PCR. anti-HA and anti-SPOP antibodies above).(see were separated by SDS–PAGE and visualized through chemiluminescence using at h 1 Thereafter, °C. 4 and buffer same samples the in twice resinwashed was 200 1 and mutants SPOP or WT (Bio-Rad, 1705061) and Fusion FX imaging platform (Vilber Lourmat). WesternSubstrateBlotting Clarity ECL 170-6516), (Bio-Rad, IgGanti-mouse through chemiluminescence using anti-HA (Sigma, A2095), HRP-coupled goat tion of SDS sample buffer. Samples were separated by SDS–PAGE and visualized 0.5 mM DTT). Reactions were incubated at 37 °C for 45 in min ubiquitinationand stopped buffer by (3 addi mM ATP, 10 mM MgCl Cdc34; 0.2 0.2 KLHL21; or KLHL13 KLHL9, SPOP, tive samples, 2 with 10 TCEP; 1× Roche protease inhibitor cocktail). Immunoprecipitates were Tris-HCl, mM (20 glycerol;mM preparedbufferNaCl; 1 5% IP mM in 150 tion 7.4; pH 10 ubiquitination reactions with a total volume of 15 7 To detect interactions of SPOP and HA-BRD3, cells were lysed in 1% NP-40 For coimmunoprecipitation experiments HA-BRD3-expressing HEK-293T cells were harvested and lysed by sonica µ l of IP buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5% glycerol) for glycerol) 5% NaCl, mM 150 7.4, Tris-HCl pH mM (20 buffer IP of l ubiquitination assay. ubiquitination µ ubiquitination and binding assays. binding and ubiquitination ∆ l of anti-HA affinity gel (Sigma) and washed with IP buffer. For respec µ ∆ µ RNA was extracted using the RNeasy kit (Qiagen) and processed processed and (Qiagen) kit RNeasy the using extracted was RNA in vitro MG132 and cycloheximide (CHX) were purchased from Sigma and M and 100 Ct method) and normalized to cyclophilin expression. The following M UbE1 (Boston Biochem); and 25 Supplementary Supplementary Figure 11 µ l of HA-BRD3 IP resin was used and supplemented with 0.3 µ µ using purified components as described previously g of anti-HA-tag antibody (9658, Sigma) or control mouse mouse control or Sigma) (9658, anti-HA-tagof antibody g l of Protein A/G magnetic beads (88803, Fisher Scientific) for BRD2 µ g/ml, respectively, in all experiments. (+)-JQ1 and OTX- frad 5-CTACGTAAGAAACCCCGGAAG-3, forward , 2 HPO µ 7 l of HA-BRD3-WT IP resin were incubated in in incubated were resin IP HA-BRD3-WT of l 293T cells were transiently transfected with with transfected transiently were cells 293T . KLHL9, KLHL13, KLHL21 and Cdc34b were Cdc34b and KLHL21 KLHL13, KLHL9, . 4 /NaH Escherichia coli µ . 2 g), and CMV-8×His-Ub (2 (2 CMV-8×His-Ub and g), PO µ in vitro 4 M CUL3–Nedd8/RBX1; 0.7 0.7 CUL3–Nedd8/RBX1; M , 10 mM imidazole; pH 8). The The 8). pH imidazole; mM 10 , Wild-type and SPOP-binding- and Wild-type µ µ l of Ni-NTA agarose (Sigma) (Sigma) agarose Ni-NTA of l M ubiquitin (BostonBiochem) 2 , 50 mM Tris-HCl pH 7.6, and µ l were assembled as follows: , , 1 as described previously as described µ M M recombinant SPOP- nature medicine nature 4 µ 0 . g). 42 h 42 g). In vitro µ µ µ g), g), 3 M M 9 - - - - .

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. rm w dfeet oot fo Bsl n Zurich and Basel from cohorts carcinosarcomas) 11 different and two carcinomas from cell clear carcinomas, 11 endometrioid carcinomas, (36 serous 26 sequencing DNA targeted by samples sue 119, 127 and 136 from the Oregon Health & UniversityScience (OHSU) and1090076AT Human Cooperative from the Tissue Network (CHTN) Cancer Center University Center University HawaiiCancer of the Center Cancer Duke the TCGA-NA-A5I1 from and Clinic Mayo the from TCGA-D1-A17D collected as follows: TCGA-D1-A0ZO, TCGA-D1-A167, TCGA-D1-A168 and recurrent annotated with literature the from identified cancer tissues were retrieved from different sources. Tissue endometrial sections Primary StV-Nr.no.of (approval 25/2007). 19cohorts tumorsboth approved Germany Regensburg, of University Pathology, of Institute the and Switzerland, Zurich, of University Pathology, specimens were collected between 1993 and 2007 from the Institute of Surgical tissue cores from two different institutes of pathology. As previously published, These analyzable. tumors histologically are a was part of tumors tissue of microarrays composed subset of a paraffin-embedded only prostate microarrays), (ref. 7 and 6 exonsof sequencing Sanger followed that assay analysis meltresolution annotated with tumors prostate mary samples. tumor Human Benjamini–Hochberg test); IIogFCI > 0.9. adjusted samples; two least at in genes: 45 > c.p.m.expressed differentially define to used were filters following the and in samples comparing by derived were Signatures Bioconductor. in package limma the of function voom the to subjected and normalized based on the number of reads acquired per sample, log were considered not expressed and were filtered out. Subsequently, counts were reads mapped <0.5 (c.p.m.) million per countsSTAR with using Genes 2.52b. done Mapping was FastQC. hg38 and to Illumina of score Phred to according v2,1 × 75 bp) was performed by Microsynth. Quality of sequencing was analyzed ing poly(A) enrichment) and RNA sequencing (Illumina NextSeq high output, Bioanalyzer. Library preparation (Illumina unstranded TruSeq Library includ using the RNeasy kit (Qiagen), and sample quality was assessed using the Agilent (500 JQ1 with treated or untreated either cells on performed RNA-seq was erated. gen were W131G) Y87C, prostate, E47K; E50K, mutant(endometrial, SPOP an SPOP-WTor overexpressing either stably cells Ishikawa mutants,isogenic with the genes expressed in human tumor tissues. lines cell the in overlaygenes robustlyofexpressed the determine to used was SPOP recurrent with tissues cancer endometrial of values RPKM median the with script per million mapped reads (RPKM) values for each cell line were correlated expression levels were determined using RNA-SeQC. Reads per kilobase of tran the human reference genome build hg19 using TopHat version 1.4, and mRNA using the Illumina TruSeq protocol for 17 CCLE cell lines. Reads were aligned to InstituteBroad atthe study.performed this was RNA sequencing addition, In mutation to determine used was data set status for SPOP included in lines cell was performed for all endometrial cancer cell lines profiled in the CCLE DNA and RNA sequencing of endometrial cell lines. ATCTTGTCC-3, reverse 5-TTGCTGGTCTTGCCATTCCT-3.GATTTTGCAGATGGG-3; cyclophilin, ( FOSL1 5-GCCCGAACTTCACTCTTTGGA-3; reverse TGTTTGCGAGTAAACC-3, reverse5-GTGTTGGGAACTTGGATAACTGG-3; TTTGACTTCGGA-3; 5-CTCCTCCTAAAAAGACGAAGA-3,forward 5-GCCCCTTCTCTT reverse 5-ATGTCGTGGTAGTCGTGCAG-3; reverse AGATGCTATCCA-3, 5-GCTTTTTCTCCAAAGCCAGTT-3;reverse nature medicine nature e hrceie a adtoa 8 piay noera cne tis cancer endometrial primary 84 additional an characterized We SPOP to response in changes output transcriptional of analysis the For µ point mutations identified in the TCGA portal M M or 2 3 frad -TCGCGGCGCA3 rvre 5-TCCGG reverse 5-CTGCAGGCGGAGACTGACAA-3, forward , ). Owing to tissue loss (a common problem encountered with tissue tissue with encountered problem common (a loss tissue to Owing ). 6 ; TCGA-N9-A4Q8 and TCGA-DI-A1NN from the MD Anderson Anderson MD the from TCGA-DI-A1NN and TCGA-N9-A4Q8 ; µ 6 ; 2001-02-G049T, 2005-08-G674T, MAD04-00646T, 1090095AT M) M) for 4 h ( TRIM24 We used a previously characterized cohort of pri of cohort characterized previously a used We Supplementary TableSupplementary 2 , forward 5-CAGCCACAAATGCCTAAGCAG-3, 4 2 . The local scientific ethics committees committees ethics scientific local The . SPOP 6 ; TCGA-FI-A2EW from WashingtonfromTCGA-FI-A2EW ; 6 PPIA ; TCGA-B5-A0JY, TCGA-B5-A0K0 TCGA-B5-A0JY,TCGA-B5-A0K0 ; mutations determined by high- by determined mutations P , owr 5CG TCCTGGC 5-CAGG forward ), value < 0.05 (according to the the to (according 0.05 < value BRD3 SPOP ). ). Total RNA was extracted 6 Whole-exome sequencing 6 forward 5-CCTCAGGG, . A threshold of 10 RPKM upeetr Tbe 2 Table Supplementary ; TCGA-BS-A0UT from TCGA-BS-A0UT ; ,forward 5-GAAATGG 43– SPOP 4 5 . Therefore, two two Therefore, . mutations were mutations 2 transformed 4 BRD4 1 5 4 . This . ; and ; - - - - - ,

(approval no. KEK-ZH-NR: 2010-0358). no. KEK-ZH-NR: (approval Zurich Hospital University Pathology, Surgical of Institute the at committee higher than fraction 0.2. allelic The study was approved by ethics the scientific in mutation of type any other nor mutations recurrent additional any harbor not did cases remaining the while cancer, serous D140Y-mutated SPOP- another Weidentified Technologies). Scientific/Life Software Fisher (Thermo Reporter Ion with analyzed then were data Sequencing run. each for Technologies) Scientific/Life Fisher (Thermo Software TorrentSuite the Fisher (Thermo Technologies).Kit Scientific/Life run Sequencing quality were metrics taken 200 from Sequencing Hi-Q PI Ion the using System Ion Proton the on sequenced and Technologies) Scientific/Life Chip Fisher PI (Thermo Ion v3 an on loaded then were samples Enriched Technologies). Life Scientific/ Fisher (Thermo Kit 200 OT2 Hi-Q PI Ion the using enrichment and for preparation template were multiplexed Twentylibraries to twenty-six the Ion Library Quantitation Kit (Thermo Fisher Scientific/Life Technologies). Kit (Thermo Fisher Technologies)Scientific/Life and quantified by qPCR with SPOP of the amplification for PCR-based applied was panel A custom instructions. manufacturer’s the to according 2.0 Kit Library Ion AmpliSeq using libraries prepare to 2.0 used was ng 20 and Technologies), Qubit with Scientific/Life Fisher Kit (Thermo Assay HS dsDNA the using quantified was DNA tions. LEV DNA Purification Kit (Promega) according Tissue to FFPE the manufacturer’s16 Maxwell instruc the using isolated was DNA and blocks, tissue ded 0.6-mm-diameter tumor tissue cylinders were punched out of paraffin-embed s availability. Data nohistochemical staining with endometrial cell line data. Kendall’s tau-b was used to test correlation of immu expression data from RNA-seqcompare to used was coefficient correlation Spearman The curves. squares ANOVA (Dunnett’s post test) was used for multiple comparisons. Extra-sum-of-ance assumption to analyze culture Two-waywas cell performed experiments. Student’s independent two-tailed unpaired, An fied. means as depicted are Data analysis. for used analysis. Statistical nuclei strongly stained (invisible nucleoli) as strong. cell nuclei weakly stained (discernable nucleoli) as weak and more than 30% of morenegative,tumoror as 30% referred tonuclei was tumorcell of 70% than more in staining detectable proteins:no BET forfollows as evaluated was ing staining with Mayer hematoxylin (Diapath, C0303). Immunohistochemicalthe ImmPACT stain DAB system (Vector, SK-4105) forVectastin 4 minABC kitat at RTa dilutionfollowed of by1:150 in nuclearPBS. Detections were performed for using30 min at RT and subsequently washed and incubated another 30 min with washed and incubated with biotinylated anti-rabbit IgG (Vector,tissue, the BRD2BA-1000) antibody was used in at PBS a dilution of 1:200. Thereafter, slides were FOSL1(Sigma-Aldrich, AV31377; 1:2,000). For microarrays of prostate cancer 1:500), BRD3 (Bethyl, A302-368A; 1:50), BRD4 (Abcam, ab128874; 1:400) and diluentreagent solution Technologies,(Life (Abcam,BRD2003118): ab13960; bated with primary antibodies at the following concentrations for 1 h 10 inmin with antibodya protein blocking solution (Dako, X0909). Then, slides were incu min with 3% H endogenousadditional10 andforanperoxidaseswereblocked min RT for20 and FOSL1 was extended to 40 min in total. Subsequently, slides were cooled to T0050). For microarrays of prostate cancer tissue, the antigen retrieval for BRD2 at 98 °C for 20 min using a citrate buffer atwere pHfirst 6dehydrated. (BETs) For antigenor retrieval,pH slides9 (FOSL1)were incubated (Diapath,in a water bath Immunohistochemistry. sible under BioProject Sequencing data have been deposited in the NCBI “MSV000080401.”public username database the andprovide are also acces “ubiquitin.”requested, word If ftp://[email protected] at accessible are and MassIVE repository proteomics public the in deposited t i t u t e gene. Libraries were labeled with the with Ion were labeled Xpress gene. Adaptors Libraries Barcode 1-96 . o F r -test was used to determine the statistical significance of dose-response g / c c l e 2 / O h o 2 CE aa r aalbe nie at online available are data CCLE m (VWR, 23615.248). After washing, slides were incubated for GraphPad Prism version 7.00 (GraphPad Software) was was Software) (GraphPad 7.00 version Prism GraphPad e SPOP . The original mass spectrometry spectra have been been have spectra spectrometry mass original The . P R ForandFOSL1ofBETproteins,detection the slides J N -mutant human tumors and BET protein levels with A 3 SPOP 5 7 9 4 2 mutation status. . hn rvdn te aa e pass set data the providing when ± s.e.m. unless otherwise speci otherwise unless s.e.m. t -test with unequal vari unequal with -test h doi: t t p 10.1038/nm.4372 : / / w w SPOP w . b r o a at an d i n ------© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. 41. 40. 39. 38. 37. doi:

Barretina, J. Barretina, R. Mosadeghi, R.I. Enchev, P.Mertins, S.C. Bendall, of anticancer drug sensitivity. drug anticancer of cycle. deneddylation ligase ubiquitin (2016). e12102 cullin-RING the and activation CSN. enrichment. serial by cells. stem embryonic 10.1038/nm.4372 Cell Rep. Cell et al. et et al. et t al. et et al. et

2 t al. et Integrated proteomic analysis of post-translational modifications post-translational of analysis proteomic Integrated , 616–627 (2012). 616–627 , The Cancer Cell Line Encyclopedia enables predictive modelling predictive enables Encyclopedia Line Cell Cancer The Prevention of amino acid conversion in SILAC experiments with experiments SILAC in conversion acid amino of Prevention tutrl ai fr rcpoa rglto bten C and SCF between regulation reciprocal a for basis Structural tutrl n kntc nlss f h COP9-signalosome the of analysis kinetic and Structural Nat. Methods Nat. Mol. Cell. Proteomics Cell. Mol. Nature

10

483 , 634–637 (2013). 634–637 , , 603–607 (2012). 603–607 ,

7 , 1587–1597 (2008). 1587–1597 , eLife

5 , 45. 44. 43. 42.

Dellas, A., Jundt, G., Sartorius, G., Schneider, M. & Moch, H. Combined PTEN and PTEN Combined H. Schneider,Moch, G., & Sartorius, M. G., Jundt, A., Dellas, Wild, P.J. A. Lebeau, A. Mortezavi, endometrial carcinomas. endometrial p27 (2012). humans. in aggressiveness tumour endometrial lesions.precursorits endometrialandcarcinoma prostatectomy. radical (2011). 1111–1121 after recurrence biochemical kip1 rti epeso pten ae soitd ih bst ad rgoi in prognosis and obesity with associated are patterns expression protein et al. t al. et t al. et p53 suppresses type II endometrial carcinomas in mice and governs etoe rcpo gn ( gene receptor Oestrogen PA epeso i a idpnet des peitr of predictor adverse independent an is expression KPNA2 Clin. Cancer Res. Cancer Clin.

15 , 2456–2462 (2009). 2456–2462 , ESR1 J. Pathol. J. MO o. Med. Mol. EMBO ) amplification is frequent in frequent is amplification ) ln Cne Res. Cancer Clin.

nature medicine nature 216 , 151–157 (2008).151–157 ,

4 808–824 ,

17 ,