Table of Contents Page - Supplemental FigureS1:Transcriptomeandtranslatomeanalysis of ENZ-sensitive 1 andr esistant cell lines - Supplemental FigureS2: Significant enrichment of sets in VCaPER and 3 VCaPCRPC transcriptomes andt ranslatomes - Supplemental Figure S3: The VCaPER ENZ-resistance model recapitulates established 5 features of drug-resistant PCa models. - Supplemental FigureS4: up- and downregulated in VCaPER assemble in 7 networks - Supplemental Figure S5: Genomicalterations in VCaPER-specificproteins are 9 linked to PCadrug resistance in patients - Supplemental Figure S6: Genomicalterations arenotalways linked to RNA expression 11 for coding for differentially expressed proteins in VCaPER - Supplemental FigureS7: HighTE ratio RNAs recapitulatekey GO-terms from the 13 translatome in VCaPER, butdonotnecessarilylead to higher protein expression. - Supplemental Figure S8: Genomicalteration inp roteins upregulated in VCaPER is 15 linked to high PCag rade, independentlyof TE ratio. - Supplemental Figure S9: Genomicalterationsforfrom VCaPER differentially expressed proteins withdifferentialTE ratiodonot always correlatewith altered mRNA 17 expression. - Supplemental Figure S10: NUDT19 expression correlates with determinants of high 19 grade and resistant PCa. - Supplemental Figure S11: lncRNAs are more associated withribosomes than expected 21 in VCaPER. - Supplemental FigureS12: lncRNAs with high TEr atiosin VCaPER are linked to 23 higherPCagrade. - Supplemental FigureS13: Genomic alterationsare nota lways linked to changes in 25 lncRNA expression. - Supplemental FigureS14: Candidate lncRNAs exhibit alternative splicing eventsin 27 PCa ENZ resistance. - Supplemental_Table_S1: Totala nd polyribosome RNAseq data - CPM values and Supplemental file EdgeR differentialE xpression - Supplemental_Table_S2: Protein abundances from massspectrometry (M.S.) and Supplemental file differentialexpression analysis - Supplemental_Table_S3: DAVID 6.8 gene ontologyterm enrichment analysis for Supplemental file Figures2Cet S2A - Supplemental_Table_S4: GSEA 4.1.0. Analysis for TOTAL RNA and RNAs highly Supplemental file boundt o in VCaPER or VCaPCRPC for Figures 2D and S2B-F - Supplemental_Table_S5: String 11.0a nd Cytoscape 3.11.1 GO termsanalysis for Supplemental file proteins differentially expressed for Figure 3B,C andS4A-C - Supplemental_Table_S6: GSEA 4.1.0. analysisfor whole proteomein VCaPER versus Supplemental file VCaPCRPC for Figure 3B - Supplemental_Table_S7: efficiency analysisfor VCaPERa nd VCaPCRPC Supplemental file - Supplemental_Table_S8: DAVID 6.8 gene ontologyterm enrichment analysis for Supplemental file Figures 4A,B and S7A - Supplemental_Table_S9: List ofputative peptides from non-coding genes inF igure5BSuppl emental file

1 Supplemental Figure S1

A VCaPCRPC VCaPCRPC B VCaPER VCaPER

M M 60S 60S 60S M 60S M 40S 40S 40S 40S LP HP LP HP LP HP LP HP Absorbance (254 nm) Absorbance (254 nm) 15% 55% 15% 55% 15% 55% 15% 55% Sucrose sedimentation Sucrose sedimentation

C VCaPCRPC ρ = 0.9571 D VCaPER ρ = 0.953 p-value = <0.0001 p-value = <0.0001 15 15

10 10

5 5 -10 -5 -10 -5 5 10 15 5 10 15 log2 (CPM translatome) -5 log2 (CPM translatome) -5

-10 -10 log2 (CPM transcriptome) log2 (CPM transcriptome)

Figure S1: Transcriptome and translatomea nalysis of ENZ-sensitive and -resistant cell lines.( A)

Polysome profiling performed in VCaPCRPC and (B)VCaP ER. Peaks show ribosomal subunits( 40Sa nd

60S), mono-ribosomes (M), light (LP) and heavy polysomes (HP), distributed along a sucrose sedimentationg radient. (C) Correlation of total mRNA levels with RNA association to ribosomes in

VCaPCRPC and (D) VCaPER cell lines. Pearson correlation coefficients (ρ) and linear regression (grey lines) are indicated. CRPC: Castration-Resistant Prostate Cancer, ER: Enzalutamide-Resistant.

2 Supplemental Figure S2

A Upregulated in VCaPER Upregulated in VCaPCRPC

Translatome -log10 (p-value) Transcriptome 12 10 8 6 420 2 4 6 8 GOBP Regulation of transcription* Angiogenesis Transcription* Response to retinoic acid Metal ion binding (+) regulation of transcription* DNA binding (-) regulation of cell proliferation Nucleic acid binding Regulation of cell proliferation GOBP Poly(A) RNA binding Rho protein signal transduction Transcription factor activity* (-) regulation of transcription* GOMF Protein binding Heart development RNA pol II core promoter* Palate development FGF-activated receptor activity Protein binding Calcium ion binding Myosin binding GOMF Heparin binding Protein binding Glucuronosyltransferase activity

B Gene sets enriched in total RNA VCaPCRPC C Gene sets enriched in polysomes VCaPCRPC Homophilic cell Cell-cell adhesion via adhesion via Microtubule organizing plasma membrane plasma membrane center organization adhesion molecules adhesion molecules

Rank Rank Rank Rank

D Gene sets enriched in total RNA VCaPER E Gene sets enriched in polysomes VCaPER ATPase-coupled Regulation of transcription ion transmembrane elongation from RNA RNA polymerase Desmosome transporter activity polymerase II promoter core enzyme binding

Rank Rank Rank Rank

F Gene sets enriched in VCaPCRPCtranscriptome Gene sets enriched in VCaPERtranscriptome ρ = 0.457, p-value = 6.7e-07 ρ = 0.489, p-value = 1.5e-17

3 Microtubule organizing 3 Desmosome 2.5 center organization 2.5 2 2 1.5 1.5 Cell-cell adhesion via 1 plasma membrane 1 ATPase-coupled 0.5 molecules 0.5 ion transmembrane transporter activity 0 0 -log2 (Translatome ES) 0.5 1 1.5 2 2.5 3 -log2 (Translatome ES) 0.5 1 1.5 2 2.5 3 -log2 (Transcriptome ES) -log2 (Transcriptome ES)

3 Figure S2: Significant enrichment of gene sets in VCaPER and VCaPCRPC transcriptomes and translatomes. (A) GO-term enrichment analysis showing top10 significant biological process (GOBP) and molecular function(GOMF) terms enriched inV CaPER or VCaPCRPC. *Full names of GO terms: regulation of transcription - DNA-templated, transcription-DNA-templated, transcription factor activity - sequence- specificDNA binding, RNA polymerase II core promoter proximal region sequence-specific DNA binding, positive regulation of transcription from RNA polymeraseI I promoter, negative regulation of transcription from RNA polymeraseI I promoter.( B) GSEA enrichment plots for selected gene sets in VCaPCRPC total

RNA,(C) VCaPCRPC poly-ribosomal fractions,( D) VCaPER total RNA and( E) VCaPER poly-ribosomal fractions.(F) S catterplots of gene set enrichment analysis (GSEA) enrichment scores (ES) for gene sets upregulated inVCaPCRPC(l eft)andVCaPER (right) transcriptome. ES scores in VCaPCRPC (blue) or VCaPER

(red) translatomes andt ranscriptomes are plotted. Spearman correlation coefficients (ρ) and linear regressions (black lines)are indicated.

4 Supplemental Figure S3

A Proteins downregulated in VCaPER B Proteins downregulated in VCaPER Proteins upregulated in VCaPER Proteins upregulated in VCaPER

16 16 14 14 SNRNP70 12 MRPS6 12 LIN7A 10 ADAR 10 EIF4B 8 ACSL5 8 RPS29

-log2 (p-value) 4EBP1 -log2 (p-value)

-3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 log2 (Fold change of protein expression VCaPER vs VCaPCRPC) log2 (Fold change of protein expression VCaPER vs VCaP)

C Proteins downregulated in VCaPER Proteins upregulated in VCaPER

16 14 12 10 8 -log2 (p-value)

-3 -2 -1 0 1 2 3 log2 (Fold change of protein expression VCaPCRPCvs VCaP)

D Proteins upregulated in VCaPER E Proteins downregulated in VCaPER Protein abundance VCaPER vs MR49F Protein abundance VCaPCRPC vs MR49F

18 ρ = 0.935 18 ρ = 0.936 p-value = 8.8E-25 p-value = 1.1E-14 16 p-value = <0.0001 16 p-value = <0.0001 14 14 12 12 10 10 8 8 6 6 4 4 2 2 0 0 log2 (Protein abundance in MR49F) 0 2 4 6 8 10 12 14 16 180.15 log2 (Protein abundance in MR49F) 0 2 4 6 8 10 12 14 16 18 0.15 Log2 (Protein abundance VCapER) Log2 (Protein abundance VCaPCRPC)

5 Figure S3: The VCaPER ENZ-resistance model recapitulates established features of drug-resistant

PCa models. (A) Volcano plots showing proteinsd ifferentially expressed between VCaPER and VCaPCRPC,

(B) VCaPER and VCaP,o r (C) VCaPCRPC and VCaP. Upregulated (red) or downregulated (blue)proteins in

VCaPER compared to VCaPCRPCare indicated.P roteins of interest are highlighted.(D ) Spearman correlation ofprotein abundances between ENZ-resistant MR49F and VCaPER or (E) VCaPCRPC. Density plots show

MR49F expression of proteins (D) upor (E) downregulated in VCaPER.

6 Supplemental Figure S4

A Network map of proteins upregulated in VCaPER B Network map of proteins downregulated in VCaPER

ANXA2 CREG1 GUSB BUD31 SNRPD2 SNRNP40

PYGB GGH SF3A1 PDIA6 FBLN2 PTGES2 ELAVL2HNRNPC IGFBP3 PKP3 DSP FAF2 PDXK AMACR PFDN5 STMN1 PPP1CA PPP1CB VCL VTN HSP90AA1 PRKAR2A RPF2 TXN2 RAB9A ERP44 CTSD SF3A3 DKC1 APOE SDC4 ATP6V1E1 PFKL IDH1 CSK LPP PFKM PFDN4 PFDN1KPNB1 CCT8 TOLLIP MYLK ARFGEF2 PTGES3 CLU A HSP90AB1 NOP2 PRDX3 RAB8A CTSH CD47 THBS1 ATP6AP1 PGRMC1 XPO1 PGM1 CCT3 CCT7 TLE3 TUBB TPM1 NDEL1 ASAH1 PRPS2 TUBA4 TUBA1A ACTN1 MYL6B

UTP18 MTX2 EXOSC6 STUB1 SUCLG2 KRT8

ASB9 MUT WDR12 APOO DAP3 MDC1 PML BAG2 FAS XRCC5 AKT1S1 HSPA13 SEC24B TNKS1BP1EIF4EBP1

CRAT PKM EIF4B CLTB GAK IGF2R NUDT19 MRPL21 MRPL53 MRPS30 MRPS25 NTPCR EIF4A2 RPL11 ABCF1 HSPA4 CAPZA2

CAT COA6 HIBADH PEX14 UQCRB HSPE1 ENO2 LSM3 CLTA CLTC COPA ALDH3A2 MRPL11 MRPL49 MRPL19 NDUFA9 NDUFS1 UQCRC1 SLC25A4CHCHD2 RPL34 ABCE1 EIF2S1 EIF3M HSPA8 PPP6R3 GADD45GIP1 SNRNP70

ECSIT CYC1 KLC2 HADHB MRPL3 MRPL13 MRPL1 MRPL38 NDUFA4 COX4I1 GRPEL1 PMPCA HSPD1 PCNA RPS29 RPLP0 EMG1 RPL31 PRCC PQBP1 RBM5 PSMD2 RAB1B ALDH6A1 SEC24C ARFGAP1

CYCS ACADM SHMT1 MRPL15 MRPL4 MRPL24 MRPL44 NDUFB9 NDUFA2 ATP5B PITRM1 MCM2KRT18RPA1 RRP9 EIF3A LSM7 HSD17B10ACADSB UQCRC2 RPL7A RPS13 NCBP1 CSTF2T CAND1 PSMC5 RAB19 PSMD11 SERPINA1

DLD OGDH SCO1 ETFB ETFA RPL9 PRCP ECHS1 OGDHL MRPS6 MRPL39MRPS33 MRPS35 NDUFV1 NDUFS2 ATP5E ATP5H ATP5S MCM6 MCM7 RPS24 RPL23 RPL18A SRSF5 PRPF31 WDR5 PSMC6 PSMD6 GOLM1 RAB3B RAB3D CHCHD5

ME2 FH PDHB SUOX AK2 MCM4 GINS4 RPL29 MRPS31 MRPL43 MRPS27 MRPL48 NDUFS3 NDUFA5 ATP5D SRP19 SEC61G

C Gene sets enriched in VCaPER Gene sets enriched in VCaPCRPC -log10 (q-value) 4 2 0 2 4 Mitochondrial translation Cytoplasmic translation Mitochondrial translational term.* mRNA catabolic process NMD* Mitochondrial gene expression Fc epsilon receptor signaling pathway Translational term.* Establishment of protein localization* Translational elong.* Cotranslational protein targeting to membrane GOBP Lipid localization Nuclear transcribed mRNA catabolic process Oxidative phosphorylation Regulation of cellular AA metabolic process Lipid oxidation TNF mediated signaling pathway Regulation of lipid localization Translational initiation Fatty acid catabolic process RNA catabolic process Mitochondrial protein complex Cytosolic ribosome Organelle inner membrane Cytosolic large ribosomal subunit Mitochondrial envelope Cytosolic small ribosomal subunit Organellar ribosome Mitochondrial matrix GOCC Mitochondrial large ribosomal* Inner mitochondrial membrane* Envelope Respirasome Respiratory chain complex Threonine type GOMF peptidase activity Peroxisome Ribosome Val Leu and Isoleu degradation Proteasome Oxidative phosphorylation KEGG Parkinsons's disease Fatty acid metabolism Alzheimers's disease Butanoate metabolism

7 Figure S4: Proteinsu p-a nd downregulated in VCaPER assemble in protein networks. (A) Network analysis of proteinsd ifferentially expressedb etween VCaPER and VCaPCRPC, showing protein clusters upregulated in VCaPER and(B) downregulated i n VCaPER. (C) GSEA shows top 10 enriched gene sets for biological processes (GOBP), cellular components (GOCC), molecularf unctions (GOMF)a ndK yoto

EncyclopediaofGenes and Genomes (KEGG) for proteins down(blue) or upregulated (pink) in VCaPER.

*Full names of gene sets: mitochondrial translational termination, translational termination, translational elongation, mitochondrial large ribosomal subunit, inner mitochondrial membrane protein complex, nuclear transcribed mRNA catabolic process nonsense mediated decay, establishmento f protein localization to endoplasmic reticulum.

8 Supplemental Figure S5

A Amplification Deep deletion Multiple alterations Mutation Fusion Proteins upregulated in VCaPER Proteins downregulated in VCaPER 600 500 400 300 200

Absolute counts 100 Mutation data CNA data NEPCProstate (Multi-InstituteProstate (Broad/CornellPRAD (Broad/CornellPRAD (MSKCC, 2016)Prostate HallmarksProstate 2012) 2020) (EurPRAD_MCSPC 2013) (CPCG-GENE,Urol (FHCRC,Prostate 2017)The MPC (MICH)PROSTATE 2016) (MSK,ProjectProstate 2017)Prostate CCR (MSKCCMSK-IMPAC(MSK 2020) (MSKCCProstate 2019)PRAD 2010) OrganoidsProstate 2014) (MSKCC/DFCIProstate (SU2C)Prostate (SU2CProstate (TCGA)Prostate2018) 2019) (TCGAProstate (TCGA PanCan (DKFZ)NEPC 2015)Prostate 2018) (Multi-InstituteProstate (Broad/CornellPRAD (Broad/CornellPRAD (MSKCC, 2016)Prostate HallmarksProstate 2012) 2020) (EurPRAD_MCSPC 2013) (CPCG-GENE,Urol (FHCRC,Prostate 2017)The MPC (MICH)PROSTATE 2016) (MSK,ProjectProstate 2017)Prostate CCR (MSKCCMSK-IMPAC(MSK 2020) (MSKCCProstate 2019)PRAD 2010) OrganoidsProstate 2014) (MSKCC/DFCIProstate (SU2C)Prostate (SU2CProstate (TCGA)Prostate2018) 2019) (TCGAProstate (TCGA PanCan (DKFZ) 2015) 2018)

T T Prostate Prostate

B Proteins upregulated in VCaPER C Proteins downregulated in VCaPER Unaltered group Altered group Unaltered group Altered group q-value = 1.648e-3 q-value = 0.0332 100% 100% 1331 patients 1331 patients 80% 225 genes 80% 260 genes 60% 60% 40% 40% 20% 20%

0% Disease-free survival 0%

Disease-free survival 0 20 40 60 80 100120140160 0 20 40 60 80 100120140160 Time (months) Time (months)

D Proteins upregulated in VCaPER E Proteins downregulated in VCaPER Unaltered group Altered group Unaltered group Altered group q-value = 2.22e-15 100% q-value = 1.07e-14 100% 1474 patients 1473 patients 80% 80% 225 genes 260 genes 60% 60% 40% 40% 20% 20% Overall survival Overall survival 0% 0% 0 40 80 120 160 200 0 40 80 120 160 200 Time (months) Time (months) F Proteins upregulated in VCaPER G Proteins downregulated in VCaPER Unaltered group Altered group Unaltered group Altered group 10 10 9 9 8 8 7 7 6 6 5+5 5+5 5+4 5+4 4+5 4+5 4+4 4+4 4+3 4+3 Gleason score Gleason score 3+5 3+5 3+4 3+4 3+3 3+3

0 0 50 50 100150200250 100150200250 # samples # samples Group Group

9 Figure S5: Genomic alterations in VCaPER-specific proteins are linked to PCa drug resistance in patients. (A) Gene alteration counts in PCa patient samples for genes coding for proteins up- or downregulated in VCaPER according to PCa type.( B) Kaplan-Meier graph of disease-free PCa patient survival for proteins up-o r (C) downregulated in VCaPER and( D) overalls urvival for proteins up-or(E) downregulated inVC aPER. (F) Distributiono f Gleason scores in PCa patients accordingt o genomic alterations inp roteins up- or (G)downregulated inV CaPER.

10 Supplemental Figure S6

A Proteins upregulated in VCaPER ADAR MRPS6 MRPL15 MRPL19 MRPL24 ve i t *** a l *** **** ** ray) 10 10 *** 10 10 r

re 15 * **** **** * s

e ** r

croa 10 i ** 5 5 5 5 m sco ( - 5 z s e l 0 0 0 0 p on

i 0 s m s a s -5 -5 -5 -5 -5 pre id

x in in in in in o loid on tion loid ion oid on ion oid oid on e l ip Ga ati e ip Ga et ipl Ga ati et ipl Ga etionipl Ga ati p D ic el D el ic el el ic i if D f D D f A l li li d w D w D w D w D Amp llo llo mp llo llo mp

o A A RN t Sha Sha Sha Sha m Putative copy-number alterations B Proteins downregulated in VCaPER EIF2S1 EIF3A EIF3M EIF4B EIF4A2 ** 15 10 *** 15 15 15 * NS NS * ** 10 * 10 10 10 5 5 5 5 5 0 ve i 0 0 t 0 0 a l ray) r re -5 -5 -5 -5 -5 s

e tion tion in on tion tion in on tion tion in on ion ion in tion tion in on r loid loid loid t t loid loid croa le e Ga ati le e Ga ati le e Ga ati e Ga le e Ga ati i ip ip ip le ip ip e el D lific e el D lific e el D lific e el D e el D lific

m p D p D p D p D

sco w D w D w D p D w D ( o mp o mp o mp w D o mp - Dee ll A Dee ll A Dee ll A ee llo Dee ll A z s ha ha ha D ha ha

e S S S S S l p on i s m LSM3 SNRNP70 RPL9 RPS29 s a s *** * pre 10 10 ** 15 10 *

id ****

x NS o e l p i

A 10 d 5 5 5 o RN t 5 m 0 0 0 0

-5 -5 -5 -5 on tion loid in on tion ti tionloid in on tion loid in on e ip Ga ati e Gain ele e ip Ga ati e ip Ga ati el D ific el Diploid el D lific el D ific l w D p D w D l low D mp llo ee llo Amp low D mp l A ha D ha l A Sha S S Sha Putative copy-number alterations

Figure S6: Genomic alterationsa re not always linked toRN A expression for genes coding for

differentially expressed proteins in VCaPER. (A) Boxplots showing mRNA expression according to gene

alteration type in PCa patient data for proteins upo r (B) downregulated in VCaPER. Kruskal-wallis test was

performed to analyse significant differences in means betweengroups (* :p<0.05,**:p<0.01, ***:p<

0.001, ****:p<0.0001).

11 Supplemental Figure S7

A LowTE ratio High TE ratio -log10 (p-value) 30 20 10 0 10 20 30 40 Extracellular exosome Nucleus Cytosol Nucleoplasm Membrane Intracellular Focal adhesion Centrosome GOCC Nucleolus Golgi membrane Actin cytoskeleton Ciliary basal body Cell-cell adherens junction Centriole Lysosomal membrane Telomeric region Endomembrane system Axoneme Protein binding Nucleic acid binding Cadherin binding* DNA binding GTPase activator activity Metal ion binding

Actin filament binding Transcription factor activity* GOMF Protein domain specific binding Poly(A) RNA binding ATP binding Helicase activity tRNA binding Protein binding RNA polymerase II core promoter*

B Proteins downregulated in VCaPER Genes with downregulated protein andlow TE ratio in VCaPER Proteins upregulated in VCaPER Genes with upregulated protein andhigh TE ratio in VCaPER 16 HSPA8 14 NUDT19 LIN7A 12 10 MRPL19 8 SHMT1 6 NUCB2 SERPINA1 -log2 (p-value) BCAM

-3 -2 -1 0 1 2 3 log2 (Fold change of protein expression VCaPER vs VCaPCRPC)

Figure S7: High TE ratio RNAs recapitulate key GO-terms fromthe translatome in VCaPER, but do

not necessarily lead to higher protein expression. (A) GO term enrichment analysis shows terms for top

10 significant cellular components (GOCC) andmolecularfunctions(GOMF) enrichedi n genes with low

or high TE ratios in VCaPER compared to VCaPCRPC. *Full names of GO terms:cadherin bindingi nvolved

in cell-cell adhesion, transcription factor activity, sequence-specific DNA binding, RNA polymerase II core

promoter proximal regions equence-specificDNA binding. (B) Volcano plot shows proteinsd ifferentially

expressed between VCaPER andV CaPCRPC. Up or downregulated in VCaPER are shown inr ed and blue

respectively. Upregulated proteins with significantly upregulated TE in VCaPER are marked in pink and

downregulated proteins with significantly downregulated TE are marked in blue.

12 Supplemental Figure S8

A Unaltered group Altered group B Unaltered group Altered group Genes with upregulated protein and Genes with downregulated protein and high TE ratio lowTE ratio q-value = 3.82E-10 100% 100% q-value = 10E-10 1474 patients 1473 patients 80% 43 genes 80% 84 genes

60% 60%

40% 40% Overall survival 20% Overall survival 20%

0% 0% 0 40 80 120 160 200 0 40 80 120 160 200 Time (Months) Time (Months) C Unaltered group Altered group D Unaltered group Altered group Genes with upregulated protein and Genes with downregulated protein and high TE ratio lowTE ratio 10 10 9 9 8 8 7 7 6 6 5+5 5+5 5+4 5+4 4+5 4+5 4+4 4+4

Gleason Score 4+3 4+3 3+5 Gleason Score 3+5 3+4 3+4 3+3 3+3 0 50 100 150 200 250 0 50 100 150 200 250 # samples # samples Group Group

Figure S8: Genomic alteration in proteins upregulated in VCaPER is linked to high PCa grade, independently of TE ratio.(A) Overall survivalforPCa patients accordingt o alterations ingenesc oding for proteins upregulatedw ithh igh TE ratios in VCaPER or (B)do wnregulated with low TE ratios.( C)

Distributiono f Gleason scores forp atients according to alterations in genesc oding for proteins upregulated withh igh TE ratiosor(D) downregulatedw ith low TE ratios.

13 Supplemental Figure S9

A Genes with upregulated proteins and high TE ratio ve i t MRPL19 NUCB2 a SHMT1 NUDT19 ray) l r 10 10 15 re **** 10 * NS NS s croa e i r 10 m ( 5 5 5 sco s - z e l 5 p on i m 0 0 0 s a s s 0 id pre o x l -5 -5 e p -5 -5 i

d in in A in on on in tion loid ti tion loid tion loid tion loid on o Ga t e ip Ga le e ip e ip Ga ati e ip Ga ati

RN el D e el D el D fic el D ic li lif m w D p D w D w D w D o o mp mp ll ee llo ll A llo A ha D ha ha ha S S S S

Putative copy-number alterations

B Genes with downregulated proteins and low TE ratio

LIN7A RPL29 SERPINA1 8 15 8 ** ** * ** 6 10 6 4 4 5 2 2 0 0 0 -2 -5 -2 in on on in on on in ve ion loid i ion oid i ion loid i t t t l t t t e ip Ga ati le e ip Ga ati le e ip Ga

a el ic ic e el ray) D e el D l if D if r l l

re w D p D w D p D w D o mp o mp o s ll A ee ll A ee ll croa e D D i r ha ha ha S S S m ( sco s - z e

l Putative copy-number alterations p on i m s a BCAM s HSPA8 s

id 15 10 pre

o NS x l * e p i d A 10

o 5 t RN

m 5 0 0

-5 -5 on in on in on ti tion loid tion loid le e ip Ga ati e ip Ga ati e el D fic el D ic li lif p D w D o mp w D mp ee ll A llo A D ha ha S S Putative copy-number alterations

14 FigureS 9: Genomicalterationsfor from VCaPER differentially expressed proteinswith differential

TE ratiodonotalways correlate with altered mRNA expression. (A) mRNA expression accordingto type of genomic alteration in PCa patients, for genes coding forp roteins upregulated and with high TE ratios in VCaPER or(C) downregulatedw ith low TEra tios. Significance was assessed with Kruskal-wallis test(*:p<0.05,**:p<0.01, ***:p<0.001, ****: p < 0.0001).

15 Supplemental Figure S10

A B VCaPCRPC VCaPER p = 2.09e-05 p = 0.013 q-value=1.32e-7 123456 2.00 0.8 42 kDa NUDT19 1.60 0.6 56 kDa TUBA1A 0.4 1.20 LNCaP MR49F 0.2 AR Score

TUBA1A 0.80 123456 0 to 42 kDa NUDT19 0.40 -0.2 56 kDa TUBA1A Fold change relative 0.00 -0.4 ER CRPC

VCaP LNCaP MR49F VCaP C D (A) 2.51-9.52 (B) 9.52-12.95 q-value = 1.21e-7 q-value = 1.263e-4 (C) 13.08-18.53(D) 18.59-41.17 0 12 NUDT19 expression bins (FPKM) -1 10 -2 8 -3 6 -4 4 -5 2 -6

Mutation Count (log2) 0 -7

Fraction Genome Altered (log2) -8 -2

(A) 1.48-9.10 (B) 9.12-13.13 (C) 13.26-18.68(D) 18.74-46.44 (A) 344.72-636.45(B) 640.06-777.86(C) 778.41-904.67 (D) 904.97-1790.75 NUDT19 expression bins (FPKM) NUDT19 expression bins (FPKM)

No correlation Correlation E q-value = 3.957e-3 F q-value = 3.515e-3 1 60 0.8 0.6 50 0.4 40 0.2 NEPC Score 0 # samples 30 -0.2

Neuroendocrin Features 20 -0.4 10

0 (A) 1.48-9.10 (B) 9.12-13.13 (C) 13.26-18.68(D) 18.74-46.44 NUDT19 expression bins (FPKM)

(A) 1.48-9.10 (B) 9.12-13.13 (C) 13.26-18.68(D) 18.74-46.44 NUDT19 expression bins (FPKM)

16 Figure S10: NUDT19 expression correlates with determinantsof highg rade and resistant PCa. (A)

Proteinexpression of NUDT19 in VCaPER, VCaPCRPC, LNCaP and MR49F, shown int riplicate lanes (left).

QuantificationofNUDT19 protein levels in VCaPER or MR49F respectively normalized to VCaPCRPC or

LNCaP (right). (B) ARs core,(C) fraction of the genome altered, (D) mutation counts, (E) NEPC score and

(F)n umber of PCapatient samples containingn euroendocrine features according to NUTD19 expression binning inPCapatients.

17 Supplemental Figure S11

A Observed Expected

mRNA lncRNA Pseudogene p = 0.344 p = 0.189 p = 0.003 p = 0.002 p = 0.221 p = 0.119 800 40 40 600 30 30 400 20 20 200 10

Gene count 10 0 0 0

ER ER ER ER ER ER CRPC CRPC CRPC CRPC CRPC CRPC

VCaP VCaP VCaP VCaP VCaP VCaP VCaP VCaP VCaP VCaP VCaP VCaP TranslatomeTranslatome TranslatomeTranslatome TranslatomeTranslatome TranscriptomeTranscriptome TranscriptomeTranscriptome TranscriptomeTranscriptome Antisense Processed transcript p = 0.556 p = 0.117 p = 0.009 p = 0.048 25 14 20 12 10 15 8 10 6 4

Gene count 5 2 0 0

ER ER ER ER CRPC CRPC CRPC CRPC

VCaP VCaP VCaP VCaP VCaP VCaP VCaP VCaP Translatome TranslatomeTranslatome Translatome TranscriptomeTranscriptome TranscriptomeTranscriptome

B Observed Expected mRNA lncRNA p-value = 5.82e-30 4000 1000 800 3000 600 2000 400

Gene count 1000 200 0 0

ER ER CRPC CRPC

High TE ratioHigh TE ratio High TE ratioHigh TE ratio inVCaP inVCaP inVCaP inVCaP

Figure S11: lncRNAs are more associated with ribosomes than expected in VCaPER. (A) Observed and

expected gene countsf or different categories of genes from both the translatome and transcriptome in

VCaPER compared toVCa PCRPC. (B)O bserved ande xpectedg ene counts of coding genes and lncRNAs

with high translation efficiencies in VCaPER compared to VCaPCRPC.

18 Supplemental Figure S12

A Amplification Multiple Alterations B Altered group Unaltered group Fusion Deep Deletion LncRNAs with LncRNAs with lncRNAs with high TE ratio lncRNAs with low TE ratio high TE ratio lowTE ratio 100% p-value=3.269e-3 100% p-value=0.0374 20% 0.1% 1470 patients 1471 patients 80% 23 genes 80% 11 genes 15% 0.08% 0.06% 60% 60% 10%

Alteration 0.04% Frequency 40% 40% 5% 0.02%

Overall survival 20% 20% Mutation data CNA data 0% 0% Prostate Prostate NeuroendocrineCRPC Prostate Prostate Prostate NeuroendocrineCRPC Prostate 0 40 80 120 160 200 0 40 80 120 160 200 AdenocarcinomaCarcinoma AdenocarcinomaCarcinoma Time (months) Time (months)

C Unaltered group D Altered group: Unaltered group High TE ratio lncRNAs Altered group: WAC-AS1 Low TE ratio lncRNAs 100% p-value = 0.659 THUMPD3-AS1 100% p-value = 3.19e-11 NOP14-AS1 1470 patients ZNF883 1471 patients SCGB1B2P 80% 80% SNHG7 11 genes DLEU2 3 genes 60% ZNF252P 60% LINC00667 40% 40% CRNDE

MRPS30-DT Overall survival Overall survival 20% 20% TMEM147-AS1 0% TRAF3IP2-AS1 0% 0 40 80 120 160 200 0 40 80 120 160 200 Time (Months) ASH1L-AS1 Time (Months)

E F Altered group Unaltered group Unaltered group Altered group: LncRNAs with LncRNAs with Low TE ratio lncRNAs high TE ratio low TE ratio 100% p-value = 0.811 POLDIP2 10 1471 patients 9 MMP24OS 8 80% 9 genes 7 WASH2P 6 60% MIR22HG 5+5 5+4 40% MTMR9LP 4+5 4+4 Overall survival PSMG3-AS1 4+3 20% Gleason Score RNASEH1-AS1 3+5 3+4 0% MCF2L-AS1 0 40 80 120 160 200 3+3 TTC28-AS1 Time (Months) 0 20 40 60 80 100 0 20 40 60 80 100 # samples (%) Group

19 FigureS 12: lncRNAswith high TE ratiosinVCaPER are linked toh igher PCa grade. (A) Alteration frequencies for lncRNAs with high or low TE ratios in VCaPER compared to VCaPCRPC according to PCa type. CNA : copy-number alteration. (B) Overall survival for PCa patients with or without alterations in lncRNAs with high (left) or low (right) TE ratios in VCaPER. (C) Overall survival for PCa patients with alterations ins ingle lncRNAs withh igh TE ratios that show no significant link to patient survival,( D) lncRNAs with lowTE that are correlatedw ithl owerp atient survival, or (E) lncRNAs with low TE ratios that show no significant link to patient survival. (F) PercentageofPCa patientsamples per Gleason score withh igh (left) or low (right) TE lncRNAs withor withouta lterations.

20 Supplemental Figure S13

A LncRNAs with high TE ratio

CRNDE OIP5-AS1 JPX TMEM147-AS1 * *** 2 4 **** 4 *** 2 * **** NS 0 2 2 * 0 -2 0 0 -2 -4 -2 -2

-6 -4 -4 -4 in on in on in tion loid on ti ion loid ti tion loid on in ray) t tion loid r e ip Ga ati le e ip Ga le e ip Ga ati Ga el D ic e el D e el D ic e ip lif lif el D w D p D w D p D w D croa mp mp i lo ee lo ee lo w D l A l l A llo

m D D

( ha ha ha S S S ha

s S e l p m a

s LINC00476 RPS10P7 CASC2 DHRS4-AS1

id 4 3 NS 3 NS 4 NS o l * p i 2 2 2 d

o 2 1 t 1 0 e

v 0 i t

a 0 -2 l 0 -1 re -4 s -2 -1 e r -2 -3 -2 -6 sco

- in in in in ion oid on ion oid on ion ion oid on ion oid z t l t l t t l t l e ip Ga ati e ip Ga ati le e ip Ga ati e ip Ga el D fic el D fic e el D fic el D on li li li i

s p D w D mp w D mp w D mp w D s llo A llo A ee llo A llo D ha ha ha ha pre S S S S x e A B LncRNAs with low TE ratio RN m SNHG7 NOP14-AS1 ve

LINC00467 i t a 3 NS l 4 NS 4 NS ray) r 2 re s

e 2 2 r croa

1 i m sco (

0 - 0 0 z s e -1 l p on i -2 -2 s m

-2 s a s

-3 pre -4 -4 id x o e in l in in ion oid on ion oid on ion ion oid on t l p t l t t l i e ip Ga ati A e ip Ga ati le e ip Ga ati el D ic d el D ic e el D ic if lif if

l o l RN w D mp t w D mp p D w D mp

m o llo A ll A ee llo A ha ha D ha S S S

Putative copy-number alterations Putative copy-number alterations

Figure S13: Genomic alterations are not alwaysl inked to changes in lncRNA expression. (A) RNA

expression accordingt o type of genomic alteration in PCa patients for lncRNAs with high TE ratios in

VCaPER or (B) low TE ratiosi n VCaPER.Significance was assessed withKruskal-wallis test(*:p<0.05,

**: p < 0.01,* **:p<0.001, ****:p<0.0001). 21 Supplemental Figure S14

A WARS2_AS1

6 73 7 166 VCaP total RNA 11 50

12 79 49 CRPC 7 VCaP total RNA

8 6 8 141 19

121 5 63 40 ER 7 7 VCaP total RNA 11 7 5 9

5 80 Number of reads 18 CRPC 6 VCaP Polysome

40 19 34

34 VCaPER Polysome 5 5 59 6 22

B VCaPCRPC VCaPCRPC VCaPER VCaPER Transcriptome Translatome Transcriptome Translatome

JPX LINC00467 Number of reads Number of reads Transcriptome 369 101 112 18 16 53 26 93 3677 Transcriptome 65 73 55 6 24 28 Translatome 89 49 21 2 2 12 4 26 533 Translatome 19 26 26 3 11 10 40% 16% *** * 30% 12% 8% 20% *** read count 4% *** 10% ** ** * % of normalized * * * 0% 0% Number of reads Number of reads Transcriptome 360 162 140 21 15 64 32 94 4071 Transcriptome 60 89 115 9 47 29 Translatome 79 44 37 0 4 5 6 17 1011 Translatome 13 21 28 7 7 11

12% 40% 30% 8% 20% * ** 4% ** read count * 10% * % of normalized 0% 0%

intron1 intron5 intron1 intron2 intron3 intron5 intron2aintron2bintron2cintron3aintron3bintron3cintron4aintron4b intron4aintron4b Figure S14:C andidate lncRNAs exhibit alternative splicing events in PCa ENZ resistance. (A)

Sashimiplots for the lncRNA WARS2_AS1 in PCa cell linesshows alternatively spliced isoforms inthe transcriptome and translatome of VCaPER and VCaPCRPC. (B) Quantification of JPX and

LINC00467 lncRNAs split reads count, corresponding to all splicing events in the transcriptome and translatomeofVCaPCRPC( blue) or VCaPER (red)(*:p<0.05,**: p < 0.01, ***: p < 0.001).

22