Supplemental Figure 1

Supplemental Table 1. A) Spearman correlation comparing RB status with other clinical correlates. Correlation with both percent RB positive and weighted RB intensity score shown. B) Association between treatment and RB positivity (top) or Ki-67 positivity (bottom) assessed through Wilcoxon rank sum testing.

A)

B)

Supplemental Figure 2. A) E2F1 binding in shCON or shRB in castrate conditions compared to previously identified E2F1 cistrome published by Ramos-Montoya et al. B) Validation of E2F1 binding in LNCaP shCON/shRB pairs, LAPC4 shCON/shRB pairs, and LNCaP vs LNCaP-abl (which lose RB during progression to castrate resistance). C) Overlap in E2F1 binding in shRB LNCaP and LNCaP abl (Xu et al 2016).

Supplemental Figure 2

9585

330210116

5924

328

782 504

RamosMontoyaetal

E2F1FBS

LNshRB

LNshCON

19049 9685 7382 LN shRB Castrate

LN abl Castrate

E2F1 Binding – LN abl vs LN shRB

B)

0

5

10

15

20

25

CITED2

ZN

F717

H3F3B

UBX

N2B

NET1

DDIT4

PCMT4

CCDC

66

RPAP

2

Rela%v

eEn

richm

ento

verInp

ut LAPC4shCON LAPC4shRB

0

5

10

15

20

25

30

CITED2

ZN

F717

H3F3B

UBX

N2B

NET1

DDIT4

PCMT4

CCDC

66

RPAP

2

Rela%v

eEn

richm

ento

verInp

ut LNCaPshCON LNCaPabl

0

5

10

15

20

25

30

CITED2

ZN

F717

H3F3B

UBX

N2B

NET1

DDIT4

PCMT4

CCDC

66

RPAP

2

Rela%v

eEn

richm

ento

verInp

ut LNCaPshCON LNCaPshRB

E2F1 Binding vs Established Dataset A)

E2F1 Binding After RB Loss in Multiple Models

C)

Motif Name P-value E2F4 1e-427 NFY 1e-330 Elk4 1.00E-297 Elk1 1.00E-283 Sp1 1.00E-278

ELF1 1.00E-269 ETS 1.00E-265 E2F6 1.00E-254 E2F1 1.00E-249 Fli1 1.00E-248

GFY-Staf 1.00E-230 GABPA 1.00E-215 E2F7 1.00E-213 CTCF 1.00E-201 GFY 1.00E-185 NRF1 1.00E-172 ETV1 1.00E-170 NRF 1.00E-164 ETS1 1.00E-153 ERG 1.00E-133

EWS:FLI1-fusion 1.00E-116 YY1 1.00E-113 KLF5 1.00E-109 E2F 1.00E-105 EHF 1.00E-102 CRE 1.00E-93

BORIS 1.00E-89 ELF5 1.00E-84

SPDEF 1.00E-68 ZNF143|STAF 1.00E-62

RFX 1.00E-56 MYB 1.00E-53 JunD 1.00E-53 E-box 1.00E-53 Stat3 1.00E-53 BMYB 1.00E-53 Rfx2 1.00E-51

Foxa2 1.00E-42 Klf4 1.00E-39 Rfx1 1.00E-38

AMYB 1.00E-37 GFX 1.00E-36

Stat3+il21 1.00E-36 Rfx5 1.00E-34 Atf2 1.00E-33

STAT4 1.00E-32 USF1 1.00E-29

Fox:Ebox 1.00E-29 CLOCK 1.00E-28 X-box 1.00E-27 Maz 1.00E-26 Atf1 1.00E-26

c-Myc 1.00E-26 bHLHE40 1.00E-25

STAT1 1.00E-25 FOXA1 1.00E-25 FOXP1 1.00E-25 FOXA1 1.00E-25 Foxo1 1.00E-24 MITF 1.00E-23

PU.1-IRF 1.00E-22 Nanog 1.00E-22

ZBTB33 1.00E-22 STAT5 1.00E-20

EWS:ERG-fusion 1.00E-20 c-Jun-CRE 1.00E-20 TATA-Box 1.00E-20

LNshCONCastrateAllBinding

Motif Name P-value CTCF 1e-2422 BORIS 1e-1489 Foxa2 1.00E-100

Fox:Ebox 1.00E-96 NF1 1.00E-86

FOXA1 1.00E-82 FOXA1 1.00E-81

NF1-halfsite 1.00E-79 FOXP1 1.00E-61

SCL 1.00E-55 Unknown-ESC-element 1.00E-48

Tlx? 1.00E-42 Tcf12 1.00E-42 Ascl1 1.00E-40

NeuroD1 1.00E-38 SPDEF 1.00E-37

REST-NRSF 1.00E-36 Foxo1 1.00E-29

Fli1 1.00E-29 ERG 1.00E-28

CTCF-SatelliteElement 1.00E-28 GRHL2 1.00E-27 Stat3 1.00E-26 Bcl6 1.00E-24 ETV1 1.00E-23

GABPA 1.00E-22 BMYB 1.00E-22

NF1:FOXA1 1.00E-21 Stat3+il21 1.00E-20 AR-halfsite 1.00E-20

LNshRBCastrateExclusiveBinding

LNshCON

LNshRB

LNshCON

LNshRB

LNshCON

LNshRB

Motif Name P-value CTCF 1e-2184 BORIS 1e-1161

Elk4 1.00E-188 Fli1 1.00E-174 Elk1 1.00E-172 ELF1 1.00E-152 NFY 1.00E-147 Sp1 1.00E-147

GABPA 1.00E-144 ETS 1.00E-139 E2F4 1.00E-129 ETV1 1.00E-123 ERG 1.00E-106

Foxa2 1.00E-95 GFY-Staf 1.00E-94 Fox:Ebox 1.00E-93

NRF 1.00E-88 ETS1 1.00E-87 GFY 1.00E-86 E2F6 1.00E-81 NRF1 1.00E-80

FOXA1 1.00E-78 NF1 1.00E-76

EWS:FLI1-fusion 1.00E-76 SPDEF 1.00E-76

EHF 1.00E-74 KLF5 1.00E-73

FOXA1 1.00E-72 NF1-halfsite 1.00E-72

YY1 1.00E-65 RFX 1.00E-64

FOXP1 1.00E-64 Rfx2 1.00E-62 E2F7 1.00E-58 ELF5 1.00E-58 BMYB 1.00E-57 Stat3 1.00E-56 AMYB 1.00E-55 Rfx1 1.00E-53 CRE 1.00E-52 E2F1 1.00E-51

CTCF-SatelliteElement 1.00E-45 Stat3+il21 1.00E-45

MYB 1.00E-44 Foxo1 1.00E-43 Tlx? 1.00E-43

STAT4 1.00E-42 E2F 1.00E-39 Rfx5 1.00E-32

E-box 1.00E-32 X-box 1.00E-32 Tcf12 1.00E-31

NF1:FOXA1 1.00E-30 Arnt:Ahr 1.00E-30 NeuroD1 1.00E-30

Unknown-ESC-element 1.00E-29 Rbpj1 1.00E-29 Klf4 1.00E-28 SCL 1.00E-26

GRHL2 1.00E-26 REST-NRSF 1.00E-26

Maz 1.00E-25 Smad3 1.00E-23

Bcl6 1.00E-23 Lhx3 1.00E-23 MafA 1.00E-23 GFX 1.00E-22

STAT1 1.00E-22 STAT5 1.00E-20

Unknown 1.00E-20

LNshRBCastrateAllBinding

Supplemental Figure 3: RB loss results in differential E2F1 binding associated motif enrichment in castrate conditions. Briefly, Homer was used to find enriched motifs using a 1kb window around the center of binding, using the binding datasets indicated by the shaded region of each venn diagram.

Supplemental Figure 3

Motif Name P-value E2F4 1.00E-106 E2F6 1.00E-103

Fox:Ebox 1.00E-90 E2F1 1.00E-83 Foxa2 1.00E-82 CTCF 1.00E-73

FOXA1 1.00E-66 FOXA1 1.00E-65 E2F7 1.00E-64

BORIS 1.00E-47 E2F 1.00E-43

FOXP1 1.00E-35

LNshCONCastrateExclusiveBinding

LNshCON

LNshRB

0

5

10

15

20

25

Rel

ativ

e En

richm

ent o

ver I

nput

MCF-7 Veh

MCF-7 E2

Supplemental Figure 4

Supplemental Figure 4. A) Immunoblot of androgen induced RB phosphorylation after 3 hours of DHT stimulation B) Estrogen treatment in MCF7 cell models elicit similar phosphorylation of RB compared to DHT treatment (left) and MCF7 cells exhibit similar expansion of E2F1 binding after E2 treatment at sites of gained E2F1 binding after DHT stimulation in LNCaP cells (right). C) E2F1 binding overlap in LNCaP shRB cells in castrate and DHT stimulated conditions.

B)

MCF7

E2F1 Binding After E2 Stimulation

21190 5241 3264

shRB Castrate shRB DHT

E2F1 Binding – shRB Castrate vs DHT C)

LNCaP

shC

ON

shR

B

A)

total Rb

GAPDH

pS780-Rb

E2F1

DHT - -+ +

shC

trl

shR

b

total Rb

GAPDH

pS780-Rb

E2F1

DHT - -+ +

shC

trl

shR

b

Supplemental Figure 5

Supplemental Figure 5. Scree plot indicating variability explained by the indicated factors. Red line indicates cumulative variability explained by each individual component.

Supplemental Figure 6 A) C)

D)

Supplemental Figure 6. A) Validation of differentially expressed targets after RB loss in multiple models of RB loss both upregulated (top), downregulated (middle), and Myc targets from GSEA enrichment (bottom). B, C) Gene set enrichment analysis utilizing previously published signature of RB loss (Ertel et al 2010) or CRPC AR targets (Sharma et al 2013). D) Transcriptional alterations for genes validated in A after CDK4/6 inhibitor treatment in castrate conditions.

GSEA – RB Loss Signature

GSEA – AR Targets

RB Loss-Induced Transcriptional Alterations Across Model Systems

Up-regulated

Down-regulated

CON shRB-LN abl shRB-LapHYLS1CDK2CDC20ADM

Myc Targets

0"

0.5"

1"

1.5"

2"

2.5"

ADM" NR4A2" TSPAN8" CELF2"

Fold%Cha

nge%

shCON"CDT"

shCON+PD"

Gene expression after CDK4/6 inhibitor treatment

B)

CON shRB-LN abl shRB-LAPC4PCNANEK1PUS1PLK1

TMSB4XCELF2TSPAN8NR4A2

211593421312052538627220028816731613427724616163402939330223224514214183221256323162156742402222812412326816610529516482287325993212552272442152382901168263120243334127147267324225150110237619256218621098928929181701121464811821114131312260532961252232933766616337326239179411025724925730881781873531425021911412815819611185155551171572792319769291262188387923010018512481332259459791242209136217462945433076340517220133209690310208175119148103202625823610822022352592923282341292802149873269132216104317170824125230883186190113782312987531818914428617121019842261641491924826671073003319306771691771064115207309182121917127629728567335251321254121802992121993043381841923413152042743394317639145311193320216813765283473191539533313850228139943132243052651319420344275122252261731401547232228195513012703271301431031161512331803032844987581333363627317420612634316027160307158426410128227165247162291521827817213534213235

0 5 10 15Value

1630

Color Keyand Histogram

Count

211593421312052538627220028816731613427724616163402939330223224514214183221256323162156742402222812412326816610529516482287325993212552272442152382901168263120243334127147267324225150110237619256218621098928929181701121464811821114131312260532961252232933766616337326239179411025724925730881781873531425021911412815819611185155551171572792319769291262188387923010018512481332259459791242209136217462945433076340517220133209690310208175119148103202625823610822022352592923282341292802149873269132216104317170824125230883186190113782312987531818914428617121019842261641491924826671073003319306771691771064115207309182121917127629728567335251321254121802992121993043381841923413152042743394317639145311193320216813765283473191539533313850228139943132243052651319420344275122252261731401547232228195513012703271301431031161512331803032844987581333363627317420612634316027160307158426410128227165247162291521827817213534213235

0 5 10 15Value

1630

Color Keyand Histogram

Count

Supplemental Figure 7

Supplemental Figure 7. RB loss results in differential E2F1 binding associated motif enrichment in castrate conditions. A) Hierarchical clustering of normalized expression data of all genes with a nearby E2F1 binding site from 144 SU2C CRPC samples (1-Pearson’s correlation coefficient for columns and Euclidean distance for rows were used as distance measures). The annotation track reports the genomic status of RB1 (black: SCNA alteration; white: no SCNA alteration). B) Hierarchical clustering of normalized expression data of differentially expressed genes (RB altered vs RB WT) identified through utilizing only SU2C expression data. C) GSEA enrichment of genes up-regulated by RB loss and exclusively bound by E2F1 in the current model within genes from analysis in B.

C)

E2F1 Comprehensive (All E2F1 Bound Genes) Differential RB Status Signature (SU2C Supervised Analysis)

SNORA5ASNORA21SNORA57TMSB10P1RNU6ATACAL031963.1PLA2G2ARDH11TMPRSS2RNU1−1RNU1−6RNU1−9RNU1−2RNU1−5RNU1−4RNU1−3CLUGAPDHFN1APLP2PKM2ENO1DDX17C12orf57PRKCSHCORO1BC22orf32ANXA2CALM1UQCRQTPI1PGK1SEC62ITM2BCREB3L4TSC22D1SLC9A3R2RNF10CTTNFAM96BKDELR1UQCRC2MRPL20NOP56STEAP2SLC44A4RBM47MCM4STMN1SHMT2HN1LCP1LBHTSC22D3CAMKK2RABEP2RGL2NECAB3C17orf28EPHB4PYGBARAFTOLLIPSF3B3TECRGPIHNRNPUL1ILF3PTBP1TRAPPC4DDX41HAGHDCAF11SNX17ADIPOR1CRTAPMRPL53SARSH2AFVCDK4SNRPBSNORD41IGHJ1SNORA68SNORA36ASCARNA8SNORA51SNORA75ZNF29PHIST1H2AMHIST1H2AIHIST2H2ABSHANK2IGF1RHIST2H4BHIST2H4AKIAA1244PHC3IQSEC1TBC1D14PRKCHCRY2WWP2SFNRP11−785H5.1.1POSTNID1SULF1MMP14SCNN1AAKAP12EYA2SEMA3CZC3H12ADAPK1AAK1TNRC6BMGAT5CCNT1NUP155ADMRECQL4MKI67TYMSBIRC5TACC3TROAPCDC20MCM2RRM2PRC1LMNB1NUSAP1PTTG1ZWINTCCNB1KPNA2TGFB3ROBO1LEF1ETS2DDX3YTTLL5TTLL4SLC16A1GRAMD4GCHFRPAFAH1B3RNASEH2AATAD2FANCGCDK2RARAKLF10WDR54PHF19POLA2DNMT1CTNNAL1C9orf30STX12NIPA2NAA38HPRT1GCATSLC10A3PEX16ILVBLDCAF12ETFDHCTPSMTUS1NUP54SLC44A1ELP3PIGCHAUS7C1orf35CARSTFE3TOP1MTTSEN54NUP85SNAP47POLR1EDCAKDADPRHL2C20orf4P4HA1RNF145KIAA1429MRPS6NARFTOR1AGSSSAE1UBA2SLBPHADHCSE1LAMZ2YEATS4RCC1NUP205RQCD1OBFC2BNUP62DAZAP1NCAPD2SLC20A1MCM3MTHFD1NUP107EXOSC2RRM1RFC2GREB1GMPRCSGALNACT1MAN1C1TMEM25ROGDIARSAACAD8VPS26BACSS1SLC37A1TAOK3RPS6KA2MAN2B2MEF2DKLHL36KIAA0556TBC1D4ATP8A1MAP3K5C6orf132ARHGAP29SLC25A16PSMD5FNDC3ARB1TRIM13AQRFAM48AANKRD13AELF1FBXW11KLHL12TXNRD2NUDT16C9orf91DECR2FRMD4ACARD8UBE2Q2WASLZNF587IFT27FBXW4STAG3L4C2CD2SOX13CC2D1BTGFBRAP1RFFLTECPR1CLEC16AZC3H13SCAF4ITSN2GIT2ERGCPNE4TRPV6PRSS8PEX10ENTPD5ALDH2BACE2ATP1B1C22orf25ATP8B2PDE4DIPUNKLSPATA13OSBPL5PHF12TTC38AMDHD2LMF1SIPA1TRABDSORBS2FAM118AFAAHMCCC1BDH1CREB3L2CPT1ATMEM135C4orf34UPF3AFUCA1CRYL1CYB5R1ESDCATMXD4VAMP2SEC22CTXNDC11PHKBABCC5DAXXICA1TMCO3FAM45ABMI1SNX19FAM214APCF11PPP6R2ELMO2PCM1CLN6MEPCEATG9AIPO9CCDC59HSPA4DPM1ARL2SWI5MRPL17C9orf142METTL11ADDX39AEIF4A3SNF8TBX3RGS10CCT2H2AFZC19orf53AC008073.5.1HMGB2PDCD5PCNARTN3VDAC3CCT6ACCT8WLSRAI14TMEM2MICAL2SRCH1FXBCS1LTBCBKDM1AYTHDF2CAND1SNRPAKHDRBS1PRPF31STXBP2PBXIP1ERLEC1POLD4CDC42SE1MGEA5N4BP2L2ARFGAP3THUMPD3JAGN1FBXO9YPEL5USP7OTUD5WDR45TMPOC8orf33MTHFD2WDR34CARHSP1MAD2L2POLR2IESYT1SF3A2PPM1BTUSC3NOP58NASPPABPC4UBE2D2DERL1HNRNPABALYREFSSRP1KHSRPMT1P3.1OR51C1POR51E2NEFHGP2LMAN1LACSM1SNHG6RPS17CBX3HMGB3LDHAMESTUBE2SUBE2CSNHG1CKS2FKBP3OSTCU1SPINK1TNNT1HSD17B6HAMPCPS1AC018816.3.1B3GNT6COL2A1CA4KLANKRD34BPTPRTLRRC31PDE8BSLC44A5SAMD13DNASE2BPLCXD2EMID2CTD−2224J9.2.1SNHG3KCTD9HAUS1POU5F1NCS1RP11−394B2.1.1TRIM59KITLGE2F3GLMNEXTL2NPPCBCHESRMSCELEPHA7NRCAMLRRN3AC092535.1VGFGHRHRAPOFDTX1FBXO2DNASE1L3ART4TAL1ADD2FSTL4AKR1E2PARK2GPR17EGR3DKK1GAD1HSPA6IL1RNCAPN13ITGB8C1orf106LAMB3BEX5MAPK4ERVMER34−1RASL11BLYPD1CYP27B1SLC16A10PLCL1STRA6C8orf73CTD−2228K2.7.1SAPCD1−AS1AMHANKRD24RP4−811H24.6.1HLA−LST8SIA1GFRA2CD96FAM124ARBKSC17orf82LETM2RP11−247A12.2.1NHSSLC2A13MCF2L2RTDR1F2RL3RELL2RP11−761I4.3.1DNMBP−AS1NUFIP1C4orf46ZNF93NUP62CLGPR137CC19orf57RP11−424C20.2.1EPHX4CHRNA5RP11−650L12.2.1FAM171A2CDK5R1C19orf40TMEM194BC9orf40LYSMD4HPSECHMLZNF165ZNF829BLMFAM54AXRCC2C5orf34CENPIBRIP1KIF14KIF18AEFNA2PANK1C5CTSL2CCNE1CDCA7SDSDLEU2RP11−22P6.3.1ZNF695FAM64ACDC25CGINS2MND1ZNF367CDC25AC1orf135CENPHSPC25RAD51AP1PBKOIP5RAD51NUF2NDC80TTKCEP55KIF11GINS1EXO1KIF15CSPG5RNFT2EME1C9orf100ATXN7L2AC034193.5.1VASH2CNIH2C17orf53C15orf42E2F8CLSPNWDR62CASC5NEIL3POLQCDCA2ORC1ESCO2KIF20BSGOL2CKAP2LCENPESGOL1SLC1A1AJUBAITGA2HBEGFSNAI1IER5LCLCF1SESN2HEXIM2FAM71E1ZNF280CZNF620SRRM5TRDMT1ZXDAZNF674DPY19L1CENPPMNS1C16orf55RNF24CEP135ERI1DENND1CSPRYD7SETDB2SWT1ZCCHC4ZNF566ZFP82ZNF567PKP1COL9A2PPYR1IGSF21KCNC4RP11−603B24.1.1IRX4PHF21BABCC8DNALI1DARCTNXBACHETMEM56FAM46CPLCG2RHOFHMHA1RHOHMYO1GSDPRCCDC69PRKCBKIF21BLCKTRAF3IP3NPR3EDARADDELFN2ACOXLLYPD6BLDHDNBEASRGAP3GRIP1KCNRGIL17RDSEMA4GITIH5FOXO1CBFA2T3HFENFATC2BAI2CDH24FGD1TYRO3GNB1LZNF34WRAP53C20orf177PPP1R3DCCNYL1CARD10MLYCDPPP2R3AZNF736ZNF671RERGZNF673CDADC1KPNA3RCBTB2MTRF1WBP4ZNF25ITM2ALIMS2RAPGEF3PDE2AGDPD5GIMAP6RAMP3CBX7FAM214BRP1−286D6.2.1HOXB13−AS1KRT10GSTM3CXADRPSPHGAR1SAP30PSMC3IPDSN1PRRT2TGFB2MFAP2GPX8PLAUECM1EGLN3ZDHHC13DONSONBTG3CCNE2NRMORC6SLC25A19CHEK2CDC7KIAA1524C1orf112GAS2L3ASPMC12orf48PRR11NCAPGWDHD1WDR76CDC6CDCA8E2F1TTLHAUS5RFWD3DSCC1PRIM1FANCD2POLE2RAD54BRBBP8FIGNL1FBXO5SLC4A4TWIST1HSD11B2ZNF703TRPM2C1QTNF6PPICELMO1DENND3PHLDB2SLC4A7GAB1SNX29MAP4K2C19orf55POLD1LIG1SLC2A12ENPP5ARHGEF38AL589988.1TAB3−AS2CRYMINHBBCAB39LMPP7KLF15ABATKCNG1CBLCZNF323PER3NFATC1KIAA0513ATXN1ATP7BFZD5PLEKHM3AC114772.1PLCB4SLC25A30PROSER1COG3VPS36COG6KIAA1704PPM1KSPIN3KIAA1797TAB3MTMR6UBR2STAMPLOD2ATF5EFNB2UFM1SUCLA2GTF2F2SUGT1PRAF2C12orf45CDKN2AIPNLFBXL3CISD1TAF12MRPL39GULP1PSMG1LSM1MED4SUPT16HCHRAC1UNGYBEYNAE1WDR5BRIX1CPSF3CUL1AP005482.1.1F2RL1CDKN3UBE2TMLF1IPCDK1KIAA0101CDKN2CMAD2L1FEN1RFC5TIMELESSAURKACCNA2CDCA3CCNB2KIF4ASPC24MELKCHEK1TRIP13POC1ACHAF1BWEE1EZH2KNTC1CDCA4MCM6CCDC99VRK1TBCEFANCIBUB1BCENPFTOP2AUHRF1ANLNASF1BHJURPFOXM1KIFC1FAM83DAURKBSPAG5KIF20ATPX2C18orf56FAM111BDTLESPL1CDT1TCF19TONSLHELLSGEN1GPSM2IQGAP3RAD54LKIF18BPSRC1CDC45BRCA1KIF2CKIF23BUB1SNX22EPHA4KIAA1958COL4A5JPH1CDC14AS1PR3SCARA3AMOTGPC4SH3D21CELSR2PFKFB4SEMA3BFCHSD1SH3TC1RHOBTB1TMEM64SLC39A8CYP2U1RIC8BTMEM38BSGCBRARBPDGFCTEAD4LHFPL2NFKBIETMEM51NETO2LONRF1MTHFD1LERO1LFAM57AC14orf129ING2POLR3DDUSP14GRAMD3TMEM39BC11orf84ASB3FANCLPPP1R21GCFC1MBIPTMEM48CSTF2NEDD1KDSRMBD2COPG2PRPF4ARHGEF9CTBSRFKRNASEH2BPHF11GLYCTKPPARAKIAA0564SLC25A15RP11−80H18.3.1CABLES2C19orf47INTS7FANCCSENP1RLFFAM49ATTC26MANEANKRFWASF3KBTBD7KBTBD6ALG11UTP14CAKAP11ZNF595RP11−106J23.2.1ZNF552WDFY2LRCH1NAA16TDRD3PIBF1DIS3DLEU1RCBTB1NHLRC3MRPS31DHRS12INTS6GPR146NAGPANAPEPLDCXorf38ARHGAP19ERMAPZNF436STK38NSUN3IKZF5RCOR1TADA2ATEX10PINX1PRR3YARS2ZNF16CEP85TUBGCP4TRIM39

0 5 10 15 20Value

4810

Color Keyand Histogram

Count

LOW

HIGH

A) B)

GSEA for E2F1 Expanded Signature (Enrichment within Supervised Analysis)

LOW

HIGH

SupplementalMethods:Primersutilizedinstudy:

ChIP Primers Forward Reverse CITED2 TTGTCCCGTTCATCTGGTGG GCTGCAGAAGCTCAACAACC ZNF717 GGAGGAAAATGGCGGAGTGG TGGGCAAAACAGAATGTCCG H3F3B ACGACGAATCTCTCGAAGCG TTCGGGGCGTCTTTCTTAGG

UBXN2B GATTTGCAGGTGAGGCGAGG ACCACCACAGTGTCAAGACC NET1 CTGTCCTACTTGAACCCAGC ATCAATCCACACCGAGTCAGC

DDIT4 AGTCCTTATAGGCTGCTCCG TAGGACCCACACACAGAAGG PCMT1 CTCAGAAAGGGACACGCAGC CAAGAGTGAGACCACCTCCC

CCDC66 AACAAACGGCATACGCAACC TGACGACCACCTTGCTTACC RPAP2 CCACAACTCCACTTACCGGC GATCCTTGCTCCTACCTGCG BUB1 TTGAAACTTGGCGGCTAGGG TCCCGTACCTACCTCAGAGC KIF24 CCTAACAGTCCCGTCAACCC AACCGATTCCTTGGATGCCC

CENPK CCGGCGATAAGGGTTTCACC CAGTCTTCTGTCAGCGTCCG RPAP2 CCACAACTCCACTTACCGGC GATCCTTGCTCCTACCTGCG SUSD5 GCTTTGGAGGAATACTGCACG AGCATGTAAATGGCTCCTGC

PRSS48 TAAACGGGATAGGAAGAGGGG ACACTGGAATATCGTGAAAAGGC PLEKHF2 ACTTTCTAAGGGCTGGTCGG TTGTCCATGTGTGAGGTCGG

BIRC2 TACGGATAGTCCCCGTTCCC ACTCTGACGCACGATGACG C1GALT1 CAGACCCCTTCGCATTAGGG CCAGATTATTCCCCGGCAGC

HMGA1 CCTATAGCAGGCTCACAAGGG TGTGTCAAAGCAGCGTTTCG TP73-AS1 CTCCCATCTAGGGATCCACACC GTTGTTGCGGGATCTCACAGG

PRDM4 TGCTTTGTTCCTCATCCGGG TGGCTGAGGATCCGGAAACG E2F1 AGGAACCGCCGCCGTTGTTCCCGT CTGCCTGCAAAGTCCCGGCCACTT

CCNB1 CGATCGCCCTGGAAACGCATTC CCAGCAGAAACCAACAGCCGTTC CDC6 GTGCAGGATCCTTCTCACGTCTCTCAC AAAGGCTCTGTGACTACAGCCAAT

mRNA Primers Forward Reverse PCNA TAGCCACATTGGAGATGCTG CAGTGGAGTGGCTTTTGTGA NEK1 AGGTGGCTCTCCATCAAAGC TCACAAGTTGACCTCCTGCC PUS1 GATTCTGGGACTGAAGCGGG ATTGTGGAAGTTGTGCGTGC PLK1 CAAGCTGGGCAACCTTTTCC GATCCTCAGCCTCCTCTTGC

TMSB4X CTCGCTTCGCTTTTCCTCCG GTACAGTGCATATTGGCGGC CELF2 AGAAGGAAGGTCCAGAGGGG GCTTGGATAGCAGCTTGTGC

TSPAN8 CAAGAAGAGTTTAAATGCTGCG AGGCACATAATTCAGGATAGTG NR4A2 ACTATTCCAGGTTCCAGGCG GGGTACGAAGTTCTGGGAGC CDH3 GTCTCAGTTCCCCCTTCAGC GACTCATAGCCTGTCTCCGC

HSPB8 CACAAAGAAAATCCAGCTTCCTGC AGAGAAGCCCTAGGGTTGGG INHBB AGCTTCGCCGAGACAGATGG CGTAGGGCAGGAGTTTCAGG

VIM TCCACGAAGAGGAAATCCA CAGGCTTGGAAACATCCAC MCM2 CAACACTGCCAATGGCTTCC CTTGCCACCTGGGTTTTTGG HYLS1 GTGGAAATGAAAGCAGAAGGTCC TTTCGGAGTCTTTGGGAGGC CDK2 CTCATCAAGAGCTATCTGTTCC TTTAAGGTCTCGGTGGAGG

CDC20 ATTTGGAACGTCTGCTCAGG CTTGGCCATGGTTGGATACT ADM CCCTGATGTACCTGGGTTCG CATCCGGACTGCTGTCTTCG

GAPDH CCAGGTGGTCTCCTCTGACTTC TCATACCAGGAAATGAGCTTGACA

ChIP-Seq Analyses: Alignment performed using Bowtie and peak calling was completed using MACS2

using a Q value cutoff of 0.01 (1). Venn Diagrams for binding overlaps generated using pybedtools

v0.7.8 and bedtools v2.24.0 (2, 3). Heatmaps for binding intensity generated using DeepTools v2.2.4

(4). Cis-regulatory element analysis performed using CEAS v1.0.2 (5). Motif analyses were generated

through Homer v4.8.3 (6). Denovo analysis was performed using a 50bp window around indicated

binding, while known analysis was performed using a 1000bp window around indicated binding.

RNA-Seq Analyses: RNA-Seq alignment was performed using STAR v2.5.2a (7). Differential gene

expression was generated using edgeR v3.16.5 (8). Gene set enrichment analysis completed through

GSEA using gene sets from the Molecular Signature Database (9). Binding to gene analysis achieved

through BETA v1.0.7 using a 30kb window around center of binding (10). Circos plot created using

Circos v0.69-3 (11).

ATAC-Seq Analyses: ATAC-Seq analysis was performed utilizing the ENCODE ATAC-Seq processing

pipeline (https://www.encodeproject.org/atac-seq/). Downstream analyses including PCA and signal

plots were obtained using DeepTools (4).

SU2C/PCF CRPC Tumor Cohort - RNA-seq data analysis: Processed SU2C/PCF (12) RNA-seq data

(n = 149) were downloaded from cBioPortal (13). Samples with neuroendocrine molecular features as

assessed by elevated Integrated NEPC Score (14) (greater than or equal to 0.25) were excluded for

downstream analysis (n=5). To nominate genes with concordant expression with respect to RB1 status

in the SU2C/PCF CRPC cohort (RB1 altered, n = 53, vs RB1 wt, n = 64) and the study model, in

addition to the same deregulation versus, at least one of the following criteria was requested to be

satisfied, i) significant differential expression (p-value < 0.1, Wilcoxon Mann Whitney test) ii) significant

association between expression level and genomic status of RB1 (wt, hemi and homozygous; p-value <

0.1, Anova test), while genes meeting this criteria were also required to exhibit the same sign FC to

ensure concordance between datasets.

SU2C/PCF CRPC Tumor Cohort - Identification of genomic status: Allele-specific copy number

genomic status of the indicated genes were assessed from the Whole Exome Sequencing (WES)

SU2C/PCF cohort as previously described (15).

Copy Number alteration and ctDNA procedure: Circulating cell free DNA from a cohort of CRPC

patients was harvested, isolated, and sequenced as previously described (16). Analysis of alteration

co-occurrence was generated using GenVisR bioconductor package(17).

Immunohistochemistry – Tampere Cohort: IHC analyses for Ki-67 and RB1 were performed on CRPC

TMAs as previously described (18, 19).

Immunohistochemistry - The Institute of Cancer Research CRPC Cohort: Tissue samples were

obtained from CRPC patients through CCR2472 -Marsden ethics committee approved protocol for

sample collection. FFPE samples were cut at 4um thick sections onto superfrost glass slides for

immunohistochemical staining. Primary anti-RB1 (mouse monoclonal, Clone G3-245, BD Biosciences,

San Jose – CA, USA) and anti-Ki67 (mouse monoclonal, Clone MIB-1, Agilent-Dako, Santa Clara – CA,

USA) antibodies were diluted 1:100. Germinal centers in associated lymphoid tissue of the appendix

was used as a positive control and striated muscle and cell line MDA-MB-468 were used as negative

controls for ki67 and RB1 respectively. Heat based antigen retrieval was performed by boiling slides in

a pressure cooker at 125ºC for 2 minutes then 90°C for 1 minute in a pH 6 citrate buffer solution.

Endogenous peroxide was blocked using a 3% H2O2 solution. Non-specific staining was blocked using

Dako protein block serum-free X0909. The Dako-Envision kit (Agilent-Dako, Santa Clara – CA, USA)

was used for reaction visualization. Ki-67 staining was semi-quantitatively scored by one pathologist

(DNR) blinded to RB1 status as a percentage determined by (proportion of positive tumor cells)/(total

number of tumor cells)x100. RB1 staining was semiquantitatively assessed by assigning

a proportion of tumour cells in each section into four tiers representing: no staining (0), weakly positive

(+1), moderately positive (+2), and strongly positive (+3).

TMA IHC Analysis - Tampere and The Institute of Cancer Research Cohort: TMA RB vs Ki67

Correlation was associated as follows. RB status was summarized in two ways. First, the total

percentage of cells staining 1 or greater, and, second, as a weighted average of the intensity scores

where the weights were the percentage of cells staining at that intensity. Spearman correlation

coefficients were calculated to assess the association between RB status and Ki67, PTEN, and AR

where applicable. Grouped scatter-plot samples were grouped by IHC score, and percent of each

tumor null for RB plotted in respective IHC score bin.

Cell Culture and Treatment: Isogenic cell lines for RB loss were generated from LNCaP cells originally

purchased from ATCC as previously described (20), with cells maintained as described previously (21).

Briefly, in castrate conditions, phenol red-free media supplemented with 5% charcoal dextran-treated

serum (CDT) was used. All experiments utilizing Dihydrotestosterone (DHT) used DHT obtained from

Sigma-Aldrich (St. Louis, MO, USA), which was dissolved in ethanol prior to use. Breast cancer MCF-7

cells were originally purchased from ATCC and were maintained and propagated in Dulbecco’s

Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% P/S. Prior to harvest, MCF-7

cells were hormone deprived for 3 days, and subsequently treated with 10 nM (final concentration)

estradiol for three hours before harvest.

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