a) b) · nrf 1.00e-164 ets1 1.00e-153 erg 1.00e-133 ews:fli1-fusion 1.00e-116 yy1 1.00e-113 klf5...
TRANSCRIPT
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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