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Figure S1. Expression of RRM2, PTTG1 and PRC1 is associated with poor prognosis in ACC. A- Correlogram shows correlation of expression of RRM2, PTTG1 and PRC1 with expression of EZH2 and E2F1 in TCGA and Michigan cohorts. B- Expression of RRM2, PTTG1 and PRC1 in normal adrenals, adrenocortical adenomas and adrenocortical carcinomas. Significance was evaluated by ANOVA. C- Expression of RRM2, PTTG1 and PRC1 in the groups of good (blue) and poor (red) prognosis in TCGA and Michigan cohorts. Significance was evaluated by Wilcoxon’s test. D- Overall (O.S) survival as a function of RRM2, PTTGI and PRC1 expression in TCGA and Michigan cohorts. E- Overall (O.S) survival as a function of RRM2, PTTG1 and PRC1 expression in all cohorts combined. Statistical significance was evaluated by the Logrank test.

Figure S2. Effect of E2F1 inhibition by HLM. A-Effect of increasing doses of HLM treatment for 72h on expression of RRM2, PTTG1 and PRC1 was evaluated by RTqPCR (graphs) and western-blot (bottom panels) in H295R cells. B- Effect of pharmacological inhibition of E2F1 on the growth of H295R cells was determined by counting live cells after 3 days of treatment with increasing amounts of HLM. C- Effect of E2F1 inhibition on expression of Cyclin coding genes in H295R cells was evaluated by RTqPCR. D- Effect of 5 uM DZNep and/or 40 µM HLM treatment for 48 h on expression of EZH2 and E2F1 was evaluated by RTqPCR (graphs) and western-blot (bottom panels) in H295R cells. E- Kinetic effect of pharmacological inhibition of RRM2 by 5 uM GW8510 on growth of H295R cells at 2, 3, 4 and 5 days. Graphs in A-E represent the mean of 4 experiments ± SEM. Statistical significance was determined by ANOVA in A-D and by multiple t tests in E. * p<0.05, ** p<0.01, *** p<0.001.

Table S1. Online sources for ChIP data analysed in the manuscript

Table S2. Antibodies used in the manuscript

Table S3. Primers used in the manuscript

Table S4. Correlation coefficients of EZH2 with all other genes in ACC patients’ transcriptome data.

Table S5. EZH2/E2F1 metagene in ACC

Table S6. Association of expression of EZH2_E2F1 metagene and different prognostic factors with overall survival in TCGA, Cochin and Michigan cohorts. Univariate and multivariate hazard ratios (HR) and 95% confidence intervals (95% CI) were determined by Cox proportional hazards regression using clinical an molecular data from the three cohorts of patients.

Table S7. Intersection of EZH2/E2F1 metagenes from ACC and prostate cancer patients

Figure S1 A Correlogram (TCGA)

E2F1

EZH2

PTTG1

RRM2

PRC1

1

E2F1

0.8

0.6

EZH2

0.4

0.2

Pearson’s R

PTTG1

0

0.2

RRM2

-0.4

-0.6

PRC1

-0.8

1

Correlogram (Michigan)

EZH2

PRC1

E2F1

RRM2

PTTG1

1

EZH2

0.8

0.6

PRC1

0.4

0.2

Pearson’s R

E2F1

0

-0.2

RRM2

-0.4

0.6

PTTG1

-0.8

1

B

Gene expression (Michigan)

RRM2

PTTG1

PRC1

13

1.9x10

.9x10T

12

2.5x10

1.9x10

14

2.1x10

11

10

12

8

9

10

ns

6

ns

ns

7

NAd

8

ACA

ACC

C

RRM2

PTTG1

PRC1

12

13

4.4x10

12

2.7x10

2.3x10

Michigan

10

12

11

11

10

8

10

9

6

9

8

Good prognosis

Poor prognosis

3.2x10

12.5

12

10

0.002

2.3×10ª

TCGA

10

11

7.5

7.5

10

5.0

5.0

9

2.5

2.5

:

8

D

RRM2 TCGA O.S.

1.0

0.8

Frequency

0.6

0.4

0.2

high: 37 cases

logrank test p-value = 1.3x104

low: 37 cases

0

0

1000

2000

3000

Overall Survival (days)

PRC1 TCGA O.S.

1.0

0.8

Frequency

0.6

0.4

high: 37 cases

low: 37 cases

0.2

logrank test

0

p-value = 4.3×10-0

0

1000

2000

3000

Overall Survival (days)

RRM2 Michigan O.S.

PTTG1 TCGA O.S.

1.0

0.8

Frequency

0.6

high: 37 cases

0.4

low: 37 cases

0.2

logrank test p-value = 2.0x104

0

0

1000

2000

3000

Overall Survival (days)

PRC1 Michigan O.S.

1.0

0.8

Frequency

0.6

high: 12 cases

low: 12 cases

0.4

0.2

logrank test

0

p-value = 0.03

0

1000

2000

3000

4000

5000

Overall Survival (days)

PTTG1 Michigan O.S.

1.0

0.8

Frequency

0.6

high: 11 cases

low. 13 cases

0.4

0.2

logrank test

0

p-value = 0.1

0

1000

2000

3000

4000

5000

Overall Survival (days)

1.0

0.8

Frequency

0.6

high: 12 cases

low: 12 cases

0.4

0.2

logrank test

0

p-value = 0.19

0

1000

2000

3000

4000

5000

Overall Survival (days)

E

RRM2 All_cohorts O.S.

PRC1 All_cohorts O.S.

1.0

0.8

Frequency

0.6

high: 72 cases

0.4

low: 74 cases

0.2

logrank test

0

p-value = 4.9x10-7

0

2000

4000

6000

8000

10000

Overall Survival (days)

1.0

0.8

Frequency

0.6

high: 72 cases

low: 74 cases

0.4

0.2

logrank test p-value = 1.1x10*

0

0

2000

4000

6000

8000

10000

Overall Survival (days)

PTTG1 All_cohorts O.S.

1.0

0.8

Frequency

0.6

high: 72 cases

0.4

low: 74 cases

0.2

logrank test

0

p-value =6.9x10-7

0

2000

4000

6000

8000

10000

Overall Survival (days)

Figure S2

A

RRM2

PTTG1

PRC1

EZH2

E2F1

**

*

*

ns

Relative mRNA accumulation

**

ns

*

*

*

**

ns

ns

ns

2.0

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

1.5

ns

ns

ns

ns

ns

ns

ns

1.0

T

0.5

0

HLM

10

20

40

60

80

100

10

20

40

60

80

100

10

20

40

60

80

100

10

20

40

60

80

100

10

20

40

60

80

100

-

-

-

-

-

-

-

-

-

-

DMSO

+

+

+

+

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

RRM2

GAPDH

TUBULIN

-

ACTIN

B

H295R Growth Rate

150

Relative Growth Rate

ns

100

L

T

50

0

HLM

10

20

40

60

80

100

-

-

DMSO

+

-

-

-

-

-

-

-

C

CyclinA

CyclinB1

CyclinB2

CyclinD1

CyclinD2

CyclinE

Relative mRNA accumulation

200

150

100

ns

50

ns

**

10-

*

8

6

ns

ns

ns

*

ns

ns

ns

ns

4

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

2

0

HLM

10

20

40

60

80

100

10

20

40

60

80

100

10

20

40

60

80

100

10

20

40

60

80

100

10

20

40

60

80

100

10

20

40

60

80

100

-

-

-

-

-

-

-

-

-

-

-

-

DMSO

+

+

+

+

+

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

D

EZH2

E2F1

E

H295R Growth Rate

ns

Relative mRNA accumulation

1.5

ns

1.5

ns

*

4

Relative Growth Rate

ttt

r

*

3

1.0

1.0

2

0.5

0.5

1

HH

DMSO

0

0

0

GW

DZNep

+

+

+

+

d0

d2

d3

d4

d5

days

-

-

-

-

-

-

HLM

+

+

+

+

-

-

-

-

-

-

DMSO

+

+

-

-

-

-

EZH2

E2F

GAPDH

ACTIN

Chipseq

Experime ntDatasets GEODatasets ENCODEInitial Data
Hela
E2F1GSM935484SRR502355.sra SRR502356.sra
H3K27m e3GSM733696SRR227472.sra
SRR227473.sra
H3K4me 3GSM733682SRR227441.sra
SRR227442.sra
EZH2GSM1003520SRR568331.sra
SRR568332.sra
GSM509051SRR036650.sra
SRR036651.sra SRR036652.sra
SRR036653.sra
InputSRR036654.sra
K562
E2F1Replicat 1 : ENCLB809EWTENCFF749JUG.fastq ENCFF942QFN.fastq
InputGSM777645SRR332096.sra
LM2
E2F 1GSM2501567SRR5282135.sra
InputGSM2501570SRR5282138.sra
LnCap ABL
E2F1GSM1656410SRR1970880.sra
InputGSM1656411SRR1970881.sra
MCF7
E2F 1GSM935477SRR1970880.sra
InputGSM594608SRR065825.sra
Raji
E2F 1GSM1976292SRR3048087.sra
InputGSM1976298SRR3048093.sra

Supplementary Table 1.

Supplementary Table 2.
AntibodySupplierReferenceDilution
EZH2Cell Signaling Technology31471/1000
E2F1Millipore05-3791/1000
RRM2Abcamab576531/400
PTTG1Abcamab795461/5000
PRC1Abcamab512481/5000
ACTINSigma AldrichA20661/500
TUBULINSigma AldrichT60741/500
GAPDHNovus BiologicalsNB300211/1000

RT-qPCR Primers

GeneOrientationSequence 5'-3'
EZH2ForwardACCAGTTTGTTGGCGGAAGCG
ReverseCCAAGTCACTGGTCACCGAACAAG
BCL-XLForwardGATCCCCATGGCAGCAGTAAAGCAAG
ReverseCCCCATCCCGGAAGAGTTCATTCACT
Birc5ForwardATTCGTCCGGTTGCGCTTTCC
ReverseCACGGCGCACTTTCTTCGCAG
BCL-2ForwardTTGTGGCCTTCTTTGAGTTCGGTG
ReverseGGTGCCGGTTCAGGTACTCAGTCA
ActinForwardCGCGAGAAGATGACCCAGATC
ReverseTCACCGGAGTCCATCACGA
RRM2ForwardGATATTCTGGCTCAAGAAACGAGGACTG
ReverseCTCTCCTCCGATGGTTTGTGTACCAG
PTTG1ForwardGCTCTGTTCCTGCCTCAGATGATG
ReverseGTCAAGGATCATGAGAGGCACTCC
PRC1ForwardGCAGAAATTCATGGAGTATGTGGCAGAAC
ReverseCGCCGCTTGCTAGGTGTTCGAG
CyclinAForwardTCCTGTCTTCCATGTCAGTGC
ReverseCAAACTCTGCTACTTCTGGGG
CyclinB1ForwardCAGTCAGACCAAAATACCTACTGGGT
ReverseACACCAACCAGCTGCAGCATCTTCTT
CyclinB2ForwardCATGTGCGTTGGCATTATGG
ReverseCAAGAGCAGAGCAGTAATCC
CyclinD1ForwardCGGTGTCCTACTTCAAATGTGTGCAGAA
ReverseGGGCTCCAGCGACAGGAAGC
CyclinD2ForwardCATTGCTCTGTGTGCCACCG
ReverseGCGAGCTCACTTCCTCATCC
CyclinEForwardCACTTTCTTGAGCAACACCC
ReverseCCGGTCAAAGAAATCTTGTGCC
Supplementary Table 3.
ChIP qPCR Primers
GeneOrientationSequence 5'-3'
PRC1_ChIP1ForwardGCGAGTCCCCACCCCGATTGCG
ReverseGGGCGAAGCCTCGTCGCG
PRC1_ChIP2ForwardGCTCCGATGAGGTAGCTGGCTTG
ReverseGGGGGATGACGTCACTGTCTGTG
PTTG1_ChIPForwardGACCCTCCCATTCACTCACGCAG
ReverseGGGCCGCGAGTTGTGGTTTAAACC
RRM2_ChIP 1ForwardGCAGACCAATGGTGGAGTAGATGC
ReverseGGCTGGTCTCAAGCGATCCTCC
RRM2_chIP 2ForwardGCGCTTGAAAATCGCGCGCGG
ReverseCGGCCCTTCCCATTGGCTGTG
00
00
00
00
00
000
00
000
00
00
00
00
00
0 0
00 0 00 0 0
0
00
00
00
0
0 0 00 0 0 00 0 0 0

0

00 0
0
0 0
0 0 0 0 0 0
0 0
00 0 0 0
0 0
0 0
00 0
0
0 0
0
00
0
0 00 0
00
00
00
00
00
00
0

0

0

0

0

00
101010 1010100
00
00
0 00 0

0

0

0

0

0

0

0

0

0

0
0
0
0
0

0

0

0

0

00
000
000
00
00
0
00
00
0 00 0 00 0 0
00 00 0
000
00
00 00 0
0
0
000
00
000
0
0 00 0 0 0 0
0
0
0
0

0

0

0

0

0

0

0

0

0 0
0 0
00 00 0
0 00 0
00
000
000
0
0 00 0
0
0
0
0

0

0

00
00
00
00
0
0 0
00 0
0 0
00 0 0 0
0 0
0
0
0
00

0

0

0

0

0

0

00
00
0 0
00
00
00
0
00
TTT
00 0
0
00
00
00
00
000
00
00
0
00
00
00
0 00 0
000
00
0 0
0 0

EZH2_E2F1_94_gene_Signature_ACC

MCM7

RRM2

DNMT1

CDCA7

POLD1

MCM2

KIAA0101

CLSPN

FANCG

PKMYT1

MCM6

FANCC

POLE2

FBXO5

POLD3

MCM3

MSH2

E2F8

GMNN

NOLC1

NASP

MCM4

CDC6

ATAD5

SNRPD1

ZNF367

MXD3

ILF3

H2AFZ

PCNA

ATAD2

CDC25A

H2AFV

SUV39H1

CTDSPL2

E2F1

TOPBP1

HNRNPD

MTF2

SMC3

EED

MAZ

PRKDC

CASP8AP2

GEN1

UBR7

SMC6

RAVER1

POLA1

HOXC10

EZH2

STMN1

CDK1

ARHGAP11A

E2F7

SASS6

SETD8

MCM8

FANCD2

RBL1

CBX3

CD3EAP

TMPO

RASAL2

RAD51

CDT1

HMGN2

POLA2

INTS7

LIG1

WEE1

KIF15

USP1

PLK4

TIPIN

MELK

CDC45

SMC2

YWHAQ

CASP2

WDR67

RPS19

NUP155

ESPL1

NCBP1

DLEU2

CCDC150

KNTC1

PRIM1

ANP32E

ANP32A

SLBP

RPA2

FUS

Supplementary Table 5.

TOGA
Univariate
EZH2_E2F1_MetageneHR95% CILog Rank
high vs low10,183,418 to 30,325,00E.07
Prognosis group
C1A vs C1B8,8562,642 to 29,692,00E.05
MKI67
high vs low10,313,514 to 30,242,00E.07
Weiss score
[4.9] vs [0-3]3,1310,8856 to 11,076,00E-02
ENSAT-rank
ENSAT (3-4] vs [1-2]6,6992,716 to 16,523,00E.06
Multivariate
EZH2_E2F1_Metagene + PrognosisHR95% CILog Rank
high vs low7,1822,247 to 22,961,00E-07
EZH2_E2F1_Metagene + MKI67
high vs low4,0131,098 to 14,671,00E-07
EZH2_E2F1_Metagene + Weiss score
high vs low20,97654,7876 to 91,912,00E.05
EZH2_E2F1_Metagene + ENSAT
high vs low8,7112,334 to 32,515,00E-08
EZH2_E2F1_Metagene + Prognosis + MK167 + Weiss + ENSAT
high vs low6,2071,18843 to 32,4207,00E.06
Adjusted
EZH2_E2F1_Metagene ("Prognosis)HR95% CILog Rank
high vs low6,1611,981 to 19,166,00E-04
EZH2_E2F1_Metagene ("MKI67)
high vs low4.3191,046 to 17,834,00E-02
EZH2_E2F1_Metagene ("Welss)
high vs low11.442,362 to 55,394,00E-04
EZH2_E2F1_Metagene ("ENSAT)
high vs low7,9312,095 to 30,031,00E.03
Overall Survival Cochin
Univariate
EZH2_E2F1_MetageneHR95% CILog Rank
high vs low4,0451,55 to 10,562,00€-03
Prognosis group
CLA v& C1B11,862,72 to 51,694,00E-05
MK167
high vs low5,6312,008 to 15,793,00E-04
Weiss score
[4-9) vs [0-3]9,8031,308 to 73,476,00E-03
ENSAT-rank
ENSAT [3-4] vs [1-2]14,884,885 to 45,31,00€-09
Multivariate
EZH2_E2F1_Metagene + PrognosisHR95% CILog Rank
high vs low2,6751,007 to 7,1083,00€-05
EZH2_E2F1_Metagene + MKI67
high vs low2,3170,813 to 6,6043,00E-04
EZH2_E2F1_Metagene + Weiss score
high vs low2,9481,469 to 28,903,00E-04
EZH2_E2F1_Metagene + ENSAT
high vs low2,8160,9908 to 8,0022,00€-09
EZH2_E2F1_Metagene + Prognosis + MKi67 + Weiss + ENSAT
high vs low1,3260,3984 to 4,4151,00E-08
Adjusted
EZH2_E2F1_Metagene ("Prognosis)HR95% CILog Rank
high vs low2,7381,029 to 7,2864,00E-02
EZH2_E2F1_Metagene (~MKI67)
high vs low2,2190,769 to 6,3961,00E-01
EZH2_E2F1_Metagene ("Weiss)
high vs low3,1061.171 to 8.2422,00E-02
EZH2_E2F1_Metagene["ENSAT)
high vs low2,60,8646 to 7,8198,00E 8,00E-02
Supplementary Table 6.
Michigan
Univariate
EZH2_E2F1_MetageneHR95% dLog Rank
high vs low2,37220,8492 to 6,6279,00E-02
Prognosis group
C1A vs C1B3,5321,251 to 9,9741,00E-02
MK 67
high vs low0,8610,3215 to 2,3068,00E-01
Weiss score
[4.9] vs [0-3]3,1110,8668 to 11,160,07
ENSAT-rank
ENSAT (3-4] vs [1-2]2,5580,9217 to 7,1026,00E-02
Multivariate
EZH2_E2F1_Metagene + PrognosisHR95% dLog Rank
high vs low1,89810,6637 to 5,4292,00E-02
EZH2_E2F1_Metagene + MKI67
high vs low2,36410,8374 to 6,6742,00E-01
EZH2_E2F1_Metagene + Weiss score
high vs low2,3810,5585 to 10,1541,00E-01
EZH2_E2F1_Metagene + ENSAT
high vs low2,1420,7485 to 6,1276,00E-02
EZH2_E2F1_Metagene + Prognosis + MK167 + Weiss + ENSAT
high vs low1,1830,2866 to 4,8851,00E-02
Adjusted
EZH2_E2F1_Metagene ("Prognosis)HR95% aLog Rank
high vs low1,85940,6258 to 5,5253,00E-01
EZH2_E2F1_Metagene ("MKI67)
high vs low2,2450,7916 to 6,3671,00E-01
EZH2_E2F1_Metagene ("Weiss)
high vs low1,6760,4947 to 5,6764,00E-01
EZH2_E2F1_Metagene("ENSAT)
high vs low2,3050,7627 to 6,9681,00E-01

EZH2_E2F1_94_gene_Signature_ACC EZH2_E2F1_gene_Signature_CrPc_XU_et_al Intersection

MCM7

ARL6IP1

ATAD2

RRM2 ATAD2

CDC6

DNMT1

BIRCS

CDK1

CDCA7

BNIP2

POLE2

POLD1

BRIX1

MCM2

BTG3

TMPO

KIAA0101

CCNA2

CLSPN

CCNE2

FANCG

CDC6

PKMYT1

CDK1

MCM6

CENPK

CHEK1 FANCC

POLE2

CKS2

FBXO5

CNOT6

POLD3

DSCC1

MCM3

FANCI

MSH2

FOXM1

E2F8

GGH

GMNN

GLOD4

NOLC1

GPSM2

NASP

GUCY1B3

MCM4

H1F0

CDC6

H1FX

ATAD5

H2AFX

SNRPD1

HMGB2

ZNF367

HMMR

MXD3

IERS

ILF3

KIF23

H2AFZ

MMD

PCNA

MND1

ATAD2

MRPL20

CDC25A

MTFR2

H2AFV

NCAPG

SUV39H1

NCAPG2

CTDSPL2

NDC1

E2F1

NME1

TOPBP1

NMU

HNRNPD

NUF2

MTF2

PA2G4

SMC3

PBK

EED

PLK1

MAZ

POLE2

PRKDC

PPIH

CASP8AP2

PRC1

GEN1

RFC2

UBR7

RFWD3

SMC6

RRM1

RAVER1

SMC2

POLA1

SMC4

HOXC10

SNRPA1

EZH2

SPAG5

STMN1

TACC3

CDK1

TMPO

ARHGAP11A

TRIP13

E2F7

TYMS

SASS6

WDR34

SETD8

MCM8

FANCD2

RBL1

CBX3

CD3EAP

TMPO

RASAL2

RAD51

CDT1

HMGN2

POLA2

INTS7

LIG1

WEE1

KIF15

USP1

PLK4

TIPIN

MELK

CDC45

SMC2

YWHAQ

CASP2

WDR67

RPS19

NUP155

ESPL1

NCBP1

DLEU2

CCDC150

KNTC1

PRIM1

ANP32E

ANP32A

SLBP

RPA2

FUS

Supplementary Table 7.

SMC2

Graph View

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