178014 2 merged 1494965256 · 6/1/2017 · (slwkholdo ryduldq fdqfhu (2& uhsuhvhqwv wkh prvw...

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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0078

http://mct.aacrjournals.org/

×

× ×

Figure 1.

A

B

D

C EDMSO JQ1 THZ1

JQ1 (μM)

0 0.1 0.3 1 3 0 0.03 0.1 0.3 1

THZ1 (μM)

CO

V 4

13

BO

VC

A420

SK

OV

3

THZ1

COV 413B

0 0.05 0.1 0.5 1

RNAPII S2

CDK7

Tubulin

RNAPII S5

RNAPII S7

RNAPII S2

CDK7

Tubulin

RNAPII S5

RNAPII S7

RNAPII S2

CDK7

Tubulin

RNAPII S5

RNAPII S7

SKOV3

0 0.05 0.1 0.5 1

OVCA420

0 0.05 0.1 0.5 1

THZ1

Dinaciclib

Flavopiridol

Romidepsin

Panobinostat

PF−04929113

AT13387

Torin 2

INK 128

BEZ235

BI 6727

BI 2536

MLN2238

Bortezomib

Dasatinib

YM155

Pemetrexed

Abitrexate

Gemcitabine

Raltitrexed

Paclitaxel

Docetaxel

Topotecan

Doxorubicin

Epirubicin

CDK

INHIBITOR PubChemID TARGET COV 413B OVCA420 SKOV3

CDK

HDAC

HSP

mTOR

PLK

Proteasome

Src

IAP

chemo

73602827

46926350

5459219

5352062

6918837

44195571

11955716

51358113

45375953

11977753

10461508

11364421

25183872

387447

3062316

46931000

394493

126941

60750

104758

36314

148124

60700

31703

41867

High

IC50 < 0.5μM

96-well plate

Imaging using ArrayScan Infinity

Ovarian cancer cells

Inhibitor library

inhib

itor a

ctiv

ity

Low

CO

V 4

13

BO

VC

A4

20

SK

OV

3

on Septem

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ssociation for Cancer R

esearch. m

ct.aacrjournals.org D

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anuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Author M

anuscript Published O

nlineFirst on June 1, 2017; D

OI: 10.1158/1535-7163.M

CT

-17-0078

Figure 2.

A

C D

E F

B

Pol II S2

cleaved PARP

CDK7

Tubulin

Pol II S5

Pol II

Pol II S7

4 8 12 16 200

300

600

900

1200

1500

1800

Vehicle

HEY xenograftA2780 xenograft

Time (days) Time (days)

THZ1

**

*

**

* **

n=10 n=10

3 6 9 12 15 18 21 24 270

200

400

600

800

1000

1200

Vehicle

THZ1

Tu

mo

r vo

lum

e (

mm

3)

A2780 COV 413B OVCA420

0 0.05 0.1 0.5 1 0 0.05 0.1 0.5 1 0 0.05 0.1 0.5 1 THZ1 (μM)

THZ1 concentration (μM)

0.010.001 0.1 1

inhibitor concentration (μM)

0.010.001 0.1 1 10

0

20

40

60

80

120

100

Ce

ll via

bili

ty (

%)

0

20

40

60

80

120

100

Ce

ll via

bili

ty (

%)

COV 362

A2780

TOV-112D

COV 413B

DOV 13

OV90

SW982

OVCA433

PA1

OVCAR5

HEY

IGROV1

SKOV3

OVCA420

OVTOKO

OVCAR8

COV 318

ES-2

SW

98

2

DMSO THZ1

OV

CA

433

PA

1O

VC

AR

5H

EY

IGR

OV

1

SK

OV

3

DMSO THZ1

OV

CA

420

OV

TO

KO

OV

CA

R8

CO

V 3

18

ES

-2

CO

V 3

62

DMSO THZ1

A2

78

0T

OV

-11

2D

CO

V 4

13

BD

OV

13

OV

90

A2

78

0C

OV

36

2E

S-2

JQ1 (μM)

0 0.1 0.3 1 103 0 0.030.01 0.1 0.3 1

THZ1 (μM)

A2780+JQ1A2780+THZ1COV 362+JQ1COV 362+THZ1ES-2+JQ1ES-2+THZ1

on Septem

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-17-0078

Figure 3.

A

C

D E

B

Pol II S2

CDK7CDK7

OVCA420

H3

CDK7

H3

SW98

2

HEY

A278

0

DO

V 13

OVT

OKO

CO

V 31

8

SKO

V3

OVC

A420

MC

AS

TOV-

112D

PA1

IGR

OV1

OVC

AR5

OVC

A433

OVC

AR8

CO

V 41

3B

ES-2

OV9

0

sgEG

FP

sgC

DK7

-1

sgC

DK7

-2

sgC

DK7

-3

Pol II S5

Pol II S7

CDK4

CDK6

CDK9

Actin

positivenegative

A2780

sgEGFP sgCDK7-1 sgCDK7-2 sgCDK7-3 sgEGFP sgCDK7-1 sgCDK7-2 sgCDK7-3

COV 413B

HEY

ES-2

SKOV3

OVCA420

sgEGFP

sgCDK7-1

sgCDK7-2

sgEGFP

sgCDK7-1

sgCDK7-2

ES-2

HEY

0 10 20 30 40 50 60

0.0

0.2

0.4

0.6

0.8

1.0

Follow up (months)

Epithelial OvCa

Ove

rall

Surv

ival

(pro

b.)

P=0.036

n=28

n=22

CDK7 negativeCDK7 positive

sgEGFP

sgCDK7-1

sgCDK7-2

sgEGFP

sgCDK7-1

sgCDK7-2

0.00

0.05

0.10

0.15

0.20

0.25

0.30 *

Tum

or w

eigh

t (g)

Tum

or w

eigh

t (g)

0.00

0.05

0.10

0.15

0.20

0.25 *

A2780

sgEG

FP

sgC

DK7

-1

sgC

DK7

-2

sgC

DK7

-3

COV 413B

sgEG

FP

sgC

DK7

-1

sgC

DK7

-2

sgC

DK7

-3

ES-2

sgEG

FP

sgC

DK7

-1

sgC

DK7

-2

sgC

DK7

-3

HEY

sgEG

FP

sgC

DK7

-1

sgC

DK7

-2

sgC

DK7

-3

SKOV3

sgEG

FP

sgC

DK7

-1

sgC

DK7

-2

sgC

DK7

-3

0.0

0.5

1.0

* * *

rela

tive

prol

ifera

tion

0.0

0.5

1.0

rela

tive

prol

ifera

tion

0.0

0.5

1.0

rela

tive

prol

ifera

tion

0.0

0.5

1.0

rela

tive

prol

ifera

tion

0.0

0.5

1.0

rela

tive

prol

ifera

tion

0.0

0.5

1.0re

lativ

e pr

olife

ratio

n

sgEGFP

sgCDK7-1

sgCDK7-2

sgCDK7-3

Figure 4.

A B

THZ1 (nM)

All

11

014 a

ctive tra

nscripts

(F

PK

M>

1)

COV 413B

Gene Expression

Log2 Fold Change

0

0-4 4

100

All

10949 a

ctive tra

nscripts

(F

PK

M>

1)

Gene Expression

Log2 Fold Change

OVCA420

0-4 4

0 100

All

9753 a

ctive tra

nscripts

(F

PK

M>

1)

Gene Expression

Log2 Fold Change

SKOV3

0-4 4

0 100

0 5 10 15 20 25 30 35 40 45

Spliceosome

Homologous recombination

Cell cycle

RNA transport

Fanconi anemia pathway

double-stranded DNA binding

poly(A) RNA binding

protein binding

centrosome

mitochondrion

nucleolus

nucleus

nucleoplasm

transcription, DNA-templated

double-strand break repair via synthesis-dependent strand annealing

organelle organization

tRNA processing

mitotic nuclear division

DNA replication

double-strand break repair via homologous recombination

gene expression

double-strand break repair

mitotic cell cycle

DNA repair

cell division

biological process

cellular component

molecular function

KEGG pathway

-Log10 FDR

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Figure 5.

A

B

C

COV 413B

LAMA5 (4)

ZMIZ1 (6)

ITGB6 (9)

TRIO (39)

SOX4 (125)

EGFR (226)

PAX8 (287)

KLF13 (301)

PAX2 (348)

FGF18 (349)

MYC (439)

HES1 (483)

CDK5 (750)

E2F3 (862)

KLF9 (528)

0.0

0 5000 10000

Ranked Enhancers Ranked Enhancers Ranked Enhancers

15000 20000 0 5000 10000 15000 0 5000 10000 15000 20000

1.0

2.0

3.0

0.0

0.5

1.0

1.5

0.0

0.5

1.0

1.5

OVCA420 SKOV3

H3

K2

7A

c s

ign

al (X

10

5)

97

9 s

up

er-

en

ha

nce

rs

75

2 s

up

er-

en

ha

nce

rs

23

7 s

up

er-

en

ha

nce

rs

COV 413B OVCA420 SKOV3

THZ1 versus DMSOTHZ1 versus DMSO THZ1 versus DMSO

En

rich

me

nt

sco

re

-0.10

-0.15

-0.20

-0.05

0.00

0.05

0.10

En

rich

me

nt

sco

re

-0.20

-0.15

-0.10

-0.25

-0.30

-0.05

0.00

0.05

En

rich

me

nt

sco

re

-0.10

-0.15

-0.20

-0.25

-0.05

0.00

0.05

FDR=0 FDR=0 FDR=0

COV 413B

OVCA420

SKOV3

−1

0

1

2

3

−1

0

1

2

3

−1

0

1

2

3

0 50 100 150 200 250 kb

ZMIZ1

rpm

/bin

0 50 100 150 200 kb

ITGB6

COV 413B

OVCA420

SKOV3

0

2

4

0

2

4

0

2

4

rpm

/bin

7550250 kb

PAX8

COV413B

OVCA420

SKOV3

−1

0

1

2

3

−1

0

1

2

3

−1

0

1

2

3

rpm

/bin

SRC (14)

LAMA5 (34)

ITGB6 (54)

BCL2L1 (65)

FGFR3 (89)

ID2 (225)

ZMIZ1 (233)

IGF1R(297)

FOSL2 (331)

SMAD3 (440)

TGFA (466)

CCND1 (497)

JUN (659)

HES1 (569)

TRIO (488)

HOXB5 (2)

ITGB6 (16)

BCL2L1 (50)

CENPA (37)

EHF (79)

SLC2A1 (60)

JUNB (87)

IGFBP4 (103)

FZD2 (112)

ZMIZ1 (154)

HNF1B (156)

SREBF1 (166)

PDGFC (209)

PAX8 (242)

LIF (199)

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Figure 6.

A

B D

C

ELK3FOSL2FOSL1MYC

MYC FOSL1 FOSL2 ELK3 MYC FOSL1 FOSL2 ELK3 MYC FOSL1 FOSL2 ELK3

sgEGFP

CO

V 4

13

BO

VC

A4

20

SK

OV

3

sgMYC-1 sgMYC-2 sgFOSL1-1 sgFOSL1-2 sgFOSL2-1 sgFOSL2-2 sgELK3-1 sgELK3-2

0.0

0

50

100

0

50

100

0

50

100

0.5

1.0

0.0

0.5

1.0

0.0 sgE

GF

P

sg

MY

C-1

sg

MY

C-2

sgF

OS

L1-1

sgF

OS

L1-2

sgF

OS

L2-1

sgF

OS

L2-2

sg

EL

K3

-1

sg

EL

K3

-2

0.5

1.0

COV 413B

OVCA420

SKOV3

−1

0

1

2

−1

0

1

2

−1

0

1

2

0 25 50 75

COV 413B

OVCA420

SKOV3

−1

0

1

2

3

−1

0

1

2

3

−1

0

1

2

3

0 10 20 30

COV 413B

OVCA420

SKOV3

0

2

4

0

2

4

0

2

4

0 10 20 30

COV 413B

OVCA420

SKOV3

−1

0

1

2

3

4

−1

0

1

2

3

4

−1

0

1

2

3

4

0 5 10 15 kb kb kb kb

rpm

/bin

COV413B

**

*

**

*

**

* ** * * *

* *

* ** *

* *

rela

tive

pro

life

ratio

n

rela

tive

gene e

xpre

ssio

n (%

)

OVCA420

rela

tive

pro

life

ratio

n

SKOV3

rela

tive

pro

life

ratio

n

COV413B OVCA420 SKOV3

DMSOTHZ1

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-17-0078

Published OnlineFirst June 1, 2017.Mol Cancer Ther Zhenfeng Zhang, Huixin Peng, Xiaojie Wang, et al. CDK7-dependent transcriptional addiction in ovarian cancerPreclinical efficacy and molecular mechanism of targeting

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