CDKN1C

ZNF467

FXYD6

NES

B

C

Targeting epigenetic dysregulation in bladder cancer through inhibition of EZH2Patricia J. Keller, Rosana Meyer, Feng Zhao, Linlin Ma, Emily Greenwald, Xinwei Han, Andrew Conery, Jennifer A. Mertz, and Patrick Trojer

Constellation Pharmaceuticals, 215 First Street, Cambridge, MA, USA 02142

EZH2EED

SUZ12

Enhancer TSS

H3K27me3

Abstract #A18

Conclusions

0.001 0.01 0.1 10

50

100

150

CPI-0209, µM

Rela

tive

Cell

Viab

ility

(%)

0.01 0.1 1 100

50

100

150

CPI-0209, µM

Rela

tive

Cell

Viab

ility

(%)

Day 6Day 12Day 18

0.001 0.01 0.1 10

50

100

150

CPI-0209, µM

Rela

tive

Cell

Viab

ility

(%)

0.0001 0.01 1 100 100000

50

100

150

HT1376

CPI-0209, nM

Ratio

H3K

27m

e3/H

3(%

)

0.0001 0.01 1 100 100000

50

100

150

HT1197

CPI-0209, nM

Ratio

H3K

27m

e3/H

3(%

)

0.0001 0.01 1 100 100000

50

100

150

T24

CPI-0209, nM

Ratio

H3K

27m

e3/H

3(%

)

0 10 20 300

500

1000

1500

2000

2500

HT1376 xenograft:tumor growth curve

days post grouping

Tum

or v

olum

e (m

m3 )

30%TGI

47%TGI

98%TGI82%TGI

0 10 20 30-15

-10

-5

0

5

10

HT1376 xenograft:body weight

days post grouping

Bod

y w

eigh

t (%

cha

nge)

Day 12 Day 310

10

20

30

40

50

HT1376 xenograft:tumor pharmacodynamic marker

Ratio

of H

3K27

me3

/H3(

%)

Vehicle10 mg/kg25 mg/kg75 mg/kg150 mg/kg

A

A

B

A B A

B

A. Biochemical selectivity panel with CPI-0209. B. H3K27me3 mark reduction. C. Maintenance of H3K27me3 mark reduction after washout. D. Efficacy, PK and PD in Karpas-422 xenograft.

References1. Kleer CG, et al. Proc Nat Acad Sci (2003) 100:116060-11611 3. Weikert S, et al. Int J Mol Med (2005) 16: 349-353 5. Robertson AG, et al. Cell (2017) 171: 540-556 2. Varambally S, et al. Nature (2002) 419:624-629 4. TCGA, Nature (2014) 507: 315-322 6. Bitler B, et al. Nature Med (2015) 21: 231-238

EZH2 is the catalytic subunit of the PRC2 complex, which methylates histone H3 to generate the transcriptionally repressive trimethylated modification state H3K27me3.

In cancers, EZH2 is mutated in hematologic malignancies and is frequently overexpressed in solid tumors, where its expression often correlates with poor prognosis due to repression of key tumor suppressors, and genes controlling cell fate and apoptosis (1-3).

Bladder cancers have frequent mutations in chromatin modifiers indicating that dysregulation of chromatin regulatory cues promotes tumor progression. Up to 89% of tumors have alterations in at least one histone modifier encoding gene, and 64% have alterations in components of the SWI/SNF (also known as BAF) chromatin remodeling complex (4,5).

Loss of function mutations in ARID1A (a SWI/SNF component) have been shown to create a dependence on EZH2 in ovarian clear cell carcinomas. ARID1A is mutated in 25% of muscle-invasive bladder cancers but it is unclear ARID1A mutations result in EZH2-dependence in bladder cancer (BLCA) (4-6).

Several first generation EZH2 inhibitors (CPI-1205, GSK126, Tazemetostat) are currently in clinical development. We have developed CPI-0209 as our second generation EZH2 inhibitor with enhanced potency in vitro and in vivo in models of both hematologic malignancies and solid tumors.

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0 100 200 300

TSC1TSC1

ASXL2ERCC2ERBB3

ELF3MLL

CREBBPSPTAN1

FAT1FGFR3ERBB2

ATMEP300

STAG2RB1

PIK3CA

KDM6AMLL2TP53

TCGA Bladder Cancer Data

number of mutations

Synin frame INDEL

Splice SiteFrameshiftNonsense

Missense

ARID1AARID1A mutations:25% of 412 cases47% are Nonsense

0.001 0.01 0.1 1 10ASH1LDNMT1

DNMT3aDNMT3b

DNMT3b/3LDOT1L

G9aGLP

MLL1 ComplexMLL2 ComplexMLL3 ComplexMLL4 Complex

NSD1NSD2

NSD2 (E1099K)NSD2 (T1150A)

NSD3PRDM9PRMT1PRMT3PRMT4

PRMT5(C449S)/MEP50 ComplexPRMT5/MEP50 Complex

PRMT6PRMT7PRMT8

SET1b ComplexSET7/9

SET8SETD2SMYD2SMYD3

SUV39H1SUV39H2

SUV420H1TV2

CPI-0209 IC50, [µM]

EZH2 (Y641F) ComplexEZH2 ComplexEZH1 Complex

DM

SO

CP

I-020

9Ta

zem

etos

tat

DM

SO

CP

I-020

9Ta

zem

etos

tat

DM

SO

CP

I-020

9Ta

zem

etos

tat

H3K27me3 EZH2 Total H3

Treated 96 hr

+ Washout 24 hr

+ Washout 48 hr

+ Washout 72 hr

C

D

0

10

20

30

40

Rat

io o

f H3K

27m

e3/H

3 (%

)

VehicleCPI-0209 25mg/kg, PO, QDTazemetostat 25 mg/kg, PO, QDTazemetostat 160 mg/kg, PO, BID

MSD ELISA

0.0001 0.01 10

50

100

150

HeLa cells:Cellular target engagement assay

Dose (µM)

Rat

io o

f H3K

27m

e3/H

3 (%

)

CPI-0209Tazemetostat

EC50:0.68 nM23.4 nM

Ovarian Cancer cell line panel

A. 18-day phenotypic assay GI50 values with an earlier 2nd Gen compound in OVCA lines (left) and CPI-0209 in BLCA lines (right). B. Representative data from 3 BLCA lines (2 ARID1Amut).

MSD ELISA

18-dayViability

CellCycle

EC50:0.147 nM

EC50:0.111 nM

EC50:0.392 nM

GI50:0.012 µM

GI50:0.003 µM

GI50:5.9 µM

Bladder Cancer cell line panel

Log2FC

HT1

376

SW78

0VM

CU

B1KU

1919

TCC

SUP

639V

T24

0 1 3.5

A. Heatmap of 90 genes upregulated in at least 4/7 BLCA cell lines after 4 days treatment with 250 nM CPI-0209. B. Time and dose-dependent upregulation of EZH2 target genes.

CPI-0209 is a potent and selective EZH2 inhibitor

Background EZH2 inhibition preferentially affects ARID1Amut bladder cancer cell models

B

A

Day 14 Day 640

5

10

15

20

25

HT1197 xenograft:tumor pharmacodynamic marker

atio

of H

3K27

me3

/H3

(%)

Vehicle75 mg/kg

0 10 20 30 40 50 600

500

1000

1500

HT1197 xenograft:tumor growth curve

days post grouping

Tum

or V

olum

e (m

m3)

82%TGI

0 10 20 30 40 50 60-20

-10

0

10

20

HT1197 xenograft:body weight

days post grouping

Body

wei

ght (

% c

hang

e)

Veh 10 25 75 1500

2

4

6

8 CDKN1C

fold

exp

ress

ion

mg/kg, PO, QD

Veh 10 25 75 1500

2

4

6

8 NES

fold

exp

ress

ion

mg/kg, PO, QD

Veh 10 25 75 1500

5

10

15 ZNF467

fold

exp

ress

ion

mg/kg, PO, QD

Veh 10 25 75 1500

20

40

60

80 FXYD6

fold

exp

ress

ion

mg/kg, PO, QD

C

CPI-0209 causes tumor regression in ARID1A mutant bladder cancer xenografts and is more efficacious than cisplatin single agent treatment

Conclusions

A. Viability and B. SubG1 effects after 7-days pre-treatment with CPI-0209 and 5-days combination treatment with cisplatin in vitro. C. Relative tumor growth for combination.

CPI-0209 is a highly potent inhibitor of EZH2, with 3-day EC50 values for cellular reduction of global H3K27me3 levels of 0.1-0.5 nM across a range of bladder cancer cell lines.

Bladder cancer cell lines harboring ARID1A mutations are preferentially sensitive to EZH2 inhibition, resulting in time- and dose-dependent cell growth inhibition that correlates with an increase in cell death.

Gene expression analysis after CPI-0209 treatment shows predominant upregulation of gene expression after 4 days of high-dose treatment. Selected genes show dose- and time-dependent upregulation in response to CPI-0209.

CPI-0209 is effective in causing tumor regression in two ARID1A mutant bladder cancer xenograft models and outperforms the standard of care chemotherapeutic cisplatin as a single agent.

Combination treatment of CPI-0209 with cisplatin enhances cell death in vitro and leads to enhanced tumor regression in vivo at well-tolerated doses.

This data supports the rationale for the development of CPI-0209 in patients with bladder cancer as a monotherapy and in combination with chemotherapeutic agents. The presence of ARID1A mutations may correlate with increased responsiveness to CPI-0209 treatment.

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CPI-0209 combines with cisplatin to enhance inhibition of BLCA cell growthCPI-0209 treatment leads to upregulation of EZH2 target genes

ARID1Amut ARID1Amut ARID1AWT

Gene induction at day 12

CPI-0209PO, QD

CPI-0209PO, QD

0 10 20 30-20

-10

0

10

HT1376 xenograft:body weight

days post grouping

body

wei

ght (

% c

hang

e)

0 10 20 300

500

1000

1500

HT1376 xenograft:tumor growth curve

days post grouping

rela

tive

tum

or v

olum

e(%

cha

nge)

82%TGI

54%TGI52%TGI

Vehicle75 mg/kg CPI-02092 mg/kg cisplatin4 mg/kg cisplatin

CPI-0209PO, QD

CisplatinIV, QW

CPI-0209sensitive

non-sensitive

0 10 20 30 40 500

500

1000

1500

2000

2500

Karpas-422 Lymphoma xenograft:tumor growth curve

days post grouping

Tum

or V

olum

e (m

m3)

VehicleCPI-0209 25 mg/kg, PO, QDTazemetostat 25 mg/kg PO, QDTazemetostat 160 mg/kg, PO, BID

Karpas-422 Lymphoma xenograft:plasma and tumor PK

Karpas-422 Lymphoma xenograft:tumor pharmacodynamic marker

HT11

97SW

780

HT13

76TC

CSU

PVM

CUB

1K

U191

964

7V T24

SW17

10JM

SU1

639V RT4

RT11

2SC

aBER J8

2Ca

l29

BFTC

905

639V

UMUC

356

37BC

3CKM

BC20.000

0.025

0.050

5

10

CPI-0

209

GI 50

,µM

ARID1A mutantARID1A WT

TOV1

12D

A278

0

TOV2

1G

CAO

V3

OVM

ANA

OV5

6

NIH

OVC

AR3

OA

W42

OVI

SE

SKO

V3

OVT

OKO OV7

CAO

V4 ES2

OV9

0

RM

G10.0

0.5

1.0

5

10

2nd

Gen

GI 50

,µM

ARID1A mutantARID1A WT

D 0.4 2 10 50 250 D 0.4 2 10 50 250 D 0.4 2 10 50 250 D 0.4 2 10 50 250

01234

10

20

30CDKN1C

fold

exp

ress

ion

CPI-0209 (nM)

D 0.4 2 10 50 250 D 0.4 2 10 50 250 D 0.4 2 10 50 250 D 0.4 2 10 50 250

012345

255075

100125

FXYD6

fold

exp

ress

ion

CPI-0209 (nM)

D 0.4 2 10 50 250 D 0.4 2 10 50 250 D 0.4 2 10 50 250 D 0.4 2 10 50 250

012345

10

20

30

40NES

fold

exp

ress

ion

CPI-0209 (nM)

D 0.4 2 10 50 250 D 0.4 2 10 50 250 D 0.4 2 10 50 250 D 0.4 2 10 50 250

012345

10

15

20

25ZNF467

fold

exp

ress

ion

CPI-0209, nM

1 day2 days

3 days4 days

D 0.004 0.008 0.016 0.031 0.063 0.125 0.25 0.5 10

50

100

CPI-0209, µM

% o

f tot

al

D 0.004 0.008 0.016 0.031 0.063 0.125 0.25 0.5 10

50

100

CPI-0209,µM

% o

f tot

al

D 0.039 0.078 0.156 0.313 0.625 1.25 2.5 5 100

50

100

CPI-0209,µM

% o

f tot

al

subG1G1SG2/M

Dosing end

Plasma D1 Plasma D12 Tumor D12100

1000

10000

100000

Plas

ma

(ng/

ml)

and

tum

or (n

g/g)

CPI-0209 25mg/kg QDTazemetostat 25 mg/kg QDTazemetostat 160 mg/kg BID

0.001 0.01 0.10

25

50

75

100 HT1376 SubG1

CPI-0209, µM

% s

ubG

1 in

crea

se

CPI-0209+ 0.123 µM+ 0.37 µM+ 1.1 µM+ 3.3 µM+ 10 µM

Cisplatin

0

25

50

75

100

Rela

tive

Cel

l Via

bilit

y(%

)

1.1 µMCisplatin

0.016 µMCPI-0209 Combo

0

25

50

75

100

% s

ubG

1 in

crea

se

1.1 µMCisplatin

0.016 µMCPI-0209 Combo

0.001 0.01 0.10

50

100

150 HT1376 viability

CPI-0209, µM

Rel

ativ

e Ce

ll Vi

abili

ty(%

) CPI-0209+ 0.123 µM+ 0.37 µM+ 1.1 µM+ 3.3 µM+ 10 µM

Cisplatin

HT1376 SubG1

HT1376 viability

Vehicle Cisplatin

+ CPI-0209

Vehicle CPI-0209

Vehicle Cisplatin

0 5 10 15 20 25 30 35 40 450

500

1000

1500

2000

days post grouping

rela

tive

tum

or v

olum

e(%

cha

nge)

0 5 10 15 20 25 30 35 40 450

500

1000

1500

2000

days post grouping

rela

tive

tum

or v

olum

e(%

cha

nge)

0 5 10 15 20 25 30 35 40 450

500

1000

1500

2000

days post grouping

rela

tive

tum

or v

olum

e(%

cha

nge)

HT1376 xenograft: tumor growth curve

0 5 10 15 20 25 30 35 40 45-10

-5

0

5

10

15

days post grouping

body

wei

ght (

% c

hang

e)

VehicleCisplatin 1.5 mg/kgCPI-0209 35 mg/kgCisplatin + CPI-0209

HT1376 xenograft: body weight

CPI-0209PO, QD

CisplatinIV, QW

+ Washout 96 hr

Upregulated in 7/7 lines

Upregulated in 6/7 lines

Upregulated in 4/7 or 5/7

lines