0.1110
LNCaP
22Rv1
VCaP
PC3
DU145
GI50 (µM CPI-1205)
0%
50%
100%
150%
200%
D4 D7 D11D14
Cell
viab
ility
Gene sets enriched :
GO_TISSUE_DEVELOPMENTGO_EPITHELIUM_DEVELOPMENT
GO_REGULATION_OF_CELL_DIFFERENTIATION
HALLMARK_ANDROGEN_RESPONSENELSON_RESPONSE_TO_ANDROGEN_UP
BENPORATH_PROLIFERATION
EZH2 inhibition as an effective treatment for metastatic castration-resistant prostate cancerWilliam D. Bradley¹, John P. McGrath¹, CC Yuan¹, Feng Zhao¹, Priyanka Sawant¹, Charlie Hatton², Xinwei Han², Barbara Bryant², Andrew Conery², and Patrick Trojer¹
Translational Sciences¹, Functional Genomics², Constellation Pharmaceuticals, 215 First Street, Cambridge, MA, USA 02142
Introduction
0
50
100
150
200
0.01 0.1 1 10
LNCaP-AR
enzalutamide (µM)
Rel
ativ
ece
llvi
abilit
y(%
) - doxycycline+ doxycycline
AR
ACTB
LNCaP-EV LNCaP-AR- 500 - 10 50 100 500 1000 ng/ml doxycycline
DayDox
22Rv1 EED KO
0+ + +- - -
4 8 14 15 18 0+ -
8
22Rv1 EED WT
- HL
LNCaP+CPI-1205
- HL
LNCaP+CPI-1205
0 2row z-score
-2
√ NELSON_RESPONSE_TO_ANDROGEN_UP √√ HALLMARK_ANDROGEN_RESPONSE √√ WONG_ADULT_TISSUE_STEM_MODULE √
SCHAEFFER_PROSTATE_DEVELOPMENT_48HR_UP √NUYTTEN_EZH2_TARGETS_UP √
√ HALLMARK_E2F_TARGETSGO_REGULATION_OF_CELL_PROLIFERATION √NUYTTEN_EZH2_TARGETS_DN √
Gene sets enriched:
enzalutamideCPI-1205
+- +
+
LNCaP
-- +
-
Enhancement ofenzalutamide effect
Maintenance ofenzalutamide effect
Maintenance ofCPI-1205 effect
EZH2 is the catalytic subunit of the PRC2 complex, and methylates histone H3 on lysine 27, resulting in suppression of gene transcription EZH2 mutations and increased expression are often observed in cancer, leading to repression of genes associated with apoptosis and differentiation (1-2)Over-expression in metastatic castration-resistant prostate cancer (mCRPC [3]), and decreased expression of target genes correlate with poor prognosis (4)While accounting for a small percentage of the overall disease incidence (5), mCRPC is very aggressive with high morbidity (6)Many mCRPC tumors remain dependent on androgen receptor signaling (ARS), but eventually develop resistance to ARS inhibitors, such as enzalutamide and abiraterone acetateRecent studies have linked EZH2 inhibition to re-sensitization to ARS inhibitors in neuroendocrine prostate cancer (NEPC) (7-9), and other PC models (10)Given this unmet need, and the link between EZH2 and advanced prostate cancer, we sought to determine if EZH2 inhibition can be utilized as an effective treatment and/or be combined with ARS inhibitor therapies to enhance their efficacyCPI-1205 is a potent, reversible, SAM-competitive small molecule inhibitor of EZH2 (11)CPI-1205 was well tolerated in a phase 1 clinical trial in patients with non-Hodgkin’s lymphoma (12), and is currently being evaluated in a phase 1b/2 trial in patients with mCRPC (ProSTAR, NCT03480646)
EZH2EED
SUZ12
Enhancer TSS
N
N
O
HNHN
O
O
FFF
CPI-1205
H3K27me3
EZH2 inhibition remains effective in cell lines with enhanced AR signaling
CPI-1205 combines with ARS inhibitors resulting insynergistic or additive cell growth inhibition
Transcriptomic analysis reveals CPI-1205 restricts PC growth in mannerdistinct from enzalutamide, yet enhances enza effect when combined
●
●
●
●
●
●
●
●●
EZH2 expression is high and polycomb repression signature low in mCRPC
Polycomb repression signatureYu, et al. (2007) Cancer Research
Comparison of RNA expression from TCGA vs. mCRPC datasets
TCG
Am
CR
PC
0
2
4
6
8 ****EZH2
TCG
Am
CR
PC
0
5
10
15
Adj L
og2
FPK
M
****AR
TCG
Am
CR
PC
-6000
-4000
-2000
0
2000
4000
ssG
SEA
pro
ject
ion
****Signature
Comparison of RNA expression data from the TCGA PRAD cohort (majority non mCRPC) to Robinson, et al. (2015) Cell and Robinson, et al. (2017) Nature mCRPC RNA sequencing data
0.01 0.1 1 100
25
50
75
100
LNCaP
CPI-1205 (µM)Rel
ativ
e ce
ll vi
abili
ty (%
)
D7D14D21D28
0.01 0.1 1 100
25
50
75
100
125PC3
CPI-1205 (µM)
Rel
ativ
e ce
ll vi
abili
ty (%
)
D7 D14 D21 D28sensitive
EZH2 inhibition effective in cell lines dependent on AR signaling
0.126
GI50:0.336
0.01 0.1 1 100
25
50
75
100
CPI-1205 (µM)
Rel
ativ
ece
llvi
abilit
y(%
)
- doxycycline+ doxycycline
0
50
100
150
200
250
300
0.01 0.1 1 10
LNCaPCSS +1 nM R1881
enzalutamide (µM)
Rel
ativ
ece
llvi
abilit
y(%
) D7D14D21D28
0.01 0.1 1 100
25
50
75
100
CPI-1205 (µM)
Rel
ativ
ece
llvi
abilit
y(%
)
0.1960.069
GI50:
LNCaP cell line engineered with doxycycline-inducible AR overexpression transgene; cell line grown in full serum for viability assays with serial cell re-seeding and drug re-application; day 14 data shown in figure
A
A
B
LNCaP cells grown in charcoal-stripped serum before stimulation with 1 nM R1881 to drive proliferation through AR; then grown in the presence of a titration of both CPI-1205 and enzalutamide, subjected to serial cell re-seeding and drug re-application
B
Specificity of CPI-1205 mediated cell growth inhibition
EED
Day: 0 4dox -
22Rv1 (EED KO +EED)dox +
6 8 10 12 14 15 17 18
EZH2AR
ACTB
H3K27me3
H3K27ac
H3
0.01 0.1 1 100
25
50
75
100
125
LNCaPdox-
CPI-1205 (µM)
Rel
ativ
e ce
ll vi
abilit
y (%
)
0.01 0.1 1 100
25
50
75
100
125
LNCaPdox+
CPI-1205 (µM)
doxycycline: - +LNCaP-EV 0.344 0.063
LNCaP-EZH2 (WT) 0.213 0.059
LNCaP-EZH2 (Y111L) 0.281 3.027
GI50 (μM)
Day 12Secondary mutations in EZH2, such as Y111L, identified in vitro using cellular models grown in the presence of EZH2 SMIs confer resistance to the inhibitor (Gibaja, et al. [2015] Oncogene)
LNCaP cell line engineered to express doxycycline-inducible expression of EZH2 WT or Y111L, then treated with CPI-1205 for 12 days
Endogenous EED-knockout 22Rv1 cell line generated with CRISPR in the presence of a doxycycline-inducible EED transgene
Doxycycline removed on day 0, and cells grown for 14 days before re-application on day 15
100.01 0.1 10
25
50
75
100
combination
CPI-1205 (µM)
Rel
ativ
e ce
ll vi
abilit
y (%
) +DMSO+0.123 µM enzalutamide+0.37 µM enzalutamide+1.111 µM enzalutamide+3.333 µM enzalutamide+10 µM enzalutamide
0.01 0.1 1 100
25
50
75
100
normalized combination
CPI-1205 (µM)
Rel
ativ
e ce
ll vi
abilit
y (%
)
Day 14 co-treatment
Single-cell RNA-sequencing (LNCaP)
“Population” RNA-sequencingBliss excess volume
0 2 2 2 1 -1 0 0 0
0 2 2 2 1 0 0 0 0
0 4 4 4 3 2 1 0 0
0 10 10 10 6 5 3 1 0
0 22 28 28 18 16 12 5 2
0 0 0 0 0 0 0 0 0
enza
CPI-1205
EZH2 inhibition is effective in prostate cancer xenograft models,and combines with enzalutamide for enhanced efficacy in LNCaP model
treatment group TGI regressions/ total
vehicle 0% 0/8
CPI-1205 35% 0/7
enzalutamide 40% 0/8
CPI-1205 + enzalutamide 65% 2/7
B
7.5
5.0
2.5
0.0
-2.5
-5.0-10 -5 0
Component 1
Com
pone
nt 2
5 10
Cell Treatment
DMSO (day 4)CPI-1205, high (day 4)CPI-1205, low (day 7)CPI-1205, high (day 7)
7.5
5.0
2.5
0.0
-2.5
-5.0-10 -5 0
Component 1
Com
pone
nt 2
5 10
Cell Cycle Phase
G1_SSG2G2_MM_G1none
LNCaP cells treated for 7 days with low (L) or high (H) dose CPI-1205 or DMSO control; heatmap shows differentially expressed genes (DEGs) for high dose CPI-1205 vs. DMSO comparison, and gene set enrichment analysis results
LNCaP dataset compared to 22Rv1 EED knockout cells (or wild type control) with re-expression of EED transgene; heatmap shows intersection of DEGs for LNCaP CPI-1205 vs. DMSO and 22Rv1 EED KO day 8 vs. 0
LNCaP cells treated as above were subjected to single cell RNA-seq (10X Genomics platform); results displayed as Monocle trajectory analysis
LNCaP cells treated with CPI-1205, enzalutamide, or combination of both followed by RNA-seq; heatmap shows DEGs for combination vs. DMSO
A
B
C
DC
A
Conclusions
D
PC cell models dependent on AR signaling are sensitive to EZH2 inhibition resulting in time- and dose-dependent cell growth inhibition, with models engineered to have elevated levels of AR signaling becoming even more sensitive to EZH2 inhibition despite reduced sensitivity to ARS inhibitorsCPI-1205 combined with ARS inhibitors, such as enzalutamide or abiraterone, in vitro and in vivo resulting in synergistic or additive cell or tumor growth inhibition, with the ability to inhibit the growth of ARS inhibitor-resistant cell linesTranscriptomic analysis revealed modulation of several specific pathways following EZH2 inhibition or EED knockout, showing modest upregulation of AR pathway genes, genes associated with prostate lineage, and importantly down-regulation of proliferation or cell cycle-associated genesDespite EZH2 inhibition modestly upregulating the AR pathway, when combined, CPI-1205 enhances the anti-androgen effect of enzalutamide while maintaining alteration of prostate lineage genes resulting in an anti-proliferative response that is more potent than using either single agent aloneThis data supports the rationale for the use of CPI-1205 in combination with ARS inhibitors in mCRPC, and importantly shows that EZH2 inhibition can overcome mechanisms of resistance to ARS inhibitors
●
●
●
●
●
D7 D14 D21 D28 D7 D14 D21 D28 D7 D14 D21 D28CPI-1205 >10 >10 >10 >10
enzalutamide >10 >10 >10 >10 0.00 0.24 0.32 0.50 na na na naabiraterone >10 >10 >10 >10 0.00 0.09 0.15 0.05 na na na na
CPI-1205 >10 >10 >10 >10enzalutamide >10 >10 >10 >10 0.00 0.08 0.19 0.23 na na na na
abiraterone >10 8.37 7.87 8.33 0.04 0.16 0.34 0.10 na na na naCPI-1205 >10 4.09 0.52 0.26
enzalutamide >10 4.52 0.92 1.01 0.69 3.36 2.80 2.17 na 0.21 0.34 0.51abiraterone >10 >10 >10 >10 0.25 3.69 3.28 3.36 na na 0.28 0.31
CPI-1205 >10 0.52 0.31 0.23enzalutamide >10 7.33 6.15 2.53 0.69 0.51 0.76 1.71 na 0.40 0.60 0.43
abiraterone >10 3.54 3.95 4.00 0.32 0.34 0.26 0.67 na 0.42 0.72 0.58CPI-1205 >10 0.95 0.42 0.20
enzalutamide 1.04 0.18 0.15 0.14 2.93 1.34 0.91 0.71 na 0.38 0.57 0.61abiraterone >10 5.10 2.06 1.79 1.81 3.05 1.67 1.59 na 0.30 0.61 0.68
CPI-1205 >10 >10 0.20 0.07enzalutamide >10 >10 3.88 3.86 0.47 3.46 5.25 2.76 na na na na
abiraterone >10 >10 >10 9.73 0.06 0.59 1.01 0.84 na na na naCPI-1205 >10 >10 0.65 0.32
enzalutamide 0.95 0.20 0.16 0.17 0.06 0.27 0.42 0.53 na na 0.44 0.56abiraterone nd nd nd nd nd nd nd nd nd nd nd nd
Bliss synergy scorecombo w/ CPI-1205
Combination indexcombo w/ CPI-1205Cell Line Treatment GI50 (μM)
LNCaP +1 nM R1881
LNCaP +60 pM R1881
VCaP
22Rv1
LNCaP
DU145
PC3
Cell lines treated with combination of CPI-1205 and either enzalutamide or abiraterone for up to 28 days
Cells were split, re-seeded, and fed fresh drug every 7 days
Combinatorial activity was determined using both Bliss and Loewe models of synergy, and combination index (CI) determined for the GI50 effect level
1. Béguelin W, et al. Cancer Cell. 2013;23(5):677–692. 2. Bradley WD, et al. Chem. Biol. 2014;21(11):1463–1475. 3. Varambally S, et al. Nature. 2002;419(6907):624–629. 4. Yu J, et al. Cancer Res. 2007;67(22):10657–10663. 5. Weiner AB, et al. Prostate Cancer Prostatic Dis. 2016;19(4):395–397. 6. Scher HI, et al. Clin. Cancer Res. 2005;11(14):5223–5232.
7. Beltran H, et al. Cancer Discov. 2011;1(6):487–495. 8. Ku SY, et al. Science. 2017;355(6320):78–83. 9. Mu P, et al. Science. 2017;355(6320):84–88. 10. Xiao L, et al. Cancer Res. 2018;78(20):5731–5740. 11. Vaswani RG, et al. J. Med. Chem. 2016;59(21):9928-41. 12. Harb W, et al. Ann. Oncol. 2018;29(suppl_3).
References
0 2 4 6 8-6000
-4000
-2000
0
2000
4000
EZH2, AdjLog2FPKM
ssG
SEA
proj
ectio
n
R2=0.51p<0.0001
TCGA datasetmCRPC dataset
Cell lines treated with titration of CPI-1205 for 28 days total with serial cell re-seeding and drug re-application every 7 days
vinculin
EZH2 (WT) EZH2 (Y111L)
dox: - ++ + + +- - - -
LNCaP
FLAGEZH2
H3K27me3
H3
μM CPI-1205: - 1.250.3 - 5
A
A
B
B
Loewe model
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.00 0.20 0.40 0.60 0.80 1.00
enza
luta
mid
e (µ
M)
CPI-1205 (µM)
Isobologram for GI50CI = 0.38
0 5 10 15 20 25 300
500
1000
1500
22Rv1 (castrated)
treatment day
tum
or v
olum
e (m
m3 )
0 5 100
200
400
600
LNCaP (intact)
treatment day
tum
or v
olum
e (m
m3 )
vehicle (PO, BID)CPI-1205 (200 mpk, PO, BID)enzalutamide (10 mpk, PO, QD)combination
vehicle (PO, BID)CPI-EZH2i (100 mpk, PO, BID)enzalutamide (10 mpk, PO, QD)
Day 50
2
4
6Tumor PD
ratio
H3K
27m
e3 /
H3
(%)
Day 280
1
2
348
12
Tumor PD
ratio
H3K
27m
e3 /
H3
(%)
treatment group TGI >50%TGI
vehicle 0% 0/8
CPI-EZH2i 75% 7/7
enzalutamide 12% 1/8
Y111EZHi
AR expre
ssion
AR mutat
ion
AR-V7 exp
ressio
n
ETS fusio
n
CPI-120
5 sen
sitive
no no no no no
no no no no no
yes no yes yes yes
yes H875Y yes no yes
yes T878A no no yes
