op-1250 is a complete estrogen receptor antagonist (ceran
TRANSCRIPT
OP-1250 is a Complete Estrogen Receptor Antagonist (CERAN) that Lacks Agonist Activity on Cell Signaling and Proliferation in Breast Cancer Cells
Leslie Hodges-Gallagher, Richard Sun, David C. Myles, Cyrus L. Harmon, Peter J. KushnerOlema Oncology, San Francisco, CA
• Recent experiments conducted by us and third parties in nonclinical models of breast cancer suggest that ER degradation, as achieved by many SERDs, on its own is not sufficient to effectively treat tumors and that the ability to completely inhibit ER function is best achieved through complete antagonism. Here we show in nonclinical studies that OP-1250 is a potent complete ER-antagonist (CERAN) that completely inhibited breast cancer cell proliferation in multiple ER+ breast cancer cell lines.
• The expression of many genes that regulate proliferation, such as cyclin D1, are dependent on activation of AF1 of the ER𝝰. Unlike partial agonists, OP-1250 completely turned off signaling through AF1 of ER𝝰. Consequently, OP-1250 lacked agonist activity on the expression of genes involved in regulating a pro-proliferative and anti-apoptotic response.
• We previously reported that OP-1250 shrinks tumors in the HCI-013 PDX model of mutant ER𝝰 that models endocrine resistance.4 Here we show that OP-1250 was a complete antagonist of AF1 on Y537S and D538G mutants of ER𝝰.
• OP-1250 is a potential new orally bioavailable agent for ER+ breast cancer. We have recently initiated a phase I/II dose escalation and expansion trial of OP-1250 in patients with recurrent, locally advanced or metastatic ER+/HER2- breast cancer whose disease has progressed on endocrine therapy.
Acknowledgements:This presentation is the intellectual property of Olema Pharmaceuticals. Contact [email protected] for permission to reprint and/or distribute.
Conclusions and Clinical Significance of Complete ER Antagonism by OP-1250
Fig. 2. Alkaline phosphatase activity was used as a measure of AF1 activity. Ishikawa endometrial cells were treated with ligands in E2-depleted media for 3 days. Absorbance was read after incubation with a chromogenic substrate. Except panel B, cells were transfected with the indicated expression plasmid. Line labeled endogenous denotes endogenous AP activity of cells treated with empty expression vector. Figures 2A and 2D were adapted from previous poster4.
CERANs like OP-1250 and Fulvestrant completely antagonize AF1 activity mediated by mutant ER𝝰
OP-1250 lacks agonist activity on estrogen-regulated pro-proliferative, anti-apoptotic genes in breast cancer cells
Cell Division (GO.0051301)
CDC6ERCC6LSPC25KIF18BSKA1NCAPGKIFC1NDC80SPC24CDCA5CDK1TACC3KIF2CBUB1BCCNA2CDCA8SKA3UBE2CNUF2CDC20KIF20ANCAPHCDC25CSPAG5BIRC5BUB1SGO1CDC25AHELLSCCNE2CENPEKIF14OIP5ZWINTFBXO5CDC7KIF11NCAPG2CCNB2CENPFMAD2L1TPX2KNTC1CENPWLIG1FAM83DCDCA3CDCA2PTTG1NEK2SMC4SGO2CCNB1AURKASEPTIN3TUBA1BCENPJCHEK2CCNE1TIMELESSVRK1NCAPD3ZWILCHMASTLTIPINMIS18ACDK2HAUS5UBE2SSPDL1PSRC1STOX1PARD6BCCND1LRRCC1BORARBBP8USP37DSN1CCSAPHAUS3HAUS6HAUS2HAUS1MIS18BP1KNSTRNNCAPD2CKS2SMC2SAC3D1TUBBHAUS8CKS1BCCNFHAUS7SMC3SMC1AHAUS4MCMBPMAD2L2TUBA1CCDC25BREEP4KMT5ANEDD1SMC5RCC1RAD21SEH1LCKAP5BUB3CNTRLCDC14ACCNG2
E2 Laso
fox
OH
.Tam
Baz
edox
RA
D19
01
AR
N.8
10
AZD
.949
6
OP.
1250
Fulv
−20246
Cell Division (GO.0051301)
CDC6ERCC6LSPC25NDC80SKA1NCAPGKIFC1KIF18BCDCA5SPC24KIF2CCDK1TACC3SKA3BUB1BCCNA2CDCA8UBE2CNUF2CDC20KIF20ANCAPHCDC25CSPAG5BIRC5BUB1SGO1CCNE2CDC25AHELLSCENPEKIF14OIP5ZWINTFBXO5CDC7KIF11NCAPG2CCNB2CENPFMAD2L1TPX2KNTC1CENPWLIG1CDCA3FAM83DPTTG1NEK2CDCA2SMC4SGO2CCNB1AURKASEPTIN3TUBA1BCENPJCHEK2CCNE1TIMELESSVRK1NCAPD3ZWILCHMASTLTIPINMIS18ACDK2HAUS5UBE2SSPDL1PSRC1LRRCC1BORARBBP8HAUS1MIS18BP1KNSTRNNCAPD2CKS2SMC2SAC3D1TUBBCKS1BCCNFHAUS8STOX1PARD6BCCND1HAUS3USP37CCSAPDSN1HAUS6HAUS2SMC3SMC1AHAUS4MCMBPHAUS7MAD2L2TUBA1CCDC25BREEP4KMT5ANEDD1SMC5RCC1RAD21SEH1LCKAP5BUB3CNTRLCDC14ACCNG2
E2 Laso
fox
OH
.Tam
Baz
edox
RA
D19
01
AR
N.8
10
AZD
.949
6
OP.
1250
SRN
.927
Fulv
−20246
Cell Cycle Arrest (GO.0007050)
CDKN2CDDIASCDKN3KIF20ABARD1TP73MTBPMYCTGFB1RASSF1CDKN2DMSH2PPP2R3BPA2G4CDKN1CCDKN1AIRF6FOXO4ING4HBP1INHATP53INP1GAS1
E2 Laso
fox
OH
.Tam
Baz
edox
AR
N.8
10
AZD
.949
6
RA
D19
01
OP.
1250
Fulv
−4−20246
Apoptosis (GO.0006915)
CDCA7PIM1MCM2BRCA1ESPL1CDK1BUB1BDDIASTRAIPMELKBUB1BIRC5DPF1TPX2PLSCR1MTFP1PIDD1SGSM1RPS6KA3PHLDA2HIP1TNFAIP8BCL2L12G2E3CSE1LKANK2CASP2KLF11TIAM1PEG3RAD21CKAP2STK17BTHOC6CTSCGRAMD4MAGI3FOXO1TGFBR2SHBMEF2DCASP9CARD14CYFIP2ING4BNIPLBIKTRAF1GADD45GCASP14RASSF6BBC3INPP5DITGB2FBXO10EPHA7TP53INP1
E2 Laso
fox
OH
.Tam
Baz
edox
AR
N.8
10
RA
D19
01
AZD
.949
6
SRN
.927
OP.
1250
Fulv
−4−20246
Apoptosis (GO.0006915)
CDCA7PIM1MCM2BRCA1ESPL1CDK1BUB1BDDIASTRAIPMELKBUB1BIRC5DPF1TPX2PLSCR1MTFP1PIDD1SGSM1RPS6KA3PHLDA2HIP1TNFAIP8BCL2L12G2E3CSE1LKANK2CASP2KLF11TIAM1PEG3RAD21CKAP2STK17BTHOC6CTSCGRAMD4MAGI3FOXO1TGFBR2SHBMEF2DCASP9CARD14CYFIP2ING4BNIPLBIKTRAF1GADD45GCASP14RASSF6BBC3INPP5DITGB2FBXO10EPHA7TP53INP1
E2 Laso
fox
OH
.Tam
Baz
edox
AR
N.8
10
RA
D19
01
AZD
.949
6
SRN
.927
OP.
1250
Fulv
−4−20246
Figs. 3A-F. Gene expression heatmaps selected for specific gene ontology terms for genes significantly regulated by E2 (log2 fold-change over vehicle ≥ 1, adj. p ≤ 0.05). Expression was determined by RNA-seq analysis on CAMA-1 cells treated with 100 pM E2 or 316 nM antiestrogen for 24 hours in E2-depleted media.
Differential gene expression in CAMA-1 cellsupregulated* downregulated*
E2 1286 905OH-Tamoxifen 372 24Lasofoxifene 404 23Bazedoxifene 250 5AZD9496 143 6RAD1901 160 5ARN-810 164 5Fulvestrant 58 11OP-1250 13 1
Table 1. RNA-seq results on CAMA-1 cells treated with 100pM E2 or 316 nM antiestrogen in E2-depleted media for 24 hours. *Significance determined by Log2 fold-change vs. vehicle ≥1 and adj. p ≤ 0.05.
C D
B C
A Partial agonists, but not CERANs, activate AF1B
Apoptosis (GO.0006915)
CDCA7PIM1MCM2BRCA1ESPL1CDK1BUB1BDDIASTRAIPMELKBUB1BIRC5DPF1TPX2PLSCR1MTFP1PIDD1SGSM1RPS6KA3PHLDA2HIP1TNFAIP8BCL2L12G2E3CSE1LKANK2CASP2KLF11TIAM1PEG3RAD21CKAP2STK17BTHOC6CTSCGRAMD4MAGI3FOXO1TGFBR2SHBMEF2DCASP9CARD14CYFIP2ING4BNIPLBIKTRAF1GADD45GCASP14RASSF6BBC3INPP5DITGB2FBXO10EPHA7TP53INP1
E2 Laso
fox
OH
.Tam
Baz
edox
AR
N.8
10
RA
D19
01
AZD
.949
6
SRN
.927
OP.
1250
Fulv
−4−20246
Apoptosis (GO.0006915)
BRCA1CDCA7ESPL1CDK1PIM1MCM2BUB1BDDIASTRAIPMELKBIRC5BUB1TPX2DPF1PLSCR1MTFP1PIDD1SGSM1PHLDA2HIP1TNFAIP8BCL2L12RPS6KA3G2E3CSE1LKANK2CASP2KLF11TIAM1PEG3RAD21CKAP2STK17BTHOC6CTSCGRAMD4MAGI3FOXO1TGFBR2SHBMEF2DCARD14CYFIP2CASP9ING4BNIPLBIKTRAF1GADD45GITGB2RASSF6BBC3INPP5DCASP14FBXO10EPHA7TP53INP1
E2 Laso
fox
OH
.Tam
Baz
edox
AR
N.8
10
RA
D19
01
AZD
.949
6
OP.
1250
Fulv
−4−20246
Cell Proliferation (GO.0008283)
TCF19UHRF1TYMSMCM10PIM1KIF15CDK1TACC3MKI67KIF2CBUB1BE2F8AURKBDLGAP5CDC25ACDC25CTRAIPSTILMELKBUB1CENPFTPX2PLK1FAM83DPCNAMCM7CKLFIGFBP4CKS2POLR3GSIX2POLA1CKS1BDTYMKCDK5R1NASPSKP2CSE1LIRS2PRKDCTFDP1SRRTPA2G4CDC14AARCITED2ERBB4COL4A3BPDACH1APC2ZFP36L2TSPAN1CHRM1EMP1ERBB2SIPA1DDIT4
E2 Laso
fox
OH
.Tam
Baz
edox
RA
D19
01
AR
N.8
10
AZD
.949
6
OP.
1250
Fulv
−4−20246
Cell Division (GO.0051301)
CDC6ERCC6LSPC25NDC80SKA1NCAPGKIFC1KIF18BCDCA5SPC24KIF2CCDK1TACC3SKA3BUB1BCCNA2CDCA8UBE2CNUF2CDC20KIF20ANCAPHCDC25CSPAG5BIRC5BUB1SGO1CCNE2CDC25AHELLSCENPEKIF14OIP5ZWINTFBXO5CDC7KIF11NCAPG2CCNB2CENPFMAD2L1TPX2KNTC1CENPWLIG1CDCA3FAM83DPTTG1NEK2CDCA2SMC4SGO2CCNB1AURKASEPTIN3TUBA1BCENPJCHEK2CCNE1TIMELESSVRK1NCAPD3ZWILCHMASTLTIPINMIS18ACDK2HAUS5UBE2SSPDL1PSRC1LRRCC1BORARBBP8HAUS1MIS18BP1KNSTRNNCAPD2CKS2SMC2SAC3D1TUBBCKS1BCCNFHAUS8STOX1PARD6BCCND1HAUS3USP37CCSAPDSN1HAUS6HAUS2SMC3SMC1AHAUS4MCMBPHAUS7MAD2L2TUBA1CCDC25BREEP4KMT5ANEDD1SMC5RCC1RAD21SEH1LCKAP5BUB3CNTRLCDC14ACCNG2
E2 Laso
fox
OH
.Tam
Baz
edox
RA
D19
01
AR
N.8
10
AZD
.949
6
OP.
1250
SRN
.927
Fulv
−20246
G2/M transition (GO.0000086)
CDK1CCNA2CDC25ACDC25CCITPLK4MELKPKMYT1FOXM1CCNB2TPX2HMMRPLK1CEP152NEK2CCNB1AURKACENPJCHEK2CDK2MASTLHAUS5CEP135CEP72HAUS1BORACCP110CEP78CHEK1TUBBHAUS8HAUS3ALMS1FGFR1OPCEP76ODF2CKAP2CNTRLHAUS6HAUS2SKP2TUBG1HAUS4CEP131TUBB4BCDC25BHAUS7CDKN1A
E2 Laso
fox
OH
.Tam
Baz
edox
RA
D19
01
AR
N.8
10
AZD
.949
6
OP.
1250
Fulv
−20246
Cell Division (GO.0051301)
CDC6ERCC6LSPC25NDC80SKA1NCAPGKIFC1KIF18BCDCA5SPC24KIF2CCDK1TACC3SKA3BUB1BCCNA2CDCA8UBE2CNUF2CDC20KIF20ANCAPHCDC25CSPAG5BIRC5BUB1SGO1CCNE2CDC25AHELLSCENPEKIF14OIP5ZWINTFBXO5CDC7KIF11NCAPG2CCNB2CENPFMAD2L1TPX2KNTC1CENPWLIG1CDCA3FAM83DPTTG1NEK2CDCA2SMC4SGO2CCNB1AURKASEPTIN3TUBA1BCENPJCHEK2CCNE1TIMELESSVRK1NCAPD3ZWILCHMASTLTIPINMIS18ACDK2HAUS5UBE2SSPDL1PSRC1LRRCC1BORARBBP8HAUS1MIS18BP1KNSTRNNCAPD2CKS2SMC2SAC3D1TUBBCKS1BCCNFHAUS8STOX1PARD6BCCND1HAUS3USP37CCSAPDSN1HAUS6HAUS2SMC3SMC1AHAUS4MCMBPHAUS7MAD2L2TUBA1CCDC25BREEP4KMT5ANEDD1SMC5RCC1RAD21SEH1LCKAP5BUB3CNTRLCDC14ACCNG2
E2 Laso
fox
OH
.Tam
Baz
edox
RA
D19
01
AR
N.8
10
AZD
.949
6
OP.
1250
SRN
.927
Fulv
−20246
G1/S transition (GO.0000082)
RRM2ORC1CDC6CDT1MCM10TYMSCDC45ORC6CDCA5CDK1IQGAP3CDKN3CDC25AMCM2CCNE2PKMYT1CDC7FBXO5MCM5PCNAMCM7POLA2POLE2MCM8MCM6MCM3CCNE1DHFRCDK2PRIM1PRIM2DBF4POLEPOLA1MCM4RPA2RBBP8RPA3SKP2RANBP1CCND1USP37CDKN2DRPA1EIF4EBP1RCC1PPATCDKN1CCDKN1A
E2 Laso
fox
OH
.Tam
Baz
edox
RA
D19
01
AR
N.8
10
AZD
.949
6
OP.
1250
Fulv
−20246
OP-1250 is a complete antagonist that turns off both AF1 and AF2 of the wild type and mutant ER
References: 1) Shang and Brown, Science, 29 Mar 2002: Vol. 295, Issue 5564, pp. 2465-2468. 2) Webb, Nguyen, and Kushner, JBC, Vol. 278, 28 Feb 2003, pp. 6912–6920. 3) Fanning, et al., Nature Comm., Jun 2018, Vol. 9:2268. 4) Hodges-Gallagher, SABCS 2019 (poster).
Fig. 1. Cell proliferation in breast cancer cells was approximated by measuring fluorescence of a DNA binding dye after treating with 100 pM E2 and/or 316 nM antiestrogen in E2-depleted media for 7-8 days.
B
C
A D E
F
Partial agonists increase, or incompletely block, breast cancer cell proliferation
AF2
Tamoxifen(partial antagonist,
During cancer progression, kinase signaling turns on AF1
Partial antagonists (such as tamoxifen) have a short duration of response for treatment of breast cancer
TAM
Myc, cyclinD1, BCL2, ...
Proliferation duringCancer Progression
DNAbinding
AF1on
mTOR
PI3K
MAPK
c-SRC
EGFR
FGFR
IGFR
AF2
CERAN(a completeantagonist)
In breast cancer and uterine cells AF1 is activated through signaling pathways
CERANs block AF1 activity, even in the presence of signaling, preventing cell proliferation
No Cancer CellProliferation
CERANs shut down both activationfunctions (AF1 and AF2) of the ER
CERAN
N-CoR
and recruit N-CoR to inactivate AF1
Myc, cyclinD1, BCL2, ...
DNAbinding
AF1
mTOR
PI3K
MAPK
c-SRC
EGFR
FGFR
IGFR
Estrogen (E2)turns on AF2
Myc, cyclinD1, BCL2, ...
AF1on
AF2
DNAbinding
on
Signalingpathways
turn on AF1Both AF1 and AF2 can
drive transcription
The Estrogen Receptor (ER) is a tripartite protein with twodistinct transcriptional activation functions (AF1 and AF2)
Proliferation duringCancer Progression
mTOR
PI3K
MAPK
c-SRC
EGFR
FGFR
IGFR
A
0
20
40
60
80
100
120
Prol
ifera
tion
(%E2
)
+E2
Veh
OH
-Tam
Endo
x
Laso
fox
Baz
edox
AZD
9496
AR
N-8
10
RA
D19
01
OP-
1250Veh
OH
-Tam
Endo
x
Laso
fox
Baz
edox
AZD
9496
AR
N-8
10
RA
D19
01
Fulv
OP-
1250
Fulv
CAMA-1
0
20
40
60
80
100
120
Prol
ifera
tion
(%E2
)
HCC1500
Veh
+E2
Veh
OH
-Tam
Endo
x
Laso
fox
Baz
edox
AZD
9496
AR
N-8
10
RA
D19
01
Fulv
OP-
1250
0
20
40
60
80
100
Prol
ifera
tion
(%E2
)
SUM44
Veh
+E2
Veh
OH
-Tam
Endo
x
Laso
fox
Baz
edox
AZD
9496
AR
N-8
10
RA
D19
01
Fulv
OP-
1250
0
25
50
75
100
125
AF-
1 ac
tivity
(% E
2)
OH
-Tam
oxife
n
Laso
foxi
fene
Endo
xife
n
Baz
edox
ifene
AZD
9496
AR
N-8
10
RA
D19
01
OP-
1250
Fulv
estr
ant
-10 -9 -8 -7 -60.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Log [M]
AF-
1 ac
tivity
D538G
endogenous
-10 -9 -8 -7 -60.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Log [M]
AF-
1 ac
tivity
Y537S
endogenous
Vehicle
RAD1901
OP-1250
Endoxifen
Fulvestrant
-10 -9 -8 -7 -60.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Log [M]
Abs
405
nm
D538G
endogenous
-10 -9 -8 -7 -60.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Log [M]
Abs
405
nm
Y537S
Vehicle
OP-1250
ARN-810AZD9496
Fulvestrant
endogenous
0 50 100 150 200 250 300
% Wild type receptor
empty vector
DBD LBD/AF2
179 595
AF1 DBD
1 282
AF1 DBD LBD/AF2
1 185 251 595
DBD LBD/AF2
179 595Y537S
AF1 DBD LBD/AF2
1 595Y537S
AF1 DBD LBD/AF2
1 595D538G
Alkaline phosphatase activity is dependent on AF1