op-1250 is a complete estrogen receptor antagonist (ceran

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 leslie@olema.com 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