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. Kushner Olema 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 ER. Unlike partial agonists, OP-1250 completely turned off signaling through AF1 of ER. 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 ER that models endocrine resistance. 4 Here we show that OP-1250 was a complete antagonist of AF1 on Y537S and D538G mutants of ER. • 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. 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 poster 4 . CERANs like OP-1250 and Fulvestrant completely antagonize AF1 activity mediated by mutant ER OP-1250 lacks agonist activity on estrogen-regulated pro-proliferative, anti-apoptotic genes in breast cancer cells Cell Division (GO.0051301) CDC6 ERCC6L SPC25 KIF18B SKA1 NCAPG KIFC1 NDC80 SPC24 CDCA5 CDK1 TACC3 KIF2C BUB1B CCNA2 CDCA8 SKA3 UBE2C NUF2 CDC20 KIF20A NCAPH CDC25C SPAG5 BIRC5 BUB1 SGO1 CDC25A HELLS CCNE2 CENPE KIF14 OIP5 ZWINT FBXO5 CDC7 KIF11 NCAPG2 CCNB2 CENPF MAD2L1 TPX2 KNTC1 CENPW LIG1 FAM83D CDCA3 CDCA2 PTTG1 NEK2 SMC4 SGO2 CCNB1 AURKA SEPTIN3 TUBA1B CENPJ CHEK2 CCNE1 TIMELESS VRK1 NCAPD3 ZWILCH MASTL TIPIN MIS18A CDK2 HAUS5 UBE2S SPDL1 PSRC1 STOX1 PARD6B CCND1 LRRCC1 BORA RBBP8 USP37 DSN1 CCSAP HAUS3 HAUS6 HAUS2 HAUS1 MIS18BP1 KNSTRN NCAPD2 CKS2 SMC2 SAC3D1 TUBB HAUS8 CKS1B CCNF HAUS7 SMC3 SMC1A HAUS4 MCMBP MAD2L2 TUBA1C CDC25B REEP4 KMT5A NEDD1 SMC5 RCC1 RAD21 SEH1L CKAP5 BUB3 CNTRL CDC14A CCNG2 E2 Lasofox OH.Tam Bazedox RAD1901 ARN.810 AZD.9496 OP.1250 Fulv −2 0 2 4 6 Cell Cycle Arrest (GO.0007050) CDKN2C DDIAS CDKN3 KIF20A BARD1 TP73 MTBP MYC TGFB1 RASSF1 CDKN2D MSH2 PPP2R3B PA2G4 CDKN1C CDKN1A IRF6 FOXO4 ING4 HBP1 INHA TP53INP1 GAS1 E2 Lasofox OH.Tam Bazedox ARN.810 AZD.9496 RAD1901 OP.1250 Fulv −4 −2 0 2 4 6 −4 −2 0 2 4 6 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 cells upregulated* downregulated* E2 1286 905 OH-Tamoxifen 372 24 Lasofoxifene 404 23 Bazedoxifene 250 5 AZD9496 143 6 RAD1901 160 5 ARN-810 164 5 Fulvestrant 58 11 OP-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 AF1 B −4 −2 0 2 4 6 Apoptosis (GO.0006915) BRCA1 CDCA7 ESPL1 CDK1 PIM1 MCM2 BUB1B DDIAS TRAIP MELK BIRC5 BUB1 TPX2 DPF1 PLSCR1 MTFP1 PIDD1 SGSM1 PHLDA2 HIP1 TNFAIP BCL2L1 RPS6KA G2E3 CSE1L KANK2 CASP2 KLF11 TIAM1 PEG3 RAD21 CKAP2 STK17B THOC6 CTSC GRAMD MAGI3 FOXO1 TGFBR SHB MEF2D CARD1 CYFIP2 CASP9 ING4 BNIPL BIK TRAF1 GADD4 ITGB2 RASSF6 BBC3 INPP5D CASP14 FBXO10 EPHA7 TP53IN E2 Lasofox OH.Tam Bazedox ARN.810 RAD1901 AZD.9496 OP.1250 Fulv Cell Proliferation (GO.0008283) TCF19 UHRF1 TYMS MCM10 PIM1 KIF15 CDK1 TACC3 MKI67 KIF2C BUB1B E2F8 AURKB DLGAP5 CDC25A CDC25C TRAIP STIL MELK BUB1 CENPF TPX2 PLK1 FAM83D PCNA MCM7 CKLF IGFBP4 CKS2 POLR3G SIX2 POLA1 CKS1B DTYMK CDK5R1 NASP SKP2 CSE1L IRS2 PRKDC TFDP1 SRRT PA2G4 CDC14A AR CITED2 ERBB4 COL4A3BP DACH1 APC2 ZFP36L2 TSPAN1 CHRM1 EMP1 ERBB2 SIPA1 DDIT4 E2 Lasofox OH.Tam Bazedox RAD1901 ARN.810 AZD.9496 OP.1250 Fulv −2 0 2 4 6 G2/M transition (GO.0000086) CDK1 CCNA2 CDC25A CDC25C CIT PLK4 MELK PKMYT1 FOXM1 CCNB2 TPX2 HMMR PLK1 CEP152 NEK2 CCNB1 AURKA CENPJ CHEK2 CDK2 MASTL HAUS5 CEP135 CEP72 HAUS1 BORA CCP110 CEP78 CHEK1 TUBB HAUS8 HAUS3 ALMS1 FGFR1O CEP76 ODF2 CKAP2 CNTRL HAUS6 HAUS2 SKP2 TUBG1 HAUS4 CEP131 TUBB4B CDC25B HAUS7 CDKN1A E2 Lasofox OH.Tam Bazedox RAD1901 ARN.810 AZD.9496 OP.1250 Fulv −2 0 2 4 6 G1/S transition (GO.0000082) RRM2 ORC1 CDC6 CDT1 MCM10 TYMS CDC45 ORC6 CDCA5 CDK1 IQGAP3 CDKN3 CDC25A MCM2 CCNE2 PKMYT1 CDC7 FBXO5 MCM5 PCNA MCM7 POLA2 POLE2 MCM8 MCM6 MCM3 CCNE1 DHFR CDK2 PRIM1 PRIM2 DBF4 POLE POLA1 MCM4 RPA2 RBBP8 RPA3 SKP2 RANBP1 CCND1 USP37 CDKN2D RPA1 EIF4EBP RCC1 PPAT CDKN1C CDKN1A E2 Lasofox OH.Tam Bazedox RAD1901 ARN.810 AZD.9496 OP.1250 Fulv 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 during Cancer Progression DNA binding AF1 on mTOR PI3K MAPK c-SRC EGFR FGFR IGFR AF2 CERAN (a complete antagonist) 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 Cell Proliferation CERANs shut down both activation functions (AF1 and AF2) of the ER CERAN N-CoR and recruit N-CoR to inactivate AF1 Myc, cyclinD1, BCL2, ... DNA binding AF1 mTOR PI3K MAPK c-SRC EGFR FGFR IGFR Estrogen (E2) turns on AF2 Myc, cyclinD1, BCL2, ... AF1 on AF2 DNA binding on Signaling pathways turn on AF1 Both AF1 and AF2 can drive transcription The Estrogen Receptor (ER) is a tripartite protein with two distinct transcriptional activation functions (AF1 and AF2) Proliferation during Cancer Progression mTOR PI3K MAPK c-SRC EGFR FGFR IGFR A 0 20 40 60 80 100 120 Proliferation (%E2) +E2 Veh OH-Tam Endox Lasofox Bazedox AZD9496 ARN-810 RAD1901 OP-1250 Veh OH-Tam Endox Lasofox Bazedox AZD9496 ARN-810 RAD1901 Fulv OP-1250 Fulv CAMA-1 0 20 40 60 80 100 120 Proliferation (%E2) HCC1500 Veh +E2 Veh OH-Tam Endox Lasofox Bazedox AZD9496 ARN-810 RAD1901 Fulv OP-1250 0 20 40 60 80 100 Proliferation (%E2) SUM44 Veh +E2 Veh OH-Tam Endox Lasofox Bazedox AZD9496 ARN-810 RAD1901 Fulv OP-1250 0 25 50 75 100 125 AF-1 activity (% E2) OH-Tamoxifen Lasofoxifene Endoxifen Bazedoxifene AZD9496 ARN-810 RAD1901 OP-1250 Fulvestrant -10 -9 -8 -7 -6 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Log [M] AF-1 activity D538G endogenous -10 -9 -8 -7 -6 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Log [M] AF-1 activity Y537S endogenous Vehicle RAD1901 OP-1250 Endoxifen Fulvestrant -10 -9 -8 -7 -6 0.0 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 -6 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Log [M] Abs 405 nm Y537S Vehicle OP-1250 ARN-810 AZD9496 Fulvestrant endogenous 0 50 100 150 200 250 300 % Wild type receptor empty vector 'B' /B'/A)2 179 595 A)1 DBD 1 22 AF1 DBD LBD/AF2 1 185 251 55 'B' /B'/A)2 179 595 <537S AF1 DBD LBD/AF2 1 55 <537S AF1 DBD LBD/AF2 1 55 D538* Alkaline phosphatase activity is dependent on AF1