The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer
- Seragon Pharmaceuticals, United States
- Genentech, United States
- Vanderbilt-Ingram Cancer Center, United States
- Vanderbilt University, United States
Figures
Figure 1 with 1 supplement
GDC-0810 induces proteasome-dependent degradation of ERα and suppresses proliferation of MCF7 cells.
(A) GDC-0810 structure. (B) MCF7 ERα In-Cell Western assay comparing GDC-0810 potency to fulvestrant and 4-hydroxytamoxifen. ERα levels are quantified by immunofluorescence assay, in triplicate, …
Figure 1—figure supplement 1
MCF7 ERα In-Cell western assay correlates with MCF7 western blot for most ER binders.
The average In-Cell Western Emax activity for GDC-0810 and ERα benchmark antagonists are plotted versus the average ligand induced ERα degradation induced by 20 hr treatment with 1 μM ligand …
Figure 2 with 1 supplement
GDC-0810 promotes an ER confirmation distinct from 4OH-tamoxifen and fulvestrant.
(A) ERα conformational profiling. A mammalian 2-hybrid assay was performed to monitor interaction of ERα with 14 conformation selective peptide probes. Luciferase signal was measured after 24 hr of …
Figure 2—figure supplement 1
GDC-0810 does not promote the binding of ERα to known ER target sites.
ERα ChIP was performed on MCF7 cells following 45 min or 4 hr compound incubation. qPCR quantification was performed using oligonsucleotides directed to 10 distinct ERα binding sites. Data for ERα …
Figure 3
GDC-0810 displays mild estrogenic activity in vitro and in vivo.
(A) Alkaline phosphatase activity in Ishikawa endometrial cells stimulated with increasing concentrations of either 4OH-tamoxifen, GDC-0810 or fulvestrant, in the absence of estrogen. (B) Uterine …
Figure 4 with 4 supplements
Antitumor activity and pharmacodynamic response of GDC-0810 in tamoxifen-sensitive breast cancer xenograft models.
(A) Tamoxifen-sensitive MCF7 tumor bearing animals were dosed with vehicle, fulvestrant (50 mg/kg on days 1, 3, 8; then 25 mg/kg 2x/week, s.c.) or GDC-0810 (1, 10, 100 mg/kg/day, p.o.) for 28 days …
Figure 4—figure supplement 1
MCF7, HCI-003 and ZR-75-1 breast cancer xenograft models.
(A) Tamoxifen and fulvestrant anti-tumor activity in MCF7 xenograft. Crl:NU-Foxn1nu mice implanted with 17-β Estradiol pellets (0.72 mg/pellet/60 days, Innovative Research of America) were injected …
Figure 4—figure supplement 2
Activity of GDC-0810 in MCF7 xenograft tumors analyzed by FES-PET.
(A) MCF7 cells were injected s.c. into athymic ovariectomized mice supplemented with a 14-day release, 0.17-mg 17β-estradiol pellets. Four weeks later, mice bearing tumors ≥250 mm3 were randomized …
Figure 4—figure supplement 3
Pharmacodynamic activity of GDC-0810 and fulvestrant in HCI-003 xenograft tumors.
(A) ER target gene transcription was analyzed in tumors 8 hr after the final treatment on day 43. qRT-PCR data from individual tumors [3 for Vehicle (+E2), Vehicle (-E2), and GDC-0810 10 and 100 …
Figure 4—figure supplement 4
GDC-0810 is not efficacious in MDA-MB-231, an ER negative human breast cancer tumor model.
Crl:NU-Foxn1nu mice injected with 5 × 106 MDA-MB-231 cells were dosed orally with Vehicle (9% Peg-400:0.5% Tween–80:0.5% Povidone:90% 0.5% Carboxymethylcellulose), tamoxifen (60 mg/kg/day), GDC-0810 …
Figure 5 with 2 supplements
Antitumor activity of GDC-0810 in a tamoxifen-resistant breast cancer xenograft model.
(A) Tamoxifen-resistant MCF7 tumors were implanted in animals supplemented with 60-day release 0.18 mg 17β-estradiol pellets. Tumor bearing animals were dosed with vehicle, tamoxifen (120 mg/kg/day …
Figure 5—figure supplement 1
Effects of estradiol pellet on ERα levels in MCF7-TamR1 xenograft tumors.
TamR1 tumor fragments were implanted into Crl:NU-Foxn1nu mice implanted with either 0.72 or 0.18 mg/60 day 17-β estradiol pellets. Once tumors were established pellets were removed from 1 group. 28 …
Figure 5—figure supplement 2
Pharmacodynamic activity of GDC-0810 in MCF7-TamR1 xenograft tumors.
(A) ERα protein levels were determined by western blot analysis of tumors 8 hr following the final compound treatment on day 27. ERα levels were normalized to α-tubulin and normalized to the average …
Figure 6 with 1 supplement
GDC-0810 antagonizes the estrogen-independent ER.Y537S mutant.
(A) A cell free, FRET-based E2 competitive binding assay (E2 present at EC80) was used to determine the binding of GDC-0810 to ER.WT, ER.Y537S and ER.D538G ligand binding domains. Shown are the IC50 …
Figure 6—figure supplement 1
Characterization of MCF7 ER.Y573S knock-in cells.
(A) Schematic detailing the strategy to engineer the ER.Y537S mutation into the endogenous ESR1 locus in MCF7 cells. (B) Y537S-specific ddPCR on genomic DNA was used to evaluate the mutant allele …
Figure 7
GDC-0810 exhibits activity in T47D ER.D538G knock-in cells.
(A) Western blot analysis of T47D ER.WT and ER.D538G cells in the presence and absence of estrogen. (B) Cell viability assays of GDC-0810 activity in ER.WT and ER.D538G cells. (C) Representative …
Figure 8
GDC-0810 antagonizes estrogen-independent ER.Y537S-expressing tumors in vivo.
(A) MCF7 HA-ER.Y537S overexpressing tumors were implanted in animals without supplemental 17β-estradiol pellets. Tumor bearing animals were dosed with vehicle, tamoxifen (60 mg/kg/day p.o.), …
Author response image 1
MCF7 cells were treated with increasing concentrations of either GDC-0810 (left) or 4OH-tamoxifen (right), for 6 hours, prior to analysis of PGR and GREB1 as ER target genes, by Taqman assays.
Gene expression was normalized to DMSO control (set to 1) either in the presence or absence of 1nM E2.
Tables
Table 1
In vitro properties of GCD-0810.
https://doi.org/10.7554/eLife.15828.004| Compound | ER binding∗ | Transcription† | Cell viability‡ | ERα degradation§ | ||||
|---|---|---|---|---|---|---|---|---|
| ERα | ERβ | 3X ERE~LUC | CellTiter-Glo | In-Cell Western | ||||
| Ki [nM] | IC50 [nM] | Emax [% E2] | IC50 [nM] | Emax [% E2] | EC50 [nM] | Emax [% Veh.] | ||
| GDC-0810 | 3.8 ± 1.6 | 3.7 ± 4.0 | 1.3 ± 0.8 | 6.1 ± 2.8 | 2.5 ± 2.1 | 24.6 ± 3.3 | 0.65 ± 0.50 | 15.3 ± 3.4 |
| 4-OH Tam | 2.2 ± 1.3 | 3.6 ± 1.7 | 6.7 ± 3.6 | 4.7 ± 2.9 | 0.53 ± 0.25 | 48.0 ± 4.7 | 0.14 ± 0.04 | 51.9 ± 2.7# |
| Fulvestrant | 13.1 ± 10.8 | 13.2 ± 7.6 | 0.3 ± 0.2 | 4.1 ± 2.6 | 0.56 ± 0.70 | 25.4 ± 3.7 | 0.39 ± 0.18 | 6.4 ± 2.0 |
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∗ Binding affinities (Ki) of GDC-0810, 4-hydroxytamoxifen (4-OHT), and fulvestrant for ERα and ERβ. Shown are the mean and standard deviation of 3–4 experiments run in duplicate.
† ERα antagonist reporter assay. Results are the mean and standard deviation of 3 experiments.
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‡ Relative cell viability after 5 d incubation with compound. Shown are the mean and standard deviation of more than 50 assays run in triplicate.
§ Relative ERα immunofluorescence activity in MCF7 In-Cell Western.
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# The apparent reduction in ERα immunoreactivity is not reproduced in western blots.
Additional files
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Supplementary file 1
Supplementary data tables related to the specificity for GDC-0810 in binding and activation of ER relative to other nuclear hormone receptors.
(A) Radioligand binding assay (B) GDC-0810 nuclear hormone receptor reporter activity; agonist mode (C) GDC-0810 nuclear hormone receptor reporter activity; antagonist mode.
- https://doi.org/10.7554/eLife.15828.021
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Supplementary file 2
GDC-0810 and fulvestrant mouse pharmacokinetic data.
(A) GDC-0810 mouse pharmacokinetics (B) Fulvestrant plasma concentrations.
- https://doi.org/10.7554/eLife.15828.022
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Supplementary file 3
Primer sequences.
(A) Transcriptional Real-time PCR Oligonucleotide Sequence (B) ER-ChIP Real-time PCR Oligonucleotide Sequence.
- https://doi.org/10.7554/eLife.15828.023
