Author response: The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer (2016)

Full text Figures and data Side by side Abstract Introduction Results Discussion Materials and methods Appendix 1 References Decision letter Author response Article and author information Metrics Abstract ER-targeted therapeutics provide valuable treatment options for patients with ER+ breast cancer, however, current relapse and mortality rates emphasize the need for improved therapeutic strategies. The recent discovery of prevalent ESR1 mutations in relapsed tumors underscores a sustained reliance of advanced tumors on ERα signaling, and provides a strong rationale for continued targeting of ERα. Here we describe GDC-0810, a novel, non-steroidal, orally bioavailable selective ER downregulator (SERD), which was identified by prospectively optimizing ERα degradation, antagonism and pharmacokinetic properties. GDC-0810 induces a distinct ERα conformation, relative to that induced by currently approved therapeutics, suggesting a unique mechanism of action. GDC-0810 has robust in vitro and in vivo activity against a variety of human breast cancer cell lines and patient derived xenografts, including a tamoxifen-resistant model and those that harbor ERα mutations. GDC-0810 is currently being evaluated in Phase II clinical studies in women with ER+ breast cancer. https://doi.org/10.7554/eLife.15828.001 Introduction Breast cancer is the most commonly diagnosed cancer and the second leading cause of death in women (Edwards et al., 2014). About 75% of breast cancers express estrogen receptor alpha (ERα), a hormone-regulated transcription factor (Dunnwald et al., 2007). ERα-positive breast cancers typically respond well to therapy that attenuates ERα signaling, either by blocking the production of estrogens via aromatase inhibitors, or antagonizing the activity of estrogens through competitive binding of ER antagonists such as tamoxifen (Puhalla et al., 2012). While estrogen disrupting therapies are often effective both in the adjuvant and metastatic setting, patients frequently relapse after prolonged therapy (Chia et al., 2008; Dowsett et al., 2010; Davies et al., 2011; Mauri et al., 2006). Though resistance to endocrine therapies frequently emerges, relapsed tumors remain dependent on ER, which is highlighted by patient responses to second and third line endocrine therapies after failure of an earlier line (Mouridsen et al., 2009; Howell et al., 2005; Lewis and Jordan, 2005; Perey et al., 2007). ER signaling re-activation in the relapsed setting can occur due to changes in ligand sensitivity and specificity, for example, resistance to tamoxifen has been proposed to be a result of the selection of tumor cells that recognize the ER:tamoxifen complex as agonistic (Gottardis and Jordan, 1988; McDonnell and Wardell, 2010; Takimoto et al., 1999). More recently however, a series of mutations in the ligand binding domain of ERα have been found at a high prevalence (25–40%) in relapsed, metastatic patients, though are very rare in untreated populations (Jeselsohn et al., 2014; Li et al., 2013; Merenbakh-Lamin et al., 2013; Robinson et al., 2013; Toy et al., 2013). These mutations have been shown in overexpression experiments to confer estrogen-independent activity to the receptor, and to reduce the potency of, for example, tamoxifen. The continued dependence of breast cancer tumors on ERα provides a strong rationale to continue to target ER in both first line and relapsed/advanced settings. Selective ER Downregulators (SERDs) are competitive ERα antagonists that also induce a conformational shift of the receptor that results in ubiquitination and subsequent degradation of ERα, via the ubiquitin-proteasome system (Berry et al., 2008; Wijayaratne and McDonnell, 2001; Wijayaratne et al., 1999). The unique dual-function of SERDs (ER antagonism and depletion) may enable them to block ER signaling in cellular settings where other endocrine agents, such as tamoxifen or aromatase inhibitors have failed. Indeed, the clinical impact of fulvestrant as a treatment of recurrent, endocrine resistant disease, supports this notion (Perey et al., 2007).Though fulvestrant has served as an important proof of concept for the SERD approach [see also (Ellis et al., 2015)], it is limited by its poor pharmaceutical properties, which necessitates administration by intramuscular injection and limits the applied dose, exposure, and receptor engagement (Robertson et al., 2001; van Kruchten et al., 2015). The fulvestrant 500 mg regimen (500 mg on day 1, 14, 28; monthly thereafter) exhibited improvement in progression free survival and overall survival over the initially approved and marketed 250 mg regimen (Di Leo et al., 2010). The 500 mg regimen achieves higher plasma concentrations and a more rapid ascent to steady-state drug levels, which in turn is thought to result in superior modulation of ER signaling (Kuter et al., 2012; Ohno et al., 2010; Pritchard et al., 2010). However, the 500 mg dose does not fully saturate ER binding in patients, as inhibition of [18F]fluoroestradiol uptake was incomplete in 38% (6/16) of patients analyzed. Importantly, this lack of receptor occupancy was associated with lack of clinical benefit (van Kruchten et al., 2015). Together, these data demonstrate that SERDs have the potential to provide effective and well-tolerated therapy for postmenopausal women with advanced breast cancer, and highlight the need for the development of SERDs with optimized bioavailability and pharmacokinetic properties. Here we describe a novel, potent non-steroidal ER antagonist and degrader, GDC-0810, that is orally bioavailable and has strong anti-tumor activity in endocrine-sensitive and -resistant models of ER+ breast cancer. Results Identification of GDC-0810, a novel SERD With the goal of creating a next generation, orally bioavailable SERD, we prospectively optimized ERα degradation, antagonist activity, as well pharmacokinetic properties, starting with a triphenylalkene ER ligand scaffold (Lai et al., 2015). These efforts resulted in the identification of an indazole series of SERDs, including GDC-0810 (Figure 1A). Compounds were assessed for their ability to modulate ERα protein levels in MCF7 breast cancer cells using a quantitative In-Cell Western (ICW) immunofluorescence assay, which enabled determination of compound potency and maximal activity in a high throughput format. GDC-0810 was prioritized for further characterization based on its ability to robustly reduce cellular ERα levels within 4 hr, to levels approaching that observed with fulvestrant, with sub-nanomolar potency (Figure 1B and Table 1). The active metabolite of tamoxifen, 4-hydroxytamoxifen (4OH-tamoxifen), exhibited some activity in this assay, though it failed to reduce ERα signal to the level achieved by fulvestrant or GDC-0810. The ability of GDC-0810 to reduce steady-state levels of ERα was confirmed using a Western Blot assay (Figure 1C). We further demonstrated that GDC-0810-mediated ERα depletion is dependent on the 26S proteasome, since addition of the proteasome inhibitor MG132, fully blocked GDC-0810 depletion of ERα, similar to the effect of MG132 treatment in preventing fulvestrant-mediated ER turnover (Figure 1C,D). Figure 1 with 1 supplement see all Download asset Open asset 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, 4-hr post compound treatment. Error bars are SEM. (C) Western blot analysis assessing the effect of GDC-0810 (100 nM), and fulvestrant (100 nM) (D) on ERα levels at 2, 4 or 6 hr of treatment, in the presence or absence of the 26S proteasome inhibitor MG132 (10 μM). (E) MCF7 cell viability assay comparing GDC-0810 activity to fulvestrant and 4OH-tamoxifen, in the absence of exogenous estradiol. Viable cells are presented as percent CellTiter-Glo luciferase activity relative to the vehicle control after 5 day compound incubation. Error bars represent standard deviation from the mean, from biological quadruplicates. (F) MCF7 cell viability assay comparing GDC-0810 activity to fulvestrant and 4OH-tamoxifen, in the presence of 0.1 nM estradiol. Viable cells are presented as percent CellTiter-Glo luciferase activity relative to the vehicle control after 5 day compound incubation. https://doi.org/10.7554/eLife.15828.002 Table 1 In vitro properties of GCD-0810. https://doi.org/10.7554/eLife.15828.004 CompoundER binding∗Transcription†Cell viability‡ERα degradation§ERαERβ3X ERE~LUCCellTiter-GloIn-Cell WesternKi [nM]IC50 [nM]Emax [% E2]IC50 [nM]Emax [% E2]EC50 [nM]Emax [% Veh.]GDC-08103.8 ± 1.63.7 ± 4.01.3 ± 0.86.1 ± 2.82.5 ± 2.124.6 ± 3.30.65 ± 0.5015.3 ± 3.44-OH Tam2.2 ± 1.33.6 ± 1.76.7 ± 3.64.7 ± 2.90.53 ± 0.2548.0 ± 4.70.14 ± 0.0451.9 ± 2.7#Fulvestrant13.1 ± 10.813.2 ± 7.60.3 ± 0.24.1 ± 2.60.56 ± 0.7025.4 ± 3.70.39 ± 0.186.4 ± 2.0 ∗ 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. ‡ 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. # The apparent reduction in ERα immunoreactivity is not reproduced in western blots. In general, data generated in the ICW assay correlated well with Western Blot assays (Figure 1—figure supplement 1). For certain ligands which showed modest activity in the ICW assay, but did not induce ERα degradation by Western Blot, such as 4OH-tamoxifen, the observed reduction of ERα signal in the ICW assay may be the result of changes in protein conformation, protein complex formation, or subcellular localization that limit the detection of the antibody epitope in the in situ ICW assay. In cell-free radio-ligand competitive binding assays, GDC-0810 binds both ERα and ERβ with low nanomolar affinity [Table 1, see also (Lai et al., 2015)]. In MCF7 cells GDC-0810 effectively antagonizes E2-mediated transcriptional activation of an ER reporter construct and inhibits cell proliferation with nanomolar potency and efficacy similar to that of fulvestrant and 4OH-tamoxifen (Table 1). GDC-0810 is selective against the other nuclear hormone receptor (NHR) family members as monitored by competitive binding and reporter activation assays (Supplementary file 1A–C). GDC-0810 induces a distinct ERα conformation versus tamoxifen and other ER therapeutics, and does not exhibit tamoxifen-like ER agonism in MCF7 cells Nuclear receptors impart biological action through ligand-induced conformational change resulting in exposure of co-regulator interaction surfaces. To assess the ERα conformation induced by GDC-0810 relative to other SERMs and SERDs, we utilized a mammalian 2-hybrid based peptide interaction assay. This assay, run in the presence of saturating concentrations of ER ligands, differentiates ligand-induced ER conformations based on the ability of the ligand-bound receptor to interact with discriminatory peptide probes (Iannone et al., 2004; Norris et al., 1999; Paige et al., 1999). We included in our analysis fulvestrant, 4OH-tamoxifen, a number of additional clinically approved ER ligands, such as bazedoxifene, and also GW7604. GW7604 is an active metabolite of GW5638, an ER ligand that entered Phase I clinical trials for breast cancer, but is not currently in further clinical development. Like GDC-0810, GW5638 is a non-steroidal dual function ER antagonist and degrader (Lai et al., 2015; Willson et al., 1994, Willson et al., 1997). The GDC-0810:ER complex recruits a distinct set of peptides relative to the other ER:ligand complexes profiled here, with the exception of GW7604 (Lai et al., 2015). GDC-0810 thus induces an ER conformational profile that is different from that induced by 4OH-tamoxifen, fulvestrant and other marketed SERMs and similar to GW7604 (Figure 2A). Since the biological activity of ERα is determined by the conformation of the receptor and its ability to interact with other regulatory proteins, this result implies differences in the response of ERα to GDC-0810 and the clinically approved ERα therapeutics. Figure 2 with 1 supplement see all Download asset Open asset 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 compound treatment (1 μM for all compounds). Interaction profiles of ERα therapies, GDC-0810 and vehicle control from biological triplicates were analyzed by hierarchical clustering using the Ward algorithm and standardized data. (B) Transcriptional activity of benchmark ERα ligands in MCF7 cells, in the absence of exogenous estrogen. Transcriptional activity was monitored using a SERM discriminatory target gene set following 24 hr 1 μM ligand treatment. Data was log2 normalized followed by standardization and hierarchical clustering. (C) Doxycycline-inducible MCF7 cells were pre-cultured in estrogen-depleted medium with 100 ng/ml doxycycline for 2 weeks. Cells were then cultured in medium containing 10, 100 and 500 nM fulvestrant or GDC-0810 for 5 days, and the effect on levels of ER, PR and Cyclin D1 were assessed by Western Blot analysis. (D, E) Doxycycline-inducible MCF7 cells were pre-cultured in estrogen-depleted medium with 10 ng/ml doxycycline for 2 weeks before treatment. Cells were treated with a range of doses of fulvestrant or GDC-0810 for 7 days in estrogen-depleted medium with or without 0.1 nM E2. Cell viability was determined by CellTiter-Glo assay. https://doi.org/10.7554/eLife.15828.005 Tamoxifen is a Selective ER Modulator (SERM), and as such can either antagonize, or agonize ER signaling, in a gene- and cell-specific manner. Though 4OH-tamoxifen primarily antagonizes estrogen-dependent ER signaling in breast cancer cells, its partial agonistic activity has been revealed through gene expression profiling in the absence of estrogen, in MCF7 cells (Wardell et al., 2013). To assess the potential agonistic activity of GDC-0810 we evaluated the consequence of GDC-0810 treatment on a previously described SERM discriminatory gene set, likewise, in the absence of estrogen in MCF7 cells (Wardell et al., 2013). In line with earlier observations, 4OH-tamoxifen induces a strong up-regulation of a gene set that includes the estrogen-responsive genes, AGR2 and RASGRP, while fulvestrant fails to do so (Figure 2B). GDC-0810 most closely resembled fulvestrant and GW7604 in this analysis, displaying little of the transcriptional agonist activity observed with the SERMs 4OH-tamoxifen, lasofoxifene, arzoxifene or endoxifene and often demonstrating inverse agonist activity on estradiol responsive genes (Figure 2B). These findings are consistent with ER-ChIP analysis demonstrating that GDC-0810 treatment, similar to fulvestrant, can reduce the levels of ERα associated with chromatin on ERα target genes (Figure 2—figure supplement 1). Wardell et al. have demonstrated that fulvestrant and bazedoxifene can inhibit ER signaling and estrogen-dependent proliferation in the absence of ER degradation (Wardell et al., 2011, Wardell et al., 2013). This was demonstrated using an experimental system whereby ER was over-expressed in an attempt to saturate the degradation machinery, enabling an uncoupling of ER degradation from ER antagonism. Likewise, we sought to determine if GDC-0810 can inhibit ER signaling in the absence ER degradation, using a similar strategy (Wardell et al., 2011, Wardell et al., 2013). Over-expression of ER in doxycycline-inducible MCF7 cells resulted in the induction of the ER target gene, PR, consistent with ligand-independent activation of the pathway. Treating these cells with fulvestrant resulted in both depletion of ER, as well as suppression of PR. In the case of GDC-0810 treatment, PR was likewise suppressed, though ER levels remained high (Figure 2C). Cell viability assays demonstrated that both fulvestrant and GDC-0810 suppress proliferation of MCF7 cells over-expressing ER, with EC50s and IC50s similar to cells expressing endogenous levels of ER, despite the retention of high ER levels in the over-expressing cells treated with GDC-0810 (Figure 2D,E). These data imply that GDC-0810 has the ability to antagonize ER function independent from its degradation activity, consistent with the demonstration that GDC-0810 displaces the co-activator PGC1α in cell free biochemical assays, in which ER degradation does not occur (see below, Figure 6A). GDC-0810 displays mild estrogenic activity in uterine models in vitro and in vivo Since the activity of ER ligands can be tissue/cell-type dependent, we next evaluated the effects of GDC-0810 in the uterus, first making use of the Ishikawa endometrial cell line, in which alkaline phosphatase expression is controlled by ER (Holinka et al., 1986; Littlefield et al., 1990). In line with previous reports, 4OH-tamoxifen robustly increases alkaline phosphatase activity, while fulvestrant shows no such effect (Figure 3A). GDC-0810 modestly increases alkaline phosphatase activity, though plateaus at low concentrations, below the level stimulated by 4OH-tamoxifen (Figure 3A). To follow up on this observation we next assessed the consequences of GDC-0810 administration on the uterus of juvenile rats. Tamoxifen at 60 mg/kg, dosed orally, once a day for 3 days, increases uterine wet weight, though not to the extent driven by the administration of 0.1 mg/kg 17α estradiol over the same time period (Figure 3B). In contrast, Fulvestrant at 50 mg/kg acts as an inverse agonist, resulting in a significant decrease in uterine weight relative to the control group. GDC-0810 dosed at 0.1 or 10 mg/kg did not significantly alter uterine wet relative to the vehicle control (Figure 3B). Importantly though, histological analysis and quantification of endometrial cell height showed that, like estradiol and tamoxifen, GDC-0810 induced a cuboidal to columnar morphological change in the epithelial cells, while the epithelia from fulvestrant-treated animals remained cuboidal (Figure 3C,D). In line with the observed morphological changes to the endometrial epithelium, gene expression analysis demonstrated that GDC-0810 induces ER target genes, including C3 (Figure 3E). These in vivo phenotypes were observed at a GDC-0810 dose of 0.1 mg/kg and were not significantly different at the 10 mg/kg dose, suggesting saturation of the effect at low doses. Together, these data demonstrate that GDC-0810 mildly stimulates ER signaling in the uterus, similar to the recently described SERD AZD9496 (Weir et al., 2016). Intriguingly, IHC analysis of ER in the uterus showed that GDC-0810 only modestly reduces ER levels in this context, similar to estradiol, while fulvestrant robustly depletes ER. Figure 3 Download asset Open asset 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 wet weight (UWW) measurements from juvenile rats treated with individual specified ER ligands; 17α-estradiol was dosed at 0.1 mg/kg as a positive control. (C) Haematoxylin and eosin (H&E) staining, and anti-ER IHC were performed on tissues dissected as in B. (D) Endometrial cell height was digitally measured from the basement membrane to the apical (luminal) surface, using a digitally scanned image at 20X magnification. Three digital measurements were taken from each section, and 2 mice per condition were scored. Results are displayed as the mean endometrial cell height from two animals ± standard error (n = 2). (E) Gene expression in the uterus, after treatment with indicated compounds, was assessed using a rat Fluidigm panel. (F) H-scores were used to quantify the ER IHC from (C). H-scores were estimated manually, incorporating both intensity and percentage of positive using the following to in and GDC-0810 tumors and suppresses estradiol uptake in breast cancer tumor models In GDC-0810 low and bioavailability (Lai et al., 2015). Importantly, GDC-0810 plasma concentrations with the applied dose and an of dosed by at 100 (Supplementary file 2A). To determine the ability of GDC-0810 to inhibit stimulated tumor in we treated MCF7 tumor mice with day with GDC-0810, from 1 to 100 Fulvestrant was by with an dose of 50 mg/kg on days 1, 3 and and subsequent of mg/kg per to similar to those achieved in the In the MCF7 GDC-0810 displayed dose dependent efficacy (Figure The 100 dose tumor an effect similar to of estrogen at the of In contrast, fulvestrant, at a clinically dose as well as at a higher dose mg/kg, 3 per only modest tumor inhibition (Figure and Figure supplement 1A). is important to that the of the fulvestrant response to tumor than or tumor was not a function of low exposure, as the plasma concentrations at the mg/kg dose were on the clinical exposure of the fulvestrant 500 mg clinical regimen et al., 2010; Pritchard et al., 2010). Gene expression analysis of MCF7 tumors demonstrated that GDC-0810 100 evaluated on day robustly ER target genes, including and (Figure Figure supplement Indeed, the gene expression changes induced by GDC-0810 are similar and in some more those induced by of the estrogen at the of the that GDC-0810 and attenuates ER signaling (Figure Figure supplement Figure 4 with 4 see all Download asset Open asset activity and response of GDC-0810 in breast cancer (A) MCF7 tumor animals were dosed with fulvestrant mg/kg on days 1, then mg/kg or GDC-0810 10, 100 for days in the presence of mg Gene expression analysis of tumors treated with 100 mg/kg GDC-0810, to tumors in the presence or absence of estrogen were on day of the this was a from that shown in GDC-0810 plasma is also = Figure supplement 1B for an of (D) of MCF7 tumors in the of mice treated with vehicle or GDC-0810 (100 were taken hr after the on the day of treatment. (E) change in after 6 days of treatment. the mean percent change in estradiol mice exhibited an of of mice treated with 10 mg/kg and 100 mg/kg exhibited a and reduction in to (F) patient derived tumors were in mice containing a 1 mg animals were dosed with fulvestrant mg/kg, GDC-0810 (10 or 100 for vehicle treated the at treatment to dependence on estradiol. an additional analysis in MCF7 xenografts, we next monitored tumor uptake by which has previously been used as a of the activity of ER in et al., To determine the ability of GDC-0810 to effectively ER in was performed on mice MCF7 tumors following a treatment with GDC-0810. to GDC-0810 administration signal was in both the vehicle and (Figure However, after the dose, GDC-0810 at 10 and 100 uptake by and percent (Figure on day the GDC-0810 treatment displayed a dose dependent decrease in tumor and in ERα and PR (Figure supplement 2). While the in reduction of uptake the 10 and 100 treatment did not each dose resulted in a uptake to that in This that maximal GDC-0810-mediated ER as a result of receptor occupancy degradation, is achieved with a dose of and in this model We next evaluated the activity of GDC-0810 in patient derived Like tumors are dependent and tamoxifen (Figure supplement 1C). GDC-0810, dosed at 10 and 100 in mice with 1 mg tumor while fulvestrant dosed at the clinical exposure induced tumor approaching that observed in vehicle treated animals estradiol were at of the period (Figure with GDC-0810 SERD activity in tumors after days of displayed ERα target gene transcription and ERα protein levels, as monitored by quantitative and Western Blot, (Figure supplement GDC-0810 also displayed anti-tumor activity in an additional ER+ breast cancer model (Figure supplement Importantly, GDC-0810 did not exhibit anti-tumor activity in an ER human breast cancer tumor model (Figure supplement of efficacy in this model is consistent with the GDC-0810 mechanism of action being through ER. GDC-0810 has anti-tumor activity in a tamoxifen-resistant MCF7 model We previously generated a tamoxifen-resistant by MCF7 tumor mice with tamoxifen a resistant tumor (Lai et al., 2015). with other models of tamoxifen treatment of the with tamoxifen stimulated tumor relative to the vehicle and et al., 2001; and Jordan, 1988; et al., 2015)]. earlier showed that treatment with GDC-0810 at 100 induced tumor in this while fulvestrant at mg/kg exhibited only modest tumor inhibition (Lai et al., 2015). These experiments were in mice with high levels of estradiol, with estradiol being via mg 60 day that are by the to and estradiol. these the high reduces the ERα protein levels, thus the of ERα protein as a (Figure supplement 1). this of the the anti-tumor and response to GDC-0810 treatment was evaluated in the model in mice with to concentrations of estradiol mg 60 day to These are to tumor but only modestly reduce ERα levels (Figure supplement 1). Figure 5 with 2 see all Download asset Open asset activity of GDC-0810 in a tamoxifen-resistant breast cancer (A) MCF7 tumors were in animals with mg animals were dosed with tamoxifen fulvestrant mg/kg, or GDC-0810 or 100 for days (B) of IHC for ERα and in tamoxifen-resistant MCF7