Introduction
The estrogen receptor (ER/
ESR1) is expressed in a majority of breast cancers, and drugs that inhibit ER signaling are the cornerstone of breast cancer pharmacotherapy for ER-positive/HER2-negative disease [
1]. These targeted approaches include the Selective Estrogen Receptor Modulator (SERM) tamoxifen that acts as a competitive ER antagonist in the breast, and aromatase inhibitors (AIs) that inhibit aromatase, the enzyme responsible for estrogen production [
2,
3]. However, the development of resistance limits the duration of meaningful therapeutic responses. Mechanisms of resistance to these endocrine therapies include cell cycle dysregulation and activation of alternative growth factor signaling pathways [
1]. For example, activation of MAPK, PI3K, and GSK-3 can result in increased phosphorylation of ER or its attendant coregulatory proteins leading to ligand-independent ER activity and resistance [
4‐
7]. Recently, genomic alterations in the ER gene itself, including amplification, translocation, and ligand binding domain mutations (most frequently ER-D538G and ER-Y537S) have emerged with AI therapy [
1,
8‐
10].
After progression during tamoxifen and AI therapy, other endocrine treatments including the steroidal selective estrogen receptor downregulator (SERD) fulvestrant (Faslodex
®) are generally used [
11]. SERDs are a class of ER antagonists that in addition to competitively displacing estrogens, also trigger ER downregulation [
12]. Although initially successful, the onset of resistance limits durable responses when used as a monotherapy. Therefore, in an effort to improve the therapeutic lifespan of endocrine treatments for metastatic breast cancer, combination regimens have been extensively studied. Clinical trials using a combination of AI or fulvestrant with pan-PI3K or mTOR inhibitors have been promising but inconclusive, and toxicity often remains an impediment to dose escalation [
13‐
16]. Therefore, CDK4/6 inhibitors have emerged as a favored option when considering combination endocrine therapies [
17‐
20]. However, the poor bioavailability of fulvestrant, coupled with its intramuscular route of administration and the long time to steady state blood levels, compromises its clinical use [
21,
22]. Indeed, even at the higher clinical dose (500 mg) of fulvestrant, pharmacodynamic imaging suggests incomplete receptor saturation [
23].
Collectively, these data highlight an unmet need for a safe, orally bioavailable SERD with appropriate pharmaceutical properties. Herein we describe the preclinical development of G1T48 (rintodestrant), an orally bioavailable, potent, and selective non-steroidal ER antagonist and downregulator [
24]. G1T48 was found to robustly inhibit ER activity in multiple in vitro models of endocrine therapy resistance, including those harboring ER mutations or growth factor activation. Importantly, G1T48 demonstrated robust antitumor activity in an animal model of early stage estrogen-dependent breast cancer and suppressed the growth of tamoxifen- and estrogen deprivation-resistant xenograft tumors with increased efficacy observed for the combination of G1T48 and lerociclib, a newly developed CDK4/6 inhibitor [
25,
26].
Methods
Reagents
Fulvestrant (CAS No: 129453–61-8, > 99% purity) was purchased from MedChem Express. Estradiol (E2) (E8875), lasofoxifene (SML1026), 4-hydroxytamoxifen (H7904), and tamoxifen (T5648) were purchased from Sigma. Raloxifene (2280) was purchased from Tocris. GDC-0810 (S7855), bazedoxifene (S2128), and AZD9496 (S8372) were purchased from Selleckchem. GW5638 (5638), GW7604 (7604), and RU 58,668 (RU) were provided by Donald McDonnell (Duke University). G1T48 was provided by G1 Therapeutics, Inc., as analytical grade compound.
RNA analysis
MCF7 cells were authenticated by short tandem repeat profiling, were tested for
Mycoplasma and were not cultured for more than three months at a time [
27]. MCF7 cells were plated in DMEM/F12 supplemented with 8% charcoal dextran treated FBS for 48 h. Cells were then treated for 24 h with ligand and RNA was isolated using the Aurum™ total RNA isolation kit (Bio-Rad, Hercules, CA). After cDNA synthesis (iScript kit, Bio-Rad) real-time PCR was performed using the Bio-Rad CFX384 real-time system. GAPDH mRNA expression was used to normalize all real-time data using the 2-ΔΔC
T method [
28]. For more detailed description of this method, please see Online Resource 1.
Proliferation
MCF7 cells were plated in DMEM/F12 supplemented with 8% charcoal dextran treated FBS in 96-well plates (5 K cells/well) for 48 h. Cells were treated with estradiol (0.1 nM) or insulin (20 μM) with or without test compound (dose response; 1.0–11 to 1.0–05 M) for 6 days. Plates were decanted and frozen at – 80°°C overnight prior to quantitation of DNA by fluorescence using Hoechst 33258.
Supplementary material
Detailed methods are available in Online Resource 1 for the following protocols: In-Cell Western, Radioactive Binding Assay, Chromatin Immunoprecipitation, Transcriptional Reporter Assays.
Murine studies
All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Duke University or South Texas Accelerated Research Therapeutics (START, San Antonio, Texas) prior to initiating the experiment. For complete details, see Online Resource 1.
Discussion
Targeting ER activity using therapies that directly oppose the mitogenic action of estrogen or that block estrogen synthesis is a proven strategy for the treatment and prevention of breast cancer. In locally advanced or metastatic disease, resistance to these therapies frequently emerges within two years, at which time treatment options are severely limited. Fulvestrant, a potent ER antagonist and downregulator, was initially approved for the treatment of endocrine therapy-resistant disease and more recently as first-line therapy for advanced ER-positive, HER2-negative breast cancer not previously treated with endocrine therapy. However, despite promising preclinical activity, the poorly controlled pharmacokinetics of fulvestrant remains a significant barrier to prolonged clinical efficacy. Clinical trials comparing high-dose (500 mg) to low-dose (250 mg) fulvestrant demonstrated superiority for the 500 mg dose in both first- and second-line settings, suggesting that increased target engagement can improve the outcome of ER degradation therapy [
54,
55]. However, given its intramuscular route of administration, continued improvements in the clinical response to fulvestrant by further dose escalation appear unlikely. Therefore, development campaigns in this area have focused on the identification of orally bioavailable SERDs. The most active SERDs share common chemical features: either (a) a steroidal backbone (e.g., fulvestrant, RU58,668) or (b) an acrylic acid side chain (GW7604, GDC-0810, AZD9496) [
29,
31,
32,
40,
56,
57]. Additional ER antagonists with novel chemical structures have also been reported to exhibit SERD properties [
35,
36,
58], but none has yet gained FDA approval and some have been discontinued due to adverse effects or for undisclosed reasons [
29‐
36,
40,
56]. We have identified a novel, orally bioavailable SERD, G1T48, that contains both a steroidal backbone and an acrylate side chain. G1T48 binds ER with low nanomolar affinity, inhibits estrogen-mediated target gene expression and breast cancer cell growth, and importantly blocks the tumor promoting effects of ER in both naïve and endocrine therapy-resistant animal models of breast cancer. Similar to AZD9496 and GDC-0810, G1T48 has good pharmacokinetic properties and maintains a more favorable side-effect profile compared to those reported for AZD9496 [
56,
59,
60].
A hallmark feature of fulvestrant differentiating it from compounds like tamoxifen is that fulvestrant is a true antagonist with no agonist activity regardless of tissue context. By contrast, tamoxifen is a Selective Estrogen Receptor Modulator (SERM), demonstrating robust antagonist activity in the breast, but mimicking the agonist effect of estrogen in bone, the endometrium, and serum lipid profiles [
61,
62]. This mechanistic difference between tamoxifen and fulvestrant can also be observed in breast cancer cells, where transcriptional profiling studies revealed that tamoxifen can regulate a subset of genes in a similar manner to estradiol. Our ER target gene regulation studies confirm the agonist activity of tamoxifen, with stimulation of
SDK2,
AGR2, and
RAPGEL1 expression similar to the effect of estrogen treatment [
42]. Compounds with SERD activity such as fulvestrant and AZD9496 did not increase these transcripts, consistent with a lack of agonist potential (i.e., pure antagonism). The transcriptional profile in breast cancer cells of G1T48 is most similar to fulvestrant and other SERDs. Interestingly, our studies revealed that there were modest differences in the transcriptional profiles even among the pure antagonist class of compounds, suggesting that they might engender different receptor conformations. The impact of these differences in ER target gene activation remain to be explored but could suggest that cross-resistance between different classes of SERDs can be avoided. Recent studies have indicated that in addition to receptor degradation, ER mobility is differentially impacted by sub-classes of SERMs and SERDs, and that compounds impeding mobility are more efficacious antagonists [
35]. The impact of G1T48 on ER mobility is not currently known; however, our studies establish that G1T48 has very low intrinsic ER agonist activity.
Acquired resistance to endocrine therapy is complex and multifactorial; however, mutations in the
ESR1 gene that result in ligand-independent receptor activity have emerged as a potential mechanism to account for approximately 30–40% of resistant disease following AI treatment [
8,
44‐
46,
48‐
51]. It is significant, therefore, that G1T48 was found to suppress both the ligand-independent cell growth and transcriptional activity attributed to the two most prevalent endocrine refractory ER mutants, ER-Y537S and ER-D538G. Intriguingly, in contrast to the reconstituted transactivation assay in SKBR3, G1T48 was found to efficiently inhibit the growth of MCF7 cells engineered to overexpress the ER-Y537S variant. Cell context may contribute to this discrepancy; differential cofactor expression patterns in the two cell lines and/or the presence of endogenous wtER in MCF7 cells may influence G1T48 efficacy.
Long-term estrogen deprivation leading to a state of estrogen hypersensitivity is another means to model aromatase inhibitor therapy resistance. We have developed a new model of resistance to estrogen deprivation without ER mutation [
53]. Using this model system, treatment with low-dose G1T48 (5 mg) resulted in incomplete tumor growth inhibition, while high-dose G1T48 (100 mg) as monotherapy resulted in tumor regression in the majority of animals, similar to fulvestrant, demonstrating the effectiveness of SERD therapy in this setting of resistant disease.
The combination of SERDs with CDK4/6 inhibitors has been evaluated clinically, most recently in the PALOMA-3 trial comparing the co-administration of the CDK4/6 inhibitor palbociclib (Ibrance
®) with fulvestrant to fulvestrant alone. The results of this study demonstrated an overall survival benefit (median survival 34.9 months compared to 28 months) and a significant progression free survival rate (9.5 months vs 4.6 months) for the combination arm [
17,
20]. These noteworthy improvements led to the 2016 FDA approval of palbociclib and fulvestrant combination therapy for ER-positive, HER-2- negative breast cancers progressing on other endocrine therapies. Further trials (MONALESSA-3 (NCT02422615) and MONARCH-2 (NCT02107703) have also demonstrated the utility of administering other CDK4/6 inhibitors with fulvestrant to improve patient outcomes [
17‐
20]. The increased efficacy observed for the combination of G1T48 and lerociclib, as compared to monotherapy administration, in multiple in vivo breast cancer models sensitive or refractory to endocrine therapy treatment supports the potential utility of this regimen as an intervention in multiple stages of breast cancer treatment. Furthermore, lerociclib has been shown to promote less myelosuppression than palbociclib [
25,
26]. Collectively, these data indicate that G1T48 has the potential to be an efficacious oral antineoplastic agent in ER+ breast cancer.
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