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Published in final edited form as: Breast Cancer Res Treat. 2020 Feb 6;180(2):359–368. doi: 10.1007/s10549-020-05558-w

GLL398, an Oral Selective Estrogen Receptor Degrader (SERD), Blocks Tumor Growth in Xenograft Breast Cancer Models

Shanchun Guo 1, Changde Zhang 1, Madhusoodanan Mottamal 1, Ahamed Hossain 1, Jiawang Liu 2, Guangdi Wang 1
PMCID: PMC7069796  NIHMSID: NIHMS1558018  PMID: 32030569

Abstract

Purpose

Selective estrogen receptor degrader (SERD) has proven clinically effective in treating advanced or metastatic breast cancer since the approval of fulvestrant by FDA in 2002. Recent expansion of indications as a first line monotherapy and as combination therapy with CDK4/6 inhibitors further extends its clinical utility as an efficacious breast cancer endocrine regimen. However, the poor pharmacokinetic properties of fulvestrant and its injection-only administration route has driven continued efforts to develop orally bioavailability SERD that could potentially improve clinical response to SERD treatment. GLL398, a boron-modified GW5638 analog, showed superior oral bioavailability while retaining both antiestrogenic activity and ER degrading efficacy at a potency level comparable to the more active metabolite of GW5638, GW7604.

Method

Here we used molecular modeling, ER (Y537S) binding assay, MCF-7 Xenograft tumor and patient derived xenograft (PDX) tumor model to conduct further studies on the pharmacology and metabolism of GLL398.

Results

Consistent with GLL398’s robust activities in breast cancer cells that are either tamoxifen resistant or express constitutively active, mutant ESR1 (Y537S), it was found to bind the mutant ERY537S at a high affinity. Molecular modeling of the binding mode of GLL398 to ER also found its molecular interactions consistent with the experimentally determined high binding affinity towards WT ER and ERY537S. To test the in vivo efficacy of GLL398, mice bearing MCF-7 derived xenograft breast tumors and patient derived xenograft tumors harboring ERY537S were treated with GLL398 which potently inhibited tumor growth in mice.

Conclusions

This study demonstrates GLL398 is an oral SERD that has therapeutic efficacy in clinically relevant breast tumor models.

Keywords: Oral SERD, Breast Cancer, PDX Breast Tumor Model, mutant ESR1, Y537S

Introduction

The selective estrogen receptor downregulator/degrader (SERD), fulvestrant was approved by FDA in 2001 as a second line endocrine therapy for breast cancer patients with progressing diseases after prior endocrine treatments such as tamoxifen or aromatase inhibitors [1,2]. The dual mode of actions of fulvestrant as a pure antiestrogen and an ER protein degrader makes the drug less susceptible to endocrine resistance [3,4], leading to clinical efficacy in patients no longer responding to previous endocrine therapies. The poor bioavailability of fulvestrant, as an intramuscular injection depot, led to further clinical trials and subsequent approval of a higher dosage of fulvestrant in 2010 [5,6,7]. A large number of studies [8,9,10,11,12], both laboratory and clinical, indicate that drug exposure of fulvestrant may be insufficient and largely motivated efforts to develop orally bioavailable SERDs in the hope that fast action and greater drug exposure offered by oral SERDs could translate to more durable clinical benefits [13,14,15,16,17,18,19].

Recent approval of fulvestrant as a first line agent for patients with advanced or metastatic breast cancer [20,21] was a result of a pivotal clinical trial (FALCON) comparing the efficacy of anastrozole and 500 mg fulvestrant in endocrine naive patients, which demonstrated that fulvestrant treated patients had a significantly longer progression free survival (PFS) and overall survival (OS) than those taking anastrozole [22,23]. Moreover, when used in combination with a CDK4/6 inhibitor, fulvestrant was shown to prolong PFS compared to fulvestrant alone as a monotherapy for advanced or metastatic breast cancer patients (PALOMA-3 phase III clinical trial; MONARCH 2 phase III trial). Thus, fulvestrant was approved for use as a combination therapy with palbociclib in 2016 [24] and with abemaciclib in 2017 [25]. These results serve to not only validate the broader clinical utility of SERDs, but also highlight the need for orally bioavailable SERDs in these expanded indications where a larger number of patients may benefit from a more efficacious oral SERD regimen.

The first observation of SERD-like properties of a nonsteroidal, tamoxifen-like compound known as GW5638 was reported in 1994[26,27,28,29], the potential clinical utility of which as an endocrine therapy for tamoxifen resistant breast cancer was subsequently tested in a phase 1 clinical trial in 2001[13]. The compound served as the prototype of nonsteroidal oral SERDs that mostly consist of a non-steroidal moiety that docks into the ligand binding domain (LBD) of ER and a side chain of acrylic acid that confers antiestrogenic and ER degrading properties. However, it would be more than a dozen years later that a structurally similar, preclinically improved oral SERD made its way to a phase 1 and subsequently a phase 2 clinical trial in 2013 [30] and 2015 [31], respectively. Other oral SERDs currently in clinical trials include AZD9496 by AstraZeneca, LSZ102 by Novartis, and RAD1901 by Radius Health. Recently, Roche/Genentech halted further clinical development of GDC-0810 in April 2017 [32] and GDC-0927 in February 2018 [33], presumably due to a combination of adverse side effects and lack of superior efficacy compared to fulvestrant. These latest developments add additional uncertainty to the clinical path of this type of nonsteroidal oral SERDs.

Reported phase 1 results so far indicate that these oral SERDs all appear to have modest oral bioavailability which made it necessary to adopt a higher phase 2 trial dosage, possibly contributing to the G.I. toxicities and lack of superior efficacy compared to fulvestrant. For instance, a dosage of 600 mg GDC-0810 per day was selected for phase II trial [34]. For GDC-0927, the best phase 1 clinical response was observed at the high dose of 1400 mg QD [35]. A twice daily dose of 600 mg was used in a phase 2 clinical trial of AZD9496 [36]. These early clinical data suggest that improved oral bioavailability in new SERDs is highly desirable.

Our laboratory has developed a boron-modified GW5638 analog, GLL398 that showed superior oral bioavailability while retaining both greater antiestrogenic activity and ER degrading efficacy at a potency level comparable to the more active metabolite of GW5638, GW7604 [37]. Here we report further studies on the pharmacology, pharmacokinetics, and metabolism of GLL398. Given GLL398’s robust activities in breast cancer cells that are either tamoxifen resistant or express constitutively active, mutant ESR1 (Y537S) [37], we first measured its binding behavior towards ERY537S. To better understand the mode of action we next performed molecular modeling of the binding of GLL398 and found its molecular interactions consistent with the experimentally determined high binding affinity towards WT ER and ERY537S. To test if the superior oral bioavailability can be translated to potent efficacy in vivo, mice bearing MCF-7 derived xenograft breast tumors and patient derived xenograft tumors harboring ERY537S mutant were treated with GLL398 via oral administration. Effect of GLL on tumor growth, ER degradation, and drug exposure level in tumor tissues were investigated to assess the therapeutic efficacy of GLL.

Materials and methods

Ethics statement

All animal studies were conducted under a protocol approved by the Institutional Animal Care and Use Committee of Xavier University of Louisiana (0080415-05CH).

Molecular Modeling

To compare the binding interactions of GW7604 and GLL398 with ERα, molecular docking studies were performed using Schrodinger’s (Suite 2015-3) Glide program.[38] The antagonistic ligands have been found to cause impairment of hormone dependent ER transactivation by inducing conformational changes on ERα and averting the binding of coactivator signal transmitting proteins [39]. Since GW7604 acts as an ER antagonist and structurally very similar to GW5638, coordinates for the structure of wild-type ERα was taken from the X-ray crystallographic structure of ERα in complex with the antagonist GW5638 (PDB entry: 1R5K) [40]. For the Y537S mutant ERα the coordinates were taken from X-ray structure of ERα in complex with H3B-9224 (PDB entry: 6CHZ). Initial structures of ERα protein were prepared by removing all the crystallographic water molecules beyond 5 Å from the crystal ligand and adding hydrogen atoms consistent with the physiologic pH of 7 using Maestro 10.3. Then the protein molecule was energy minimized with an RMSD cutoff value of 0.3 Å for all heavy atoms. Compounds GW7604 and GLL398 were prepared using the builder of Schrodinger followed by energy minimization. The antagonist binding site-based receptor grid was generated for the ER-ligand docking. All the docking calculations were performed with Glide 6.8 using the default parameters under the extra precision (XP) mode for procuring the best docked representative structure. The binding free energies of the complexes were also obtained using the MM/GBSA method with the OPLS/AA force field and a GB/SA continuum solvent model.

ER (Y537S) binding assay

The recombinant Estrogen Receptor Alpha (ER alpha) LBD GST protein containing a Y537S mutation was prepared based on the Life Technologies ER alpha LBD GST construct (amino acids 282-595, accession Number: NP_000116.2). The construct was expressed in insect cells and the protein purified using Life Technologies proprietary methods. The final purified protein was tested in a LanthaScreen® TR-FRET ER Alpha Competitive Binding assay and in a LanthaScreen® TRFRET ER Alpha Coactivator Assay. GLL398, fulvestrant, and AZD9496 were profiled in the binding assay. The concentration of the GST-ER α (Y537S) was optimized for the LanthaScreen® TRFRET Competitive Binding Assay by titrating the GST-ER α (Y537S) in the assay from 1 μM to 3 pM (data not shown). The EC80 concentration of the GST-ER α (Y537S) was 8 nM. This was the final concentration of GST-ER α (Y537S) used in the standard assay. Compounds were run in the LanthaScreen® TRFRET GST-ER α (Y537S) Competitive Binding Assay and IC50 values for each compound are reported.

Efficacy Study in an MCF-7 Xenograft Tumor Model

Four to six weeks old female ovariectomized Nu/Nu mice were purchased from Charles River Laboratories (Wilmington, MA). MCF-7 cells were cultured and harvested in the exponential growth phase using a PBS/EDTA solution. The animals were injected bilaterally in the mammary fat pad (MFP) with 5x106 viable cells suspended in 50 μL sterile PBS mixed with 100 μL Matrigel (reduced factor; BD Biosciences, Bed- ford, MA). 17b-Estradiol pellets (0.72 mg, 60 day release; Innovative Research of America, Sarasota, FL) were implanted subcutaneously in the lateral area of the neck using a precision trochar (10 gages) at the time of cell injection. After the tumor formed and became palpable, the animals were randomized into three groups, and treated with vehicle, GLL398 at 5 mg/kg, or GLL398 at 20 mg/kg by oral gavage. Tumor sizes were monitored and recorded every other day for three weeks of treatment duration.

Efficacy Study in a Patient Derived Xenograft (PDX) Tumor Model

WHIM20, a highly characterized, HER2-, ER+, PR+ PDX model of breast cancer which derived from a skin metastasis of breast cancer patient [41]. This line possesses a Y537S mutation in ESR1, a C182X mutation in p53, and an E542K mutation in PIK3CA. The tumor tissue was engrafted in mice with homozygous for Foxn1mutation (Jackson Lab). After the tumor formed and became palpable, the animals were randomized into four groups, and treated with vehicle, GLL398 at 5 mg/kg, or 20 mg/kg by oral gavage. Tumor sizes were monitored and recorded every other day for three weeks of treatment duration.

Immunohistochemistry

IHC for ERα (SC-53493, Santa Cruz Biotechnology, TX, USA), Ki67 (Ab 16667, ABCAM) were performed using standard protocols according to the manufacturers’ instructions (Santa Cruz Biotechnology, TX, USA), where the tumor tissues were sampled from both treatment and control groups at each imaging time point. IHC analysis for quantitative antigen expression was based on standard procedures for breast cancer. The total proportion of cells positively stained with any intensity was scored as follows: 0, no cells stained; 1, 1%–25% cells stained; 2, 26%–50% cells stained; 3, 50%–75% cells stained; and 4, >75% cells stained.

Western blot analysis

Xenograft tissues were lysed in accordance with standardized protocols. Protein lysates (50 μg) were resolved by SDS-PAGE, and electrophoretically transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). After blocking in 5% BSA, membranes were hybridized overnight at 4°C with primary antibodies specific for the detection of ERα, PgR (MA5-14505, Invitrogen), and β-actin (Proteintech). Mouse and rabbit horseradish peroxidase–conjugated secondary antibodies (Amersham Biosciences) were used at 1:5,000 dilution in TBS-Tween solution. Protein-antibody complexes were detected by chemiluminescence with the SuperSignal West Dura Extended Duration Substrate (Millipore corperation, Billerica, MA, USA). The experiments were repeated at least three times.

Results

Binding of GLL398 to ERY537S

In a previous study we found that GLL398 competitively bound to the estrogen receptor with an IC50 value of 1.14 nM which compared favorably with GW7604 (IC50=13.8 nM). For an efficacious oral SERD that could be used in a clinical setting where the recurring disease upon prior endocrine treatment may harbor mutant ER, we sought to determine the potency with which GLL398 could bind to ERY537S. A recombinant estrogen receptor alpha ligand binding domain GST protein containing a Y537S mutation was used to test in a LanthaScreen® TR-FRET ER Alpha Competitive Binding assay. GLL398, fulvestrant, and AZD9496 were profiled in the binding assay at 15 concentrations from 0.5 nM to 10 μM. As shown in Figure 1, GLL398 exhibits a strong dose-dependent binding profile for the ER with a Y537S point mutation. The ER binding IC50 for GLL398 was determined to be 29.5 nM. In comparison, fulvestrant and AZD9496 show similar binding profiles to the mutant ER, with IC50 values measuring 19.3 nM and 14.8 nM, respectively.

Figure 1.

Figure 1.

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Titration curves for GLL398, AZD9496, and fulvestrant in the LanthaScreen® TRFRET GST-ERα (Y537S) Competitive Binding assay.

Molecular Modeling

We next examined the mode of binding by GLL398 to both wild type and mutant ER using molecular docking method. Given the experimental observation that the binding affinity of GLL3 98 toward the wild type ERα was 10-fold higher than that of GW7604 (IC50 1.14 nM vs IC50 13.8 nM), we first compared how the two compounds bind into the antagonistic ligand binding site of ERα and found that both exhibit similar binding mode with that of co-crystallized GW5638 [40], which lacks the phenolic hydroxyl group at position 4. As shown in Figure 2, GW5638 and GW7604 possess the same structural scaffold, and the only difference between them is the presence of a hydroxyl group at C-4 in GW7604, which was found to interact with Glu353 and Arg394 (Figure 2). Like GW7604, which is structurally analogous to 4-hydorxytamoxifen (4-OHT), 4-OHT also has a hydroxyl group at the fourth position and the crystal structure (3ERT.pdb)[42] has shown this hydroxyl group interacting with Glu353 and Arg394 of ERα. Such interactions were also observed in the crystal structure of ERα in complex with (E)-3-[4-(2-Oxo-3-Aryl-Chromen-4Yl)Oxyphenyl] Acrylic Acid. [43] Superposition of these crystal structures has shown similar binding mode for these compounds. In fact, hydrogen bonds with Glu353 and Arg394 are conserved in many ER-antagonist and ER-agonist complexes, including the above described compounds. [42,43,44,45,46] The phenylacrylic acid moieties of GW7604 and the crystal ligand GW5638 lie in the same position and the end carboxylic acid group makes hydrogen bond interactions with Asp351 and a water molecule. Similarly the α-ethylstilbene moiety of both compounds lie in the same position in the hydrophobic environment of the pocket established by many hydrophobic residues as shown in Figure 2.

Figure 2.

Figure 2.

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Binding postures of GW7604 (green), GLL398 (dark cyan) and the co-crystal GW5638 (orange) in the antagonistic binding site of wild-type ERα. Important amino acids in the binding pockets are shown in stick models, among them the grey sticks are the hydrophobic residues.

GLL398 also binds to the wild-type ERα in a similar mode as GW7604 (Figure 2). Though the hydroxyl group was replaced with a boronic acid group, placement of the main core of the molecules was not altered drastically due to the small size of boron. In addition to the hydrogen bonds with Glu353 and Arg394, the boronic acid group in GLL398 formed a hydrogen bond with the backbone carbonyl oxygen of Leu387, making GLL398 a more stable complex than GW7604. Docking scores also showed that GLL398 (−15.79 kcal/mol) has a stronger ERα binding affinity than GW7604 (−13.75 kcal/mol). Similarly the free energies of binding obtained by MMGB/SA calculations further showed higher ERα binding energy for GLL398 (−97.99 kcal/mol) than GW7604 (−95.58 kcal/mol).

A comparison of the binding of GL398 to the wild-type and Y537S mutant ERα is depicted in Figure 3. In both cases the boronic acid group was found to make hydrogen bond interactions with Glu353, Arg394 and Leu387. The main difference is the loss of hydrogen bond interaction with Asp351 in the mutant ERα. This is due to the structural change observed in the C-terminal end of the mutant ERα, as evidenced from the distance between Cα atoms of D351 and Y/S-537 in the wild-type (6.8 Å) and mutant (14.0 Å) systems. Thus the binding of GLL398 with the mutant ERα is weaker than with the wild-type ERα. The docking score (−14.02 kcal/mol) and the binding free energy (−96.45) of GLL398 in the mutant system were also found to be higher than the same in the wild-type ERα (−15.79 kcal/mol and −97.99 kcal/mol, respectively).

Figure 3.

Figure 3.

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A close-up view comparison of the binding of GLL398 to the wild-type (A) and Y537S mutant (B) ERα. Important amino acids in the binding pockets are shown in stick models, and the hydrogen bond interactions are shown with magenta dashed lines.

GLL398 is orally efficacious in inhibiting MCF-7 breast cancer xenograft

Having previously shown that GLL398 is active in binding to ER, inhibiting ER mediated transcriptional activities, downregulating ER expression, and inhibiting the growth of ER+ breast cancer cells37, we now seek to determine the efficacy of GLL398 in blocking tumor growth in vivo. Mice bearing MCF-7 xenografts supplemented with estrogen-releasing pellets were treated with 5 mg/kg or 20 mg/kg dose of GLL398 by daily oral gavage for three weeks to investigate the effect of GLL398 on tumor growth. The initial tumor size of each group was around 110 mm3. Tumor volumes in the untreated group, as shown in Figure 4A, grow exponentially. In mice treated with oral daily doses of GLL398, tumor growth was effectively blocked. While both dose levels were efficacious in the inhibition of tumor growth, the 20 mg/kg dose group exhibited greater efficacy in blocking tumor growth in the final ten days of treatment (Figure 4A). To investigate the drug exposure level in xenograft tumors, tissues were collected at end of treatment on day 23 and analyzed for drug and its major active metabolite (GW7604)31 concentrations. As shown in Figure 4B, significant drug exposure in tumor tissue is achieved with the total drug concentration exceeding 1000 ng/g, a level that is consistent with the observed tumor inhibition efficacy of GLL398 treatment.

Figure 4.

Figure 4.

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A. Nude mice bearing MCF-7 breast cancer xenograft were treated with vehicle or GLL398 at two different doses PO. Treatment continued for three weeks before the animals were sacrificed and plasma and tumor tissues were collected. A. tumor volumes were plotted vs. days of drug treatment; B. concentration of GLL398 and its active metabolite GW7604 in tumor tissue samples at end of study.

GLL398 inhibits patient derived xenograft breast tumor expressing mutant (Y537S) ER

We next investigated the in vivo efficacy of GLL398 in a patient derived xenograft model (WHIM20) that expresses a mutant (Y537S) and constitutively active estrogen receptor. The initial tumor size of each group was around 150 mm3. Tumor volumes are measured over the treatment period of 23 days in mice either administered with vehicle, 5 mg/kg GLL398 oral gavage, or 20 mg/kg GLL398 oral gavage. Near complete growth inhibition of the PDX tumors was observed with the 5 mg/kg treatment group, whereas treatment with 20 mg/kg resulted in significant tumor regression over the course of GLL398 treatment (Figure 5A and 5B). Analysis of tumor samples collected at end of study indicate robust drug exposure as measured by the total active drug concentration (sum of GLL398 and its active metabolite, GW7604) (Figure 5C). Western blot analysis of tumor samples revealed that treatment of GLL398 resulted in loss of ER expression and to a less degree downregulated the expression of PgR protein.

Figure 5.

Figure 5.

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A. Inhibition of PDX WHIM 20 breast tumors by GLL398 orally administered to mice at 5 and 20 mg/kg, respectively; B. Average tumor weight of each treatment group; C. Drug concentration in tumor tissue and final plasma of mice at end of study; and D. Downregulation of ERα in tumor tissues treated by GLL398 at 5mg/kg or 20 mg/kg, respectively.

The degradation of ER in tumor tissues by the action of GLL398 as a potent SERD is confirmed in the tissue immunohistochemical analysis. As shown in Figure 6, untreated breast tumor stained with strong expression of ER with the staining intensity scored at the level of 4 (Figure 6A), whereas the tumor tissue from mice treated with 20 mg/kg GLL398 showed no ER staining ( staining intensity score 0) (Figure 6C). Furthermore, elevated Ki-67 expression found in untreated tumor samples (staining intensity score 4) (Figure 6B) was markedly downregulated in GLL398 treated tumors (staining intensity score 0) (Figure 6D), indicating the highly efficacious antitumor effect of GLL398 against the WHIM20 tumor line.

Figure 6.

Figure 6.

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WHIM20 tumor tissue immunohistochemical staining, A. untreated tumor, ER staining, staining intensity score 4. B. untreated tumor, Ki67 staining, staining intensity score 4. C. tumor treated with GLL398 20mg/kg/day, ER staining, staining intensity score 0. D. tumor treated with GLL398 20mg/kg/day, Ki67 staining, staining intensity score 0.

Discussion

Our modeling study shows that GW7604 and GLL398 can bind to the antagonistic binding site of wild-type ERα and their binding affinities are consistent with the trend observed by the IC50 calculations. Having found that both GW7604 and GLL398 can adopt similar binding mode in the active site of ERα, and the binding affinity of GLL398 is stronger than GW7604, consistent with the observed IC50 values, computer modeling study also support the hypothesis that the boronic acid group may be acting as a bioisostere of hydroxyl group in these compounds. Since they possess similar binding mode, both compounds may be functioning through the same mechanism of action. Similarly modeling studies also show that the binding affinity of GLL398 for the Y537S mutant ERα is weaker than that for the wild-type ERα, consistent with reports that antiestrogens such as 4-hydroxytamoxifen and fulvestrant all exhibited reduced binding potency in point-mutated estrogen receptor [47].

Indeed, competitive binding assay of GLL398 to the Y537S mutant ERα confirmed a moderately reduced binding affinity of the molecule towards the mutant receptor. Binding IC50 of GLL398 decreased from 1.14 nM [37] with wild type ER to 29.5 nM with Y537S mutant ER. Consistent with this observation, the known SERD, fulvestrant showed a binding affinity to ER at 0.8 nM [16] and a reduced binding affinity to Y537S mutant ER at 19.3 nM. Similarly the nonsteroidal SERD, AZD9496, which is under clinical evaluation, binds more strongly to the wild type ER than the mutant counterpart [16]. Breast cancer cells harboring the point mutation at Y537 in the ER ligand binding domain has been shown to have decreased response to 4-hydroxytamoxifen and fulvestrant and require higher doses of the antiestrogens to achieve the inhibition of cell proliferation similar to breast cancer cells expressing WT ER [47]. The attenuated binding affinity of potent SERDs such as fulvestrant and GLL398 towards mutant ER may partially explain the resistance of breast cancer cells expressing mutant ER (Y537S) to endocrine treatment. Moreover, these results suggest that the ER mutants would be relatively resistant to established clinical doses of endocrine therapies and higher doses of antiestrogens or SERDs with enhanced pharmacokinetic properties are needed to inhibit mutant ER activity.

The pharmacokinetic profile of the boron-modified GLL398 was previously shown to afford high drug exposure in vivo, with AUC value exceeding 36000 ng.hr/mL at a single dose of 10 mg/mL (37). In this study we show that such high oral bioavailability of the drug is translatable to in vivo efficacy. In the first efficacy study where mice bearing MCF-7 xenograft were treated with GLL398 at 5 and 20 mg/mL via oral gavage, effective inhibition of tumor growth seen in both treatment groups closely correlated with high drug exposure in tumor tissues. In the 5 mg/kg treatment group, the concentration of GLL398 reached 740 ng/g. A significant level of the active metabolite, GW7604 was also detected at 340 ng/g, bringing the total drug concentration to a level of 1080 ng/g. In the 20 mg/kg group, the total active drug level reached 1314 ng/g. These tissue drug exposure levels are significantly above the IC50 of GLL398 in antiproliferative activity [37], and likely responsible for the efficacy in the xenograft tumor model.

The efficacy of GLL398 in ER mutant, endocrine resistant breast cancer was tested in a patient derived xenograft model harboring Y537S mutant ER. Mice bearing WHIM20 tumors were treated with GLL398 via daily oral administration, which was seen to completely block tumor growth during the three weeks of treatment. While ESR1 mutation (Y537S) constitutively activates ER, tumor cells remain dependent on the ER mediated pathways to grow and proliferate. The continued efficacy of a SERD in an ESR1 mutant breast tumor is in part attributable to its ability to degrade the receptor, as the constitutively active ER is no longer affected by the antiestrogenic activity of the SERD. The effective downregulation of ER in tumor tissues by GLL398, as seen in Figure 5D, provides strong support to the mechanism of SERD action in estrogen independent, endocrine resistant breast cancer.

In conclusion, the orally bioavailable SERD, GLL398 was found to bind to the mutant estrogen receptor with high affinity to exert its ER degrading effect in a clinically relevant, ESR1 mutant model, leading to robust anti-tumor efficacy. The high drug exposure of GLL398, made possible by its boronic structure known to enhance oral bioavailability, contributed to the in vivo efficacy of the oral SERD.

Acknowledgement

This study was supported in part by NIH grants U54MD007595 from NIMHD, 1R43CA213462, and 2R44CA213462 from NCI, and by Louisiana Cancer Research Consortium (LCRC).

Footnotes

Conflict of interest No authors on this manuscript declare a conflict of interest.

Publisher's Disclaimer: This Author Accepted Manuscript is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication but has not been copyedited or corrected. The official version of record that is published in the journal is kept up to date and so may therefore differ from this version.

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