ERα Mutant Research Articles

Effect of estrogen receptor α on cardiopulmonary adaptation to chronic developmental hypoxia in a rat model.

Humans living at high-altitude (HA) have adapted to this environment by increasing pulmonary vascular and alveolar growth. RNA sequencing data from a novel murine model that mimics this phenotypical response to HA suggested estrogen signaling via estrogen receptor alpha (ERα) may be involved in this adaptation. We hypothesized ERα was a key mediator in the cardiopulmonary adaptation to chronic hypoxia and sought to delineate the mechanistic role ERα contributes to this process by exposing novel loss-of-function ERα mutant (ERαMut) rats to simulated HA. ERα mutant or wild-type (wt) rats were exposed to normoxia or hypoxia starting at conception and continued postnatally until 6 wk of age. Both wt and ERαMut animals born and raised in hypoxia exhibited lower body mass and higher hematocrits, total alveolar volumes (Va), diffusion capacities of carbon monoxide (DLCO), pulmonary arteriole (PA) wall thickness, and Fulton indices than normoxia animals. Right ventricle adaptation was maintained in the setting of hypoxia. Although no major physiologic differences were seen between wt and ERαMut animals at either exposure, ERαMut animals exhibited smaller mean linear intercepts (MLI) and increased PA total and lumen areas. Hypoxia exposure or ERα loss-of-function did not affect lung mRNA abundance of vascular endothelial growth factor, angiopoietin 2, or apelin. Sexual dimorphisms were noted in PA wall thickness and PA lumen area in ERαMut rats. In summary, in room air-exposed rats and rats with peri- and postnatal hypoxia exposure, ERα loss-of-function was associated with decreased alveolar size (primarily driven by hypoxic animals) and increased PA remodeling.NEW & NOTEWORTHY By exposing novel loss-of-function estrogen receptor alpha (Erα) mutant rats to a novel model of human high-altitude exposure, we demonstrate that ERα has subtle but inconsistent effects on endpoints relevant to cardiopulmonary adaptation to chronic hypoxia. Given that we observed some histologic, sex, and genotype differences, further research into cell-specific effects of ERα during hypoxia-induced cardiopulmonary adaptation is warranted.

Abstract 6576: Discovery of potent and selective novel small molecule drugs to target estrogen receptor mutants in endocrine-resistant breast cancer

Abstract Introduction: The acquisition of estrogen receptor alpha (ERα) gene (ESR1) mutations is a key driver for the development of resistance to current endocrine therapy in breast cancer. Clinical studies have shown that ESR1 mutations are frequently observed in metastatic ER-positive breast cancer patients and are associated with poor survival. It is well appreciated that activating ESR1 somatic mutations, especially Y537S and D538G, can drive estrogen-independent activities. In addition, these ESR1 mutations diminish the potency of the current standard-of-care agents (tamoxifen and fulvestrant) that bind ERα directly. Therefore, it is an unmet need to develop next-generation antiestrogens that inhibit ERα mutant signaling in breast cancer to improve patient survival. Here, we search for small molecule inhibitors against ERα mutants Y537S and D538G using DNA-encoded chemical library (DEL). Methods: ERα ligand binding domain (LBD) proteins corresponding to wild type (WT), Y537S and D538G mutants were expressed in E. coli and purified by Ni-NTA, anion exchange, and size exclusion chromatography. The ability of these purified proteins to bind ligands was tested in biochemical assays to validate their use in DEL selections. We conducted a DEL affinity selection using our in-house collection of 6 billion small molecule compounds against WT, Y537S and D538G mutants ER LBD proteins in the presence and absence of estradiol. Selection hits that enriched with these targets were resynthesized off-DNA and tested in biochemical and functional studies using CRISPR-Cas9 knock-in Y537S or D538G mutant breast cancer cells. Results: We have successfully purified microgram amounts of ERα LBD for WT, Y537S, and D538G proteins. In our biochemical assay, the SRC3 peptide binds to the WT ERα LBD in the presence of estradiol, whereas Y537S and D538G LBDs bind the SRC3 peptide in the absence of estradiol, consistent with these mutants constitutively binding to SRC3. Our multibillion small molecule collections of DELs can screen and identify several hits in WT and mutant ERα LBDs. To confirm the selection output, we synthesized off-DNA compounds and validated these in biochemical and cell-based studies. Compounds CDD-1274 and CDD-1802 dramatically decrease the WT and mutant ERα protein levels in multiple ER+ breast cancer cells. These compounds greatly reduce protein levels for GREB1, TFF1, c-MYC, E2F1, and survivin in wildtype and mutant breast cancer cells, and they increase levels of cleaved PARP, a marker for apoptosis in wildtype and mutant ER+ breast cancer cells but not in ER-negative breast cancer cells. Conclusion: Our study rapidly identified potent and novel small molecule drugs to target estrogen receptor mutants using our DNA-encoded chemical library platform. Acknowledgments: Funding sources (NIH/NCI R03 CA259664 and CPRIT RP220524 to MP). Citation Format: Anil Kumar Devakrishnan, Chandrashekhar Madasu, Suzanne A.W Fuqua, Martin M. Matzuk, Murugesan Palaniappan. Discovery of potent and selective novel small molecule drugs to target estrogen receptor mutants in endocrine-resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6576.

Discovery of a Novel Class of PROTACs as Potent and Selective Estrogen Receptor α Degraders to Overcome Endocrine-Resistant Breast Cancer In Vitro and In Vivo.

The estrogen receptor (ER) is a well-established target for endocrine therapies of ER-positive breast cancer (ER+ BC), but endocrine resistance limits the efficacy of clinical drugs. Using proteolysis targeting chimera (PROTAC) technology to degrade ERα may be an effective alternative to endocrine therapies. Herein, we disclose a novel series of potent and selective ERα PROTACs based on an oxabicycloheptane sulfonamide (OBHSA) scaffold, with no associated ERβ degradation. These PROTACs showed significant antiproliferation and ERα degradation activities against a broad spectrum of ER+ BC cells including tamoxifen-resistant and ERα mutant cell lines. Genomics analysis confirmed that these PROTACs inhibited the nascent RNA synthesis of ERα target genes and impaired genome-wide ERα binding. Compound ZD12 exhibited excellent antitumor potency and ERα degradation activity in both tamoxifen-sensitive and -resistant BC mice models, which are superior to fulvestrant. This study demonstrates the potential of these PROTACs as novel drug candidates for endocrine-resistant BC treatment.

17⍺-Estradiol Protects against HIV-1 Tat-Induced Endolysosome Dysfunction and Dendritic Impairments in Neurons.

HIV-1 Tat continues to play an important role in the development of HIV-associated neurocognitive disorders (HAND), which persist in 15-55% of people living with HIV even with virological control. In the brain, Tat is present on neurons, where Tat exerts direct neuronal damaging effects by, at least in part, disrupting endolysosome functions, a pathological feature present in HAND. In this study, we determined the protective effects of 17α-estradiol (17αE2), the predominant form of estrogen in the brain, against Tat-induced endolysosome dysfunction and dendritic impairment in primary cultured hippocampal neurons. We demonstrated that pre-treatment with 17αE2 protected against Tat-induced endolysosome dysfunction and reduction in dendritic spine density. Estrogen receptor alpha (ERα) knockdown impairs the ability of 17αE2 to protect against Tat-induced endolysosome dysfunction and reduction in dendritic spine density. Furthermore, over-expressing an ERα mutant that fails to localize on endolysosomes impairs 17αE2's protective effects against Tat-induced endolysosome dysfunction and reduction in dendritic spine density. Our findings demonstrate that 17αE2 protects against Tat-induced neuronal injury via a novel ERα-mediated and endolysosome-dependent pathway, and such a finding might lead to the development of novel adjunct therapeutics against HAND.

Estrogen receptor α K303R mutation reorganizes its binding to forkhead box protein A1 regions and induces chromatin opening

BackgroundEstrogen receptor alpha (ERα) is a frequently mutated gene in breast cancer (BC). While many studies have investigated molecular dysregulation by hotspot mutations at Y537 and D538, which exhibit an estrogen-independent constitutively active phenotype, the functional abnormalities of other mutations remain obscure. The K303R mutation in primary invasive BC has been implicated with endocrine resistance, tumor size, and lymph node positivity. However, the impact of the K303R mutation on the cell epigenome is yet unknown.Methods and resultsWe introduced the K303R ERα mutant in ERα-negative MDA-MB-453 cells to monitor ERα-dependent transactivation and to perform epigenomic analyses. ATAC-seq and ChIP-Seq analyses indicated that both wild-type (WT) and the K303R mutant associated with Forkhead box (Fox) protein family motif regions at similar rates, even without an ERα-binding sequence, but only the K303R mutant induced chromatin opening at those regions. Biochemical analyses demonstrated that the WT and the K303R mutant can be tethered on DNA by FoxA1 indirectly, but only the K303R/FoxA1/DNA complex can induce associations with the nuclear receptor cofactor 2 (NCOA2).ConclusionsThese findings suggest that the K303R mutant induces chromatin opening at the Fox binding region through the FoxA1-dependent associations of the K303R mutant to NCOA2 and then probably disrupts the regulation of Fox-target genes, resulting in K303R-related BC events.

A Novel Mouse Model to Analyze Non-Genomic ERα Physiological Actions.

Nongenomic effects of estrogen receptor α (ERα) signaling have been described for decades. Several distinct animal models have been generated previously to analyze the nongenomic ERα signaling (eg, membrane-only ER, and ERαC451A). However, the mechanisms and physiological processes resulting solely from nongenomic signaling are still poorly understood. Herein, we describe a novel mouse model for analyzing nongenomic ERα actions named H2NES knock-in (KI). H2NES ERα possesses a nuclear export signal (NES) in the hinge region of ERα protein resulting in exclusive cytoplasmic localization that involves only the nongenomic action but not nuclear genomic actions. We generated H2NESKI mice by homologous recombination method and have characterized the phenotypes. H2NESKI homozygote mice possess almost identical phenotypes with ERα null mice except for the vascular activity on reendothelialization. We conclude that ERα-mediated nongenomic estrogenic signaling alone is insufficient to control most estrogen-mediated endocrine physiological responses; however, there could be some physiological responses that are nongenomic action dominant. H2NESKI mice have been deposited in the repository at Jax (stock no. 032176). These mice should be useful for analyzing nongenomic estrogenic responses and could expand analysis along with other ERα mutant mice lacking membrane-bound ERα. We expect the H2NESKI mouse model to aid our understanding of ERα-mediated nongenomic physiological responses and serve as an in vivo model for evaluating the nongenomic action of various estrogenic agents.

Endolysosome Localization of ERα Is Involved in the Protective Effect of 17α-Estradiol against HIV-1 gp120-Induced Neuronal Injury.

Neurotoxic HIV-1 viral proteins contribute to the development of HIV-associated neurocognitive disorder (HAND), the prevalence of which remains high (30-50%) with no effective treatment available. Estrogen is a known neuroprotective agent; however, the diverse mechanisms of estrogen action on the different types of estrogen receptors is not completely understood. In this study, we determined the extent to which and mechanisms by which 17α-estradiol (17αE2), a natural less-feminizing estrogen, offers neuroprotection against HIV-1 gp120-induced neuronal injury. Endolysosomes are important for neuronal function, and endolysosomal dysfunction contributes to HAND and other neurodegenerative disorders. In hippocampal neurons, estrogen receptor α (ERα) is localized to endolysosomes and 17αE2 acidifies endolysosomes. ERα knockdown or overexpressing an ERα mutant that is deficient in endolysosome localization prevents 17αE2-induced endolysosome acidification. Furthermore, 17αE2-induced increases in dendritic spine density depend on endolysosome localization of ERα. Pretreatment with 17αE2 protected against HIV-1 gp120-induced endolysosome deacidification and reductions in dendritic spines; such protective effects depended on endolysosome localization of ERα. In male HIV-1 transgenic rats, we show that 17αE2 treatment prevents the development of enlarged endolysosomes and reduction in dendritic spines. Our findings demonstrate a novel endolysosome-dependent pathway that governs the ERα-mediated neuroprotective actions of 17αE2, findings that might lead to the development of novel therapeutic strategies against HAND.SIGNIFICANCE STATEMENT Extranuclear presence of membrane-bound estrogen receptors (ERs) underlie the enhancing effect of estrogen on cognition and synaptic function. The estrogen receptor subtype ERα is present on endolysosomes and plays a critical role in the enhancing effects of 17αE2 on endolysosomes and dendritic spines. These findings provide novel insight into the neuroprotective actions of estrogen. Furthermore, 17αE2 protected against HIV-1 gp120-induced endolysosome dysfunction and reductions in dendritic spines, and these protective effects of 17αE2 were mediated via endolysosome localization of ERα. Such findings provide a rationale for developing 17αE2 as a therapeutic strategy against HIV-associated neurocognitive disorders.

A protein-fragment complementation assay reveals that celastrol and gambogic acid suppress ERα mutants in breast cancer

Somatic gain-of-function mutations within estrogen receptor alpha (ERα) are highly associated with hormone therapy resistance in breast cancer. However, current understanding of abnormal activity of ERα mutants and their relevant targeted intervention is still very limited. Herein, we developed a new, real-time, and reliably Gaussia luciferase-based protein-fragment complementation assay (GLPCA) for evaluating ERα mutants activities. We found that, compared with ER WT, ERα mutants (Y537S/N and D538G) exhibit high ligand-independent activity, suggesting the gain-of-function phenotype of these ERα mutants. Notably, Y537S, the most common ERα mutant type, has the highest intrinsic activation. We then collected and screened a natural product library for potential ERα antagonists via GLPCA and identified celastrol and gambogic acid as new antagonists of the ERα Y537S mutant. Moreover, interactions between these two compounds and the ERα Y537S mutant were confirmed by molecular docking and cellular thermal shift assay. Importantly, we further demonstrated that celastrol and gambogic acid exhibit synergistic antiproliferative and pro-apoptotic effects when combined with an approved CDK4/6 inhibitor abemaciclib in breast cancer cells expressing ERα Y537S. In summary, GLPCA provides a powerful platform for exploring innovative functional biology and drug discovery of antagonists targeting ERα mutants.

Real-Time Challenging of ERα Y537S Mutant Transcriptional Activity in Living Cells

Metastatic estrogen receptor α (ERα)-expressing breast cancer (BC) occurs after prolonged patient treatment with endocrine therapy (ET) (e.g., aromatase inhibitors—AI; 4OH-tamoxifen—4OH-Tam). Often these metastatic BCs express a mutated ERα variant (e.g., Y537S), which is transcriptionally hyperactive, sustains uncontrolled proliferation, and renders tumor cells insensitive to ET drugs. Therefore, new molecules blocking hyperactive Y537S ERα mutation transcriptional activity are requested. Here we generated an MCF-7 cell line expressing the Y537S ERα mutation stably expressing an estrogen-responsive element (ERE) promoter, which activity can be monitored in living cells. Characterization of this cell line shows both hyperactive basal transcriptional activity with respect to normal MCF-7 cells, which stably express the same ERE-based promoter and a decreased effect of selective ER downregulators (SERDs) in reducing Y537S ERα mutant transcriptional activity with respect to wild type ERα transcriptional activity. Kinetic profiles of Y537S ERα mutant-based transcription produced by both drugs inducing receptor degradation and siRNA-mediated depletion of specific proteins (e.g., FOXA1 and caveolin1) reveals biphasic dynamics of the inhibition of the receptor-regulated transcriptional effects. Overall, we report a new model where to study the behavior of the Y537S ERα mutant that can be used for the identification of new targets and pathways regulating the Y537S ERα transcriptional activity.

Abstract PS12-23: Development of H3B-6545, a first-in-class oral selective ER covalent antagonist (SERCA), for the treatment of ERaWT and ERaMUT breast cancer

Abstract Mutations in the ligand-binding domain of estrogen receptor alpha (ERα) are detected in up to 30% of patients (pts) who have relapsed or progressed during endocrine therapy. By favoring the agonistic conformation in ERα, these hotspot mutations promote ligand-independent activation of ERα and confer partial resistance to ER-directed therapies. Of the various hotspot mutations, Y537S is the most constitutively active, promotes the greatest resistance phenotype to current endocrine therapies, and is associated with the worst prognosis relative to other ERα mutations. The fact that current ER-directed therapies have limited activity in the ERα mutant setting emphasizes the critical need to develop the next generation of high affinity ER antagonists that can overcome the aberrant activity of mutant ERα. H3B-6545 is a first-in-class selective ERα covalent antagonist (SERCA) which inactivates both wild-type and mutant ERα by irreversibly engaging cysteine-530. Biophysical and biochemical analyses confirm the long residence time achieved by covalent binding, and cellular analyses confirm the selectivity and single-digit nanomolar potency of H3B-6545 across a panel of ERαWT and ERαMUT breast cancer cell lines. H3B-6545 as a monotherapy demonstrates superior anti-tumor activity relative to fulvestrant across a set of CDK4/6 inhibitor naïve ERαWT and ERαY537S cell line-derived xenograft (CDX)/patient-derived xenograft (PDX) models, with regressions being noted in both the ERαWT and ERαMUT settings. Furthermore, H3B-6545 continues to demonstrate single agent activity in CDK4/6 inhibitor-resistant ERαWT and ERαY537S PDX models, in which fulvestrant fails to demonstrate significant anti-tumor activity. Lastly, improved activity and duration of response are noted when H3B-6545 is combined with several targeted therapies, including CDK4/6 inhibitors palbociclib and abemaciclib across a range of ERαWT and ERαY537S CDX/PDX models. The phase I-II trial (NCT03250676) enrolled 130 heavily pretreated pts with ER+, HER2- metastatic breast cancer, including 12 pts harboring high allele frequency clonal ESR1 Y537S circulating tumor DNA (ctDNA). Median number of prior therapy in the metastatic setting was 3 (range: 1-10). Consistent with the preclinical data, H3B-6545 demonstrated promising clinical activity among these pts with clonal Y537S mutations, with a median progression free survival of 7.3 months and an overall response rate of 25% (3 confirmed partial responses). In summary, these compelling preclinical data coupled with emerging clinical activity in heavily pretreated poor prognosis pts support further development of H3B-6545 as monotherapy or combination treatment. Citation Format: Manav Korpal, Craig Furman, Xiaoling Puyang, Zhaojie Zhang, Zhenhua Wu, Deepti Banka, Subhasree Das, Benoit Destenaves, Lei Gao, Erika Hamilton, Ming-Hong Hao, Sean Irwin, Stephen Johnston, Jaya J Joshi, Dejan Juric, Amy Kim, Tuong-Vi Nguyen, Marc Pipas, Timothy Pluard, Victoria Rimkunas, Nathalie Rioux, Joanne Schindler, Peter Smith, Michael Thomas, John Wang, Judy S Wang, Markus Warmuth, Huilan Yao, Shihua Yao, Lihua Yu, Frédéric H Vaillancourt, David M Bolduc, Nicholas A Larsen, GuoZhu Zheng, Sudeep Prajapati, Tarek Sahmoud, Antonio Gualberto, Ping Zhu. Development of H3B-6545, a first-in-class oral selective ER covalent antagonist (SERCA), for the treatment of ERaWT and ERaMUT breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS12-23.

Abstract 922: Preclinical anticancer activity of LSZ102, a novel oral selective estrogen receptor degrader targeting wild-type and mutant ER

Abstract Estrogen receptor (ER) blockade is a well-established therapeutic approach in ER+ breast cancer. A novel oral selective estrogen receptor degrader (SERD), LSZ102, is in development. LSZ102 induces proteasome-mediated degradation of both wild type and mutant ERα in MCF-7 cells. LSZ102 also inhibits transcription of ERα target genes and results in a decrease in cell proliferation in a dose dependent manner. Expression of ERα Y537S results in a shift in inhibition of cell proliferation upon incubation with either LSZ102 or fulvestrant; however, the shift with LSZ102 was less severe. Similarly, LSZ102 induced a more pronounced level of ERα degradation than fulvestrant in the Y537S mutant MCF7 cells. Overall, LSZ102 is effective in the wild type and Y537S ERα mutant setting in vitro. In vivo, LSZ102 treatment of ER+ breast cancer xenografts resulted in inhibition of ERα regulated transcripts and a decrease in ERα protein levels. In the MCF7 xenograft model, expression of ERα Y537S resulted in reduced activity by fulvestrant, but not by LSZ102. In vivo, LSZ102 exhibited single agent and combination efficacy upon co-administration with the CDK4/6 inhibitor ribociclib and the alpha-specific PI3K inhibitor alpelisib. LSZ102 is currently in a Phase I clinical trial in patients with ER+ breast cancer in which it is tested as a single agent, and in combination with either ribociclib or alpelisib. Citation Format: L. Alex Gaither, Choi Lai Tiong Yip, Chunrong Wang, Weiyi Toy, Qing Sheng, Jinyun Chen, Yuji Mishina, Rita Das, Stefan Peukert, Alice Loo, Sarat Chandarlapaty, Adam Crystal, Tinya J. Abrams. Preclinical anticancer activity of LSZ102, a novel oral selective estrogen receptor degrader targeting wild-type and mutant ER [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 922.

Novel mechanism for estrogen receptor alpha modulation of murine lupus

Female sex is a risk factor for lupus. Sex hormones, sex chromosomes and hormone receptors are implicated in the pathogenic pathways in lupus. Estrogen receptor alpha (ERα) knockout (KO) mice are used for defining hormone receptor effects in lupus. Prior studies of ERα KO in lupus have conflicting results, likely due to sex hormone levels, different lupus strains and different ERα KO constructs. Our objective was to compare a complete KO of ERα vs. the original functional KO of ERα (expressing a short ERα) on disease expression and immune phenotype, while controlling sex hormone levels. We studied female lupus prone NZM2410 WT and ERα mutant mice. All mice (n = 44) were ovariectomized (OVX) for hormonal control. Groups of each genotype were estrogen (E2)-repleted after OVX. We found that OVXed NZM mice expressing the truncated ERα (ERα short) had significantly reduced nephritis and prolonged survival compared to both wildtype and the complete ERαKO (ERα null) mice, but surprisingly only if E2-repleted. ERα null mice were not protected regardless of E2 status. We observed significant differences in splenic B cells and dendritic cells and a decrease in cDC2 (CD11b+CD8−) dendritic cells, without a concomitant decrease in cDC1 (CD11b-CD8a+) cells comparing ERα short to ERα null or WT mice. Our data support a protective role for the ERα short protein. ERα short is similar to an endogenously expressed ERα variant (ERα46). Modulating its expression/activity represents a potential approach for treating female-predominant autoimmune diseases.

Abstract P1-10-08: Development of a first-in-class oral selective ERα covalent antagonist (SERCA) for the treatment of ERαWT and ERαMUT breast cancer

Abstract Mutations in estrogen receptor alpha (ERα) are detected in up to 30% of breast cancer patients who have relapsed during endocrine therapy. ERα mutations functionally confer resistance to existing classes of endocrine therapies, likely through gaining constitutive activity. The fact that current ER-directed therapies are only partially effective in the ERα mutant setting, and that a significant proportion of resistant breast cancer metastases continue to remain dependent on ERα signaling for growth/survival, highlights the critical need to develop the next generation of ERα antagonists that can overcome aberrant ERα activity. Using structure-based drug design approaches we have identified a novel class of ERα antagonist referred to as Selective ERα Covalent Antagonist (SERCA) that inactivate both wild-type and mutant ERα by targeting a unique cysteine residue that is not conserved among other steroid hormone receptors. Biophysical, biochemical and cellular analyses confirm the covalent mechanism of action, specific binding to ER and selective inhibition of ERα-dependent transcription of SERCAs. H3B-6545 is a highly selective SERCA that potently antagonizes wild-type and mutant ERα in biochemical and cell based assays demonstrating increased potency over standard of care and other experimental agents. In vivo, H3B-6545 shows superior efficacy to fulvestrant in the MCF-7 xenograft model with once daily oral dosing, achieving maximal antitumor activity at doses >10x below the maximum tolerated dose in mice. In addition, H3B-6545 shows superior antitumor activity to both tamoxifen and fulvestrant in patient derived xenograft models of breast cancer carrying estrogen receptor mutations. In summary, H3B-6545 is a first-in-class, orally available and selective ER covalent antagonist with a compelling pre-clinical profile that is being developed for the treatment of ERα positive breast cancer. Citation Format: Korpal M, Puyang X, Furman C, Zheng GZ, Banka D, Wu J, Zhang Z, Thomas M, Mackenzie C, Yao H, Rimkunas V, Kumar P, Caleb B, Karr C, Subramanian V, Irwin S, Larsen N, Vaillancourt F, Nguyen T-V, Davis A, Chan B, Hao MH, O'Shea M, Prajapati S, Agoulnik S, Kuznetsov G, Kumar N, Yu Y, Lai G, Hart A, Eckley S, Fekkes P, Bowser T, Joshi JJ, Selvaraj A, Wardell S, Norris J, Smith S, Reynolds D, Mitchell L, Wang J, Yu L, Kim A, Rioux N, Sahmoud T, Warmuth M, Smith PG, Zhu P. Development of a first-in-class oral selective ERα covalent antagonist (SERCA) for the treatment of ERαWT and ERαMUT breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-08.

Abstract DDT01-04: Discovery and development of H3B-6545: A novel, oral, selective estrogen receptor covalent antagonist (SERCA) for the treatment of breast cancer

Abstract Mutations in the estrogen receptor (ER) are detected in up to 30% of patients that initially respond but subsequently relaps to anti-endocrine therapies. ERα mutations, likely through constitutively activating ERα, can functionally confer resistance to existing classes of endocrine therapies. Current endocrine therapies are only partially effective in the ERα mutant setting and a significant proportion of endocrine-therapy resistant breast cancer metastases continue to remain dependent on ERα signaling for growth/survival indicating a critical need to develop the next generation of ERα antagonists that can overcome ERα wild-type and mutant activity. Here we describe a novel series of compounds with a unique mode of inhibition that potently target both wild-type and mutant ERα. These compounds are Selective Estrogen Receptor Covalent Antagonists (SERCAs) that inactivate the estrogen receptor by targeting a cysteine that is not present in other nuclear hormone receptors, leading to a unique biological and activity profile differentiated from Selective Estrogen Receptor Modulators (SERMs) and Selective Estrogen Receptor Degraders (SERDs). Using structure-based drug design approaches we have identified a first-in-class clinical candidate, H3B-6545. H3B-6545 is a highly selective small molecule that potently antagonizes wild-type and mutant ERα in biochemical and cell based assays. In vitro comparisons with standard of care and other experimental agents confirm increased cell potency of H3B-6545 under continuous as well as washout treatment conditions. In vivo, once daily oral dosing of H3B-6545 shows potent activity and superior efficacy to fulvestrant in the MCF-7 xenograft model with maximal antitumor activity at doses >10x below the maximum tolerated dose in mice. In addition, H3B-6545 shows superior antitumor activity to tamoxifen and fulvestrant in patient derived xenograft models of estrogen receptor positive breast cancer including models carrying ERα mutations In non-clinical safety studies in rat and monkeys, H3B-6545 is well tolerated across a broad dose range and at exposures that significantly exceed those required for efficacy in mouse xenograft models. In summary, H3B-6545 is a first-in-class, orally available and potent selective estrogen receptor covalent antagonist with a compelling preclinical efficacy and safety profile that is being developed for the treatment of breast cancer. Citation Format: Peter G. Smith, Xiaoling Puyang, Craig Furman, Guo Zhu Zheng, Deepti Banka, Michael Thomas, Vanitha Subramanian, Sean Irwin, Nicholas Larsen, Benjamin Caleb, Craig Karr, Jeremy Wu, Morgan O’Shea, Joyce Yang, Allison Davis, Amy Kim, Nathalie Rioux, Victoria Rimkunas, Huilan Yao, Crystal MacKenzie, Pavan Kumar, Sherri Smith, Sean Eckley, Andrew Hart, George Lai, Christopher Rowbottom, Peter Fekkes, Silvia Buonamici, Dominic Reynolds, Lihua Yu, Tarek Sahmoud, Markus Warmuth, Lorna Mitchell, Ping Zhu, Manav Korpal. Discovery and development of H3B-6545: A novel, oral, selective estrogen receptor covalent antagonist (SERCA) for the treatment of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr DDT01-04. doi:10.1158/1538-7445.AM2017-DDT01-04

Abstract DDT01-03: Discovery and pre-clinical pharmacology of AZD9496: An oral, selective estrogen receptor down-regulator (SERD)

Abstract With over 70% of breast cancers expressing estrogen receptor alpha protein (ERα), treatment with either anti-hormonal therapies that directly block ERα function (e.g. tamoxifen) or therapies that block the production of estrogen itself (e.g. anastrozole) have proven to be effective treatments for the disease. Following the discovery of the ERα antagonist tamoxifen in the 1960s, identification of the selective estrogen receptor down-regulator (SERD) fulvestrant represented a further step forward in the treatment of advanced ER+ breast cancer, especially in the endocrine resistance setting where ERα appears to be activated by a ligand independent route through other growth factor signaling pathways. In addition, fulvestrant has also shown significant overall survival (OS) results in the FIRST trial comparing 500 mg fulvestrant with anastrozole in first line advanced ER+ve patients where the majority of patients had not received prior endocrine therapy. Given fulvestrant's low bioavailability following intramuscular injection and the levels of ERα protein in clinical samples after treatment, the question remains as to whether an agent that could achieve higher steady state levels of drug more rapidly and drive further decreases in ERα levels would give enhanced clinical benefit. We have identified a novel, potent, non-steroidal SERD that can be administered orally and could yield improved exposure and clinical benefit. This presentation will describe the discovery and pre-clinical pharmacology of AZD9496, a small molecule that can antagonise ERα and induce receptor degradation in breast cancer cell lines at picomolar concentrations. The good oral pharmacokinetic properties of the compound in pre-clinical species led to significant tumor growth inhibition in an endocrine sensitive MCF-7 xenograft model at a dose of 5 mg/kg and >90% reduction in ER-regulated, progesterone receptor (PR) levels. Tumor regressions were seen in a long term estrogen deprived (LTED) in vivo model, representing the aromatase resistant setting, and corresponded with significant reductions in ERα protein levels, >90% at 5 mg/kg dose. AZD9496 also showed antagonist and down-regulation activity against ERα mutant protein both in vitro and in vivo. These findings strongly supported selection of AZD9496 as a clinical candidate for the treatment of ER+ve breast cancer and the drug is now under evaluation in a Phase 1 clinical trial. Citation Format: Hazel Weir, Mandy Lawson, Rowena Callis, Michael Hulse, Michael Tonge, Gareth Davies, Graeme Walker, Rachel Rowlinson, Jon Curwen, Zena Wilson, Steve Powell, Robert Bradbury, Alfred Rabow, Craig Donald, David Buttar, Richard Norman, Camila de Almeida, Peter Ballard, Gordon Currie, David Andrews, Graham Richmond, Anne Marie Mazzola, Ermira Pazolli, Brendon Ladd, Celina D'Cruz, Chris De Savi. Discovery and pre-clinical pharmacology of AZD9496: An oral, selective estrogen receptor down-regulator (SERD). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr DDT01-03. doi:10.1158/1538-7445.AM2015-DDT01-03

Estrogen Receptor Alpha Binding to ERE is Required for Full Tlr7- and Tlr9-Induced Inflammation.

We previously found that a maximum innate inflammatory response induced by stimulation of Toll-like receptors (TLRs) 3, 7 and 9 requires ERα, but does not require estrogen in multiple cell types from both control and lupus-prone mice. Given the estrogen-independence, we hypothesized that ERα mediates TLR signaling by tethering to, and enhancing, the activity of downstream transcription factors such as NFκB, rather than acting classically by binding EREs on target genes. To investigate the mechanism of ERα impact on TLR signaling, we utilized mice with a knock-in ERα mutant that is unable to bind ERE. After stimulation with TLR ligands, both ex vivo spleen cells and bone marrow-derived dendritic cells (BM-DCs) isolated from mutant ERα ("KIKO") mice produced significantly less IL-6 compared with cells from wild-type (WT) littermates. These results suggest that ERα modulation of TLR signaling does indeed require ERE binding for its effect on the innate immune response.

Estrogen Receptor α L543A,L544A Mutation Changes Antagonists to Agonists, Correlating with the Ligand Binding Domain Dimerization Associated with DNA Binding Activity

A ligand-dependent nuclear transcription factor, ERα has two transactivating functional domains (AF), AF-1 and AF-2. AF-1 is localized in the N-terminal region, and AF-2 is distributed in the C-terminal ligand-binding domain (LBD) of the ERα protein. Helix 12 (H12) in the LBD is a component of the AF-2, and the configuration of H12 is ligand-inducible to an active or inactive form. We demonstrated previously that the ERα mutant (AF2ER) possessing L543A,L544A mutations in H12 disrupts AF-2 function and reverses antagonists such as fulvestrant/ICI182780 (ICI) or 4-hydoxytamoxifen (OHT) into agonists in the AF2ER knock-in mouse. Our previous in vitro studies suggested that the mode of AF2ER activation is similar to the partial agonist activity of OHT for WT-ERα. However, it is still unclear how antagonists activate ERα. To understand the molecular mechanism of antagonist reversal activity, we analyzed the correlation between the ICI-dependent estrogen-responsive element-mediated transcription activity of AF2ER and AF2ER-LBD dimerization activity. We report here that ICI-dependent AF2ER activation correlated with the activity of AF2ER-LBD homodimerization. Prevention of dimerization impaired the ICI-dependent ERE binding and transcription activity of AF2ER. The dislocation of H12 caused ICI-dependent LBD homodimerization involving the F-domain, the adjoining region of H12. Furthermore, F-domain truncation also strongly depressed the dimerization of WT-ERα-LBD with antagonists but not with E2. AF2ER activation levels with ICI, OHT, and raloxifene were parallel with the degree of AF2ER-LBD homodimerization, supporting a mechanism that antagonist-dependent LBD homodimerization involving the F-domain results in antagonist reversal activity of H12-mutated ERα.

Selective Mutations in Estrogen Receptor α D-domain Alters Nuclear Translocation and Non-estrogen Response Element Gene Regulatory Mechanisms

The three main mechanisms of ERα action are: 1) nuclear, genomic, direct DNA binding, 2) nuclear, genomic, "tethered"-mediated, protein-protein interactions, and 3) non-nuclear, non-genomic, rapid action responses. Reports suggest the D-domain or hinge region of ERα plays an important role in mechanisms 1 and 2 above. Studies demonstrating the functionality of the ERα hinge region have resected the full D-domain; therefore, site directed mutations were made to attribute precise sequence functionality to this domain. This study focuses on the characterization and properties of three novel site directed ERα- D-domain mutants. The Hinge 1 (H1) ERα mutant has disrupted nuclear localization, can no longer perform tethered mediated responses and has lost interaction with c-Jun, but retains estrogen response element (ERE)-mediated functions as demonstrated by confocal microscopy, reporter assays, endogenous gene expression and co-immunoprecipitation. The H2 ERα mutant is non-nuclear, but translocates to the nucleus with estradiol (E2) treatment and maintains ERE-mediated functionality. The H2+NES ERα mutant does not maintain nuclear translocation with hormone binding, no longer activates ERE-target genes, functions in ERE- or tethered-mediated luciferase assays, but does retain the non-genomic, non-nuclear, rapid action response. These studies reveal the sequence(s) in the ERα hinge region that are involved in tethered-mediated actions as well as nuclear localization and attribute important functionality to this region of the receptor. In addition, the properties of these ERα mutants will allow future studies to further dissect and characterize the three main ERα mechanisms of action and determine the mechanistic role each action has in estrogen hormone regulation.

Non-nuclear Estrogen Receptor Signaling in the Endothelium

In addition to the classical function of estrogen receptors (ER) as transcription factors, evidence continues to accumulate that they mediate non-nuclear processes in numerous cell types, including the endothelium, in which they activate endothelial NO synthase. Non-nuclear ER signaling entails unique post-translational modifications and protein-protein interactions of the receptor with adaptor molecules, kinases, and G proteins. Recent in vitro and in vivo studies in mice using an estrogen-dendrimer conjugate that is excluded from the nucleus indicate that non-nuclear ER activation underlies the migration and growth responses of endothelial cells to estrogen but not the growth responses of endometrial or breast cancer cells to the hormone. In this minireview, the features of ERα and protein-protein interactions that enable it to invoke extranuclear signaling in the endothelium and the consequences of that signaling are discussed.

Estrogen receptor-alpha-interacting cytokeratins potentiate the antiestrogenic activity of fulvestrant

Fulvestrant (ICI 182, 780) is a selective estrogen receptor downregulator (SERD) and potent antiestrogen. In estrogen receptor alpha positive ERα(+) breast cancer, the drug immobilizes ERα in the nuclear matrix, inducing receptor polyubiquitination and subsequent degradation via the 26S proteasome. We previously reported that fulvestrant-induced ERa degradation depends on the interaction of ERα with cytokeratins 8 and 18 (CK8/CK18). Here we further investigate the role of these two cytokeratins in the antagonistic activity of the SERD. Using ER-responsive reporter assays, we demonstrate greater antiestrogenic activity of fulvestrant in CK8/CK18(+) vs. CK8/CK18(-) cancer cells and loss of CK8/CK18 expression was observed in a breast cancer cell model for acquired fulvestrant resistance. In contrast, the presence of CK8/CK18 had no effect on the antiestrogenic activity of 4-hydroxytamoxifen, which was unable to induce an interaction between these CKs and ERα. By utilizing the ligand activity inversion ERα mutant L540Q to further examine the mechanism of fulvestrant action, we demonstrate that the ERα mutant does not interact with CK8/CK18 in the presence of fulvestrant and L540Q is not immobilized to the nuclear matrix after antiestrogen treatment. In transcription assays, fulvestrant displayed agonist activity, stimulating L540Q-mediated gene expression. In addition, fulvestrant did not induce an ERβ interaction with CK8/CK18 and subsequent ERb degradation. Collectively, these results suggest that CK8/18 play an important role in the antiestrogenic action of fulvestrant in breast cancer cells and that these two cytokeratins could serve as prognostic markers for SERD therapy response in breast cancer patients.