Mutant Estrogen Receptor Research Articles

Abstract GS6-05: A joint atlas of single-cell and bulk RNA-seq in metastatic breast cancer allows inference of oncogenic and drug-resistant transcriptional programs in malignant cells and the tumor microenvironment

Abstract While recent studies have begun to elucidate the genomics of metastatic breast cancer (MBC), the transcriptional programs (progs) that drive the drug-resistant phenotype remain poorly understood. We prospectively collected biopsies from patients with MBC with detailed clinicopathologic features, including treatment and response characteristics. We profiled 29 biopsies by single-cell RNA seq, as well as bulk RNA-seq and whole exome sequencing on an additional 207 biopsies (analyzed snapshot, ongoing study). We analyzed these data to generate an atlas, delineating the cell-types, cell-states, and transcriptional progs. We profiled 100,470 single cell transcriptomes and generated a comprehensive MBC atlas of the tumor and tumor microenvironment (TME). We next inferred cell types and progs associated with clinicopathologic characteristics. For example, we found significant differences in the TME of liver metastases compared to other sites, consistent with immunosuppression in the hepatic space. In particular, liver metastases were depleted in activated B-cells (lower CD69, TCL1A, VPREB3, BCNP1, and several activation-related chemokines - CXCR4/5/6/7), and their infiltrating T-cells expressed lower levels of effector and cytotoxicity markers (table=T) including CD8, CD3, beta chemokines CCL4/CCL5, IFI16, PRF1, granzymes, and GNLY, and exceptionally low expression of antigen processing genes (T). To increase our power to make clinically relevant associations, we performed a joint analysis of the single-cell and bulk RNA-Seq data, to identify malignant cell progs related to specific oncogenic mutations, with implications for metastatic and drug-resistance phenotypes. For example, we characterized the oncogenic prog associated with activating estrogen receptor mutation (ESR1-mut). As expected, ESR1-mut prog overlaps with many known ER and luminal B markers (T). ESR1-mut prog also included specific Interferon-stimulated genes (ISGs) - IFI6, ISG15, IFIT1, STAT1, which are associated with tamoxifen resistance (T) and ECM-mediated regulation of apoptosis (T). These ISGs are predictive of poor prognosis among endocrine-treated patients (HR=1.69. p=1e-04, n=929, kmplotter). The ESR1-mut prog also included genes associated with cell-migration (SOX9, AGR2, TXNIP and several S100 genes). This suggests a role for ESR1 mutation in pathogenicity, beyond ligand-independent activation of ER signaling. A second malignant cell prog was associated with RB-null. While most of these genes were highly correlated with cell-cycle (R=0.4), some do not (HINT1, RHOC, SNRPB, ANAPC11, TMEM208, POLR2J). In spite of decoupling from proliferation, these still predict of poor outcome across breast cancer (HR=1.41, p= 6.0e-10, n=3951, kmplotter). We characterized the prog of high-grade tumors in our cohort, and found a strong association with RB-null prog (T). Similarly, we recovered additional mutation-specific oncogenic progs, including for TP53, GATA3, FOXA1, HER2, and FGFR mutants, forming a compendium of in-vivo oncogenic signatures. To the best of our knowledge these data represent the first integration of single cell and bulk RNA-seq data in MBC, resulting in a comprehensive single-cell-resolution transcriptional atlas, and a catalog of drug-resistance oncogenic progs with implications for immunotherapy and precision-oncology. Single-cell In-vivo programComparison Gene setAssociationP-value(top genes)(MSigDB name, PubMed ID, when applicable)Odds-ratio(Fisher''''s Exact test, two-sided)Depleted in liver T-cellsEffectors-cells markers4.077.29E-07(compared to other metastatic site - Bone, Skin, Chest-wall, Lymph node, Breast-met)(PMID: 28052254)""Antigen processing markers32.17.26E-07(PMID: 28052254)ESR1-mutVANTVEER_BREAST_CANCER_ESR1_UP5.82.30E-11(Enriched in ESR1-mutatated)(PMID: 11823860)“SMID_BREAST_CANCER_LUMINAL_B_UP5.898.10E-12“”DOANE_BREAST_CANCER_ESR1_UP6.30812.10E-09""BOWIE_RESPONSE_TO_TAMOXIFEN10.360.00036(PMID 17016442)""BECKER_TAMOXIFEN_RESISTANCE_UP7.81.20E-06(PMID 15657362)""BOWIE_RESPONSE_TO_EXTRACELLULAR_MATRIX8.240.0028(PMID 17016442)Enriched in RB-nullEnriched in High-grade (from our study)23.97.50E-05 Citation Format: Ofir Cohen, Daniel Abravanel, Michal Slyper, Johanna Klughammer, Judit Jane-Valbuena, Karla Helvie, Laura Dellostritto, Allison Frangieh, Sebastien Vigneau, Jingyi Wu, Angie Mayorga, Julia Waldman, Lan Nguyen, Danielle Dionne, Orr Ashenberg, Matan Hofree, Mike Cuoco, Christopher Rodman, Eric P Winer, Nancy Lin, Bruce Johnson, Asaf Rotem, Orit Rozenblatt-Rosen, Aviv Regev, Nikhil Wagle. A joint atlas of single-cell and bulk RNA-seq in metastatic breast cancer allows inference of oncogenic and drug-resistant transcriptional programs in malignant cells and the tumor microenvironment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS6-05.

Early Enrichment of ESR1 Mutations and the Impact on Gene Expression in Presurgical Primary Breast Cancer Treated with Aromatase Inhibitors.

To investigate the presence of ESR1 mutations in primary estrogen-receptor-positive (ER+) breast cancer treated with extended (>4 weeks) neoadjuvant (presurgical) aromatase inhibitor (NAI) therapy and to identify patients who may gain less benefit from aromatase inhibition (AI) alone based upon on-treatment changes in gene expression. We evaluated ER, progesterone receptor, and Ki67 by immunostaining, ESR1 mutations by droplet-digital PCR and expression of over 800 key breast cancer genes in paired pre- and post-NAI tumor samples from 87 ER+ breast cancer patients. Cell proliferation and estrogen-regulated genes (ERG) remained suppressed in most tumors indicative of persistent response to NAI. Enrichment of ESR1 mutations was found in five tumors and predominantly in patients receiving therapy for >6 months. ESR1-mutant tumors showed increased expression of ESR1 transcript and limited suppression of ERGs and proliferation-associated genes in response to NAI. ESR1 wild-type tumors with high residual proliferation (Ki67r ≥10%; 15/87 tumors) showed lower ESR1/ER expression pre- and post-therapy and lower ERGs. Tumors with ESR1 mutations or Ki67r ≥10% showed less inhibition of estrogen response, cell cycle, and E2F-target genes. Ligand-independent ER signaling, as a result of ESR1 mutation or reduced ER dependence, identified after extended NAI therapy, can guide early selection of patients who would benefit from combination therapy.

Androgen Receptor Is a Non-canonical Inhibitor of Wild-Type and Mutant Estrogen Receptors in Hormone Receptor-Positive Breast Cancers.

SummarySustained treatment of estrogen receptor (ER)-positive breast cancer with ER-targeting drugs results in ER mutations and refractory unresponsive cancers. Androgen receptor (AR), which is expressed in 80%–95% of ER-positive breast cancers, could serve as an alternate therapeutic target. Although AR agonists were used in the past to treat breast cancer, their use is currently infrequent due to virilizing side effects. Discovery of tissue-selective AR modulators (SARMs) has renewed interest in using AR agonists to treat breast cancer. Using translational models, we show that AR agonist and SARM, but not antagonist, inhibit the proliferation and growth of ER-positive breast cancer cells, patient-derived tissues, and patient-derived xenografts (PDX). Ligand-activated AR inhibits wild-type and mutant ER activity by reprogramming the ER and FOXA1 cistrome and rendering tumor growth inhibition. These findings suggest that ligand-activated AR may function as a non-canonical inhibitor of ER and that AR agonists may offer a safe and effective treatment for ER-positive breast cancer.

An AND-Gated Drug and Photoactivatable Cre-loxP System for Spatiotemporal Control in Cell-Based Therapeutics.

While engineered chimeric antigen receptor (CAR) T cells have shown promise in detecting and eradicating cancer cells within patients, it remains difficult to identify a set of truly cancer-specific CAR-targeting cell surface antigens to prevent potentially fatal on-target off-tumor toxicity against other healthy tissues within the body. To help address this issue, we present a novel tamoxifen-gated photoactivatable split-Cre recombinase optogenetic system, called TamPA-Cre, that features high spatiotemporal control to limit CAR T cell activity to the tumor site. We created and optimized a novel genetic AND gate switch by integrating the features of tamoxifen-dependent nuclear localization and blue-light-inducible heterodimerization of Magnet protein domains (nMag, pMag) into split Cre recombinase. By fusing the cytosol-localizing mutant estrogen receptor ligand binding domain (ERT2) to the N-terminal half of split Cre(2-59aa)-nMag, the TamPA-Cre protein ERT2-CreN-nMag is physically separated from its nuclear-localized binding partner, NLS-pMag-CreC(60-343aa). Without tamoxifen to drive nuclear localization of ERT2-CreN-nMag, the typically high background of the photoactivation system was significantly suppressed. Upon blue light stimulation following tamoxifen treatment, the TamPA-Cre system exhibits sensitivity to low intensity, short durations of blue light exposure to induce robust Cre-loxP recombination efficiency. We finally demonstrate that this TamPA-Cre system can be applied to specifically control localized CAR expression and subsequently T cell activation. As such, we posit that CAR T cell activity can be confined to a solid tumor site by applying an external stimulus, with high precision of control in both space and time, such as light.

Circulating oestrogen receptor mutations and splice variants in advanced prostate cancer

To characterize circulating oestrogen receptor ( ER) mutants and splice variants in men with advanced prostate cancer. Sequential blood samples were obtained from men with advanced prostate cancer, and from healthy controls. Blood-derived RNA samples were analysed using droplet digital PCR for the presence of six ERα mutations (E380Q, L536Q, Y537C, Y537S, Y537N and D538G), and six ERα and ERβ splice variants (ERα-66, ERα-36, ERβ1, ERβ2, ERβ4 & ERβ5). A total of 94 samples were collected from 42 men with advanced prostate cancer. Four mutations (E380Q, L536Q, Y537S and D538G) and all six splice variants were detected in patient samples. Splice variants were detectable in non-cancer control samples. The presence of ER mutations was associated with bone metastases and castration resistance. ERβ splice variant concentrations decreased after successive lines of treatment. The ER mutations were detectable in plasma from patients with advanced prostate cancer. ER splice variants were frequently detected in both men with and without prostate cancer.

1895P - Breast cancer organoids model treatment response of HER2 targeted therapy in HER2-mutant breast cancer

BackgroundTo facilitate cancer precision medicine, breast cancer organoids have recently emerged as a useful pre-clinical model for retaining sufficient fidelity regarding histology, the transcriptome and genome. However, their potential to predict clinical treatment responses remains unclear. MethodsWe generated breast cancer organoids from breast cancer tissues and performed drug sensitivity test on these organoids based on genomic analysis data. ResultsA total of 25 fresh breast cancer tissues and biopsies from 22 patients were processed between November 2017 and February 2019. Breast cancer organoids were grown successfully from 10 out of 25 patients (40%). We performed histopathological analysis of H&E stained tissues and organoid sections and confirmed that the phenotypes of organoids matched the original histological breast cancer types. We also performed whole genome DNA sequencing (WGS) and RNA sequencing (RNA-seq) on breast cancer organoids and paired breast cancer tissues. One of these breast cancer organoids had HER2 mutations (previously shown to be an activating mutation) and retained expression of estrogen receptor (ER) and progesterone receptor (PR) (Luminal A subtype). In our previous work, we identified activating HER2 mutations (S310F D769Y V777L 778insGSP) in ER positive/HER2-amplification negative breast cancer, who had developed resistance to multi-line endocrine therapy. Two patients achieved a durable partial response (approximately 1 years) to trastuzumab combined with everolimus. We also showed that HER2 mutations were constitutively active, and T47D and MCF7 overexpressing HER2 mutations were sensitive to HER2 targeted therapies combined with everolimus. Hence, we tested the effects of trastuzumab and everolimus on this HER2-mutant breast cancer organoid in vitro and in vivo. HER2-targeted therapies combined with everolimus produced regression of the HER2-mutated organoid. ConclusionsThe breast cancer organoids may serve as a high-fidelity platform and recapitulate clinical treatment responses in personalized medicine. These data provide a strong preclinical rationale for combining HER2-targeted therapies with everolimus in HER2-mutant/ HER2-amplification negative breast cancer. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

P2.09-08 Correlation Between Hormone Receptor Expression and EGFR Gene Mutation in Lung Cancer Patients with Simultaneous Primary Breast Cancer

Double primary breast cancer (BC) and lung cancer (LC) is not uncommon but research is limited. To decipher the inner pathogenesis, relationship between hormone receptor (HR) protein expression and EGFR gene mutation was explored in the present study. Clinicopathological characteristics of 400 female patients with double primary BC and LC were analyzed, while another 114 patients with single LC were compared correspondingly. Tissue samples were obtained from enrolled subjects to detect EGFR mutation status by gene sequencing analysis, and estrogen receptor (ER) and progesterone receptor (PR) expression was determined by immunohistochemistry. Among 169 patients, synchronous and metachronous double primary BC-LC cases accounted for 39.1% and 61.0%, respectively. For most female LC patients with simultaneous primary BC, adenocarcinoma was the dominant subtype (95.1%). The positivity rates were 13% for ER and 13% for PR in lung tumor tissues of 200 double BC-LC patients, slightly higher than those in single LC patients. Among BC-LC patients with mutant EGFR, 48.2% were either ER-positive (30.6%) or PR-positive (30.6%). But for those without EGFR mutation, both ER and PR could not be detected in lung tumor tissue samples. χ2 testfurther confirmed a significantly positive correlation between ER, PR expression and EGFR mutation in lung tumor tissues in double primary patients (P < 0.05). However, such relationship could not be similarly observed in single LC cases. Besides, the presence of family tumor history was associated with the onset time of the two primary cancers. Double primary BC-LC patients have distinctive clinicopathological features. Expression of HRs (both ER and PR) significantly correlated with EGFR mutation status in their lung tumor tissues.

Early-onset complete ovarian failure and lack of puberty in a woman with mutated estrogen receptor β (ESR2)

Early-onset complete ovarian failure and lack of puberty in a woman with mutated estrogen receptor β (ESR2)

Structural, Thermodynamic, and Kinetic Traits of Antiestrogen-Compounds Selectively Targeting the Y537S Mutant Estrogen Receptor α Transcriptional Activity in Breast Cancer Cell Lines.

The most frequently diagnosed cancers in women are the estrogen receptor (ER)-positive breast cancer subtypes, which are characterized by estrogen dependency for their growth. The mainstay of clinical treatment for this tumor relies on the modulation of ERα action or on the suppression of estrogen biosynthesis via the administration of Selective ERα Modulators/Down-regulators (SERMs/SERDs) or aromatase inhibitors, respectively. Nevertheless, de novo and acquired resistance to these therapies frequently occurs and represents a major clinical concern for patient survival. Recently, somatic mutations affecting the hormone-binding domain of ERα (i.e., Y537S, Y537N, D538G) have been associated with endocrine resistance, disease relapse and increased mortality rates. Hence, devising novel therapies against these ERα isoforms represents a daunting challenge. Here, we identified five molecules active on recurrent Y537S ERα polymorphism by employing in silico virtual screening on commercial databases of molecules, complemented by ER-transactivation and MTT assays in MCF7 and MDA-MB-231 breast cancer cells expressing wild type or mutated ERα. Among them, one molecule selectively targets Y537S ERα without inducing any cytotoxicity in breast cell lines. Multi-microseconds (4.5 μs) of biased and unbiased molecular dynamics provided an atomic-level picture of the structural, thermodynamics (i.e., binding free energies) and the kinetic (i.e., dissociation free energy barriers) of these active ligands as compared to clinically used SERM/SERDs upon binding to wild type and distinct ERα variants (Y537S, Y537N, D538G). This study contributes to a dissection of the key molecular traits needed by drug-candidates to hamper the agonist (active)-like conformation of ERα, normally selected by those polymorphic variants. This information can be useful to discover mutant specific drug-candidates, enabling to move a step forward toward tailored approaches for breast cancer treatment.

Estrogen-independent molecular actions of mutant estrogen receptor 1 in endometrial cancer.

Estrogen receptor 1 (ESR1) mutations have been identified in hormone therapy–resistant breast cancer and primary endometrial cancer. Analyses in breast cancer suggest that mutant ESR1 exhibits estrogen-independent activity. In endometrial cancer, ESR1 mutations are associated with worse outcomes and less obesity, however, experimental investigation of these mutations has not been performed. Using a unique CRISPR/Cas9 strategy, we introduced the D538G mutation, a common endometrial cancer mutation that alters the ligand binding domain of ESR1, while epitope tagging the endogenous locus. We discovered estrogen-independent mutant ESR1 genomic binding that is significantly altered from wild-type ESR1. The D538G mutation impacted expression, including a large set of nonestrogen-regulated genes, and chromatin accessibility, with most affected loci bound by mutant ESR1. Mutant ESR1 is distinct from constitutive ESR1 activity because mutant-specific changes are not recapitulated with prolonged estrogen exposure. Overall, the D538G mutant ESR1 confers estrogen-independent activity while causing additional regulatory changes in endometrial cancer cells that are distinct from breast cancer cells.

Glucocorticoid receptor modulation decreases ER-positive breast cancer cell proliferation and suppresses wild-type and mutant ER chromatin association

BackgroundNon-ER nuclear receptor activity can alter estrogen receptor (ER) chromatin association and resultant ER-mediated transcription. Consistent with GR modulation of ER activity, high tumor glucocorticoid receptor (GR) expression correlates with improved relapse-free survival in ER+ breast cancer (BC) patients.MethodsIn vitro cell proliferation assays were used to assess ER-mediated BC cell proliferation following GR modulation. ER chromatin association following ER/GR co-liganding was measured using global ChIP sequencing and directed ChIP analysis of proliferative gene enhancers.ResultsWe found that GR liganding with either a pure agonist or a selective GR modulator (SGRM) slowed estradiol (E2)-mediated proliferation in ER+ BC models. SGRMs that antagonized transcription of GR-unique genes both promoted GR chromatin association and inhibited ER chromatin localization at common DNA enhancer sites. Gene expression analysis revealed that ER and GR co-activation decreased proliferative gene activation (compared to ER activation alone), specifically reducing CCND1, CDK2, and CDK6 gene expression. We also found that ligand-dependent GR occupancy of common ER-bound enhancer regions suppressed both wild-type and mutant ER chromatin association and decreased corresponding gene expression. In vivo, treatment with structurally diverse SGRMs also reduced MCF-7 Y537S ER-expressing BC xenograft growth.ConclusionThese studies demonstrate that liganded GR can suppress ER chromatin occupancy at shared ER-regulated enhancers, including CCND1 (Cyclin D1), regardless of whether the ligand is a classic GR agonist or antagonist. Resulting GR-mediated suppression of ER+ BC proliferative gene expression and cell division suggests that SGRMs could decrease ER-driven gene expression.

Abstract 1004: Leveraging an expanding tool box for ER+ breast cancer: exploring breast cancer dependencies to support optimal therapeutic strategies

Abstract Aromatase inhibitors (AIs), the Estrogen Receptor (ER) modulator tamoxifen and the full ER antagonist fulvestrant each target ER signaling and constitute the backbone of standard of care for ER+ breast cancer. The growing appreciation of liabilities associated with each of these modalities, which may limit their clinical potential, has triggered a wave of activity around next-generation ER therapeutics, and there are currently more than 10 new molecular entities targeting ER being evaluated in Phase 1 clinical trials. In addition to a collection of ER-targeted therapeutics either approved or under evaluation, the emergence of CDK4/6 inhibitors as combination partners has meaningfully contributed to the oncologist’s tool box for the treatment of breast cancer, while also adding complexity to the treatment landscape. Although direct ER antagonists are expected to be superior to AIs in patients with tumors expressing constitutively activating ER mutations (e.g. ER.Y537S), it is currently unclear if a subset of ER wildtype patients may likewise derive superior benefit from direct ER targeting. It is also unclear which ER+ tumors require CDK4/6 inhibitor/ER antagonist combinations for maximal anti-tumor activity, versus those where single-agent ER antagonist treatment may achieve full cell cycle control. To address these key questions, we used a panel of 22 ER+ cell lines to evaluate the relative anti-proliferative and cytotoxic effects of estrogen (E2) withdrawal (modeling AIs), ER modulation, and full ER inhibition, as single agents and in combination with the CDK4/6 inhibitor Palbociclib. We find a number of cell lines in which direct ER antagonism is superior to E2 withdrawal and is associated with E2-independent ER signaling not attributable to ER mutations. The impact of E2 withdrawal on cell cycle control and proliferation is significantly enhanced by concurrent CDK4/6 inhibition, in line with clinical observations, however, direct ER antagonism in combination with CDK4/6 inhibition remains most efficacious. We are integrating our cell response data with detailed molecular analyses and transcriptional profiling to 1) identify cellular contexts, beyond ER mutations, where direct ER antagonism is superior to targeting E2 synthesis, 2) identify tumors where full cell cycle control and anti-tumor efficacy is achievable through single-agent ER targeting, and 3) better understand the basis for the co-operativity of endocrine suppression with CDK4/6 inhibition. Deconvolution of the ER+ breast cancer response to distinct modes of ER signaling inhibition alone and in combination with CDK4/6 inhibitors has the potential to guide clinical decision-making for personalized patient care. Citation Format: Wei Zhou, Jane Guan, Georgia Hatzivassiliou, Anneleen Daemen, Marc Hafner, Ciara Metcalfe. Leveraging an expanding tool box for ER+ breast cancer: exploring breast cancer dependencies to support optimal therapeutic strategies [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 1004.

Abstract 21: Generation and characterization of potent analogues of ERX-11

Abstract Breast cancer (BC) is the most common malignancy in women. Approximately 70% BC are Estrogen receptor (ER) positive, However, most patients develop resistance to current therapies and progress to incurable metastases. We had earlier reported a novel therapeutic agent, ERX-11, that modulates estrogen receptor coregulator interactions. For lead optimization, we designed, synthesized, and tested over 500 analogs of ERX-11 in multiple models of BC. Methods: In vitro activity was tested using cell titer glo, MTT, and apoptosis assays. The utility of the ERX analogs in treating therapy resistant ER-positive BC was evaluated using models with acquired resistance (Tamoxifen, Letrozole), and engineered models that express ER mutations. Xenografts, patient derived xenografts (PDX), and patient derived xenograft explants (PDEx) were used for testing the utility of ERX analogs. Results: Our screening studies identified several ERX analogs with potent activity against BC cells. Subtle changes in the ERX analogs appear to have significant ramifications on both their potency against ER-positive BC cell lines and against other tumors types. Some analogs like ERX-41 were more potent than ERX-11 in their ability to block the proliferation of multiple ER-positive BC cell lines (IC50 ranging from 50-200 nM). Other analogs like ERX-208 showed similar activity as ERX-11 against ER-positive BC cell lines but had potent activity (IC50 ranging from 50 -100 nM) against ovarian cancer cell lines. Through iterative changes, we have identified lead compounds with significant activity against other cancers, including in gliomas, ovarian and pancreatic cancers. Although all these compounds were designed to better target the ligand binding pocket of ER, CRISPR-Cas9 based KO screening revealed that these active compounds do not all target ER and appear to target other proteins, including other nuclear receptors. Some compounds for example have activity in ER-negative BC. In several xenograft and models, including pancreatic cancer and ER-negative BC, the activity of the compounds have been confirmed by oral administration of the ERX analogs in vivo. We have also validated the activity of these analogs using PDX and PDEX models. Conclusions: From our studies to develop a more potent ERX-11 lead analog, we have identified multiple analogs with activities against multiple cancers. While the intended target of these analogs was ER, our library of analogs has potent activity against both ER-positive and ER-negative tumors. In collaboration, we are pursuing further leads in multiple cancers to further delineate the mechanism of action of these various analogs. Citation Format: Suryavathi Viswanadhapalli, Mengxing Li, Shi-Hong Ma, Gangadhara Reddy Sareddy, Tae-Kyung Lee, Mei Zhou, Yiliao Liu, Xihui Liu, Dede N. Bahun-Wilson, Kara Kassees, Jung-Mo Ahn, Ganesh V. Raj, Ratna K. Vadlamudi. Generation and characterization of potent analogues of ERX-11 [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 21.

Modeling the Effect of the Metastatic Microenvironment on Phenotypes Conferred by Estrogen Receptor Mutations Using a Human Liver Microphysiological System

Reciprocal coevolution of tumors and their microenvironments underlies disease progression, yet intrinsic limitations of patient-derived xenografts and simpler cell-based models present challenges towards a deeper understanding of these intercellular communication networks. To help overcome these barriers and complement existing models, we have developed a human microphysiological system (MPS) model of the human liver acinus, a common metastatic site, and have applied this system to estrogen receptor (ER)+ breast cancer. In addition to their hallmark constitutive (but ER-dependent) growth phenotype, different ESR1 missense mutations, prominently observed during estrogen deprivation therapy, confer distinct estrogen-enhanced growth and drug resistant phenotypes not evident under cell autonomous conditions. Under low molecular oxygen within the physiological range (~5–20%) of the normal liver acinus, the estrogen-enhanced growth phenotypes are lost, a dependency not observed in monoculture. In contrast, the constitutive growth phenotypes are invariant within this range of molecular oxygen suggesting that ESR1 mutations confer a growth advantage not only during estrogen deprivation but also at lower oxygen levels. We discuss the prospects and limitations of implementing human MPS, especially in conjunction with in situ single cell hyperplexed computational pathology platforms, to identify biomarkers mechanistically linked to disease progression that inform optimal therapeutic strategies for patients.

Defining the molecular pharmacology of disease relevant estrogen receptor mutations for effective therapeutic targeting in breast cancer

The Estrogen Receptor (ER/ESR1) is expressed in the majority of luminal breast cancers and drugs that inhibit ER‐signaling, (e.g. the Selective Estrogen Receptor Modulator (SERM) tamoxifen and aromatase inhibitors like letrozole, or anastrozole) are the cornerstone of breast cancer pharmacotherapy. However, in the adjuvant setting, the clinical success of these drugs are limited by the development of resistance. Whereas the mechanisms underlying resistance are varied and complex, it is now clear that gain of function point mutations within the ligand binding domain (LBD) of ESR1 likely contribute to the development of resistance to aromatase inhibitors. Although extremely rare in primary breast tumors, mutations in the ESR1 LBD occur in up to 40% of metastatic lesions. Preclinically, it has been demonstrated that these mutations drive ER− dependent transcription, proliferation and tumor cell migration in the absence of hormone. Importantly, several reports have demonstrated that these mutant ERs may display resistance to standard hormonal therapies, a finding which defines the need for new antiestrogens.The goal of this study is to understand the molecular basis for the altered pharmacology of the most clinically relevant ESR1 mutations and to use this information to inform the selection of new or existing drugs that would be expected to inhibit the activity of these receptors. In cell based models of breast cancer, we made the important observation that when compared to wild‐type ER, the pharmacology (most notably potency) of these mutants was significantly impacted by cell context and receptor expression level. This was apparent in assays that measured ER‐transcriptional activation and cell proliferation. Specifically, although we and others have observed that the potency of existing ER antagonists are reduced when ESR1 mutants are evaluated, such differences are dependent on the expression level of the receptors and are most apparent under conditions of overexpression. These data highlight the need to define the relationship between ER expression level and ligand potency/efficacy in the development of pharmaceutical approaches to target ESR1 mutants.Importantly, we observed that the potency and efficacy of lasofoxifene, a very high affinity SERM, did not differ significantly in any of the model systems studied. We believe that this activity of lasofoxifene can most likely be explained by its increased affinity as it was not shown to have any significant differences from other modulators in its ability to regulate ER stability or conformational state. This data suggest that the pharmacology of the most prevalent ESR1 mutants is dependent on both receptor expression level and cell context, and that these differences are not significant when a high affinity ER‐antagonist is employed. These findings have near term clinical implications for the treatment of endocrine therapy resistant breast cancers.Support or Funding InformationFunding was provided by NIH R01 DK048807 (DPM) and the Pharmaceutical Sciences Training Program (T32 GM 007105) and NCI F31 CA220978‐01 (KJA)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exploring the PROTAC degron candidates: OBHSA with different side chains as novel selective estrogen receptor degraders (SERDs)

As the mutant estrogen receptor (ER) continues to be characterized, breast cancer is becoming increasingly difficult to cure when treated with hormone therapy. In this regard, a strategy to selectively and effectively degrade the ER might be an effective alternative to endocrine therapy for breast cancer. In a previous study, we identified a novel series of 7-oxabicyclo[2.2.1]heptene sulfonamide (OBHSA) compounds as full ER antagonists while lacking the prototypical ligand side chain that has been widely used to induce antagonism of ERα. Further crystal structure studies and phenotypic assays revealed that these compounds are selective estrogen receptor degraders (SERDs) with a new mechanism of action. However, from a drug discovery point of view, there still is room to improve the potency of these OBHSA compounds. In this study, we have developed new classes of SERDs that contain the OBHSA core structure and different side chains, e.g., basic side chains, long alkyl acid side chains, and glycerol ether side chains, to simply mimic the degrons of proteolysis targeting chimera (PROTAC) and then investigated the structure-activity relationships of these PROTAC-like hybrid compounds. These novel SERDs could effectively inhibit MCF-7 cell proliferation and demonstrated good ERα degradation efficacy. Among the SERDs, compounds 17d, 17e and 17g containing a basic side chain with a N-trifluoroethyl substituent and a para methoxyl group at the phenyl group of the sulfonamide turned out to be the best candidates for ER degraders. A further docking study of these compounds with ERα elucidates their structure-activity relationships, which provides guidance to design new PROTAC degrons targeting ER for breast cancer therapy. Lastly, easy modification of these PROTAC-like SERDs enables further fine-tuning of their pharmacokinetic properties, including oral availability.

Impaired flow-mediated vasodilatation in mutated estrogen receptor alpha (ERα) mouse reveals a role of unliganted membrane, but not nuclear, ERα in shear stress-dependent mechanotransduction

Flow-mediated dilation (FMD) of resistance arteries (RAs) plays a key role in tissue perfusion. A reduced FMD is the hallmark of the endothelium dysfunction occurring early in cardiovascular and metabolic disorders. Estrogen therapy improves FMD in diseased conditions without major effect in healthy subjects. Nevertheless, estrogen receptor alpha (ERα) mutation in a man was associated with severe and selective FMD reduction. Thus, we evaluated the role of ERα in FMD in healthy young male mice. FMD was determined in vitro in mesenteric RAs isolated from mice lacking ERα (ERα−/−), the ERα nuclear activation function AF2 (AF2-ERα−/−) or the ERα palmitoylation site (C451A-ERα−/−, inactivation of the membrane effects or ERα), compared to littermate wild-type (WT or+/+) mice. We found that FMD was significantly reduced in ERα−/− compared to ERα+/+ male and female (ovariectomized or not) mice. Exogenous ERα stimulation or blockade did not affect FMD. FMD was not affected in AF2-ERα−/− mice whereas it was significantly decreased in C451A-ERα−/− mice. NO-synthesis blockade with L-NNA strongly reduced FMD in WT and AF2-ERα−/− mice without major effect in ERα-/- and C451A-ERα−/− mice. Acetylcholine-mediated relaxation, not affected by the absence of ERα was similarly reduced by L-NNA in all groups. After ROS reduction in vivo, NO-dependent FMD was equivalent in C451A-ERα−/− and C451A-ERα+/+ mice. Thus, membrane ERα is important for FMD through activation of eNOS activity and reduction of ROS production, possibly in a ligand-independent manner.

Abstract P5-04-18: ARV-471, an oral estrogen receptor PROTAC degrader for breast cancer

Abstract ARV-471, an estrogen receptor (ER) alpha PROTAC, is a hetero-bifunctional molecule that facilitates the interactions between estrogen receptor alpha and an intracellular E3 ligase complex, leading to the ubiquitylation and subsequent degradation of estrogen receptors via the proteasome. ARV-471 robustly degrades ER in ER-positive breast cancer cell lines with a half-maximal degradation concentration (DC50) of ˜ 2 nM. PROTAC-mediated ER degradation decreases the expression of classically-regulated ER-target genes (PR, GREB1, TFF) and inhibits cell proliferation of ER-dependent cell lines (MCF7, T47D). Additionally, ARV-471 degrades clinically-relevant ESR1 variants (Y537S and D538G) and inhibits growth of cell lines expressing those variants. In an immature rat uterotrophic model, ARV-471 degrades rat uterine ER and demonstrates no agonist activity. Daily, oral-administration of single agent ARV-471 (3, 10, and 30 mpk) leads to significant tumor volume regressions of estradiol-dependent MCF7 xenografts and concomitant tumor ER protein reductions of &amp;gt;90% at study termination. Moreover, when a CDK4/6 inhibitor is combined with ARV-471 in the MCF7 model, even more pronounced tumor growth inhibition is observed (˜130% TGI), accompanied by significant reductions in ER protein levels. In an ESR1 Y537S, hormone-independent patient-derived xenograft model, ARV-471 at 10 mpk completely inhibited growth and also reduced mutant ER protein levels. Taken together, the preclinical data of ARV-471 supports its continued development as a best-in-class oral ER PROTAC-degrader. Citation Format: Flanagan JJ, Qian Y, Gough SM, Andreoli M, Bookbinder M, Cadelina G, Bradley J, Rousseau E, Willard R, Pizzano J, Crews CM, Crew AP, Taylor I, Houston J. ARV-471, an oral estrogen receptor PROTAC degrader for breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-18.

Acquired HER2 mutations in ER+ metastatic breast cancer confer resistance to estrogen receptor-directed therapies.

Seventy percent of breast cancers express the estrogen receptor (ER), and agents that target the ER are the mainstay of treatment. However, virtually all people with ER+ breast cancer develop resistance to ER-directed agents in the metastatic setting. Beyond mutations in the ER itself, which occur in 25-30% of people treated with aromatase inhibitors1-4, knowledge about clinical resistance mechanisms remains incomplete. We identified activating HER2 mutations in metastatic biopsies from eight patients with ER+ metastatic breast cancer who had developed resistance to aromatase inhibitors, tamoxifen or fulvestrant. Examination of treatment-naive primary tumors in five patients showed no evidence of pre-existing mutations in four of five patients, suggesting that these mutations were acquired under the selective pressure of ER-directed therapy. The HER2 mutations and ER mutations were mutually exclusive, suggesting a distinct mechanism of acquired resistance to ER-directed therapies. In vitro analysis confirmed that the HER2 mutations conferred estrogen independence as well as-in contrast to ER mutations-resistance to tamoxifen, fulvestrant and the CDK4 and CDK6 inhibitor palbociclib. Resistance was overcome by combining ER-directed therapy with the irreversible HER2 kinase inhibitor neratinib.

Genes and Pathways Promoting Long-Term Liver Repopulation byEx VivohYAP-ERT2 Transduced Hepatocytes and Treatment of Jaundice in Gunn Rats.

Hepatocyte transplantation is an attractive alternative to liver transplantation. Thus far, however, extensive liver repopulation by adult hepatocytes has required ongoing genetic, physical, or chemical injury to host liver. We hypothesized that providing a regulated proliferative and/or survival advantage to transplanted hepatocytes should enable repopulation in a normal liver microenvironment. Here, we repopulated livers of DPPIV− (dipeptidyl peptidase‐4) rats and Ugt1a1 (uridinediphosphoglucuronate glucuronosyltransferase 1a1)‐deficient Gunn rats (model of Crigler‐Najjar syndrome type 1), both models without underlying liver injury, for up to 1 year by transplanting lenti‐hYAP‐ERT2 (mutated estrogen receptor ligand‐binding domain 2)‐transduced hepatocytes (YAP‐Hc). Yap (yes‐associated protein) nuclear translocation/function in YAP‐Hc was regulated by tamoxifen. Repopulating YAP‐Hc and host hepatocytes were fluorescence‐activated cell sorting–purified and their transcriptomic profiles compared by RNAseq. After 1 year of liver repopulation, YAP‐Hc clusters exhibited normal morphology, integration into hepatic plates and hepatocyte‐specific gene expression, without dysplasia, dedifferentiation, or tumorigenesis. RNAseq analysis showed up‐regulation of 145 genes promoting cell proliferation and 305 genes suppressing apoptosis, including hepatocyte growth factor and connective tissue growth factor among the top 30 in each category and provided insight into the mechanism of cell competition that enabled replacement of host hepatocytes by YAP‐Hc. In Gunn rats transplanted with YAP‐Hc+tamoxifen, there was a 65%‐81% decline in serum bilirubin over 6 months versus 8%‐20% with control‐Hc, representing a 3‐4‐fold increase in therapeutic response. This correlated with liver repopulation as demonstrated by the presence of Ugt1a1‐positive hepatocyte clusters in livers and western blot analysis of tissue homogenates. Conclusion: Tamoxifen‐regulated nuclear translocation/function of hYAP‐ERT2 enabled long‐term repopulation of DPPIV−/− and Gunn rat livers by hYAP‐ERT2‐transduced hepatocytes without tumorigenesis. This cell transplantation strategy may offer a potential therapy for most of the inherited monogenic liver diseases that do not exhibit liver injury.