Estrogen Receptor Ligand-binding Domain Research Articles

Abstract PO3-26-06: A self-replicating RNA Precision Immunotherapeutic for Overcoming Resistance to Endocrine Therapy in Estrogen Receptor Positive Breast Cancer (ER+BC)

Abstract Advanced or metastatic ER+HER2- breast cancer (ER+BC) is an incurable disease. Standard 1L therapy (SOC) utilizes endocrine blockade with a nonsteroidal aromatase inhibitor (NSAI) combined with a CDK4/6 inhibitor (CDK4/6i). While most patients respond and disease remains controlled for over 2 years (mPFS ~25-28 months), virtually all patients progress over time, requiring more toxic therapies with decreasing periods of disease control. Prolonging the time that the tumor remains under endocrine control is therefore of significant medical value. The most common molecular drivers of endocrine resistance are well characterized and include mutations in ESR1 and bypass mechanisms in select signaling pathways. RBI-1000 is a self-replicating RNA (srRNA) precision immunotherapeutic to generate robust immunity directed against acquired resistance mutations that develop in ER+ BC in response to endocrine blockade by NSAI. RBI-1000 includes 6 on-target mutations within the estrogen receptor ligand binding domain, and bypass mutations either in the form of activating mutations in PI3K kinase or amplifications of HER2/HER3. Precision immunotherapy (PIO), combined with SOC targeted therapy, educates the patient’s immune system to eliminate tumor cells that express acquired resistance mutations. Thus, the tumor is in a lose-lose situation where it is either eliminated by the targeted therapy, or if it develops acquired resistance mutations to the targeted therapy, it is killed by the PIO. Specifically for endocrine resistance, RBI-1000 PIO offers an attractive therapeutic approach to prevent/delay endocrine resistance specifically, by generating an effective immune response against those tumor cells that express the resistance-associated molecules that arise while on NSAI+CDK4/6i. The ability to encode multiple targets in a single therapeutic srRNA molecule, alongside the prolonged expression of srRNA in host cells (>49 days) creates a versatile and powerful cancer drug development platform. srRNA precision immunotherapeutics are delivered intramuscularly (i.e. a jab in the non-dominant deltoid) and generate an effective immune response via the same pathways and mechanisms as a traditional vaccine. We have demonstrated that this srRNA encapsulated in a lipid nanoparticle primes polyfunctional CD4 and CD8 T cells and antibody responses, leading to tumor growth inhibition and improved survival in a mouse model expressing the targeted acquired resistance mutations. Priming of T cells against acquired mutations is also confirmed in human HLA-transgenic mice. The immune cell-mediated elimination of clones expressing the acquired resistance mutations is predicted to prolong endocrine control of ER+BC, in an analogous manner to small molecule or monoclonal antibody targeted therapies, but with a more favorable dosing and adverse event profile due to precise immunologic targeting and no DDI. RBI-1000 is anticipated to enter clinical studies in the first half of 2024. Citation Format: Zelanna Goldberg, Christian Maine, Gabrielle Dailey, Christine Domingo, Gaelle Picarda, Hunter Little, Annie Chou, Jessica Sparks, Darina Spasova, Shiegeki Miyake-Stoner, Zachary C Hartman, Christopher Rabiola, Erika Crosby, Herbert Lyerly, Nathaniel Wang, Parinaz Aliahmad. A self-replicating RNA Precision Immunotherapeutic for Overcoming Resistance to Endocrine Therapy in Estrogen Receptor Positive Breast Cancer (ER+BC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-06.

Abstract PO3-18-09: OPERA-01: A randomized, open-label, phase 3, study of palazestrant (OP-1250) vs standard-of-care treatment for ER+, HER2- advanced or metastatic breast cancer after endocrine and CDK4/6 inhibitor therapy

Abstract Background: In estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (MBC), adding a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor to endocrine therapy (ET) has improved outcomes and is the current standard-of-care treatment in the first-line setting. However, resistance to first-line treatment develops in most patients (pts), which is often attributed to acquired mutations in ESR1. Limited data exist for ET-based treatment options after progression on prior ET and CDK4/6 inhibitors, and most pts will transition to chemotherapy for further treatment. Therefore, there is a significant unmet need for more effective ET in pts with ER-positive, HER2-negative MBC to improve outcomes and delay time to chemotherapy. Palazestrant (OP-1250) is a small molecule oral complete ER antagonist (CERAN) and selective ER degrader (SERD) that binds the ligand binding domain of ER and completely blocks ER-driven transcriptional activity in both wild-type (ESR1-wt) and mutant (ESR1-mut) forms of ER. In preclinical studies, palazestrant demonstrated better tumor shrinkage in ESR1-wt and ESR1-mut models when compared to fulvestrant; it also showed efficacy in brain metastasis models. In a phase 1/2 monotherapy study in pts with heavily pretreated ER-positive, HER2-negative advanced breast cancer (BC) or MBC (NCT04505826), palazestrant showed a tolerable safety profile, promising antitumor efficacy, and favorable pharmacokinetics (PK) supporting once a day (qd) dosing. The recommended Phase 2 dose of palazestrant is 120 mg qd. Methods: The OPERA-01 trial is an international, multicenter, randomized, open-label, phase 3 clinical trial comparing the efficacy and safety of palazestrant as a single agent to standard-of-care ET (fulvestrant, anastrozole, letrozole, or exemestane) in pts with ER-positive, HER2-negative advanced BC or MBC that has relapsed or progressed on 1 or 2 prior lines of ET for MBC, which includes a CDK4/6 inhibitor. Eligible pts are women (pre- or post-menopausal) or men who have a confirmed diagnosis of ER-positive, HER2-negative locally advanced evaluable BC or MBC not amenable to curative therapy. ER and HER2 status are determined according to the American Society of Clinical Oncology/College of American Pathologists guidelines. Patients must have Eastern Cooperative Oncology Group performance status 0 or 1. Prior treatments must include 1 or 2 prior lines of ET as monotherapy or in combination with a CDK4/6 inhibitor for MBC; must have received CDK4/6 inhibitor in combination with ET and have disease progression during or within 28 days of completion of each line of prior treatment for MBC; most recent ET given for ≥6 months. Prior chemotherapy in the metastatic setting is not permitted. Patients (N∼500) are randomized to palazestrant or standard-of-care ET monotherapy, and are stratified by ESR1 mutation status, prior lines of ET for advanced disease (1 vs 2 lines), and presence or absence of visceral disease. The primary endpoint of progression-free survival will be assessed by blinded independent central review in pts with and without ESR1 mutations in the intent-to-treat population. Secondary endpoints include overall survival, antitumor activity (objective response rate, clinical benefit rate, and duration of response), safety, patient-reported outcomes, and PK in pts with and without ESR1 mutations. The study is planned to be conducted globally. Citation Format: Arlene Chan, Jane Meisel, Komal Jhaveri, Joo Hyuk Sohn, Meritxell Bellet- Ezquerra, Jason Schroeder, Mark Shilkrut, Barbara Pistilli. OPERA-01: A randomized, open-label, phase 3, study of palazestrant (OP-1250) vs standard-of-care treatment for ER+, HER2- advanced or metastatic breast cancer after endocrine and CDK4/6 inhibitor therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-18-09.

Abstract PO4-04-12: A Phase 1b/2 study of palazestrant (OP-1250) in combination with ribociclib or alpelisib in patients with estrogen receptor-positive, human epidermal growth factor receptor 2-negative, advanced and/or metastatic breast cancer

Abstract Background: Endocrine therapy (ET) when administered with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors has improved outcomes in patients (pts) with estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer (BC) or metastatic breast cancer (MBC) and is the current standard of care for first-line treatment. Addition of the PIK3 inhibitor alpelisib to ET significantly improved progression-free survival in pts with PIK3CA-mutated, ER-positive, HER2-negative MBC, and alpelisib + fulvestrant is the standard of care for second or later-line treatment. Most pts will acquire resistance to ET, with mutations in ESR1 constituting the most common mechanism of resistance. Palazestrant (OP-1250) is a small molecule oral complete ER antagonist (CERAN) and selective ER degrader (SERD) that binds the ligand binding domain of ER and completely blocks ER-driven transcriptional activity in both wild-type (ESR1-wt) and mutant (ESR1-mut) forms of ER. In preclinical studies, palazestrant in combination with ribociclib demonstrated activity in both ESR1-wt and ESR1-mut xenograft models and showed efficacy in brain metastasis xenograft models. In a Phase 1/2 monotherapy study in pts with ER-positive, HER2-negative MBC, palazestrant was well tolerated with demonstrated antitumor efficacy and favorable pharmacokinetics (PK) supporting once a day (qd) dosing at the recommended Phase 2 dose (RP2D) of 120 mg. The aim of this study is to evaluate the safety, PK, and antitumor activity of palazestrant + ribociclib or alpelisib in pts with ER-positive, HER2-negative advanced BC or MBC. Methods: Eligible pts have evaluable ER-positive, HER2-negative advanced BC or MBC; ≤2 prior ETs (prior CDK4/6 inhibitors allowed); and ≤1 prior line of chemotherapy. In the alpelisib arm, pts must have a PIK3CA mutation in the tumor or ctDNA. Patients are administered oral palazestrant at escalating doses of 30, 60, and 120 mg qd in combination with the approved dose of oral ribociclib (600 mg qd; days 1–21 of 28-day cycle) or approved dose of oral alpelisib (300 mg qd) in a 3+3 design to identify the RP2D. The dose-expansion part will assess additional safety and PK parameters and the antitumor activity of palazestrant in combination with ribociclib or alpelisib (NCT05508906). Results: As of June 15, 2023, 10 pts have received 30 mg and 60 mg doses of palazestrant in combination with ribociclib (n=6) or alpelisib (n=4). No dose-limiting toxicities have been observed. After administration in the first two combination dose levels of 30 and 60 mg, palazestrant exposure was consistent with monotherapy administration; exposure data show no drug–drug interactions (DDI) when compared to published exposure parameters. Most reported treatment-emergent adverse events (TEAEs) were grade 1 or 2 and consistent with the known safety profiles of all 3 drugs. In the ribociclib arm, the TEAEs occurring in ≥2 pts included grade 1 nausea (n=4), grade 1 constipation (n=2), and neutropenia (grade 2, n=1; grade 3, n=3). For the 3 pts in the 30 mg palazestrant + alpelisib cohort, the only TEAE occurring in ≥2 pts was grade 1 diarrhea. One pt had grade 1 hyperglycemia. Conclusions: In this ongoing study, palazestrant in combination with ribociclib or alpelisib was well tolerated, and enrollment to the palazestrant 120 mg dose with ribociclib or alpelisib is ongoing. No new safety signals were observed for the 3 drugs, and no clinically significant DDIs were observed between palazestrant and ribociclib or alpelisib at the doses evaluated. Exposure of each drug was consistent with observed monotherapy exposure levels. Updated data will be presented. Citation Format: Virginia Borges, Carlos Alemany, Nancy Lin, Sara Nunnery, Cynthia Ma, Margaret Tonda, Morena Shaw, Arlene Chan. A Phase 1b/2 study of palazestrant (OP-1250) in combination with ribociclib or alpelisib in patients with estrogen receptor-positive, human epidermal growth factor receptor 2-negative, advanced and/or metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-04-12.

Abstract PS15-04: A Phase 1b/2 study of palazestrant (OP-1250), an oral complete estrogen receptor antagonist (CERAN) and selective ER degrader (SERD), with palbociclib in ER-positive, HER2-negative, advanced or metastatic breast cancer patients

Abstract Background: Palazestrant (OP-1250) is a small molecule oral complete estrogen receptor (ER) antagonist (CERAN) and selective ER degrader (SERD) that binds the ligand binding domain of ER and completely blocks ER-driven transcriptional activity in both wild-type (ESR1-wt) and mutant (ESR1-mut) forms of ER. In preclinical studies, palazestrant demonstrated improved tumor growth inhibition and shrinkage when combined with a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor. In a Phase 1/2 monotherapy clinical study in patients with ER-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer, palazestrant was well tolerated with demonstrated antitumor efficacy and favorable pharmacokinetics (PK) supporting once a day (qd) dosing at the recommended Phase 2 dose of 120 mg. In this study, pts received palazestrant at escalating doses of 30, 60, 90, and 120 mg qd in combination with palbociclib (125 mg qd) and no dose-limiting toxicities were observed. Palazestrant did not affect palbociclib PK and no drug–drug interactions (DDIs) occurred. Here we report updates on safety, efficacy, and PK from the ongoing study of palazestrant in combination with palbociclib. Methods: Patients with advanced or metastatic breast cancer with progression on or after ≤1 line of endocrine therapy (prior CDK4/6 inhibitor therapy and ≤1 chemotherapy allowed) were enrolled to receive oral palazestrant 120 mg qd with oral palbociclib 125 mg qd for 21 of 28 days (NCT0526610). Results: As of May 12, 2023, 33 patients have been treated with palazestrant in combination with palbociclib; 12 pts were dosed in the dose-escalation phase and 21 patients were dosed in the dose-expansion phase of the study. Twenty-two patients (67%) received prior CDK4/6 inhibitor therapy; 15 patients received prior palbociclib. Thirteen of 25 patients (52%) with available baseline ctDNA had an ESR1 mutation. The most common (≥20%) treatment-emergent adverse events (TEAEs) were neutropenia, nausea, vomiting, constipation, and diarrhea. Most of these events were grade 1 or 2. The most common grade 3 or 4 related AE was neutropenia (18/33 [55%] were grade 3; 3/33 [9%] were grade 4). The steady-state exposure of palazestrant when dosed with 125 mg palbociclib was consistent with the monotherapy study. Palbociclib exposure at steady-state was comparable to published monotherapy data when combined with palazestrant at all dose levels tested. As of the data cut-off, there were 4 partial responses (2 confirmed) out of 19 response-evaluable patients, with a clinical benefit rate (CBR) of 42% (8/19) across all patients and 67% (6/9) in patients with ESR1 mutations. Conclusions: Palazestrant and palbociclib dosed in combination were well tolerated with a safety profile consistent with the individual profiles of each drug as monotherapy, and there were no DDIs. Tumor responses and clinical benefit were observed in this population of patients, including those who received prior CDK4/6 inhibitors. Expanding on our previous report, these data provide the rationale to continue advancing the clinical development of palazestrant with the approved dose of palbociclib. Updated data will be presented. Citation Format: Arlene Chan, Daphne Day, Phuong Dinh, Michael Slancar, Janine Lombard, Vinod Ganju, Nicole McCarthy, Rosalind Wilson, Demiana Faltaos, Gurpreet Mathauda-Sahota, Caitlin Murphy. A Phase 1b/2 study of palazestrant (OP-1250), an oral complete estrogen receptor antagonist (CERAN) and selective ER degrader (SERD), with palbociclib in ER-positive, HER2-negative, advanced or metastatic breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS15-04.

Abstract PO2-24-04: Exploring the role and therapeutic potential of Ubiquitin Specific Peptidase 11 (USP11) in estrogen receptor positive breast cancer

Abstract Dysregulated estrogen receptor (ER) function is a key feature of 70% of breast cancers. In this setting ER primarily functions as a growth-controlling transcription factor driving pro-tumorigenic properties, thus there is a need to identify novel modulators of ER transcriptional activity as alternative therapeutic options. Previously, we showed a significant association between high ubiquitin specific protease 11 (USP11) expression and poor survival in ER+ breast cancer patients, but not in ER- patients, indicating a role for USP11 in ER+ subtypes. However, the precise role of USP11 in controlling ER function remains undescribed. To interrogate USP11 function in ER+ breast cancer we generated a CRISPR USP11 knockout MCF7 breast cancer cell line. Our results showed for the first time that USP11 knockout significantly reduces proliferation, induces G2 cell cycle arrest and apoptosis, abrogates ERα transcriptional activity, significantly impairs cell migration and invasion, whilst also impairing MCF7 cancer stem-cell like properties and malignant transformation in vitro. In addition, we also observed significant dysregulation of epithelial-mesenchymal transition-related markers including E-cadherin, N-cadherin, Snail and Slug. Using RNA-seq of MCF7 cells +/- estradiol, +/- stable USP11 knockdown we identified dysregulated pathways in protein binding, cell adhesion molecule binding and cell cycle regulation following USP11 modulation. Of note, gene expression analysis indicated NCOA3 as the most differentially expressed coding gene. Further validation confirmed a significant reduction in NCOA3- encoded Steroid receptor coactivator- 3 (SRC-3) protein expression following USP11 modulation with immunocytochemical analysis also indicating co-localisation within the nucleus. Frequently upregulated in breast cancer and associated with poor outcome, SRC-3 promotes ER transcriptional activity and co-activates a wide range of additional pro-tumorigenic transcription factors. Thus, SRC-3 modulation offers an attractive therapeutic target in breast cancer. This finding may present a novel role for USP11 in controlling SRC-3 and therefore associated ER transcriptional activity. We then aimed to explore the therapeutic potential of USP11. Although rare in primary breast cancer, activating mutations in the ER ligand-binding domain have recently been linked to recurrent, anti-endocrine resistant disease and metastasis. As such cancers remain dependent on ER-signalling for proliferation, but are resistant to ER targeted therapies, we identified USP11 as a potential novel therapeutic target. Using CRISPR knock-in ER mutant MCF7 cell lines expressing the four most common ER activating mutations, we demonstrated that USP11 knockdown continues to significantly reduce proliferation and abrogates mutant ERα transcriptional activity. We then explored amphipathic cell penetrating peptide RALA encapsulation as a delivery method for siRNA targeting USP11. Optimisation of nanoparticle delivery in the Y537S ER-mutant MCF7 breast cancer cell line achieved significant knockdown of USP11 and ER- related target genes including TFF1, PgR, GREB1 and PKIB in 2D with minimal associated cellular toxicity. To overcome the siRNA-related issues of rapid clearance upon systemic delivery, we then explored the use of a freeze-dried collagen-based scaffold model as an in vitro 3D RALA-encapsulated siRNA delivery system. Here we observed successful delivery of nanoparticle to MCF7 breast cancer cells, with a significant reduction in USP11 mRNA expression. Bio-compatible scaffold- facilitated delivery may therefore offer a more feasible translation of this siRNA-based therapeutic. Overall, this study highlights a novel role for USP11 in various oncogenic pathways. USP11 may therefore offer a novel therapeutic target to be exploited for the management of ER-positive breast cancer, including ER mutant breast cancer. Citation Format: Rachel Moore, Anna Blümel, Elspeth Ward, Elizabeth Sainsbury, Emer Conroy, Fergal O’Brien, Caroline Curtin, Simak Ali, Helen McCarthy, William Gallagher, Darran O'Connor. Exploring the role and therapeutic potential of Ubiquitin Specific Peptidase 11 (USP11) in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-04.

A phase 1b/2 dose escalation and expansion study of OP-1250 in combination with ribociclib or alpelisib in patients with advanced and/or metastatic estrogen receptor–positive (ER+)/HER2-negative (HER2-) breast cancer.

TPS1127 Background: Therapies targeting the estrogen receptor (ER) are standard of care in the management of ER+/HER2- metastatic breast cancer (MBC) and when administered concurrently with cyclin-dependent kinase (CDK) inhibitors are the most effective first-line therapy. CDK 4/6 inhibitors plus endocrine therapy improve progression-free survival and, for some agents, overall survival for patients with MBC. However, most patients will acquire resistance, highlighting an unmet need for more effective endocrine options in ER+ disease. OP-1250 is a small molecule oral complete estrogen receptor antagonist (CERAN) and selective estrogen receptor degrader (SERD) that binds the ligand binding domain of ER and completely blocks ER-driven transcriptional activity. OP-1250 potently inactivates both wild-type and mutant forms of ER, the latter of which confers ligand independent activity as a mechanism of resistance to standard of care endocrine therapies. In preclinical studies, OP-1250 in combination with CDK4/6 inhibitors demonstrated synergistic activity in models of both wild-type ER and those with ESR1 (the gene that encodes ER) activating mutations, and in brain metastasis. OP-1250 is orally bioavailable with a favorable pharmacokinetic (PK) profile supportive of once-daily dosing, and a low probability of drug:drug interactions, making it an attractive agent for patients with ER+/HER2- MBC. Methods: The study (NCT05508906) is an open-label, multicenter, 2-part study of OP-1250 in combination with either the CDK4/6 inhibitor ribociclib or the PI3K inhibitor alpelisib for patients with MBC. Eligibility includes adults with evaluable (measurable or non-measurable) ER+/HER2- recurrent, locally advanced or MBC confirmed by histology/cytology, ≤2 prior hormonal regimens (prior CDK4/6 inhibitors allowed) and ≤ 1 prior line of chemotherapy. Patients in the alpelisib arm must have a PIK3CA mutation. A total of 30 patients will be enrolled in each arm. The dose escalation cohorts will evaluate the safety and PK of varied OP-1250 doses (30mg, 60mg, or 120mg) administered orally (PO) every day (QD) in combination with approved dosages of either ribociclib 600 mg PO QD or alpelisib 300 mg PO QD to identify the recommended phase 2 doses (RP2D). The dose expansion cohorts will assess additional safety and PK parameters and further explore the anti-tumor activity of OP-1250 in combination with ribociclib or alpelisib. Tolerability, safety and PK of OP-1250 in combination with ribociclib or alpelisib constitute the primary endpoints. Overall response rate, clinical benefit rate and duration of response are key secondary endpoints. Enrollment at approximately 20 sites in the United States and Australia is ongoing. Clinical trial information: NCT05508906 .

Abstract 6403: A self-replicating RNA precision medicine approach to overcoming resistance to endocrine therapy in ER+BC

Abstract Drug resistance remains the major driving factor behind the clinical failure of targeted therapeutics. Current oncology precision medicine approaches rely on targeting known acquired resistance mutations, such as EGFR T790M or ALK/ROS mutations in NSCLC with 2nd and 3rd generation molecules designed to overcome or prevent resistance. These next generation targeted therapeutic approaches have increasingly long and complex drug development timelines and burdensome toxicities from off target effects (e.g. wild-type receptor targeting) or drug-drug interactions (DDI). The toxicities limit tolerability, compliance and combinability of different targeted therapeutics. RNA-based immunotherapy approaches offer an increasingly attractive alternative to next generation small molecule targeted therapeutics approaches: (1) RNA-based approaches only require a known acquired resistance sequence, (2) drug development timelines, cost and complexity can be meaningfully condensed, and (3) multiple acquired resistance mutations can be targeted with the same candidate. RBI-1000 is a candidate using a novel type of self-replicating RNA (srRNA) to generate robust immunity directed against acquired resistance mutations that develop in ER+ breast cancer (ER+ BC) in response to endocrine therapy. RBI-1000 includes on-target mutations within the estrogen receptor ligand binding domain, and bypass mutations either in the form of activating mutations in the PI3K kinase domain or amplifications of HER2/HER3. Here, we demonstrate that this srRNA encapsulated in a lipid nanoparticle primes polyfunctional CD4 and CD8 T cells leading to tumor growth inhibition and improved survival in a mouse model expressing the targeted acquired resistance mutation. Priming of T cells against acquired mutations is also confirmed in human HLA-transgenic mice. The immune cell-mediated elimination of clones expressing the acquired resistance mutations is predicted to prolong endocrine control of ER+BC, in an analogous manner to small molecule or monoclonal antibody targeted therapies, but with a more favorable dosing and adverse event profile due to precise immunologic targeting and no DDI. Citation Format: Zelanna Goldberg, Christian Maine, Gabrielle P. Dailey, Christine Domingo, Gaelle Picarda, Hunter Little, Annie Chou, Jessica Sparks, Darina Spasova, Shigeki Miyake-Stoner, Zachary C. Hartman, Christopher A. Rabiola, Erika J. Crosby, Herbert K. Lyerly, Nathaniel Wang, Parinaz Aliahmad. A self-replicating RNA precision medicine approach to overcoming resistance to endocrine therapy in ER+BC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6403.

Abstract 4354: An exosome-based ESR1 monitoring RT-qPCR technology that rapidly and accurately detects circulating tumor acquired resistance variants at ≤0.1% frequency in liquid biopsy samples

Abstract Introduction: Hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) breast cancer is the most common type of breast cancer. Patients with HR+/HER2- metastatic breast cancer (mBC) often become resistant to aromatase inhibitors commonly used in endocrine therapy (ET). Estrogen receptor (ESR1) ligand binding domain mutations are frequently detected in HR+ mBC and have been reported to be associated with ET resistance, noting up to 60% patients with mBC will develop resistance to treatment via acquired resistance in ESR1. Recent studies have shown that monitoring of ESR1 mutations in plasma may serve as a predictive biomarker of acquired resistance to ET, showcasing a strong need for sensitive nucleic acid-based assays. We describe a comprehensive methodology for targeted clinical RT-qPCR monitoring of ESR1 mutations in plasma that utilizes both exosomal nucleic acids and cfDNA to report multiple mutations, provide a streamlined workflow, and accommodate a range of inputs from clinically relevant samples. Methods: Exosomal nucleic acids (exosomal DNA and RNA) and circulating cell-free DNA (cfDNA) were co-isolated using the ExoLution Plus Isolation Kit (Exosome Diagnostics). RT-qPCR-based target enrichment was performed using modified QuantideX reagents (Asuragen) and mutations were confirmed on the QuantStudio qPCR Platform (ThermoFisher). Bioinformatic analyses were conducted using custom software. Results: We developed novel technologies and tested greater than samples across key ET resistance mutations, utilizing contrived samples as needed due to availability. Preliminary studies demonstrated that ESR1 exosomal RNA + cfDNA ranged from approximately 9,500 copies to 64,000 copies in a set of 2mL female presumed normal plasma samples; these results suggest that even 2mL plasma could provide sufficient exosomal RNA/DNA + cfDNA for mutation detection at variant frequencies less than or equal to 0.01%. The ESR1 RT-qPCR showed greater than 90% analytical sensitivity (at less than or equal to 1% mutant allele frequency) and greater than90% analytical specificity. Further, the technology detected less than 10 mutant copies for most tested mutants, showing an LOD of at least 0.1% (5 mutant copies in a background of 5,000 WT copies). Conclusion: A fast, efficient, and sensitive ESR1 RT-qPCR panel approach was developed and evaluated, demonstrating the reliable and specific detection of rare variants in liquid biopsy specimens. Importantly, this prototype technology has potential to address several challenges associated with mutation monitoring in liquid biopsies by expanding detection of mutant analytes (exosomal RNA and DNA), improving analytical sensitivity (novel reagents and software), and increasing accessibility (qPCR instrument install base). Citation Format: Sarah Statt, Julie Thibert, Kurt Franzen, Aquiles Sanchez, Liangjing Chen, Megan Yociss, Elliot Hallmark, Melissa Church, Stela Filipovic-Sadic, Gary Latham, Johan Skog. An exosome-based ESR1 monitoring RT-qPCR technology that rapidly and accurately detects circulating tumor acquired resistance variants at ≤0.1% frequency in liquid biopsy samples. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4354.

Abstract P6-10-12: Discovery and Development of Next-Generation Estrogen Receptor Mutant Inhibitors using DNA-Encoded Chemical Library Screening

Abstract Background: Activating somatic ESR1 mutations Y537S and D538G occur more frequently in endocrine therapy-resistant metastatic breast cancer, which is associated with an aggressive phenotype and poor survival in breast cancer patients. These gain of function mutant receptors are constitutively active and allow resistance to first-line endocrine therapies. Therefore, the development of next-generation small molecule drugs targeting mutant estrogen receptor (ER) is an important priority. Here, we searched the small molecule inhibitors for Y537S and D538D ER mutants using DNA-encoded chemical library screening. Methods: Wild type (WT) and mutant ER ligand binding domain (LBD) proteins were expressed in E. coli. The soluble proteins were purified by Ni-NTA chromatography followed by anion exchange and size exclusion chromatography. Homogeneous time-resolved fluorescence (HTRF) and fluorescent polarization (FP) assays were performed in these purified proteins. We employed a DNA-encoded chemical library affinity selection using our in-house collection of 6 billion compounds. Hit compounds were resynthesized and validated in biochemical assays. Finally, we have performed functional studies in CRISPR-Cas9 knock-in of Y537S and D538G mutant MCF-7 breast cancer cells. Results: We have successfully purified microgram amounts of ERα LBD of WT, Y537S, and D538G proteins. To test whether the purified WT and mutant proteins are active, HTRF and FP assays were performed in the presence and absence of estradiol and 4OH tamoxifen. Steroid receptor coactivator 3 (SRC3) peptide binding to the WT ER protein occurred only in the presence of estradiol. However, Y537S and D538G proteins are recruited by the SRC3 peptide in the absence of estradiol, indicating that these mutants are constitutively active and bind to SRC3. Furthermore, an in vitro biochemical FP assay was also established for WT and mutants in the presence of estradiol and 4OH tamoxifen. The screen of our multibillion small molecule collection of DNA-encoded chemical libraries identified several hits in WT and mutant ER. To confirm the selection output, we synthesized off-DNA compounds and validated these in biochemical and cell-based studies. We have identified that the compounds, CDD-1272 and CDD-1274, are active in HTRF and FP assays. Furthermore, these compounds inhibit WT and mutant cell growth in the presence of estradiol. More importantly, CDD-1274 degrades ER mutant and cyclin D1 proteins. In addition, CDD-1274 induced p21 protein expression in WT and mutant cells. Conclusions: We have identified potent novel ER mutant binders by using our DNA-encoded chemical library platform. Our compounds are active in biochemical and ER mutant cell lines, suggesting these molecules are potential chemical probes to explore in in vivo models of breast cancer. Support: NIH/NCI R03 CA259664 and CPRIT RP220524 to MP. Citation Format: Murugesan Palaniappan, Kurt M. Bohren, Yong Wang, Damian W. Young, Suzanne A. Fuqua, Martin M. Matzuk. Discovery and Development of Next-Generation Estrogen Receptor Mutant Inhibitors using DNA-Encoded Chemical Library Screening [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-12.

Characterization of a microglia-specific humanized P2X7 receptor knock-out mouse line: Implications for translational psychoneuroimmunology

IntroductionDepression is a common psychiatric disorder and chronic stress is considered its main environmental risk factor. Recently, immune processes including adenosine triphosphate mediated P2X7 receptor (P2X7R) signalling via microglia and macrophages (M/Ms) were found to play a critical role in depression genesis, by linking environmental stress to depression biology and symptoms.ObjectivesTo characterize the role of human P2X7R (hP2X7R) in psychosocial and immune stress conditions, both in vitro and in vivo.MethodsSeveral, custom designed mouse lines expressing the loxP-flanked, hP2X7R-sequence in the murine P2X7R locus were established. In addition, these mice possess a Cre-sensitive reporter and express a Cre recombinase fused to a mutant estrogen receptor ligand-binding domain in M/Ms. This enables conditional, tamoxifen-inducible hP2X7R inactivation and simultaneous tdTomato expression. First, we established primary microglia cell cultures and characterized them at baseline and following immune stimulation. Next, we performed behavioural assessment of hP2X7Rwt and microglia-specific hP2X7RKO mice following chronic psychosocial stress. Last, we developed a novel in vivo two-photon microscopy (TPM) approach by use of frontolimbic cranial windows.ResultsPrimary hP2X7RKO microglia displayed significantly lower IL-1β production, increased survival and decreased morphological activation upon immune stimulation. Although hP2X7RKO mice showed a significant increase of locomotor activity at baseline, there was no impact on anxiety- and depressive-like phenotypes. Longitudinal in vivo TPM enabled morphometric characterization of cortical M/Ms over several weeks.ConclusionsOur results illustrate the great potential of this humanized mouse line for translational psychiatry. In the future, this system could proof useful to evaluate immunomodulatory approaches in chronic stress and depression.DisclosureNo significant relationships.

Bisacodyl in Stimulant Laxatives Can Induce Estrogen Agonist/Antagonist Activity in Breast Cancer Cells in Culture

Exposure to xenobiotic estrogens has the potential to induce estrogen activity that may contribute to a range of undesired physiological effects including the stimulation of estrogen responsive tumors. We have previously determined that extracts of OTC medications containing the stimulant laxative bisacodyl induce estrogenic activity in tissue culture bioassays. In this study, we tested the hypothesis that bisacodyl is responsible for the estrogen activity of these extracts and then characterized these effects. Ethanol extracts and dilutions were prepared from OTC medications containing Bisacodyl (1 gm:1 ml). The estrogen agonist and antagonist activity of each extract, as well as bisacodyl and then metabolite DA-bisacodyl was determined using the T47dkbluc estrogen reporter gene and the MCF-7 E3 estrogen responsive proliferation assays. LC-MS analysis was used to determine bisacodyl and DA-bisacodyl concentration in extracts as well as to trace the metabolism of bisacodyl to DA-bisacodyl in the cell culture bioassays. Molecular modeling “docking” simulations of the interactions of bisacodyl and the metabolite DA-bisacodyl with the estrogen receptor (ER) ligand binding domain was performed using MOE from Chemical Computing Group. Bisacodyl and the metabolite DA-bisacodyl induced mixed agonist/antagonist activity in MCF-7 E3 estrogen responsive proliferation assay similar to 4OH-tamoxifen. At the same time, both compounds stimulated only minimal estrogen activity in the T47dkbluc estrogen reporter gene assay. LC-MS analysis determinations identified that almost all bisacodyl was converted to the dihydroxy metabolite DA-bisacodyl in cell culture bioassays. Molecular modeling “docking” simulations determined that while bisacodyl does not fit into the agonist (estradiol) or antagonist (4OH-tamoxifen) conformations of the estrogen receptor ligand binding site, the metabolite DA-bisacodyl may fit into the antagonist induced binding pocket of the ER in a reasonable way. This study characterizes the observed estrogen activity of extracts from OTC medications containing bisacodyl as resulting from the bisacodyl metabolite DA-bisacodyl interacting with the estrogen receptor. Thus, bisacodyl is an OTC medication active ingredient that has potential to induce side effects and or toxicity involving estrogen signalling. The capacity for medications containing bisacodyl or other estrogenic substances to induce estrogen activity in patients is unclear. At the same time, consumers and practitioners should be aware of the potential estrogen activity of bisacodyl containing products.

In vivo and in silico analyses of estrogenic potential of equine estrogens in medaka (Oryzias latipes)

Equine estrogens (EEs) are widely used in hormone replacement therapy pharmaceuticals for postmenopausal women. Previous studies have shown that EEs occur in the aquatic environment; however, the potential estrogenicity and risk of EEs in aquatic organisms, including fish, have yet to be studied in detail. Therefore, we evaluated the estrogenic potential of major EEs, namely equilin (Eq), 17α-dihydroequilin (17α-Eq), 17β-dihydroequilin (17β-Eq), equilenin (Eqn), 17α-dihydroequilenin (17α-Eqn), and 17β-dihydroequilenin (17β-Eqn), on medaka (Oryzias latipes) using in vivo and in silico assays. Quantitative real-time RT-PCR analyses revealed that expression levels of choriogenin L (ChgL) and choriogenin H (ChgH) in medaka embryos responded to various types and concentrations of EEs in a concentration-dependent manner, whereas transcription levels of vitellogenin 1 were not significantly affected by any of the EEs in the concentration range tested. The order of the in vivo estrogenic potencies of EEs was as follows: 17β-Eq > Eq > 17β-Eqn > Eqn > 17α-Eqn > 17α-Eq. Additionally, the 50% effective concentrations (EC50) of 17β-Eq was lower than that of 17β-estradiol. We also investigated the interaction potential of EEs with medaka estrogen receptor (ER) subtypes in silico using a three-dimensional model of the ligand-binding domain (LBD) for each ER and docking simulations. All six EEs were found to interact with the LBDs of ERα, ERβ1, and ERβ2. The order of the in silico interaction potentials of EEs with each ER LBD was as follows: 17β-Eq > 17α-Eq > Eq > 17β-Eqn > 17α-Eqn > Eqn. Furthermore, we identified the key amino acids that interact with EEs in each ER LBD; our findings suggest that amino acids and/or their hydrogen bonding may be responsible for the ligand-specific interactions with each ER. This study is the first to comprehensively analyze the estrogenic potential of EEs in medaka both in vivo and in silico.

Plasma-Based Longitudinal Evaluation of ESR1 Epigenetic Status in Hormone Receptor-Positive HER2-Negative Metastatic Breast Cancer.

BackgroundEndocrine therapy (ET) is the mainstay of treatment for hormone receptor-positive human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer; however, adaptive mechanisms emerge in about 25–30% of cases through alterations in the estrogen receptor ligand-binding domain, with a consequent ligand-independent estrogen receptor activity. Epigenetic-mediated events are less known and potentially involved in alternative mechanisms of resistance. The aim of this study was to test the feasibility of estrogen receptor 1 (ESR1) epigenetic characterization through liquid biopsy and to show its potential longitudinal application for an early ET sensitivity assessment.MethodsA cohort of 49 women with hormone receptor-positive HER2-negative MBC was prospectively enrolled and characterized through circulating tumor DNA using methylation-specific droplet digital PCR (MS-ddPCR) before treatment start (BL) and after 3 months concomitantly with computed tomography (CT) scan restaging (EV1). ESR1 epigenetic status was defined by assessing the methylation of its main promoters (promA and promB). The most established cell-free tumor DNA (ctDNA) factors associated with ET resistance [ESR1 and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations] were assessed through next-generation sequencing. Associations were tested through Mann–Whitney U test, matched pairs variations through Wilcoxon signed rank test, and survival was analyzed by log-rank test.ResultsThe ET backbone was mainly based on aromatase inhibitors (AIs) (70.83%) in association with CDK4/6 inhibitors (93.75%). Significantly lower promA levels at baseline were observed in patients with liver metastases (P = 0.0212) and in patients with ESR1 mutations (P = 0.0091). No significant impact on PFS was observed for promA (P = 0.3777) and promB (P = 0.7455) dichotomized at the median while a ≥2-fold increase in promB or in either promA or promB at EV1 resulted in a significantly worse prognosis (respectively P = 0.0189, P = 0.0294). A significant increase at EV1 was observed for promB among patients with PIK3CA mutation (P = 0.0173). A trend was observed for promB in ESR1 wild-type patients and for promA in the ESR1 mutant subgroup.ConclusionThe study proofed the concept of an epigenetic characterization strategy based on ctDNA and is capable of being integrated in the current clinical workflow to give useful insights on treatment sensitivity.

OR12-07 Full Antagonism of Breast Cancer Cell Proliferation Can Result from Many Ligand-Induced Conformational Distortions of the Estrogen Receptor Ligand Binding Domain

Although most estrogen receptor alpha (ERα)-positive breast cancers initially respond well to endocrine therapies using aromatase inhibitors (AIs) or antiestrogens, after varying time periods the cancer frequently recurs as metastatic disease. A significant fraction of these recurrences are driven by ERs that have acquired activating mutations in their ligand binding domains (LBDs), giving them constitutive activity and thus resistance to AIs. Because these mutations also reduce the affinity and potency of SERMs and SERDs, expanded efforts have been made to vary the structure of antiestrogens to make them more potent.Typical antiestrogens are comprised of a core element that binds securely in the ligand binding pocket and from which extends a single ring (ring E) having a side chain that sterically interferes with the position of helix-12 by direct antagonism, reorienting it so that it occludes the activation function 2 (AF2) hydrophobic groove for coactivator binding. Through structural studies, we found that bridged oxabicycloheptene-sulfonamide (OBHS-N) core ligands have two rings (E and F) that can be poised to engage in both “direct antagonism” and “indirect antagonism”, the latter of which disrupts the orientation of helix-12 by impinging on helix-11 and the helix-11–12 loop.In this study, we have placed typical antiestrogen side chains on either the E or the F ring of OBHS-N core ligands and characterized their activities in ERα-positive breast cancer cells. All compounds have full antiproliferative activity and reverse estrogen-regulated gene expression, with the antiproliferative potency of each type of side chain having a distinct preference for E- vs F-ring attachment. Conformational analysis using a multiplexed coregulator peptide interaction assay shows that compounds with an E-ring substitution have interaction profiles similar to 4-hydroxytamoxifen and fulvestrant, whereas the F-ring substitution gives a very different pattern, suggesting that the antagonist activity of the two classes rely on different sets of coregulator proteins. A large number of high resolution (better than 2 Å) X-ray crystal structures reveal that this set of novel ER antagonists disrupt the conformation of the ER LBD in a variety of ways, several of which are distinct from those seen with previous antiestrogens such as Tamoxifen and Fulvestrant.Our findings expand design concepts by which ERα ligands can block the activity of this receptor and illustrate how direct and indirect modes of ER antagonism can be combined to facilitate the development of more efficacious antiestrogens for breast cancer treatment and possibly for regulating ER-mediated activities in other estrogen target tissues.

The Structure-Function Relationship of Angular Estrogens and Estrogen Receptor Alpha to Initiate Estrogen-Induced Apoptosis in Breast Cancer Cells.

High-dose synthetic estrogen therapy was the standard treatment of advanced breast cancer for three decades until the discovery of tamoxifen. A range of substituted triphenylethylene synthetic estrogens and diethylstilbestrol were used. It is now known that low doses of estrogens can cause apoptosis in long-term estrogen deprived (LTED) breast cancer cells resistant to antiestrogens. This action of estrogen can explain the reduced breast cancer incidence in postmenopausal women over 60 who are taking conjugated equine estrogens and the beneficial effect of low-dose estrogen treatment of patients with acquired aromatase inhibitor resistance in clinical trials. To decipher the molecular mechanism of estrogens at the estrogen receptor (ER) complex by different types of estrogens—planar [17β-estradiol (E2)] and angular triphenylethylene (TPE) derivatives—we have synthesized a small series of compounds with either no substitutions on the TPE phenyl ring containing the antiestrogenic side chain of endoxifen or a free hydroxyl. In the first week of treatment with E2 the LTED cells undergo apoptosis completely. By contrast, the test TPE derivatives act as antiestrogens with a free para-hydroxyl on the phenyl ring that contains an antiestrogenic side chain in endoxifen. This inhibits early E2-induced apoptosis if a free hydroxyl is present. No substitution at the site occupied by the antiestrogenic side chain of endoxifen results in early apoptosis similar to planar E2. The TPE compounds recruit coregulators to the ER differentially and predictably, leading to delayed apoptosis in these cells.SIGNIFICANCE STATEMENTIn this paper we investigate the role of the structure-function relationship of a panel of synthetic triphenylethylene (TPE) derivatives and a novel mechanism of estrogen-induced cell death in breast cancer, which is now clinically relevant. Our study indicates that these TPE derivatives, depending on the positioning of the hydroxyl groups, induce various conformations of the estrogen receptor’s ligand-binding domain, which in turn produces differential recruitment of coregulators and subsequently different apoptotic effects on the antiestrogen-resistant breast cancer cells.

Amyloid Fibril Formation of α‐Synuclein Is Modulated via the Estrogen Receptor Ligand Binding Domain of Estrogen Receptor α Bound with Tamoxifen‐based Small Molecules

Alpha‐synuclein (αSyn) forms amyloid fibrils in the brain of patients suffering from Parkinson's disease. Aiming at the development of a new therapy via regulating amyloid fibril formation of αSyn, we studied estrogen receptor ligand‐binding domain (ERLBD) of estrogen receptor α (ΕRα). The ERLBD encoding gene was cloned, expressed by IPTG induction, and successfully purified by Ni2+‐affinity chromatography followed by anion‐exchange chromatography. In the thioflavin T (ThT) fluorescence assay, we examined the kinetics of amyloid fibril formation of αSyn in the presence of ERLBD protein. Dose‐dependent inhibition of amyloid fibril formation of αSyn by ERLBD protein was observed, indicating that ERLBD protein binds to αSyn, thereby blocking amyloid fibril formation. The kinetics of amyloid fibril formation of αSyn were compared in the absence and presence of a small molecule, such as 17β‐estradiol (E2, agonist) or tamoxifen (TMX, antagonist) of ΕRα. Regardless of the presence of E2, we found that ERLBD protein inhibits αSyn from forming amyloid fibrils. On the other hand, TMX reversed the inhibition effect of ERLBD protein on amyloid fibril formation of αSyn. Interestingly, the photo‐reactive hybrid ligand 2‐phenylquinoline‐4‐hydroxytamoxifen (PQ‐OHT) was expected to exhibit the anti‐inhibitory effect of ERLBD on amyloid fibril formation of αSyn. Upon illuminating with a UV lamp, PQ‐OHT was found to degrade ERLBD protein as well. These observations suggest the examination of the possibility that a complex of ERLBD‐αSyn amyloid fibril can be eliminated via the ligand PQ‐OHT upon exposure to light for therapeutic purposes.

Prevalence of ESR1 Mutation in Chinese ER-Positive Breast Cancer.

BackgroundESR1 mutation and its possible relation to endocrine therapy resistance in ER-positive breast cancers have been studied with respect to genetic sequencing data from Western patients but rarely from Chinese patients. This study aimed to investigate the prevalence of ESR1 mutation in Chinese primary and metastatic ER-positive breast cancer.MethodsTumor samples from 297 primary breast cancer (PBC) patients and blood samples from 43 metastatic breast cancer (MBC) patients were obtained to perform whole exon sequencing of the ESR1 gene through next-generation sequencing (NGS). Clinicopathological features of MBC patients were listed and grouped to explore potential factors in ESR1 mutations.ResultsA total of 15 ESR1 variations, including 11 point mutations, 1 in-frame deletion mutation, 1 synonymous mutation, and 2 amplifications were identified in 13 patients. The ESR1 mutation rate was 1% (3/297) in PBC patients and 18.6% (8/43) in MBC patients. All ESR1 point mutations occurred in the estrogen receptor ligand-binding domain. Six (54.5%) of the 11 point mutations were hotspot mutations. Among all MBC patients, the ESR1 mutation rate in those who had a treatment history using aromatase inhibitors (AI) was significantly higher than those who did not (25.8% versus 0%, P=0.015). Moreover, the ESR1 mutation rate in those who received AI treatment over a period of 12 months was significantly higher than in those whose treatment lasted less than 12 months [36.3% versus 0%, P<0.001].ConclusionESR1 mutations were more frequently observed in the circulating cell-free DNA of MBC patients than in PBC patients among the Chinese cohort, and higher among those pretreated with AI, suggesting that such mutations may undergo selection during AI treatment.

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.

Comparative in Vitro and in Vivo Evaluation of the Estrogenic Effect of Hexafluoropropylene Oxide Homologues.

As alternatives to perfluorooctanoic acid (PFOA), hexafluoropropylene oxide (HFPO) homologues, including hexafluoropropylene oxide dimer acid (HFPO-DA), hexafluoropropylene oxide trimer acid (HFPO-TA), and hexafluoropropylene oxide tetramer acid (HFPO-TeA), have been used in the fluoropolymer industry for a long period of time. These compounds have attracted widespread attention in recent years due to their environmental ubiquity and high bioaccumulation capability, as well as their toxicity. In our study, we evaluated the potential estrogenic effects of HFPOs in comparison to PFOA by ligand binding, transcriptional activity, and in vivo assays. Fluorescence ligand binding assays showed that both HFPO-TA and HFPO-TeA exhibited higher binding affinity to estrogen receptor ligand binding domains (ER-LBDs) than PFOA, with 2.5- and 57.5-fold higher affinity to ERα-LBD and 2.6- and 41.8-fold higher affinity to ERβ-LBD, respectively, whereas HFPO-DA exhibited weaker binding affinity than PFOA. Unlike PFOA, HFPO-TA and HFPO-TeA exhibited antagonistic activity toward the ERs' signaling pathway, with HFPO-TeA displaying the strongest potency. In silico study revealed that while PFOA binds with ERs in a similar fashion as 17β-estradiol, the HFPOs display an antagonistic binding mode. Using a zebrafish model, we further found that exposure to HFPO homologues significantly altered the levels of sex steroid hormones and vitellogenin. In general, both in vivo and in vitro results indicate that HFPO homologues might exert higher estrogenic effects than PFOA.

Abstract ES10-1: Recurrent ESR1 mutations activating mutations in breast cancer

Abstract Endocrine treatments are the cornerstone treatments in estrogen receptor positive (ER+) breast cancer. Despite their efficacy, resistance to endocrine treatments remains an important clinical challenge. A number of years ago, recurrent estrogen receptor ligand binding domain (LBD) mutations were identified as the most common genomic mechanism of acquired resistance to endocrine treatment in metastatic breast cancer. These mutations were found in about 20% of ER+ metastatic tissue specimens and in more than 30% of patients with metastatic ER+ breast cancer when testing circulating tumor DNA. Pre-clinical studies showed that these mutations lead to constitutional ligand independent transcriptional activity and tumor growth. Recently, we showed that the ER mutations have neomorphic properties that promote metastases. Clinical data, derived mainly from retrospective analyses of clinical trials, indicate that the ER mutations confer resistance to aromatase inhibitors and fulvestrant, and decreased overall survival. A recent study also showed that the Y537S LBD ER mutation is acquired during fulvestrant and palbociclib treatment. Collectively, the ER mutations are important drivers of resistance standard endocrine treatments and confer worse outcomes in ER+ metastatic breast cancer. Clinical trials stratifying patients by ER mutant status or dedicated to patients with the ER mutations are needed to identify therapeutic strategies to target these mutations. Citation Format: Jeselsohn R. Recurrent ESR1 mutations activating mutations in 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 ES10-1.