Mutant Estrogen Receptor Research Articles

Characterization of estrogen receptor mutant breast cancer in 3D cell culture.

14 Background: Breast cancer is a leading cancer in women worldwide. Many primary breast cancers are estrogen receptor positive (ER+) and responsive to anti-estrogenic therapies. These tumors can mutate estrogen receptors to survive, allowing the tumors to become more triple-negative-like and therefore more dangerous. Triple-negative breast cancer (TNBC) has been particularly challenging to treat due to its lack of estrogen (ER), progesterone (PR), and human epidermal growth factor (HER2) receptors. Due to the lack of treatment options, TNBC has a poor prognosis and contributes to a significant percentage of breast cancer mortalities. These ER mutants act more like TNBC, resulting in worse clinical outcomes. Current research on these ER mutants has been conducted using two-dimensional (2D), monolayer cell culture, which does not translate effectively in animal models, and ultimately, humans. Three-dimensional (3D) cell culture, which allows for the formation of spheroids, mimics actual tumors and provides results more consistent with tumor treatment in vitro. Due to the lack of research on these ER mutants in 3D culture, they must be characterized to determine baseline gene expression and behavior. After characterization, identifying changes resulting from drug treatment will be possible. Methods: Parental, ER+ MCF7-Luc cells, and daughter D538G and Y537S mutants were seeded at a density of 3000 cells/well in a low-attachment, round-bottomed 96-well plate. 48 hours and 7 days post-seeding, newly-formed spheroids were imaged. Using ImageJ analysis software, diameter, area, perimeter, circularity, aspect ratio, roundness, and solidity were determined. After 7 days in culture, spheres were collected for RNA extraction. Next, cDNA was synthesized, and qRT-PCR was performed to assess gene expression differences. Results: The wild-type ER+ spheres have smaller diameters, spherocity values closer to one, and are more compact. They express different levels of EMT markers from the controls, indicating alterations to signaling pathways. These 3D cultures vary in expression from the 2D cultures of the same cell lines. Conclusions: MCF-7 ER+ breast cancer cells aggregate more readily than ER+ mutants. The ER must be involved in signaling that promotes aggregation, as reduced ER signaling decreases the ability for spheroid formation. This phenomenon and the differences in gene expression may explain why mutants tend to behave in a more triple-negative manner. Cells cultured in 3D express some genes to different extents, confirming the importance of 3D culture for identifying future therapies – cells behave differently in different culturing contexts, 3D being more consistent with tumor behavior. Therefore, characterizing ER+ mutants prior to drug treatment studies is crucial to understanding how compounds affect cancer cells, as well as for identifying differences in various ER+ mutants for better treatment outcomes.

Pure estrogen receptor antagonists potentiate capecitabine activity in ESR1-mutant breast cancer

The ESR1 ligand binding domain activating mutations are the most prevalent genetic mechanism of acquired endocrine resistance in metastatic hormone receptor-positive breast cancer. These mutations confer endocrine resistance that remains estrogen receptor (ER) dependent. We hypothesized that in the presence of the ER mutations, continued ER blockade with endocrine therapies that target mutant ER is essential for tumor suppression even with chemotherapy treatment. Here, we conducted comprehensive pre-clinical in vitro and in vivo experiments testing the efficacy of adding fulvestrant to fluorouracil (5FU) and the 5FU pro-drug, capecitabine, in models of wild-type (WT) and mutant ER. Our findings revealed that while this combination had an additive effect in the presence of WT-ER, in the presence of the Y537S ER mutation there was synergy. Notably, these effects were not seen with the combination of 5FU and selective estrogen receptor modulators, such as tamoxifen, or in the absence of intact P53. Likewise, in a patient-derived xenograft (PDX) harboring a Y537S ER mutation the addition of fulvestrant to capecitabine potentiated tumor suppression. Moreover, multiplex immunofluorescence revealed that this effect was due to decreased cell proliferation in all cells expressing ER and was not dependent on the degree of ER expression. Taken together, these results support the clinical investigation of the combination of ER antagonists with capecitabine in patients with metastatic hormone receptor-positive breast cancer who have experienced progression on endocrine therapy and targeted therapies, particularly in the presence of an ESR1 activating mutation.

GOG3069: Phase 2 study of alpelisib and fulvestrant for PIK3CA-mutated estrogen receptor (ER)-positive endometrioid endometrial cancers.

TPS5639 Background: Treatment options for persistent, metastatic, or recurrent endometrial cancer continues to evolve as we learn more about the molecular landscape of this disease. Recent studies have shown a benefit to endocrine-based combinations such as aromatase inhibitors with mTOR or CDK4/6 inhibitors especially in patients who had not received prior chemotherapy ( 1– 3). The presence of oncogenic PIK3CA mutations have been shown to impact response to aromatase inhibitors in breast cancer. The combination of alpelisib and fulvestrant was approved for the treatment of metastatic hormone receptor-positive, HER2-negative breast cancer harboring oncogenic mutations in the PIK3CA gene that progressed on prior endocrine therapy( 4). PIK3CA mutations are commonly identified in endometrial cancers and may similarly confer endocrine resistance. GOG3069 is a phase II trial designed to evaluate the efficacy and safety of alpelisib and fulvestrant in a biomarker-selected endometrial cancer patient population. Methods: This is a phase II non-randomized, open label, multicenter, two-stage study. Eligible patients must have advanced (FIGO 2014 Stage III or IV), persistent, or recurrent endometrial carcinoma with measurable disease by RECISTv1.1 criteria. Patients cannot be curable by surgery or radiotherapy. Disease must be of endometrioid histology with positive expression of estrogen receptor (defined as ≥1% of tumor cells demonstrating positive nuclear staining by immunohistochemistry) and oncogenic PIK3CA mutation. Patients may have received no more than 3 prior lines of therapy including 1 prior line of systemic chemotherapy. Enrolled patients will receive treatment with alpelisib 300mg orally daily and fulvestrant 500mg IM on Day 1 and Day 15 of Cycle 1, then 500mg IM on Day 1 of each 28-day cycle. The primary endpoint is overall response rate as measured by RECISTv1.1-defined tumor response. Secondary endpoints include safety, tolerability, progression-free survival, duration of response, and 24-month overall survival rate. GOG3069 activated in November 2023 and expects to enroll across 15 US sites. The statistical plan uses a conditional stratified design factoring differing probabilities of response based on whether the patient had prior chemotherapy exposure. ClinicalTrials.gov Identifier: NCT05154487

Oral Estrogen Receptor PROTAC Vepdegestrant (ARV-471) Is Highly Efficacious as Monotherapy and in Combination with CDK4/6 or PI3K/mTOR Pathway Inhibitors in Preclinical ER+ Breast Cancer Models.

Estrogen receptor (ER) alpha signaling is a known driver of ER-positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-) breast cancer. Combining endocrine therapy (ET) such as fulvestrant with CDK4/6, mTOR, or PI3K inhibitors has become a central strategy in the treatment of ER+ advanced breast cancer. However, suboptimal ER inhibition and resistance resulting from the ESR1 mutation dictates that new therapies are needed. A medicinal chemistry campaign identified vepdegestrant (ARV-471), a selective, orally bioavailable, and potent small molecule PROteolysis-TArgeting Chimera (PROTAC) degrader of ER. We used biochemical and intracellular target engagement assays to demonstrate the mechanism of action of vepdegestrant, and ESR1 wild-type (WT) and mutant ER+ preclinical breast cancer models to demonstrate ER degradation-mediated tumor growth inhibition (TGI). Vepdegestrant induced ≥90% degradation of wild-type and mutant ER, inhibited ER-dependent breast cancer cell line proliferation in vitro, and achieved substantial TGI (87%-123%) in MCF7 orthotopic xenograft models, better than those of the ET agent fulvestrant (31%-80% TGI). In the hormone independent (HI) mutant ER Y537S patient-derived xenograft (PDX) breast cancer model ST941/HI, vepdegestrant achieved tumor regression and was similarly efficacious in the ST941/HI/PBR palbociclib-resistant model (102% TGI). Vepdegestrant-induced robust tumor regressions in combination with each of the CDK4/6 inhibitors palbociclib, abemaciclib, and ribociclib; the mTOR inhibitor everolimus; and the PI3K inhibitors alpelisib and inavolisib. Vepdegestrant achieved greater ER degradation in vivo compared with fulvestrant, which correlated with improved TGI, suggesting vepdegestrant could be a more effective backbone ET for patients with ER+/HER2- breast cancer.

Abstract PO3-26-11: Enhancing endoplasmic reticulum stress for treating endocrine therapy resistant breast cancers driven by mutant estrogen receptors

Abstract Background: Estrogen receptor alpha (ERα) mutations are common (30-40%) in metastatic endocrine therapy-resistant breast cancers (ETR-BC), enable resistance to endocrine therapies and are the molecular drivers of ETR-BC. We had previously shown that an oligobenzamide, ERX-41, could enhance endoplasmic reticulum stress in ETR-BCs driven by mutant (MT) ERα, resulting in cancer cell death in vitro and and in vivo. To enable clinical translation of ERX-41, we performed lead optimization, followed by preclinical and IND-enabling studies. Methods: Over 2000 oligo-benzamides were designed, synthesized and tested in multiple BC models including those that express WT-ERα (MCF7, and ZR75) and BC models with acquired resistance (MCF7-Tam, and MCF7-LTLT) and engineered models that express MT-ERα (MCF7-ERα-D538G, MCF7-ERα-Y537S, ZR75-ERα-D538G, ZR75-ERα-Y537S). For our lead compound, mechanistic validation studies were performed using CRISPR LIPA mutants, RT-qPCR and Western blotting. Explants, organoids, cell line-(CDX) and patient-derived (PDX) xenografts were used to test the ex vivo and in vivo effectiveness of our lead compound as a monotherapy and in combination with abemaciclib. Results: Testing of >2000 synthesized oligo-benzamides identified a lead compound, ERX-315, that had broad and potent activity (IC50 between 20-100 nM) against both WT and MT (mutant) ERα-driven BC cells in in vitro assays. CRISPR KO of LIPA (which encodes lysosomal acid lipase (LAL) in BC cells abrogated cytotoxic response to ERX-315, validating LIPA as the molecular target of ERX-315. Ultrastructural and molecular studies confirm that ERX-315 induces significant endoplasmic reticulum stress, leading to a shutdown of de novo protein synthesis and apoptotic cell death in BC. Importantly, ERX-315 does not induce endoplasmic reticulum stress or cell death in normal cells and is non-toxic in animal models. We have shown that this capacity of ERX-315 to induce endoplasmic reticulum stress is unique among drugs targeting ERα, including selective ERα modulators and degraders, such as GDC-0180, AZD-9496 and fulvestrant. ERX-315 has potent anti-proliferative activity against MT-ERα-driven BC, as seen in genetically modified cell lines both grown as monolayer or spheroids in vitro, patient derived explants (PDEs) ex vivo and cell line derived (CDX) and patient-derived (PDX) xenografts in vivo. The combination of ERX-315 and CDK4/6 inhibitor abemaciclib was synergistic in decreasing the proliferation of both endocrine therapy-sensitive and endocrine therapy-resistant BC cells, in vitro, in xenograft models in vivo, and in primary patient-derived explants ex vivo. We are currently optimizing both the synthesis of ERX-315 using GMP process for kilogram scale production and validated methods for pharmacokinetic studies. We have developed formulations for both intravenous and oral administration with favorable pharmacokinetic parameters and proven utility of the formulated ERX-315 against CDX and PDX tumors in vivo. Toxicity studies in dogs and rodents have demonstrated >8-fold therapeutic to toxicity window. A phase I clinical trial is planned to be open to enrollment by Q1 2024. Conclusions: We have identified a lead compound ERX-315, which represents a novel class of agent that induce catastrophic levels of ER stress resulting in cancer cell death and that can effectively work against multiple forms of ETR-BC, including those driven by MT-ERα. Preclinical studies, GMP manufacturing, formulation and IND-enabling studies are being completed in time for the commencement of the phase I clinical trial by Q1 2024. Citation Format: Suryavathi Viswanadhapalli, Karla Parra, Tae-Kyung Lee, Rahul Gopalam, Kara Kassees, Tanner Reese, Gaurav Sharma, Xihui Liu, Xue Yang, ChiaYuan Chen, Carlos Roggero, Liping Chen, Sautam Bhattacharya, Uday Pratap, Russell Hayward, Sharron Gargosky, Jung-Mo Ahn, Ganesh V. Raj, Ratna Vadlamudi. Enhancing endoplasmic reticulum stress for treating endocrine therapy resistant breast cancers driven by mutant estrogen receptors [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 PO3-26-11.

Abstract PO4-27-05: A phase I open-label dose escalation trial of FWD1802 as monotherapy and in combination with palbociclib in patients with ER+/HER2- unresectable locally advanced or metastatic breast cancer with or without ESR1 mutations

Abstract Background: Estrogen receptor (ER) signaling sits at center of the tumor biology in ER+/HER2- breast cancers. Current standard of care involves hormone therapy with agents including selective estrogen receptor modulators (SERMs), selective estrogen receptor degraders (SERDs), and aromatase inhibitors (AIs). Unfortunately, most patients eventually develop resistance to these therapies. Mutations in estrogen receptor 1 (ESR1) are partially responsible for the acquired resistance. Mutated estrogen receptors are still sensitive to fulvestrant but with a much less sensitivity. In January 2023, elacestrant received its first approval for the treatment of postmenopausal women or adult men with breast cancer harboring ESR1 mutations in US. However, there is still great unmet medical need for this population. FWD1802 is a third-generation oral SERD for ER-positive breast cancer patients. It binds competitively to ER, with a much higher affinity than that of fulvestrant. Preclinically, FWD1802 alone and in combination with palbociclib exhibits significant anti-tumor activity in the ER+/HER2- models and in ESR1-mutated models in vitro and in vivo. This ongoing phase I study is to assess the safety, tolerability, pharmacokinetics (PK) and clinical activity of FWD1802 as monotherapy and in combination with palbociclib in ER+/HER2- unresectable locally advanced or metastatic breast cancer with or without ESR1 mutations. Methods: This clinical study (CTR20232130) consists of three parts: monotherapy dose escalation (Part A), dose escalation in combination with palbociclib (Part B) and monotherapy dose expansion in ESR1-mutated patients (Part C). A conventional “3+3” design is used for this study. Approximately a total of 129 patients will be enrolled. FWD1802 is administered orally daily. In monotherapy dose escalation study (Part A), at each dose level, a single ascending dose (SAD) is followed by a 28-day continuous multiple ascending dose (MAD) period (defined as 1 cycle). In Part B dose, palbociclib (125 mg daily) is administered on a 28-day treatment cycle with 21-day on treatment and 7-day off treatment, along with the 28-day treatment cycle of FWD1802. The primary objectives include dose limiting toxicity (DLT), maximum tolerated dose (MTD), safety, tolerability, and the recommended phase 2 dose (RP2D) of FWD1802. Secondary objectives include pharmacokinetics characteristics of FWD1802 and its major metabolite as monotherapy and in combination with palbociclib. Preliminary anti-tumor activity including objective response rate (ORR), duration of response (DoR), disease control rate (DCR) and progression-free survival (PFS) will also be evaluated. DLT evaluation will be conducted after the first cycle of multi-dose period, and tumor assessment will be conducted every 8 weeks by investigators according to RECIST version 1.1. Patients with ER+/HER2- unresectable locally advanced or metastatic breast cancer with or without ESR1 mutation are eligible for the enrollment. Enrolled patients should have progressed disease, or failed from previous therapy, or intolerable or inaccessible to standard therapy. In addition, patients with ECOG score ≤1 and with adequate renal, cardiac, hepatic, and hematologic function are eligible for enrollment. The monotherapy dose escalation (Part A) is currently ongoing. The dose selection for Part B and Part C will be based on the data from the ongoing Part A. Clinical trial information: CTR20232130. Research Sponsor: Shenzhen Forward Pharmaceuticals Co., Ltd. (FORWARD PHARMA). Citation Format: Ling Zeng, Jin Liu, Dan Wu, Huiwen Ge, Zirui Bian, Chenggang Zhu. A phase I open-label dose escalation trial of FWD1802 as monotherapy and in combination with palbociclib in patients with ER+/HER2- unresectable locally advanced or metastatic breast cancer with or without ESR1 mutations [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 PO4-27-05.

Abstract PO3-05-08: Updated results from VERITAC evaluating vepdegestrant, a PROteolysis TArgeting Chimera (PROTAC) estrogen receptor (ER) degrader, in ER–positive/human epidermal growth factor receptor 2 (HER2)–negative advanced breast cancer

Abstract Background: Vepdegestrant (ARV-471) is an oral PROTAC ER degrader with activity toward wild-type and mutant ER. The phase 2 expansion (VERITAC) of a phase 1/2 study (NCT04072952) tested 2 vepdegestrant doses (200 mg once daily [QD] and 500 mg QD) in heavily pretreated patients with ER+/HER2- advanced breast cancer. Vepdegestrant 200 mg QD was selected as the phase 3 monotherapy dose based on comparable efficacy and favorable tolerability vs 500 mg QD and robust ER degradation (data cutoff: Jun 6, 2022). We present updated data for the vepdegestrant 200 mg QD cohort after 12 additional months of follow-up. Methods: Vepdegestrant was administered to patients with ER+/HER2- locally advanced/metastatic breast cancer who had received ≥1 prior endocrine therapy for ≥6 months, ≥1 cyclin-dependent kinase (CDK)4/6 inhibitor, and ≤1 chemotherapy regimen. The primary endpoint was clinical benefit rate (CBR; rate of confirmed complete response, partial response, or stable disease ≥24 weeks). Results: As of Jun 6, 2023, 35 patients received vepdegestrant 200 mg QD; 34 (97.1%) were female and median age was 63 y (range: 42–79). Patients had received a median of 4 prior regimens (range: 1–9) in all settings and 3 prior regimens (range: 0–7) in the metastatic setting; 100% had prior CDK4/6 inhibitors, 88.6% had prior aromatase inhibitors, 74.3% had prior fulvestrant, and 74.3% had prior chemotherapy (45.7% in metastatic setting). CBR with vepdegestrant 200 mg QD was 37.1% (95% CI: 21.5–55.1) in all evaluable patients (n=35) and the objective response rate was 8.3% (95% CI: 1.0–27.0) among 24 patients with measurable disease at baseline. Median progression-free survival in all evaluable patients was 3.5 months (95% CI: 1.8–8.2). As of the data cutoff date, 14 patients in the 200 mg QD cohort received treatment for ≥24 weeks (4 for ≥48 weeks) with 1 patient ongoing. No patients required a dose reduction due to a treatment-emergent adverse event (TEAE); 2 (5.7%) patients had a TEAE that led to vepdegestrant discontinuation (grade 3 QT prolongation and grade 3 anemia). The most common treatment-related adverse events (≥10%) were fatigue (43%), hot flush (20%), nausea (17%), arthralgia (11%), increased aspartate aminotransferase (11%), and increased blood alkaline phosphatase (11%); all were grade 1/2. Substantial on-treatment decreases in mutant ESR1 circulating tumor DNA levels were observed with vepdegestrant 200 mg QD and sustained for multiple treatment cycles. Conclusions: With 12 months of additional follow-up from the first data report, durable clinical activity with vepdegestrant 200 mg QD was seen in heavily pretreated patients with ER+/HER2- advanced breast cancer, in addition to sustained reduction in circulating mutant ESR1 tumor DNA levels. Vepdegestrant 200 mg QD continued to show a favorable safety profile. The ongoing global, randomized phase 3 VERITAC-2 study (NCT05654623) is evaluating vepdegestrant 200 mg QD vs intramuscular fulvestrant in patients with ER+/HER2- advanced breast cancer after prior combination CDK4/6 inhibitor therapy and endocrine therapy. Citation Format: Sara Hurvitz, Anne Schott, Cynthia Ma, Rita Nanda, George Zahrah, Natasha Hunter, Antoinette Tan, Melinda Telli, Jesus Anampa Mesias, Rinath Jeselsohn, Pamela Munster, Eric Zhi, Elizabeth Duperret, Cecile Mather, Erika Hamilton, Hyo Han. Updated results from VERITAC evaluating vepdegestrant, a PROteolysis TArgeting Chimera (PROTAC) estrogen receptor (ER) degrader, in ER–positive/human epidermal growth factor receptor 2 (HER2)–negative advanced 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 PO3-05-08.

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.

Abstract LB179: Identification of the highly potent and orally available ER-targeting PROTAC degrader HP568 for the treatment of breast cancer

Abstract Breast cancer remains an unmet medical need. It has become the most occurring and deadliest cancer for women globally. Estrogen receptor (ER) is expressed in 75% of breast cancers and has the key role in tumorigenesis and progression of ER+ breast cancers. Endocrine treatments that target ER are the mainstay therapies for the ER+ breast cancers, yet drug resistance unavoidably develops due to mutations in the ER gene. The ER-targeting proteolysis targeting chimera (PROTAC) degrader is an emerging new modality that interrupts the ER signaling through degradation of ER and offers unprecedented potential for solving the endocrine resistance. We have discovered HP568, an ER-targeting PROTAC that is highly potent against both the wild-type ER and ER mutants. HP568 has a favorable ADME profile that is superior to known competitor ER-targeting PROTAC. In the head-to-head comparison efficacy studies, HP568 has demonstrated the dose-dependent and superior efficacy that is well consistent with the drug exposure profile and PD biomarkers. HP568 also shows a synergetic effect in combination with a CDK4/6 inhibitor. HP568 is clean in the secondary pharmacology profiling, and well tolerated in animal tox studies. HP568 is under the preclinical development and has the potential to be the best-in-class ER-targeting degrader. HP568 structure will not be disclosed. Citation Format: Wu Du, Zhilin Tu, Dekun Qin, Haibo Li, Jingyi Duan, Shijuan Liu, Meng Zhang, Zhenyan Nie, Zhihui Yuan, Jing Li, Xinghai Li. Identification of the highly potent and orally available ER-targeting PROTAC degrader HP568 for the treatment of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB179.

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

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

Abstract B026: Substratification of mismatch repair deficient (MMRd) endometrial cancers can provide prognostic and predictive refinement

Abstract Background: Mismatch repair deficient (MMRd) endometrial cancer (EC) represents one third of all ECs. Identifying patients with MMRd EC enables testing for Lynch Syndrome (LS) and access to FDA-approved immune checkpoint blockade (ICB) therapy. Recent data have highlighted the diversity within MMRd tumors, suggesting worse outcomes and lower response to ICB in patients with MLH1 loss. Our aim was to characterize a cohort of MMRd ECs to elucidate if clinically meaningful substratification of this molecular subtype (MLH1 loss vs. other MMRd) could be achieved. Methods: MMRd ECs were identified from retrospective institutional and population-based cohorts (1994-2016) with clinicopathologic data, immunohistochemistry (IHC) assessment of estrogen receptor (ER) and L1CAM and CTNNB1 mutation status recorded. Multiplex IHC for immune markers (CD3, CD8, CD79a, CD138, PD-1, PD-L1, FoxP3, IDO-1) was assessed and compared in patients with MLH1 loss or PMS2/MLH1 loss (when 2 antibody MMRd testing performed) vs. isolated MSH2, MSH6, PMS2 or MSH2/MSH6 loss (remainder of MMRd). Results: 655 MMRd ECs were identified, 52% of cases assessed with 4 antibody IHC (MLH1, PMS2, MSH2, MSH6) and 48% with 2 (PMS2 and MSH6). This included 32 (5%) patients with MLH1 loss and 488 (75%) with PMS2/MLH1 loss (together 80% of MMRd cohort), 1% (n= 9) MSH2, 11% (n=75) MSH6, 4% (n=24) PMS2, and 4% (n=24) with loss of MSH2/MSH6). 76 cases were confirmed to have MLH1 hypermethylation but 67% of cases with loss of MLH1 or PMS2/MLH1 did not have methylation testing performed. The majority MMRd ECs were FIGO stage I (75%) and endometrioid histotype (90%). Patients with loss of MLH1 or PMS2/MLH1 were older (p<0.001), had higher BMI (p<0.001) and had tumors with more LVI (p=0.031), deep myoinvasion (p<0.001) and grade 3 (p=0.046) compared to remainder of MMRd EC. 9% of the MMRd cohort were ER negative, 8% with loss of MLH1 or PMS2/MLH1 and 16% in remainder of MMRd. Inferior outcomes were observed for progression-free survival and overall survival in patients with MLH1 or PMS2/MLH1 loss compared with the remainder of MMRd. ER, L1CAM and CTNNB1 status were not associated with clinical outcomes across the total MMRd cohort, nor within MLH1 or PMS2/MLH1 loss. There was diversity in the immune landscape within MMRd EC with significantly lower CD8 levels in MLH1 or PMS2/MLH1 loss compared to the remainder of MMRd EC. Furthermore, 25% of the MLH1 or PMS2/MLH1 loss group were tumor infiltrating lymphocyte (TIL) ‘low’/immune cold by cluster analysis compared to 11% in the remainder of MMRd EC. Only 16 patients (2.4%) were identified as having LS but 82% of these MMRd patients had not been tested (31% non-LS testing attributable to identification of hypermethylation of MLH1). Conclusion: Substratification within MMRd ECs can provide prognostic and predictive information, with loss of MLH1 and/or its dimer partner PMS2 identifying a subset of MMRd EC with inferior outcomes that may be attributed to lower TIL. ER, L1CAM, and CTNNB1 mutation status do not add prognostic refinement within MMRd EC. Citation Format: Amy Jamieson, Jennifer Pors, Samuel Leung, Derek Chiu, Stefan Kommoss, Aline Talhouk, David G. Huntsman, Naveena Singh, Blake Gilks, Jessica N. McAlpine. Substratification of mismatch repair deficient (MMRd) endometrial cancers can provide prognostic and predictive refinement [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B026.

Estrogen Receptor Mutations as Novel Targets for Immunotherapy in Metastatic Estrogen Receptor-positive Breast Cancer.

Estrogen receptor (ESR1) mutations have emerged as a key factor in endocrine therapy resistance. We identified and validated five novel, immunogenic ESR1-derived peptides that could be targeted through vaccine-based immunotherapy.

Copper Chaperone for Superoxide Dismutase Subtypes as a Prognostic Marker in Luminal B Breast Cancer.

Copper chaperone for superoxide dismutase (CCS) is an essential component of the oxidation-reduction system. In breast cancer cells, CCS expression is highly up-regulated, which contributes to cellular proliferation and migration. Breast cancer is a multifaceted disease with different tumor prognoses and responses to clinical treatments, which may be associated with multiple molecular subtypes of CCS. The CCS expression patterns in breast cancer were investigated by TNMplot, cBioPortal, and HPA network database. The correlation of CCS expression with clinicopathological parameters was analyzed using the UALCAN database. The Cancer Genome Atlas (TCGA) data set was used to analyze the Clinical characteristics of CCS in luminal B patients. The bc-GenExMiner database was used to analyze the effects of BReast-CAncer susceptibility gene (BRCA)1/2, TP53 mutation status, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER) expression on CCS expression. The survival curves and prognostic value of CCS in luminal B breast cancer were performed through Kaplan-Meier curves, univariate and multivariate Cox regression using the PrognoScan, bc-GenExMiner, and Clinical bioinformatics analysis platform. We found that CCS expression was associated with patient age, race, ER, and PR status. We also discovered that BRCA1/2 mutations had an effect on CCS expression. The luminal B subtype had the highest CCS expression, which was linked to poor survival compared with other subtypes. In addition, Kaplan-Meier curve analysis showed that luminal B patients with high CCS mRNA expression showed a poor survival and the CCS gene is an independent predictor of outcome in patients with luminal B breast cancer by univariate and multivariate Cox regression. Our findings emphasize the significant expression of CCS in luminal B breast cancer and its potential as an autonomous prognostic determinant for this specific molecular subtype. These findings suggest that CCS holds promise as a prospective marker for the treatment of luminal B breast cancer.

Estrogen Receptor Signaling in Breast Cancer.

Estrogen receptor (ER) signaling is a critical regulator of cell proliferation, differentiation, and survival in breast cancer (BC) and other hormone-sensitive cancers. In this review, we explore the mechanism of ER-dependent downstream signaling in BC and the role of estrogens as growth factors necessary for cancer invasion and dissemination. The significance of the clinical implications of ER signaling in BC, including the potential of endocrine therapies that target estrogens' synthesis and ER-dependent signal transmission, such as aromatase inhibitors or selective estrogen receptor modulators, is discussed. As a consequence, the challenges associated with the resistance to these therapies resulting from acquired ER mutations and potential strategies to overcome them are the critical point for the new treatment strategies' development.

Simultaneous isolation of hormone receptor–positive breast cancer organoids and fibroblasts reveals stroma-mediated resistance mechanisms

Recurrent hormone receptor-positive (HR+) breast cancer kills more than 600,000 women annually. Although HR+ breast cancers typically respond well to therapies, approximately 30% of patients relapse. At this stage, the tumors are usually metastatic and incurable. Resistance to therapy, particularly endocrine therapy is typically thought to be tumor intrinsic (e.g., estrogen receptor mutations). However, tumor-extrinsic factors also contribute to resistance. For example, stromal cells, such as cancer-associated fibroblasts (CAFs), residing in the tumor microenvironment, are known to stimulate resistance and disease recurrence. Recurrence in HR+ disease has been difficult to study due to the prolonged clinical course, complex nature of resistance, and lack of appropriate model systems. Existing HR+ models are limited to HR+ cell lines, a few HR+ organoid models, and xenograft models that all lack components of the human stroma. Therefore, there is an urgent need for more clinically relevant models to study the complex nature of recurrent HR+ breast cancer, and the factors contributing to treatment relapse. Here, we present an optimized protocol that allows a high take-rate, and simultaneous propagation of patient-derived organoids (PDOs) and matching CAFs, from primary and metastatic HR+ breast cancers. Our protocol allows for long-term culturing of HR+ PDOs that retain estrogen receptor expression and show responsiveness to hormone therapy. We further show the functional utility of this system by identifying CAF-secreted cytokines, such as growth-regulated oncogene α , as stroma-derived resistance drivers to endocrine therapy in HR+ PDOs.

ESR1 mutational landscape and impact of co-existing resistance variants on clinical outcomes in patients with metastatic breast cancer.

1074 Background: Mutations in estrogen receptor 1 ( ESR1) confer resistance to aromatase inhibitors but may retain sensitivity to selective estrogen receptor degraders (SERD). Recently, elacestrant, an oral SERD, was approved for patients with HR+/HER2- ESR1mut metastatic breast cancer (MBC). In this study, we evaluated the genomic landscape of ESR1 alterations from a genomic database and also evaluated the impact of resistance-associated alterations on clinical outcomes. Methods: A large de-identified database of mutations detected in plasma cell-free DNA (cfDNA) from patients with HR+/HER2- MBC was assessed for ESR1 mutations and resistance-associated co-alterations. We used the Guardant 360 assay, a 70- to 74-gene targeted next generation sequencing panel and applied a breast cancer subtype classifier based on detected mutations (Bardia A, SABCS 2020). In addition, clinical outcomes were evaluated from a clinically annotated institutional dataset, linking genomic alterations with progression-free survival (PFS) and overall survival (OS) to single-agent SERD therapy for patients with MBC. Results: Among 11,456 patients with MBC and detectable cfDNA, 4,694 were classified as likely HR+/HER2-, of which 2,708 (58%) had ESR1 missense mutations, the majority of which occurred in the ligand binding domain (n = 2,690, 99%). Among the latter, alterations included 204 unique mutations, of which 55 were variants of uncertain significance (VUS). Multiple ESR1 mutations were detected in 979 (36%) patients. Among 567 patients classified as HR+/HER2- that underwent serial sampling, 136 (24%) had detectable ESR1 missense mutations that were previously undetectable. Within the ESR1mut population, 1,990 (74%) had co-existing non-synonymous, non-VUS alterations at genes associated with SERD resistance: PIK3CA (n = 1,330; 49%), ARID1A (n = 314; 12%), PTEN (n = 201; 7%), ERBB2 (n = 180; 7%), AKT1 (n = 139; 5%), and NF1 (n = 103; 4%); top amplifications were FGFR1 (n = 522; 19%), EGFR (n = 277; 10%), and MYC (n = 177; 7%). Among a clinically annotated HR+/HER2- subgroup (n = 350), 51 patients received SERD monotherapy, either fulvestrant (n = 9) or oral SERD on clinical trial (n = 42); among 28 patients with cfDNA variants associated with SERD resistance, median PFS and OS were lower compared to the 23 patients without resistance mutations (PFS: 2.4 vs 11.3 mo; HR = 0.44; 95% CI, 0.23-0.81; p = 0.008; and OS: 21.1 vs 31.4 mo; HR 0.47; 95% CI, 0.22-0.98; p = 0.045). Conclusions: ESR1 mutations that qualify patients to receive approved SERD therapy are detectable by plasma-based genotyping and can associate with various genomic co-alterations, including variants that may impact clinical outcomes. Further research is needed to confirm the impact of co-alterations in additional datasets and evaluate the role of SERD-based combination therapy to further improve clinical outcomes of patients with MBC.

(114) Vaginal/Vulvar Symptoms with Lasofoxifene versus Fulvestrant in ESR1-mutated, ER+/HER2- Metastatic Breast Cancer Patients

Abstract Introduction Endocrine therapy (ET), specifically aromatase inhibitors (AIs) that lower estrogen to subphysiologic levels and estrogen receptor (ER) degraders that non-selectively and negatively impact ER function, may induce genitourinary syndrome of menopause (GSM) in breast cancer patients. Novel therapies with positive impacts are desirable. Lasofoxifene reduced breast cancer incidence in a large clinical study, while demonstrating ER agonist activity on the urogenital system and bone in clinical trials, including two phase 3 studies in postmenopausal women with moderate to severe vulvovaginal atrophy. Lasofoxifene is currently under investigation as a targeted ET in patients with disease progression on prior ET for metastatic breast cancer (mBC) harboring ER mutations (mESR1). Prevalence of vaginal/vulvar symptoms and potential impact of endocrine treatments on GSM in this patient population are unclear, thus data were gathered from one of the largest studies of lasofoxifene in this patient population to assess its potential positive impact in this area. Objective To investigate changes in vaginal/vulvar symptoms with lasofoxifene versus fulvestrant in women with mESR1, ER+/HER2- mBC in ELAINE 1. Methods Women in the multinational (US, Canada, Israel), phase 2, ELAINE 1 study were randomized to oral lasofoxifene 5 mg (daily) or IM fulvestrant 500 mg (days 1, 15, and 29, then every 28 days), until disease progression/severe toxicity. Vaginal/vulvar symptoms were evaluated as an exploratory endpoint using the vaginal (VAS) and vulvar (VuAS) assessment scales, instruments validated in breast cancer patients to assess dryness, soreness, irritation, and pain using a 4-point scale (0=none, 1=mild, 2=moderate, 3=severe). English-speaking patients completed the VAS/VuAS at baseline and every 8 weeks until disease progression. Changes in the mean composite VAS/VuAS score (average of all reported scores for a patient) and the mean score for the most bothersome symptom (the symptom with the greatest baseline score for a patient) over 16 weeks of treatment were analyzed. Results Of 103 enrolled patients, 39/52 (75%) lasofoxifene patients and 33/51 (65%) fulvestrant patients completed the VAS/VuAS; 9/39 (23%) and 10/33 (30%) had ≥1 moderate/severe symptom, respectively (Table). Among those who completed the VAS/VuAS (median age 61.5 years), the mean composite score changed from baseline to week 16 by −74% in lasofoxifene patients versus +36% in fulvestrant patients; the mean score for most bothersome symptom changed by −65% with lasofoxifene and by −5% with fulvestrant. In women with ≥1 moderate/severe symptom at baseline, percent changes from baseline to week 16 in the mean composite VAS/VuAS score were −72% with lasofoxifene versus +32% with fulvestrant. Conclusions In this exploratory analysis in mBC patients unselected for GSM symptoms, lasofoxifene 5 mg/day numerically improved vaginal/vulvar symptoms relative to fulvestrant, albeit with a small sample size. Baseline prevalence of moderate/severe GSM symptoms (20–30%) appears to have been under-reported in this population, which may be due to patients minimizing symptoms less important than mBC disease control or being embarrassed to communicate sexual concerns to their oncologists. Further studies are warranted to evaluate potential clinical benefits of lasofoxifene on GSM symptoms and sexual health in patients with breast cancer. Disclosure Yes, this is sponsored by industry/sponsor: Sermonix Pharmaceuticals. Clarification: Industry initiated, executed and funded study Any of the authors act as a consultant, employee or shareholder of an industry for: Sermonix Pharmaceuticals.

202P A phase Ib/II study of OP-1250, an oral complete estrogen receptor (ER) antagonist (CERAN) and selective ER degrader (SERD) with palbociclib in patients (pts) with advanced or metastatic ER-positive, HER2-negative breast cancer (MBC)

OP-1250 is small molecule CERAN/SERD that binds to and completely blocks transcriptional activity of wild-type and mutant ER. OP-1250 was well tolerated in a phase I/II monotherapy study (OP-1250-001), and the recommended phase II dose is 120 mg once a day (qd). OP-1250 with palbociclib showed synergistic activity in preclinical models. Here we report updates of pharmacokinetics (PK), drug-drug interactions (DDI), safety and efficacy from a study of OP-1250 with palbociclib (OP-1250-002). Pts with advanced or MBC with progression on or after ≤1 line of endocrine therapy (prior CDK4/6 inhibitors and chemotherapy were allowed) were enrolled into sequential cohorts to receive escalating doses of OP-1250 PO qd with palbociclib 125 mg PO qd for 21 of 28 days, using a 3+3 design, followed by dose expansion. As of January 23, 2023, 20 pts have been treated with palbociclib and OP-1250 doses of 30/60/90/120 mg (n=3/3/3/11). Fourteen received prior CDK4/6 inhibitor; 11 received prior palbociclib. No DLTs occurred. The most common (≥4 pts) treatment emergent adverse events (AEs) were neutropenia, nausea, vomiting, anemia, gastroesophageal reflux, constipation, and thrombocytopenia (all were Grade 1-2, except neutropenia). Grade 3 neutropenia occurred in 11 pts (55%). No Grade 4 AEs occurred. The exposure of OP-1250 (n=18) was consistent with the monotherapy study. Palbociclib exposure at steady state was comparable to published monotherapy data when combined with OP-1250 at all dose levels tested. Anti-tumor activity has been observed including partial responses. OP-1250 did not affect palbociclib PK and no DDIs have been observed with this combination. OP-1250 and palbociclib combination was well tolerated, safety was consistent with individual profiles of each drug as a monotherapy. Tumor responses were observed in this heavily pretreated population. Expanding on our previous report (SABCS 2022), these data provide rationale to continue exploring OP-1250 with the approved dose of palbociclib. Updated data will be presented. (NCT0526610).

256TiP TACTIVE-E: Phase Ib study of ARV-471, a PROteolysis TArgeting Chimera (PROTAC) estrogen receptor (ER) degrader, in combination with everolimus in ER+/human epidermal growth factor receptor 2 (HER2)- advanced breast cancer

ARV-471 is an oral PROTAC ER degrader that binds to and degrades both wild-type ER and ESR1 mutants. ARV-471 was well tolerated and showed clinical activity in a phase I/II study in heavily pretreated patients (pts) with ER+/HER2- advanced breast cancer. Everolimus, an inhibitor of mammalian target of rapamycin (mTOR), is approved with exemestane for pts with ER+/HER2- breast cancer after progression on aromatase inhibitors and has shown clinical activity after cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor treatment. In pts with prior CDK4/6 inhibitor therapy, the combination of ARV-471 and everolimus may offer additional advantages as ARV-471 can degrade mutant forms of ER, and ESR1 mutations are enriched in this setting. Preclinical studies in ER-expressing breast cancer cell lines showed evidence of cell growth inhibition with ARV-471 plus everolimus, including in cells expressing Y537S or D538G ESR1 mutations. ARV-471 plus everolimus demonstrated greater tumor growth inhibition in a xenograft breast cancer model than either monotherapy. Here we describe the open-label, phase Ib TACTIVE-E study (NCT05501769) of ARV-471 plus everolimus in pts with ER+/HER2- advanced breast cancer. Eligible pts (aged ≥18 years) have histologically or cytologically confirmed ER+/HER2- metastatic, recurrent, or unresectable breast cancer; received 1–3 prior lines systemic therapy in the advanced/metastatic setting, including ≥1 endocrine therapy and ≤1 chemotherapy; and progression on or intolerance to a CDK4/6 inhibitor. Pts may not have received prior ARV-471 or an mTOR-targeting therapy. ARV-471 combined with everolimus is administered orally in 28-day cycles. The primary endpoints are dose-limiting toxicities to determine the recommended phase II dose for ARV-471 in combination with everolimus, and type, frequency, and severity of adverse events and laboratory abnormalities. Secondary endpoints are preliminary antitumor activity (overall response rate, clinical benefit rate, and duration of response) and pharmacokinetic parameters of ARV-471 plus everolimus. NCT05501769. Medical writing support: Justine Lempart, PhD, of Apollo Medical Communications, and funded by Arvinas Operations, Inc. Arvinas Estrogen Receptor, Inc.

Abstract 3075: Enhanced efficacy of ARV-471, a novel PROTAC® estrogen receptor degrader, in combination with targeted agents in estrogen receptor-positive (ER+) breast cancer models

Abstract ARV-471 is a selective, orally bioavailable PROteolysis-TArgeting Chimera (PROTAC®) small molecule that induces wild-type and mutant estrogen receptor (ER) alpha degradation via the ubiquitin-proteasome system. ARV-471 demonstrates superior ER degradation and antitumor activity compared to fulvestrant in endocrine sensitive and resistant xenograft models and has shown significant ER degradation and promising clinical benefit in late-line ER+ breast cancer patients. Dual pathway inhibition combining ER targeting agents with CDK4/6 or PIK3CA/mTOR pathway inhibitors is now a central strategy for treatment of advanced ER+ breast cancer. However, resistance to aromatase inhibitors resulting from ESR1 gene mutations, suboptimal ER degradation and the intramuscular route of administration of fulvestrant underscore a need for superior orally bioavailable endocrine therapy backbones for these combinations. Here, we assessed the effects of ARV-471 in combination with CDK4/6 or PIK3CA/mTOR pathway inhibitors in preclinical models of ER+ breast cancer. In vitro studies revealed evidence of synergistic interactions between ARV-471 and the CDK4/6 inhibitors abemaciclib and ribociclib, the mTOR inhibitor everolimus, or the PIK3CA inhibitors alpelisib and inavolisib in ER+ breast cancer cells. Evidence of synergistic effects between ARV-471 and everolimus was also observed in ER+ breast cancer cells expressing ER Y537S or D538G mutations. ARV-471 in combination with CDK4/6, PIK3CA or mTOR inhibitors led to tumor regressions in MCF7 xenografts when compared to single agents alone. ARV-471 also displayed greater anti-tumor activity in combination with abemaciclib, ribociclib or inavolisib than that observed with fulvestrant in combination with these agents. Taken together, these data suggest the potential utility of ARV-471 as a combination partner for clinically relevant targeted agents for treatment of early and late-stage ER+ disease. Citation Format: Jessica Teh, Elizabeth Bortolon, Jennifer Pizzano, Melissa Pannone, Sean Landrette, Richard Gedrich, Ian Taylor. Enhanced efficacy of ARV-471, a novel PROTAC® estrogen receptor degrader, in combination with targeted agents in estrogen receptor-positive (ER+) breast cancer models [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 3075.