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Estrogen receptor alpha (ER/ESR1) mutations occur in 30% to 40% of endocrine resistant ER-positive (ER+) breast cancer. Forkhead box A1 (FOXA1) is a key pioneer factor mediating ER–chromatin interactions and endocrine response in ER+ breast cancer, but its role in ESR1-mutant breast cancer remains unclear. Our previous FOXA1 chromatin immunoprecipitation sequencing (ChIP-seq) identified a large portion of redistributed binding sites in T47D genome-edited Y537S and D538G ESR1–mutant cells. Here, we further integrated FOXA1 genomic binding profile with the isogenic ER cistrome, accessible genome, and transcriptome data of T47D cell model. FOXA1 redistribution was significantly associated with transcriptomic alterations caused by ESR1 mutations. Furthermore, in ESR1-mutant cells, FOXA1-binding sites less frequently overlapped with ER, and differential gene expression was less associated with the canonical FOXA1–ER axis. Motif analysis revealed a unique enrichment of retinoid X receptor (RXR) motifs in FOXA1-binding sites of ESR1-mutant cells. Consistently, ESR1-mutant cells were more sensitive to growth stimulation with the RXR agonist LG268. The mutant-specific response was dependent on two RXR isoforms, RXR-α and RXR-β, with a stronger dependency on the latter. In addition, T3, the agonist of thyroid receptor (TR) also showed a similar growth-promoting effect in ESR1-mutant cells. Importantly, RXR antagonist HX531 blocked growth of ESR1-mutant cells and a patient-derived xenograft (PDX)-derived organoid with an ESR1 D538G mutation. Collectively, our data support the evidence for a stronger RXR response associated with FOXA1 reprograming in ESR1-mutant cells, suggesting development of therapeutic strategies targeting RXR pathways in breast tumors with ESR1 mutation. Implications: It provides comprehensive characterization of the role of FOXA1 in ESR1-mutant breast cancer and potential therapeutic strategy through blocking RXR activation.

Background: Previous studies have shown that rapid autopsies (RA) provide a unique opportunity for tissue collection from patients who succumb to the disease. Because cancer patients are unable to donate their organs to other people, this program provides the patient an opportunity to leave a legacy by donating their body to research. These donations are vital for advancing breast cancer research. The UPMC/Pitt RA group revamped an existing program in 2018 through the formation of a larger multidisciplinary team that includes breast cancer laboratory and clinical researchers, pathologists, nurses, bioinformaticians, and tissue bankers. Because recruitment to the RA program was a challenge, we recently added patient advocates to the team to provide their essential perspective, and a dedicated research coordinator who serves as an ambassador for the program. Methods: Autopsy is performed by the Autopsy and Forensic Pathology Center of Excellence/Decedent Affairs Service of UPMC. Samples are banked in the Pitt Biospecimen Core (PBC), in addition to immediate processing including preparing of samples for sequencing and growing of organoids in the laboratory. Immunohistochemical (IHC) analysis is performed by UPMC/Magee Pathology. Results: The research coordinator quickly became an integral part of the program and closely interacts with care providers, patients and their families, pathologists on call, and manages interactions with transport services. Five breast cancer advocates have been instrumental in advising on additional changes to the program. The advocates attend regular team meetings and have formulated patient considerations for the the RA program, including appropriate and sensitive recruitment of patients, the role of physicians in decision making by the patient, registration for more than one RA program, potential issues with transporting a body across state lines and more. The advocates also developed the name for the program - “Hope for OTHERS” with Others standing for “Our Tissue Helping Enhance Research & Science”. As of June 2022, the team has completed 26 autopsies, and an additional 20 patients have consented to the program. The completed autopsies include patients with breast tumors representing different molecular and histological classes, ethnicities, and genders. The average disease-free survival and overall survival of patients that underwent autopsy was 81.6 and 127.8 months, respectively. Most patients passed outside the hospital (86%), with 62% in home hospice and 24% in inpatient hospice. Average time between death and start and end of autopsy was 4.56 hrs and 7.09 hours, respectively. The most common metastatic sites from which specimens are collected are liver, lung and lymph nodes. Per patient we collect on average specimens from 4 different organs. In addition to the metastatic lesions, we have access to primary tumor tissue and liquid biopsies obtained during the breast cancer disease progression for 44% and 73% of the patients, respectively. For a subset of the patients, tissue has been grown as patient-derived organoids or xenograft models. Preliminary IHC and sequencing analysis has provided insight into inter- and intra-patient and intra-tumor heterogeneity. Further molecular studies are ongoing. Conclusion In summary, over the last 5+ years, we have established a successful post-mortem tissue collection program, by addressing a series of barriers through the formation and work of a multi-disciplinary well-coordinated team. We are currently expanding our omics studies using state-of-the-art technologies to improve understanding how intra- and inter-tumor heterogeneity play a role in the clinical course of advanced breast cancer, to increase diversity of the patients enrolled in the RA program, and to support the successful implementation of other RA programs nationwide and worldwide. Citation Format: Steffi Oesterreich, Lori Miller, Margaret Q. Rosenzweig, Tanner L. Bartholow, Megan Yates, Ashuvinee Elangovan, Laura Savariau, Allison N. Casey, Nolan Priedigkeit, Kai Ding, Abdalla Wedn, Jie Bin Liu, Daniel D. Brown, Tara Hyder, Geoffrey Pecar, Neil Carleton, Humberto Trejo Bittar, Daniel Geisler, Oscar Lopez-Nunez, Amanda M. Clark, Alan Wells, Partha Roy, Shannon Puhalla, Naomi Howard, Christine Needles, Susan Trent, Stephanie Walker, Christine Hodgdon, Rohit Bhargava, Jennifer M. Atkinson, Adrian V. Lee. Hope for OTHERS – An organ donation program for metastatic breast cancer research [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 P6-14-02.

Estrogen receptor alpha (ER/ESR1) mutations occur in 30% to 40% of endocrine resistant ER-positive (ER+) breast cancer. Forkhead box A1 (FOXA1) is a key pioneer factor mediating ER–chromatin interactions and endocrine response in ER+ breast cancer, but its role in ESR1-mutant breast cancer remains unclear. Our previous FOXA1 chromatin immunoprecipitation sequencing (ChIP-seq) identified a large portion of redistributed binding sites in T47D genome-edited Y537S and D538G ESR1–mutant cells. Here, we further integrated FOXA1 genomic binding profile with the isogenic ER cistrome, accessible genome, and transcriptome data of T47D cell model. FOXA1 redistribution was significantly associated with transcriptomic alterations caused by ESR1 mutations. Furthermore, in ESR1-mutant cells, FOXA1-binding sites less frequently overlapped with ER, and differential gene expression was less associated with the canonical FOXA1–ER axis. Motif analysis revealed a unique enrichment of retinoid X receptor (RXR) motifs in FOXA1-binding sites of ESR1-mutant cells. Consistently, ESR1-mutant cells were more sensitive to growth stimulation with the RXR agonist LG268. The mutant-specific response was dependent on two RXR isoforms, RXR-α and RXR-β, with a stronger dependency on the latter. In addition, T3, the agonist of thyroid receptor (TR) also showed a similar growth-promoting effect in ESR1-mutant cells. Importantly, RXR antagonist HX531 blocked growth of ESR1-mutant cells and a patient-derived xenograft (PDX)-derived organoid with an ESR1 D538G mutation. Collectively, our data support the evidence for a stronger RXR response associated with FOXA1 reprograming in ESR1-mutant cells, suggesting development of therapeutic strategies targeting RXR pathways in breast tumors with ESR1 mutation.Implications:It provides comprehensive characterization of the role of FOXA1 in ESR1-mutant breast cancer and potential therapeutic strategy through blocking RXR activation.

S1. FOXA1 redistribution in ESR1 mutant cells causes distinct ER-FOXA1 interaction.S2. FOXA1 gained peaks annotated genes are significantly associated with novel target genes.S3. FOXA1 redistribution in ESR1 mutant cells confers potential RXR binding sites.S4. RXR agonist LG100268 can promote colonization and growth of ESR1 mutant cells.S5. Clonogenic survival assay and 2D growth assay to test RXR response in Park and Gertz MCF7 cell models.S6. FOXA1 knockdown drastically attenuated the base line growth of both ESR1 WT and mutant cells.S7. RXR antagonist HX531 can inhibit the response of ESR1 mutant cells to RXR agonist.S8. RXR antagonist HX531 can inhibit the basal and agonist promoted growth of IPM-PDXO-073 organoid.

Table S1. Data Sets UsedTable S2. FOXA1 and ER Chip-seq peaksTable S3. DE genesTable S4. Enriched FOXA1 peak motifsTable S5. Enriched IPA pathways

Obesity and metabolic diseases, such as insulin resistance and type 2 diabetes (T2D), are associated with metastatic breast cancer in post-menopausal women. Here, we investigated the critical cellular and molecular factors behind this link. We found that primary human adipocytes shed extracellular vesicles—specifically exosomes—that induced the expression of genes associated with epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) traits in cocultured breast cancer cell lines. Transcription of these genes was further increased in cells exposed to exosomes shed from T2D patient-derived adipocytes or insulin-resistant adipocytes and required the epigenetic reader proteins BRD2 and BRD4 in recipient cells. The thrombospondin family protein TSP5, which is associated with cancer, was more abundant in exosomes from T2D or insulin-resistant adipocytes and partially contributed to EMT in recipient cells. Bioinformatic analysis of breast cancer patient tissue showed that greater co-expression of COMP (which encodes TSP5) and BRD2 or BRD3 correlated with poorer prognosis, specifically decreased distant metastasis-free survival. Our findings reveal a mechanism of exosome-mediated crosstalk between metabolically abnormal adipocytes and breast cancer cells that may promote tumor aggressiveness in T2D patients.

Therapeutic strategies aiming to leverage anti-tumor immunity are being intensively investigated as they show promising results in cancer therapy. The PD-1/PD-L1 pathway constitutes an important target to restore functional anti-tumor immune response. Here, we report that BET protein inhibition suppresses PD-1/PD-L1 in triple-negative breast cancer. BET proteins control PD-1 expression in T cells, and PD-L1 in breast cancer cell models. BET protein targeting reduces T cell-derived interferon-γ production and signaling, thereby suppressing PD-L1 induction in breast cancer cells. Moreover, BET protein inhibition improves tumor cell-specific T cell cytotoxic function. Overall, we demonstrate that BET protein targeting represents a promising strategy to overcome tumor-reactive T cell exhaustion and improve anti-tumor immune responses, by reducing the PD-1/PD-L1 axis in triple-negative breast cancer.

Background Androgen deprivation therapies for the hormone-dependent stages of prostate cancer have become so effective that new forms of chemoresistant tumors are emerging in clinical practice, and require new targeted therapies in the metastatic setting. Yet there are important gaps in our understanding of the relevant transcriptional networks driving this process. Progression from localized to metastatic castration resistant prostate cancer (mCRPC) occurs as a result of accumulated resistance mechanisms that develop upon sustained androgen receptor (AR) suppression. Critical to this progression is the plastic nature by which prostate tumor cells transition from epithelial to mesenchymal states (EMT). Methods Here, using prostate cancer cell lines with different AR composition, we systematically manipulated somatic proteins of the Bromodomain and ExtraTerminal (BET) family (BRD2, BRD3 and BRD4) to determine which BET proteins influence EMT. We used the TCGA repository to correlate the expression of individual BET genes with key EMT genes and determined biochemical recurrence in 414 patients and progression free survival in 488 patients. Results We found that only BRD4 – and not BRD2 or BRD3 – regulates the expression of SNAI1 and SNAI2, and that the downregulation of these EMT transcription factors significantly increases E-cadherin expression. Furthermore, of the BET genes, only BRD4 correlates with survival outcomes in prostate cancer patients. Moreover, selective degradation of BRD4 protein with MZ1 ablates EMT (transcriptionally and morphologically) induced by TGFß signaling. Conclusions Many relapsed/refractory tumors share a neuroendocrine transcriptional signature that had been relatively rare until highly successful antiandrogen drugs like abiraterone and enzalutamide came into widespread use. New therapeutic targets must therefore be developed. Our results identify key EMT genes regulated by BRD4, and offers a novel druggable target to treat mCRPC. BRD4-selective protein degraders offer a promising next generation approach to treat the emerging forms of chemoresistance in advanced prostate cancer.