Endocrine Therapy Resistance Research Articles

Abstract 5693: Cancer cells subvert fibroblast function to promote a growth factor enriched tumor microenvironment in endocrine therapy resistant ER+ breast cancer

Abstract While endocrine therapy (ET) has considerably improved Stage II/III ER+ breast cancer survival rates, resistance emerges in 30-50% of patients. Recent advances have uncovered numerous cancer intrinsic and tumor-microenvironment mediated mechanisms contributing to ET resistance. However, the predominant mechanisms that prevent cure and ultimately lead to development of terminal metastatic cancer remain unknown. To reveal the leading causes of ET resistance in early-stage ER+ breast cancers, we used single-cell RNA sequencing to profile serial tumor biopsies from a larger cohort of patients. Patients received either endocrine therapy (letrozole) alone or in combination with a CDK4/6 cell cycle inhibitor (ribociclib). We examined the evolution of tumor composition, cellular communication, and the phenotypic diversity of tumor-associated cancer and non-cancer cells throughout treatment. Our analyses reveal that ET-resistant tumors are fibroblast- and endothelial-enriched and consistently exhibit broad upregulation of growth factor signaling to cancer cells, with consistent activation of ERBB communication across patient tumors. Fibroblasts emerged as the primary source of these additional growth factor signals. Upregulation of fibroblast growth factor signaling (via NRG1/2, EGF and IGF) was commensurate with differentiation to a mesenchymal cancer-associated-fibroblast (myCAF) phenotype, which was itself stimulated by cancer cells through EGF and TGF signaling. In response to growth factor enrichment, cancer cells of ET-resistant tumors showed activation of MAPK signal transduction and downstream transcription factors promoting proliferation including FOS, JUN and TRIB1. These results indicate that ET resistance emerges in patient tumors when cancer cells subvert fibroblast function to promote a growth factor enriched tumor microenvironment permitting estrogen independent cancer proliferation. Citation Format: Jason I. Griffiths, Patrick A. Cosgrove, Eric Medina Castaneda, Aritro Nath, Jinfeng Chen, Frederick R. Adler, Jeffrey T. Chang, Qamar J. Khan, Andrea H. Bild. Cancer cells subvert fibroblast function to promote a growth factor enriched tumor microenvironment in endocrine therapy resistant ER+ breast cancer. [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 5693.

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 3084: Progesterone receptor mediated crosstalk between cancer associated fibroblasts (CAFs) and ER+/PR+ breast cancer cells through CAF-secreted FGF2

Abstract Late recurring ER+ breast cancer emerges in post-menopausal women despite low/no hormone conditions. Cancer associated fibroblasts (CAFs) represent a major player in the promotion of metastasis, stemness and chemotherapy resistance in breast cancer. Progesterone receptors (PR) are emerging as drivers of stemness and metastasis in ER+ breast cancers. We propose that CAFs provide a mechanism to turn on ligand-independent PR signaling pathways through the secretion of paracrine factors. Methods: Tumorsphere assays were used to measure stem cell expansion. Cell clustering assays were used to measure CAF/tumor cell interactions. ChIP assays were used to measure PR direct binding to stemness gene regulatory elements. Results: Using the ER(+) T47D cell series (i.e., PR-null variants or cells expressing either endogenous PR (T47DCO) or PR-A or PR-B isoforms), we observed that CAF conditioned media (CM) increased tumorsphere formation in PR+ cells relative to PR-null and controls. Similar results were obtained with MCF7L cell models. CAF CM activated MAPK/ERK kinases and induced rapid phosphorylation of PR at Ser294. Physical interaction between CAFs and PR expressing T47D and MCF7L cells in clustering assays demonstrated PR driven co-cluster formation. CAF-derived FGF2 increased PR dependent tumorsphere and co-cluster formation. Blockage of FGF2 with the inhibitor, PD173074, decreased CAF CM-induction of tumorspheres and co-cluster formation. Previous data demonstrated that the cell adhesion molecule, CD44, is implicated in CAF/BC co-cluster formation. Both FGF2 and progestins increased CD44 expression in ER+ breast cancer cell models. Chromatin immunoprecipitation (ChIP) assays demonstrated that both PR isoforms are recruited to PRE-containing regions in the CD44 gene in response to progestin. Finally, we compared the gene expression profiles of breast cancer cells from T47DCO mono-clusters and CAF/T47DCO co-clusters using Single Cell RNA Seq. Among the differentially expressed genes identified were two genes implicated in tamoxifen resistance, BAMBI and NR2F2. Conclusion: We propose a model in which clustering between CAFs and ER+/PR+ breast cancer cells is promoted by the CAF secreted factor, FGF2. FGF2 promotes PR activation and upregulation of CD44 expression, which may further increase physical cell-cell interactions. Importantly, CAF-derived FGF2 can also act through PR to initiate MAPK/ERK signaling pathways that drive stemness and endocrine therapy resistance. Our studies of the interactions between PR+ breast cancer cells with CAFs either in the primary tumor or in the circulation (as CAF/CTC clusters) suggest the need to include anti-progestins as a component of endocrine therapy in late recurring ER+ breast cancer. Citation Format: Angela K. Spartz, Caroline H. Diep, Carol A. Lange, Dorraya El-Ashry. Progesterone receptor mediated crosstalk between cancer associated fibroblasts (CAFs) and ER+/PR+ breast cancer cells through CAF-secreted FGF2 [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 3084.

Abstract 3439: Evaluating nuclear receptor heterodimerization using live cell proximity assays: Implications for novel therapeutic approaches for hormone driven cancers

Abstract Breast and prostate cancer are the leading cancer diagnoses for women and men, respectively, second only to cutaneous cancers. Breast and prostate cancer arise from aberrant estrogen/estrogen receptor (ER) and testosterone/androgen receptor signaling (AR), respectively. These sex hormone receptors are members of the steroid hormone receptor family, which also includes the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and progesterone receptor (PR). Although these receptors canonically function through homodimerization, recent evidence has suggested that receptor crosstalk can amplify or dampen the actions of heterologous receptors. However, these interactions have not been extensively studied in all possible combinations of steroid hormone receptor crosstalk. In this project, we hypothesize that heterodimerization may amplify or repress the transcriptional response(s) elicited by the various receptor complexes depending upon conditions such as concentration of ligands, type of ligand, physiological states, and the types/functions of promoter regions. To test this hypothesis, we investigated the significance of ligand-induced interactions between different homo- and heterodimer combinations of AR, ARV7, GR, MR, PR, and ERα in HEK293 cells using IncuCyte live-cell imaging and NanoBRET technology. Ultimately, the combination of transfection experiments, live-cell imaging, and NanoBRET assays provided insight into which combination of steroid receptors successfully interact to amplify (or dampen) the HaloTag expression, luminescence intensity at 450nM (i.e., NanoLuc expression), and NanoBRET ratio (i.e., acceptor emission luminescence at 610nM divided by donor emission luminescence at 450nM) in HEK293 cells. Our studies revealed that heterodimerization of ERα-PR, ERα-MR, AR-GR, AR-PR, ARV7-MR, ARV7-PR, and GR-MR produced significant ligand-induced changes in signaling intensity compared to no ligand conditions for each fusion protein construct. Additionally, to demonstrate the value of the NanoBRET method for modelling clinically relevant disease conditions, we assessed the physical interaction of ERα and PR in the context of the ERα Y537S mutation - a mutation associated with endocrine therapy resistance in breast cancer that results in a constitutively active ERα. The results showed that ERα-Y537S mutation increases ERα/PR physical interaction upon PR-B stimulation in a NanoBRET proximity-based assay. Future directions include testing these interactions in breast and prostate cancer cell lines and identifying the receptors’ localization within the cell, the results of which may be of clinical importance. Citation Format: Amira Ishag-Osman, David Hosfield, Rosemary Huggins, Geoffrey Greene. Evaluating nuclear receptor heterodimerization using live cell proximity assays: Implications for novel therapeutic approaches for hormone driven cancers [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 3439.

Abstract 3734: Neuroendocrine differentiation (ND) in sensitivity of neuroendocrine tumor (NET) cells to ONC201/TIC10 cancer therapeutic

Abstract Neuroendocrine tumors (NETs) harbor neuroendocrine differentiation (ND) with specific markers including protein gene product 9.5 (PGP9.5) and Chromogranin A (CgA). In prostate cancers (PC), ND is induced by BRN2/SOX2 transcription factors. NET-like cells with low or absent androgen receptor (AR) signaling cause hormone therapy resistance and poor prognosis in PC. Small cell lung carcinoma (SCLC), a high-grade NET, presents with metastasis early and has poor survival. ONC201/TIC10 is a small molecule inducer of TRAIL signaling in clinical trials. ONC201 antagonizes dopamine D2 or D3 receptors (DRD2/DRD3) and is an agonist of mitochondrial caseinolytic protease P (ClpP) resulting in activation of DR5/TRAIL-dependent apoptosis involving the integrated stress response (ISR). ONC201 is active in various malignancies including H3K27M-mutated glioma and NETs expressing high levels of DRD2. We hypothesized that altered BRN2/SOX2 may impact NET apoptosis by ONC201 through the ISR and TRAIL/DR5. We analyzed the expression of neuroendocrine markers PGP9.5, CgA, SOX2, and BRN2, as well as markers of TRAIL signaling pathway markers ATF4, DR5, ClpP, ClpX, and DRD2/DRD3 in PC and SCLC cell lines (N=6) ± treatment with ONC201. Specifically, we compared pre-treatment protein expression levels with the IC50. Our results reveal that DU145 (IC50=3.11μM), PC3 (IC50=3.02μM), and LNCaP (IC50=1.33μM) are ONC201 sensitive. H1417 SCLC expresses CgA, unlike PC3 and DU145. PGP9.5 is expressed in these lines. PGP9.5 is expressed in PC3, DU145, H1417, and H1048 but not in LNCaP and 22RV1. BRN2 is expressed in PC3, H1417, and H1048 but not DU145, LNCaP, or 22RV1. ClpX is expressed in all 6 lines but at lower levels in SCLC. ClpP is expressed in the 6 lines. DR5 is expressed at higher levels in PC3, DU145, LNCaP, and 22RV1 PC versus H1417 and H1048 SCLC. SOX2 is expressed at high levels in H1417 cells. These results are establishing the landscape of ND in PC and SCLC lines for further experimentation and testing of our hypothesis. To characterize the association of BRN2 dysregulation with ONC201 sensitivity, we are performing BRN2/SOX2 knockdown experiments using siRNA and evaluating effects towards ONC201 sensitivity. Our results provide insights into molecular mechanisms of ND in PC and SCLC sensitivity to ONC201. We are also currently working to overexpress BRN2 and SOX2 in cell lines to analyze the impact on neuroendocrine differentiation and ONC201 sensitivity. Citation Format: Elizabeth C. Ding, Wafik S. El-Deiry. Neuroendocrine differentiation (ND) in sensitivity of neuroendocrine tumor (NET) cells to ONC201/TIC10 cancer therapeutic. [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 3734.

Abstract 5916: Weight loss intervention triggered gut microbiota modulation can positively impact obesity associated endocrine therapy resistance

Abstract Background: 41.9% of adult US population is obese and every 5-unit BMI increase is associated with 12 percent increase in breast cancer risk; it is an independent predictor of poor outcomes in breast cancer. Microbiome has emerged as a central regulator of obesity and we recently discovered that microbes can not only potentiate breast cancer growth and metastasis but also affect therapeutic response. We showed weight-loss by Diet/Exercise/Behavioral intervention (POWER-remote trial) exerts favorable effects. Here, we examine molecular underpinnings of poor therapy response in obese state and explore the gut microbiome to mitigate the same. Methods: We co-implanted lineage determined human White preAdipocytes (hWAT) with MCF7 breast cancer cells in the mammary glands of mice. Changes in tumor microenvironment and tumor cells were evaluated ex situ by RNA sequencing, flow cytometry, Raman spectroscopy, Quantitative phase microscopy, PCR, immunoblotting and functional assays. To inspect the gut microbiome, 16 obese breast cancer patients were subjected to weight loss intervention over 14 weeks. Gut microbiome was analyzed at baseline and post-intervention by microbial shotgun sequencing. Patients were divided into responders and non-responders based on % weight-loss. Results: We found that human breast cancer cells co-implanted with hWAT formed more aggressive tumors compared to controls. RNA sequencing revealed significant upregulation of multiple oncogenic pathways along with robust innate immune response. Tumor dissociated cells from hWAT-MCF7 tumors showed extensive molecular reprogramming rendering them more proliferative, motile and stem-like along with dramatic physiological changes like increased intercellular lipid accumulation. hWAT-MCF7 cells attained resistance to multiple drugs including Tamoxifen, Fulvestrant, Letrozole and Palbociclib. Simultaneous targeting of multiple signaling pathways using pharmacological inhibitors is infeasible in clinical setting hence we explored the obesity-microbiome axis. Obese breast cancer patients underwent POWER-remote intervention and their gut microbiome analysis showed significant differential expression of many microbes. Based on abundance and metabolite profile, F. prausnitzii was selected as the candidate of interest. On treatment with F. prausnitzii growth media, acquired multi-drug resistance in hWAT-MCF7 cells could be reversed suggesting that increase in gut population of the bacterium could be helpful in sensitizing patients unresponsive to therapy. Conclusion: Direct interaction between breast cancer cells and WAT rendered them resistant to multiple drugs. Metabolites produced by weight-loss-associated bacterium F. prausnitzii reversed adiposity-induced multi-drug resistance, hence, can potentially have therapeutic benefits in obese breast cancer patients. Citation Format: Sheetal Parida, Sumit Siddharth, Nethaji Muniraj, Zhenhui Liu, Ishan Barman, Michael Smith, Jenni Sheng, Vered Stearns, Janelle W. Coughlin, Dipali Sharma. Weight loss intervention triggered gut microbiota modulation can positively impact obesity associated endocrine therapy resistance [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 5916.

CircPVT1 promotes ER‐positive breast tumorigenesis and drug resistance by targeting ESR1 and MAVS

The molecular mechanisms underlying estrogen receptor (ER)‐positive breast carcinogenesis and endocrine therapy resistance remain incompletely understood. Here, we report that circPVT1, a circular RNA generated from the lncRNA PVT1, is highly expressed in ERα‐positive breast cancer cell lines and tumor samples and is functionally important in promoting ERα‐positive breast tumorigenesis and endocrine therapy resistance. CircPVT1 acts as a competing endogenous RNA (ceRNA) to sponge miR‐181a‐2‐3p, promoting the expression of ESR1 and downstream ERα‐target genes and breast cancer cell growth. Furthermore, circPVT1 directly interacts with MAVS protein to disrupt the RIGI–MAVS complex formation, inhibiting type I interferon (IFN) signaling pathway and anti‐tumor immunity. Anti‐sense oligonucleotide (ASO)‐targeting circPVT1 inhibits ERα‐positive breast cancer cell and tumor growth, re‐sensitizing tamoxifen‐resistant ERα‐positive breast cancer cells to tamoxifen treatment. Taken together, our data demonstrated that circPVT1 can work through both ceRNA and protein scaffolding mechanisms to promote cancer. Thus, circPVT1 may serve as a diagnostic biomarker and therapeutic target for ERα‐positive breast cancer in the clinic.

437 Novel Targeting of Semaphorin 7a-Mediated Survival Signaling in ER+ Breast Cancer to Mitigate Therapy Resistance

OBJECTIVES/GOALS: Estrogen receptor-positive (ER+) breast cancers (BC) comprise >70% of all BC and cause the most BC-related deaths in women worldwide. Despite available therapies against ER+ BC, recurrence often arises due to development of endocrine therapy resistance. Thus, our goal is to identify novel biomarkers that could serve as therapeutic targets for ER+ BC. METHODS/STUDY POPULATION: We have identified a neuroimmune molecule, Semaphorin 7a (SEMA7A), as a potential biomarker for endocrine therapy resistance, relapse, and poor survival in ER+ BC patients. SEMA7A promotes tumor growth, angiogenesis, epithelial-to-mesenchymal transition, metastasis, and endocrine therapy resistance in our pre-clinical models. Specifically, using in vivo models we have shown that SEMA7A+ MCF7 tumors result in lung metastases that do not respond to fulvestrant, in part via downregulation of ER, posing the need to identify novel, druggable targets for SEMA7A+ ER+ BC patients. SEMA7A is a membrane-bound protein that can inhibit tumor cell death via integrin-mediated PI3K/Akt pro-survival signaling. Thus, we hypothesized that SEMA7A+ ER+ BC may be sensitive to PI3K inhibition in combination with fulvestrant. RESULTS/ANTICIPATED RESULTS: Our preliminary studies confirmed that high SEMA7A expression associates with increased phospho-Akt levels and decreased apoptosis of tumor cells in forced suspension conditions. We also observed that human MCF7 ER+ SEMA7A overexpressing (OE) cells are sensitive to the PI3K (P110ï ¡ï€©ï€ inhibitor, Alpelisib. Also, the combination of Alpelisib with fulvestrant inhibited tumor cell viability in MCF7 cells, which was further enhanced in the SEMA7A OE counterparts. The combination also decreased proliferation and tumor sphere formation. There are currently no therapies that directly target SEMA7A, and here I propose an innovative hypothesis that PI3K inhibition will block SEMA7A signaling. DISCUSSION/SIGNIFICANCE: The role of SEMA7A has been studied in several cancer types, but its function in ER+ BC remains less well understood. In future studies, we will explore the mechanisms by which SEMA7A signals in the cell and to promote tumor cell survival. Delineating these mechanisms will help optimize treatment combinations to improve BC patient survival.

Abstract P4-02-08: Towards Precision Radiation Oncology: Endocrine Therapy Resistance as a Biomarker for Radiation Resistance in ER-Positive Breast Cancer

Abstract Pre-operative endocrine therapy use in post-menopausal women with localized, ER-positive breast cancer affords comparable rates of response and breast preservation, but lower toxicity relative to chemotherapy. Pre-operative endocrine therapy exerts selective pressure on cancer cells and promote evolution and/or enrichment of pathogenic alternations such as ESR1 mutations and other cellular adaptations. How such endocrine therapy-induced adaptations alter response to radiation therapy remains poorly defined. In this study, we show that diverse mechanisms that confer endocrine therapy resistance also drive radiation resistance by reprogramming of DNA repair pathways. We also show that BRD4, a member of bromodomain and extraterminal domain (BET) family of proteins, mediates such DNA repair reprogramming. BRD4 also plays a key role in trascriptional reprogramming in ER-positive breast cancer cells that confers endocrine therapy resistance. Thus, OTX015, a BET inhibitor with a favorable safety profile in early stage clinical trials, reverses both endocrine therapy resistance and radiation resistance in ER-positive breast cancer cells and tumors. Our findings are also consistent with reports that tamoxifen-resistant breast cancer cells are resistant to DNA damaging chemotherapeutic agents such as adriamycin and cisplatin. Overall, our findings suggest that endocrine therapy resistance in the pre-operative setting serves as a biomarker for reduced response to adjuvant radiation therapy, and that pharmacological BET inhibition reverses radiation resistance in such patients. The increasing use of “window of opportunity” studies to assess response to endocrine therapy in the pre-operative setting will further facilitate personalization of radiation treatments in these patients based on their response to endocrine therapy. Thus, we provides a therapeutic rationale for personalization of radiation treatments in breast cancer patients based on their response to endocrine therapy. We also provide a molecularly targeted approach for reversing radiation resistance in such patients. Thus, our study provides a framework for advancing Precision Radiation Oncology in breast cancer patients. Citation Format: SM Nashir Udden, Asal Rahimi, Dong W. Nathan Kim, Prasanna Alluri. Towards Precision Radiation Oncology: Endocrine Therapy Resistance as a Biomarker for Radiation Resistance in ER-Positive Breast Cancer [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 P4-02-08.

Abstract PD10-09: PD10-09 Multiomics analysis of matched ER+ primary and recurrent breast cancers on or after adjuvant endocrine therapy

Abstract Background: 80% of all breast cancers (BCs) are ER-positive (ER+). Not all respond to adjuvant endocrine therapy (aET) and a significant number develop endocrine resistance and recur. The basis for primary and acquired endocrine resistance is poorly understood. A multiomics analysis of primary ER+ BCs matched with recurrences on or after completion of aET has been performed. Patients: A unique cohort of 520 women with matched primary and recurrent ER+, HER2-negative (HER2-) BC is being analysed. In the first subset of 75, all had surgery to clear margins, followed by aET. The endocrine therapy given was tamoxifen (66%), aromatase inhibitors (AI) (28%: 17% letrozole, 6% anastrozole, 2% exemestane, and 3% a succession of 2 different AIs), or a combination of tamoxifen and an AI (6%). aET duration was 5 years, unless the patient stopped treatment or developed a recurrence sooner. 16/75 patients (21%) had positive lymph nodes. All patients developed recurrences: local in 59/75, concurrent local and nodal in 13/75 and lymph node-only in 3/75. Median time to recurrence was 4.1 years (range: 0.7-29 years). 62% of patients were on aET at the time of recurrence. All patients have long-term follow-up. Methods: DNA and RNA were extracted from matched primary and recurrence BC tissue samples. Targeted DNA-exome and whole-genome expression analyses were performed. A custom targeted DNA panel was used to study genes implicated in endocrine therapy resistance (ETR): this included 73 different targets, selected based on our previous full-exome sequencing of sequential ET recurrences and those implicated in the literature and in curated somatic and cancer mutation databases. Somatic mutations and copy number alterations (CNA) were determined. Differential gene expression analysis was performed using two-class unpaired Significance Analysis of Microarrays (SAM). Validation of pathways implicated in ETR using NanoString GeoMx protein analysis is ongoing. Results: Targeted DNA-exome profiling identified 1 or 2 potential driver mutations in all but a few primary samples. Multiple aberrations and a highly diverse mutational landscape were observed in all the recurrences. Matched breast and lymph node samples from synchronous recurrences had very similar somatic profiles. Changes significantly enriched in recurrent samples included somatic aberrations in well-established drivers such as MAP3K1, PIK3CA, TP53 and CDH1, as well as ESR1. Aberrations were also common in PTEN and in ER-associated factors FOXA1 and GATA3. Transcriptomic analysis revealed a number of pathways implicated in resistance, including ER, HER2, GATA3, AKT, RAS and p63 signalling. A panel-based, targeted DNA sequencing approach for mutational profiling allowed capture of relevant mutational profiles linked to ETR in a cost-effective manner compared with traditional whole-exome sequencing. Ongoing analysis has linked mutational profiles to specific endocrine agents and has allowed us to demonstrate significant differences between recurrences on aET compared with those after completion of aET. Multiomics profiling of the remaining samples in the cohort is underway. Discussion: This multiomics study provides the largest cohort to-date of matched early and recurrent ER+/HER2- BCs. It has shed new light on how different adjuvant endocrine agents can affect primary drivers and lead to complex somatic and transcriptomic changes in recurrent disease. This work confirms that the mechanisms of endocrine resistance are diverse and has already identified mechanisms underlying ETR and clinically meaningful biomarkers of ETR, including potentially actionable mutations and targets. Citation Format: Carlos Martinez-Perez, Charlene Kay, James Meehan, J Michael Dixon, Arran K Turnbull. PD10-09 Multiomics analysis of matched ER+ primary and recurrent breast cancers on or after adjuvant endocrine therapy [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 PD10-09.

Abstract P3-11-03: Novel Targeting of Semaphorin 7a-Mediated Survival Signaling in ER+ Breast Cancer

Abstract Endocrine receptor-positive (ER+) breast cancers (BC) comprise over 70% of all breast cancers and are the leading cause of BC-related deaths in women. Despite available targeted therapies against ER+ BC, recurrence arises primarily due to the development of endocrine therapy resistance and metastases. Thus, there is an unmet need to identify novel biomarkers for treating ER+ BC patients with metastases. We have identified a neuroimmune molecule, Semaphorin 7a (SEMA7A), as a potential biomarker for endocrine therapy resistance, increased relapse and decreased overall survival in ER+ BC patients. We have also found that SEMA7A promotes tumor growth, angio- and lymphangiogenesis, epithelial-to-mesenchymal transition (EMT), metastasis and endocrine therapy resistance in pre-clinical models. Specifically, we have shown that SEMA7A+ ER+ MCF7 tumors result in lung metastases that are resistant to the endocrine therapy, fulvestrant, in part, via downregulation of ER in vivo – posing the need to identify novel, druggable targets for SEMA7A+ ER+ BC. SEMA7A is a membrane-bound protein that we have shown can inhibit tumor cell death via integrin-mediated activation of PI3K/Akt pro-survival signaling. Thus, we hypothesized that that SEMA7A+ ER+ BC cells may be sensitive to PI3K inhibition in combination with fulvestrant. Our results demonstrate that SEMA7A+ MCF7 cells are sensitive to the combination of fulvestrant and PI3K inhibition by alpelisib, which is FDA approved for ER+ BC patients with activating mutations in PI3K; this combination also inhibited tumor cell growth and decreased tumor sphere formation. In complementary studies, we identified that Singleminded 2s (SIM2s), which is expressed in breast epithelial cells and inhibits EMT, is downregulated SEMA7A+ MCF7 tumors. Additionally, our studies show that while ER-mediated signaling turns on SEMA7A expression prolonged expression of SEMA7A results in downregulation of ER, and the loss of SIM2s causes downregulation of ER expression and upregulation of SEMA7A in MCF7 cells. Further, while SEMA7A-overexpressing (OE) tumor cells exhibit increased tumor-promoting PI3K/Akt survival signaling, we found that SIM2s plays a role in suppression of PI3K/Akt survival signaling. Therefore, we are investigating the interactions between SIM2s, SEMA7A and the survival signaling pathways involved in their crosstalk. Our recent results show that MCF7 SIM2-knockout (KO) cells exhibit increased SEMA7A promoter activity, SEMA7A expression, activation of Akt signaling, EMT phenotypes and decreased ER expression. Also, addition of exogenous SEMA7A protein reduces SIM2s expression and promoter activity. Since SIM2s correlates with inhibition of PI3K/Akt signaling and decreased SEMA7A, and SEMA7A promotes survival signaling, we are investigating additional therapeutic strategies to prevent endocrine therapy resistance via direct inhibition of SEMA7A and/or restoration of SIM2s, including inhibition of COX-2 and Akt signaling. Citation Format: Rachel Steinmetz, Garhett Wyatt, Traci Lyons, Weston Porter, Lyndsey Crump. Novel Targeting of Semaphorin 7a-Mediated Survival Signaling in ER+ Breast Cancer [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 P3-11-03.

Abstract P3-11-06: Chromatin landscape analysis based on ATAC-seq and RNA- seq reveals that GRHL2 is a novel key transcription factor for endocrine therapy resistance

Abstract Current data using gene expression and DNA modification profiles yield limited information for evaluating the potential resistance to therapies. Recent studies show that the chromatin landscape is dynamic and defines cell identity and disease status. Thus, changes in open chromatin sites could provide important information on disease progression and identify potential therapeutic targets. The majority of breast cancers express estrogen receptor (ER) and initially respond to endocrine therapies blocking ER activity. However, endocrine therapy resistance (ETR) occurs de novo or follows an initial response. Recent studies have identified new drivers of ETR, such as mTOR and CDK4/6. Therapy with these inhibitors in combination with endocrine therapy have improved patient survival, but the molecular mechanism of ETR is not fully understood and additional therapeutic options for ETR are needed. To investigate the molecular mechanisms of ETR, we established in vitro Long-Term Estrogen Deprivation (LTED) model using multiple human ER-positive breast cancer cell lines. These cells showed differential time-dependent patterns of ESR1 expression upon LTED with ESR1 upregulation in some cell lines and downregulation in others. LTEDs with ESR1 upregulation were sensitive to fulvestrant, while the others were resistant. We analyzed time-course of chromatin landscape transitions during acquisition of ETR in ESR1 up- and downregulated LTEDs cells by Assay for Transposase Accessible Chromatin (ATAC-seq) combined with RNA-seq. Because transcription factors play a primary key role in gene expression and in cell-fate changes including response to therapies, we performed bioinformatic analysis to uncover differential open chromatin sites, select enhancer signatures and identify digital footprints of transcription factors. Unlike ChIP-seq, which identifies binding sites for a known transcription factor, ATAC-seq permits analysis of all sites potentially accessible to the transcriptional machinery and predict occupancy by ~700 different transcription factors at once. With these algorithms, we found that Grainyhead-like 2 (GRHL2) is associated with development of both ESR1 overexpressed and downregulated types of LTEDs. Furthermore, RNA-seq analysis showed that GRHL2 target genes are upregulated in LTEDs. Furthermore, in the METABRIC database, high GRHL2 expression is significantly associated with poor outcome in ER+ breast cancer patients treated with endocrine therapy. Taken together, these data suggest that GRHL2 may be a novel important transcription factor in endocrine therapy resistant breast cancer regardless of fulvestrant sensitivity. Our studies provide evidence that chromatin landscape analysis, coupled with the transcription factor network algorithm, is capable of identifying novel biomarkers or therapeutic targets, This approach will expand the range of translational research as it is applicable to many cancers and diseases. Citation Format: Saori Fujiwara, Songjoon Baek, Kaustubh Wagh, Diana Stavreva, Lyuba Varticovski, Gordon Hager. Chromatin landscape analysis based on ATAC-seq and RNA- seq reveals that GRHL2 is a novel key transcription factor for endocrine therapy resistance [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 P3-11-06.

Abstract P3-11-11: Impaired Succinate Metabolism Supports Endocrine Therapy Resistance in ER Positive Breast Cancers

Abstract Estrogen receptor positive (ER+) breast cancer is the most common subtype among all breast cancers and is responsible for most breast cancer related deaths. Endocrine therapies, such as selective estrogen receptor modulators (SERM) and aromatase inhibitors (AI), often given in combination with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors, are initially effective but the majority of advanced ER+ breast cancers eventually become refractory to endocrine therapies. Therefore, understanding the underlying molecular mechanisms that enable or promote resistance to endocrine therapies may lead to novel therapeutic targets. Cancer cells consume greater quantities of glucose to meet their energy and anabolic demands to support cell growth and proliferation. In this study, we used breast cancer cell models that are endocrine therapy sensitive and resistant and investigated the metabolites by growing the cells in the presence of the stable glucose isotope, UC13-Glucose. We measured the C13-labelled metabolites of glycolysis and the TCA cycle. Glucose consumption was higher in endocrine therapy resistant MCF7/LCC9 (LCC9) cells compared with parental MCF7 (MCF7) and estrogen independent, endocrine therapy sensitive MCF7/LCC1 (LCC1) cells. Notably, in LCC9 cells the m+4 and m+6 isotopomer of C-13 labelled citrate was absent with a concurrent high succinate:fumarate ratio. Furthermore, in LCC9 cells, most of the fumarate was unlabeled suggesting that fumarate was not generated from glucose. These results suggest that the TCA cycle is impaired in LCC9 cells and the conversion of succinate to fumarate is dysregulated. Succinate dehydrogenase (SDH) enzyme is responsible for reversible catalytic conversion of succinate to fumarate. SDH is a multimeric protein comprised of four different subunits and SDH assembly factor 2 (SDHAF2), which is a tumor suppressor gene. Comparable SDH enzyme activity was observed in LCC9 and LCC1 cells. Intriguingly, when SDH activity was inhibited using dimethylmalonate, the LCC9 cells were re-sensitized to both Fulvestrant and 4-hydroxytamoxifen. Succinate accumulation was accompanied with HIF 1-alpha stabilization and SDH inhibition led to lower levels of HIF1-alpha. This indicated that SDH may function in the conversion of fumarate to succinate in LCC9 cells. Our study further investigates the source of fumarate in LCC9 cells. Overall, this study suggests that targeting succinate metabolism may help to restore sensitivity to Fulvestrant and tamoxifen in endocrine therapy resistance in ER+ breast cancer cells. Citation Format: Anil Yadav, Karla Andrade de Oliveira, Lu Jin, Robert Clarke, Surojeet Sengupta. Impaired Succinate Metabolism Supports Endocrine Therapy Resistance in ER Positive Breast Cancers [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 P3-11-11.

Abstract P5-14-11: Rearrangements in CDH1, ESR1, and ERBB2 are commonly observed in breast cancer and may influence diagnosis and treatment

Abstract Background The status of CDH1, ERBB2, and ESR1 is important for the diagnostic and treatment workup for patients with breast cancer. Most alterations in these genes occur in the form of short variants (eg missense and indel alterations) or copy number alterations (eg amplifications of ERBB2 or deletions in CDH1). The prevalence and characteristics of rearrangement events have been understudied. Materials and Methods: Comprehensive genomic profiling using a hybrid-capture based approach was performed on 44,842 breast carcinomas during the course of routine clinical care (FoundationOne® or FoundationOne®CDx) examining all classes of alterations in up to 395 genes, including CDH1, ESR1, and ERBB2. All rearrangements were included in the analysis (known/likely pathogenic and variants of uncertain significance). Estrogen receptor status was extracted from pathology reports for a subset of samples. Results: Rearrangements in CDH1, ESR1, and ERBB2 were observed in 0.26% (115/44842), 0.34% (153/44842), and 1.33% (598/44842) of breast cancer samples, respectively. As expected, CDH1 rearrangements were most common in invasive lobular carcinoma (ILC) (0.64%; 16/2516) though events were observed in samples originally submitted as invastive ductal carcinoma (IDC) (0.16%; 26/15,836), suggesting possible misdiagnosis. CDH1 rearrangements were predominantly loss of function consisting of large deletions, inversions, and truncation events. ESR1 rearrangements were observed at the highest frequency in ER+/HER2- tumors (0.58%) and were never seen in ER-/HER2- and ER-/HER2+ tumors. ESR1 rearrangements were observed with a variety of partners, with recurrent events with CCDC170, SYNE1, RMND1, PLEKHG1, ARMT1, MTHFD1L, and ZBTB2. Consistent with a possible role in therapy resistance, ESR1 rearrangements were enriched in metastatic samples relative to those biopsied from the breast (OR = 2.25; p = 8E-05). ERBB2 rearrangement events were commonly observed in HER2 amplified tumors (13.4%) and rarely in other subtypes (0.12% in ER+/HER2- and 0.28% in ER-/HER2-). Most of the events were intra-chromosomal and typically represented non-fusion duplication fragments that may have generated the ERBB2 amplification. Fusions were much rarer. Out of the 598 rearrangement events, only 18 were predicted to create in-frame and in-strand fusion products retaining the HER2 kinase domain following manual review of the events. Most fusion events were unique, though a fusion with IKZF3 was seen recurrently (n=2). Conclusions Rearrangement events in diagnostically important breast cancer genes (CDH1, ESR1, and ERBB2) were commonly observed in breast cancer with subtype-specific enrichment. Since these alterations have implications in disease diagnosis and therapy response (eg endocrine therapy resistance), comprehensive genomic profiling can provide value in breast cancer care. Citation Format: Ethan Sokol, Dexter Jin, Jeffrey S. Ross, ADRIAN V. LEE, Steffi Oesterreich. Rearrangements in CDH1, ESR1, and ERBB2 are commonly observed in breast cancer and may influence diagnosis and treatment [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 P5-14-11.

Abstract PD10-01: PD10-01 Impact of ESR1 mutations on Selective Estrogen Receptor Degraders and Modulators: an integrated liquid-biopsy and pharmacodynamics approach

Abstract Background: ESR1 hotspot mutations (HS) (i.e. 380, 536, 537, and 538) are important drivers of resistance to aromatase inhibitors, but the differential impact of genomic variants (HS vs non-HS) on response to endocrine therapies (ET) under clinical development, such as novel oral Selective Estrogen Receptor Degraders and Modulators (SERDs and SERMs), is not known. The aim of the study was to evaluate the impact of non-HS ESR1 mutations on the pharmacodynamics of SERDs and SERMs as an additional ET resistance mechanism. Materials and Methods: The study analyzed a multi-institutional cohort of 1008 patients with hormone receptor positive metastatic breast cancer characterized by circulating tumor DNA (ctDNA). Pathway classification was defined based on previous work (i.e. RTK, RAS, RAF, MEK, NRF2, ER, WNT, MYC, p53, Cell Cycle, Notch, PI3K). Single nucleotide variations (SNVs) were annotated through OncoKB; co-occurrence was tested by Fisher’s exact test. A structure-based computational strategy was used to create 3D-models of ESR1 mutants and predict changes in binding affinity (dAff) across approved and experimental drugs. A positive dAff reflects a lower affinity of the drug for mutant ESR1 compared with wild type and thus a potential for a reduced response. Results: Among the total 680 detected ESR1 mutations, 633 were missense, and 631 were gain-of-function. The most frequent mutations were in codon 537 (N=305), followed by 538 (N=224). No significant MAF differences were observed across ESR1 variants (P=0.0829). The L391F mutation resulted in an increased binding affinity for Lasofoxifene (LAS) (dAff -0.34), Giredestrant (GIR) (dAff -0.18), Elacestrant (ELA) (dAff -0.08) and Amcenestrant (AMC) (dAff -0.41), while a decreased binding affinity was observed for 4OH-Tamoxifen (TAM) (dAff 0.01), Imlunestrant (IML) (dAff 0.15), Fulvestrant (FUL) (dAff 0.43), and Camizestrant (CAM) (dAff 0.02). V392F decreased binding affinity for TAM (dAff 0.05), LAS (dAff 0.13), IML (dAff 0.11), GIR (dAff 0.11), FUL (dAff 0.04), CAM (dAff 0.05), AMC (dAff 0.06) but not for ELA (dAff -0.01). F404L decreased binding affinity for FUL (dAff 0.07), ELA (dAff 0.73), and CAM (dAff 0.26), while it increased binding affinity for TAM (dAff -0.27), LAS (dAff -0.02), IML (dAff -0.05), GIR (dAff -0.69), and AMC (dAff -2.01). G415E increased binding affinity for LAS, (dAff -0.15) GIR (dAff -0.02) and ELA (dAff -0.08), while it decreased binding affinity for TAM (dAff 0.11), IML (dAff 0.09), FUL (dAff 0.29), CAM (dAff 0.19) and AMC (dAff 0.10). Mutations in codon 537 did not affect dAff for TAM, GIR, and ELA; a significant decrease in binding affinity was observed for FUL and AMC, whereas it was increased for LAS. Mutational co-occurrence was tested between ESR1 mutations in FUL docking sites and oncogenic pathways. Significant associations were observed for cell cycle SNVs (P=0.047), Notch SNVs (P=0.020), and ER SNVs (P< 0.001). Within these pathways, significant single-gene associations were observed for FBXW7 SNVs (P=0.020), ESR1 SNVs (P< 0.001), and GATA3 SNVs (P= 0.016). Given the highly significant co-occurrence of non-HS with other ESR1 mutations, combined models were examined. The Y537/F404 combination resulted in decreased binding affinity for FUL and increased binding affinity for LAS, while L536/F404 decreased binding affinity for TAM and increased binding affinity for IML, ELA, and AMC. Notably, L540/F404 restored the FUL-ESR1 interaction resulting in an increased binding affinity (dAff -2.1). Conclusions: The study suggests that genomic variability in drug targets detectable through ctDNA may modulate therapeutic response. Preclinical models are under development to investigate the combined endocrine resistance mechanism suggested by the significant co-occurrence between ESR1 mutations in SERDs/SERMs docking sites and ESR1 hotspot mutations and provide valuable additional insights for drug development and future treatment algorithms. Citation Format: Lorenzo Gerratana, Rossana Roncato, Mattia Sturlese, Andrew A. Davis, Marko Velimirovic, Carolina REDUZZI, Katherine K. Clifton, Whitney L. Hensing, Ami N. Shah, Charles S. Dai, Paolo D’Amico, Arielle J. Medford, Alessandra Franzoni, Linda Cucciniello, Firas Wehbe, Seth A. Wander, Barbara Belletti, William Gradishar, Amir Behdad, Giuseppe Damante, Cynthia Ma, Fabio Puglisi, Aditya Bardia, Massimo Cristofanilli. PD10-01 Impact of ESR1 mutations on Selective Estrogen Receptor Degraders and Modulators: an integrated liquid-biopsy and pharmacodynamics approach. [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 PD10-01.

Abstract PD10-06: PD10-06 Clinical outcomes and exploratory gene expression analysis of OPPORTUNE: a phase II window-of-opportunity study to evaluate pictilisib+anastrozole versus anastrozole alone in ER-positive breast cancer

Abstract Background: Endocrine therapy (ET) is the mainstay of ER+ BC treatment. However; up to 20% of ER+ BC tumors progress into metastatic disease and develop ET resistance, underscoring the need for combination therapies. Preclinical data suggest that combining phosphatidylinositol 3-kinase (PI3K) inhibitors with ET may overcome resistance. OPPORTUNE, a preoperative phase II window trial, evaluated whether the combination of the PI3K inhibitor pictilisib with anastrozole (PIC+ANA) can increase the antitumor effect of ANA in newly diagnosed operable ER+ BC. Early results showed greater suppression of tumor Ki67 in patients treated with PIC+ANA versus ANA alone. Here, we present gene expression analysis from tumors collected pre- and post-treatment, and their associations with Ki67 outcomes. Methods: Postmenopausal women with newly diagnosed operable ER+/HER2-negative BC were randomly allocated (2:1, favoring the combination) to 2 weeks of preoperative treatment with ANA 1 mg once per day (n = 47) or the combination of ANA 1 mg with PIC 260 mg once per day (n = 89). The primary end point was inhibition of tumor cell proliferation measured by change in Ki67 protein expression via IHC between tumor samples taken pre- and post-treatment. Samples were analyzed by RNA-sequencing—ER pathway activity, PAM50 intrinsic subtypes, and pathway analyses were assessed by Ki67 outcomes. Elastic net regression analysis and transcription factor activity inference with Dorothea were performed to identify features strongly associated with Ki67 outcomes. Results: 124 patients (ANA, n=43; PIC+ANA, n=81) had paired tumor samples at baseline/week 2 that were evaluable for both Ki67 and RNA-seq. PIC+ANA showed improved suppression of Ki67 compared to ANA alone (-83.78% vs -73.85%, p=0.012) and a greater proportion of patients achieved complete cell cycle arrest (CCCA, as defined by Ki67< 2.7%) in the PIC+ANA arm (45.68% vs 36.59%). ER pathway activity suppression was comparable between treatments. PAM50 classification based on RNA-seq showed that 83.0% of tumors were luminal (Lum) A at baseline and 12.9% were LumB, with the remainder being classified as Normal-like. PIC+ANA showed greater suppression of Ki67 in LumB tumors compared to LumA (-92.29% vs -81.62%). This effect was much greater in the PIC+ANA arm compared to LumB tumors treated with ANA alone (-92.29% vs -37.87%); however, given the low number of LumB tumors in the ANA arm (n=3), we could not determine statistical significance. Bioinformatic analysis of RNA-seq from baseline specimens showed that MYBL2 activity was associated with resistance to ANA (as defined by Ki67≥7.5% at week2). Tumors in the top quartile of MYBL2 activity at baseline showed improved Ki67 outcomes in the PIC+ANA arm compared to ANA alone (-91.71% vs -51.44%), while no differences were observed between treatments for tumors with lower MYBL2 activity (-79.16% vs -76.72%). Single-nucleus (sn)-RNA-seq from untreated ER+ BC tumors showed that MYBL2 was enriched in a single cluster of tumor cells, which also had the highest expression of a subset of actionable targets (including CDK1, CDK2, CDK4, EZH2, and AKT1), as compared to other tumor cells. Expression data from DepMap and drug-sensitivity data from ER+ BC cell lines show a positive trend between high MYBL2 expression and sensitivity to the ER degrader, giredestrant. Conclusions: PIC exhibited greater antiproliferative effects in combination with ANA in ER+/HER2- early BC compared to ANA alone, particularly in LumB and MYBL2-high tumors. Furthermore, the transcriptional profile and in vitro response of tumor cells with high MYBL2 expression suggest potential sensitivity to other combination therapies. Citation Format: Alejandro Martinez Chibly, Alice Shia, Radia Johnson, Michael S. Hwang, Marc Hafner, Ciara Metcalfe, Kalpit Shah, Michal Slyper, Chris Bolen, Sarah E. Pinder, Alastair M. Thompson, Steven Gendreau, Peter Schmid. PD10-06 Clinical outcomes and exploratory gene expression analysis of OPPORTUNE: a phase II window-of-opportunity study to evaluate pictilisib+anastrozole versus anastrozole alone in ER-positive breast cancer [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 PD10-06.

Abstract OT1-01-02: EMBER-4: A phase 3 adjuvant trial of imlunestrant vs standard endocrine therapy (ET) in patients with ER+, HER2- early breast cancer (EBC) with an increased risk of recurrence who have previously received 2 to 5 years of adjuvant ET

Abstract Background: Adjuvant ET has been the standard of care for patients with estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-negative (HER2-) EBC. A significant proportion of patients with increased risk still experience disease relapse despite available ET and more optimum ET is needed to prevent patients developing incurable metastatic cancer. Distant recurrence risk ranges from 20% to 40% after 5 years of adjuvant ET, depending on clinicopathological (clin-path) features at diagnosis. Consequently, there is a need to further optimize adjuvant treatment, particularly in those patients who are at increased risk of recurrence. Imlunestrant is an orally bioavailable selective estrogen receptor degrader (SERD) with pure antagonistic properties and the potential to overcome ET resistance. In early phase trials, imlunestrant monotherapy showed favorable safety with pharmacokinetic (PK) exposures exceeding fulvestrant and preliminary efficacy in ER+, HER2- advanced breast cancer patients (EMBER, Jhaveri 2022) along with robust biological/pharmacodynamic activity and tolerability in EBC (EMBER-2, Neven). Trial Design: EMBER-4 is a randomized, open-label, global phase 3 study comparing imlunestrant versus physicians’ choice of ET, in patients who are at an increased risk of recurrence based on clin-path features and who have received 2 to 5 years of standard adjuvant ET. Approximately 6,000 patients will be randomized 1:1 to receive imlunestrant (400 mg daily) for 5 years or physicians’ choice of adjuvant ET (tamoxifen or an aromatase inhibitor, AI, dosed per label). Study treatment duration is 5 years. Males and pre-/peri-menopausal women will receive concomitant treatment with a GnRH agonist if receiving imlunestrant or an AI. Stratification factors include time from initial adjuvant ET, use of prior adjuvant cyclin dependent kinase 4/6 inhibitors, nodal status, menopausal status, and geographic region. Eligibility criteria: Eligible patients are adult males and females (pre-, peri- or postmenopausal) with ER+, HER2- EBC who have completed definitive locoregional therapy and have received 2 to 5 years of prior adjuvant ET without disease recurrence, but who are at increased risk of recurrence based on clin-path features at diagnosis. Prior (neo) adjuvant chemotherapy and/or targeted therapy with a CDK4/6- or PARP- inhibitor is permitted. Study endpoints: The primary endpoint is invasive disease-free survival (IDFS), excluding second non-breast primary invasive cancers. Key secondary endpoints include distant relapse-free survival, overall survival, IDFS including second non-breast primary invasive cancers, safety, PK and patient reported outcomes. Recruitment for EMBER-4 begins globally in Q4 2022. Citation Format: Komal Jhaveri, Joyce O’Shaughnessy, Fabrice Andre, Matthew P. Goetz, Nadia Harbeck, Miguel Martín, Francois-Clement Bidard, Zachary M. Thomas, Suzanne Young, Roohi Ismail-Khan, Lillian M. Smyth, Michael Gnant. EMBER-4: A phase 3 adjuvant trial of imlunestrant vs standard endocrine therapy (ET) in patients with ER+, HER2- early breast cancer (EBC) with an increased risk of recurrence who have previously received 2 to 5 years of adjuvant ET [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 OT1-01-02.

Abstract P3-11-01: Increasing DAXX Expression in ER+ Breast Cancer Cells to Overcome Endocrine Therapy Resistance

Abstract Background: New treatment paradigms are needed to overcome resistance to endocrine therapy (ET; tamoxifen or aromatase inhibitors, AI) in ER+ breast cancer (BC). ET resistance is due to survival of breast cancer stem cells (BCSCs) that contribute to relapse of ER+ BC. Notch signaling drives BCSCs. In order to identify Notch specific biomarkers for the purpose of patient selection for anti-Notch therapy, we conducted a pre-surgical biomarker window study combining ET plus MK-0752, a -secretase inhibitor (GSI). Death Associated Protein 6 (DAXX) was discovered to be a novel Notch1 target gene and necessary for GSI-mediated inhibition of BCSCs. Subsequently, we found that DAXX alone was sufficient to inhibit BCSCs. In this current study, we investigated the mechanism by which high DAXX expression inhibited growth of ET resistant ER+ BC cells in vitro and in vivo. Methods: Isogenic ER+ BC cell lines (parental MCF-7, ET resistant MCF-7/5C, parental T47D, and ET resistant T47D-ED) were used. Cells were cultured in estrogen-deprived medium for more than 1 year to mimic AI use. DAXX was depleted using siRNA or overexpressed using a pCMV-expression vector. Bulk cell proliferation was analyzed in response to estrogen depletion or increasing concentrations of 17-estradiol. BCSC survival was measured using the mammosphere-forming assay. Tumor onset and burden were measured by injecting DAXX-expressing or depleted mammospheres into mammary fat pads of female, athymic nude mice. Recurrence of an ER+ PDX tumor (BCM 5097) was measured after withdrawal of estrogen. RNA sequencing identified enriched genes and pathways that required DAXX. Based on these results, cell death was assessed using Annexin V/7-AAD flow cytometry, PARP-1 and Caspase 8 cleavage, phosphorylation of JNK, and expression of apoptotic protein regulators, BIM, BAX, Bcl-2, and Bcl-xL. JNK signaling was inhibited using SB600125. Results: Estradiol stimulated proliferation of parental, ER+ MCF-7 and T47D cells. In contrast, estradiol inhibited proliferation of isogenic ET resistant MCF-7/5C and T47D-ED cells in a concentration and ER-dependent manner. Estradiol induced the DAXX protein in both ET sensitive and resistant cells. DAXX was required for BCSC survival in ET sensitive cells. However, once ER+ cells acquired resistance to ET, DAXX was necessary and sufficient to inhibit both bulk cell proliferation and BCSC survival, suggesting that increasing DAXX might be a novel approach to overcome ET resistance. In mice, high DAXX expression significantly inhibited tumor onset and burden of ET resistant tumors compared to DAXX-depleted tumors. Low DAXX expression was significantly associated with recurrence of an ER+ PDX tumor (BCM 5097) after withdrawal of estrogen. RNA sequencing revealed that DAXX activated an anti-neoplastic gene signature, including transcription factors that regulate cell death genes including the Bcl2-family. DAXX was required for high BIM expression and low levels of Bcl-xL. DAXX was necessary and sufficient to induce apoptosis, PARP-1 cleavage, and phosphorylation of JNK in ET resistant cells. A selective JNK inhibitor, SB600125 rescued DAXX-mediated inhibition of ET resistant bulk cell proliferation and BCSC survival, suggesting that high DAXX expression activates JNK signaling to regulate apoptotic proteins to induce cell death of BCSCs-derived from ET resistant BC. Conclusions: Expressing high DAXX levels is a potent method to inhibit ET-resistant BC cell proliferation and BCSC survival. The mechanism by which DAXX inhibits ET-resistant BC is through activation of JNK signaling, regulation of pro-apoptotic genes, and induction of apoptosis. The translational impact of this research is to identify novel agents that can increase DAXX expression and test them pre-clinically and in clinical trials for patients with ET-resistant breast cancer. Citation Format: Clodia Osipo, Kathy S. Albain, Daniel Peiffer, Debra Wyatt. Increasing DAXX Expression in ER+ Breast Cancer Cells to Overcome Endocrine Therapy Resistance [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 P3-11-01.

Abstract GS3-04: GS3-04 Capivasertib and fulvestrant for patients with aromatase inhibitor-resistant hormone receptor-positive/human epidermal growth factor receptor 2-negative advanced breast cancer: results from the Phase III CAPItello-291 trial

Abstract Background: AKT pathway activation has been implicated in the development of endocrine therapy resistance in hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2–) advanced breast cancer (ABC). In the Phase II, placebo (PBO)-controlled FAKTION trial, the addition of the pan-AKT inhibitor capivasertib to fulvestrant significantly improved progression-free survival (PFS) and overall survival in postmenopausal women with aromatase inhibitor (AI)-resistant HR+/HER2– ABC. The Phase III, randomized, double-blind, PBO-controlled CAPItello-291 trial (NCT04305496) investigated the efficacy and safety of capivasertib + fulvestrant in patients with AI-resistant HR+/HER2– ABC. Methods: Eligible pre/peri or postmenopausal women or men with HR+/HER2– ABC that had recurred or progressed on or after AI therapy with or without a cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor were randomized 1:1 to receive fulvestrant (per standard dosing schedule) with either PBO or capivasertib (400 mg twice daily; 4 days on, 3 days off). Randomization was stratified by the presence of liver metastases, prior use of CDK4/6 inhibitors, and geographic location. AKT pathway alteration status (at least one qualifying PIK3CA, AKT1, or PTEN alteration) was determined using next-generation sequencing in tumor tissue. The dual primary endpoint was investigator-assessed PFS in the overall population and in patients with AKT pathway-altered tumors. Results: A total of 708 patients were randomized: 355 to capivasertib + fulvestrant and 353 to PBO + fulvestrant. Overall, 41% of patients had AKT pathway-altered tumors (48% [n=289/602] of patients with tumor sequencing results), 22% were pre/perimenopausal and 77% postmenopausal, with 1% male. Prior therapy for advanced disease included: 87% of patients with ≥1 line of prior treatment, 69% with a prior CDK4/6 inhibitor, and 18% with prior chemotherapy. Demographic and baseline characteristics were broadly balanced between the overall and altered populations and by treatment groups. At primary analysis (data cut-off Aug 15, 2022), 551 and 236 PFS events had occurred in the overall and pathway-altered populations, respectively. Overall, the median PFS was 7.2 months with capivasertib + fulvestrant and 3.6 months with PBO + fulvestrant (hazard ratio [HR] 0.60; 95% confidence interval [CI] 0.51–0.71; p<0.001). In patients with AKT pathway-altered tumors, median PFS was 7.3 months with capivasertib + fulvestrant and 3.1 months with PBO + fulvestrant (HR 0.50; 95% CI 0.38–0.65; p<0.001). The objective response rate in patients with measurable disease was 22.9% for capivasertib + fulvestrant vs 12.2% for PBO + fulvestrant overall and 28.8% vs 9.7% in the AKT pathway-altered population. The most frequent all-grade adverse events (AEs) with capivasertib + fulvestrant were diarrhea (72.4% vs 20.0% PBO + fulvestrant arm), rash (group term of rash, rash macular, rash maculo-papular, rash papular, rash pruritic; 38.0% vs 7.1%) and nausea (34.6% vs 15.4%). The most frequently reported grade ≥3 AEs were rash (group term; 12.1% vs 0.3%), diarrhea (9.3% vs 0.3%), and hyperglycemia (2.3% vs 0.3%); grade ≥3 stomatitis was 2.0% vs 0%. AEs leading to discontinuation of capivasertib/placebo were reported in 13.0% and 2.3% of patients, respectively. Conclusions: Capivasertib + fulvestrant significantly improved PFS compared to fulvestrant alone in the overall population, and in patients with AKT pathway-altered tumors, and may become a future treatment option in this setting. The safety profile of capivasertib + fulvestrant was generally manageable and consistent with prior data. Funding: CAPItello-291 is sponsored by AstraZeneca. Editorial acknowledgment: AstraZeneca-funded medical writing support was provided by Suzanne Patel, Ph.D., from BOLDSCIENCE Inc. Capivasertib was discovered by AstraZeneca subsequent to a collaboration with Astex Therapeutics (and its collaboration with the Institute of Cancer Research and Cancer Research Technology Limited). Citation Format: Nicholas Turner, Mafalda Oliveira, Sacha J. Howell, Florence Dalenc, Javier Cortés, Henry Gomez, Xichun Hu, Komal Jhaveri, Sibylle Loibl, Serafin Morales Murillo, Zbigniew Nowecki, Meena Okera, Yeon H. Park, Masakazu Toi, Lyudmila Zhukova, Chris Yan, Gaia Schiavon, Andrew Foxley, Hope Rugo. GS3-04 Capivasertib and fulvestrant for patients with aromatase inhibitor-resistant hormone receptor-positive/human epidermal growth factor receptor 2-negative advanced breast cancer: results from the Phase III CAPItello-291 trial [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 GS3-04.

Abstract P6-10-05: Mutations in the RNA Splicing Factor SF3B1 drive endocrine therapy resistance and confer a targetable replication stress response defect through PARP inhibition

Abstract Background: Heterozygous hotspot mutations in the RNA splicing factor SF3B1, occur in 3% of unselected breast cancers and are associated with oestrogen receptor (ER+) breast cancer (BC) where they are enriched in metastatic disease and are associated with a poor clinical outcome. SF3B1 mutations drive distinct signatures of alternative splicing through cryptic 3’ splice site selection leading to global transcriptomic and proteomic changes. The functional consequences of the mis-splicing events and resultant genetic vulnerabilities are poorly understood and precision medicine approaches that exploit these characteristics are not clinically available (Table 1). Methods: To understand the role of SF3B1 mutations in ER+ BC, we generated a series of SF3B1 mutant (SF3B1MUT) isogenic cell lines which were characterised using RNA-sequencing and high content mass-spectrometry proteomic profiling. SF3B1 interactome analysis was also performed using immunoprecipitation of SF3B1 followed by mass-spectrometry. The molecular consequences of aberrant splicing were investigated using a targeted screening approach of 280 genes predicted to be alternatively spliced in SF3B1MUT BC, while high-throughput drug screens were used to identify novel therapeutic options for patients with SF3B1MUT breast cancer using isogenic cells. Hits were validated in vitro and in vivo using cell line and patient derived xenografts. Results: Transcriptomic and proteomic profiling of SF3B1MUT cells identified global alternative 3’ splice site selection and subsequent proteomic changes induced by the mutations. Investigation of the SF3B1K700E interactome identified an enrichment of SF3B1K700E binding with ER, aberrant splicing of ER target genes, global rewiring of ER chromatin binding and resistance to endocrine therapy. Silencing of the aberrantly spliced candidate genes PPIH, TRIM37, HIGD1A, BRD9, and PHKG2 significantly enhanced the growth of the SF3B1 mutant cells, suggestive of a dose dependent tumour suppressive effect. Through synthetic-lethal drug screens we found that SF3B1MUT cells are selectively sensitive to PARP inhibitors. SF3B1MUT cells display a defective response to PARPi induced replication stress. Mechanistically, this occurs via defective ATR signalling in SF3B1MUT cells, which upon PARPi exposure leads to increased replication origin firing and loss of pChk1 (S317) induction. The resultant replication stress leads to failure to resolve DNA replication intermediates via the endonuclease MUS81 and cell cycle stalling at the G2/M checkpoint. These defects can be further targeted by ATM, CDK7 or FACT inhibition, when used in combination with PARPi treatment. This SF3B1MUT selective PARPi sensitivity is preserved across multiple cell lines and patient derived tumour models. In vivo, PARPi produce profound anti-tumour effects in multiple SF3B1MUT cancer models and eliminate distant metastases. Conclusions: Our integrative analysis reveals mechanistic insight into the role of SF3B1 mutations in endocrine therapy response in ER+ breast cancers, where altered SF3B1 induces ER-transcriptional re-programming. We further identified a robust synthetic-lethal relationship of mutant SF3B1 with PARP inhibition that is caused by a defective response to PARPi induced replication stress. Furthermore, we identified several potential selective combination strategies together with PARPi that are selective for SF3B1MUT cells. Together, these data provide the pre-clinical and mechanistic rationale for assessing already-approved PARPi in a biomarker-defined subset of advanced ER+ BC. Table 1. Identified potential therapies for SF3B1 mutant cancers from this study and the literature Citation Format: Phil Bland, Harry Saville, Abigail Read, Patty Wai, Gareth Muirhead, Lucinda Curnow, Jadwiga Nieminuszczy, Nivedita Ravindran, Marie John, Somaieh Hedayat, Holly Barker, James Wright, Lu Yu, Ioanna Mavrommati, Barrie Peck, Mark Allen, Patrycja Gazinska, Helen Pemberton, Aditi Gulati, Sarah Nash, Farzana Noor, Naomi Guppy, Ioannis Roxanis, Samantha Barlow, Helen Kalirai, Sarah Coupland, Ronan Broderick, Samar Alsafadi, Alexandre Houy, Marc-Henri Stern, Stephen Pettit, Jyoti Choudhary, Syed Haider, Wojciech Niedzwiedz, Christopher Lord, Rachael Natrajan. Mutations in the RNA Splicing Factor SF3B1 drive endocrine therapy resistance and confer a targetable replication stress response defect through PARP inhibition. [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 P6-10-05.