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Background Despite the remarkable activity of CDK4/6 inhibitors (CDK4/6i) in the treatment of estrogen receptor positive (ER+) metastatic breast cancer (BC), most patients eventually develop resistance to these drugs. The ctDNA analysis of the PALOMA-3 trial showed that the estrogen receptor (ER) mutation Y537S is a potential mechanism of acquired resistance to the combination of endocrine therapy (ET) with CDK4/6i. To date, the role of the ER mutations in the clonal evolution and the mechanisms of acquired resistance to CDK4/6i is unknown. Moreover, it is not known if the development of resistance to CDK4/6i in the presence or absence of ER mutations is due to the expansion of pre-existing resistant clones or to the de novo acquisition of resistance mechanisms. Methods To explore the clonal evolution and the mechanisms of resistance to CDK4/6i in ER-wild type (ER-WT) and ER-mutant (ER-Mut) BC, we transduced doxycycline (DOX)-inducible Y537S ER-Mut MCF7 cells with the ClonTracer library, a high-complexity DNA barcode library, and cultured the barcoded cells without DOX (MCF7), or with DOX to induce the expression of the Y537S ER mutation (MCF7-YS). To develop Palbociclib (Palbo)-resistant (PDR) and Abemaciclib (Abema)-resistant (ABR) cell models, the barcoded MCF7 and MCF7-YS cells were passaged in culture with increasing concentrations of Palbo and Abema until the acquisition of resistance. The clonal dynamics and the molecular characteristics of the PDR and ABR models were investigated by barcode sequencing, whole-exome sequencing (WES), bulk and single cell RNA sequencing (RNAseq) and protein analyses. Finally, using an ER-Mut barcoded mice model, we compared the in vitro clonal evolution of ER-Mut CDK4/6i-resistant cells with the in vivo clonal evolution of ER-Mut metastases. Results The analysis of the barcodes revealed that during the acquisition of resistance to either Palbo or Abema there is a strong clonal selection of pre-existing resistant clones. The PDR clones were different in the presence of the Y537S mutation versus WT-ER. In contrast, the clones enriched in the ABR cells were comparable between WT and mutant ER. Furthermore, the ER mutations led to decreased diversity of the enriched clones in the PDR but not in the ABR cells. Interestingly, the barcodes enriched in the PDR and ABR models did not overlap. Unsupervised analyses showed that the samples clustering based on the barcodes fractions and the mutations were similar, suggesting that the clonal selection was driven by cellular populations with specific mutational landscapes. All the ER-WT and ER-Mut resistant models had different transcriptional profiles and by single-cell RNAseq showed various degrees of intra-sample heterogeneity. At the protein level, the PDR and the ABR cells displayed downregulation of ER, Rb and p27 and upregulation of p21. In the ER-Mut conditions Cyclin D1 was upregulated in the PDR cells, while Cyclin E was upregulated in the ABR cells. Finally, the barcode sequencing of the mice metastases revealed that the clonal selection in ER-Mut metastases and in ER-Mut CDK4/6i-resistant cells is different. Conclusion Our study suggests that the development of resistance to CDK4/6i is due to the selection of pre-existing resistant clones. We also demonstrate that the expression of the Y537S ER mutation impacts the clonal evolution and the mechanisms of acquired resistance to Palbo but not to Abema. Finally, we show that the clonal evolution and mechanisms are disparate in Palbo and Abema resistance. These results support the addition of a third drug to CDK4/6i and ET, early in treatment, to delay the selection of pre-existing resistant clones and prolong the response to treatment and highlight differences between Palbo and Abema. Citation Format: Cristina Guarducci, Simona Cristea, Avery Feit, Sergey Naumenko, Agostina Nardone, Wen Ma, Douglas Russo, Gabriella Cohen Feit, Ariel Feiglin, Francisco Hermida-Prado, Shira Sherman, Myles Brown, Franziska Michor, Rinath Jeselsohn. GS3-07 Clonal evolution and mechanisms of acquired resistance to CDK4/6 inhibitors in ER-wild type and ER-mutant 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 GS3-07.

Purpose: Sensitivity to endocrine therapy (ET) is critical for the clinical benefit from the combination of palbociclib plus ET in hormone receptor–positive/HER2-negative (HR+/HER2−) advanced breast cancer. Bazedoxifene is a third-generation selective estrogen receptor (ER) modulator and selective ER degrader with activity in preclinical models of endocrine-resistant breast cancer, including models harboring ESR1 mutations. Clinical trials in healthy women showed that bazedoxifene is well tolerated. Patients and Methods: We conducted a phase Ib/II study of bazedoxifene plus palbociclib in patients with HR+/HER2− advanced breast cancer who progressed on prior ET (N = 36; NCT02448771). Results: The study met its primary endpoint, with a clinical benefit rate of 33.3%, and the safety profile was consistent with what has previously been seen with palbociclib monotherapy. The median progression-free survival (PFS) was 3.6 months [95% confidence interval (CI), 2.0–7.2]. An activating PIK3CA mutation at baseline was associated with a shorter PFS (HR = 4.4; 95% CI, 1.5–13; P = 0.0026), but activating ESR1 mutations did not impact the PFS. Longitudinal plasma circulating tumor DNA whole-exome sequencing (WES; N = 68 plasma samples) provided an overview of the tumor heterogeneity and the subclonal genetic evolution, and identified actionable mutations acquired during treatment. Conclusions: The combination of palbociclib and bazedoxifene has clinical efficacy and an acceptable safety profile in a heavily pretreated patient population with advanced HR+/HER2− breast cancer. These results merit continued investigation of bazedoxifene in breast cancer.

Most invasive lobular breast cancers (ILC) are of the luminal A subtype and are strongly hormone receptor–positive. Yet, ILC is relatively resistant to tamoxifen and associated with inferior long-term outcomes compared with invasive ductal cancers (IDC). In this study, we sought to gain mechanistic insights into these clinical findings that are not explained by the genetic landscape of ILC and to identify strategies to improve patient outcomes. A comprehensive analysis of the epigenome of ILC in preclinical models and clinical samples showed that, compared with IDC, ILC harbored a distinct chromatin state linked to gained recruitment of FOXA1, a lineage-defining pioneer transcription factor. This resulted in an ILC-unique FOXA1–estrogen receptor (ER) axis that promoted the transcription of genes associated with tumor progression and poor outcomes. The ILC-unique FOXA1–ER axis led to retained ER chromatin binding after tamoxifen treatment, which facilitated tamoxifen resistance while remaining strongly dependent on ER signaling. Mechanistically, gained FOXA1 binding was associated with the autoinduction of FOXA1 in ILC through an ILC-unique FOXA1 binding site. Targeted silencing of this regulatory site resulted in the disruption of the feed-forward loop and growth inhibition in ILC. In summary, ILC is characterized by a unique chromatin state and FOXA1–ER axis that is associated with tumor progression, offering a novel mechanism of tamoxifen resistance. These results underscore the importance of conducting clinical trials dedicated to patients with ILC in order to optimize treatments in this breast cancer subtype. Significance: A unique FOXA1–ER axis in invasive lobular breast cancer promotes disease progression and tamoxifen resistance, highlighting a potential therapeutic avenue for clinical investigations dedicated to this disease. See related commentary by Blawski and Toska, p. 3668

see Legend

Legend to Supplemental Figures

The surface glycoprotein THY is a marker of myoepithelial precursor cells, which are basal cells with epithelial–mesenchymal intermediate phenotype originating from the ectoderm. Myoepithelial precursor cells are lost during progression from in situ to invasive carcinoma. To define the functional role of Thy1-positive cells within the myoepithelial population, we tracked Thy1 expression in human breast cancer samples, isolated THY1-positive myoepithelial progenitor cells (CD44+/CD24low/CD90+), and established long-term cultures (parental cells). Parental cells were used to generate a xenograft model to examine Thy1 expression during tumor formation. Post-transplantation cell cultures lost THY1 expression through methylation at the THY1 locus and this is associated with an increase in EGFR and NOTCH1 transcript levels. Thy1-low cells are sensitive to the EGFR/HER2 dual inhibitor lapatinib. High THY1 expression is associated with poorer relapse-free survival in patients with breast cancer. THY1 methylation may track the shift of bipotent progenitors into differentiated cells. Thy1 is a good candidate biomarker in basal-like breast cancer.Implications:Our findings provide evidence that THY1 expression is lost in xenografts due to promoter methylation. Thy1-low cells with increased EGFR and Notch1 expression are responsive to target therapy. Because DNA methylation is often altered in early cancer development, candidate methylation markers may be exploited as biomarkers for basal-like breast cancer.

List of primers

Supplemental Figure 1: Consort diagram

Supplementary tables 1-4 and supplementary figure legend

Supplemental Figure 5: SSNVs detected in the cancer-related genes across 68 ctDNA WES samples

Supplemental Figure 2: Plasma circulating tumor DNA (ctDNA) collection and analysis

Purpose:Sensitivity to endocrine therapy (ET) is critical for the clinical benefit from the combination of palbociclib plus ET in hormone receptor–positive/HER2-negative (HR+/HER2−) advanced breast cancer. Bazedoxifene is a third-generation selective estrogen receptor (ER) modulator and selective ER degrader with activity in preclinical models of endocrine-resistant breast cancer, including models harboring ESR1 mutations. Clinical trials in healthy women showed that bazedoxifene is well tolerated.Patients and Methods:We conducted a phase Ib/II study of bazedoxifene plus palbociclib in patients with HR+/HER2− advanced breast cancer who progressed on prior ET (N = 36; NCT02448771).Results:The study met its primary endpoint, with a clinical benefit rate of 33.3%, and the safety profile was consistent with what has previously been seen with palbociclib monotherapy. The median progression-free survival (PFS) was 3.6 months [95% confidence interval (CI), 2.0–7.2]. An activating PIK3CA mutation at baseline was associated with a shorter PFS (HR = 4.4; 95% CI, 1.5–13; P = 0.0026), but activating ESR1 mutations did not impact the PFS. Longitudinal plasma circulating tumor DNA whole-exome sequencing (WES; N = 68 plasma samples) provided an overview of the tumor heterogeneity and the subclonal genetic evolution, and identified actionable mutations acquired during treatment.Conclusions:The combination of palbociclib and bazedoxifene has clinical efficacy and an acceptable safety profile in a heavily pretreated patient population with advanced HR+/HER2− breast cancer. These results merit continued investigation of bazedoxifene in breast cancer.

Supplemental Figure 4: Overview of SSNVs in 68 ctDNA WES samples

Supplemental Figure 6: Evolutionary analysis of longitudinal plasma ctDNA samples

Supplementary Table from A Distinct Chromatin State Drives Therapeutic Resistance in Invasive Lobular Breast Cancer

Supplementary Figure S1. ER, PR, and HER2 mRNA and protein levels across endocrine-sensitive (parental) and endocrine-resistant BC cell lines. Supplementary Figure S2. Reduced sensitivity to CDK4/6 inhibition is associated with IFN-signaling in ER+ and endocrine-resistant derivative BC cell lines. Supplementary Figure S3. The cyclin D-CDK4/6-Rb axis is altered in ER+ cell lines with acquired resistance to palbociclib. Supplementary Figure S4. Effect of acute IFN-signaling activation on sensitivity to palbociclib in ER+ BC cell lines. Supplementary Figure S5. Proteomic profiles of MCF7 P, MCF7 EDR, and their PalboR derivatives. Supplementary Figure S6. Relationship between IFN-signaling and Rb status in ER+/HER2- BC. Supplementary Figure S7. DNMT1 mRNA levels in palbociclib-sensitive and PalboR MCF7 and T47D cell lines. Supplementary Figure S8. Mutations of DNA damage response genes are not associated with IFN-signaling in ER+ BC. Supplementary Figure S9. Levels of ER and ER signalling in palbociclib-sensitive and PalboR MCF7 and T47D cell lines. Supplementary Figure S10. HALLMARK gene sets significantly up-regulated in ER+/HER2- BC patients stratified by the status (+/-) of RBsig and IRPS. Supplementary Figure S11. Impact of selected IRPS genes on relapse-free survival of ER+ BC patients.

Supplementary Figure from A Distinct Chromatin State Drives Therapeutic Resistance in Invasive Lobular Breast Cancer

Supplementary Figure 2. ER expression changes upon neoadjuvant treatment with trastuzumab

Purpose: To investigate the direct effect and therapeutic consequences of epidermal growth factor receptor 2 (HER2)-targeting therapy on expression of estrogen receptor (ER) and Bcl2 in preclinical models and clinical tumor samples.Experimental design: Archived xenograft tumors from two preclinical models (UACC812 and MCF7/HER2-18) treated with ER and HER2-targeting therapies and also HER2+ clinical breast cancer specimens collected in a lapatinib neoadjuvant trial (baseline and week 2 posttreatment) were used. Expression levels of ER and Bcl2 were evaluated by immunohistochemistry and Western blot analysis. The effects of Bcl2 and ER inhibition, by ABT-737 and fulvestrant, respectively, were tested in parental versus lapatinib-resistant UACC812 cells in vitro.Results: Expression of ER and Bcl2 was significantly increased in xenograft tumors with acquired resistance to anti-HER2 therapy compared with untreated tumors in both preclinical models (UACC812: ER P = 0.0014; Bcl2 P < 0.001 and MCF7/HER2-18: ER P = 0.0007; Bcl2 P = 0.0306). In the neoadjuvant clinical study, lapatinib treatment for 2 weeks was associated with parallel upregulation of ER and Bcl2 (Spearman coefficient: 0.70; P = 0.0002). Importantly, 18% of tumors originally ER-negative (ER−) converted to ER+ upon anti-HER2 therapy. In ER−/HER2+ MCF7/HER2-18 xenografts, ER reexpression was primarily observed in tumors responding to potent combination of anti-HER2 drugs. Estrogen deprivation added to this anti-HER2 regimen significantly delayed tumor progression (P = 0.018). In the UACC812 cells, fulvestrant, but not ABT-737, was able to completely inhibit anti–HER2-resistant growth (P < 0.0001).Conclusions: HER2 inhibition can enhance or restore ER expression with parallel Bcl2 upregulation, representing an ER-dependent survival mechanism potentially leading to anti-HER2 resistance. Clin Cancer Res; 21(17); 3995–4003. ©2015 AACR.

Purpose: Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies.Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition.Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2–irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivo.Conclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123–34. ©2017 AACR.

Most invasive lobular breast cancers (ILC) are of the luminal A subtype and are strongly hormone receptor–positive. Yet, ILC is relatively resistant to tamoxifen and associated with inferior long-term outcomes compared with invasive ductal cancers (IDC). In this study, we sought to gain mechanistic insights into these clinical findings that are not explained by the genetic landscape of ILC and to identify strategies to improve patient outcomes. A comprehensive analysis of the epigenome of ILC in preclinical models and clinical samples showed that, compared with IDC, ILC harbored a distinct chromatin state linked to gained recruitment of FOXA1, a lineage-defining pioneer transcription factor. This resulted in an ILC-unique FOXA1–estrogen receptor (ER) axis that promoted the transcription of genes associated with tumor progression and poor outcomes. The ILC-unique FOXA1–ER axis led to retained ER chromatin binding after tamoxifen treatment, which facilitated tamoxifen resistance while remaining strongly dependent on ER signaling. Mechanistically, gained FOXA1 binding was associated with the autoinduction of FOXA1 in ILC through an ILC-unique FOXA1 binding site. Targeted silencing of this regulatory site resulted in the disruption of the feed-forward loop and growth inhibition in ILC. In summary, ILC is characterized by a unique chromatin state and FOXA1–ER axis that is associated with tumor progression, offering a novel mechanism of tamoxifen resistance. These results underscore the importance of conducting clinical trials dedicated to patients with ILC in order to optimize treatments in this breast cancer subtype.Significance:A unique FOXA1–ER axis in invasive lobular breast cancer promotes disease progression and tamoxifen resistance, highlighting a potential therapeutic avenue for clinical investigations dedicated to this disease.See related commentary by Blawski and Toska, p. 3668

Supplementary Table S1. Details of antibodies used in this study. Supplementary Table S2. Q-PCR primers. Supplementary Table S3. HALLMARK gene-set enrichment analysis (GSEA) of baseline tumor samples from the NeoPalANA trial. Supplementary Table S4. Palbociclib IC50 values in MCF7 P, MCF7 EDR, T47D P-LM, and T47D EDR palbociclib-resistant (PalboR)-LM cell lines. Supplementary Table S5. Top-20 Gene Ontology (GO) terms significantly enriched in PalboR cell lines. Supplementary Table S6. HALLMARK gene sets significantly up-regulated in cell lines with acquired resistance to palbociclib. Supplementary Table S7. RPPA of MCF7 P, MCF7 EDR, and their PalboR derivatives.

Supplemental Materials and Methods and Materials present a complete description of methods, reagents, and statistics required to reproduce the experiments in the paper.

Supplementary Data from A Distinct Chromatin State Drives Therapeutic Resistance in Invasive Lobular Breast Cancer

Supplementary Figures S1-S8

Supplementary figure legend. Legend of supplementary Figure 1 and 2

supplementary methods and figure legends

Supplementary Figure 1. Lapatinib clinical trial scheme

Purpose:Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) are highly effective against estrogen receptor–positive (ER+)/HER2− breast cancer; however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes.Experimental Design:Parental breast cancer cells and their endocrine-resistant derivatives (EndoR) were used. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen deprivation–resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. Gene expression data from CDK4/6i neoadjuvant trials and publicly available datasets were interrogated for correlations of gene signatures and patient outcomes.Results:Parental and EndoR breast cancer lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high IFN signaling and reduced CDK4/6i sensitivity; thus an “IFN-related palbociclib-resistance Signature” (IRPS) was derived. In two neoadjuvant trials of CDK4/6i plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. PalboR derivatives displayed dramatic activation of IFN/STAT1 signaling compared with their short-term treated or untreated counterparts. In primary ER+/HER2− tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis.Conclusions:Aberrant IFN signaling is associated with intrinsic resistance to CDK4/6i. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN pathway, warranting additional studies to clarify its involvement in resistance to CDK4/6i.

Supplementary tables S1-S4

Tamoxifen is widely used in the adjuvant treatment of estrogen receptor–positive (ER+) breast cancer and is an important drug for pre-menopausal women and post-menopausal patients who cannot tolerate aromatase inhibitors. Despite the clear clinical benefit in improving relapse-free and overall survival in these patients, an adverse effect of tamoxifen is a 2- to 7-fold increased risk of uterine cancer (UC) after 2-5 years of treatment. To date, the mechanism of tamoxifen-driven tumorigenesis is not well understood, and preventive approaches are lacking. Here, to molecularly characterize tamoxifen-associated uterine cancers (TA-UCs) and gain insights into their unique evolution, we performed whole-exome sequencing of 21 TA-UCs (discovery cohort) and droplet digital PCR (ddPCR) of an additional 40 TA-UCs (validation cohort) obtained from the ‘Tamoxifen Associated Malignancies: Aspects of Risk’ (TAMARISK) study. In addition, we used in vivo mouse models to: (i) further investigate tamoxifen-activated molecular pathways that may be involved in TA-UC tumorigenesis; and (ii) offer mechanistic insights. Overall, we discovered that TA-UCs were genomically similar to non–TA-UCs from The Cancer Genome Atlas (TCGA) project, with one profound exception: TA-UCs are characterized by a lower-than-expected frequency of mutations in two highly prevalent UC driver genes in the PI3K pathway: PIK3CA (14% [3/21] vs 48% [265/554] in non–TA-UC; P=0.003, Fisher’s exact test; Q=0.02, Benjamini-Hochberg FDR) and PIK3R1 (0%, [0/21] vs 31% [174/554]; P=0.001; Q=0.01). We used ddPCR in the independent TA-UC validation cohort and confirmed the low frequency of mutations in PIK3CA (7.5% [3/40] vs 21% [144/685] in control UCs from the Dana-Farber contribution to the AACR GENIE project; P=0.04). We next performed mouse in vivo studies and demonstrated that tamoxifen activated the PI3K pathway and increased cell proliferation in normal mouse uterine tissue through paracrine and autocrine effects, both of which were abrogated by the PI3K inhibitor alpelisib. Taken together, we describe a distinct and novel pathway of carcinogenesis in which tamoxifen acts as a driver event in the uterus and promotes tumor development in a mutation-independent manner. Indeed, tamoxifen may increase the risk of UC by activating the PI3K pathway, which can substitute for the early acquisition of oncogenic PIK3CA or PIK3R1 mutations observed in non–TA-UC tumors. Furthermore, the ability of a PI3K inhibitor to reduce cell proliferation in our mouse model raises the possibility that downregulating the PI3K pathway may prevent or significantly reduce TA-UC development, offering a potential future therapeutic and prevention strategy for specific high-risk patients undergoing tamoxifen therapy. Citation Format: Kirsten Kubler, Agostina Nardone, Shankara Anand, Daniel Gorvich, Marjolein Droog, Francisco Hermida-Prado, Tara Akshi, Avery S Feit, Gabriella Cohen, Gwen Dackus, Matthew Pun, Yanan Kuang, Justin Cha, Mendy Miller, William J Gibson, Cloud P Paweletz, Eliezer M Van Allen, Flora E van Leeuwen, Petra Nederlof, Harry Hollema, Quang-Dé Nguyen, Marian JE Mourits, Ignaty Leshchiner, Chip Stewart, Ursula A Matulonis, Wilbert Zwart, Yosef E Maruvka, Gad Getz, Rinath Jeselsohn. Tamoxifen instigates uterine cancer development by activating PI3K signaling and supersedes PIK3CA driver mutations [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS2-09.

Background: Aberrant activation of the pioneer transcription factor (TF) FOXA1 contributes to endocrine resistance and metastasis in ER+ breast cancer (BC) by promoting genome-wide enhancer and transcriptional reprogramming that engages the AP-1 TF complex. Identification of central transcriptional nexuses in this deregulated network is key for developing new therapeutic interventions focusing on transcriptional programs. We previously identified FRA1, among the AP-1 components, as the top super-enhancer target forming a FOXA1/FRA1-centered transcriptional axis to activate genes enriched in luminal B-subtype BC and ER+ metastases. To further dissect the FOXA1/FRA1-centered transcriptional axis and its potential therapeutic role, we employed integrative multi-omics data analysis and functional studies targeting FRA1 using our MCF7-parental (P) and FOXA1-amplifed tamoxifen-resistant (TamR) preclinical models. Methods: RNA-seq data were obtained in MCF7-P and TamR cells with FOXA1 and/or AP-1 (FRA1 and c-Jun) perturbation via overexpression and/or si/shRNA knockdown (KD). Differential gene expression was analyzed using DESeq2 or the limma-voom R package. ChIP-seq-based genome-wide FOXA1 binding sites were further refined by intersection with promoter-tethered regions (PTRs) denoting Hi-C-mapped chromatin looping. Genomic binding of FOXA1/c-Jun and H3K27ac modification at target gene loci were aligned and visualized by IGV. Significance of gene set enrichment was determined using a chi-square test adjusted for multiple comparisons. Clinical relevance was examined using an RNA-seq dataset of ER+ metastatic BC (SABCS19-GS2-02). Clonogenicity, soft agar, and wound-healing assays were performed using MCF7-TamR cell derivatives engineered for doxycycline (Dox)-inducible KD of FRA1 or NS shRNA. Significance of the KD effects in functional assays was determined using a linear mixed-effects model. Results: We found that FRA1 and the two embryonic TFs SOX9 and KLF4 that harbor FOXA1-bound PTRs were commonly down-regulated in TamR cells upon KD of FOXA1, FRA1, or c-Jun. We observed increased c-Jun binding at the FOXA1-bound super-enhancer and the PTR looping to the FRA1 gene locus in TamR vs. P cells, suggesting a feed-forward mechanism by which FRA1 transcription is strengthened by the AP-1-engaged super-enhancer in TamR cells. Among the FOXA1-activated genes in TamR cells, a subset of secretory protein-encoding genes was more enriched in the genes commonly dependent on both FRA1 and c-Jun, vs. the genes depending on either FRA1 or c-Jun alone. This FOXA1/FRA1/c-Jun-activated secretome is enriched for multiple biological processes engaged in tumor metastasis and associated microenvironmental niches, and was upregulated in clinical ER+ metastases vs. primary tumors. A larger proportion of this secretome, including CXCL8 and S100P, also relies on ER preferentially in TamR vs. P cells (40% vs. 16%, respectively). FRA1 KD using two different shRNA sequences in TamR cells reduced SOX9 and KLF4 expression levels, and significantly diminished clonogenicity, soft agar colony formation, and wound healing, compared to the control NS KD. Conclusions: Our integrative bioinformatics analyses reveal a feed-forward mechanism on FRA1 activation in amplifying high-FOXA1-induced transcriptional reprogramming in endocrine resistance. A prometastatic secretome activated by FRA1/c-Jun may represent a main transcriptional output of the FOXA1/FRA1-dependent nexus in promoting ER+ disease progression. These identified key transcriptional nexuses may present network hotspots susceptible to therapeutic interventions in which FRA1 inhibition could be used as a new transcriptional program-oriented therapy to treat advanced ER+ BC. Citation Format: Chia Chia Liu, Lanfang Qin, Carmine De Angelis, Sarmistha Nanda, Resel Pereira, Martin J. Shea, Agostina Nardone, Rinath Jeselsohn, Ofir Cohen, Nikhil Wagle, Zhijie Liu, Mothaffar F. Rimawi, C. Kent Osborne, Rachel Schiff, Xiaoyong Fu. Targeting the FRA1-dependent transcriptional nexus in high FOXA1-driven endocrine-resistant and metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-05.

Purpose: Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) are highly effective against estrogen receptor–positive (ER+)/HER2− breast cancer; however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes. Experimental Design: Parental breast cancer cells and their endocrine-resistant derivatives (EndoR) were used. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen deprivation–resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. Gene expression data from CDK4/6i neoadjuvant trials and publicly available datasets were interrogated for correlations of gene signatures and patient outcomes. Results: Parental and EndoR breast cancer lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high IFN signaling and reduced CDK4/6i sensitivity; thus an “IFN-related palbociclib-resistance Signature” (IRPS) was derived. In two neoadjuvant trials of CDK4/6i plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. PalboR derivatives displayed dramatic activation of IFN/STAT1 signaling compared with their short-term treated or untreated counterparts. In primary ER+/HER2− tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis. Conclusions: Aberrant IFN signaling is associated with intrinsic resistance to CDK4/6i. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN pathway, warranting additional studies to clarify its involvement in resistance to CDK4/6i.

Background: We have previously identified two potentially predictive signatures of palbociclib resistance: the RBsig, composed of E2F1/E2F2 dependent genes, which is correlated with genetic loss of RB1, and the ratio between the gene expression levels of CCNE1 to RB1 (CCNE1/RB1). Both signatures have been previously tested in vitro and in neoadjuvant studies with palbociclib. The present analysis aims to explore the prognostic and predictive role of RBsig and CCNE1/RB1 in the pivotal phase III randomized trials PALOMA-2 and PALOMA-3. Materials and methods: Gene expression data from the PALOMA-2 and PALOMA-3 datasets were generated using the HTG EdgeSeq Oncology BM Panel, as previously described. Of the 87 genes composing RBsig, 46 were available within the EdgeSeq dataset and were used for the analyses; CCNE1 and RB1 were both available. RBsig was calculated as the mean of the Z-score scaled gene expression (log) of the 46 genes; CCNE1/RB1 was computed as the log ratio between the mRNA expression of CCNE1 and RB1. High and low values of RBsig and CCNE1/RB1 were defined based on the third quartile (Q3) as cutoff or as continuous variables. The prognostic/predictive effect of the signatures in terms of PFS was tested using Cox proportional hazard models and the Wald test. Results: The 46-genes RBsig versus the original signature showed excellent correlation in the METABRIC dataset (R=0.99), confirming its reliability as a surrogate of the original RBsig using EdgeSeq data. In both PALOMA-2 and PALOMA-3, RBsig high was significantly associated with a worse outcome compared to RBsig low in the palbociclib arm but not in the control arm [PALOMA-2: HR 1.4 (95% CI 1.0, 2.0) p=0.029 for palbociclib arm; HR 1.1 (95% CI 0.7, 1.6) p=0.71 for control arm. PALOMA-3: HR 1.7 (95% CI 1.1, 2.6) p=0.01 for palbociclib arm; HR 1.2 (95% CI 0.7, 1.9) p= 0.49 for control arm]. However, in both studies RBsig was not predictive of palbociclib resistance both when considered as a continuous variable and when dichotomized at Q3. Similarly to RBsig, in PALOMA- 3 patients with CCNE1/RB1 high tumors treated in the palbociclib arm showed a significantly worse outcome compared to those with CCNE1/RB1 low but this effect was not observed in those treated in the control arm [HR 1.6 (95% CI 1.1- 2.5) p= 0.03 for palbociclib arm; HR 1.2 (95% CI 0.7, 1.9) p=0.5 for control arm]. In addition, CCNE1/RB1 as a continuous variable was predictive of palbociclib benefit in PALOMA-3 (interaction p= 0.047). These effects were not observed in PALOMA-2. Conclusions: RBsig is a prognostic biomarker in patients treated with palbociclib, suggesting it may help in patients’ risk stratification. CCNE1/RB1 is predictive of palbociclib benefit in PALOMA-3, but not in PALOMA-2 probably due to the different patient populations and characteristics. Further studies of these biomarkers in patients treated with CDK4/6 inhibitors in the metastatic as well in the adjuvant setting are warranted. Citation Format: Luca Malorni, Matteo Benelli, Yuan Liu, Shibing Deng, Zhe Zhang, Cristina Guarducci, Angela Leo, Agostina Nardone, Francesca Galardi, Emanuela Risi, Erica Moretti, Dario Romagnoli, Chiara Biagioni, Marta Paoli, Laura Biganzoli, Ilenia Migliaccio. Prognostic and predictive role of RBsig and CCNE1/RB1 gene-expression signatures in patients with advanced breast cancer treated with palbociclib in combination with endocrine therapy in the PALOMA-2 and 3 trials [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 P5-02-17.

Forkhead box A1 (FOXA1) is a pioneer factor that facilitates chromatin binding and function of lineage-specific and oncogenic transcription factors. Hyperactive FOXA1 signaling due to gene amplification or overexpression has been reported in estrogen receptor-positive (ER + ) endocrine-resistant metastatic breast cancer. However, the molecular mechanisms by which FOXA1 up-regulation promotes these processes and the key downstream targets of the FOXA1 oncogenic network remain elusive. Here, we demonstrate that FOXA1 overexpression in ER + breast cancer cells drives genome-wide enhancer reprogramming to activate prometastatic transcriptional programs. Up-regulated FOXA1 employs superenhancers (SEs) to synchronize transcriptional reprogramming in endocrine-resistant breast cancer cells, reflecting an early embryonic development process. We identify the hypoxia-inducible transcription factor hypoxia-inducible factor-2α (HIF-2α) as the top high FOXA1-induced SE target, mediating the impact of high FOXA1 in activating prometastatic gene sets and pathways associated with poor clinical outcome. Using clinical ER + /HER2 − metastatic breast cancer datasets, we show that the aberrant FOXA1/HIF-2α transcriptional axis is largely nonconcurrent with the ESR1 mutations, suggesting different mechanisms of endocrine resistance and treatment strategies. We further demonstrate the selective efficacy of an HIF-2α antagonist, currently in clinical trials for advanced kidney cancer and recurrent glioblastoma, in reducing the clonogenicity, migration, and invasion of endocrine-resistant breast cancer cells expressing high FOXA1. Our study has uncovered high FOXA1-induced enhancer reprogramming and HIF-2α–dependent transcriptional programs as vulnerable targets for treating endocrine-resistant and metastatic breast cancer.

Despite effective strategies, resistance in HER2+ breast cancer remains a challenge. While the mevalonate pathway (MVA) is suggested to promote cell growth and survival, including in HER2+ models, its potential role in resistance to HER2-targeted therapy is unknown. Parental HER2+ breast cancer cells and their lapatinib-resistant and lapatinib + trastuzumab–resistant derivatives were used for this study. MVA activity was found to be increased in lapatinib-resistant and lapatinib + trastuzumab–resistant cells. Specific blockade of this pathway with lipophilic but not hydrophilic statins and with the N-bisphosphonate zoledronic acid led to apoptosis and substantial growth inhibition of R cells. Inhibition was rescued by mevalonate or the intermediate metabolites farnesyl pyrophosphate or geranylgeranyl pyrophosphate, but not cholesterol. Activated Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) and mTORC1 signaling, and their downstream target gene product Survivin, were inhibited by MVA blockade, especially in the lapatinib-resistant/lapatinib + trastuzumab–resistant models. Overexpression of constitutively active YAP rescued Survivin and phosphorylated-S6 levels, despite blockade of the MVA. These results suggest that the MVA provides alternative signaling leading to cell survival and resistance by activating YAP/TAZ–mTORC1–Survivin signaling when HER2 is blocked, suggesting novel therapeutic targets. MVA inhibitors including lipophilic statins and N-bisphosphonates may circumvent resistance to anti-HER2 therapy warranting further clinical investigation. Implications: The MVA was found to constitute an escape mechanism of survival and growth in HER2+ breast cancer models resistant to anti-HER2 therapies. MVA inhibitors such as simvastatin and zoledronic acid are potential therapeutic agents to resensitize the tumors that depend on the MVA to progress on anti-HER2 therapies.

Background: CDK4/6 inhibitors (CDK4/6i) palbociclib (palbo) and abemaciclib (abema) have significantly improved outcomes in patients with hormonal receptor positive (HR+)/human epidermal growth factor receptor negative (HER2-) breast cancer. Yet almost 20% of patients never respond to treatment and all patients eventually develop acquired resistance. In vitro palbo and abema have different affinities for CDK4/6 and other cellular kinases, and in vivo they have different toxicity profiles. At present there are no clinically validated biomarkers to determine CDK4/6i sensitivity or resistance. Further it is not clear if and how to choose between the CDK4/6i for optimal outcomes. In this study, using 13C-isotopically enriched glucose we sought to identify metabolic changes associated with palbo and abema treatment in sensitive and resistant cell lines. Methods: MCF-7 cells (wild-type), palbociclib resistant cells (PDR) and abemaciclib resistant cells (ABR) treated with DMSO, palbo or abema were incubated for 24hrs in DMEM supplemented with 13C-glucose. After the incubation, cells were harvested, metabolites were extracted and analyzed via 1D and 2D NMR spectroscopy. Biological triplicates were prepared for each condition. Results: In wild-type MCF-7 cells treated with palbo or abema significant metabolite changes were observed compared to DMSO. In both palbo and abema treatment a marked decrease in nucleotide metabolites was observed, which is consistent with the ability of CDK4/6i to prevent cell cycle progression. In abema treated cells there was also significant decrease in metabolites involved in the serine-glycine pathway, which was not observed in the palbo treated cells. Palbo and abema treated cells were easily distinguishable via hierarchical clustering. PDR and ABR also had distinct metabolic profiles when they were maintained with palbo and abema, respectively or if the drugs were removed. Notably the serine-glycine pathway was increased in ABR cells but decreased in PDR cells. In PDR cells we observed an increase in glucose-1-phosphate and shift towards galactose/glycogen metabolism. Conclusions: Using cell line models we were able to demonstrate that glucose metabolism is altered in palbo and abema treatment and that resistance to each drug may activate different metabolic pathways. To the best of our knowledge this is the first direct evidence of palbo-specific and abema-specific metabolite signatures. We are assessing whether the results of these in vitro studies translate to clinical samples. If so, the altered metabolites could lead to biomarkers that correlate with response and resistance for specific CDK4/6i. Further, we are exploring if by targeting the pathways associated with resistance, we can restore CDK4/6i sensitivity. This could lead to novel therapeutic targets or treatment regimens that improve patient outcomes. Citation Format: Chandrashekhar Honrao, Kanchan Sonkar, Cristina Guarducci, Agostina Nardone, Wen Ma, Srihari Raghavendra Rao, Chen Dong, Leo Rodrigues, Rinath Jeselsohn, Elizabeth O'Day. Isotopic tracing reveals distinct metabolic changes with palbociclib or abemaciclib treatment & resistance in breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5802.

BACKGROUND: The oestrogen receptor (ER) is an important therapeutic target in ER-positive (ER+) breast cancer. The selective ER degrader (SERD), fulvestrant, is effective in patients with metastatic breast cancer, but its intramuscular route of administration and low bioavailability are major clinical limitations. METHODS: Here, we studied the pharmacology of a new oral SERD, AZD9496, in a panel of in vitro and in vivo endocrine-sensitive and -resistant breast cancer models. RESULTS: In endocrine-sensitive models, AZD9496 inhibited cell growth and blocked ER activity in the presence or absence of oestrogen. In vivo, in the presence of oestrogen, short-term AZD9496 treatment, like fulvestrant, resulted in tumour growth inhibition and reduced expression of ER-dependent genes. AZD9496 inhibited cell growth in oestrogen deprivation-resistant and tamoxifen-resistant cell lines and xenograft models that retain ER expression. AZD9496 effectively reduced ER levels and ER-induced transcription. Expression analysis of short-term treated tumours showed that AZD9496 potently inhibited classic oestrogen-induced gene transcription, while simultaneously increasing expression of genes negatively regulated by ER, including genes potentially involved in escape pathways of endocrine resistance. CONCLUSIONS: These data suggest that AZD9496 is a potent anti-oestrogen that antagonises and degrades ER with anti-tumour activity in both endocrine-sensitive and endocrine-resistant models.

Steroid hormones are pivotal modulators of pathophysiological processes in many organs, where they interact with nuclear receptors to regulate gene transcription. However, our understanding of hormone action at the single cell level remains incomplete. Here, we focused on estrogen stimulation of the well-characterized GREB1 and MYC target genes that revealed large differences in cell-by-cell responses, and, more interestingly, between alleles within the same cell, both over time and hormone concentration. We specifically analyzed the role of receptor level and activity state during allele-by-allele regulation and found that neither receptor level nor activation status are the determinant of maximal hormonal response, indicating that additional pathways are potentially in place to modulate cell- and allele-specific responses. Interestingly, we found that a small molecule inhibitor of the arginine methyltransferases CARM1 and PRMT6 was able to increase, in a gene specific manner, the number of active alleles/cell before and after hormonal stimulation, suggesting that mechanisms do indeed exist to modulate hormone receptor responses at the single cell and allele level.

PURPOSE: CDK4/6 inhibitors are highly effective against ER+/HER2- breast cancer (BC); however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i’s may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes. EXPERIMENTAL DESIGN: Parental BC cells and their endocrine-resistant derivatives (EndoR) were used. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen-deprivation resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. Gene expression data from CDK4/6i neoadjuvant trials and publicly available datasets were interrogated for correlations of gene signatures and patient outcomes. RESULTS: Parental and EndoR BC lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high interferon (IFN) signaling and reduced CDK4/6i sensitivity; thus an ‘IFN-Related Palbociclib-Resistance Signature’ (IRPS) was derived. In two neoadjuvant trials of CDK4/6i plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. PalboR derivatives displayed dramatic activation of IFN/STAT1-signaling compared to their short-term treated or untreated counterparts. In primary ER+/HER2- tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis. CONCLUSION: Aberrant IFN-signaling is associated with intrinsic resistance to CDK4/6i. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN-pathway, warranting additional studies to clarify its involvement in resistance to CDK4/6i.

Circulating tumor cell clusters (CTCcl) have a higher metastatic potential compared to single CTCs and predict long-term outcomes in breast cancer (BC) patients. Because of the rarity of CTCcls, molecular characterization of primary tumors that give rise to CTCcl hold significant promise for better diagnosis and target discovery to combat metastatic BC. In our study, we utilized the reverse-phase protein array (RPPA) and transcriptomic (RNA-Seq) data of 10 triple-negative BC patient-derived xenograft (TNBC PDX) transplantable models with CTCs and evaluated expression of upregulated candidate protein Bcl2 (B-cell lymphoma 2) by immunohistochemistry (IHC). The sample-set consisted of six CTCcl-negative (CTCcl&minus, ) and four CTCcl-positive (CTCcl+) models. We analyzed the RPPA and transcriptomic profiles of CTCcl&minus, and CTCcl+ TNBC PDX models. In addition, we derived a CTCcl-specific gene signature for testing if it predicted outcomes using a publicly available dataset from 360 patients with basal-like BC. The RPPA analysis of CTCcl+ vs. CTCcl&minus, TNBC PDX tumors revealed elevated expression of Bcl2 (false discovery rate (FDR) &lt, 0.0001, fold change (FC) = 3.5) and reduced acetyl coenzyme A carboxylase-1 (ACC1) (FDR = 0.0005, FC = 0.3) in CTCcl+ compared to CTCcl&minus, tumors. Genome-wide transcriptomic analysis of CTCcl+ vs. CTCcl&minus, tumors revealed 549 differentially expressed genes associated with the presence of CTCcls. Apoptosis was one of the significantly downregulated pathways (normalized enrichment score (NES) = &minus, 1.69, FDR &lt, 0.05) in TNBC PDX tumors associated with CTCcl positivity. Two out of four CTCcl+ TNBC PDX primary tumors had high Bcl2 expression by IHC (H-score &gt, 34), whereas, only one of six CTCcl&minus, TNBC PDX primary tumors met this criterion. Evaluation of epithelial-mesenchymal transition (EMT)-specific signature did not show significant differences between CTCcl+ and CTCcl&minus, tumors. However, a gene signature associated with the presence of CTCcls in TNBC PDX models was associated with worse relapse-free survival in the publicly available dataset from 360 patients with basal-like BC. In summary, we identified the multigene signature of primary PDX tumors associated with the presence of CTCcls. Evaluation of additional TNBC PDX models and patients can further illuminate cellular and molecular pathways facilitating CTCcl formation.

Background Breast cancer patient-derived xenograft (BC-PDX) models represent a continuous and reproducible source of circulating tumor cells (CTCs) for studying their role in tumor biology and metastasis. We have previously shown the utility of BC-PDX models in the study of CTCs by immunohistochemistry (IHC) on serial paraffin sections and manual microscopic identification of cytokeratin-positive cells, a method that is both low-throughput and labor-intensive. We therefore aimed to identify and characterize CTCs from small volume mouse blood samples and examined its practical workflow in a study of BC-PDX mice treated with chemotherapy using an automated imaging platform, the AccuCyte®–CyteFinder® system. Methods CTC analysis was conducted using blood from non-tumor bearing SCID/Beige mice spiked with human breast cancer cells, BC-PDX-bearing mice, and BC-PDX mice treated with vehicle or chemotherapeutic agent(s). After red blood cell lysis, nucleated cells were mixed with transfer solution, processed onto microscope slides, and stained by immunofluorescence. The CyteFinder automated scanning microscope was used to identify CTCs, defined as nucleated cells that were human cytokeratin-positive, and mouse CD45-negative. Disaggregated primary BC-PDX tumors and lung metastatic nodules were processed using the same immunostaining protocol. Collective expression of breast cancer cell surface markers (EpCAM, EGFR, and HER2) using a cocktail of target-specific antibodies was assessed. CTCs and disaggregated tumor cells were individually retrieved from slides using the CytePicker® module for sequence analysis of a BC-PDX tumor-specific PIK3CA mutation. Results The recovery rate of human cancer cells spiked into murine blood was 83 ± 12%. CTC detection was not significantly different from the IHC method. One-third of CTCs did not stain positive for cell surface markers. A PIK3CA T1035A mutation present in a BC-PDX tumor was confirmed in isolated single CTCs and cells from dissociated metastatic nodules after whole genome amplification and sequencing. CTC evaluation could be simply implemented into a preclinical PDX therapeutic study setting with substantial improvements in workflow over the IHC method. Conclusions Analysis of small volume blood samples from BC-PDX-bearing mice using the AccuCyte–CyteFinder system allows investigation of the role of CTCs in tumor biology and metastasis independent of surface marker expression. Electronic supplementary material The online version of this article (10.1186/s12885-019-5382-1) contains supplementary material, which is available to authorized users.

Background: Forkhead box A1 (FOXA1) is an essential pioneer transcription factor (TF) evoking other key TFs-mediated lineage-specific transcriptional programs in several endoderm-derived organs. Aberrant FOXA1 augmentation, via genetic alterations, occurs in 10-15% of ER+ primary and metastatic breast cancer (BC). We have recently shown that high levels of FOXA1 (H-FOXA1) induces enhancer and transcriptional reprogramming to promote endocrine-resistant (EndoR) and pro-metastatic phenotypes. Using the core transcriptional regulatory circuitry (CRC) mapping method, we identified the AP-1 TF JUNB as a key CRC component in BC cells expressing H-FOXA1. In this study, we aimed to further characterize key AP-1 components that play a role in mediating H-FOXA1-induced transcriptional reprogramming in EndoR and metastatic BC. Methods: The ROSE and HOMER tools were used to identify super-enhancers (SEs) and the predicted SE-harboring TFs in MCF7-parental (P) cells with ectopic FOXA1 overexpression (OE), and in MCF7 tamoxifen-resistant (TamR) cells with endogenous FOXA1 amplification and OE. Cell growth, migration, and co-immunoprecipitation assays, were performed using ER+ BC P cells with ectopic FRA1 OE. A FOXA1 core gene signature (GS) was deduced using the BETA.plus algorithm to analyze our previously reported RNA-seq and ChIP-seq data derived from MCF7-P cells with ectopic FOXA1 OE. Additional RNA-seq analyses include MCF7-P cells with ectopic FRA1 OE, FOXA1 OE and simultaneous FRA1 siRNA knockdown (KD), and MCF7-TamR cells with FRA1 KD. We identified a FOXA1/FRA1-centered GS and its clinical relevance was examined using expression profiles of TCGA, METABRIC, and a metastatic biopsy study from cohort of patients with ER+ metastatic BC from Dana-Farber Cancer Institute.Results: We identified FRA1 as one of the top TFs selectively harboring SEs at their gene loci in MCF7-TamR vs. P cells. Both FRA1 and JUNB expression was elevated in TamR vs. P cells and altered concordantly with FOXA1 in P and TamR cells upon FOXA1 OE or KD, respectively. As we identified JUNB as a CRC component with binding sites enriched at the SEs in BC cells expressing H-FOXA1, we hypothesized that FRA1 and JUNB form a feed-forward transcriptional axis amplifying H-FOXA1-induced enhancer reprogramming. We found that JUNB co-immunoprecipitated with FRA1 in MCF7-TamR and MCF7-P cells with ectopic FRA1 OE, suggesting that FRA1 forms a heterodimer with JUNB to exert AP-1 activity. Ectopic FRA1 OE reduced P cell endocrine sensitivity, increased cell migration, and elicited a transcriptome enriched for the FOXA1-induced core GS. In P cells with ectopic FOXA1 OE, we identified a FRA1-dependent GS (n = 27) that is highly enriched for interferon signaling. This FOXA1/FRA1 GS was highly expressed in luminal B vs. A subtype of primary tumors, further elevated in ER+ metastases, where its expression was positively correlated with FRA1 mRNA levels. Notably, this FOXA1/FRA1 GS was not dependent on FRA1 in P cells without FOXA1 OE, suggesting its relevance in the context of H-FOXA1.Conclusions: Here we show that a FOXA1/FRA1-centered transcriptional axis induces an interferon signaling-enriched GS associated with poor outcome of ER+ BC and metastasis. A FRA1/JUNB AP-1 complex may form a feed-forward transcriptional axis to amplify H-FOXA1 signaling. The FOXA1/FRA1-centered GS could be used to stratify patients with ER+ BC who may need additional targeted therapies. Further studies are warranted to elucidate the interplay between FOXA1 and FRA1/JUNB in regulating interferon signaling, which may guide approaches to improve patient outcomes, possibly with immunotherapy using immune checkpoint inhibitors. Citation Format: Xiaoyong Fu, Resel Pereira, Lanfang Qin, Carmine De Angelis, Sarmistha Nanda, Martin Shea, Agostina Nardone, Rinath Jeselsohn, Ofir Cohen, Nikhil Wagle, Mothaffar Rimawi, C Kent Osborne, Rachel Schiff. A FOXA1/FRA1-centered transcriptional axis regulates interferon signaling in high FOXA1-associated endocrine-resistant and metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD8-03.