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Background: About half of all breast cancers exhibit low HER2 expression. Despite lack of ERBB2 amplification, HER2-low tumors respond to trastuzumab deruxtecan (T-DXd), leading to the NCCN recommendation of T-DXd both for patients with HER2+ and HER2-low metastatic breast cancer (MBC). It remains however unclear if HER2-low represents a distinct molecular entity, as compared to HER2-0 MBC. Here, we compare the genomic landscape of HER2-low versus HER2-0 breast cancers in a large, single institution cohort. Methods: We identified consecutive patients with MBC seen at Dana-Farber Cancer Institute between 07/2013 and 12/2020. Patients were included if they had HER2-negative MBC per ASCO/CAP Guidelines and had undergone next generation sequencing (NGS) testing with a targeted, tumor-only platform (OncoPanel). Based on the HER2 status of the specimen tested by NGS, patients were divided into 2 groups: (i) HER2-low if immunohistochemistry (IHC) 1+ or 2+ non-amplified, or (ii) HER2-0 if IHC 0. Mutations of interest detected on NGS were classified as oncogenic using the OncoKB tool and additional annotation. Genomic profiles of HER2-low and HER2-0 tumors were compared using Chi-Square and Kruskal-Wallis tests. To determine genomic event enrichment between the two HER2 groups, logistic regression models were used, accounting for background rate and estrogen receptor (ER) expression. ERBB2 copy counts were calculated for tumors with recorded histology-estimated purities and copy-number segmentation using a simple model of allelic gain/loss. Results: Among 1847 patients with HER2-negative MBC, 1043 underwent NGS testing on a HER2-low (n=489, 47%) or HER2-0 sample (n=554, 53%). Most samples were metastatic (71%, n=743) while 29% (n=300) were from primary tumors. 73% had ductal histology, 13% were lobular and 14% had mixed or other histology. ER expression was enriched among HER2-low vs. HER2-0 tumors (76% vs. 60%; p< 0.001). Focusing on the most commonly occurring genetic mutations, no major differences were observed in HER2-low vs. HER2-0 tumors, after correcting for ER status (Table 1). Among all mutational events, any mutation in MPL, CYLD, and MAP3K and oncogenic mutations in TP53 and NF1 were more common in HER2-0, while any mutation in MTOR, RAD21, DNMT3A, and PDGFRA were enriched in HER2-low patients, when controlling for ER status and background mutational rate (p< 0.05). However, no mutation reached significance after accounting for multiple hypothesis testing. Similarly, no deep deletion or high amplification CNV events reached significance for either group. Analysis of tumor mutational burden in HER2-low vs. HER-0 tumors revealed no significant differences (median: 7.26 muts/Mb vs. 7.60 muts/Mb, p=1.00), including when accounting for ER status. Finally, among tumors with sufficient tumor purity for ERBB2 copy count analysis (n=374 and 419 for HER2-low and HER2-0, respectively), HER2-low tumors had a significantly higher number of ERBB2 alleles as compared to HER2-0 (< 2 copies, 15.0% vs. 30.9%, 2 copies 67.4% vs. 60.5%, and >2 copies, 17.6% vs. 8.6%; p< 0.001 by Kruskal-Wallis). Conclusions: To our knowledge, this is the largest comprehensive genomic analysis of HER2-low MBC to date. In our cohort of patients with HER2-negative MBC, the genomic landscape of HER2-low and HER2-0 tumors did not differ significantly, apart from a higher number of ERBB2 alleles. These data further support the notion that HER2-low, as currently defined, is not a distinct molecular subtype of breast cancer. Citation Format: Paolo Tarantino, Hersh V. Gupta, Melissa E. Hughes, Janet L. Files, Sarah Strauss, Gregory Kirkner, Anne-Marie Feeney, Yvonne Y. Li, Ana C. Garrido-Castro, Romualdo Barroso-Sousa, Brittany Bychkovsky, Laura MacConaill, Neal Lindeman, Bruce Johnson, Matthew Meyerson, Sheheryar Kabraji, Rinath Jeselsohn, Xintao Qiu, Rong Li, Henry W. Long, Eric Winer, Deborah A. Dillon, Giuseppe Curigliano, Andrew Cherniack, Sara Tolaney, Nancy U. Lin. HER2-05 Comprehensive genomic characterization of HER2-low 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 HER2-05.

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.

Several endocrine therapies are currently available for the treatment of estrogen receptor (ER) positive breast cancer, but the clinical benefit of these agents is limited by endocrine therapy drug resistance. A common mechanism of endocrine therapy resistance is

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

Time to progression with E2 and Tam +/- Dox in xenografts of MCF7 Tet-off Vector control clone 3.

The estrogen receptor (ER/ESR1) is expressed in a majority of breast cancers and drugs that inhibit ER signaling are the cornerstone of breast cancer pharmacotherapy. Currently, aromatase inhibitors are the frontline endocrine interventions of choice although their durability in metastatic disease is limited by activating point mutations within the ligand-binding domain of ESR1 that permit ligand-independent activation of the receptor. It has been suggested that the most commonly occurring ESR1 mutations would likely compromise the clinical activity of selective estrogen receptor downregulators and selective estrogen receptor modulators (SERMs) when used as second-line therapies. It was unclear, however, how these mutations, which are likely coexpressed in cells with ERWT, may impact response to ER ligands in a clinically meaningful manner. To address this issue, we dissected the molecular mechanism(s) underlying ESR1-mutant pharmacology in models relevant to metastatic disease. These studies revealed that the response of ESR1 mutations to ligands was dictated primarily by the relative coexpression of ERWT in cells. Specifically, dysregulated pharmacology was only evident in cells in which the mutants were overexpressed relative to ligand-activated ERWT; a finding that highlights the role of allelism in determining ER-mutant pharmacology. Importantly, we demonstrated that the antagonist activity of the SERM, lasofoxifene, was not impacted by mutant status; a finding that has led to its clinical evaluation as a treatment for patients with advanced ER-positive breast cancer whose tumors harbor ESR1 mutations.

Genes up-regulated in Tam-R and putatively associated with EGF-induced ER binding sites (genes containing an AP-1 motif are shown in bold)

The transcription factor AP-1 is downstream of growth factor (GF) receptors (GFRs) and stress-related kinases, both of which are implicated in breast cancer endocrine resistance. Previously, we have suggested that acquired endocrine resistance is associated with increased activity of AP-1 in an in vivo model. In this report, we provide direct evidence for the role of AP-1 in endocrine resistance. First, significant overlap was found between genes modulated in tamoxifen resistance and a gene signature associated with GF-induced estrogen receptor (ER) cistrome. Interestingly, these overlapping genes were enriched for key signaling components of GFRs and stress-related kinases and had AP-1 motifs in their promoters/enhancers. Second, to determine a more definitive role of AP-1 in endocrine resistance, AP-1 was inhibited using an inducible dominant-negative (DN) cJun expressed in MCF7 breast cancer cells in vitro and in vivo. AP-1 blockade enhanced the antiproliferative effect of endocrine treatments in vitro, accelerated xenograft tumor response to tamoxifen and estrogen deprivation in vivo, promoted complete regression of tumors, and delayed the onset of tamoxifen resistance. Induction of DN-cJun after the development of tamoxifen resistance resulted in dramatic tumor shrinkage, accompanied by reduced proliferation and increased apoptosis. These data suggest that AP-1 is a key determinant of endocrine resistance by mediating a global shift in the ER transcriptional program.Implications: AP-1 represents a viable therapeutic target to overcome endocrine resistance. Mol Cancer Res; 14(5); 470–81. ©2016 AACR.

Supplementary Table 1: Genes up-regulated in Tam-R and putatively associated with EGF-induced ER binding sites.

Time to response, time to complete response, and rate of complete response to endocrine treatment +/- Dox in xenografts of MCF7 Tet-off Vector Control clone 3.

Genes down-regulated in Tam-R and putatively associated with EGF-induced ER binding sites (genes containing an AP-1 motif are shown in bold)

Full gel

Legend for supplementay figures

Supplemental Figure 1: Structures of clinically relevant compounds used in this study. Supplemental Figure 2: Cells expressing both the ERWT and ERmuts have similar pharmacological responses to antiestrogens when compared to cells only expressing ERWT. Supplemental Figure 3: Albeit differences in clonal variability, there is indistinguishable differences in T47D proliferation in response to antiestrogens. Supplemental Figure 4: Downregulation of ER in MCF7B cells is unaffected by the presence of the mutant receptor. Supplemental Figure 5: Validation of ER mutation status in engineered cell lines. Supplemental Figure 6: Protein levels of transfected constructs in SKBR3 cells are consistent with MCF7 expression level. Supplemental Figure 7: ER expression in MCF7I cells is titratable with doxycycline treatment. Supplemental Figure 8: Overexpression of ERY537S or ERD538G, not ERWT, in MCF7I cells changes ligand potency. Supplemental Figure 9: Western blot validation of relative ER expression levels in SKBR3. Supplemental Figure 10: Agonist activation of relative expression levels of ERWT and ERmuts in SKBR3 cells. Supplemental Figure 11: In-Cell Western validation of ER expression levels for siRNA knockdown and cDNA overexpression in MCF7i. Supplemental Figure 12: Agonist activation of relative expression levels of ERWT and ERmuts in MCF7i cells. Supplemental Figure 13: The altered potency of fulvestrant against the ERmuts on endogenous ER target genes is only evident when expressed at a level higher than the WT receptor.

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

Purpose: We undertook this study to determine the prevalence of estrogen receptor (ER) α (ESR1) mutations throughout the natural history of hormone-dependent breast cancer and to delineate the functional roles of the most commonly detected alterations.Experimental Design: We studied a total of 249 tumor specimens from 208 patients. The specimens include 134 ER-positive (ER+/HER2−) and, as controls, 115 ER-negative (ER−) tumors. The ER+ samples consist of 58 primary breast cancers and 76 metastatic samples. All tumors were sequenced to high unique coverage using next-generation sequencing targeting the coding sequence of the estrogen receptor and an additional 182 cancer-related genes.Results: Recurring somatic mutations in codons 537 and 538 within the ligand-binding domain of ER were detected in ER+ metastatic disease. Overall, the frequency of these mutations was 12% [9/76; 95% confidence interval (CI), 6%–21%] in metastatic tumors and in a subgroup of patients who received an average of 7 lines of treatment the frequency was 20% (5/25; 95% CI, 7%–41%). These mutations were not detected in primary or treatment-naïve ER+ cancer or in any stage of ER− disease. Functional studies in cell line models demonstrate that these mutations render estrogen receptor constitutive activity and confer partial resistance to currently available endocrine treatments.Conclusions: In this study, we show evidence for the temporal selection of functional ESR1 mutations as potential drivers of endocrine resistance during the progression of ER+ breast cancer. Clin Cancer Res; 20(7); 1757–67. ©2014 AACR.

This file contains eight supplementary figures depicting the experiments validated in different cell lines and cohorts, as well as the results supportive to the main text.

Supplemental Figure Legends

QC analysis

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.

Cox proportional hazards analyses

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

Resistance to cancer treatment can be driven by epigenetic reprogramming of specific transcriptomes in favor of the refractory phenotypes. Here we discover that tamoxifen resistance in breast cancer is driven by a regulatory axis consisting of a master transcription factor, its cofactor, and an epigenetic regulator. The oncogenic histone methyltransferase EZH2 conferred tamoxifen resistance by silencing the expression of the estrogen receptor α (ERα) cofactor GREB1. In clinical specimens, induction of DNA methylation of a particular CpG-enriched region at the GREB1 promoter negatively correlated with GREB1 levels and cell sensitivity to endocrine agents. GREB1 also ensured proper cellular reactions to different ligands by recruiting distinct sets of ERα cofactors to cis-regulatory elements, which explains the contradictory biological effects of GREB1 on breast cancer cell growth in response to estrogen or antiestrogen. In refractory cells, EZH2-dependent repression of GREB1 triggered chromatin reallocation of ERα coregulators, converting the antiestrogen into an agonist. In clinical specimens from patients receiving adjuvant tamoxifen treatment, expression levels of EZH2 and GREB1 were correlated negatively, and taken together better predicted patient responses to endocrine therapy. Overall, our work suggests a new strategy to overcome endocrine resistance in metastatic breast cancer by targeting a particular epigenetic program.Significance: This study suggests a new strategy to overcome endocrine resistance in metastatic breast cancer by targeting a particular epigenetic program defined within. Cancer Res; 78(3); 671–84. ©2017 AACR.

Supplemental Materials & Methods and Figures 1-6. Sup. Figure 1. The effect of antiestrogens against ER+ BrCa spheroid cultures is attenuated by BMSCs. Sup. Figure 2. BMSCs attenuate the antiestrogen response of BrCa xenografts in vivo. Sup. Figure 3. BMSCs attenuate hormone-induced gene expression in cancer spheroids with varying effect on HR expression. Sup. Figure 4. BMSCs paracrine effect induces HT resistance in PrCa/BrCa 3D spheroids via IL-6 secretion or in IL-6-independent manner. Sup. Figure 5. Transcriptional signatures of genes downregulated (log2FC< -1) or upregulated (log2FC > 1) in MCF7 spheroids cocultured (8 days) with HS5 (vs. monoculture) do not correlate with relapse-free survival of 342 ER-negative BrCa patients (stratified using the upper tertile of the respective signature as cutoff). Sup. Figure 6. BrCa spheroids in coculture with BMSC acquire increased dependence on growth factor receptor and downstream signaling.

PFS and OS of PAM50 and OncotypeDx risk categories

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.