Endocrine-resistant Breast Cancer Research Articles

Rezultati multicentričnog testiranja somatskih mutacija gena PIK3CA kod uznapredovalog luminalnog raka dojke s negativnim HER2 receptorom

Aim: Activating somatic mutations in p110α catalytic subunit of PIK3-kinase (PIK3CA) are present in about 40% of breast cancers (BC), and are involved in oncogenesis and cancer growth. PIK3CA inhibitor therapy is approved for patients with advanced HR+/HER2- endocrine-resistant BC with somatic PIK3CA mutations. Such targeted therapy improved patient disease-free survival. Method: Analyses of PIK3CA gene mutations were performed in five University Hospital Centers in Croatia. Qualitative detection of PIK3CA gene mutations was performed on DNA extracted from 507 breast cancer samples, by the real-time PCR method using Cobas® PIK3CA mutation test. Results: PIK3CA gene mutations were detected in 209 (42.2%) out of 495 successfully analyzed cases. In 12 cases (2.4%), the tumor tissue was not of sufficient quality for analysis. Most mutations were detected in the helical (41.2%) and kinase domains (46.9%) of the PIK3CA protein. The most frequently detected mutations were H1047L/R/Y (46.4%), E545A/D/G/K (26.3%) and E542K (13.9%), followed by N345K (8.1%). PIK3CA mutations were detected in 44.8% of primary BC cases and in 39.6% of metastatic lesions (χ2=0.647; P=0.421). The age of the tissue did not significantly affect the percentage of detected mutations (χ2=0.543; P =0.461). Institutions differed in the number of analyzed cases, but not in the percentage of detected PIK3CA mutations (χ2=6.23; df=4; P=0.183) nor in the frequency of the most common mutations (χ2=2.65; df=4; P=0.618). Conclusion: The frequencies of PIK3CA mutations correspond to those reported in the literature. We have shown that the tissue of primary BC as well as metastatic lesions are suitable for analysis, and that the age of the tissue is not a significant obstacle in the analysis.

Inhibition of FASN as a potential treatment of advanced endocrine therapy-resistant breast cancer.

e13077 Background: The past decade has seen significant advancement in increasing survival estrogen receptor alpha (ERα) positive breast cancer. The use of selective estrogen receptor down-regulators and modulators (SERMs) and cyclin-dependent kinase inhibitors aided to extend overall survival of breast cancer patients. However, resistance to endocrine therapy is a common occurrence and patients will eventually succumb to their disease. Fatty acid synthase (FASN), a key enzyme in lipid biosynthesis, is overexpressed in more aggressive and therapy-resistant tumors, including breast cancers. FASN inhibitor, TVB-2640, has been evaluated in multiple tumor cell lines and in a phase 1 clinical study, and showed partial responses in 5 patients and multiple patients with prolonged stable disease (≥16 weeks). Methods: We generated therapy-resistant cells by long term exposure to tamoxifen (MCF7/TamR cells), and fulvestrant (MCF7/FR cells). Palbociclib-resistant (MCF7/RB1Crispr and ZR75/RB1Crispr) cells were generated through CRISPR/Cas9 knockout of the retinoblastoma (RB) gene. We assessed the impact of TVB-3166 inhibitor in these cells on proliferation, viability, cell cycle, apoptosis, ERα expression in patient derived explants, and tumor growth in xenografts. RNA sequencing of tamoxifen- and fulvestrant-resistant cells was performed to investigate gene expression. Subcellular localization of ERα was assessed using subcellular fractionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels were analyzed by western blot after treatment with TVB with or without the addition of palmitate or BAPTA. Results: TVB treatment leads to a marked inhibition of proliferation in tamoxifen-, fulvestrant- and palbociclib-resistant cells compared to the parental cells. RNA sequencing of explants of patients with ERα positive disease showed down regulation of ESR1 related genes and genes involved in invasiveness. RNA sequencing of fulvestrant-resistant cells showed that treatment with TVB results in down regulation of EMT and E2F target genes, cholesterol homeostasis genes and mTORC1 signaling. Additionally, TVB significantly inhibited tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition significantly reduced ERα levels most prominently in endocrine resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERα expression upon TVB treatment is mediated through the induction of endoplasmic reticulum stress. Conclusions: Our preclinical data provide evidence that FASN inhibition presents a promising therapeutic strategy for treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine resistant breast cancer should be considered.

Discovery of novel covalent selective estrogen receptor degraders against endocrine-resistant breast cancer

Endocrine-resistance remains a major challenge in estrogen receptor α positive (ERα+) breast cancer (BC) treatment and constitutively active somatic mutations in ERα are a common mechanism. There is an urgent need to develop novel drugs with new mode of mechanism to fight endocrine-resistance. Given aberrant ERα activity, we herein report the identification of novel covalent selective estrogen receptor degraders (cSERDs) possessing the advantages of both covalent and degradation strategies. A highly potent cSERD 29c was identified with superior anti-proliferative activity than fulvestrant against a panel of ERα+ breast cancer cell lines including mutant ERα. Crystal structure of ERα‒29c complex alongside intact mass spectrometry revealed that 29c disrupted ERα protein homeostasis through covalent targeting C530 and strong hydrophobic interaction collied on H11, thus enforcing a unique antagonist conformation and driving the ERα degradation. These significant effects of the cSERD on ERα homeostasis, unlike typical ERα degraders that occur directly via long side chains perturbing the morphology of H12, demonstrating a distinct mechanism of action (MoA). In vivo, 29c showed potent antitumor activity in MCF-7 tumor xenograft models and low toxicity. This proof-of-principle study verifies that novel cSERDs offering new opportunities for the development of innovative therapies for endocrine-resistant BC.

Discovery of a Novel Class of PROTACs as Potent and Selective Estrogen Receptor α Degraders to Overcome Endocrine-Resistant Breast Cancer In Vitro and In Vivo.

The estrogen receptor (ER) is a well-established target for endocrine therapies of ER-positive breast cancer (ER+ BC), but endocrine resistance limits the efficacy of clinical drugs. Using proteolysis targeting chimera (PROTAC) technology to degrade ERα may be an effective alternative to endocrine therapies. Herein, we disclose a novel series of potent and selective ERα PROTACs based on an oxabicycloheptane sulfonamide (OBHSA) scaffold, with no associated ERβ degradation. These PROTACs showed significant antiproliferation and ERα degradation activities against a broad spectrum of ER+ BC cells including tamoxifen-resistant and ERα mutant cell lines. Genomics analysis confirmed that these PROTACs inhibited the nascent RNA synthesis of ERα target genes and impaired genome-wide ERα binding. Compound ZD12 exhibited excellent antitumor potency and ERα degradation activity in both tamoxifen-sensitive and -resistant BC mice models, which are superior to fulvestrant. This study demonstrates the potential of these PROTACs as novel drug candidates for endocrine-resistant BC treatment.

FDXR drives primary and endocrine-resistant tumor cell growth in ER+ breast cancer via CPT1A-mediated fatty acid oxidation.

The majority of breast cancers (BCs) expressing estrogen receptor (ER) have shown endocrine resistance. Our previous study demonstrated that ferredoxin reductase (FDXR) promoted mitochondrial function and ER+ breast tumorigenesis. But the underlying mechanism is not clear. Liquid chromatography (LC) tandem mass spectrometry (MS/MS)-based metabolite profiling was utilized to reveal the metabolites regulated by FDXR. RNA microarray was utilized to determine the potential downstream targets of FDXR. Seahorse XF24 analyzer was performed to analyze the FAO-mediated oxygen consumption rate (OCR). Q-PCR and western blotting assays were used to measure expression levels of FDXR and CPT1A. MTS, 2D colony formation and anchorage-independent growth assays were used to evaluate the effects of FDXR or drug treatments on tumor cell growth of primary or endocrine-resistant breast cancer cells. We found that depletion of FDXR inhibited fatty acid oxidation (FAO) by suppressing CPT1A expression. Endocrine treatment increased the expression levels of both FDXR and CPT1A. Further, we showed that depletion of FDXR or FAO inhibitor etomoxir treatment reduced primary and endocrine-resistant breast cancer cell growth. Therapeutically, combining endocrine therapy with FAO inhibitor etomoxir synergistically inhibits primary and endocrine-resistant breast cancer cell growth. We reveal that the FDXR-CPT1A-FAO signaling axis is essential for primary and endocrine-resistant breast cancer cell growth, thus providing a potential combinatory therapy against endocrine resistance in ER+ breast cancer.

Structure-guided identification of novel dual-targeting estrogen receptor α degraders with aromatase inhibitory activity for the treatment of endocrine-resistant breast cancer

Drug resistance is a major challenge in conventional endocrine therapy for estrogen receptor (ER) positive breast cancer (BC). BC is a multifactorial disease, in which simultaneous aromatase (ARO) inhibition and ERα degradation may effectively inhibit the signal transduction of both proteins, thus potentially overcoming drug resistance caused by overexpression or mutation of target proteins. In this study, guided by the X-ray structure of a hit compound 30a in complex with ER-Y537S, a structure-based optimization was performed to get a series of multiacting inhibitors targeting both ERα and ARO, and finally a novel class of potent selective estrogen receptor degraders (SERDs) based on a three-dimensional oxabicycloheptene sulfonamide (OBHSA) scaffold equipped with aromatase inhibitor (AI) activity were identified. Of these dual-targeting SERD-AI hybrids, compound 31q incorporating a 1H-1,2,4-triazole moiety showed excellent ERα degradation activity, ARO inhibitory activity and remarkable antiproliferative activity against BC resistant cells. Furthermore, 31q manifested efficient tumor suppression in MCF-7 tumor xenograft models. Taken together, our study reported for the first time the highly efficient dual-targeting SERD-AI hybrid compounds, which may lay the foundation of translational research for improved treatment of endocrine-resistant BC.

Abstract 4852: Metabolomic rewiring in endocrine therapy resistant estrogen receptor positive breast cancer

Abstract Breast cancer (BC) is one of the most commonly diagnosed cancers worldwide and the most common in women in the United States. The majority of the BC are estrogen receptor alpha positive (ER+) and are likely to respond to endocrine therapy. Tamoxifen and fulvestrant are the most widely used hormonal treatments for ER+ BC. However, nearly half of patients receiving endocrine therapy suffer from the risk of recurrence due to either intrinsic or acquired resistance. Thus, it is vital to understand the mechanisms of resistance to endocrine therapy in ER+ BC. Metabolic reprogramming is one of the significant hallmarks of cancer and it is now known that many tumors utilize mitochondrial oxidative phosphorylation (OXPHOS) for their energy needs. Our lab previously reported that metastatic triple-negative breast cancer (TNBC), which does not express ER, has a high energy dependency on mitochondrial fatty acid beta-oxidation (FAO). In this project, we developed a gene signature by integrating the transcriptomic, metabolomic, and lipidomic data from FAO rate-limiting gene (CPT1) modulated TNBC cells. Our signature represented the FAO-regulated gene set relevant to metabolic or lipidomic changes. We discovered a significant reliance on this TNBC signature in endocrine-resistant ER+ BC. Clinical data suggests that our gene signature predicts the survival of endocrine-treated ER+ BC patients. Though increased fatty acid consumption is reported in tamoxifen-resistant cells, the role of FAO in endocrine treatment resistance is largely unclear. We then analyzed the metabolic reprogramming in ER+ BC cell lines after tamoxifen or fulvestrant therapy. Molecular, genetic, and metabolic analyses suggested that endocrine therapy-induce AMPK-FAO-OXPHOS signal activation in ER+ BC cells. Furthermore, the knockdown of CPT1, the rate-limiting enzyme of FAO, or treatment with FAO inhibitors significantly enhanced the response to endocrine therapies. We have also reported that FAO induces the autophosphorylation of the c-Src proto-oncogene in TNBC tumors. Thus, we analyzed this phenomenon in the endocrine-resistant ER+ BC cells. As expected, endocrine therapy-induced FAO activated the Src pathway in ER+ BC also. Moreover, in vitro, and in vivo studies confirmed that endocrine-resistant ER+ BC cells have increased sensitivity to FAO, OXPHOS, and Src inhibitors. Finally, analysis of clinical data suggests that low expression of FAO rate-limiting genes in the ER+ primary tumors have better recurrence-free and distant metastasis-free survival after endocrine therapy. Overall, our findings suggest that the gene signature generated from FAO-modulated TNBC cells predicts response to endocrine therapy in ER+ BC. Moreover, metabolic reprogramming in endocrine-resistant ER+ tumors induce FAO, OXPHOS, and Src pathways, providing potential targets to overcome endocrine-resistance ER+ BC patients. Citation Format: Songyeon Ahn, Junhyoung Park, Sandra L. Grimm, Badrajee WB Piyarathna, Nagireddy Putluri, Gokul Das, Cristian Coarfa, Benny A. Kaipparettu. Metabolomic rewiring in endocrine therapy resistant estrogen receptor positive 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 4852.

Abstract 3889: The role of CXCL1 in crosstalk between breast cancer cells with ESR1 mutations and lymphatic endothelial cells

Abstract Various endocrine therapies have been developed such as selective estrogen receptor modulation (SERM), selective estrogen receptor downregulator (SERD) and ligand deprivation using aromatase inhibitors (AI). Tamoxifen, fulvestrant, and AIs are the most used adjuvant treatment for pre- and postmenopausal women with early-stage estrogen receptor positive breast cancer. Tamoxifen and AIs have widespread anti-neoplastic and chemopreventive effects, yet despite this, many breast cancers that are initially receptive ultimately become resistant and recur. There are many known underlying molecular processes that result in resistance, but no overarching pattern has yet been identified. Our previous research demonstrates that the crucial molecular and cellular players secreted from a crosstalk between stromal cells and breast cancer cells can control the development of the tumor and the metastatic process in breast cancer. Breast cancer metastasis involves lymphatic dissemination in addition to hematogenous spreading. Although stromal lymphatic vessels (LVs) serve as initial metastatic routes, roles of organ-residing LVs are underinvestigated. In this report, we demonstrated that crosstalk between ESR1 mutant cells (Y537S and D538G), and lymphatic endothelial cells (LECs), a component of lymphatic vessels, promoted ESR mutant cell growth and migration. To identify a critical secreted factor in this crosstalk, we performed the cytokine array in secretome of crosstalk between ESR1 mutant cells and LECs, and found that CXCL1 is highly secreted from LECs. Knockdown of CXCR2 in ESR1 mutant cells attenuated ESR1 cell migration in the conditioned media of LECs crosstalked with ESR1 mutant cells. To investigate a potential impact given the current clinical landscape of endocrine resistant breast cancer with ESR1 mutations, we performed a cell viability assay and migration assay using reparixin, CXCR1/2 inhibitor in co-culture with ESR1 mutant cells and LECs. Significantly, reparixin decreased ESR1 mutant cell viability and migration compared to vehicle. We found that the combination treatment of reparixin and Palbociclib and ribociclib significantly inhibited ESR1 mutant cell growth and migration compared the single treatment. These findings implicate CXCL1-CXCR1/2 signaling as a critical event in the growth of breast cancer with ESR1 mutation and metastasis. Therefore, we have provided evidence that supports the hypothesis that functional inhibition of the CXCL1 and CDK4/6 signaling pathway has the potential to circumvent endocrine resistant tumor growth and metastasis. Citation Format: Daniel Galke, Kideok Jin. The role of CXCL1 in crosstalk between breast cancer cells with ESR1 mutations and lymphatic endothelial cells. [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 3889.

Abstract 2051: MDN1, a novel potential early biomarker for endocrine resistant breast cancer

Abstract Midasin (MDN1) is a chaperone protein required for maturation and nuclear export of pre-ribosomal RNA. Recent investigation regarding the mutational status of MDN1 in breast cancer (BC) revealed an increased tumor mutational burden in BC patients. Interestingly, our previously published work demonstrated significantly high MDN1 protein levels in AI-resistant mammospheres. Therefore, we investigated the overall status of MDN1 in the TCGA database in breast cancer tumors and identified MDN1 expression in breast cancer cells that were sensitive or resistant to AIs. The purpose of this study was to evaluate whether there is an association between MDN1 expression or mutation status to examine if MDN1 functions as an early high-risk indicator for breast cancers resistant to AIs or endocrine therapy. Using the cBioportal for Cancer Genomics, in silico analysis was conducted and BC patients with altered MDN1 status demonstrated several clinical attributes that were significantly associated with higher mutation counts (Kruskal Wallis test, p value = 1.41 × 10-10 and q value = 4.11 × 10-9); MSI sensor score, (p value = 2.74 × 10-5 and q value = 2.27 × 10-4); and Fraction Altered Genome score (p value = 2.37 × 10-3 and q value = 0.011). Next, aneuploidy in breast cancer significantly evolved and was positively correlated with higher MDN1 expression (Spearman: 0.64, p value = 3.92 × 10-122). Compared to the other BC subtypes, MDN1 was significantly elevated in basal breast cancer (Chi square test p value < 10.0 × 10-10, q value = 4.11 × 10-9) and significantly higher in deceased versus living BC patients. The DepMap breast cancer cell line database analysis also revealed significantly increased MDN1 expression in both ER+/HER2- cell lines (p value = 7.93 × 10-3; 12 cell lines) and in ER-/HER2- cell lines (p value = 2.88 × 10-2; 31 cell lines). Primary and metastatic BC cell lines were compared and MDN1 expression was significantly higher in metastatic BC cells (p value = 8.04 × 10-5; 33 metastatic cell lines). Likewise, AI-resistant cell lines exhibited altered MDN1 expression compared to their sensitive counterparts. Additionally, there was a statistically significant association between higher ER copy number and increased MDN1 expression in BC cell lines (p value = 1.44 × 10-3; 61 cell lines). Further in-depth analysis of proteins that were coexpressed in MDN1-altered tumors revealed a panel of genes (MSH2, MSH6, RBM15, FOXM1,CCNE2, and CHEK1) that could represent a signature of early markers of AI-resistant BC since there was a positive correlation between MDN1 expression and associated pathways. In conclusion, increased expression of MDN1 in BC tumors or cells may serve as preliminary marker to predict resistance to endocrine therapy due to its significant association with genes such as MSH2, MSH6 and FOXM1 that are strongly established as indicators of endocrine resistance. This work was supported by NIH grant # SC1GM126617 and U54MD007582. Citation Format: Afia Ohemeng, Akash Gupta, Bipika Banjara, Mounika M. Pamukuntla, Manasa Kotina, A Michael Davidson, Syreeta L. Tilghman. MDN1, a novel potential early biomarker for endocrine resistant 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 2051.

Abstract 3875: Evaluation of resistance mechanisms to ARV471, an ER-targeted PROTAC

Abstract Estrogen receptor alpha (ER) is a ligand activated transcription factor and a driver of ER+ breast cancer. The discovery of activating mutations in ER (gene name ESR1), in patients with advanced endocrine-resistant ER+ breast cancer, has ignited a large pharmaceutical effort to develop next-generation ER antagonists that also target the mutant protein. Here, we profiled the potency and selectivity of many next-generation ER antagonists currently in clinical trials, including ER-targeted proteolysis-targeting chimeras (PROTACs), selective ER modulators (SERMs), and selective ER degraders (SERDs). To contextualize these antagonists, we evaluated their activity against the only FDA-approved SERD, Fulvestrant, at the level of chromatin, transcriptome, proteome, and cell viability, using cells expressing wild-type ER as well as activating mutant ER (ERY537S and ERD538G). Overall, we find that many of the novel oral SERDs are potent ER antagonists, including against mutant ER. To anticipate resistance mechanisms to these next-generation ER antagonists, we developed a strategy initially focused on ARV471 (the most clinically advanced ER PROTAC), with two approaches. First, we performed whole-genome CRISPR/Cas9-knockout screening in ER+ breast cancer cell lines treated with ARV471. Among the enriched gRNAs were PTEN, NF2, and CRBN. Loss of PTEN and NF2 results in activation of the PI3K and MAPK pathways, respectively, and CRBN is the ubiquitin E3 ligase required for ER protein degradation by ARV471. PTEN and NF2 are known mechanisms of endocrine resistance but it is not yet known if CRBN loss in response to PROTACs is a predominant clinical resistance mechanism. Therefore, in a second approach we generated ARV471 resistant breast cancer cell lines through long term exposure (LTE-ARV471). These cells developed an ER-independent resistance mechanism, with decreased ER expression and increased expression of pathways associated with Ras/MAPK and EMT. Therefore, as more potent ER degraders enter the clinic, such as PROTACs and oral SERDs, it may be important to monitor resistance mechanisms that involve loss of ER. Currently, we are following up our findings with LTE-ARV471 cells and performing CRISPR screening to identify targetable dependencies and combination therapies for endocrine resistant breast cancer. Citation Format: Delia Margaret Friel, Zachary Sandusky, Yijia Jiang, Xintao Qiu, Rong Li, Henry Long, Rinath Jeselsohn, Myles Brown. Evaluation of resistance mechanisms to ARV471, an ER-targeted PROTAC. [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 3875.

Pembrolizumab in Lymphopenic Metastatic Breast Cancer Patients Treated with Metronomic Cyclophosphamide: A Clinical and Translational Prospective Study.

Metastatic endocrine-resistant breast cancer (MBC) is a disease with poor prognosis and few treatment options. Low lymphocyte count is associated with limited overall survival. In a prospective cohort of lymphopenic patients with HER-2 negative MBC, we assessed the clinical and biological impact of pembrolizumab combined with metronomic cyclophosphamide. This multicenter Phase II study evaluated the safety and clinical activity of pembrolizumab (intravenous (IV), 200mg, every 3 weeks) combined with metronomic cyclophosphamide (50mg/day, per os) in lymphopenic adult patients with HER2-negative MBC previously treated by at least one line of chemotherapy in this setting according to a Simon's minimax two-stage design. Blood and tumor samples were collected to assess the impact of the combined treatment on circulating immune cells and the tumor immune microenvironment through multiparametric flow cytometry and multiplex immunofluorescence analyses. Primary endpoint was the clinical benefit rate at 6 months of treatment (CBR-6M). Secondary endpoints were objective response rate (ORR), duration of response, progression free survival (PFS), and overall survival (OS). Two out of the twenty treated patients presented clinical benefit (one Tumor Mutational Burden (TMB)-high patient with complete response (CR) and one patient with objective response (OR) per Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST V1.1) associated with a strong increase of cytokine-producing and proliferating CD4+ T cells and higher CD8+ T cells to macrophage ratios in the tumor. This impact on CD4+ and CD8+ T cell polyfunctionality was still observed more than one year for the patient with CR. A decreased in their absolute number of CD4+ and CD8+ memory T cells was observed in other patients. Pembrolizumab combined with metronomic cyclophosphamide was well tolerated, and displayed limited anti-tumoral activity in lymphopenic MBC. Correlative translational data of our trial advocates for additional studies with other chemotherapy combinations.

A phase I trial of palbociclib and bosutinib with fulvestrant in patients with metastatic hormone receptor positive and HER2 negative (HR+ HER2-) breast cancer refractory to an aromatase inhibitor and a CDK4/6 inhibitor

Standard treatment for metastatic hormone positive (HR+) breast cancer includes a combination of a CDK4/6 inhibitor and antiestrogen therapy. Despite durable responses, eventual endocrine resistance results in disease progression. The Src/Abl pathway has been shown to mediate endocrine resistance in breast cancer, thus providing a promising target for novel therapies. Bosutinib is a tyrosine kinase inhibitor that targets the Src/Abl pathway, which has been studied in hematologic malignancies. Preclinical data suggests that the addition of bosutinib to a CDK4/6 inhibitor and antiestrogen therapy has the potential to reverse endocrine resistance.This is a phase I, single arm, open-label clinical trial in which we evaluate the combination of palbociclib and fulvestrant with bosutinib in metastatic HR+ breast cancer. Patients with confirmed advanced HR+/HER2- breast cancer who have received no more than three lines of chemotherapy and have progressed on at least one aromatase inhibitor and one CDK4/6 inhibitor will be enrolled. Participants will be given a combination of palbociclib, fulvestrant and bosutinib over 28-day cycles. The primary objective of this study is to assess the safety and tolerability of bosutinib in combination with palbociclib and fulvestrant in the study population. Secondary objectives are to 1) determine the anti-tumor effect of this therapeutic combination by assessing overall response rate (ORR) and clinical benefit rate (CBR) after 6 months of treatment, 2) to determine the clinical pharmacology parameters of bosutinib in this regimen, and 3) to build a tissue repository at Georgetown Lombardi Comprehensive Cancer Center for further translational study.

Abstract PD10-03: PD10-03 Super-enhancer-oriented integrative bioinformatics identifies aberrant KLF4 signaling in endocrine-resistant breast cancer (BC)

Abstract Background: Recent studies suggest that enhancer reprogramming underlies heterogeneity and disease progression in estrogen receptor-positive (ER+) BC. Cell-type/state specific transcription is governed by high-order assemblies of master transcription factors (TFs) and epigenetically defined regulatory regions including super-enhancers (SEs). We previously showed that aberrant activation of the pioneer TF FOXA1 promotes enhancer and transcriptional reprogramming in endocrine-resistant BC, involving the ER and the AP-1 FRA1 and c-JUN TFs. As SEs maintain a robust cell-type/state specific core transcriptional regulatory circuitry (CRC) in developmental and tumorigenic processes, we sought to identify key additional TFs in SE/FOXA1-driven CRCs in endocrine resistance, which could serve as attractive therapeutic targets. Methods: TF binding motif at the shared SEs (mapped by H3K27ac ChIP-seq) between MCF7-parental (P) cells with ectopic FOXA1 overexpression (OE) and the endogenous FOXA1-amplifed tamoxifen-resistant (TamR) cells was analyzed by HOMER. ER-bound SEs distinguishing TamR vs. P cells were defined by integrating the SEs with our prior ER ChIP-seq data (PMID 28507152). KLF4 motif within these ER-bound SEs was scanned using FIMO and linked to nearby genes by intersection with the previously defined promoter-tethered regions (PTRs) (PMID 24141950). Differential gene expression in MCF7-TamR cells upon KLF4 knockdown (KD) by 3 unique siRNAs was analyzed using limma from edgeR. The biological and clinical significance of the KLF4-dependent genes was analyzed using Gene Ontology and survival modeling with METABRIC and the ER+ metastatic BC cohort (SABCS19-GS2-02). Cell migration was assessed by the wound-healing assay. Results: We identified KLF4 among the top enriched TF binding motifs at the shared SEs in FOXA1-overexpressing MCF7-P cells and the FOXA1-amplified TamR cells. Analysis of our prior RNA-seq data of MCF7-P and TamR cells upon OE or siRNA KD of FOXA1, FRA1, or c-JUN (PMIDs 27791031, 31826955, 32424275, SABCS21-PD1-05) revealed KLF4, the Yamanaka factor for induced pluripotent stem cells, as a common target activated by the FOXA1/FRA1/c-JUN axis. We next identified 44 genes commonly down-regulated upon KLF4 KD in the MCF7-TamR cells. This KLF4-dependent 44-gene set was enriched in biological processes of embryonic development and tumor progression, preferentially dependent on ER in MCF7-TamR vs. P cells, highly elevated in ER+ metastases vs. primary tumors, and associated with poor outcome in ER+ BC treated with endocrine therapy. KLF4 KD, using the 2 siRNAs that generated similar pathway perturbations in MCF7-TamR cells, reduced TamR cell migration. Notably, among the genes co-dependent on KLF4 and ER in TamR cells, PYGB was the only gene with a PTR residing in an ER-bound SE established in TamR but not P cells. Glycogen phosphorylase B, encoded by PYGB, is the rate-limiting enzyme in glycogen degradation and plays a role in the progression of various tumors. Expression of KLF4, FOXA1, and FRA1 are commonly activated during the differentiation of human embryonic stem cells into foregut endoderm and in the inner core of fibroblasts of first-trimester human placenta villi, suggesting a unique role of KLF4 in mediating lineage-specific CRC, possibly by engaging PYGB and the glycogen metabolic pathway in advanced ER+ disease. Conclusions: Using SE-oriented integrative bioinformatics, we identified KLF4 as a potential novel target in the FOXA1/AP-1 transcriptional axis. As KLF4 binding motif resides in the unique ER-bound SEs of TamR cells, KLF4 likely forms an auto-regulated loop amplifying CRC in transcriptional reprogramming, among which the PYGB/glycogen metabolic pathway merits further investigation in endocrine-resistant ER+ disease. Citation Format: Chia Chia Liu, Lanfang Qin, Shanunak Sathe, Sarmistha Nanda, Jamunarani Veeraraghavan, Ofir Cohen, Nikhil Wagle, Mothaffar Rimawi, C. Kent Osborne, Xiaoyong Fu, Rachel Schiff. PD10-03 Super-enhancer-oriented integrative bioinformatics identifies aberrant KLF4 signaling in endocrine-resistant breast cancer (BC) [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 PD10-03.

Apoptosis-modulatory miR-361-3p as a novel treatment target in endocrine-responsive and endocrine-resistant breast cancer.

Breast cancer (BC) is the most diagnosed cancer in women worldwide. In estrogen receptor (ER)-positive disease, anti-estrogens and aromatase inhibitors (AI) improve patient survival; however, many patients develop resistance. Dysregulation of apoptosis is a common resistance mechanism; thus, agents that can reinstate the activity of apoptotic pathways represent promising therapeutics for advanced drug-resistant disease. Emerging targets in this scenario include microRNAs (miRs). To identify miRs modulating apoptosis in drug-responsive and -resistant BC, a high-throughput miR inhibitor screen was performed, followed by high-content screening microscopy for apoptotic markers. Validation demonstrated that miR-361-3p inhibitor significantly increases early apoptosis and reduces proliferation of drug-responsive (MCF7), plus AI-/antiestrogen-resistant derivatives (LTED, TamR, FulvR), and ER- cells (MDA-MB-231). Importantly, proliferation-inhibitory effects were observed in vivo in a xenograft model, indicating the potential clinical application of miR-361-3p inhibition. RNA-seq of tumour xenografts identified FANCA as a direct miR-361-3p target, and validation suggested miR-361-3p inhibitor effects might be mediated in part through FANCA modulation. Moreover, miR-361-3p inhibition resulted in p53-mediated G1 cell cycle arrest through activation of p21 and reduced BC invasion. Analysis of publicly available datasets showed miR-361-3p expression is significantly higher in primary breast tumours vspaired normal tissue and is associated with decreased overall survival. In addition, miR-361-3p inhibitor treatment of BC patient explants decreased levels of miR-361-3p and proliferation marker, Ki67. Finally, miR-361-3p inhibitor showed synergistic effects on BC growth when combined with PARP inhibitor, Olaparib. Together, these studies identify miR-361-3p inhibitor as a potential new treatment for drug-responsive and -resistant advanced BC.

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 P2-24-03: Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation

Abstract Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation Sayantani Sarkar Bhattacharya1, Taylor L. Witter1, Anh Q. T. Cong1, Elizabeth Bruinsma2, Swaathi Jayaraman2, Matthew P. Goetz2, John R. Hawse1, and Matthew J. Schellenberg1 1Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN USA 55905 2Department of Oncology, Mayo Clinic, Rochester, MN USA 55905 Background: Z-Endoxifen (ENDX), the active metabolite of tamoxifen (TAM) and a selective estrogen receptor modulator (SERM), exhibited high antitumor activity in endocrine-resistant hormone receptor-positive breast and other gynecologic cancer. ENDX has also been shown to be a protein kinase C (PKC) inhibitor. PKCs participate in diverse cellular functions and their activity is often elevated in breast tumors. Guided by mechanistic insights from our recently determined crystal structure of PKCβI, we sought to determine the effects of ENDX on PKCβI in a breast cancer cell line model. Methods: To determine how ENDX regulates PKC activity, we used a Z’LYTE kinase activity assay. This Fluorescence Resonance Energy Transfer (FRET) based biochemical method can detect differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. We probed changes in activity for conventional and novel PKCs, as well as the purified catalytic domain of conventional PKCs in vitro. Alongside, as kinase activity of PKC relies on its spatial assembly, therefore we studied its intracellular localization using live cell confocal imaging. MCF7 cells expressing YFP-tagged PKCβI were grown in a glass bottom chamber and treated with ENDX and other modulators for relevant time and doses for this study. Images were taken using Zeiss LSM 780 confocal laser scanning microscope and analyzed in Zeiss-ZEN microscope software and GraphPad Prism 9. Results: Our data from an in vitro kinase assay indicates that ENDX inhibits the kinase activity of conventional (PKCβI) and novel (PKC𝛿) PKC isoforms with a similar IC50, however PKCβI catalytic domain is less sensitive to ENDX. We also identified a multi-domain mechanism of PKC inhibition through an allosteric inhibitory mechanism. Our live cell imaging study demonstrated that ENDX promotes the recruitment of PKCβI to the cell membrane in both a dose and time-dependent manner. Moreover, this translocation can also be mitigated by co-treatment with inhibitors of the PKC-regulator PHLPP phosphatases. Conclusion: Taken together, these results suggest the allosteric effects of ENDX trigger PKCβI recruitment to the cell membrane, yet since ENDX also inhibits kinase activity it suggests that ENDX triggers a non-productive interaction with the enzyme and "breaks" the well-known mechanism of PKC activation upon binding the cell membrane. Furthermore, ENDX is likely to trigger dephosphorylation and ultimately degradation, suggesting that ENDX represents a new mechanistic basis for targeting and downregulating PKC in cancer cells. Hence, the current study provides an integrated pattern of highly specific treatments regimen that can exploit PKCβI as a repurposing clinical target in breast cancer. Citation Format: Sayantani Sarkar Bhattacharya, Taylor L. Witter, Anh T. Cong, Elizabeth S. Bruinsma, Swaathi Jayaraman, Matthew P. Goetz, John Hawse, Matthew Schellenberg. Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation [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 P2-24-03.

Abstract P3-11-07: Inhibition of FASN as a potential treatment of advanced endocrine therapy-resistant breast cancer

Abstract INTRODUCTION: The past decade has seen significant advancement in increasing survival in estrogen receptor alpha (ERα) positive breast cancer. The use of selective estrogen receptor down-regulators and modulators (SERMs) (e.g. fulvestrant and tamoxifen), mTOR inhibitors (e.g. everolimus), aromatase inhibitors (AIs), and cyclin-dependent kinase inhibitors (e.g. palbociclib, ribociclib, and abemaciclib) have helped to extend overall survival of breast cancer patients. Unfortunately, resistance to endocrine therapy is a common occurrence and all patients will eventually succumb to their disease. Fatty acid synthase (FASN) is a key enzyme in lipid biosynthesis and is overexpressed in more aggressive and therapy-resistant tumors, including breast cancers. FASN inhibitor, TVB-2640, has been evaluated in multiple tumor cell lines and in a phase 1 clinical study, and showed partial responses in 5 patients and multiple patients with prolonged stable disease (≥16 weeks). METHODS: We generated tamoxifen- and fulvestrant-resistant MCF7 cells by long term exposure to tamoxifen (MCF7/TamR cells) and fulvestrant (MCF7/FR cells), and palbociclib-resistant (MCF7/RB1Crispr and ZR75/RB1Crispr) cells were generated through CRISPR/Cas9 knockout of the retinoblastoma (RB) gene. We assessed the impact of TVB-3166 inhibitor (and analog of TVB-2640 with slightly lower molecular weight for in vitro use) on proliferation, viability, cell cycle, and apoptosis in these cells. We evaluated the impact of TVB on proliferation and ERα expression in patient derived explants, and tumor growth in xenografts. RNA sequencing of tamoxifen- and fulvestrant-resistant cells was performed to investigate alterations in gene expression. Subcellular localization of ERα was assessed using subcellular fractionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels were analyzed by western blotting after treatment with TVB with or without the addition of palmitate or BAPTA. RESULTS: TVB treatment leads to a marked inhibition of proliferation in tamoxifen-, fulvestrant- and palbociclib-resistant cells compared to the parental cells. RNA sequencing of explants of patients with ERα positive disease showed down regulation of ESR1 related genes and genes involved in invasiveness. RNA sequencing of fulvestrant-resistant cells showed that treatment with TVB results in down regulation of EMT and E2F target genes, cholesterol homeostasis genes and mTORC1 signaling. Additionally, TVB significantly inhibited tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition significantly reduced ERα levels most prominently in endocrine resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERα expression upon TVB treatment is mediated through the induction of endoplasmic reticulum stress in tamoxifen-resistant cells. CONCLUSION: Our preclinical data provide evidence that FASN inhibition by TVB-3166 presents a promising therapeutic strategy for treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine resistant breast cancer should be considered. Citation Format: Henriette Balinda. Inhibition of FASN as a potential treatment of advanced endocrine therapy-resistant 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 P3-11-07.

GDNF-RET signaling and EGR1 form a positive feedback loop that promotes tamoxifen resistance via cyclin D1

BackgroundRearranged during transfection (RET) tyrosine kinase signaling has been previously implicated in endocrine resistant breast cancer, however the mechanism by which this signaling cascade promotes resistance is currently not well described. We recently reported that glial cell-derived neurotrophic factor (GDNF)-RET signaling appears to promote a positive feedback loop with the transcription factor early growth response 1 (EGR1). Here we investigate the mechanism behind this feedback loop and test the hypothesis that GDNF-RET signaling forms a regulatory loop with EGR1 to upregulate cyclin D1 (CCND1) transcription, leading to cell cycle progression and tamoxifen resistance.MethodsTo gain a better understanding of the GDNF-RET-EGR1 resistance mechanism, we studied the GDNF-EGR1 positive feedback loop and the role of GDNF and EGR1 in endocrine resistance by modulating their transcription levels using CRISPR-dCAS9 in tamoxifen sensitive (TamS) and tamoxifen resistant (TamR) MCF-7 cells. Additionally, we performed kinetic studies using recombinant GDNF (rGDNF) treatment of TamS cells. Finally, we performed cell proliferation assays using rGDNF, tamoxifen (TAM), and Palbociclib treatments in TamS cells. Statistical significance for qPCR and chromatin immunoprecipitation (ChIP)-qPCR experiments were determined using a student’s paired t-test and statistical significance for the cell viability assay was a one-way ANOVA.ResultsGDNF-RET signaling formed a positive feedback loop with EGR1 and also downregulated estrogen receptor 1 (ESR1) transcription. Upregulation of GDNF and EGR1 promoted tamoxifen resistance in TamS cells and downregulation of GDNF promoted tamoxifen sensitivity in TamR cells. Additionally, we show that rGDNF treatment activated GDNF-RET signaling in TamS cells, leading to recruitment of phospho-ELK-1 to the EGR1 promoter, upregulation of EGR1 mRNA and protein, binding of EGR1 to the GDNF and CCND1 promoters, increased GDNF protein expression, and subsequent upregulation of CCND1 mRNA levels. We also show that inhibition of cyclin D1 with Palbociclib, in the presence of rGDNF, decreases cell proliferation and resensitizes cells to TAM.ConclusionOutcomes from these studies support the hypotheses that GDNF-RET signaling forms a positive feedback loop with the transcription factor EGR1, and that GDNF-RET-EGR1 signaling promotes endocrine resistance via signaling to cyclin D1. Inhibition of components of this signaling pathway could lead to therapeutic insights into the treatment of endocrine resistant breast cancer.

The Role of ESRP1 in the Regulation of PHGDH in Estrogen Receptor–Positive Breast Cancer

Resistance to hormone therapy leads to a recurrence of estrogen receptor–positive breast cancer. We have demonstrated that the epithelial splicing regulatory protein 1 (ESRP1) significantly affects cell/tumor growth and metabolism and is associated with a poor prognosis in this breast cancer subtype. In this study, we aimed to investigate the ESRP1 protein-messenger RNA (mRNA) interaction in hormone therapy–resistant breast cancer. RNA-binding protein immunoprecipitation (RIP) followed by Clariom D (Applied Biosystems/Thermo Fisher Scientific) transcriptomics microarray (RIP-Chip) was performed to identify mRNA-binding partners of ESRP1. The integration of RIP-Chip and immunoprecipitation-mass spectrometry analyses identified phosphoglycerate dehydrogenase (PHGDH), a key metabolic enzyme, as a binding partner of ESRP1 in hormone-resistant breast cancer. Bioinformatic analysis showed ESRP1 binding to the 5′ untranslated region of PHGDH. RNA electrophoresis mobility shift assay and RIP-quantitative reverse transcription–polymerase chain reaction further validated the ESRP1-PHGDH binding. In addition, knockdown of ESRP1 decreased PHGDH mRNA stability significantly, suggesting the posttranscriptional regulation of PHGDH by ESRP1. The presence or absence of ESRP1 levels significantly affected the stability in tamoxifen-resistant LCC2 and fulvestrant-resistant LCC9 cells. PHGDH knockdown in tamoxifen-resistant cells further reduced the oxygen consumption rate (ranging from P = .005 and P = .02), mimicking the effects of ESRP1 knockdown. Glycolytic parameters were also altered (ranging P = .001 and P = .005). ESRP1 levels did not affect the stability of PHGDH in T-47D cells, although knockdown of PHGDH affected the growth of these cells. In conclusion, to our knowledge, this study, for the first time, reports that ESRP1 binds to the 5′ untranslated region of PHGDH, increasing its mRNA stability in hormone therapy-resistant estrogen receptor–positive breast cancer. These findings provide evidence for a novel mechanism of action of RNA-binding proteins such as ESRP1. These new insights could assist in developing novel strategies for the treatment of hormone therapy–resistant breast cancer.

Targeting hormone-resistant breast cancer cells with docetaxel: a look inside the resistance.

Aim: The study aims to analyze the effect of long-term incubation of ERα-positive MCF7 breast cancer cells with 4-hydroxytamoxifen (HT) on their sensitivity to tubulin polymerization inhibitor docetaxel. Methods: The analysis of cell viability was performed by the MTT method. The expression of signaling proteins was analyzed by immunoblotting and flow cytometry. ERα activity was evaluated by gene reporter assay. To establish hormone-resistant subline MCF7, breast cancer cells were treated with 4-hydroxytamoxifen for 12 months. Results: The developed MCF7/HT subline has lost sensitivity to 4-hydroxytamoxifen, and the resistance index was 2. Increased Akt activity (2.2-fold) and decreased ERα expression (1.5-fold) were revealed in MCF7/HT cells. The activity of the estrogen receptor α was reduced (1.5-fold) in MCF7/HT. Evaluation of class III β-tubulin expression (TUBB3), a marker associated with metastasis, revealed the following trends: higher expression of TUBB3 was detected in triple-negative breast cancer MDA-MB-231 cells compared to hormone-responsive MCF7 cells (P < 0.05). The lowest expression of TUBB3 was found in hormone-resistant MCF7/HT cells (MCF7/HT < MCF7 < MDA-MB-231, approximately 1:2:4). High TUBB3 expression strongly correlated with docetaxel resistance: IC50 value of docetaxel for MDA-MB-231 cells was greater than that for MCF7 cells, whereas resistant MCF7/HT cells were the most sensitive to the drug. The accumulation of cleaved PARP (a 1.6-fold increase) and Bcl-2 downregulation (1.8-fold) were more pronounced in docetaxel-treated resistant cells (P < 0.05). The expression of cyclin D1 decreased (2.8-fold) only in resistant cells after 4 nM docetaxel treatment, while this marker was unchanged in parental MCF7 breast cancer cells. Conclusion: Further development of taxane-based chemotherapy for hormone-resistant cancer looks highly promising, especially for cancers with low TUBB3 expression.