FOXA1 Binding Research Articles

12421 Reduced ZMPSTE24 Expression Leads To Prelamin Accumulation And Development Of Steatosis In MASLD Patients

Abstract Disclosure: J. Schinderle: None. I. Bochkis: None. A. Wu: None. Reduced ZMPSTE24 expression leads to prelamin accumulation and development of steatosis in MASLD patients. Joseph D. Schinderle[1],[2], Anqi Wu[1],[2], Irina M. Bochkis[1],[2]* [1] Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15261 [2] Pittsburgh Liver Research Center, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15261 We have previously observed changes at the nuclear lamina in MASLD patients that led to redistribution of lamina-associated domains and reshaping of FOXA2 binding with progression of steatosis. Here we report that the changes at the nuclear envelope are caused by downregulation of ZMPSTE24, an enzyme that processes prelamin to mature lamin A, leading to accumulation of prelamin in MASLD patients. Mutations of LMNA and ZMPSTE24 have been associated with fatty liver. In addition, Zmpste24 mutant mice develop hepatic steatosis and Zmpste24 deficiency is linked to upregulation of p53 target genes. Ingenuity Pathway Analysis determined upregulated p53 activity in genes differentially expressed in both male and female MASLD patients. Furthermore, upstream analysis identified p53 as a regulator of genes bound by FOXA2 in these individuals, corresponding to observations in Zmpste24 knockout animals. Additional regulator analysis of RNA-Seq determined that genes regulated by glucose and insulin to be downregulated in MASLD patients, suggesting altered glucose metabolism and insulin resistance, hallmarks of type 2 diabetes (T2D). Hence, our genomics data shows that MASLD patients with severe steatosis that have yet to progress to MASH are already suffering from severe metabolic consequences and underscores the need for treatment at this stage of the disease. Presentation: 6/2/2024

The xenobiotic transporter ABCC4/MRP4 promotes epithelial mesenchymal transition in pancreatic cancer.

The xenobiotic transporter ABCC4/MRP4 is highly expressed in pancreatic ductal adenocarcinoma (PDAC) and correlates with a more aggressive phenotype and metastatic propensity. Here, we show that ABCC4 promotes epithelial-mesenchymal transition (EMT) in PDAC, a hallmark process involving the acquisition of mesenchymal traits by epithelial cells, enhanced cell motility, and chemoresistance. Modulation of ABCC4 levels in PANC-1 and BxPC-3 cell lines resulted in the dysregulation of genes present in the EMT signature. Bioinformatic analysis on several cohorts including tumor samples, primary patient-derived cultured cells, patient-derived xenografts, and cell lines, revealed a positive correlation between ABCC4 expression and EMT markers. We also characterized the ABCC4 cistrome and identified four candidate clusters in the distal promoter and intron one that showed differential binding of pro-epithelial FOXA1 and pro-mesenchymal GATA2 transcription factors in low ABCC4-expressing HPAF-II and high ABCC4-expressing PANC-1 xenografts. HPAF-II xenografts showed exclusive binding of FOXA1, and PANC-1 xenografts exclusive binding of GATA2, at ABCC4 clusters, consistent with their low and high EMT phenotype respectively. Our results underscore ABCC4/MRP4 as a valuable prognostic marker and a potential therapeutic target to treat PDAC subtypes with prominent EMT features, such as the basal-like/squamous subtype, characterized by worse prognosis and no effective therapies.

Systematic dissection of sequence features affecting binding specificity of a pioneer factor reveals binding synergy between FOXA1 and AP-1

Despite the unique ability of pioneer factors (PFs) to target nucleosomal sites in closed chromatin, they only bind a small fraction of their genomic motifs. The underlying mechanism of this selectivity is not well understood. Here, we design a high-throughput assay called chromatin immunoprecipitation with integrated synthetic oligonucleotides (ChIP-ISO) to systematically dissect sequence features affecting the binding specificity of a classic PF, FOXA1, in human A549 cells. Combining ChIP-ISO with invitro and neural network analyses, we find that (1) FOXA1 binding is strongly affected by co-binding transcription factors (TFs) AP-1 and CEBPB; (2) FOXA1 and AP-1 show binding cooperativity invitro; (3) FOXA1's binding is determined more by local sequences than chromatin context, including eu-/heterochromatin; and (4) AP-1 is partially responsible for differential binding of FOXA1 in different cell types. Our study presents a framework for elucidating genetic rules underlying PF binding specificity and reveals a mechanism for context-specific regulation of its binding.

Ductal carcinoma in situ: Molecular and cellular basis of malignancy.

e12582 Background: The goal of our study is to improve the diagnosis and treatment of DCIS through the identification of the factors driving its invasion and metastasis. Methods: We utilized the Mouse INtraDuctal (MIND) model to investigate the mechanism(s) by which some DCIS become invasive while others remain indolent. This model closely replicates the human condition by injecting non-invasive epithelial cells from patients into mouse mammary ducts to form in situ lesions. In some cases, these cells will display invasive behavior by overcoming the natural barriers of myoepithelium. To compare these two processes, we performed single-cell (sc) RNA-sequencing analysis of 60,000 cells from the pre-transplant time-point representing 17 DCIS (11 became invasive, 6 remained non-invasive). We also performed scATAC/RNA sequencing of a DCIS with associated IDC. Results: Our analysis revealed that the proportion of endothelial cells was higher in cases that became invasive, while the proportion of plasma and T cells was higher in those that remained non-invasive (t-test; P<0.05). This finding suggested that invasive cells reprogrammed the immune microenvironment to exclude immune cells as a means of evading immune surveillance. Additionally, we performed cell-cell interaction (CCI) analysis by CellPhoneDB. This analysis revealed highly ranked CCIs in invasive cases, including luminal cell interactions with pericytes via Wnt5A-SFRP1. On the other hand, highly ranked CCIs in non-invasive cases included HR-positive luminal cell interactions with T cells via CD226-Nectin-2 and CD96-Nectin1. Notably, CD226 and CD96 are T cell receptors that enhance CD8+ T cell activity. These results support the potential role of T-cell interactions with DCIS luminal cells in preventing their invasive transition. We conducted additional analysis of sc-RNA-sequencing data using a stem cell-based gene signature algorithm called CytoTRACE. Our analysis revealed that invasive cells were enriched in cell clusters with significantly higher stem cell scores than non-invasive cells. In another analysis of a patient DCIS with associated IDC using sc-ATAC and RNA sequencing, we discovered that the open chromatin regions in the stem cell cluster were enriched for FOXA1 binding motifs. This cluster also had the highest expression of two pioneer factors (FOXA1 and GRHL2), NEAT1 (paraspeckle protein), ERBB2, 3,4, and ANKRD30A (cell surface protein). Our findings suggest that the pioneer factors FOXA1 and GRHL2 play a crucial role as transcriptional drivers of stemness. Additionally, the ANKRD30A could be used to isolate stem cell clusters to validate their self-renewal potential. Conclusions: DCIS progression is facilitated by interactions between stromal, immune, and epithelial cells, which lead to epigenetic and transcriptional changes. This results in the formation of stem-like cells that are characterized by their plasticity and invasive capacity.

FOXA1 regulates ribosomal RNA transcription in prostate cancer.

Ribosome biogenesis is excessively activated in tumor cells, yet it is little known whether oncogenic transcription factors (TFs) are involved in the ribosomal RNA (rRNA) transactivation. Nucleolar proteomics data and large-scale immunofluorescence were re-analyzed to jointly identify the proteins localized at nucleolus. RNA-Seq data of five prostate cancer (PCa) cohorts were combined and integrated with multi-dimensional data to define the upregulated nucleolar TFs in PCa tissues. Then, ChIP-Seq data of PCa cell lines and two PCa clinical cohorts were re-analyzed to reveal the TF binding patterns at ribosomal DNA (rDNA) repeats. The TF binding at rDNA was validated by ChIP-qPCR. The effect of the TF on rRNA transcription was determined by rDNA luciferase reporter, nascent RNA synthesis, and global protein translation assays. In this study, we reveal the role of oncogenic TF FOXA1 in regulating rRNA transcription within nucleolar organization regions. By analyzing human TFs in prostate cancer clinical datasets and nucleolar proteomics data, we identified that FOXA1 is partially localized in the nucleolus and correlated with global protein translation. Our extensive FOXA1 ChIP-Seq analysis provides robust evidence of FOXA1 binding across rDNA repeats in prostate cancer cell lines, primary tumors, and castration-resistant variants. Notably, FOXA1 occupancy at rDNA repeats correlates with histone modifications associated with active transcription, namely H3K27ac and H3K4me3. Reducing FOXA1 expression results in decreased transactivation at rDNA, subsequently diminishing global protein synthesis. Our results suggest FOXA1 regulates aberrant ribosome biogenesis downstream of oncogenic signaling in prostate cancer.

Conserved methylation signatures associate with the tumor immune microenvironment and immunotherapy response

BackgroundAberrant DNA methylation is a major characteristic of cancer genomes. It remains unclear which biological processes determine epigenetic reprogramming and how these processes influence the variants in the cancer methylome, which can further impact cancer phenotypes.MethodsWe performed pairwise permutations of 381,900 loci in 569 paired DNA methylation profiles of cancer tissue and matched normal tissue from The Cancer Genome Atlas (TCGA) and defined conserved differentially methylated positions (DMPs) based on the resulting null distribution. Then, we derived independent methylation signatures from 2,465 cancer-only methylation profiles from the TCGA and 241 cell line-based methylation profiles from the Genomics of Drug Sensitivity in Cancer (GDSC) cohort using nonnegative matrix factorization (NMF). We correlated DNA methylation signatures with various clinical and biological features, including age, survival, cancer stage, tumor immune microenvironment factors, and immunotherapy response. We inferred the determinant genes of these methylation signatures by integrating genomic and transcriptomic data and evaluated the impact of these signatures on cancer phenotypes in independent bulk and single-cell RNA/methylome cohorts.ResultsWe identified 7,364 differentially methylated positions (2,969 Hyper-DMPs and 4,395 Hypo-DMPs) in nine cancer types from the TCGA. We subsequently retrieved three highly conserved, independent methylation signatures (Hyper-MS1, Hypo-MS1, and Hypo-MS4) from cancer tissues and cell lines based on these Hyper and Hypo-DMPs. Our data suggested that Hypo-MS4 activity predicts poor survival and is associated with immunotherapy response and distant tumor metastasis, and Hypo-MS4 activity is related to TP53 mutation and FOXA1 binding specificity. In addition, we demonstrated a correlation between the activities of Hypo-MS4 in cancer cells and the fractions of regulatory CD4 + T cells with the expression levels of immunological genes in the tumor immune microenvironment.ConclusionsOur findings demonstrated that the methylation signatures of distinct biological processes are associated with immune activity in the cancer microenvironment and predict immunotherapy response.

Clinically-observed FOXA1 mutations upregulate SEMA3C through transcriptional derepression in prostate cancer

FOXA1 is a pioneer transcription factor that is frequently mutated in prostate, breast, bladder, and salivary gland malignancies. Indeed, metastatic castration-resistant prostate cancer (mCRPC) commonly harbour FOXA1 mutations with a prevalence of 35%. However, despite the frequent recurrence of FOXA1 mutations in prostate cancer, the mechanisms by which FOXA1 variants drive its oncogenic effects are still unclear. Semaphorin 3C (SEMA3C) is a secreted autocrine growth factor that drives growth and treatment resistance of prostate and other cancers and is known to be regulated by both AR and FOXA1. In the present study, we characterize FOXA1 alterations with respect to its regulation of SEMA3C. Our findings reveal that FOXA1 alterations lead to elevated levels of SEMA3C both in prostate cancer specimens and in vitro. We further show that FOXA1 negatively regulates SEMA3C via intronic cis elements, and that mutations in FOXA1 forkhead domain attenuate its inhibitory function in reporter assays, presumably by disrupting DNA binding of FOXA1. Our findings underscore the key role of FOXA1 in prostate cancer progression and treatment resistance by regulating SEMA3C expression and suggest that SEMA3C may be a driver of growth and tumor vulnerability of mCRPC harboring FOXA1 alterations.

Abstract A076: The role of PARP-1-mediated FoxA1 ADP-ribosylation in breast cancer biology

Abstract Breast cancer is the most common cancer diagnosed in women. At the time of diagnosis, approximately 70% of breast cancers express Estrogen Receptor alpha (ERα), a key signature of luminal subtype tumors. Equally important in these cases is the expression of FoxA1, a subtype-specific transcription factor (TF) essential for ERα-mediated cell proliferation. FoxA1 is a key pioneer TF that assists ERα with the assembly of enhancers and the transcription of estrogen (E2)-regulated target genes. Together, the FoxA1-ERα axis is a major driver of cancer cell growth. Our recently published work implicates poly(ADP-ribose) polymerase 1 (PARP-1) and ADP-ribosylation (ADPRylation) in the regulation of E2-dependent transcriptional responses in ERα+ breast cancer cells. To determine the underlying mechanisms, we identified FoxA1 as a substrate of PARP-1-mediated ADPRylation. We hypothesize that (1) PARP-1 elicits its effects on E2 signaling and ERα enhancer function, in part, by ADPRylating FoxA1 at specific residues and (2) Loss of these regulatory ADPRylation events alters FoxA1 activity, which in turn can alter the assembly of ERα enhancers and transform the breast cancer cell transcriptome. Indeed, the sites of FoxA1 ADPRylation are recurrently mutated in breast cancers and are highly enriched in metastatic cases. Using an integrated set of biochemical, molecular, cell based, genomic and proteomic approaches, we have validated the ADPRylation of FoxA1 by PARP-1 in vitro and in vivo. We selected two FoxA1 ADPRylation sites, D226 and E255, for further analysis due to their positions in a mutation ‘hot spot’ region corresponding to the C-terminal forkhead DNA-binding domain. Mutation of these sites blocks FoxA1 ADPRylation and enhances cell proliferation in MCF-7 human breast cancer cells. Additionally, preliminary ChIP-seq analysis indicates that the binding of both ERα and FoxA1 are globally reduced at ERα enhancers in MCF-7 cells expressing the FoxA1 ADPRylation site mutants compared to wild-type but may also increase both at selected enhancers. While there is a global reduction in mutant FoxA1 binding near the canonical estrogen-regulated genes, interestingly, we observe an overall enhanced binding of FoxA1 D226N compared to wild-type outside of this canonical program. Collectively, these studies will (1) reveal new molecular endpoints modulated by PARP-1, providing new insights about the use of PARPi to treat ERα+ breast cancers, and (2) suggest new therapeutic strategies to disrupt the aberrant signaling pathways associated with mutant FoxA1 luminal breast cancers, and more importantly metastatic cases, which is of utmost clinical significance. Citation Format: Cristel V. Camacho, Aishwarya Sundaresan, Tulip Nandu, W. Lee Kraus. The role of PARP-1-mediated FoxA1 ADP-ribosylation in breast cancer biology [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A076.

Pioneer Factor Foxa2 Mediates Chromatin Conformation Changes for Activation of Bile Acid Targets of FXR

Transcription factors regulate gene expression that orchestrates liver physiology. Many bind at distal enhancers and chromatin looping is required to activate their targets. Chromatin architecture has been linked to essential functions of the liver, including metabolism and sexually dimorphic gene expression. We have previously shown that pioneer factor Foxa2 opens chromatin for binding of nuclear receptors farnesoid X receptor (FXR) and liver X receptor-α during acute ligand activation. FXR is activated by bile acids and deletion of Foxa2 in the liver results in intrahepatic cholestasis. We hypothesized that Foxa2 also enables chromatin conformational changes during ligand activation and performed genome-wide studies to test this hypothesis. We performed Foxa2 HiChIP (Hi-C and ChIP) to assess Foxa2-dependent long-range interactions in mouse livers treated with either vehicle control or FXR agonist GW4064. HiChIP contact analysis shows that global chromatin interactions are dramatically increased during FXR activation. Ligand-treated livers exhibit extensive redistribution of topological associated domains and substantial increase in Foxa2-anchored loops, suggesting Foxa2 is involved in dynamic chromatin conformational changes. We demonstrate that chromatin conformation, including genome-wide interactions, topological associated domains, and intrachromosomal and interchromosomal Foxa2-anchored loops, drastically changes on addition of FXR agonist. Additional Foxa2 binding in ligand-activated state leads to formation of Foxa2-anchored loops, leading to distal interactions and activation of gene expression of FXR targets. Ligand activation of FXR, and likely of related receptors, requires global changes in chromatin architecture. We determine a novel role for Foxa2 in enabling these conformational changes, extending its function in bile acid metabolism.

Abstract A056: AR/FOXA1-mediated active gene repression as a mechanism of prostate carcinogenesis

Abstract The identity of the prostate gland is defined by transcription factors (TF) like the androgen receptor (AR), FOXA1, and NKX3.1 that bind regulatory elements early in development to activate the lineage gene program. For instance, KLK3, is a canonical target gene activated by AR. While the mechanistic interactions between pioneer factors, chromatin remodelers, and epigenetic proteins required for gene activation is well understood, repressive activity of lineage-defining TFs remain largely uncharacterized. Here, we document and mechanistically describe the active, repressive transcriptional functions of AR and FOXA1 in the context of prostate tumorigenesis.We identified a shared repertoire of repressed gene targets of AR and FOXA1 by using transcriptomic profiles (RNA-seq) of PCa cells treated with dihydrotestosterone (DHT) or TF targeting siRNA. Chromatin binding of AR and/or FOXA1 (ChIPseq) at cis-regulatory elements (dCREs) within the gene’s topologically-associated domain was used as primary evidence for direct regulation. Here, the KLK3 and DEPTOR gene loci emerged as candidate-activated or repressed AR/FOXA1 targets in PCa cells. DEPTOR, a tumor suppressor gene, negatively regulates the mTORC1/2 complexes by inhibiting their kinase activity. In contrast to the passive model of repression, where sequestration of co-activators ensues in transcriptional down-regulation of genes, we found ligand-bound AR and FOXA1 bind CREs within DEPTORs’ TAD establishing looping interactions (H3K3me3 HiChIP-seq) with the DEPTOR gene promoter. Functional inactivation of AR or FOXA1 led to a 2-4 fold upregulation of DEPTOR in VCaP, LNCaP, and 22RV1 cells. Instead, DHT-stimulation of AR led to DEPTOR downregulation. To assess the rapidness of this effect, we used a SWI/SNF ATPase PROTAC, AU15330, which triggers instant compaction of CREs and dislodges AR/FOXA1 from the chromatin. Treatment of PCa cells with AU15330 for 4 hours led to an immediate and significant upregulation of DEPTOR, substantiating the direct transcriptional repression by the AR/FOXA1 complex. Consistently, AU-15330 treatment also abolished FOXA1 and AR binding and 3D loop formation into the DEPTOR promoter. Using an orthogonal genetic approach, CRISPR-KRAB-targeted heterochromatinization of the AR/FOXA1 repressive CREs led to increased production of the nascent DEPTOR transcript. Finally, monoclones devoid of the CRE had elevated DEPTOR levels. Altogether, our findings position DEPTOR as an actively repressed target of the AR/FOXA1 transcriptional complex, suggesting the “silencer” duties of driver TF oncogenes to play a critical role in prostate tumorigenesis. Furthermore, the repertoire of activated and repressed gene targets identified in this study serve as exciting loci to study differences in the co-factor machinery and/or other cis or trans-attributes of the encompassing TADs that regulate downstream transcriptional outcomes. We posit the active mode of gene repression plays a driver role in prostate carcinogenesis. Citation Format: Sanjana Eyunni, Abhijit Parolia, Eleanor Young, James George, Mustapha Jaber, Lanbo Xiao, Fengyun Su, Rui Wang, Xuhong Cao, Arul Chinnaiyan. AR/FOXA1-mediated active gene repression as a mechanism of prostate carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A056.

Abstract A019: Enzalutamide resistance is driven by adaptations that enhance AR splice variant and FOXA1 activities

Abstract Prostate cancer (PCa) that progresses on therapy with an androgen receptor (AR) inhibitor such as enzalutamide (Enz) generally continues to express high levels of transcriptionally active AR, but mechanisms driving this persistent AR activity remain to be established. These tumors also generally express increased levels of AR splice variants that lack the ligand binding domain and hence have androgen-independent activity (with ARv7 being the most common), but the extent to which these variants contribute to AR activity, and their dependence on the full length AR (ARfl), also remain unclear. We found Enz treatment initially suppressed AR activity in VCaP cells, despite an increase in ARv7 splice variant, while Enz-resistant VCaP cells (VCaPEnzR) generated by prolonged culture in Enz had restoration of AR transcriptional activity. Notably, this restored AR activity was not associated with further increases in ARv7 expression. However, by ChIP-seq we found it was associated with increased ARv7 chromatin binding, with ARv7 binding at a subset of the sites occupied by ARfl, but not at any unique sites. Moreover, approaches using RNAi and an AR degrader targeting ARfl showed that ARv7 was the major driver of AR transcriptional activity in the VCaPEnzR cells, despite much higher levels of ARfl expression, and was not dependent on ARfl. ATAC-seq showed there was a global increase in chromatin accessibility in the VCaPEnzR cells, with the greatest increase being at ARv7 sites. This suggested these sites had decreased nucleosome occupancy, which was confirmed by MNase-seq. As FOXA1 can act as a pioneer factor to open chromatin, we next carried out FOXA1 ChIP-seq. This showed increased FOXA1 binding across all FOXA1 sites in the VCaPEnzR cells versus parental VCaP, with the greatest increases being at ARv7 binding sites and at sites that showed the greatest increases in accessibility by ATAC-seq. Notably, frameshift mutations in the C-terminus of FOXA1 are found at increased frequency in castration-resistant prostate cancer (CRPC) and may enhance FOXA1 activity, but FOXA1 was not mutated in the VCaPEnzR versus parental VCaP cells. Mass spectrometry then identified posttranslational modifications (PTMs) in FOXA1 that have been previously reported in PCa, including K270 methylation that can impair FOXA1 chromatin binding, but this was not consistently decreased in VCaPEnzR cells. In contrast, we found consistent increases in phosphorylation at C-terminal sites including T334 and Y429. Notably, the Y429 phosphorylation was reported previously to enhance FOXA1 chromatin binding and estrogen receptor activity. Together these findings indicate that progression to Enz-resistance is dependent on adaptations that enhance ARv7 activity. These adaptations include increasing chromatin accessibility at ARv7 binding sites, which may be driven by PTMs in FOXA1. Citation Format: Betul Ersoy-Fazlioglu, Larysa Poluben, Mannan Nouri, Henry W. Long, Joshua W. Russo, Steven P. Balk. Enzalutamide resistance is driven by adaptations that enhance AR splice variant and FOXA1 activities [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A019.

Abstract 4729: LSD1 activates oncogenic super-enhancers in castration-resistant prostate cancer by forming nuclear condensates with BRD4

Abstract Lysine-specific demethylase (LSD1) was initially identified as a transcriptional repressor through demethylation of mono/di-methylated histone 3 lysine 4 (H3K4me1/2). Subsequent work suggests that LSD1 may trigger gene activation via demethylating H3K9me1/2 in the context of steroid receptors. Our previous studies have confirmed that LSD1 functions broadly as a coactivator of androgen receptor (AR)-regulated enhancers but retains its H3K4 demethylation function at these sites. Importantly, our recent work revealed that LSD1 regulates the accessibility of active enhancers through the interaction with FOXA1, which functions as a pioneer transcription factor by facilitating AR access to chromatin LSD1 inhibition disrupts the global binding of FOXA1 by blocking the demethylation of K270 of FOXA1, leading to inhibition of AR binding and transcriptional activity. However, in addition to AR signaling, it remains largely unknown whether LSD1 regulates other critical oncogenic programs during prostate cancer (PCa) progression. In this study, we aimed to fully understand the functional targets of LSD1 in castration-resistant PCa (CRPC) models. We performed a large transcriptomic profiling of CRPC patient-derived xenograft (PDX) models that are sensitive to LSD1 inhibitors. Our analyses indicated that LSD1 inhibition targeted multiples oncogenic programs, including previously known pathways such as AR, FOXA1, and E2F, and a previously undefined pathway, MYC signaling. Through single-cell RNA-seq analysis in a CRPC PDX model, we confirmed that MYC signaling was decreased in the responsive tumor cells and identified a previously unknown molecular subtype, AR+/FOXA1+/LSD1low/RB1−, which was resistant to LSD1 inhibition. It is well known that MYC is driven by super-enhancers (SEs), which are regulated by BRD4 (bromodomain and extraterminal (BET) protein). We further evaluated LSD1, FOXA1 and BRD4 ChIP-seq and defined SEs by using H3K27ac ChIP-seq followed by ROSE analysis. We found that LSD1, FOXA1, and BRD4 co-occupied at MYC SEs and BRD4 was recruited by unmethylated FOXA1 but not K270-methylated FOXA1. In addition, we observed the puncta-like formation of LSD1/BRD4 marked nuclear condensates under confocal immunofluorescence microscopy. By examining the public datasets of H3K27ac ChIP-seq of samples with different stages of PCa development, we identified a subset of SEs which were distinctly activated in CRPC. Using multiple CRPC PDXs, we revealed that combining LSD1 inhibitors with BET inhibitors exhibited strong synergy possibly via disrupting the CRPC-specific SEs. In summary, this study reveals a novel oncogenic function of LSD1 in driving PCa progression by activating SE-driven oncogenic programs, such as MYC signaling. These results provide a strong therapeutic potential of combining inhibitors of LSD1 and BRD4 in treating CRPC. Citation Format: Mingyu Liu, Muqing Li, Dong Han, Zifeng Wang, Wanting Han, Housheng Hansen He, Shuai Gao, Changmeng Cai. LSD1 activates oncogenic super-enhancers in castration-resistant prostate cancer by forming nuclear condensates with BRD4. [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 4729.

Abstract 4753: LSD1 &8211mediated FOXA2/AP1 transcription program drives lineage plasticity in prostate cancer

Abstract Background: While castration-resistant PCa (CRPC) can be further treated with second-generation androgen deprivation therapies (ADTs), the tumor can quickly generate resistance through multiple mechanisms. One critical mechanism is that tumor cells can progress to AR-indifferent stem cell-like (SCL-PCa or SCLPC) or neuroendocrine-like CRPC (NE-PCa or NEPC) through lineage plasticity. However, the underlying molecular basis remains to be determined, and clinical treatment options for these aggressive CRPC subtypes are currently limited. FOXA1 and FOXA2, which are members of the FOXA (Forkhead Box A) protein family, are pioneer transcription factors. While FOXA1 is well known for its function as a critical pioneer factor of AR and pivotal for maintaining AR signaling, the molecular function of FOXA2 in PCa cells is poorly understood despite that FOXA2 is known to be overexpressed in NEPC. Moreover, since FOXA2, like FOXA1, is currently undruggable in the clinic, there is an urgent need to decipher the molecular basis for the chromatin binding of FOXA2 and identify druggable targets that regulate FOXA2 binding and activity in the aggressive FOXA2-positive CRPC. Methods: In the current study, we first examined the role of FOXA2 in tumorigenesis and metastasis by using various in vitro and in vivo assays including mouse subcutaneous injection and zebrafish embryo injection approaches. We then conducted integrated whole genomic transcriptome and cistrome analyses in SCLPC and NEPC cell line models to characterize the activity of FOXA2 on chromatin and to identify its collaborating transcription factors that may play specific functions in promoting SCLPC or NEPC using a series of biochemical and bioinformatic approaches. Results: We found that FOXA2 silencing decreased the growth and metastasis of SCLPC and NEPC cells. Importantly, we discovered that FOXA2 chromatin binding is tightly associated with binding of JUN family proteins (primarily c-Jun) and FOXA2 silencing dramatically interrupted the global chromatin binding of c-Jun and FOSL1. The transcription targets of FOXA2/AP-1 are highly enriched for neuroendocrine and plasticity associated genes and associated with poor clinical outcomes. Furthermore, we also found that FOXA2 chromatin binding is globally enhanced by an epigenetic factor LSD1, and LSD1 inhibition can repress FOXA2/AP-1 activity in multiple CRPC models. Conclusion: Overall, our data indicate that FOXA2 functions to maintain tumor growth and metastasis in SCLPC and NEPC models. Mechanistically, FOXA2 can act as a pioneer factor of AP-1 (c-Jun/FOSL1) and reprogram AP-1 transcription activity. This FOXA2/AP-1 axis is regulated by LSD1 via demethylating FOXA2 protein and LSD1 inhibition represses FOXA2-dependent CRPC tumor progression. These findings provide novel mechanistic insights into the molecular mechanisms for PCa lineage plasticity and treatment resistance. Citation Format: Zifeng Wang, Mingyu Liu, Songqi Zhang, Muqing Li, Susan Patalano, Jill A. Macoska, Dong Han, Shuai Gao, Hansen He, Changmeng Cai. LSD1 &8211mediated FOXA2/AP1 transcription program drives lineage plasticity in prostate 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 4753.

Abstract P2-24-04: The ER antagonist giredestrant induces profound chromatin remodeling including activation of cis-regulatory elements bound by FOXA1 and GATA3 in HR+ breast cancer models

Abstract The progression of HR+ breast cancers is governed by ER signaling which makes the development of optimized ER-targeting agents a high priority. Among current therapeutics, selective ER antagonists and degraders (SERDs) have emerged as a particularly attractive class. Fulvestrant is the only SERD that is currently approved, but its clinical potential is hypothesized to be somewhat limited by poor physicochemical properties, dosage and exposure. We identified giredestrant as an orally bioavailable, highly potent non-steroidal ER antagonist, immobilizer and degrader with robust anti-proliferative activity. Giredestrant is currently being evaluated in phase III clinical trials for the treatment of ER+ breast cancer, after showing evidence of clinical activity in phase I and II trials. Here, we set out to investigate the impact of giredestrant on the transcriptome and chromatin landscape of ER+ breast cancer cells in vitro and in vivo. RNA-seq data from MCF-7 cells in vitro and three patient-derived xenograft (PDX) models in vivo showed that giredestrant inversely regulates the RNA levels of E2-regulated genes. Transcriptome wide, the expression of genes affected by giredestrant are strongly correlated (r2=0.8) with those affected by siRNA-mediated silencing of ESR1, consistent with on-target ER antagonism. Performing ATAC-seq in the three PDX models, including one ESR1 mutant model, reveals that giredestrant reduces accessibility in up to 18,173 chromatin regions that are associated with genes induced by E2. These regions are also enriched in the ER binding motif. In addition, we found up to 17,127 chromatin regions with increased accessibility upon giredestrant treatment. These regions are associated with genes suppressed by E2, and are enriched in the FOXA1 binding motif. To probe giredestrant’s role in functional modulation of cis-regulatory elements (CREs), we performed ChIP-seq targeting FOXA1 and GATA3, as key pioneer factors for ER, as well as ER itself and H3K27ac as a marker of active enhancers in MCF-7 cells. We identified 3,195 regions with significantly reduced H3K27ac signals that also exhibit significant reduction of both ER and FOXA1 binding upon giredestrant treatment, indicating that giredestrant deactivates these CREs concomitantly with suppressing ER and FOXA1 binding. ChIP-seq analysis further revealed 2,292 regions with a significant increase in H3K27ac signals upon giredestrant treatment. Interestingly, the GATA3 motif was identified as the most enriched motif in these activated CREs. Further, GATA3 and FOXA1 ChIP-seq signals were stronger in the activated CREs compared to the deactivated ones. Similar profiling of both tamoxifen, an approved selective ER modulator (SERM), as well an GNE-858, a SERM from the same chemical series as giredestrant, showed their epigenetic effects to be highly distinct from those observed for giredestrant, suggesting that the high degree of chromatin remodeling is likely related to giredestrant’s mechanism of action. Our studies here show that giredestrant profoundly alters ER binding and remodels the chromatin landscape, thus changing the transcriptional profile of cells in a manner that is associated with an anti-proliferative response. While deactivation of CREs with ER binding is perhaps in line with expectations, giredestrant treatment also leads to the activation of a number of regions associated with pioneer factors, potentially through their redistribution. This raises the question of the functional consequences of CRE activation and if this is relevant for the anti-proliferative effects of giredestrant. Citation Format: Musaddeque Ahmed, Jane Guan, Wei Zhou, Xiaosai Yao, Ciara Metcalfe, Marc Hafner. The ER antagonist giredestrant induces profound chromatin remodeling including activation of cis-regulatory elements bound by FOXA1 and GATA3 in HR+ breast cancer models [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 P2-24-04.

Abstract P3-11-10: Estrogen receptor reprogramming by HOXB13 and GATA3 in ER-positive breast cancer cells

Abstract Background: The Breast Cancer Index (BCI) is a gene expression-based signature comprising two functional biomarker panels. The Molecular Grade Index (MGI) is composed of five genes that measure tumor proliferation. BCI (H/I) is a ratio of the HOXB13 and IL17BR genes and measures estrogen signaling. Integration of MGI and BCI (H/I) provides a single prognostic score that quantifies the risk of both late (5-10 years) and overall (0-10 years) distant recurrence. BCI (H/I) has been validated to predict benefit from extended endocrine therapy across multiple adjuvant endocrine treatment backgrounds in several prospective-retrospective studies. Hormone receptor responses are dependent on genome-wide hormone receptor binding patterns. In hormone-dependent cancers, nuclear receptor binding patterns are frequently reprogrammed by other transcription factors. The homeobox transcription factor HOXB13 has previously been shown to reprogram genome-wide binding of the androgen receptor (AR) during prostate cancer tumorigenesis, where it colocalizes with FOXA1 at reprogrammed AR binding sites. Similarly, the transcription factor GATA3 was found to mediate enhancer accessibility at regulatory regions involved in estrogen receptor (ER)-mediated transcription. We have shown previously that HOXB13 overexpression reprograms and expands the ER binding pattern in breast cancer cells. In this study, we have further characterized the role of HOXB13 in reprograming the ER cistrome and evaluated potential interactions with GATA3. Methods: HOXB13 was expressed in MCF-7 and T47D cells by electroporating HOXB13 mRNA or eGFP mRNA as control. Cells were harvested after 18 hours and analyzed by western blot and chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) using antibodies against ER, HOXB13, FOXA1, GATA3 and histone H3K27ac. Chromatin accessibility was assessed by ATAC-seq. After aligning reads with Bowtie2, peak calling, data integration and motif enrichment analysis was performed using HOMER v4.10. Results: ChIP-seq analysis of T47D cells expressing HOXB13 confirmed previous results in MCF-7 cells, where binding of HOXB13 to a significant number of genomic binding sites induced changes in binding of ER, FOXA1 and GATA3 compared to control cells. Integrative analysis of ATAC-seq and ChIP-seq data revealed that HOXB13-induced reprogramming results in open chromatin that frequently exhibits increased acetylation of histone H3K27, a hallmark of transcriptional activation. While both increased chromatin accessibility and H3K27 acetylation were associated with HOX and AP-1 motif enrichment of the underlying DNA sequences, newly open chromatin was specifically co-enriched for motifs for pioneering factors such as FOXA1 and GRHL1. Conclusion: This study lends further support to a model of HOXB13-mediated reprogramming of the ER cistrome in breast cancer. Motif analysis of HOXB13-induced chromatin opening suggests interactions with other pioneer transcription factors including FOXA1 and GRHL1, while HOXB13-induced transcriptional activation is associated with motifs for activating factors such as AP-1. These results will be expanded to inducible cell lines to further characterize the effects of HOXB13 expression on ER binding and function. Citation Format: Kai Treuner, Camila De Arruda Saldanha, Sven Heinz. Estrogen receptor reprogramming by HOXB13 and GATA3 in ER-positive breast cancer cells [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 P3-11-10.

Abstract P6-12-08: Identifying FOXA1 Binding Partners using Proximity Labeling

Abstract Approximately 75% of breast cancers are driven by the estrogen receptor alpha (ER), and despite the advent of endocrine therapy to block ER signaling pathways, a significant portion of women develop resistance to these drugs. The pioneer factor FOXA1 has been shown to facilitate nearly all DNA-binding events of ER in response to estrogen in ER+ breast cancer (ER+BC). Notably, up-regulation of FOXA1 is a hallmark of endocrine-resistant phenotypes and has been shown to reprogram enhancer elements, leading to an altered transcriptome. However, FOXA1 is a critical pioneer factor for multiple nuclear hormone receptors, aside from ER, and is implicated in regulation of important factors such as HER2 and the androgen receptor (AR). With the diverse array of breast cancer molecular subtypes displaying complex interplay between ER, HER2, AR, PR, and other hormone receptors, describing the complete ensemble of FOXA1 binding partners in various contexts, such as endocrine-resistant tumors, is of increasing importance. To define a comprehensive catalog of FOXA1 binding partners under basal conditions, we generated MCF-7 cell lines stably expressing constructs of FOXA1 fused at its N- or C-terminus to the biotin ligase miniTurbo. Using proximity labeling coupled with mass-spectrometry, we have comprehensively cataloged binding partners of FOXA1, including many expected proteins such as ER, AR, MLL3, YAP1, and GATA-3. Moreover, we have discovered more than 150 previously unidentified binding partners of FOXA1, which may exert profound effects on FOXA1 function. Importantly, high hazard ratios and significant dependencies are associated with several of these new binding partners, such as subunits of a previously described histone deacetylase (HDAC) complex containing genetic suppressor element 1 (GSE1) and lysine-specific histone demethylase 1A (KDM1A). Genomic approaches are currently underway to characterize where in the genome FOXA1 is interacting with these novel proteins and to guide future exploration into the physiological significance of these interactions. Integrating biochemical, molecular, and genomic approaches, we have potentially highlighted new mechanisms of FOXA1, which could have significant clinical impact in the future. Citation Format: Rosemary N. Plagens, Shen Li, Christine A. Mills, Laura Herring, Hector L. Franco. Identifying FOXA1 Binding Partners using Proximity Labeling [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-08.

Estrogen receptor α K303R mutation reorganizes its binding to forkhead box protein A1 regions and induces chromatin opening

BackgroundEstrogen receptor alpha (ERα) is a frequently mutated gene in breast cancer (BC). While many studies have investigated molecular dysregulation by hotspot mutations at Y537 and D538, which exhibit an estrogen-independent constitutively active phenotype, the functional abnormalities of other mutations remain obscure. The K303R mutation in primary invasive BC has been implicated with endocrine resistance, tumor size, and lymph node positivity. However, the impact of the K303R mutation on the cell epigenome is yet unknown.Methods and resultsWe introduced the K303R ERα mutant in ERα-negative MDA-MB-453 cells to monitor ERα-dependent transactivation and to perform epigenomic analyses. ATAC-seq and ChIP-Seq analyses indicated that both wild-type (WT) and the K303R mutant associated with Forkhead box (Fox) protein family motif regions at similar rates, even without an ERα-binding sequence, but only the K303R mutant induced chromatin opening at those regions. Biochemical analyses demonstrated that the WT and the K303R mutant can be tethered on DNA by FoxA1 indirectly, but only the K303R/FoxA1/DNA complex can induce associations with the nuclear receptor cofactor 2 (NCOA2).ConclusionsThese findings suggest that the K303R mutant induces chromatin opening at the Fox binding region through the FoxA1-dependent associations of the K303R mutant to NCOA2 and then probably disrupts the regulation of Fox-target genes, resulting in K303R-related BC events.

Redistribution of lamina-associated domains reshapes binding of pioneer factor FOXA2 in development of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in type 2 diabetes mellitus and the elderly, impacting 40% of individuals over 70. Regulation of heterochromatin at the nuclear lamina has been associated with aging and age-dependent metabolic changes. We previously showed that changes at the lamina in aged hepatocytes and laminopathy models lead to redistribution of lamina-associated domains (LADs), opening of repressed chromatin, and up-regulation of genes regulating lipid synthesis and storage, culminating in fatty liver. Here, we test the hypothesis that change in the expression of lamina-associated proteins and nuclear shape leads to redistribution of LADs, followed by altered binding of pioneer factor FOXA2 and by up-regulation of lipid synthesis and storage, culminating in steatosis in younger NAFLD patients (aged 21-51). Changes in nuclear morphology alter LAD partitioning and reduced lamin B1 signal correlate with increased FOXA2 binding before severe steatosis in young mice placed on a western diet. Nuclear shape is also changed in younger NAFLD patients. LADs are redistrubted and lamin B1 signal decreases similarly in mild and severe steatosis. In contrast, FOXA2 binding is similar in normal and NAFLD patients with moderate steatosis and is repositioned only in NAFLD patients with more severe lipid accumulation. Hence, changes at the nuclear lamina reshape FOXA2 binding with progression of the disease. Our results suggest a role for nuclear lamina in etiology of NAFLD, irrespective of aging, with potential for improved stratification of patients and novel treatments aimed at restoring nuclear lamina function.

FOXA1 regulates alternative splicing in prostate cancer.

SummaryDysregulation of alternative splicing in prostate cancer is linked to transcriptional programs activated by AR, ERG, FOXA1, and MYC. Here, we show that FOXA1 functions as the primary orchestrator of alternative splicing dysregulation across 500 primary and metastatic prostate cancer transcriptomes. We demonstrate that FOXA1 binds to the regulatory regions of splicing-related genes, including HNRNPK and SRSF1. By controlling trans-acting factor expression, FOXA1 exploits an “exon definition” mechanism calibrating alternative splicing toward dominant isoform production. This regulation especially impacts splicing factors themselves and leads to a reduction of nonsense-mediated decay (NMD)-targeted isoforms. Inclusion of the NMD-determinant FLNA exon 30 by FOXA1-controlled oncogene SRSF1 promotes cell growth in vitro and predicts disease recurrence. Overall, we report a role for FOXA1 in rewiring the alternative splicing landscape in prostate cancer through a cascade of events from chromatin access, to splicing factor regulation, and, finally, to alternative splicing of exons influencing patient survival.

A Distinct Chromatin State Drives Therapeutic Resistance in Invasive Lobular Breast Cancer.

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.