Pioneer Transcription Factor Research Articles

Roles of KLF4 and AMPK in the inhibition of glycolysis by pulsatile shear stress in endothelial cells

Vascular endothelial cells (ECs) sense and respond to hemodynamic forces such as pulsatile shear stress (PS) and oscillatory shear stress (OS). Among the metabolic pathways, glycolysis is differentially regulated by atheroprone OS and atheroprotective PS. Studying the molecular mechanisms by which PS suppresses glycolytic flux at the epigenetic, transcriptomic, and kinomic levels, we have demonstrated that glucokinase regulatory protein (GCKR) was markedly induced by PS in vitro and in vivo, although PS down-regulates other glycolysis enzymes such as hexokinase (HK1). Using next-generation sequencing data, we identified the binding of PS-induced Krüppel-like factor 4 (KLF4), which functions as a pioneer transcription factor, binding to the GCKR promoter to change the chromatin structure for transactivation of GCKR. At the posttranslational level, PS-activated AMP-activated protein kinase (AMPK) phosphorylates GCKR at Ser-481, thereby enhancing the interaction between GCKR and HK1 in ECs. In vivo, the level of phosphorylated GCKR Ser-481 and the interaction between GCKR and HK1 were increased in the thoracic aorta of wild-type AMPKα2+/+ mice in comparison with littermates with EC ablation of AMPKα2 (AMPKα2-/-). In addition, the level of GCKR was elevated in the aortas of mice with a high level of voluntary wheel running. The underlying mechanisms for the PS induction of GCKR involve regulation at the epigenetic level by KLF4 and at the posttranslational level by AMPK.

FOXA1 and adaptive response determinants to HER2 targeted therapy in TBCRC 036

Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.

Interplay of two transcription factors for recruitment of the chromatin remodeling complex modulates fungal nitrosative stress response

Nitric oxide (NO) is a diffusible signaling molecule that modulates animal and plant immune responses. In addition, reactive nitrogen species derived from NO can display antimicrobial activities by reacting with microbial cellular components, leading to nitrosative stress (NS) in pathogens. Here, we identify FgAreB as a regulator of the NS response in Fusarium graminearum, a fungal pathogen of cereal crops. FgAreB serves as a pioneer transcription factor for recruitment of the chromatin-remodeling complex SWI/SNF at the promoters of genes involved in the NS response, thus promoting their transcription. FgAreB plays important roles in fungal infection and growth. Furthermore, we show that a transcription repressor (FgIxr1) competes with the SWI/SNF complex for FgAreB binding, and negatively regulates the NS response. NS, in turn, promotes the degradation of FgIxr1, thus enhancing the recruitment of the SWI/SNF complex by FgAreB.

Constrained chromatin accessibility in PU.1-mutated agammaglobulinemia patients.

The pioneer transcription factor (TF) PU.1 controls hematopoietic cell fate by decompacting stem cell heterochromatin and allowing nonpioneer TFs to enter otherwise inaccessible genomic sites. PU.1 deficiency fatally arrests lymphopoiesis and myelopoiesis in mice, but human congenital PU.1 disorders have not previously been described. We studied six unrelated agammaglobulinemic patients, each harboring a heterozygous mutation (four de novo, two unphased) of SPI1, the gene encoding PU.1. Affected patients lacked circulating B cells and possessed few conventional dendritic cells. Introducing disease-similar SPI1 mutations into human hematopoietic stem and progenitor cells impaired early in vitro B cell and myeloid cell differentiation. Patient SPI1 mutations encoded destabilized PU.1 proteins unable to nuclear localize or bind target DNA. In PU.1-haploinsufficient pro-B cell lines, euchromatin was less accessible to nonpioneer TFs critical for B cell development, and gene expression patterns associated with the pro- to pre-B cell transition were undermined. Our findings molecularly describe a novel form of agammaglobulinemia and underscore PU.1's critical, dose-dependent role as a hematopoietic euchromatin gatekeeper.

Epithelial Transcription Factor FOXA1 Regulates Prostate Cancer Immune Response

Background: While localized prostate cancer (PCa) can be mitigated by surgery and radiation, metastatic PCa remains a challenge to treat. Androgen deprivation therapies and androgen receptor (AR) pathway inhibitors are mainstay treatments for advanced PCa. Yet, resistance often develops leading to castration-resistant prostate cancer (CRPC). Forkhead Box A1 (FOXA1) is a pioneer transcription factor that plays pivotal roles in regulating AR activity and promoting epithelial differentiation. Studies have shown that FOXA1 is frequently downregulated in CRPC tumors. Congruently, FOXA1 loss is reported to induce aberrant AR signaling, epithelial-mesenchymal transition, and PCa de-differentiation. However, the role of FOXA1 in regulating PCa immune response, an area of much interest recently, has not been reported. CRPC has shown poor response to immune checkpoint inhibitors, due to its immunosuppressive nature. A better understanding of the tumor intrinsic mechanisms regulating PCa tumor immunity will inform the design of better targeted immunotherapeutic approaches. Methods: We performed RNA-seq, ChIP-seq, qPCR, western blot, and ELISA analyses to evaluate how FOXA1 regulates inflammatory response genes. We utilized an in vitro macrophage infiltration transwell assay, in which M2-like macrophages were added to the upper chamber and PCa cells were plated in the lower chamber, to examine how perturbations to PCa cells affect macrophage migration. Finally, we performed bioinformatic analyses of patient datasets to confirm the clinical relevance of FOXA1 repression of inflammatory genes in PCa. Results: Through integration of RNA-seq and ChIP-seq data, we uncovered a novel function of FOXA1 in suppressing inflammatory response pathways. In accordance, patient data analyses revealed that inflammatory response genes were upregulated in FOXA1-low PCa tumors. Mechanistically, we showed that FOXA1 proteins bound an intragenic enhancer of Hypoxia-inducible factor 1-alpha (HIF1A) gene to directly repress its expression, such that FOXA1 loss induced HIF1A upregulation. We further showed that Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) became upregulated upon FOXA1 depletion in a HIF1A-dependent manner. This led to infiltration by immunosuppressive, tumor promoting M2-like macrophages. Inhibiting this HIF1A-CCL2 axis with a HIF1A inhibitor or CCL2 neutralizing antibody blocked macrophage infiltration. Future studies using immunocompetent mouse models are needed to confirm the effect of FOXA1 on macrophage infiltration in vivo and evaluate the preclinical potential of targeting the FOXA1-HIF1A-CCL2 axis in CRPC. Conclusion: This study proposes a novel role for FOXA1 loss in promoting macrophage infiltration via the HIF1A-CCL2 axis. Moreover, our findings suggest that targeting this axis may be a promising approach for the treatment of FOXA1-low CRPC tumors.

Bayesian Identification of Transcription Factors that Regulate Aqp2 Transcription

Background Renal collecting duct and connecting tubule cells selectively express the water channel aquaporin-2 (AQP2) and Aqp2 gene transcription is strongly regulated by vasopressin. However, the transcription factors (TFs) responsible for regulation of expression of AQP2 remain largely unknown. Here, we used Bayesian data integration techniques to identify these TFs. Method The general strategy is to use Bayes’ Rule to integrate several -omic datasets to stratify a curated list of 1344 TFs present in the mouse genome with regard to probability of regulating Aqp2 gene transcription. First, existing proteomic and transcriptomic data were used to prioritize TFs according to expression level in mpkCCD and native IMCD cells. Second, we used our existing ATAC-Seq, histone H3K27-acetylation ChIP-Seq, and RNA-polymerase II ChIP-Seq data to identify enhancer regions in the CTCF loop surrounding the Aqp2 gene. The sequences within these enhancers were analyzed to identify recognized TF binding motifs within them; and these motifs were matched to TFs on the Bayesian list to identify the TFs most likely to bind Aqp2 regulatory regions. Beyond this, we use additional -omic datasets to prioritize TFs that are regulated by vasopressin. Finally, we carried out new RNA-Seq experiments mapping the time course of vasopressin induced changes in the transcriptome of mouse mpkCCD cells to further prioritize TFs that change in tandem with AQP2. Results The analysis identified 21 TFs out of 1344 in the mouse genome that are most likely to be involved in regulation of Aqp2 gene transcription. These TFs included nine that have been proposed in prior studies to play a role in Aqp2 regulation, viz. Cebpb, Elf1, Elf3, Ets1, Hes1, Jun, Junb, Nfkb1, and Sp1. The remaining 12 represent new candidates for future studies (Atf1, Cebpg, Fosl2, Irf3, Klf5, Klf6, Mef2d, Nfib, Nfyb, Nfyc, Nr2f6, Stat3). Conspicuously absent is CREB, i.e. Creb1, which has been widely proposed to mediate vasopressin-induced regulation of Aqp2 gene transcription. Instead, another CREB-like TF, Atf1, with a pKID PKA-phosphorylation domain, ranked second among all transcription factors, representing an apt target for future studies. The RNA-Seq time course experiments in mpkCCD cells showed a rapid increase in Aqp2 mRNA, within 3 hour of vasopressin exposure. This response was matched by an equally rapid increase in the abundance of the mRNA coding for Cebpb, a so-called pioneer TF, which we have shown by ChIP-seq studies to bind to an enhancer 938 bp downstream from the Aqp2 gene. Conclusions The Bayesian analysis has identified the TFs most likely to bind to these regions and likely to be regulated by vasopressin stimulation, providing a roadmap for future studies to understand regulation of Aqp2 gene expression.

Oncogenic KRAS Recruits an Expansive Transcriptional Network through Mutant p53 to Drive Pancreatic Cancer Metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal and characterized by early metastasis. Oncogenic KRAS mutations prevail in 95% of PDAC tumors and co-occur with genetic alterations in the TP53 tumor suppressor in nearly 70% of patients. Most TP53 alterations are missense mutations that exhibit gain-of-function phenotypes that include increased invasiveness and metastasis, yet the extent of direct cooperation between KRAS effectors and mutant p53 remains largely undefined. We show that oncogenic KRAS effectors activate CREB1 to allow physical interactions with mutant p53 that hyperactivate multiple prometastatic transcriptional networks. Specifically, mutant p53 and CREB1 upregulate the prometastatic, pioneer transcription factor FOXA1, activating its transcriptional network while promoting WNT/β-catenin signaling, together driving PDAC metastasis. Pharmacologic CREB1 inhibition dramatically reduced FOXA1 and β-catenin expression and dampened PDAC metastasis, identifying a new therapeutic strategy to disrupt cooperation between oncogenic KRAS and mutant p53 to mitigate metastasis. SIGNIFICANCE: Oncogenic KRAS and mutant p53 are the most commonly mutated oncogene and tumor suppressor gene in human cancers, yet direct interactions between these genetic drivers remain undefined. We identified a cooperative node between oncogenic KRAS effectors and mutant p53 that can be therapeutically targeted to undermine cooperation and mitigate metastasis.This article is highlighted in the In This Issue feature, p. 1861.

Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during early Drosophila development.

Acquisition of cell fate is thought to rely on the specific interaction of remote cis-regulatory modules (CRMs), for example, enhancers and target promoters. However, the precise interplay between chromatin structure and gene expression is still unclear, particularly within multicellular developing organisms. In the present study, we employ Hi-M, a single-cell spatial genomics approach, to detect CRM-promoter looping interactions within topologically associating domains (TADs) during early Drosophila development. By comparing cis-regulatory loops in alternate cell types, we show that physical proximity does not necessarily instruct transcriptional states. Moreover, multi-way analyses reveal that multiple CRMs spatially coalesce to form hubs. Loops and CRM hubs are established early during development, before the emergence of TADs. Moreover, CRM hubs are formed, in part, via the action of the pioneer transcription factor Zelda and precede transcriptional activation. Our approach provides insight into the role of CRM-promoter interactions in defining transcriptional states, as well as distinct cell types.

Molecular Mechanisms of Oncogenesis through the Lens of Nucleosomes and Histones.

At the cellular level, cancer is the disease of both the genome and the epigenome, and the interplay between genetic mutations and epigenetic states may occur at the level of elementary chromatin units, the nucleosomes. They are formed by a segment of DNA wrapped around an octamer of histone proteins. In this review, we survey various mechanisms of cancer etiology and progression mediated by histones and nucleosomes. In particular, we discuss the effects of mutations in histones, changes in their expression and slicing on epigenetic dysregulation and carcinogenesis. The links between cancer phenotypes and differential expression of histone variants and isoforms are summarized. Finally, we discourse the geometric and steric effects of DNA compaction in nucleosomes on DNA mutation rate, interactions with transcription factors, including pioneer transcription factors, and prospects of cancer cells' genome and epigenome editing.

CRISPR‐based knock‐in mutagenesis of the pioneer transcription factor FOXA1: optimization of strategies for multi‐allelic proteins in cancer cells

Precise genome engineering of living cells has been revolutionized by the introduction of the highly specific and easily programmable properties of the clustered regularly interspaced short palindromic repeats (CRISPR) technology. This has greatly accelerated research into human health and has facilitated the discovery of novel therapeutics. CRISPR‐Cas9 is most widely employed for its ability to inactivate or knockout specific genes, but can be also used to introduce subtle site‐specific substitutions of DNA sequences that can lead to changes in the amino acid composition of proteins. Despite the proven success of CRISPR‐based knock‐in strategies of genes in typical diploid cells (i.e., cells containing two sets of chromosomes), precise editing of cancer cells, that typically have unstable genomes and multiple copies of chromosomes, is more challenging and not adequately addressed in the literature. Herein, we detail our methodology for replacing endogenous proteins with intended knock‐in mutants in polyploid cancer cells and discuss our experimental design, screening strategy, and facile allele frequency estimation methodology. As proof of principle, we performed genome editing of specific amino acids within the pioneer transcription factor FOXA1, a critical component of estrogen and androgen receptor signaling, in MCF‐7 breast cancer cells. We confirm mutant FOXA1 protein expression and intended amino acid substitutions via western blotting and mass spectrometry. In addition, we show that mutant allele frequency estimation is easily achieved by topoisomerase‐based cloning combined with allele‐specific PCR, which we later confirmed by next‐generation RNA‐sequencing. Typically, there are 4 ‐ 5 copies (alleles) of FOXA1 in breast cancer cells, making the editing of this protein inherently challenging. As a result, most studies that focus on FOXA1 mutants rely on ectopic overexpression of FOXA1 from a plasmid. Therefore, we provide an optimized methodology for replacing endogenous wild‐type FOXA1 with precise knock‐in mutants to enable the systematic analysis of its molecular mechanisms within the appropriate physiological context.

GAF is essential for zygotic genome activation and chromatin accessibility in the early Drosophila embryo.

Following fertilization, the genomes of the germ cells are reprogrammed to form the totipotent embryo. Pioneer transcription factors are essential for remodeling the chromatin and driving the initial wave of zygotic gene expression. In Drosophila melanogaster, the pioneer factor Zelda is essential for development through this dramatic period of reprogramming, known as the maternal-to-zygotic transition (MZT). However, it was unknown whether additional pioneer factors were required for this transition. We identified an additional maternally encoded factor required for development through the MZT, GAGA Factor (GAF). GAF is necessary to activate widespread zygotic transcription and to remodel the chromatin accessibility landscape. We demonstrated that Zelda preferentially controls expression of the earliest transcribed genes, while genes expressed during widespread activation are predominantly dependent on GAF. Thus, progression through the MZT requires coordination of multiple pioneer-like factors, and we propose that as development proceeds control is gradually transferred from Zelda to GAF.

Interplay of BAF and MLL4 promotes cell type-specific enhancer activation

Cell type-specific enhancers are activated by coordinated actions of lineage-determining transcription factors (LDTFs) and chromatin regulators. The SWI/SNF chromatin remodeling complex BAF and the histone H3K4 methyltransferase MLL4 (KMT2D) are both implicated in enhancer activation. However, the interplay between BAF and MLL4 in enhancer activation remains unclear. Using adipogenesis as a model system, we identify BAF as the major SWI/SNF complex that colocalizes with MLL4 and LDTFs on active enhancers and is required for cell differentiation. In contrast, the promoter enriched SWI/SNF complex PBAF is dispensable for adipogenesis. By depleting BAF subunits SMARCA4 (BRG1) and SMARCB1 (SNF5) as well as MLL4 in cells, we show that BAF and MLL4 reciprocally regulate each other’s binding on active enhancers before and during adipogenesis. By focusing on enhancer activation by the adipogenic pioneer transcription factor C/EBPβ without inducing cell differentiation, we provide direct evidence for an interdependent relationship between BAF and MLL4 in activating cell type-specific enhancers. Together, these findings reveal a positive feedback between BAF and MLL4 in promoting LDTF-dependent activation of cell type-specific enhancers.

The LEAFY floral regulator displays pioneer transcription factor properties

Pioneer transcription factors (TFs) are a special category of TFs with the capacity to bind to closed chromatin regions in which DNA is wrapped around histones and may be highly methylated. Subsequently, pioneer TFs are able to modify the chromatin state to initiate gene expression. In plants, LEAFY (LFY) is a master floral regulator and has been suggested to act as a pioneer TF in Arabidopsis. Here, we demonstrate that LFY is able to bind both methylated and non-methylated DNA using a combination of in vitro genome-wide binding experiments and structural modeling. Comparisons between regions bound by LFY in vivo and chromatin accessibility data suggest that a subset of LFY bound regions is occupied by nucleosomes. We confirm that LFY is able to bind nucleosomal DNA in vitro using reconstituted nucleosomes. Finally, we show that constitutive LFY expression in seedling tissues is sufficient to induce chromatin accessibility in the LFY direct target genes APETALA1 and AGAMOUS. Taken together, our study suggests that LFY possesses key pioneer TF features that contribute to launching the floral gene expression program.

Relative DNA Methylation and Demethylation Efficiencies during Postnatal Liver Development Regulate Hepatitis B Virus Biosynthesis.

Hepatitis B virus (HBV) transcription and replication increase progressively throughout postnatal liver development with maximal viral biosynthesis occurring at around 4 weeks of age in the HBV transgenic mouse model of chronic infection. Increasing viral biosynthesis is associated with a corresponding progressive loss of DNA methylation. The loss of DNA methylation is associated with increasing levels of 5-hydroxymethylcytosine (5hmC) residues which correlate with increased liver-enriched pioneer transcription factor Forkhead box protein A (FoxA) RNA levels, a rapid decline in postnatal liver DNA methyltransferase (Dnmt) transcripts, and a very modest reduction in ten-eleven translocation (Tet) methylcytosine dioxygenase expression. These observations are consistent with the suggestion that the balance between active HBV DNA methylation and demethylation is regulated by FoxA recruitment of Tet in the presence of declining Dnmt activity. These changes lead to demethylation of the viral genome during hepatocyte maturation with associated increases in viral biosynthesis. Consequently, manipulation of the relative activities of these two counterbalancing processes might permit the specific silencing of HBV gene expression with the loss of viral biosynthesis and the resolution of chronic HBV infections.IMPORTANCE HBV biosynthesis begins at birth and increases during early postnatal liver development in the HBV transgenic mouse model of chronic infection. The levels of viral RNA and DNA synthesis correlate with pioneer transcription factor FoxA transcript plus Tet methylcytosine dioxygenase-generated 5hmC abundance but inversely with Dnmt transcript levels and HBV DNA methylation. Together, these findings suggest that HBV DNA methylation during neonatal liver development is actively modulated by the relative contributions of FoxA-recruited Tet-mediated DNA demethylation and Dnmt-mediated DNA methylation activities. This mode of gene regulation, mediated by the loss of DNA methylation at hepatocyte-specific viral and cellular promoters, likely contributes to hepatocyte maturation during liver development in addition to the postnatal activation of HBV transcription and replication.

Abstract PD8-03: A FOXA1/FRA1-centered transcriptional axis regulates interferon signaling in high FOXA1-associated endocrine-resistant and metastatic breast cancer

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

MAPK4 promotes prostate cancer by concerted activation of androgen receptor and AKT.

Prostate cancer (PCa) is the second leading cause of cancer death in American men. Androgen receptor (AR) signaling is essential for PCa cell growth/survival and remains a key therapeutic target for lethal castration-resistant PCa (CRPC). GATA2 is a pioneer transcription factor crucial for inducing AR expression/activation. We recently reported that MAPK4, an atypical MAPK, promotes tumor progression via noncanonical activation of AKT. Here, we demonstrated that MAPK4 activated AR by enhancing GATA2 transcriptional expression and stabilizing GATA2 protein through repression of GATA2 ubiquitination/degradation. MAPK4 expression correlated with AR activation in human CRPC. Concerted activation of both GATA2/AR and AKT by MAPK4 promoted PCa cell proliferation, anchorage-independent growth, xenograft growth, and castration resistance. Conversely, knockdown of MAPK4 decreased activation of both AR and AKT and inhibited PCa cell and xenograft growth, including castration-resistant growth. Both GATA2/AR and AKT activation were necessary for MAPK4 tumor-promoting activity. Interestingly, combined overexpression of GATA2 plus a constitutively activated AKT was sufficient to drive PCa growth and castration resistance, shedding light on an alternative, MAPK4-independent tumor-promoting pathway in human PCa. We concluded that MAPK4 promotes PCa growth and castration resistance by cooperating parallel pathways of activating GATA2/AR and AKT and that MAPK4 is a novel therapeutic target in PCa, especially CRPC.

Development: Keeping Time with Transcription

A new study shows that the pioneer transcription factor BLMP-1 governs the cyclic transcriptional output of hundreds of genes during Caenorhabditis elegans larval development. BLMP-1 is also critical for restarting the proper developmental dynamics of gene expression after nutritionally induced developmental arrest.

Regulation of CEACAM5 and Therapeutic Efficacy of an Anti-CEACAM5-SN38 Antibody-drug Conjugate in Neuroendocrine Prostate Cancer.

Neuroendocrine prostate cancer (NEPC) is an aggressive form of castration-resistant prostate cancer (CRPC) for which effective therapies are lacking. We previously identified carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) as a promising NEPC cell surface antigen. Here we investigated the scope of CEACAM5 expression in end-stage prostate cancer, the basis for CEACAM5 enrichment in NEPC, and the therapeutic potential of the CEACAM5 antibody-drug conjugate labetuzumab govitecan in prostate cancer. The expression of CEACAM5 and other clinically relevant antigens was characterized by multiplex immunofluorescence of a tissue microarray comprising metastatic tumors from 34 lethal metastatic CRPC (mCRPC) cases. A genetically defined neuroendocrine transdifferentiation assay of prostate cancer was developed to evaluate mechanisms of CEACAM5 regulation in NEPC. The specificity and efficacy of labetuzumab govitecan was determined in CEACAM5+ prostate cancer cell lines and patient-derived xenografts models. CEACAM5 expression was enriched in NEPC compared with other mCRPC subtypes and minimally overlapped with prostate-specific membrane antigen, prostate stem cell antigen, and trophoblast cell surface antigen 2 expression. We focused on a correlation between the expression of the pioneer transcription factor ASCL1 and CEACAM5 to determine that ASCL1 can drive neuroendocrine reprogramming of prostate cancer which is associated with increased chromatin accessibility of the CEACAM5 core promoter and CEACAM5 expression. Labetuzumab govitecan induced DNA damage in CEACAM5+ prostate cancer cell lines and marked antitumor responses in CEACAM5+ CRPC xenograft models including chemotherapy-resistant NEPC. Our findings provide insights into the scope and regulation of CEACAM5 expression in prostate cancer and strong support for clinical studies of labetuzumab govitecan for NEPC.

LEAFY is a pioneer transcription factor and licenses cell reprogramming to floral fate

Master transcription factors reprogram cell fate in multicellular eukaryotes. Pioneer transcription factors have prominent roles in this process because of their ability to contact their cognate binding motifs in closed chromatin. Reprogramming is pervasive in plants, whose development is plastic and tuned by the environment, yet little is known about pioneer transcription factors in this kingdom. Here, we show that the master transcription factor LEAFY (LFY), which promotes floral fate through upregulation of the floral commitment factor APETALA1 (AP1), is a pioneer transcription factor. In vitro, LFY binds to the endogenous AP1 target locus DNA assembled into a nucleosome. In vivo, LFY associates with nucleosome occupied binding sites at the majority of its target loci, including AP1. Upon binding, LFY ‘unlocks’ chromatin locally by displacing the H1 linker histone and by recruiting SWI/SNF chromatin remodelers, but broad changes in chromatin accessibility occur later. Our study provides a mechanistic framework for patterning of inflorescence architecture and uncovers striking similarities between LFY and animal pioneer transcription factor.

Clinical and Biological Features of Neuroendocrine Prostate Cancer.

Neuroendocrine prostate cancer (NEPC) is an aggressive histologic subtype of prostate cancer that most commonly arises in later stages of prostate cancer as a mechanism of treatment resistance. The poor prognosis of NEPC is attributed in part to late diagnosis and a lack of effective therapeutic agents. Here, we review the clinical and molecular features of NEPC based on recent studies and outline future strategies and directions. NEPC can arise "de novo" but most commonly develops as a result of lineage plasticity whereby prostate cancer cells adopt alternative lineage programs as a means to bypass therapy. Dependence on androgen receptor (AR) signaling is lost as tumors progress from a prostate adenocarcinoma to a NEPC histology, typically manifested by the downregulation of AR, PSA, and PSMA expression in tumors. Genomic analyses from patient biopsies combined with preclinical modeling have pointed to loss of tumor suppressors RB1 and TP53 as key facilitators of lineage plasticity. Activation of oncogenic drivers combined with significant epigenetic changes (e.g., EZH2 overexpression, DNA methylation) further drives tumor proliferation and expression of downstream neuronal and neuroendocrine lineage pathways controlled in part by pioneer and lineage determinant transcription factors (e.g., SOX2, ASCL1, BRN2). These biologic insights have provided a framework for the study of this subgroup of advanced prostate cancers and have started to provide rationale for the development of biomarker-driven therapeutic strategies. Further study of the dynamic process that leads to NEPC is required for the development of effective strategies to identify and treat patients developing lineage plasticity as a mechanism of treatment resistance.