AP-1 Family Of Transcription Factors Research Articles

Genome-wide CRISPR/Cas9 screen reveals JunB downmodulation of HIV co-receptor CXCR4

HIV-1 relies extensively on host cell machinery for replication. Identification and characterization of these host-virus interactions is vital to our understanding of viral replication and the consequences of infection in cells. Several prior screens have identified host factors important for HIV replication but with limited replication of findings, likely due to differences in experimental design and conditions. Thus, unidentified factors likely exist. To identify novel host factors required for HIV-1 infection, we performed a genome-wide CRISPR/Cas9 screen using HIV-induced cell death as a partitioning method. We created a gene knockout library in TZM-GFP reporter cells using GeCKOv2, which targets 19,050 genes, and infected the library with a lethal dose of HIV-1NL4-3. We hypothesized that cells with a knockout of a gene critical for HIV infection would survive while cells with a knockout of a non-consequential gene would undergo HIV-induced death and be lost from the population. Surviving cells were analyzed by high throughput sequencing of the integrated CRISPR/Cas9 cassette to identify the gene knockout. Of the gene targets, an overwhelming majority of the surviving cells harbored the guide sequence for the AP-1 transcription factor family protein, JunB. Upon the generation of a clonal JunB knockout cell line, we found that HIV-1NL4-3 infection was blocked in the absence of JunB. The phenotype resulted from downregulation of CXCR4, as infection levels were recovered by reintroduction of CXCR4 in JunB KO cells. Thus, JunB downmodulates CXCR4 expression in TZM-GFP cells, reducing CXCR4-tropic HIV infection.

Epigenetic Memory of Intestinal Epithelial Cells in Inflammatory Bowel Disease

Background: The pathogenesis of human Inflammatory Bowel Disease (IBD) is a multifactorial process including interaction between immune, epithelial, and environmental factors. Growing evidence suggests that epithelial cell defects play major role in the pathogenesis of IBD. Epigenetic memory involves lasting, cell-type specific modifications to chromatin at multiple scales, which impacts transcriptional programs required for normal cell functions. A major gap in knowledge are the mechanisms involved in establishing and maintaining memory in cell types and diseases of the intestine. The objective of this study was to determine if intestinal epithelial cells (IECs) retain a memory of altered chromatin architectures and resultant regulation following inflammation. The hypothesis was that the IECs would show lasting altered chromatin structure landscapes (epigenetic memory) in response to inflammation. Methods: We have developed a multicellular model system of IBD using adult stem cell derived organoids from humans with IBD, from regions of the colon with and without active inflammation. We performed ATAC-seq, bulk RNA-seq and single cell RNA-seq on these organoids derived from the uninflamed and inflamed tissues of IBD patients, specifically Ulcerative Colitis (UC). Results: Regions of increased chromatin accessibility are enriched in organoids derived from areas of inflammation in UC patients compared to organoids from patient-matched, uninflamed regions. These open chromatin regions are associated with proinflammatory genes, with modest transcriptional alterations, pointing to an epigenetic inflammatory memory. Motif analysis for accessibly peaks in UC showed an enrichment of the AP-1 family of transcription factors and chromatin remodelers (SMARCA4). This data is consistent with other studies of inflammatory memory from other epithelial cell types, but we show this for the first time in IECs in the context of IBD. Conclusions: This study presents a novel line of investigation and development of model systems to examine epigenetic memory of IECs in human IBD. We expect that our studies will identify new chromatin-based mechanisms of IEC function that are impacted by inflammation. This research was funded by American Gastroenterological Association Research Scholar Award and KL2 Scholar Career Development Award (KL2 TR002379, National Center for Advancing Translational Science). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Integrative single-cell chromatin and transcriptome analysis of human plasma cell differentiation

AbstractPlasma cells (PCs) are highly specialized cells representing the end stage of B-cell differentiation. We have shown that PC differentiation can be reproduced in vitro using elaborate culture systems. The molecular changes occurring during PC differentiation are recapitulated in this in vitro differentiation model. However, a major challenge exists to decipher the spatiotemporal epigenetic and transcriptional programs that drive the early stages of PC differentiation. We combined single cell (sc) RNA sequencing (RNA-seq) and scATAC-seq to decipher the trajectories involved in PC differentiation. ScRNA-seq experiments revealed a strong heterogeneity of the preplasmablastic and plasmablastic stages. Among genes that were commonly identified using scATAC-seq and scRNA-seq, we identified several transcription factors with significant stage specific potential importance in PC differentiation. Interestingly, differentially accessible peaks characterizing the preplasmablastic stage were enriched in motifs of BATF3, FOS and BATF, belonging to the AP-1 transcription factor family that may represent key transcriptional nodes involved in PC differentiation. Integration of transcriptomic and epigenetic data at the single cell level revealed that a population of preplasmablasts had already undergone epigenetic remodeling related to PC profile together with unfolded protein response activation and are committed to differentiate in PC. These results and the supporting data generated with our in vitro PC differentiation model provide a unique resource for the identification of molecular circuits that are crucial for early and mature PC maturation and biological functions. These data thus provide critical insights into epigenetic- and transcription–mediated reprogramming events that sustain PC differentiation.

Abstract 3940: Nuclear translocation of HER3 promotes breast cancer progression and dissemination recruiting immune cells via CXCL1 and CXCL8

Abstract Breast cancer is a highly complex multifactorial disease, which can be driven by the aberrant regulation of different signaling pathways including receptor tyrosine kinase (RTK) signaling. Notably, while RTKs have been classically described as transmembrane receptors, they can also translocate to the nucleus, even though the functional importance of this process remains largely unexplored for most RTKs. Here, we report a novel role for the nuclear form of the RTK human epidermal growth factor receptor 3 (HER3) in driving primary breast cancer growth and metastasis. Firstly, using different patient-derived organoids (PDOs) established from metastatic breast cancer patients, we demonstrated the robust translocation of the activated phosphorylated HER3 receptor (pHER3) from the plasma membrane to the nucleus in response to its ligand Neuregulin 1 (NRG1). Interestingly, the nuclear translocation was observed not only in the HER2-positive breast cancer subtype but also in luminal and triple-negative breast cancer-derived cells. In order to decipher the functional role of nuclear HER3 (nHER3), we identified and mutated its nuclear localization signal, resulting in decreased nuclear translocation of the receptor while its membrane form remained intact. This reduction in nHER3 remarkably impeded primary tumor growth and metastatic spread in different patient-derived xenograft (PDX) models. Conversely, selective overexpression of HER3 in the nucleus led to increased primary tumor growth and metastatic burden in vivo. In order to decipher the mechanism of action of nHER3, we performed co-immunoprecipitation followed by global mass spectrometric analysis to identify the protein binding partners of the receptor in the nucleus. Specifically, nHER3 was found to interact with different transcription factors such as the AP-2 family of transcription factors and with major chromatin remodeling complexes like the nucleosome remodeling and deacetylase (NuRD) complex, implicating its role in transcriptional regulation. Furthermore, gene expression analysis of PDOs expressing the wild-type or the mutated form of HER3 revealed the modulation of several key growth regulators such as early growth response 3 (EGR3) by nHER3. The nHER3-EGR3 axis further controlled downstream effectors like the immune chemoattractants CXCL1 and CXCL8. Accordingly, PDX from breast cancer cells overexpressing nHER3 showed significantly increased immune infiltration, suggesting a possible role of nHER3 in regulating the tumor microenvironment. Altogether, we report here a novel non-canonical role of the nuclear form of HER3 receptor in driving breast tumorigenesis, with key implications for our understanding and targeting of RTK signaling in breast cancer. Citation Format: Tasneem Cheytan, Roberto Würth, Nina Hahnen, Elisa Donato, Corinna Klein, Rebecca Weber, Daniele Colombo, Jeroen Krijgsveld, Martin Sprick, Andreas Trumpp. Nuclear translocation of HER3 promotes breast cancer progression and dissemination recruiting immune cells via CXCL1 and CXCL8 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3940.

Expansion of memory Vδ2 T cells following SARS-CoV-2 vaccination revealed by temporal single-cell transcriptomics

γδ T cells provide rapid cellular immunity against pathogens. Here, we conducted matched single-cell RNA-sequencing and γδ-TCR-sequencing to delineate the molecular changes in γδ T cells during a longitudinal study following mRNA SARS-CoV-2 vaccination. While the first dose of vaccine primes Vδ2 T cells, it is the second administration that significantly boosts their immune response. Specifically, the second vaccination uncovers memory features of Vδ2 T cells, shaped by the induction of AP-1 family transcription factors and characterized by a convergent central memory signature, clonal expansion, and an enhanced effector potential. This temporally distinct effector response of Vδ2 T cells was also confirmed in vitro upon stimulation with SARS-CoV-2 spike-peptides. Indeed, the second challenge triggers a significantly higher production of IFNγ by Vδ2 T cells. Collectively, our findings suggest that mRNA SARS-CoV-2 vaccination might benefit from the establishment of long-lasting central memory Vδ2 T cells to confer protection against SARS-CoV-2 infection.

Chromatin profiling reveals TFAP4 as a critical transcriptional regulator of bovine satellite cell differentiation

BackgroundSatellite cells are myogenic precursor cells in adult skeletal muscle and play a crucial role in skeletal muscle regeneration, maintenance, and growth. Like embryonic myoblasts, satellite cells have the ability to proliferate, differentiate, and fuse to form multinucleated myofibers. In this study, we aimed to identify additional transcription factors that control gene expression during bovine satellite cell proliferation and differentiation.ResultsUsing chromatin immunoprecipitation followed by sequencing, we identified 56,973 and 54,470 genomic regions marked with both the histone modifications H3K4me1 and H3K27ac, which were considered active enhancers, and 50,956 and 59,174 genomic regions marked with H3K27me3, which were considered repressed enhancers, in proliferating and differentiating bovine satellite cells, respectively. In addition, we identified 1,216 and 1,171 super-enhancers in proliferating and differentiating bovine satellite cells, respectively. Analyzing these enhancers showed that in proliferating bovine satellite cells, active enhancers were associated with genes stimulating cell proliferation or inhibiting myoblast differentiation whereas repressed enhancers were associated with genes essential for myoblast differentiation, and that in differentiating satellite cells, active enhancers were associated with genes essential for myoblast differentiation or muscle contraction whereas repressed enhancers were associated with genes stimulating cell proliferation or inhibiting myoblast differentiation. Active enhancers in proliferating bovine satellite cells were enriched with binding sites for many transcription factors such as MYF5 and the AP-1 family transcription factors; active enhancers in differentiating bovine satellite cells were enriched with binding sites for many transcription factors such as MYOG and TFAP4; and repressed enhancers in both proliferating and differentiating bovine satellite cells were enriched with binding sites for NF-kB, ZEB-1, and several other transcription factors. The role of TFAP4 in satellite cell or myoblast differentiation was previously unknown, and through gene knockdown and overexpression, we experimentally validated a critical role for TFAP4 in the differentiation and fusion of bovine satellite cells into myofibers.ConclusionsSatellite cell proliferation and differentiation are controlled by many transcription factors such as AP-1, TFAP4, NF-kB, and ZEB-1 whose roles in these processes were previously unknown in addition to those transcription factors such as MYF5 and MYOG whose roles in these processes are widely known.

A Pan-Cancer Single-Cell Transcriptomic Atlas of Natural Killer (NK) Cells Reveals Intrinsic and Extrinsic Mediators of NK Cell Anti-Tumor Functions

Background: Natural killer (NK) cells are critical mediators of anti-tumor immunity, engaging in direct cytotoxic responses and promoting pro-inflammatory immune microenvironments. The molecular mechanisms underlying these functions, however, have yet to be fully elucidated. This is due in part to a lack of understanding of the phenotypic and functional diversity of NK cells across cancer types. Here, we present a single-cell transcriptomic atlas of >250,000 NK cells spanning 340 patients across 17 cancer types to advance our understanding of tumor-infiltrating NK cells. Methods: We re-analyzed publicly available single-cell RNA-sequencing (scRNA-seq) datasets of primary, therapy-naïve tumors, as well as matching peripheral blood samples and healthy donor blood samples. We employed systematic computational analyses to deconvolute the phenotypic and functional diversity of tumor-infiltrating NK cells and define key regulators of NK cell functions in both a cancer type-specific and pan-cancer manner. Feature expression module scoring was used to infer phenotypic and functional characteristics of NK cell clusters in each cancer type. Transcription factor regulon and cell-cell communication prediction algorithms were applied to understand the mechanistic drivers of NK cell populations within tumors. Results: Tumor-infiltrating NK cells display distinct patterns of phenotypic and functional features across cancer types. Nonetheless, we identify consensus NK cell populations across cancers that demonstrate the utility and performance of our gene module-based phenotypic and functional annotations. Interestingly, we observe the enrichment of adaptive-like and CD56 bright NK cell populations in tumors that exhibit features of tissue residency, including the upregulation of ITGA1, ITGAE, and CD69 alongside the downregulation of FCG3RA. Notably, these tissue-resident populations display enhanced chemokine and cytokine activity compared to their recruited counterparts, suggesting the significance of tissue-resident NK cell populations in immune recruitment to tumors. We find the phenotypic composition of intra-tumoral NK cells is vastly different than that of circulating NK cells which harbor predominantly CD56 dim populations, as well as a significant proportion of adaptive-like NK cells in several cancer types. Leveraging the paired nature of several tumor and peripheral blood samples, we performed temporal comparative analyses that revealed the robust expression of KLF2 and S1PR5 in circulation which is then lost in paired tumor tissues. While KLF2 and S1PR5 have been previously implicated in the homing and migration of immune cells, their roles in the trafficking of NK cells to tumors have yet to be defined. In modeling transitions from canonical to stressed NK cell states, we not only identify well-established NK cell checkpoints ( TIM-3, LAG3, and CISH) but also identify novel putative checkpoints yet to be investigated in NK cells, such as the AP-1 family of transcription factors. Additionally, we examine extrinsic factors contributing to intra-tumoral NK cell phenotypes, detecting significant crosstalk between NK cells and immunomodulatory macrophages and CD4+ T cells mediated by galectin-9 and TGFβ interactions. Conclusions: We define the phenotypic and functional pan-cancer landscape of NK cells and uncover both intrinsic and extrinsic factors influencing their anti-tumor functions. We envision our NK cell atlas will facilitate the development of strategies to enhance NK cell-based immunotherapies against cancer.

A TGF-β signaling-related lncRNA signature for prediction of glioma prognosis, immune microenvironment, and immunotherapy response.

The dysregulation of TGF-β signaling is a crucial pathophysiological process in tumorigenesis and progression. LncRNAs have diverse biological functions and are significant participants in the regulation of tumor signaling pathways. However, the clinical value of lncRNAs related to TGF-β signaling in glioma is currently unclear. Data on glioma's RNA-seq transcriptome, somatic mutation, DNA methylation data, and clinicopathological information were derived from the CGGA and TCGA databases. A prognostic lncRNA signature was constructed by Cox and LASSO regression analyses. TIMER2.0 database was utilized to deduce immune infiltration characteristics. "ELMER v.2" was used to reconstruct TF-methylation-gene regulatory network. Immunotherapy and chemotherapy response predictions were implemented by the TIDE algorithm and GDSC database, respectively. Invitro and invivo experiments were conducted to verify the results and clarify the regulatory mechanism of lncRNA. In glioma, a TGF-β signaling-related 15-lncRNA signature was constructed, including AC010173.1, HOXA-AS2, AC074286.1, AL592424.1, DRAIC, HOXC13-AS, AC007938.1, AC010729.1, AC013472.3, AC093895.1, AC131097.4, AL606970.4, HOXC-AS1, AGAP2-AS1, and AC002456.1. This signature proved to be a reliable prognostic tool, with high risk indicating an unfavorable prognosis and being linked to malignant clinicopathological and genomic mutation traits. Risk levels were associated with different immune infiltration landscapes, where high risk was indicative of high levels of macrophage infiltration. In addition, high risk also suggested better immunotherapy and chemotherapy response. cg05987823 was an important methylation site in glioma progression, and AP-1 transcription factor family participated in the regulation of signature lncRNA expression. AGAP2-AS1 knockdown in invitro and invivo experiments inhibited the proliferation, migration, and invasion of glioma cells, as well as the growth of glioma, by downregulating the expression levels of NF-κB and ERK 1/2 in the TGF-β signaling pathway. A prognostic lncRNA signature of TGF-β signaling was established in glioma, which can be used for prognostic judgment, immune infiltration status inference, and immunotherapy response prediction. AGAP2-AS1 plays an important role in glioma progression.

An 840 kb distant upstream enhancer is a crucial regulator of catecholamine-dependent expression of the Bdnf gene in astrocytes.

Brain-derived neurotrophic factor (BDNF) plays a fundamental role in the developing and adult nervous system, contributing to neuronal survival, differentiation, and synaptic plasticity. Dysregulation of BDNF synthesis, secretion or signaling has been associated with many neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. Although the transcriptional regulation of the Bdnf gene has been extensively studied in neurons, less is known about the regulation and function of BDNF in non-neuronal cells. The most abundant type of non-neuronal cells in the brain, astrocytes, express BDNF in response to catecholamines. However, genetic elements responsible for this regulation have not been identified. Here,we investigated four potential Bdnf enhancer regions and based on reporter gene assays, CRISPR/Cas9 engineering and CAPTURE-3C-sequencing we conclude that a region 840 kb upstream of the Bdnf gene regulates catecholamine-dependent expression of Bdnf in rodent astrocytes. We also provide evidence that this regulation is mediated by CREB and AP1 family transcription factors. This is the first report of an enhancer coordinating the transcription of Bdnf gene in non-neuronal cells.

Transcriptional variation in Babesia gibsoni (Wuhan isolate) between in vivo and in vitro cultures in blood stage

BackgroundBabesia gibsoni, the causative agent of canine babesiosis, belongs to the phylum Apicomplexa. The development of in vitro culture technology has driven research progress in various kinds of omics studies, including transcriptomic analysis of Plasmodium spp. between in vitro and in vivo environments, which has prompted the observation of diagnostic antigens and vaccine development. Nevertheless, no information on Babesia spp. could be obtained in this respect, which greatly hinders the further understanding of parasite growth and development in the blood stage.MethodsIn this study, considerable changes in the morphology and infectivity of continuous in vitro cultured B. gibsoni (Wuhan isolate) were observed compared to in vivo parasites. Based on these changes, B. gibsoni (Wuhan isolate) was collected from both in vivo and in vitro cultures, followed by total RNA extraction and Illumina transcriptome sequencing. The acquired differentially expressed genes (DEGs) were validated using qRT-PCR, and then functionally annotated through several databases. The gene with the greatest upregulation after in vitro culture was cloned from the genome of B. gibsoni (Wuhan isolate) and characterized by western blotting and indirect immunofluorescence assay for detecting the native form and cellular localization.ResultsThrough laboratory cultivation, multiple forms of parasites were observed, and the infectivity of in vitro cultured parasites in dogs was found to be lower. Based on these changes, Illumina transcriptome sequencing was conducted, showing that 377 unigenes were upregulated and 334 unigenes were downregulated. Notably, an AP2 transcription factor family, essential for all developmental stages of parasites, was screened, and the transcriptional changes in these family members were tested. Thus, the novel AP2 transcription factor gene (BgAP2-M) with the highest upregulated expression after in vitro adaptation was selected. This gene comprises an open reading frame (ORF) of 1989 base pairs encoding a full-length protein of 662 amino acids. BgAP2-M contains one AP2 domain and one ACDC conserved domain, which may be involved in the nuclear biology of parasites. The prepared polyclonal antibodies against the BgAP2-M peptides further detected a native size of ~ 73 kDa and were localized to the nuclei of B. gibsoni.ConclusionThis study presents a thorough transcriptome analysis of B. gibsoni in vivo and in vitro for the first time, contributing to a detailed understanding of the effects of environmental changes on the growth and development of parasites in the blood stage. Moreover, it also provides a deeper investigation for the different members of the ApiAP2 transcription factor family as various life stage regulators in Babesia spp.Graphical

Abstract P2114: Chromatin Accessibility Governs Mechanosensitive Gene Expression In Cardiac Fibroblasts

Objective: We recently demonstrated regionalized myocardial fibrosis in patients undergoing surgical repair for Mitral Valve Prolapse. The location of fibrosis suggests a mechanically driven process wherein mechanical stress alters the phenotype of ventricular fibroblasts to promote an increase in tissue stiffness, eventually causing establishment of a fibrotic scar. We sought to better understand how mechanical stiffness affected cellular programs. Methods: We cultured primary human cardiac fibroblasts derived from the same patient on a mechanically stiff (TC Plastic, ~2GPa), as well as a mechanically soft (2kPa) substrate and then performed immunofluorescence, western blotting, RNA Seq, and ATAC Seq. Following differential analysis of omics data, we performed an integration analysis by filterinig differentially accessible chromatin to only include sites that fell within 10 kB of opened/closed chromatin. Finally, we performed a motif enrichment analysis using the online bioinformatics package Homer. Results: Immunofluorescence and western blotting revealed that cells cultured on a stiff substrate expressed canonical markers of fibroblast activation compared with soft substrates (alpha SMA, COL1A1, P<0.0001). RNA Sequencing revealed differential changes in 1615 genes in fibroblasts (FDR adjusted P<1.6*10 -6 ), several of which were subsequently validated using western blot. ATAC Seq identified 5855 regions across the genome which opened/closed in response to different mechanical environments in fibroblasts (adjusted P<0.05). Integrating these datasets revealed 803 Chromatin changes that fell within 10kb of differentially expressed genes, with AP1 and TEAD family transcription factors predicted to be master regulators of closing and opening chromatin, respectively. Proof-of-concept motif validation conducted on differentially expressed chromatin within intron 12 of the MYH10 gene demonstrated presence of a transcriptional repressor with evidence of JunD as a binding partner for this site. Conclusions: The mechanical environment plays a major role in cellular phenotype and regulates chromatin organization and expression of pro-fibrotic genes through changes in chromatin accessibility.

Overexpression of REDUCED WALL ACETYLATION C increases xylan acetylation and biomass recalcitrance in Populus.

Plant lignocellulosic biomass, i.e. secondary cell walls of plants, is a vital alternative source for bioenergy. However, the acetylation of xylan in secondary cell walls impedes the conversion of biomass to biofuels. Previous studies have shown that REDUCED WALL ACETYLATION (RWA) proteins are directly involved in the acetylation of xylan but the regulatory mechanism of RWAs is not fully understood. In this study, we demonstrate that overexpression of a Populus trichocarpa PtRWA-C gene increases the level of xylan acetylation and increases the lignin content and S/G ratio, ultimately yielding poplar woody biomass with reduced saccharification efficiency. Furthermore, through gene coexpression network and expression quantitative trait loci (eQTL) analysis, we found that PtRWA-C was regulated not only by the secondary cell wall hierarchical regulatory network but also by an AP2 family transcription factor HARDY (HRD). Specifically, HRD activates PtRWA-C expression by directly binding to the PtRWA-C promoter, which is also the cis-eQTL for PtRWA-C. Taken together, our findings provide insights into the functional roles of PtRWA-C in xylan acetylation and consequently saccharification and shed light on synthetic biology approaches to manipulate this gene and alter cell wall properties. These findings have substantial implications for genetic engineering of woody species, which could be used as a sustainable source of biofuels, valuable biochemicals, and biomaterials.

A Carboxy-terminal Smarcb1 Point Mutation Induces Hydrocephalus Formation and Affects AP-1 and Neuronal Signalling Pathways in Mice

The BAF (BRG1/BRM-associated factor) chromatin remodelling complex is essential for the regulation of DNA accessibility and gene expression during neuronal differentiation. Mutations of its core subunit SMARCB1 result in a broad spectrum of pathologies, including aggressive rhabdoid tumours or neurodevelopmental disorders. Other mouse models have addressed the influence of a homo- or heterozygous loss of Smarcb1, yet the impact of specific non-truncating mutations remains poorly understood. Here, we have established a new mouse model for the carboxy-terminal Smarcb1 c.1148del point mutation, which leads to the synthesis of elongated SMARCB1 proteins. We have investigated its impact on brain development in mice using magnetic resonance imaging, histology, and single-cell RNA sequencing. During adolescence, Smarcb11148del/1148del mice demonstrated rather slow weight gain and frequently developed hydrocephalus including enlarged lateral ventricles. In embryonic and neonatal stages, mutant brains did not differ anatomically and histologically from wild-type controls. Single-cell RNA sequencing of brains from newborn mutant mice revealed that a complete brain including all cell types of a physiologic mouse brain is formed despite the SMARCB1 mutation. However, neuronal signalling appeared disturbed in newborn mice, since genes of the AP-1 transcription factor family and neurite outgrowth-related transcripts were downregulated. These findings support the important role of SMARCB1 in neurodevelopment and extend the knowledge of different Smarcb1 mutations and their associated phenotypes.

Impact of supraphysiologic MDM2 expression on chromatin networks and therapeutic responses in sarcoma

Amplification of MDM2 on supernumerary chromosomes is a common mechanism of P53 inactivation across tumors. Here, we investigated the impact of MDM2 overexpression on chromatin, gene expression, and cellular phenotypes in liposarcoma. Three independent regulatory circuits predominate in aggressive, dedifferentiated tumors. RUNX and AP-1 family transcription factors bind mesenchymal gene enhancers. P53 and MDM2 co-occupy enhancers and promoters associated with P53 signaling. When highly expressed, MDM2 also binds thousands of P53-independent growth and stress response genes, whose promoters engage in multi-way topological interactions. Overexpressed MDM2 concentrates within nuclear foci that co-localize with PML and YY1 and could also contribute to P53-independent phenotypes associated with supraphysiologic MDM2. Importantly, we observe striking cell-to-cell variability in MDM2 copy number and expression in tumors and models. Whereas liposarcoma cells are generally sensitive to MDM2 inhibitors and their combination with pro-apoptotic drugs, MDM2-high cells tolerate them and may underlie the poor clinical efficacy of these agents.

AP-1 is a regulatory transcription factor of inflammaging in the murine kidney and liver.

Aging is characterized by chronic low-grade inflammation in multiple tissues, also termed "inflammaging", which represents a significant risk factor for many aging-related chronic diseases. However, the mechanisms and regulatory networks underlying inflammaging across different tissues have not yet been fully elucidated. Here, we profiled the transcriptomes and epigenomes of the kidney and liver from young and aged mice and found that activation of the inflammatory response is a conserved signature in both tissues. Moreover, we revealed links between transcriptome changes and chromatin dynamics through integrative analysis and identified AP-1 and ETS family transcription factors (TFs) as potential regulators of inflammaging. Further in situ validation showed that c-JUN (a member of the AP-1 family) was mainly activated in aged renal and hepatic cells, while increased SPI1 (a member of the ETS family) was mostly induced by elevated infiltration of macrophages, indicating that these TFs have different mechanisms in inflammaging. Functional data demonstrated that genetic knockdown of Fos, a major member of the AP-1 family, significantly attenuated the inflammatory response in aged kidneys and livers. Taken together, our results revealed conserved signatures and regulatory TFs of inflammaging in the kidney and liver, providing novel targets for the development of anti-aging interventions.

Discovery of a molecular clock that controls CD8+ T cell function and exhaustion

Abstract During cancer and chronic viral infections, the persistence of antigen progressively causes CD8+ T cells to differentiate into a dysfunctional PD1+ “exhausted” state, with reduced production of inflammatory cytokines relative to effector cells that form during acute infections. Antigen and costimulation signals activate kinase cascades to induce distinct T cell transcription programs, but how T cells distinguish acute and chronic signals to program the exhausted or effector transcriptional states remains poorly understood. We found that members of the protein kinase C (PKC) family function together as a “molecular clock,” sensing acute or chronic agonism to drive distinct transcriptional programs. Continuous stimulation of PKC induces many features of T cell exhaustion, including a loss of production of the cytokines IFNγ and TNF, upregulation of inhibitory receptors and TOX, and altered expression of the proteins in the AP-1 family. Mechanistically, CD8+ T cells express several different PKC proteins, and chronic agonism of PKC leads to degradation of multiple family members and selective maintenance of only one PKC protein, PKC-η. This “PKC switch” alters downstream signaling to support the transcriptional reprogramming of T cells into a terminally exhausted state. In summary, continuous signaling through PKCs causes changes in the output from these kinases initially at the protein level, driving transcriptional changes downstream of PKC targets in the AP-1 transcription factor family and thus allowing further widespread transcriptional and functional changes that characterize T cell exhaustion. Supported by a Damon Runyon Cancer Research Fellowship (DRG-2358-19) and an NIH training grant, T32-CA009370.

Antigen experience history directs distinct functional states of CD8+ CAR T cells during the anti-leukemia response

Abstract Chimeric antigen receptor (CAR) T cells are highly effective in treating hematologic cancers, but patients can relapse due to downregulation of the CAR-targeted antigen, leading to the hypothesis that CAR T cells require high density of target antigen to be effective. Non-engineered CD8+ memory T cells generate more rapid and robust effector T cell responses upon secondary infection, relative to their naïve counterparts, and can display higher antigen sensitivity. Our objective was to characterize the role of prior CD8+ T cell antigen experience (AgEx) in dictating functional CAR-mediated anti-tumor responses. To study the effect of T cell AgEx history on CD8+ CAR T cell (CAR8) function in vivo, we used a vaccine model to control prior T cell AgEx history, and a syngeneic adoptive transfer model with murine anti-CD19 CAR8 targeting murine leukemia engineered to express a low or high level of CD19. We analyzed CAR8 expansion and leukemia burden using flow cytometry and performed ATACseq and RNAseq at timepoints pre- and post-CAR transduction and after reinfusion of CAR8 into leukemia-bearing mice to ask whether CAR8 function was determined by the epigenetic and/or transcriptomic state of the starting cell population. We found that starting cell state dictated in vivo CAR8 effector functions, expansion, and differential chromatin accessibility in regions containing binding motifs for AP-1, Runx, Tcf and other transcription factor (TF) families. Motif chromatin accessibility and gene expression of many TFs showed distinct patterns temporally. We conclude that CAR8 maintain functional traits epigenetically imprinted in the starting cell and predict these analyses could inform methods to transcriptionally modulate CAR T cell function.

Abstract NG10: Chromatin profiles classify castration-resistant prostate cancers suggesting therapeutic targets

Abstract Untreated prostate cancers rely on androgen receptor (AR) signaling for growth and survival, forming the basis for the initial efficacy of androgen deprivation therapy (ADT). Yet the disease can relapse and progress to a lethal stage termed castration-resistant prostate cancer (CRPC). Reactivation of AR signaling represents the most common driver of CRPC growth, and next-generation AR signaling inhibitors (ARSIs) are now used in combination with ADT as first-line therapy. However, ARSIs can result in selective pressure, thereby generating AR-independent tumors. The transition from AR dependence frequently accompanies a change in a phenotype resembling developmental transdifferentiation or “lineage plasticity”. Neuroendocrine prostate cancer, which lacks a defined pathologic classification, is the most studied type of lineage plasticity. However, most AR-null tumors do not exhibit neuroendocrine features and are classified as “double-negative prostate cancer”, the drivers of which are poorly defined. Lineage plasticity studies in CRPC are limited by the lack of genetically defined patient-derived models that recapitulate the disease spectrum. To address this, we developed a biobank of organoids generated from patient biopsies to study the landscape of metastatic CRPC and allow for functional validation assays. Proteins called transcription factors (TFs) are drivers of tumor lineage plasticity. To identify the key TFs that drive the growth of AR-independent tumors, we integrated epigenetic and transcriptomic data generated from CRPC models. We generated ATAC-seq (assay for transposase-accessible chromatin sequencing) and RNA-seq data from 22 metastatic human prostate cancer organoids, six patient-derived xenografts (PDXs), and 12 derived or traditional cell lines. We classified the 40 models into four subtypes and predicted key TFs of each subtype. Besides the well-characterized AR-dependent (CRPC-AR) and neuroendocrine subtypes (CRPC-NE), we identified two novel AR-negative/low groups, including a Wnt-dependent subtype (CRPC-WNT), driven by TCF/LEF TFs, and a stem cell-like (SCL) subtype (CRPC-SCL), driven by the AP-1 family of TFs. To apply the subtype classification to patient samples, we derived RNA-seq signatures from the organoids and applied them to 366 patient samples from two independent CRPC cohorts. The generated signatures recapitulated the four-subtype classification and revealed that CRPC-SCL is the second most prevalent group. Patients from CRPC-SCL are also associated with shorter time under ARSI treatment compared to CRPC-AR, indicating that the ARSI treatments were less effective for CRPC-SCL patients. Additional chromatin immunoprecipitation sequencing (ChIP-seq) analysis indicated that AP-1 (FOSL1) collaboratively binds with TEAD and transcription coactivators, YAP and TAZ. Knocking down of AP-1 (FOSL1), YAP/TAZ decreased cell growth of CRPC-SCL and showed a decrease of chromatin accessibility at CRPC-SCL-specific open chromatin sites and down-regulation of YAP/TAZ target gene expression. In addition, the expression of AP-1 (FOSL1) decreased upon YAP/TAZ knockdown suggesting a positive feedback loop as well as YAP/TAZ as actional targets in CRPC-SCL. We used two small-molecule inhibitors, verteporfin and T-5224, that act on the YAP/TAZ/AP-1 pathway for their potential use as therapeutics for CRPC-SCL tumors, both inhibited the growth of samples from CRPC-SCL but not CRPC-AR. By overexpressing an AP-1 family gene (FOSL1) in AR-high cells, we observed an increase in chromatin accessibility at CRPC-SCL-specific open chromatin sites as well as significant up-regulation of CRPC-SCL signature genes, suggesting that AP-1 functions as a pioneering factor and master regulator for CRPC-SCL. All this work was recently published in Science (Tang, Xu et al. Science, 2022) where I am the co-first author. In summary, by using a diverse biobank of organoids, PDXs, and cell lines that recapitulate the heterogeneity of metastatic prostate cancer, we created a map of the chromatin accessibility and transcriptomic landscape of CRPC. We validated the CRPC-AR and CRPC-NE subtypes and report two novel subtypes of AR-negative/low samples, CRPC-SCL and CRPC-WNT, as well as their respective key TFs. Additional analysis revealed a model in which YAP, TAZ, TEAD, and AP-1 function together and drive oncogenic growth in CRPC-SCL samples. In addition, we proposed small inhibitors of YAP and TAZ that can potentially be used to treat CRPC-SCL patients. Overall, our results show how the stratification of CRPC patients into four subtypes using their transcriptomes can potentially inform appropriate clinical decisions. Citation Format: Fanying Tang, Duo Xu, Shangqian Wang, Chen Khuan Wong, Alexander Martinez-Fundichely, Cindy J. Lee, Sandra Cohen, Jane Park, Corinne E. Hill, Kenneth Eng, Rohan Bareja, Teng Han, Eric Minwei Liu, Ann Palladino, Wei Di, Dong Gao, Wassim Abida, Shaham Beg, Loredana Puca, Maximiliano Meneses, Elisa de Stanchina, Michael F. Berger, Anuradha Gopalan, Lukas E. Dow, Juan Miguel Mosquera, Himisha Beltran, Cora N. Sternberg, Ping Chi, Howard I. Scher, Andrea Sboner, Yu Chen, Ekta Khurana. Chromatin profiles classify castration-resistant prostate cancers suggesting therapeutic targets. [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 NG10.

Abstract 1431: Sprouty 2 expression attenuates proliferation in nras-driven acute myeloid leukemia cell lines

Abstract Oncogenic mutations in the NRAS gene are found in many cancers including a subset of acute myeloid leukemias (AML). Established AML cell lines exhibiting hyperactive signaling through the RAS-RAF-MEK-ERK pathway show constitutive ERK activation generating a transcriptome that mediates escape from programmed cell death and continuous proliferation. It has been established that exposure of many AML cell lines to phorbol esters such as phorbol-12-myristate-13-acetate (PMA) mediates cell cycle arrest, myeloid differentiation, and apoptosis. This effect seems counterintuitive given that PMA irreversibly activates protein kinase C (PKC), which enhances ERK activity leading to transcriptional upregulation of target genes controlled by ERK activated transcription factors, many of which promote proliferation. Here we define a set of PMA response genes in AML cell lines driven by oncogenic NRAS that encode negative regulators of RAS-RAF-MEK-ERK signaling, including SPRY2, RASA1, DUSP 5, and DUSP6. Additionally, we show that overexpression of SPRY2 alone attenuates proliferation by inhibiting ERK activation and altering the AML cell transcriptome through the activation of genes encoding members of the AP-1 transcription factor family, FOS, JUNB, and JUND, and the cyclin-CDK inhibitors CDKN1A and CDKN2B. We hypothesize that oncogenic NRAS signaling is overridden by PMA treatment through the activation of ERK target genes that ultimately inhibit the MAPK-ERK pathway, such as SPRY2. Finally, we show that SPRY 2 expression in AML patient samples represents a survival prognostic indicator where higher expression levels correlate with improved overall survival. Citation Format: Michael Roberts, Sher Bahadur, Coltin Albitz, Jae Chung, Samantha Garcia, Cuong Nguyen, Sophia Kovatsis. Sprouty 2 expression attenuates proliferation in nras-driven acute myeloid leukemia cell lines [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 1431.

Tonic-signaling chimeric antigen receptors drive human regulatory T cell exhaustion

Regulatory T cell (Treg) therapy is a promising approach to improve outcomes in transplantation and autoimmunity. In conventional T cell therapy, chronic stimulation can result in poor invivo function, a phenomenon termed exhaustion. Whether or not Tregs are also susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. To "benchmark" exhaustion in human Tregs, we used a method known to induce exhaustion in conventional T cells: expression of a tonic-signaling chimeric antigen receptor (TS-CAR). We found that TS-CAR-expressing Tregs rapidly acquired a phenotype that resembled exhaustion and had major changes in their transcriptome, metabolism, and epigenome. Similar to conventional T cells, TS-CAR Tregs upregulated expression of inhibitory receptors and transcription factors such as PD-1, TIM3, TOX and BLIMP1, and displayed a global increase in chromatin accessibility-enriched AP-1 family transcription factor binding sites. However, they also displayed Treg-specific changes such as high expression of 4-1BB, LAP, and GARP. DNA methylation analysis and comparison to a CD8+ T cell-based multipotency index showed that Tregs naturally exist in a relatively differentiated state, with further TS-CAR-induced changes. Functionally, TS-CAR Tregs remained stable and suppressive invitro but were nonfunctional invivo, as tested in a model of xenogeneic graft-versus-host disease. These data are the first comprehensive investigation of exhaustion in Tregs and reveal key similarities and differences with exhausted conventional T cells. The finding that human Tregs are susceptible to chronic stimulation-driven dysfunction has important implications for the design of CAR Treg adoptive immunotherapy strategies.