SE-associated Genes Research Articles

Super-Enhancer-Driven Syndecan-4 Regulates Intercellular Communication in Hypoxic Pulmonary Hypertension.

Unveiling pro-proliferation genes involved in crosstalk between pulmonary artery endothelial cells and pulmonary artery smooth muscle cells (PASMCs) are important to improving the therapeutic outcome of pulmonary hypertension (PH). Although growing studies have shown that super-enhancers (SEs) play a pivotal role in pathological and physiological processes, the SE-associated genes in PH and their impact on PASMC proliferation remain largely unexplored. We used serotype 5 adenovirus-associated virus to interfere with syndecan-4 and constructed an SU5416 combined with hypoxia-PH model. Chromatin immunoprecipitation sequencing analysis, chromatin immunoprecipitation quantitative polymerase chain reaction, and bioinformatics were used to confirm early growth response 1 was involved in regulating syndecan-4-associated SE in PASMCs. The effects of syndecan-4 and its underlying mechanisms were subsequently elucidated using Western blot, coimmunoprecipitation, and cell coculture assays. Herein, we identified a novel SE-associated gene, syndecan-4, in hypoxia-exposed PASMCs. Syndecan-4 was transcriptionally driven by early growth response 1 via an SE and was significantly overexpressed in hypoxic PASMCs and plasma from patients with PH. Mechanism studies revealed that syndecan-4 induces PASMC proliferation by interacting and regulating protein kinase C α ubiquitination. In addition, syndecan-4 was enriched in exosomes secreted from hypoxic PASMCs, which subsequently transported and led to pulmonary artery endothelial cell dysfunction. Syndecan-4 inhibition in hypoxia by serotype 5 adenovirus-associated virus treatment attenuated the pulmonary artery remodeling and development of PH invivo. Taken together, our results demonstrate that an SE-driven syndecan-4 modulates crosstalk of PASMCs and pulmonary artery endothelial cells and promotes vascular remodeling via the protein kinase C α and exosome pathway, thus providing potential targets for the early diagnosis and treatment of hypoxic PH.

SMC3 contributes to heart development by regulating super-enhancer associated genes

Abnormal cardiac development has been observed in individuals with Cornelia de Lange syndrome (CdLS) due to mutations in genes encoding members of the cohesin complex. However, the precise role of cohesin in heart development remains elusive. In this study, we aimed to elucidate the indispensable role of SMC3, a component of the cohesin complex, in cardiac development and its underlying mechanism. Our investigation revealed that CdLS patients with SMC3 mutations have high rates of congenital heart disease (CHD). We utilized heart-specific Smc3-knockout (SMC3-cKO) mice, which exhibit varying degrees of outflow tract (OFT) abnormalities, to further explore this relationship. Additionally, we identified 16 rare SMC3 variants with potential pathogenicity in individuals with isolated CHD. By employing single-nucleus RNA sequencing and chromosome conformation capture high-throughput genome-wide translocation sequencing, we revealed that Smc3 deletion downregulates the expression of key genes, including Ets2, in OFT cardiac muscle cells by specifically decreasing interactions between super-enhancers (SEs) and promoters. Notably, Ets2-SE-null mice also exhibit delayed OFT development in the heart. Our research revealed a novel role for SMC3 in heart development via the regulation of SE-associated genes, suggesting its potential relevance as a CHD-related gene and providing crucial insights into the molecular basis of cardiac development.

KLF7 regulates super-enhancer-driven IGF2BP2 overexpression to promote the progression of head and neck squamous cell carcinoma

BackgroundHead and neck squamous carcinoma (HNSCC) is known for its high aggressiveness and susceptibility to cervical lymph node metastasis, which greatly contributes to its poor prognosis. During tumorigenesis, many types of cancer cells acquire oncogenic super-enhancers (SEs) that drive the overexpression of oncogenes, thereby maintaining malignant progression. This study aimed to identify and validate the role of oncogenic SE-associated genes in the malignant progression of HNSCC.MethodsWe identified HNSCC cell-specific SE-associated genes through H3K27Ac ChIP-seq and overlapped them with HNSCC-associated genes obtained from The Cancer Genome Atlas (TCGA) dataset and Gene Expression Omnibus (GEO) datasets using weighted gene coexpression network analysis (WGCNA) to identify hub genes. The expression of IGF2BP2 and KLF7 in HNSCC was detected using clinical samples. To determine the biological role of IGF2BP2, we performed CCK-8, colony formation assay, Transwell migration assay, invasion assay, and orthotopic xenograft model experiments. Furthermore, we utilized a CRISPR/Cas9 gene-editing system, small-molecule inhibitors, ChIP-qPCR, and dual-luciferase reporter assays to investigate the molecular mechanisms of IGF2BP2 and its upstream transcription factors.ResultsOur study identified IGF2BP2 as a hub SE-associated gene that exhibited aberrant expression in HNSCC tissues. Increased expression of IGF2BP2 was observed to be linked with malignant progression and unfavorable prognosis in HNSCC patients. Both in vitro and in vivo experiments confirmed that IGF2BP2 promotes the tumorigenicity and metastasis of HNSCC by promoting cell proliferation, migration, and invasion. Mechanistically, the IGF2BP2-SE region displayed enrichment for H3K27Ac, BRD4, and MED1, which led to the inhibition of IGF2BP2 transcription and expression through deactivation of the SE-associated transcriptional program. Additionally, KLF7 was found to induce the transcription of IGF2BP2 and directly bind to its promoter and SE regions. Moreover, the abundance of KLF7 exhibited a positive correlation with the abundance of IGF2BP2 in HNSCC. Patients with high expression of both KLF7 and IGF2BP2 showed poorer prognosis. Lastly, we demonstrated that the small molecule inhibitor JQ1, targeting BRD4, attenuated the proliferation and metastatic abilities of HNSCC cells.ConclusionsOur study reveals the critical role of IGF2BP2 overexpression mediated by SE and KLF7 in promoting HNSCC progression. Targeting SE-associated transcriptional programs may represent a potential therapeutic strategy in managing HNSCC.

The BET inhibitor GNE-987 effectively induces anti-cancer effects in T-cell acute lymphoblastic leukemia by targeting enhancer regulated genes.

T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematologic malignancy originating from T progenitor cells. It accounts for 15% of childhood and 25% of adult ALL cases. GNE-987 is a novel chimeric molecule developed using proteolysis-targeting chimeras (PROTAC) technology for targeted therapy. It consists of a potent inhibitor of the bromodomain and extraterminal (BET) protein, as well as the E3 ubiquitin ligase Von Hippel-Lindau (VHL), which enables the effective induction of proteasomal degradation of BRD4. Although GNE-987 has shown persistent inhibition of cell proliferation and apoptosis, its specific antitumor activity in T-ALL remains unclear. In this study, we aimed to investigate the molecular mechanisms underlying the antitumor effect of GNE-987 in T-ALL. To achieve this, we employed technologies including RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq) and CUT&Tag. The degradation of BET proteins, specifically BRD4, by GNE-987 has a profound impact on T-ALL cell. In in vivo experiments, sh-BRD4 lentivirus reduced T-ALL cell proliferation and invasion, extending the survival time of mice. The RNA-seq and CUT&Tag analyses provided further insights into the mechanism of action of GNE-987 in T-ALL. These analyses revealed that GNE-987 possesses the ability to suppress the expression of various genes associated with super-enhancers (SEs), including lymphoblastic leukemia 1 (LCK). By targeting these SE-associated genes, GNE-987 effectively inhibits the progression of T-ALL. Importantly, SE-related oncogenes like LCK were identified as critical targets of GNE-987. Based on these findings, GNE-987 holds promise as a potential novel candidate drug for the treatment of T-ALL.

Prognostic and predictive value of super-enhancer-derived signatures for survival and lung metastasis in osteosarcoma

BackgroundRisk stratification and personalized care are crucial in managing osteosarcoma due to its complexity and heterogeneity. However, current prognostic prediction using clinical variables has limited accuracy. Thus, this study aimed to explore potential molecular biomarkers to improve prognostic assessment.MethodsHigh-throughput inhibitor screening of 150 compounds with broad targeting properties was performed and indicated a direction towards super-enhancers (SEs). Bulk RNA-seq, scRNA-seq, and immunohistochemistry (IHC) were used to investigate SE-associated gene expression profiles in osteosarcoma cells and patient tissue specimens. Data of 212 osteosarcoma patients who received standard treatment were collected and randomized into training and validation groups for retrospective analysis. Prognostic signatures and nomograms for overall survival (OS) and lung metastasis-free survival (LMFS) were developed using Cox regression analyses. The discriminatory power, calibration, and clinical value of nomograms were evaluated.ResultsHigh-throughput inhibitor screening showed that SEs significantly contribute to the oncogenic transcriptional output in osteosarcoma. Based on this finding, focus was given to 10 SE-associated genes with distinct characteristics and potential oncogenic function. With multi-omics approaches, the hyperexpression of these genes was observed in tumor cell subclusters of patient specimens, which were consistently correlated with poor outcomes and rapid metastasis, and the majority of these identified SE-associated genes were confirmed as independent risk factors for poor outcomes. Two molecular signatures were then developed to predict survival and occurrence of lung metastasis: the SE-derived OS-signature (comprising LACTB, CEP55, SRSF3, TCF7L2, and FOXP1) and the SE-derived LMFS-signature (comprising SRSF3, TCF7L2, FOXP1, and APOLD1). Both signatures significantly improved prognostic accuracy beyond conventional clinical factors.ConclusionsOncogenic transcription driven by SEs exhibit strong associations with osteosarcoma outcomes. The SE-derived signatures developed in this study hold promise as prognostic biomarkers for predicting OS and LMFS in patients undergoing standard treatments. Integrative prognostic models that combine conventional clinical factors with these SE-derived signatures demonstrate substantially improved accuracy, and have the potential to facilitate patient counseling and individualized management.

Genetic Determinants of Selenium Availability, Selenium-Response, and Risk of Polycystic Ovary Syndrome.

Selenium is a trace element and its deficiency has been associated with the risk of PCOS, a multifactorial syndrome that affects a large number of women worldwide. Several databases and literature were searched to find out genetic variants of the genes involved in selenium uptake, metabolism, and regulation which may be significantly associated with the risk of PCOS through Se-related pathways. Genes that require selenium for their biological actions to perform were also shortlisted. A total of eighteen significantly associated genes with forty-four variants were identified as candidate variants that could play a potential role in the modulation of PCOS risk among the study population. The genetic variant distribution data was available in-house and was obtained through a GWAS study of the North India population. In silico tools were applied to understand the functional impact of these variants. Three variants namely LDLR (rs2228671), TNF (rs1041981), and SAA2 (rs2468844) are strongly associated with PCOS risk and have a functional impact on encoded protein. Certain variants of Se uptake genes such as DIO1, GPX2, TXNRD1,DIO2and GPX3 are also significantly associated with the risk of PCOS development. "C" allele of the Se transporter gene SELENOP (rs9686343) significantly increases PCOS risk. Other potential genes require selenium for their biological actions and are involved in the inflammatory, antioxidant response, and energy homeostasis signaling pathways. Thus, genetic variants of the population may affect the Se availability in the body. Also, deficiency of Se effects may get modulated due to underlying genetic polymorphism of Se-associated genes. This information may be helpful in dosage adjustment of Se supplementation for a population in order to get maximum benefits.

Super-Enhancer-Driven IGF2BP2 and IGF2BP3 Promote DDX21 Expression in Acute Myeloid Leukemia

Background : Acute myeloid leukemia (AML) is a hematological malignancy with a median overall survival (OS) of 8.5 months and a 5-year OS rate of 24.0%. Considering that relapse and drug resistance are major obstacles affecting the survival of AML patients, it is urgent to investigate the deeper pathogenesis of AML. Epigenetic modifications are extensively involved in the leukemogenesis of AML, especially N6-methyladenosine (m 6A) RNA methylation and super-enhancers (SEs). However, the relationship between m 6A and SEs in AML has not been elucidated. Methods : Chromatin immunoprecipitation (ChIP) sequencing data from Gene Expression Omnibus (GEO) cohort were analyzed to search SE-related genes. The mechanisms of m 6A-binding proteins IGF2BP2 and IGF2BP3 on DDX21 were explored via methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP) and luciferase reporter assays. The potential roles of DDX21 in AML were verified through functional assays in vitro and in vivo. Results: Two SE-associated genes, IGF2BP2 and IGF2BP3, were identified in AML. There were high enrichment of H3K27ac, H3K4me1 and BRD4 in IGF2BP2 and IGF2BP3. BRD4 inhibitor JQ1 or BRD4 knockdown could lead to the suppression of IGF2BP2/3 expression, indicating the activation by SE machinery on the transcription of IGF2BP2/3. As m 6A-binding proteins, IGF2BP2 and IGF2BP3 enhanced the stability of DDX21 mRNA in an m 6A-dependent manner. DDX21 was highly expressed in AML patients, and elevated DDX21 expression suggested a poor survival. Functionally, DDX21 deficiency inhibited proliferation, facilitated apoptosis and arrested cell cycle of AML cells in vitro . I n vivo, knockdown of DDX21 substantially reduced tumor load and prolonged survival of recipient mice. Conclusions: Dysregulation of SE-IGF2BP2/IGF2BP3-DDX21 axis promotes the progression of AML, which paves the way to develop more effective therapeutic strategies.

A body map of super-enhancers and their function in pig.

Super-enhancers (SEs) are clusters of enhancers that act synergistically to drive the high-level expression of genes involved in cell identity and function. Although SEs have been extensively investigated in humans and mice, they have not been well characterized in pigs. Here, we identified 42,380 SEs in 14 pig tissues using chromatin immunoprecipitation sequencing, and statistics of its overall situation, studied the composition and characteristics of SE, and explored the influence of SEs characteristics on gene expression. We observed that approximately 40% of normal enhancers (NEs) form SEs. Compared to NEs, we found that SEs were more likely to be enriched with an activated enhancer and show activated functions. Interestingly, SEs showed X chromosome depletion and short interspersed nuclear element enrichment, implying that SEs play an important role in sex traits and repeat evolution. Additionally, SE-associated genes exhibited higher expression levels and stronger conservation than NE-associated genes. However, genes with the largest SEs had higher expression levels than those with the smallest SEs, indicating that SE size may influence gene expression. Moreover, we observed a negative correlation between SE gene distance and gene expression, indicating that the proximity of SEs can affect gene activity. Gene ontology enrichment and motif analysis revealed that SEs have strong tissue-specific activity. For example, the CORO2B gene with a brain-specific SE shows strong brain-specific expression, and the phenylalanine hydroxylase gene with liver-specific SEs shows strong liver-specific expression. In this study, we illustrated a body map of SEs and explored their functions in pigs, providing information on the composition and tissue-specific patterns of SEs. This study can serve as a valuable resource of gene regulatory and comparative analyses to the scientific community and provides a theoretical reference for genetic control mechanisms of important traits in pigs.

MDB-26. CDK8 IS AN ONCOGENE IN GROUP 3 MEDULLOBLASTOMA THAT IMPACTS PROTEIN TRANSLATION BY REGULATING THE TRANSCRIPTIONAL ACTIVITY OF RIBOSOMAL GENES

Abstract Medulloblastoma (MB) is one of the most common malignant childhood brain tumors. Single-cell RNA-seq analysis of human medulloblastoma patients revealed that Group 3 MBs are dominated by undifferentiated progenitor-like cells with elevated expression of ribosomal genes, suggesting that overactive ribosome biogenesis is a hallmark of Group 3 MB. Using CRISPR-Cas9 screen, we found that Cyclin-dependent kinase 8 (CDK8) is an essential gene for MYC MB viability. We showed that CDK8 facilitates MYC-driven transcriptional programs and is necessary for sustaining expression of MYC. Furthermore, the loss of CDK8 resulted in a reduction in the phosphorylation of RNA polymerase II CTD and caused significant changes in genome transcription. Using cut&run, we found CDK8 directly regulated ribosomal gene transcription activity. CDK8 inhibition impaired ribosome biosynthesis suppressed protein translation and led to a striking reduction in phospho-Pol II binding at the promoter regions and gene bodies of SE-associated genes. The combination of an mTOR inhibitor with CDK8 inhibitors showed remarkable synergy in suppressing ribosome biogenesis, decreasing cell viability and attenuating tumor growth in vitro and in multiple models of MB. This proposal outlines the mechanistic consequences of CDK8 inhibition on ribosome biogenesis and provides pre-clinical validation of CDK8 inhibitor treatment as a novel therapeutic approach in MYC MB.

Integrated high-throughput analysis identifies super enhancers in metastatic castration-resistant prostate cancer.

Background: Metastatic castration-resistant prostate cancer (mCRPC) is a highly aggressive stage of prostate cancer, and non-mutational epigenetic reprogramming plays a critical role in its progression. Super enhancers (SE), epigenetic elements, are involved in multiple tumor-promoting signaling pathways. However, the SE-mediated mechanism in mCRPC remains unclear. Methods: SE-associated genes and transcription factors were identified from a cell line (C4-2B) of mCRPC by the CUT&Tag assay. Differentially expressed genes (DEGs) between mCRPC and primary prostate cancer (PCa) samples in the GSE35988 dataset were identified. What's more, a recurrence risk prediction model was constructed based on the overlapping genes (termed SE-associated DEGs). To confirm the key SE-associated DEGs, BET inhibitor JQ1 was applied to cells to block SE-mediated transcription. Finally, single-cell analysis was performed to visualize cell subpopulations expressing the key SE-associated DEGs. Results: Nine human TFs, 867 SE-associated genes and 5417 DEGs were identified. 142 overlapping SE-associated DEGs showed excellent performance in recurrence prediction. Time-dependent receiver operating characteristic (ROC) curve analysis showed strong predictive power at 1year (0.80), 3years (0.85), and 5years (0.88). The efficacy of his performance has also been validated in external datasets. In addition, FKBP5 activity was significantly inhibited by JQ1. Conclusion: We present a landscape of SE and their associated genes in mCPRC, and discuss the potential clinical implications of these findings in terms of their translation to the clinic.

Abstract 1478: Broad downregulation of normal-specific super-enhancer associated genes in lung adenocarcinoma

Abstract Background Lung adenocarcinoma (LUAD) is the most common histologic subtype of lung cancer and a highly heterogeneous disease whose mechanism is not fully uncovered. Recently, Super-enhancers (SEs), tissue-specific clusters of enhancer elements, were found to drive cell identity genes and play an important role in tumorigenesis. However, current studies focus on SE in lung tumors but largely neglect those specific in normal lung tissues, which also provide critical data for understanding transformation of normal cells into cancer cells. For the first time, this study characterized normal-specific SE-associated genes (SEGs) in LUAD by dysregulation in tumor, prognostic significance and tumor microenvironment (TME) interactions via a comprehensive analysis of 22 bulk and single-cell transcriptomic datasets. Methods We analyzed TCGA datasets of 10 cancer types, and additional datasets comprising 4 datasets of LUAD tumor and normal paired samples, 5 datasets of LUAD tumors with survival information, and 3 single-cell transcriptomic datasets. For each dataset, standard methods were used for data preprocessing and normalization according to its generation platforms (e.g., microarray, bulk RNA-seq and scRNA-seq). For downstream analyses, paired t-test and linear model were used to identify dysregulated SEGs; Cox Proportional Hazards Regression was used to evaluate their prognostic significance and LASSO was used to select features for building multivariate prognostic models; bulk data deconvolution and correlation analysis were applied to assess the association of TME cell composition and SEG expression; Gene set enrichment analysis was applied for functional analysis. Results We found normal-lung SEGs were significantly disproportionally downregulated in LUAD tumors (Chi-squared test, p < 0.01), and associated with poor prognosis of patients. The patterns were consistently found in discovery (TCGA) and independent validation datasets. Interestingly, the effect appears specific to lung as such downregulation of normal-lung SEGs was also found in lung squamous cell carcinoma but not observed in most of other 8 cancer types. The downregulated SEGs were significantly involved in cell differentiation, apoptosis, proliferation, and stem cell pluripotency, and were enriched in tumor suppressors. Also, their downregulation correlate with reduced endothelial cells and lower immune activity with reduced B and CD8 T cells and inhibited immune target genes. Conclusions Our study characterized the genes associated to normal-specific SEs in LUAD, shedding sheds light on their role in tumorigenesis of lung cancers. Their broad downregulation indicates a significant effect of SE aberrancy in the transformation of normal cell into cancer cells with a modulation of immune microenvironment; further functional studies are needed to elucidate the underlying mechanism. Citation Format: Guoshuai Cai, Yihan Liu, Xiang Cui, Christopher Amos, Feifei Xiao. Broad downregulation of normal-specific super-enhancer associated genes in lung adenocarcinoma [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 1478.

CDK9 inhibition induces epigenetic reprogramming revealing strategies to circumvent resistance in lymphoma

Diffuse large B-cell lymphoma (DLBCL) exhibits significant genetic heterogeneity which contributes to drug resistance, necessitating development of novel therapeutic approaches. Pharmacological inhibitors of cyclin-dependent kinases (CDK) demonstrated pre-clinical activity in DLBCL, however many stalled in clinical development. Here we show that AZD4573, a selective inhibitor of CDK9, restricted growth of DLBCL cells. CDK9 inhibition (CDK9i) resulted in rapid changes in the transcriptome and proteome, with downmodulation of multiple oncoproteins (eg, MYC, Mcl-1, JunB, PIM3) and deregulation of phosphoinotiside-3 kinase (PI3K) and senescence pathways. Following initial transcriptional repression due to RNAPII pausing, we observed transcriptional recovery of several oncogenes, including MYC and PIM3. ATAC-Seq and ChIP-Seq experiments revealed that CDK9i induced epigenetic remodeling with bi-directional changes in chromatin accessibility, suppressed promoter activation and led to sustained reprograming of the super-enhancer landscape. A CRISPR library screen suggested that SE-associated genes in the Mediator complex, as well as AKT1, confer resistance to CDK9i. Consistent with this, sgRNA-mediated knockout of MED12 sensitized cells to CDK9i. Informed by our mechanistic findings, we combined AZD4573 with either PIM kinase or PI3K inhibitors. Both combinations decreased proliferation and induced apoptosis in DLBCL and primary lymphoma cells in vitro as well as resulted in delayed tumor progression and extended survival of mice xenografted with DLBCL in vivo. Thus, CDK9i induces reprogramming of the epigenetic landscape, and super-enhancer driven recovery of select oncogenes may contribute to resistance to CDK9i. PIM and PI3K represent potential targets to circumvent resistance to CDK9i in the heterogeneous landscape of DLBCL.

Super-enhancer-associated transcription factors collaboratively regulate trophoblast-active gene expression programs in human trophoblast stem cells.

The placenta is an essential organ that supports the growth and development of the fetus during pregnancy. However, cell type-specific enhancers and transcription factors (TFs), and the mechanisms underlying the maintenance and differentiation of trophoblast stem cell (TSC) populations in the human placenta remain elusive. Here, using human TSCs as a model system, we identify 31,362 enhancers that are enriched with the motifs of previously reported TSC-pivotal TFs, including TEAD4, GATA2/3 and TFAP2C. Subsequently, we identify 580 super-enhancers (SEs) and 549 SE-associated genes. These genes are robustly expressed in the human placenta and include numerous TFs, implying that SE-associated TFs (SE-TFs) may play crucial roles in placental development. Additionally, we identify the global binding sites of five TSC-pivotal SE-TFs (FOS, GATA2, MAFK, TEAD4 and TFAP2C), revealing that they preferentially co-occupy enhancers, regulate each otherand form a trophoblast-active gene regulatory network. Loss-of-function studies unveil that the five TFs promote self-renewal of TSCs by activating proliferation-associated genes while repressing developmental genes. We further reveal that the five TFs exert conserved and unique functions on placental development between humans and mice. Our study provides important insights into the roles of human TSC-pivotal TFs in regulating placenta-specific gene expression programs.

Epigenetic Regulation of MAP3K8 in EBV-Associated Gastric Carcinoma.

Super-enhancers (SEs) regulate gene expressions, which are critical for cell type-identity and tumorigenesis. Although genome wide H3K27ac profiling have revealed the presence of SE-associated genes in gastric cancer (GC), their roles remain unclear. In this study, ChIP-seq and HiChIP-seq experiments revealed mitogen-activated protein kinase 8 (MAP3K8) to be an SE-associated gene with chromosome interactions in Epstein-Barr virus-associated gastric carcinoma (EBVaGC) cells. CRISPRi mediated repression of the MAP3K8 SEs attenuated MAP3K8 expression and EBVaGC cell proliferation. The results were validated by treating EBVaGC cells with bromodomain and the extra-terminal motif (BET) inhibitor, OTX015. Further, functional analysis of MAP3K8 in EBVaGC revealed that silencing MAP3K8 could inhibit the cell proliferation, colony formation, and migration of EBVaGC cells. RNA-seq and pathway analysis indicated that knocking down MAP3K8 obstructed the notch signaling pathway and epithelial-mesenchymal transition (EMT) in EBVaGC cells. Further, analysis of the cancer genome atlas (TCGA) and GSE51575 databases exhibited augmented MAP3K8 expression in gastric cancer and it was found to be inversely correlated with the disease-free progression of GC. Moreover, Spearman's correlation revealed that MAP3K8 expression was positively correlated with the expressions of notch pathway and EMT related genes, such as, Notch1, Notch2, C-terminal binding protein 2 (CTBP2), alpha smooth muscle actin isotype 2 (ACTA2), transforming growth factor beta receptor 1 (TGFβR1), and snail family transcriptional repressors 1/2 (SNAI1/SNAI2) in GC. Taken together, we are the first to functionally interrogate the mechanism of SE-mediated regulation of MAP3K8 in EBVaGC cell lines.

The super-enhancer repertoire in porcine liver.

The transcriptional initiation of genes is inextricably bound with the functions of cis-regulatory sequences. The pig is one of the most important livestock species and an ideal animal model for biomedical studies. At the same time, the liver is a critical organ with diverse and complex metabolic functions. Here, we performed Cleavage Under Targets and Tagmentation (CUT&Tag) coupled with high-throughput sequencing to profile the chromatin landscape of histone H3 lysine 27 acetylation (H3K27ac), histone H3 lysine 4 monomethylation (H3K4me1), and CCAAT enhancer-binding protein β (C-EBPβ) in the 70-d-old porcine liver, compared the different profiles among the three markers and their associated stitched-enhancers by stitching and sorting the peaks within 12.5 kb (Pott and Lieb, 2015) and generated the porcine liver-specific super-enhancers (SEs) by the combination of three markers. Compared to typical enhancers (TEs) and other stitched-enhancers, liver-specific SEs showed a higher density of cis-motifs and SNPs, which may recruit more tissue-specific vital TFs. The expression profiles in fetal and 70-d-old pigs proved that a large proportion of SE-associated genes were up-regulated and were more related to hepatic metabolisms and detoxification pathways. Our results illustrated the difference and connection among promoter and enhancer markers, identified the features of liver SEs and their associated genes, and provided novel insight into cis-element identification, function, and liver transcriptional regulation.

Cyclin‐dependent kinase 12‐mediated super‐enhancers as promising targets in cancer therapy

Cyclin‐dependent kinase 12‐mediated super‐enhancers as promising targets in cancer therapy

Abstract 2350: Foxj1 is a new master regulator of activated PI3K pathway pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second leading cause of cancer mortality within a decade with overall 5-year survival of 8% for all stages combined. Currently, it is well documented that mechanisms driving PDAC progression involve epigenetic and transcriptional rewiring. Here we combined assay for transposase-accessible chromatin using sequencing (ATAC-seq) and enrichment for H3K27 acetylation chromatin immunoprecipitation (H3K27ac ChIP-seq) measures to explore the epigenetic landscape of different mouse primary pancreatic tumor (PPT) cell lines. Methods: Kras-driven (n=36) and PI3K-driven PPT cell lines (n=9) were cultured in DMEM medium (Gibco). DNA was extracted using manufacture protocols (Qiagen, MinElute PCR Purification Kit) then DNA libraries and high-throughput sequencing were performed. Bioinformatics analysis (ROSE2 Python script) was conducted on H3k27ac ChIP-seq data to define super-enhancer (SEs) and SE-associated genes. Then, ATAC-seq data was explored using Coltron Python package to distinguish enriched Transcription Factor (TF) motifs into SEs. Transcriptomic data was used to slim down the list of potential cis-regulatory elements. We developed knockout (ko) PPT cell lines using CRISPR/CAS9 gene editing method to better characterize the role of Foxj1 as a novel potential master regulator in pancreatic cancer. Lastly, immunohistochemistry (IHC) staining for FOXJ1 was conducted on human PDAC cohort. Results: By k-means clustering, we identified 463 SE-associated genes. Many of them are associated with Kras-driven (epithelial or mesenchymal) or PI3K-driven cell lines exclusively. Surprisingly, we found Foxj1 as SE-associated TF exclusively in PI3K-driven PPT cell lines. Consistent with the epigenetic data, transcriptomic analysis confirmed higher expression of Foxj1 in PI3K-driven PPT cell lines. Then, RNA-seq data revealed downregulation of predicted Foxj1 target genes and enhanced EMT and Wnt/β-catenin signatures in Foxj1 ko cells. These data suggest that epithelial properties of PDAC cells are stabilized by Foxj1 activity. Consistent with these results we detect a higher potential of TGFβ treatment to induce mesenchymal features in Foxj1 ko cells. Furthermore, overexpression of β-catenin protein was confirmed by immunofluorescence. Enhanced Wnt/β-catenin signaling could be responsible for the higher proliferation of Foxj1 ko cells as revealed by proliferation assay. Finally, we investigated FOXJ1 protein level in our PDAC human cohort. Interestingly, we found high nuclear FOXJ1 expression in 23% of cases which is linked with better overall survival. Conclusions: In summary, our data revealed Foxj1 as a novel PDAC associated TF with the ability to reduce the cancer aggressiveness blocking epithelial to mesenchymal transition and β-catenin activity elucidating the better prognosis into the FOXJ1 high expressed patients. Citation Format: Andrea Terrasi, Swathi Subramanian, Christine Klement, Sruthi Ramesh, Heike Bollig, Chiara Falcomatà, Katja Steiger, Rupert Öllinger, Dieter Saur, Roland Rad, Maximilian Reichert, Günter Schneider, Gunnar Schotta. Foxj1 is a new master regulator of activated PI3K pathway pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2350.

Abstract 2956: Superenhancer-drvien SMC4 promote cell growth and proliferation in multiple myeloma

Abstract Background Multiple myeloma (MM) is an incurable malignancy characterized by complex heterogeneous cytogenetic abnormalities. Defined as large clusters of cis-acting enhancers, super-enhancers (SEs) could promote oncogenic transcription to which cancer cells highly addicted. Structural maintenance of chromosome 4 (SMC4) is a core subunit of condensin complexes, which contributes to chromosome condensation and segregation. Here, we identified SMC4 as a novel SE-associated oncogene of myeloma cells, whose functional roles in MM remain largely elusive. Methods We performed H3K27ac ChIP-seq and RNA-seq on primary MM patient samples, MM cell lines, normal CD138+ plasma cell and lymphoma cell lines as the controls, then the ROSE analysis was used to annotate SEs and their associated genes. THZ1 as a transcriptional CDK7 inhibitor was used to further filter THZ1-responsive genes. Combined analysis of THZ1-sensitive and SE-associated gene uncovered a number of promising MM oncogenes as SMC4. The public data mining, RNA interference, CRISPR/Cas9 gene editing system, overexpression and a variety of cellular functional assays were performed to determine the oncogenic effects of SMC4 on MM malignant. Transcriptome analysis with SMC4 silencing and overexpression, and the underlying molecular mechanism of SMC4 oncogenic role in MM are ongoing. Results We have demonstrated SMC4 as one of the SE-associated genes specific to MM patient samples and cell lines. SMC4 was related to myelomagenesis with increased expression from MGUS, SMM to MM cases, and overexpression of SMC4 predicted poor overall survival of MM patients, suggesting its clinical relevance. SMC4 silencing in MM cells suppressed cell proliferation and lead to cell apoptosis. Repression of the SMC4-SE region also led to impairment of cell viability and cell growth, consistent with an oncogenic function. Conclusion In summary, our integrative approached by combing H3H27ac ChIP-seq, RNA-seq and THZ1-sensitive transcripts identified the landscape of SEs and novel oncogenic transcripts as SMC4 in MM. Mapping these acquired SEs and their associated genes may provide novel insight into understanding MM biology, and SMC4 may serve as novel therapeutic targets in MM. Citation Format: Yunlu Jia, Jianbiao Zhou, Tze-King Tan, Tae-Hoon Chung, Yongxia Chen, Enfan Zhang, Zhen Cai, Takaomi Sanda, Wee-Joo Chng. Superenhancer-drvien SMC4 promote cell growth and proliferation in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2956.

A Novel Defined Super-Enhancer Associated Gene Signature to Predict Prognosis in Patients With Diffuse Large B-Cell Lymphoma

Background: Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease that can have profound differences in survival outcomes. A variety of powerful prognostic factors and models have been constructed; however, the development of more accurate prognosis prediction and targeted treatment for DLBCL still faces challenges. An explosion of research on super-enhancer (SE)–associated genes provide the possibility to use in prognostication for cancer patients. Here, we aimed to establish a novel effective prognostic model using SE-associated genes from DLBCL. Methods: A total of 1,105 DLBCL patients from the Gene Expression Omnibus database were included in this study and were divided into a training set and a validation set. A total of 11 SE-associated genes (BCL2, SPAG16, PXK, BTG1, LRRC37A2, EXT1, TGFBR2, ANKRD12, MYCBP2, PAX5, and MYC) were initially screened and identified by the least absolute shrinkage and selection operator (Lasso) penalized Cox regression, univariate and multivariate Cox regression analysis. Finally, a risk score model based on these 11 genes was constructed. Results: Kaplan–Meier (K–M) curves showed that the low-risk group appeared to have better clinical survival outcomes. The excellent performance of the model was determined via time-dependent receiver operating characteristic (ROC) curves. A nomogram based on the polygenic risk score was further established to promote reliable prognostic prediction. This study proposed that the SE-associated-gene risk signature can effectively predict the response to chemotherapy in DLBCL patients. Conclusion: A novel and reliable SE-associated-gene signature that can effectively classify DLBCL patients into high-risk and low-risk groups in terms of overall survival was developed, which may assist clinicians in the treatment of DLBCL.

Super-Enhancer-Driven PPP1R15B As an Oncogenic and Potential Therapeutic Target in Multiple Myeloma

Background: Multiple myeloma (MM) is a hematological malignancy arising from immunoglobulin-secreting plasma cells. Despite recent progress in stem-cell transplantation, high-dose chemotherapy and novel therapies, MM remains incurable and most patients experience relapse. In addition of genetic alterations, growing evidence has suggested that alterations in epigenetic landscape contribute to pathogenesis of MM. Super enhancers (SE) are large clusters of putative enhancers with aberrantly strong binding of mediators and transcription-regulating proteins. MM cells are highly dependent on unfolded protein response (UPR) signaling pathways due to high level of endoplasmic reticulum (ER) stress. Phosphorylation of eIF2α can attenuate protein translation. The PPP1R15B (denoted as R15B hereafter) gene encodes a regulatory subunit of an eIF2α-specific phosphatase complex. In this study, we identified SE-driven oncogenes specific in MM with a particular focus on a candidate SE-associated gene R15B, whose functional roles in MM remain largely elusive.Methods: We performed H3K27Ac ChIP-seq on MM cell lines, primary MM patient samples and normal CD138+ plasma cells, memory B cells (controls). ROSE analysis was used to systematically annotate SEs and their associated genes between tumors and controls. A combination of public data mining, RNA interference (RNAi), overexpression and CRISPR/Cas9 technologies followed by functional assays were conducted to determine the oncogenic effects of R15B in MM. Transcriptome analysis of MM cell line NCI-H929 with R15B knockdown and scrambled control was performed. To further study the interactions between SE and its promoters, we are currently working on highly integrative chromatin immunoprecipitation (HiChIP) followed by sequencing.Results: We have identified R15B as one of the SE-associated genes specific to MM patient samples and MM cell lines. The expression of R15B was 6- to 50-fold higher in a panel of 8 MM cell lines than normal controls. SE activity was correlated with the expression level of R15B. Higher expression of R15B predicted poor overall survival of MM patients, suggesting its clinical relevance in MM pathogenesis. Knockdown and knockout of R15B significantly reduced cell viability, clonogenicity and induced G2/M phase cell cycle arrest. Gene Ontology (GO) enrichment and pathway analysis of downregulated genes in response to R15B knockdown indicates that these genes are significantly associated with G2/M checkpoint hallmark, oxidative phosphorylation, unfolded protein response and E2F1-related pathways. Loss of R15B may induce apoptosis via activation of UPR pro-apoptotic executors PUMA and NOXA. ChIP-qPCR assays indicates that transcription factor C/EBP-β is strongly enriched at R15B SE region. Furthermore, we examined the therapeutic effects of Salubrinal, a selective inhibitor of eIF2α phosphorylation, on MM cells. Salubrinal inhibited MM cell proliferation in a dose-dependent manner, and MM cell lines with higher R15B expression were more sensitive to Salubrinal than those with lower R15B level.Conclusions: Our integrative approaches identified R15B as a novel SE-driven oncogene, and may form a positive feedback loop with C/EBP-β. Salubrinal selectively inhibits proliferation of MM cells with high R15B expression. Hence, we propose that targeting R15B may serve as a new approach for effective anti-myeloma therapy, which warrants further clinical investigation. DisclosuresChng: Novartis: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Abbvie: Honoraria; Amgen: Honoraria, Research Funding.