Super-enhancer Research Articles

Super-Enhancer Reprograming Driven by SOX9 and TCF7L2 Represents Transcription-Targeted Therapeutic Vulnerability for Treating Gallbladder Cancer.

Gallbladder cancer (GBC) is a highly aggressive malignancy lacking clinically available targeted therapeutic agents. Super-enhancers (SEs) are crucial epigenetic cis-regulatory elements whose extensive reprogramming drives aberrant transcription in cancers. To study SE in GBC, the genomic distribution of H3K27ac is profiled in multiple GBC tissue and cell line samples to establish the SE landscape and its associated core regulatory circuitry (CRC). The biliary lineage factor SOX9 and Wnt pathway effector TCF7L2, two master transcription factor (TF) candidates identified by CRC analysis, are verified to co-occupy each other's SE region, forming a mutually autoregulatory loop to drive oncogenic SE reprogramming in a subset of GBC. The SOX9/TCF7L2 double-high GBC cells are highly dependent on the two TFs and enriched of SE-associated gene signatures related to stemness, ErbB and Wnt pathways. Patients with more such GBC cells exhibited significantly worse prognosis. Furthermore, SOX9/TCF7L2 double-high GBC preclinical models are found to be susceptible to SE-targeted CDK7 inhibition therapy in vitro and in vivo. Together, this study provides novel insights into the epigenetic mechanisms underlying the oncogenesis of a subset of GBCs with poorer prognosis and illustrates promising prognostic stratification and therapeutic strategies for treating those GBC patients in future clinical trials.

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.

Super-Enhancer-Driven IRF2BP2 is Activated by Master Transcription Factors and Sustains T-ALL Cell Growth and Survival.

Super enhancers (SEs) are large clusters of transcriptional enhancers driving the expression of genes crucial for defining cell identity. In cancer, tumor-specific SEs activate key oncogenes, leading to tumorigenesis. Identifying SE-driven oncogenes in tumors and understanding their functional mechanisms is of significant importance. In this study, a previously unreported SE region is identified in T-cell acute lymphoblastic leukemia (T-ALL) patient samples and cell lines. This SE activates the expression of interferon regulatory factor 2 binding protein 2 (IRF2BP2) and is regulated by T-ALL master transcription factors (TFs) such as ETS transcription factor ERG (ERG), E74 like ETS transcription factor 1 (ELF1), and ETS proto-oncogene 1, transcription factor (ETS1). Hematopoietic system-specific IRF2BP2 conditional knockout mice is generated and showed that IRF2BP2 has minimal impact on normal T cell development. However, in vitro and in vivo experiments demonstrated that IRF2BP2 is crucial for T-ALL cell growth and survival. Loss of IRF2BP2 affects the MYC and E2F pathways in T-ALL cells. Cleavage under targets and tagmentation (CUT&Tag) assays and immunoprecipitation revealed that IRF2BP2 cooperates with the master TFs of T-ALL cells, targeting the enhancer of the T-ALL susceptibility gene recombination activating 1 (RAG1) and modulating its expression. These findings provide new insights into the regulatory network within T-ALL cells, identifying potential new targets for therapeutic intervention.

Analyzing super-enhancer temporal dynamics reveals potential critical enhancers and their gene regulatory networks underlying skeletal muscle development.

Super-enhancers (SEs) govern the expression of genes defining cell identity. However, the dynamic landscape of SEs and their critical constituent enhancers involved in skeletal muscle development remains unclear. In this study, using pig as a model, we employed CUT&Tag to profile the enhancer-associated histone modification marker H3K27ac in skeletal muscle across two prenatal and three postnatal stages and investigated how SEs influence skeletal muscle development. We identified three SE families with distinct temporal dynamics: continuous (Con, 397), transient (TS, 434), and de novo (DN, 756). These SE families are associated with different temporal gene expression trajectories, biological functions, and DNA methylation levels. Notably, several lines of evidence suggest a potential prominent role of Con SEs in regulating porcine muscle development and meat traits. To pinpoint key cis-regulatory units in Con SEs, we developed an integrative approach that leverages information from eRNA annotation, GWAS signals and high-throughput capture STARR-seq experiments. Within Con SEs, we identified 20 candidate critical enhancers with meat and carcass-associated DNA variations that affect enhancer activity and inferred their upstream TFs and downstream target genes. As a proof of concept, we experimentally validated the role of one such enhancer and its potential target gene during myogenesis. Our findings reveal the dynamic regulatory features of SEs in skeletal muscle development and provide a general integrative framework for identifying critical enhancers underlying the formation of complex traits.

Super-enhancer-driven ZFP36L1 promotes PD-L1 expression in infiltrative gastric cancer

Gastric cancer (GC) is a major cause of cancer-related mortality worldwide. Despite the widespread recognition of tumor immunotherapy in treating unresectable GC, challenges, including ineffective immunotherapy and drug resistance, persist. Therefore, understanding the regulatory mechanisms of PD-L1, particularly in the context of super-enhancers (SEs) and zinc finger protein 36 ring finger protein-like 1 (ZFP36L1) RNA-binding protein, is crucial. In this study, we performed H3K27ac Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing, investigated the heterogeneity of SEs between two GC subtypes with differential growth patterns, and revealed the immune escape signatures driven by ZFP36L1-SE in infiltrative GC through SEs inhibitors treatment. The regulation of ZFP36L1 to PD-L1 was evaluated by quantitative PCR, western blot, flow cytometry, and immunohistochemistry. Furthermore, we explored its regulatory mechanisms using a combination of molecular biology techniques, including luciferase reporter assay, GST/RNA pull-down, chromatin immunoprecipitation (ChIP)/RIP experiments, and in vivo functional assays. We demonstrated that ZFP36L1, driven by an SE, enhances IFN-γ-induced PD-L1 expression, with SPI1 identified as the specific transcription factor binding to ZFP36L1-SE. Mechanistically, ZFP36L1 binds to the adenylate uridylate-rich element in the 3ʹ untranslated region (3ʹUTR) of HDAC3 mRNA, exacerbating its mRNA decay, and thereby facilitating PD-L1 abnormal transcriptional activation. Collectively, our findings provide mechanistic insights into the role of the SPI1-ZFP36L1-HDAC3-PD-L1 signaling axis in orchestrating immune escape mechanisms in GC, thereby offering valuable insights into the potential targets for immune checkpoint therapy in GC management.

Super-enhancer-driven ZFP36L1 promotes PD-L1 expression in infiltrative gastric cancer.

Gastric cancer (GC) is a major cause of cancer-related mortality worldwide. Despite the widespread recognition of tumor immunotherapy in treating unresectable GC, challenges, including ineffective immunotherapy and drug resistance, persist. Therefore, understanding the regulatory mechanisms of PD-L1, particularly in the context of super-enhancers (SEs) and zinc finger protein 36 ring finger protein-like 1 (ZFP36L1) RNA-binding protein, is crucial. In this study, we performed H3K27ac Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing, investigated the heterogeneity of SEs between two GC subtypes with differential growth patterns, and revealed the immune escape signatures driven by ZFP36L1-SE in infiltrative GC through SEs inhibitors treatment. The regulation of ZFP36L1 to PD-L1 was evaluated by quantitative PCR, western blot, flow cytometry, and immunohistochemistry. Furthermore, we explored its regulatory mechanisms using a combination of molecular biology techniques, including luciferase reporter assay, GST/RNA pull-down, chromatin immunoprecipitation (ChIP)/RIP experiments, and in vivo functional assays. We demonstrated that ZFP36L1, driven by an SE, enhances IFN-γ-induced PD-L1 expression, with SPI1 identified as the specific transcription factor binding to ZFP36L1-SE. Mechanistically, ZFP36L1 binds to the adenylate uridylate-rich element in the 3' untranslated region (3'UTR) of HDAC3 mRNA, exacerbating its mRNA decay, and thereby facilitating PD-L1 abnormal transcriptional activation. Collectively, our findings provide mechanistic insights into the role of the SPI1-ZFP36L1-HDAC3-PD-L1 signaling axis in orchestrating immune escape mechanisms in GC, thereby offering valuable insights into the potential targets for immune checkpoint therapy in GC management.

7324 Regulation of Super Enhancers by Phosphorylated Progesterone Receptors Drives Breast Cancer Stem Cell Expansion

Abstract Disclosure: N. Gillis: None. C.A. Lange: None. Current treatments for estrogen receptor (ER) positive breast cancer largely target rapidly dividing tumor cells. However, aggressive breast cancers often contain long-lived and weakly proliferative cancer stem cells (CSCs) that readily escape treatments aimed at cycling cells. We previously reported that progesterone receptors (PR) and their target genes aid the growth of these CSCs and help them evade anti-estrogen therapy. As proliferation and stemness are directed by separate signaling pathways that lead to the expression of distinct gene programs, it's crucial to understand how PR facilitates this cellular transformation at the genomic level. Establishing a gene expression program that may limit proliferation but encourages stemness requires coordination between transcription factors and their coregulators to alter the nuclear microenvironment and chromatin landscape.To reveal these mechanisms, we profiled PR and ER genomic binding using CUT&RUN in the T47D luminal breast cancer cell line grown in ultra-low attachment (3D) mammosphere conditions. Our analysis of genomic binding profiles displayed a pattern of PR/ER co-occupancy at distant sites more than 10 kilobases away from the nearest transcriptional start site. We used Rank Ordering of Super Enhancer (ROSE) analysis on these PR/ER binding sites to predict which are likely super enhancer elements capable of coordinating the regulation of multi-gene subsets. We employed a genetic approach to directly test the function of novel enhancer sites linked to PR-target genes by disrupting the PR/ER binding with a dCas9-KRAB construct directed to the predicted enhancer elements. We propose that abnormal growth factor signaling in breast cancer cells permits PR/ER complex formation which promotes expression of genes that induce endocrine resistance and CSC expansion through regulation of super enhancer elements. Further studies to elucidate these mechanisms are ongoing. Understanding the intricacies of PR/ER crosstalk is the crucial next step in developing new therapies that target PR-driven processes in ER+ breast cancers. Presentation: 6/3/2024

HOXDeRNA activates a cancerous transcription program and super enhancers via genome-wide binding

The role of long non-coding RNAs (lncRNAs) in malignant cell transformation remains elusive. We previously identified an enhancer-associated lncRNA, LINC01116 (named HOXDeRNA), as a transformative factor converting human astrocytes into glioma-like cells. Employing a combination of CRISPR editing, chromatin isolation by RNA purification coupled with sequencing (ChIRP-seq), in situ mapping RNA-genome interactions (iMARGI), chromatin immunoprecipitation sequencing (ChIP-seq), HiC, and RNA/DNA FISH, we found that HOXDeRNA directly binds to CpG islands within the promoters of 35 glioma-specific transcription factors (TFs) distributed throughout the genome, including key stem cell TFs SOX2, OLIG2, POU3F2, and ASCL1, liberating them from PRC2 repression. This process requires a distinct RNA quadruplex structure and other segments of HOXDeRNA, interacting with EZH2 and CpGs, respectively. Subsequent transformation activates multiple oncogenes (e.g., EGFR, miR-21, and WEE1), driven by the SOX2- and OLIG2-dependent glioma-specific super enhancers. These results help reconstruct the sequence of events underlying the process of astrocyte transformation, highlighting HOXDeRNA's central genome-wide activity and suggesting a shared RNA-dependent mechanism in otherwise heterogeneous and multifactorial gliomagenesis.

Dual activity of Minnelide chemosensitize basal/triple negative breast cancer stem cells and reprograms immunosuppressive tumor microenvironment

Triple negative breast cancer (TNBC) subtype is characterized with higher EMT/stemness properties and immune suppressive tumor microenvironment (TME). Women with advanced TNBC exhibit aggressive disease and have limited treatment options. Although immune suppressive TME is implicated in driving aggressive properties of basal/TNBC subtype and therapy resistance, effectively targeting it remains a challenge. Minnelide, a prodrug of triptolide currently being tested in clinical trials, has shown anti-tumorigenic activity in multiple malignancies via targeting super enhancers, Myc and anti-apoptotic pathways such as HSP70. Distinct super-enhancer landscape drives cancer stem cells (CSC) in TNBC subtype while inducing immune suppressive TME. We show that Minnelide selectively targets CSCs in human and murine TNBC cell lines compared to cell lines of luminal subtype by targeting Myc and HSP70. Minnelide in combination with cyclophosphamide significantly reduces the tumor growth and eliminates metastasis by reprogramming the tumor microenvironment and enhancing cytotoxic T cell infiltration in 4T1 tumor-bearing mice. Resection of residual tumors following the combination treatment leads to complete eradication of disseminated tumor cells as all mice are free of local and distant recurrences. All control mice showed recurrences within 3 weeks of post-resection while single Minnelide treatment delayed recurrence and one mouse was free of tumor. We provide evidence that Minnelide targets tumor intrinsic pathways and reprograms the immune suppressive microenvironment. Our studies also suggest that Minnelide in combination with cyclophosphamide may lead to durable responses in patients with basal/TNBC subtype warranting its clinical investigation.

Super-enhancer profiling reveals ThPOK/ZBTB7B, a CD4 + cell lineage commitment factor, as a master regulator that restricts breast cancer cells to a luminal non-migratory phenotype.

Despite efforts to understand breast cancer biology, metastatic disease remains a clinical challenge. Identifying suppressors of breast cancer progression and mechanisms of transition to more invasive phenotypes could provide game changing therapeutic opportunities. Transcriptional deregulation is central to all malignancies, highlighted by the extensive reprogramming of regulatory elements that underlie oncogenic programs. Among these, super-enhancers (SEs) stand out due to their enrichment in genes controlling cancer hallmarks. To reveal novel breast cancer dependencies, we integrated the analysis of the SE landscape with master regulator activity inference for a series of breast cancer cell lines. As a result, we identified T - h elper-inducing Poxviruses and Zinc-finger ( PO Z)/ K rüppel-like factor (ThPOK, ZBTB7B ), a CD4 + cell lineage commitment factor, as a breast cancer master regulator that is recurrently associated with a SE. ThPOK expression is highest in luminal breast cancer but is significantly reduced in the basal subtype. Manipulation of ThPOK levels in cell lines shows that its repressive function restricts breast cancer cells to an epithelial phenotype by suppressing the expression of genes involved in the epithelial-mesenchymal transition (EMT), WNT/β-catenin target genes, and the pro-metastatic TGFβ pathway. Our study reveals ThPOK as a master transcription factor that restricts the acquisition of metastatic features in breast cancer cells.

Integrated multi-omics profiling reveals the ZZZ3/CD70 axis is a super-enhancer-driven regulator of diffuse large B-cell lymphoma cell-natural killer cell interactions.

Tumor immune microenvironment is crucial for diffuse large B-cell lymphoma (DLBCL) development. However, the mechanisms by which super-enhancers (SEs) regulate the interactions between DLBCL cells and tumor-infiltrating immune cells remains largely unknown. This study aimed to investigate the role of SE-controlled genes in regulating the interactions between DLBCL cells and tumor-infiltrating immune cells. Single-cell RNA-seq, bulk RNA-seq and H3K27ac ChIP-seq data were downloaded from the Heidelberg Open Research Data database and Gene Expression Omnibus database. HOMER algorithm and Seurat package in R were used for bioinformatics analysis. Cell proliferation and lactate dehydrogenase (LDH) release was detected by MTS and LDH release assays, respectively. Interaction between B cell cluster and CD8+ T cell and NK cell cluster was most obviously enhanced in DLBCL, with CD70-CD27, MIF-CD74/CXCR2 complex, MIF-CD74/CD44 complex and CCL3-CCR5 interactions were significantly increased. NK cell sub-cluster showed the strongest interaction with B cell cluster. ZZZ3 upregulated the transcription of CD70 by binding to its SE. Silencing CD70 in DOHH2 cells significantly promoted the proliferation of co-cultured NK92 cells and LDH release from DOHH2 cells, which was counteracted by ZZZ3 overexpression in DOHH2 cells. CD70 silencing combined with PD-L1 blockade promoted LDH release from DOHH2 cells co-cultured with NK92 cells. In conclusion, DLBCL cells inhibited the proliferation and killing of infiltrating NK cells by regulating ZZZ3/CD70 axis. Targeting ZZZ3/CD70 axis combined with PD-L1 blockade is expected to be a promising strategy for DLBCL treatment.

Elevated choline drives KLF5-dominated transcriptional reprogramming to facilitate liver cancer progression.

An increase in the total choline-containing compound content is a common characteristic of cancer cells, and aberrant choline metabolism in cancer is closely associated with malignant progression. However, the potential role of choline-induced global transcriptional changes in cancer cells remains unclear. In this study, we reveal that an elevated choline content facilitates hepatocellular carcinoma (HCC) cell proliferation by reprogramming Krüppel-like factor 5 (KLF5)-dominated core transcriptional regulatory circuitry (CRC). Mechanistically, choline administration leads to elevated S-adenosylmethionine (SAM) levels, inducing the formation of H3K4me1 within the super-enhancer (SE) region of KLF5 and activating its transcription. KLF5, as a key transcription factor (TF) of CRC established by choline, further transactivates downstream genes to facilitate HCC cell cycle progression. Additionally, KLF5 can increase the expression of choline kinase-α (CHKA) and CTP:phosphocholine cytidylyltransferase (CCT) resulting in a positive feedback loop to promote HCC cell proliferation. Notably, the histone deacetylase inhibitor (HDACi) vorinostat (SAHA) significantly suppressed KLF5 expression and liver tumor growth in mice, leading to a prolonged lifespan. In conclusion, these findings highlight the epigenetic regulatory mechanism of the SE-driven key regulatory factor KLF5 conducted by choline metabolism in HCC and suggest a potential therapeutic strategy for HCC patients with high choline content.

Heat shock transcription factor 1 facilitates liver cancer progression by driving super-enhancer-mediated transcription of MYCN.

Heat shock transcription factors (HSFs) play crucial roles in the development of malignancies. However, the specific roles of HSFs in hepatocellular carcinoma (HCC) have yet to be fully elucidated. To explore the involvement of the HSF family, particularly HSF1, in the progression and prognosis of HCC. We conducted a thorough analysis of HSF expression and copy number variations across various cancer datasets. Specifically focusing on HSF1, we examined its expression levels and prognostic implications in HCC. Invitro and invivo experiments were carried out to evaluate the impact of HSF1 on liver cancer cell proliferation. Additionally, we utilized CUT&Tag, H3K27 acetylation enrichment, and RNA sequencing (RNA-seq) to investigate the super-enhancer (SE) regulatory landscapes of HSF1 in liver cancer cell lines. HSF1 expression is elevated in HCC and is linked to poor prognosis in several datasets. HSF1 stimulates liver cancer cell proliferation both invitro and invivo, partly through modulation of H3K27ac levels, influencing enhancer distribution. Mechanistically, our findings demonstrate that HSF1 transcriptionally activates MYCN expression by binding to its promoter and SE elements, thereby promoting liver cancer cell proliferation. Moreover, increased MYCN expression was detected in HCC tumors and correlated with unfavorable patient outcomes. Our study sheds light on previously unexplored aspects of HSF1 biology, identifying it as a transcription factor capable of shaping the epigenetic landscape in the context of HCC. Given HSF1's potential as an epigenetic regulator, targeting the HSF1-MYCN axis could open up new therapeutic possibilities for HCC treatment. The HSF1-MYCN axis constitutes a transcription-dependent regulatory mechanism that may function as both a prognostic indicator and a promising therapeutic target in liver cancer. Further exploration of this axis could yield valuable insights into novel treatment strategies for HCC.

RUNX1 expression is regulated by a super-enhancer and is a therapeutic target in adult T-cell leukemia/lymphoma

Super-enhancers (SEs) play an important role in regulating tumor-specific gene expression. JQ1, a Bromodomain-containing protein 4 (BRD4) inhibitor, exerts antitumor effects by disrupting SE-mediated regulation of gene expression. We investigated the anti-adult T-cell leukemia/lymphoma (ATL) effects of JQ1. JQ1 induced apoptosis and inhibited ATL cell proliferation. JQ1 suppressed RUNX1expression through the disruption of SE-mediated gene regulation. In the previous reports, it was shown that IC50s of AI-10-104 and Ro5-3335, RUNX1 inhibitors were 1-10 µM for lymphoblastic leukemia cell lines carrying RUNX1 mutations. In the present study, we demonstrated that IC50s of AI-10-104 and Ro5-3335 were also 1-10 µM or lower for ATL cell lines. Simultaneously, AI-10-104 suppressed MYC proto-oncogene (c-MYC) expression. RUNX1 is a potential therapeutic target for ATL that promotes c-MYC expression. We showed that RUNX1 expression is regulated via SEs in ATL and that RUNX1 may be a novel therapeutic target for ATL.

Super-Enhancer Driven LIF/LIFR-STAT3-SOX2 Regulatory Feedback Loop Promotes Cancer Stemness in Head and Neck Squamous Cell Carcinoma.

Super-enhancers (SEs) have been recognized as key epigenetic regulators underlying cancer stemness and malignant traits by aberrant transcriptional control and promising therapeutic targets against human cancers. However, the SE landscape and their roles during head and neck squamous cell carcinoma (HNSCC) development especially in cancer stem cells (CSCs) maintenance remain underexplored yet. Here, we identify leukemia inhibitory factor (LIF)-SE as a representative oncogenic SE to activate LIF transcription in HNSCC. LIF secreted from cancer cells and cancer-associated fibroblasts promotes cancer stemness by driving SOX2 transcription in an autocrine/paracrine manner, respectively. Mechanistically, enhancer elements E1, 2, 4 within LIF-SE recruit SOX2/SMAD3/BRD4/EP300 to facilitate LIF transcription; LIF activates downstream LIFR-STAT3 signaling to drive SOX2 transcription, thus forming a previously unknown regulatory feedback loop (LIF-SE-LIF/LIFR-STAT3-SOX2) to maintain LIF overexpression and CSCs stemness. Clinically, increased LIF abundance in clinical samples correlate with malignant clinicopathological features and patient prognosis; higher LIF concentrations in presurgical plasma dramatically diminish following cancer eradication. Therapeutically, pharmacological targeting LIF-SE-LIF/LIFR-STAT3 significantly impairs tumor growth and reduces CSC subpopulations in xenograft and PDX models. Our findings reveal a hitherto uncharacterized LIF-SE-mediated auto-regulatory loop in regulating HNSCC stemness and highlight LIF as a novel noninvasive biomarker and potential therapeutic target for HNSCC.

Predicting the Effect of miRNA on Gene Regulation to Foster Translational Multi-Omics Research-A Review on the Role of Super-Enhancers.

Gene regulation is crucial for cellular function and homeostasis. It involves diverse mechanisms controlling the production of specific gene products and contributing to tissue-specific variations in gene expression. The dysregulation of genes leads to disease, emphasizing the need to understand these mechanisms. Computational methods have jointly studied transcription factors (TFs), microRNA (miRNA), and messenger RNA (mRNA) to investigate gene regulatory networks. However, there remains a knowledge gap in comprehending gene regulatory networks. On the other hand, super-enhancers (SEs) have been implicated in miRNA biogenesis and function in recent experimental studies, in addition to their pivotal roles in cell identity and disease progression. However, statistical/computational methodologies harnessing the potential of SEs in deciphering gene regulation networks remain notably absent. However, to understand the effect of miRNA on mRNA, existing statistical/computational methods could be updated, or novel methods could be developed by accounting for SEs in the model. In this review, we categorize existing computational methods that utilize TF and miRNA data to understand gene regulatory networks into three broad areas and explore the challenges of integrating enhancers/SEs. The three areas include unraveling indirect regulatory networks, identifying network motifs, and enriching pathway identification by dissecting gene regulators. We hypothesize that addressing these challenges will enhance our understanding of gene regulation, aiding in the identification of therapeutic targets and disease biomarkers. We believe that constructing statistical/computational models that dissect the role of SEs in predicting the effect of miRNA on gene regulation is crucial for tackling these challenges.

Targeting Super-Enhancer-Driven Transcriptional Dependencies Suppresses Aberrant Hedgehog Pathway Activation and Overcomes Smoothened Inhibitor Resistance.

Aberrant activation of the Hedgehog (Hh) signaling pathway plays important roles in oncogenesis and therapeutic resistance in several types of cancer. The clinical application of FDA-approved Hh-targeted smoothened inhibitors (SMOi) is hindered by the emergence of primary or acquired drug resistance. Epigenetic and transcriptional-targeted therapies represent a promising direction for developing improved anti-Hh therapies. In this study, we integrated epigenetic/transcriptional-targeted small-molecule library screening with CRISPR/Cas9 knockout library screening and identified CDK9 and CDK12, two transcription elongation regulators, as therapeutic targets for antagonizing aberrant Hh activation and overcoming SMOi resistance. Inhibition of CDK9 or CDK12 potently suppressed Hh signaling and tumor growth in various SMOi responsive or resistant Hh-driven tumor models. Systemic epigenomic profiling elucidated the Hh-driven super-enhancer (SE) landscape and identified IRS1, encoding a critical component and cytoplasmic adaptor protein of the insulin-like growth factor (IGF) pathway, as an oncogenic Hh-driven SE target gene and effective therapeutic target in Hh-driven tumor models. Collectively, this study identifies SE-driven transcriptional dependencies that represent promising therapeutic vulnerabilities for suppressing the Hh pathway and overcoming SMOi resistance. As CDK9 and IRS inhibitors have already entered human clinical trials for cancer treatment, these findings provide comprehensive preclinical support for developing trials for Hh-driven cancers. Significance: Dissecting transcriptional dependencies driven by super-enhancers uncovers therapeutic targets in Hedgehog-driven cancers and identifies strategies for overcoming resistance to smoothened inhibitors.

Super enhancer acquisition drives expression of oncogenic PPP1R15B that regulates protein homeostasis in multiple myeloma

Multiple myeloma is a hematological malignancy arising from immunoglobulin-secreting plasma cells. It remains poorly understood how chromatin rewiring of regulatory elements contributes to tumorigenesis and therapy resistance in myeloma. Here we generate a high-resolution contact map of myeloma-associated super-enhancers by integrating H3K27ac ChIP-seq and HiChIP from myeloma cell lines, patient-derived myeloma cells and normal plasma cells. Our comprehensive transcriptomic and phenomic analyses prioritize candidate genes with biological and clinical implications in myeloma. We show that myeloma cells frequently acquire SE that transcriptionally activate an oncogene PPP1R15B, which encodes a regulatory subunit of the holophosphatase complex that dephosphorylates translation initiation factor eIF2α. Epigenetic silencing or knockdown of PPP1R15B activates pro-apoptotic eIF2α-ATF4-CHOP pathway, while inhibiting protein synthesis and immunoglobulin production. Pharmacological inhibition of PPP1R15B using Raphin1 potentiates the anti-myeloma effect of bortezomib. Our study reveals that myeloma cells are vulnerable to perturbation of PPP1R15B-dependent protein homeostasis, highlighting a promising therapeutic strategy.

Abstract We084: Deciphering the Molecular Code Underlining Postnatal Cardiomyocyte Maturation by an RNA Splicing Factor RBFox1

Mammalian hearts undergo major changes after birth during the perinatal period. While several extrinsic factors such as mechanical load, electrical stimulation, hormones, and nutrients have been implicated in this process, the intrinsic regulatory circuit governing cardiomyocyte postnatal maturation is largely unknown. Importantly, stem-cell derived cardiomyocytes commonly exhibit immaturity which is a major limiting factor for their application as disease models or cell therapy agents. Therefore, uncovering the intrinsic regulatory mechanism of mammalian cardiomyocyte maturation has significant implications in both basic cardiac biology and cell-based therapy and disease modeling. In our recent analysis of the global transcriptome transition from neonatal to adult rat hearts, we found RNA splicing among the top changed pathways. Additionally, we found that expression of RBFox1, a cardiac enriched RNA splicing regulator, was significantly increased during the postnatal transition to adolescence. We further demonstrate that RBFox1 mediated post-transcriptional regulation has a potent effect to promote neonatal and stem-cell derived cardiomyocyte maturation. RNA-seq followed by Gene Ontology enrichment analysis showed that Rbfox1 led to differentially expressed genes enriched in cardiac contraction and conduction, sarcomere organization, K + ion import and muscle filament sliding, as well as differential isoform switches enriched in cardiac contraction and sarcomere structure. An evolutionarily conserved, temporal specific super enhancer exists upstream of cardiac Rbfox1 , which is occupied by active epigenetic markers in the postnatal period. We have identified the tissue and temporal transcriptional activity of this super enhancer in vitro and in vivo . Several binding motifs of transcription factors are predicted within it and a key DNA fragment was identified to be essential for enhancer activity. A transgenic mouse strain has been established to facilitate the tracing of myocyte maturation in vivo by constructing a reporter system driven by a 4xSuperEnhancer-Rbfox1Promoter-tdTomato construct. In conclusion, we propose a mechanism that RBFox1 is an intracellular regulator with tissue- and temporal-specific expression patterns that regulates cardiac maturation by promoting alternative splicing that fine tunes the genetic code.

The BET PROTAC inhibitor GNE-987 displays anti-tumor effects by targeting super-enhancers regulated gene in osteosarcoma

BackgroundOsteosarcoma (OS) is one of the most common primary malignant tumors of bone in children, which develops from osteoblasts and typically occurs during the rapid growth phase of the bone. Recently, Super-Enhancers(SEs)have been reported to play a crucial role in osteosarcoma growth and metastasis. Therefore, there is an urgent need to identify specific targeted inhibitors of SEs to assist clinical therapy. This study aimed to elucidate the role of BRD4 inhibitor GNE-987 targeting SEs in OS and preliminarily explore its mechanism.MethodsWe evaluated changes in osteosarcoma cells following treatment with a BRD4 inhibitor GNE-987. We assessed the anti-tumor effect of GNE-987 in vitro and in vivo by Western blot, CCK8, flow cytometry detection, clone formation, xenograft tumor size measurements, and Ki67 immunohistochemical staining, and combined ChIP-seq with RNA-seq techniques to find its anti-tumor mechanism.ResultsIn this study, we found that extremely low concentrations of GNE-987(2–10 nM) significantly reduced the proliferation and survival of OS cells by degrading BRD4. In addition, we found that GNE-987 markedly induced cell cycle arrest and apoptosis in OS cells. Further study indicated that VHL was critical for GNE-987 to exert its antitumor effect in OS cells. Consistent with in vitro results, GNE-987 administration significantly reduced tumor size in xenograft models with minimal toxicity, and partially degraded the BRD4 protein. KRT80 was identified through analysis of the RNA-seq and ChIP-seq data. U2OS HiC analysis suggested a higher frequency of chromatin interactions near the KRT80 binding site. The enrichment of H3K27ac modification at KRT80 was significantly reduced after GNE-987 treatment. KRT80 was identified as playing an important role in OS occurrence and development.ConclusionsThis research revealed that GNE-987 selectively degraded BRD4 and disrupted the transcriptional regulation of oncogenes in OS. GNE-987 has the potential to affect KRT80 against OS.