Targeting Epstein-Barr Virus super-enhancers

 描述（由申请人提供）：EB病毒（EBV）在HIV感染和免疫抑制人群中引起淋巴瘤和淋巴增生性疾病。这些肿瘤细胞经常表达潜伏III期EBV核抗原（EBNA）和潜伏膜蛋白（LMP）。在体外，EBV通过表达相同的病毒蛋白将静息B淋巴细胞（RBL）转化为持续增殖的淋巴母细胞细胞系（LCL）。因此，EBV将RBL转化为LCL是一个相关的模型，可以通过遗传操作来研究EBV在生长转化中的作用。LCL的生长依赖于EBV转录因子（TF）EBNA 2、EBNALP、EBNA 3A、EBNA 3C和LMP 1激活的NF-κB。最近，我们发现所有必需的EBNAs和LMP 1激活的NF-κ B亚基都集中在少数增强子位点。在这些增强子中，187个具有显著更高和更宽的组蛋白H3 K27 ac信号，这是超级增强子的特征，并被称为“EBV超级增强子（ESE）”。“超级增强子（SE）控制细胞转录，发育，表型和肿瘤发生。我们发现ESE相关基因包括MYC和BCL 2癌基因。ESE富集B细胞TF基序，并具有高的STAT 5A和NFAT共占有率。ESE相关基因的表达高于其他LCL基因。通过布罗莫结构域抑制剂JQ 1破坏ESS或有条件地灭活EBV癌蛋白或NF-κ B降低MYC或BCL 2表达并阻止LCL生长。为了进一步表征ESE，我们将检查它们的（1）DNA元件，（2）蛋白质组成和（3）相关的增强子RNA（eRNA）。我们将使用报告基因分析和重复的规则间隔短回文重复序列（CRISPR）来鉴定ESE活性所必需的DNA元件。我们将使用BioTAP-XL交联，亲和纯化，然后用质谱法来表征ESE蛋白质组组分。我们还将使用Global Run On，然后进行深度测序（GRO-seq）来鉴定受EBV超级增强子影响的增强子RNA，并使用发夹RNA（shRNA），短干扰RNA（siRNA）或锁核酸反义寡核苷酸（LNA）敲除来确定其意义。本实验采用综合方法来阐明ESE激活关键致癌因子的分子机制。这些研究将确定治疗干预的机会。

 DESCRIPTION (provided by applicant): Epstein-Barr Virus (EBV) causes lymphomas and lymphoproliferative diseases in HIV infected and immune suppressed people. These tumor cells frequently express Latency III EBV Nuclear Antigens (EBNAs) and Latent Membrane Proteins (LMP). In vitro, EBV converts Resting B Lymphocytes (RBLs) to continuously proliferating Lymphoblasts Cell Lines (LCLs) by expressing the same viral proteins. EBV conversion of RBLs to LCLs is therefore a relevant model that can be genetically manipulated to investigate EBV's role in growth transformation. LCL growth depends on EBV transcription factors (TFs) EBNA2, EBNALP, EBNA3A, EBNA3C and LMP1 activated NF-κB. Recently, we found that all the essential EBNAs and LMP1 activated NF-kB subunits converge to a small number of enhancers sites. Of these enhancers, 187 had markedly higher and broader histone H3K27ac signals, characteristic of super-enhancers, and were designated "EBV super-enhancers (ESE)." Super-enhancers (SE) govern cell transcription, development, phenotype, and oncogenesis. We found ESE- associated genes included the MYC and BCL2 oncogenes. ESEs were enriched for B cell TF motifs and had high STAT5A and NFAT co-occupancy. ESE associated genes were more highly expressed than other LCL genes. Disrupting ESEs by the bromodomain inhibitor JQ1 or conditionally inactivating an EBV oncoprotein or NF-kB decreased MYC or BCL2 expression and arrested LCL growth. To further characterize the ESEs, we will examine their (1) DNA elements, (2) protein compositions, and (3) associated enhancer RNAs (eRNAs). We will use reporter assays and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to identify the DNA elements essential for ESE activity. We will use BioTAP-XL crosslinking, affinity purification followed by mass spectrometry to characterize the ESE proteomic components. We will also use Global Run On followed by deep sequencing (GRO-seq) to identify enhancer RNAs affected by EBV super-enhancers and useshort hairpin RNAs (shRNAs), short interferring RNAs (siRNAs) or Locked Nucleic Acid anti-sense oligonucleotides (LNAs) knock down to determine their significance. The experiments here in use integrative approaches to elucidate the molecular mechanism by which ESEs activate key oncogenic divers. These studies will identify opportunities for therapeutic intervention.