The molecular function of the oncogenic NAB2-STAT6 fusion protein

项目摘要 孤立性纤维瘤（SFT）是一种间叶肿瘤类型，估计影响10，000例 美国人每年这些肿瘤中有10-20%会变成恶性，对治疗无反应。的 SFT的发病机制目前尚不清楚，因为在已知的肿瘤抑制因子中没有复发突变 基因或致癌基因。在SFT中鉴定的唯一重复突变是NAB 2-STAT 6之间的基因融合， 产生融合蛋白。NAB 2和STAT 6都是转录调节因子。NAB 2是早期生长抑制因子 应答转录因子（EGFR 1/2）和STAT 6是应答转录程序的激活因子。 细胞因子这两种蛋白质也有助于增强子激活。尽管这两种蛋白质都有已知的功能， NAB 2-STAT 6影响SFT中的基因表达是未知的。 使用基因集富集分析（GSEA）的一个大的微阵列数据集的SFT，我们发现 NAB 2和EGR 1靶点的表达在SFT中显著上调。但 STAT 6靶点的表达没有变化。我们表达NAB 2-STAT 6并进行ChIP-seq分析 并发现NAB 2-STAT 6定位于远端活性转录增强子。最后，我们分析了RNA-seq 发现近2000个基因在恶性肿瘤和良性肿瘤中差异表达， 几个基因，这是由充分表征的转录增强子调控。我们假设NAB 2- STAT 6异常激活EGR 1靶点以增加增殖并提高转录因子的活性。 促进恶性肿瘤的增强剂。 我们已经产生了负责NAB 2-STAT 6表达的染色体内倒位。 良性肺成纤维细胞IMR 90细胞系，其使用CRISPR-Cas9复制SFT的间充质起源。在 目的：1、研究NAB 2-STAT 6在基因表达调控中的作用。我们将描述 NAB 2-STAT 6的基因组结合谱以及其对基因表达和增殖的影响。在目标2中， 将研究NAB 2-STAT 6重编程转录增强子以促进恶性肿瘤的能力。第一、 我们将开发一个可诱导的NAB 2-STAT 6系统来测量NAB 2-STAT 6重编程的能力， 转录增强子。然后，我们将验证我们的结果在主要的SFT和检查的差异， 恶性肿瘤与良性肿瘤。最后，我们将检查转录增强子的变化影响的能力， 通过分析初级SFT的RNA-seq的基因表达。总的来说，这些目标将确立以下职能： NAB 2-STAT 6在促进SFTs肿瘤发生中的作用

Project Summary Solitary Fibrous Tumors (SFTs) are a mesenchymal tumor type that affects an estimated 10,000 Americans each year. 10-20% of these tumors become malignant and unresponsive to treatments. The pathogenesis of SFTs is currently unknown, as there are no recurring mutations in known tumor suppressors genes or oncogenes. The only recurring mutation identified in SFTs is a gene fusion between NAB2-STAT6 that results in a fusion protein. NAB2 and STAT6 are both transcription regulators. NAB2 is repressor of early growth response transcription factors (EGR1/2) and STAT6 is an activator of transcriptional programs in response to cytokines. Both proteins also contribute to enhancer activation. Despite both proteins' known functions, how NAB2-STAT6 affects gene expression in SFTs is unknown. Using the gene set enrichment analysis (GSEA) of a large microarray data set of SFTs we have found that the expression of both NAB2 and EGR1 targets were significantly upregulated in SFTs. However, the expression of STAT6 targets was unchanged. We expressed NAB2-STAT6 and performed ChIP-seq analysis and found that NAB2-STAT6 localizes to distal active transcriptional enhancers. Lastly, we analyzed RNA-seq and saw that almost 2000 genes were differentially expressed in Malignant tumors vs Benign tumors including several genes, which are regulated by well characterized transcriptional enhancers. We hypothesize that NAB2- STAT6 aberrantly activates EGR1 targets to increase proliferation and highjacks the activity of transcriptional enhancers to promote malignancy. We have generated the intra-chromosomal inversion responsible for NAB2-STAT6 expression in the benign lung fibroblast IMR90 cell line, which replicates the mesenchymal origin of SFTs using CRISPR-Cas9. In Aim 1 we will establish NAB2-STAT6’s role in directing aberrant gene expression. We will characterize the genomic binding profile of NAB2-STAT6 as well as its effect on gene expression and proliferation. In Aim 2 we will investigate the ability of NAB2-STAT6 to reprogram transcriptional enhancers to promote malignancy. First, we will develop an inducible NAB2-STAT6 system to measure the ability of NAB2-STAT6 to reprogram transcriptional enhancers. Then, we will validate our results in primary SFTs and examine differences in malignant vs benign tumors. Finally, we will examine the ability of transcriptional enhancer changes to affect gene expression through analysis of RNA-seq of primary SFTs. Overall, these aims will establish the function of NAB2-STAT6 in promoting tumorigenesis in SFTs