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