Most patients with small intestinal neuroendocrine tumors (SI-NETs) present distant metastases at diagnosis, however the underlying mechanisms that contribute to metastasis are not yet fully understood. Previous studies have demonstrated that SI-NETs harbor fewer mutations than other cancers, but are highly epigenetically dysregulated, which suggest the great potential of epigenetic-targeted therapy for these tumors. During metastatic cascade, tumor cells acquire the ability to invade and migrate, and to rapidly adopt to the changing microenvironment. The global transcriptional reprogramming involved in this process implies that reversible epigenetic mechanisms, rather than permanent genetic mutations, are the potential key regulators of metastasis. Therefore, understanding the underlying epigenetic mechanisms involved in metastatic progression is the main concern of this project. Multiple primary tumors and metastases from a single patient will be used to perform Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) analysis. This method uses an engineered, hyperactive Tn5 transposase that cleaves and adds adopters into open regions of chromatin. The resulting fragmented DNA will be then sequenced. The open chromatin regions are often cell-type or -state specific and are enriched with transcription factor (TF) binding motifs and regulatory elements. First, the peaks identified by ATAC-seq will be compared between primary tumors and metastases to find differentially accessible regions. Second, TF motif analysis will be performed to identify enriched TFs in the differentially accessible regions. These results will allow us to find metastatic specific TF, their specific binding sites and target genes, and will help us to identify putative TF or gene targets that have a key role in metastatic progression. The identified potential targets will be further studied experimentally using a validation cohort from our biobank, the neuroendocrine cell lines, and the SI-NET xenograft mouse model. Luciferase assay, CRISPR-cas9 gene editing tool, quantitative RT-PCR, immunostaining and western blotting will be applied. In this project, we will combine a unique neuroendocrine tumor biobank, state-of-the-art sequencing technologies and bioinformatics tools to uncover the epigenetic drivers of metastasis in SI-NETs. Our invaluable tissue and blood biobank provides excellent material for the planned study. By applying ATAC-seq we will be able to identify regulatory elements, TFs and potential target genes that are involved in the metastatic progression. Evaluation of these potential targets using in vitro and in vivo models can ultimately unlock specific therapeutic options and improve prognostic biomarkers. Possibly, these findings will be translatable to other NETs due to their similarities, thus potentially benefiting more patients.