SRP328720 PRJNA747310 GSE180281 In vivo CRISPR screening identifies ISL2 as a tumor suppressor and regulator of metabolic gene expression in pancreatic cancer Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers with no targeted or personalized therapeutics. PDAC cells reprogram their transcriptional and metabolic state to sustain their energetic needs in the nutrient-poor tumor microenvironment. Identifying molecular regulators of PDAC growth is a critical step towards an improved understanding of the disease and new therapeutic approaches. We employed in vivo CRISPR screening in an orthotopic PDAC model to identify transcription factors (TFs) and chromatin regulators (CRs) whose inhibition promotes aggressive growth of PDAC cells in vivo. The screening identified a previously uncharacterized ISL LIM homeobox 2 (ISL2) gene as a candidate tumor suppressor among some anticipated growth modulators. We confirmed that depletion of ISL2 leads to enhanced cell proliferation and tumor growth in vitro and in vivo. Conversely, the exogenous expression of ISL2 or CRISPR-mediated epigenetic upregulation of the endogenous loci led to reduced cell proliferation, supporting the hypothesis that ISL2 is a candidate tumor suppressor. Importantly, we find that ISL2 is a nuclear, chromatin-associated transcription factor that is epigenetically silenced through DNA methylation in a significant fraction of PDAC tumors. Critically, higher DNA methylation of ISL2 or its reduced expression correlates with poor patient survival. Mechanistically, ISL2 binds to thousands of genes, and its depletion increases antioxidant capacity, oxidative phosphorylation (OXPHOS) and fatty acid metabolism. Critically, ISL2 depleted cells have heightened stemness potential, and we find PPAR? as a critical modulator of cell proliferation in ISL2 depleted cells. Notably, ISL2 depleted cells are sensitive to small molecule inhibitors of mitochondrial complex I in vitro and in vivo. Collectively, these findings nominate ISL2 as a putative tumor suppressor candidate whose inactivation leads to an aggressive PDAC growth through increased mitochondrial metabolism and antioxidant capacity, which creates a potentially exploitable therapeutic vulnerability. Overall design: Flag tagged ISL2 expressing mPanc96 cells were targeted by Flag Ab and H3K27ac Ab for CHIP seq with two replicates. GSE180281