Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death and has the lowest survival rate of all major cancers (3-5%). Despite our advanced understanding of disease pathology and underlying genetics, there are no effective chemotherapeutic or targeted treatment options, and the median survival time remains unchanged at only 6 months. PDAC arises from complex and heterogeneous genetic aberrations, which in a single case typically involve >50 genes. Besides four hallmark mutations affecting KRAS, TP53, CDKN2A and SMAD4, aberrations of the chromatin regulatory machinery are common in PDAC. Chromatin-associated genes are particularly attractive as drug targets, since resulting epigenetic aberrations are reversible and often amendable to drug-based modulation. However, the mechanistic role of chromatin aberrations, as well as their potential as therapeutic targets have not been explored in PDAC. In order to accelerate the search for more effective therapies for this devastating disease, this project will combine genetically engineered PDAC mouse models, multiplexed RNAi screening and advanced in vivo RNAi technologies to systematically analyze chromatin-regulatory genes in tumorigenesis and maintenance of PDAC. As tractable experimental system for in vivo RNAi studies, I will generate a Tet-on competent PDAC mouse model based on endogenous KrasG12D and homozygous loss of Trp53. Following its characterization, the model will be employed in multiplexed in vitro and in vivo RNAi screens using a new chromatin shRNA library. Selected hits will be functionally characterized using innovative in vivo RNAi approaches. Overall, these studies aim to advance our mechanistic understanding of chromatin (dys)regulation in PDAC, identify new target molecules for the development of more effective therapeutic, and establish a powerful in vivo system that is generally applicable for the pre-clinical evaluation of targeted therapies in PDAC.