PROJECT SUMMARY/ ABSTRACT Characterization of the regulatory landscape of pancreatic cancer subtypes. The goal of this proposal is to investigate regulatory differences between pancreatic ductal adenocarcinoma (PDAC) molecular subtypes. PDAC tumors are heterogenous, containing tumor epithelial cells as well as components of the tumor microenvironment. PDAC molecular subtypes which correlate with prognosis and treatment response have been identified through gene expression analysis. Identifying effective treatments for these diverse subtypes will require a deeper understanding of differences between the subtypes and how they evolve, which, in turn, requires understanding of differential gene regulation between subtypes and computational methods to identify relevant differences in layers of multi-omic regulatory data. This proposal provides a career transition plan for Dr. Deborah Weighill that will equip her with the additional training necessary to conduct her own multi-omic cancer studies and experimentally validate regulatory relationships hypothesized by her gene regulatory network analysis. During the mentored phase of the award (K99), she will leverage existing large, multi-omic PDAC datasets to construct tumor-specific gene regulatory networks (GRNs), and use these networks and network topology/comparison methods to identify differentially regulated genes between basal-like and classical subtypes (Aim 1), while training in multiple next-generation sequencing techniques, cell culture, and gene knockdowns experiments. This critical phase of training will be co- supervised by Dr. Jeh Jen Yeh (UNC Chapel Hill) and Dr. John Quackenbush (Harvard T.H. Chan School of Public Health), who are both experts in GRN analysis and PDAC cancer biology, respectively. During the transition to independence (K99/R00), she will perform whole genome sequencing, RNA-seq, chromatin accessibility and DNA methylation assays for 20 PDAC tumors, prioritize differentially regulated genes disrupted by multiple mechanisms, and assess the congruence of differential gene regulation between tumors and in vitro model systems (Aim 3). The final step in achieving independence (R00) will involve validating key regulatory relationships experimentally using ChIP-seq and gene knockdowns and identifying the source compartment of the differential regulation (tumor or stroma) computationally using non-negative matrix factorization methods and experimentally using GeoMx digital spatial profiling of tumors (Aim 3). This three- phase transition plan will illuminate our basic understanding of differential gene regulation between PDAC subtypes and provide an extensible computational/experimental platform for further investigation of therapeutic vulnerabilities. These aims are highly congruent with the NCI’s priorities of understanding the mechanisms of cancer, and of detecting and diagnosing cancer, as it will illuminate how gene regulation contributes to PDAC subtypes, treatment and outco...