PROJECT SUMMARY/ABSTRACT Individual cells within a given cancer type are capable of expressing a diversity of phenotypic states resulting from an underlying heterogeneity of genetic, epigenetic, transcriptomic, and molecular features. How this diversity evolves and influences therapeutic response is an essential question in cancer biology. One emerging mechanism of developing such heterogeneity is the evolution of microenvironmental niches within tumors that support a cancer stem cell (CSC) state. CSCs are defined by their functional capabilities such as long-term self- renewal, the capacity to give rise to a range of differentiated cell types, and enhanced tumor-forming ability. They are also believed to drive resistance to anti-tumor therapies, such as radiation therapy. Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, with a 5-year overall survival of <10% and a dire need for novel therapeutic strategies. Radiation is an integral part of PDAC therapy, however not all cancer cells respond. Identifying mechanisms of radioresistance would transform the clinical management of PDAC. Using pancreatic tumor models, our preliminary results suggest that secreted Wnt ligands produced by one cancer cell subset drive a Lgr5+ stem-like state in another cancer cell subset, in essence forming a supportive niche that promotes stemness within pancreatic tumors. In this proposal, I will test the central hypothesis that Wnt-driven cellular phenotypic heterogeneity and stemness promote radiotherapy resistance in pancreatic adenocarcinoma. I will examine the role of pancreatic tumor cell subpopulations in radiation resistance, including the Lgr5+ cells (Aim 1) and the Wnt producing niche (Aim 2). Under Aim 1, I will characterize the cancer stem cell properties of Lgr5+ cells in pancreatic cancer, their relative resistance to radiation therapy, and their role in tumor repopulation. These results will determine whether Lgr5+ cells are CSCs and drivers of radioresistance in established tumors and inform their molecular characteristics, which may provide added means to target these cells. Under Aim 2, I will investigate the targeting of the Wnt-producing niche in combination with radiation. Specifically, I will use genetic or pharmacologic perturbation of the Wnt pathway in combination with radiation and assay tumor response and animal survival. These efforts will test the therapeutic potential of Wnt inhibitors as radiosensitizers in PDAC. Collectively, this work will facilitate basic mechanistic insights into both cellular heterogeneity and radioresistance, with the ultimate goal of translating these discoveries and developing improved treatment strategies for PDAC patients.