PROJECT SUMMARY The goal of our proposal is to identify therapeutic vulnerabilities associated with nutrient-poor conditions in tumors. Pancreatic ductal adenocarcinoma (PDAC) remains a major clinical challenge and is characterized by a dense stroma and paucity of blood vessels. The resulting hypoxia and scarcity of nutrients forces cancer cells to seek alternative sources of nutrients for growth. Paradoxically, cells residing in these nutrient- deprived microenvironments are among the most resistant to therapy as a result of poor drug diffusion and reduced cell proliferation (rendering DNA-damaging agents less effective). To target these cells, therefore, a better understanding of the adaptive mechanisms by which they cope with nutrient deprivation is urgently needed. In preliminary studies, we developed a culture protocol – limiting for oxygen, glucose, amino acids, and serum – to model the “arid conditions” of pancreatic tumors. Under such conditions, tumor cells slow down proliferation and metabolic activity. Using this system, we performed a whole genome CRISPR screen to identify genes required under arid conditions but dispensable under nutrient-replete (“fertile”) conditions. This revealed a strong dependency on the TCA cycle and oxidative phosphorylation for survival, but also suggested that genes involved in many biosynthetic activities (e.g. translation and cell division) are detrimental under arid conditions. Based on these results, we hypothesize that cells survive severe nutrient and oxygen deprivation by maximizing energy generation from limited fuel supplies while minimizing the use of such resources for macromolecule production. We further hypothesize that nutrient and oxygen deprivation alters the dynamic crosstalk between cancer cells and other cells comprising the tumor microenvironment (TME). Importantly, individual tumors may respond to these metabolic changes in different ways, contributing to inter-tumoral heterogeneity. Our proposal will explore these ideas to develop strategies that target therapy-resistant cancer cells residing in these nutrient-deprived niches. Aim 1. Characterize the heterogeneity in metabolic adaptations to arid conditions. Aim 2. Determine how arid conditions influence cellular crosstalk in the TME. Aim 3. Test the activity of metabolic inhibitors against cells in arid conditions in vitro and in vivo.