Abstract: Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal human malignancies and is responsible for over 48,000 deaths per year in the United States. PDAC tumors have several genetic and physiological properties that promote the generation of highly toxic reactive oxygen species (ROS), which can interact destructively with cellular macromolecules. As a result, these tumors rely heavily on ROS detoxification programs which employ thiol groups to mitigate the electron imbalances that define ROS. Those thiols are ultimately derived from cysteine, a semi-essential amino acid with a thiol-containing side chain. As cysteine is ultimately sourced from dietary sources, the import of exogenous cysteine is a critical step in the ROS detoxification process. In the first term of this grant, we tested the hypothesis that PDAC depends on cysteine import for survival. We found that depletion of cysteine, either through depletion from the media or by inhibition of cysteine uptake, resulted in the induction of ferroptosis, a form of regulated necrosis that occurs due to oxidative damage to cellular membranes. We demonstrated that cysteine depletion could selectively induce ferroptosis in PDAC tumors in vivo using both genetic and pharmacological approaches and we expanded the mechanistic understanding of ferroptosis by demonstrating that depletion of coenzyme A (a cysteine derivative) is necessary for the induction of ferroptosis in pancreatic tumor cells. Here we will extend these findings in order to potentiate the effects of cysteine depletion in PDAC. First, we will study the mechanistic difference between inhibition of system xC– versus degradation of exogenous cystine with the engineered enzyme cyst(e)inase. We will then directly compare the preclinical efficacy of these approaches in a genetically engineered models of PDAC, making use of a new, highly potent system xC– inhibitor. Second, we will pursue our earlier findings on the role of coenzyme A in regulating ferroptosis by studying whether high levels of coenzyme A in the tumor microenvironment might help suppress ferroptosis in malignant epithelial cells. This leads to a strategy of targeting ENPP proteins, which are necessary for the (indirect) import of exogenous coenzyme A. Third, we will determine whether the release of free iron from ferritin through via autophagy can promote ferroptosis in the context of cysteine depletion. This leads to a strategy of activating autophagy via MEK inhibition to increase lipid ROS production and sensitize to ferroptosis in combination with cysteine depletion. For each of these topics, we will pursue both basic science mechanisms and translational strategies. The studies will make use of genetically engineered mouse models of PDAC as well as novel tumor explant models developed by our group in which human or murine PDAC samples are cultured intact in an engineered system for up to a week. Together, these studies will further elucidate the mechanis...