Ferroptosis is a form of cell death that results from the catastrophic accumulation of lipid reactive oxygen species (ROS). Oncogenic signaling elevates lipid ROS production in many tumor types and is counteracted by metabolites that are derived from the amino acid cysteine. In this work, we show that the import of oxidized cysteine (cystine) via system xC– is a critical dependency of pancreatic ductal adenocarcinoma (PDAC), which is a leading cause of cancer mortality. PDAC cells used cysteine to synthesize glutathione and coenzyme A, which, together, down-regulated ferroptosis. Studying genetically engineered mice, we found that the deletion of a system xC– subunit, Slc7a11, induced tumor-selective ferroptosis and inhibited PDAC growth. This was replicated through the administration of cyst(e)inase, a drug that depletes cysteine and cystine, demonstrating a translatable means to induce ferroptosis in PDAC.

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Pancreatic ductal adenocarcinoma (PDA) is the third-leading cause of cancer mortality in the US and is highly resistant to classical, targeted, and immune therapies. We show that human PDA cells are dependent on the provision of exogenous cystine to avert a catastrophic accumulation of lipid reactive oxygen species (ROS) that, left unchecked, leads to ferroptotic cell death, both in vitro and in vivo. Using a dual-recombinase genetically engineered model, we found that acute deletion of Slc7a11 led to tumorselective ferroptosis, tumor stabilizations/regressions, and extended overall survival. The mechanism of ferroptosis induction in PDA cells required the concerted depletion of both glutathione and coenzyme A, highlighting a novel branch of ferroptosis-relevant metabolism. Finally, we found that cystine depletion in vivo using the pre-IND agent cyst(e)inase phenocopied Slc7a11 deletion, inducing tumor-selective ferroptosis and disease stabilizations/regressions in the well-validated KPC model of PDA.One Sentence Summary: Genetic and pharmacological targeting of cystine import induces pancreatic cancer-selective ferroptosis in vivo. Main Body:The cancer-selective induction of apoptosis through cytotoxic chemotherapy is a foundation of modern oncology. However, some cancers, such as PDA, have proven highly resistant to traditional therapeutic strategies (1). In addition to proliferative signals, activating mutations in KRAS (found in >90% of human pancreatic tumors) induce both an increase in the generation of ROS as well as compensatory antioxidant programs (2-4). We hypothesize that in this state of elevated ROS flux, ROS detoxification programs become a critical metabolic dependency.Cellular ROS are neutralized primarily by thiols derived from the semi-essential amino acid cysteine (5-10). Antioxidant molecules such as glutathione and thioredoxin serve to position the uniquelyreactive sulfhydryl group of cysteine to catalyze ROS detoxification reactions. As cysteine is ultimately derived from extracellular sources, we examined the effects of culturing PDA cell lines in the absence of Author Contributions

PTEN is the second most frequently altered tumor suppressor found in human cancer, and is downregulated in a majority of pancreatic tumors. PTEN-Long is an endogenously expressed translational variant of this tumor suppressor that is identical to PTEN with the exception of an additional N-terminal region that gives this variant the ability to cross the cell membrane. Purified PTEN-Long can be used therapeutically to restore lost PTEN function, and has provoked profound regressions in PTEN null xenograft models. Here we explore the effect of PTEN-Long therapy in pancreatic cancer, both in the presence and absence of endogenous PTEN. In preliminary data, we found that combining PTEN-Long with chemotherapy extended overall survival in a clinically predictive genetically engineered mouse model of PDA (K-rasLSL.G12D/+; p53LSL.R172H/+;PdxCre, called KPC). To explore PTEN-Long’s ability to enter various tissues we performed pharmacokinetic and pharmacodynamic experiments. We also utilized a surgical biopsy technique to obtain paired pre- and post-treatment tumor samples. These experiments demonstrated that PTEN-Long is able to alter canonical PTEN/PI3K signaling within pancreatic tumor tissues. Finally, we treated PTEN deficient mice (K-rasLSL.G12D/+; PTENFlox/+; PdxCre) with PTEN-Long in an attempt to model the therapeutic response of the ~5% of human pancreatic tumors that are genetically PTEN null. Longitudinal ultrasound imaging of the first of these mice suggests that these mice have more significant and prolonged regressions compared to KPC mice following PTEN-Long treatment. This abstract is also presented as Poster A86. Citation Format: Roshan Ara Ahmed, Benjamin Hopkins, Carmine Palermo, Steve A. Sastra, Zachary Rapp, Ramon Parsons, Kenneth P. Olive. PTEN-Long and gemcitabine combination treatment as a therapeutic for pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr PR14.

This abstract is also being presented as a short talk in Session 8: Clinical Science Trials. A full abstract is printed in the Proffered Abstracts section (PR14) of the Conference Proceedings. Citation Format: Roshan Ara Ahmed, Benjamin Hopkins, Carmine Palermo, Steve A. Sastra, Zachary Rapp, Ramon Parsons, Kenneth P. Olive. PTEN-Long and gemcitabine combination treatment as a therapeutic for pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A86.