Abstract 105: Depletion of extracellular cystine and cysteine by a mutagenized human enzyme causes ROS mediated cytotoxicity in pancreatic cancer cells (2017)

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival rate at 7%. The oncogene KRAS that is mutationally activated in over 90% of PDACs, has been shown to be involved in regulating redox homeostasis. Hence, perturbation of oxidative balance might provide a therapeutic window to effectively treat pancreatic cancer. One strategy to achieve this is to target the tripeptide glutathione (GSH) – a major intracellular antioxidant. Cysteine (Cys), which has the functional moiety of GSH, can either be synthesized de novo or imported, predominantly as cystine (CSSC) that is then reduced intracellularly to Cys. In cancer, intracellular Cys synthesis has to be supplemented with extracellular import in order to fulfil the excessive metabolic demand of proliferation, which includes maintenance of oxidative balance through GSH synthesis. We hypothesize that this increased requirement for Cys/CSSC import in tumor cells will make them selectively sensitive to prolonged depletion of these amino acids in the serum by a genetically engineered human enzyme called Cyst(e)inase. In addition, we believe that combining Cyst(e)inase with other redox balance perturbing agents will produce a synergistic therapeutic effect. In our study, Cyst(e)inase treatment of cultured pancreatic cancer cell lines (Panc1, MIA-PaCa2, BxPC3) decreased intracellular Cys and GSH, and inhibited cell growth. Sensitivity to Cyst(e)inase was correlated with ROS accumulation (Panc1&amp;gt;MIA-PaCa2&amp;gt;BxPC3). Panc1 cells exhibited G2-arrest and apoptotic cell death following 24 hours of treatment whereas BxPC3 cells underwent only a G1-arrest and no cell death even after 72 hours of treatment. Further mechanistic investigation showed activation of AMP kinase and other stress related kinases (p38, ERK and JNK), and DNA damage signaling (ATM) only in the more sensitive cell lines. Inhibition of the mTORC1-p70S6K-S6 ribosomal protein signaling pathway was observed in all 3 cell lines. Cyst(e)inase displayed synergistic cytotoxicity when combined with buthionine sulfoximine (GSH synthesis inhibitor), auranofin (thioredoxin reductase inhibitor), sulfasalazine (inhibitor of cystine import) and the natural compound curcumin known to increase intracellular ROS, indicating that concurrently inhibiting alternative cellular antioxidant pathways or directly increasing intracellular ROS might improve the anti-tumor efficacy of Cyst(e)inase. The effect of biweekly intraperitoneal Cyst(e)inase treatment on growth of pancreatic cancer cell xenografts in nude mice will also be reported. Collectively, the current data suggest that depletion of extracellular Cys/CSSC using Cyst(e)inase may have utility either as a monotherapy or a combination therapy for pancreatic cancer. Citation Format: Sabin Kshattry, Achinto Saha, Shira Cramer, John DiGiovanni, Stefano Tiziani, Nathalie Munoz, George Georgiou, Everett Stone. Depletion of extracellular cystine and cysteine by a mutagenized human enzyme causes ROS mediated cytotoxicity in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 105. doi:10.1158/1538-7445.AM2017-105