Abstract 367: Assessing the therapeutic efficacy of Cyst(e)inase to induce oxidative stress mediated cytotoxicity in pancreatic cancer cells

Abstract

Abstract Pancreatic ductal adenocarcinoma is the only cancer in the US with a dismal one-digit 5-year survival rate (7%). Its oncogenic program relies on upregulated ROS antioxidant mechanisms to maintain oxidative balance. Hence, perturbation of this balance might be an effective therapeutic strategy for pancreatic cancer. A major intracellular antioxidant is the tripeptide glutathione (GSH). Cysteine (Cys), which has the functional moiety of GSH, can either be synthesized de novo or imported [predominantly as cystine (CSSC) that is reduced intracellularly to Cys]. In cancer, de novo synthesis of Cys has to be supplemented with extracellular import in order to fulfil the excessive metabolic demand of proliferation. This intricate balance between ROS and antioxidants, and the excessive requirement for Cys/CSSC import in tumor cells, in part to maintain oxidative balance through GSH synthesis, potentially provides a therapeutic window. We believe that prolonged depletion of the serum Cys and CSSC pool by a genetically engineered human enzyme called Cyst(e)inase may provide a pancreatic cancer therapeutic avenue either as a monotherapy or in combination with other agents with low or minimal toxicity. In this study, Cyst(e)inase treatment (3-500 nM) of cultured pancreatic cancer cell lines (MIA-PaCa2, BxPC3, Panc1) caused a dose-dependent decrease in survival and intracellular GSH content. Further mechanistic investigation showed an increase in intracellular ROS, activation of AMP kinase and autophagy signaling, as well as inhibition of the mTORC1-p70S6K-S6 ribosomal protein signaling pathway. There was also a concentration dependent reduction in cyclin D1 after treatment with the enzyme. Cyst(e)inase displayed synergistic cytotoxicity when combined with the natural compound curcumin. Notably, a single intraperitoneal 100 mg/kg dose of PEGylated Cysteinase in mice was able to deplete serum CSSC below 5 μM (normal is ∼ 70 μM) for over 3 days with a half-life of ∼4 days. Longer treatment with Cyst(e)inase at this dose given 4 times/week for 6 weeks did not produce significant signs of toxicity. This in vivo dose also inhibits the growth of prostate cancer cells in xenograft tumor models. Ongoing experiments to evaluate the in vivo efficacy of Cyst(e)inase for inhibition of pancreatic tumor growth as well as further mechanistic studies will be presented. 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 several cancers including pancreatic cancer. Citation Format: Sabin Kshattry, Achinto Saha, Shira Cramer, Everett M. Stone, George Georgiou, John DiGiovanni. Assessing the therapeutic efficacy of Cyst(e)inase to induce oxidative stress mediated cytotoxicity in pancreatic cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 367.