Abstract 3963: Cystine depleting human enzyme combined with auranofin synergistically inhibits pancreatic cancer cell growth in vitro and in vivo

Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest 5-year survival rates of all cancers at 8%. Upregulated mechanisms to curb reactive oxygen species (ROS) accumulation are thought to help the growth of PDACs. Hence, perturbation of oxidative balance might be an effective therapeutic strategy for pancreatic cancer. To achieve this end, we used a human enzyme called Cyst(e)inase that degrades extracellular cysteine (Cys) and cystine (CSSC) - substrates that are necessary for synthesis of the antioxidant glutathione (GSH). Since tumor cells need to supplement intracellular Cys synthesis with extracellular import in order to fulfil their increased demand for GSH and protein synthesis, Cys/CSSC depletion can provide a therapeutic window. We validated this idea in our previous study where Cyst(e)inase treatment markedly inhibited tumor growth in several cancer models in vivo without any toxicity. In this study, we assessed the efficacy of Cyst(e)inase alone as well as in combination with auranofin in reducing pancreatic tumor growth. Cyst(e)inase treatment of cultured pancreatic cancer cells (Panc1, MIA-PaCa2, BxPC3) decreased intracellular Cys and GSH, and inhibited cell growth. Panc1 was very sensitive to Cyst(e)inase in vitro, exhibiting ROS accumulation and activation of stress-related signaling as well as G2-arrest and apoptotic cell death whereas BxPC3 and MIA-PaCa2 showed only a G1-arrest. Inhibition of the mTORC1-p70S6K-S6 ribosomal protein signaling pathway via the eIF2α-ATF4-Sestrin2 axis was observed in all 3 cell lines. In addition, MIA-PaCa2 but not Panc1 exhibited a rewiring of mitochondrial metabolism upon Cyst(e)inase treatment that bypassed succinate dehydrogenase. Combining auranofin and Cyst(e)inase caused synergistic increase in mitochondrial ROS and apoptotic cell death in the resistant cell lines MIA-PaCa2 and BxPC3 with associated activation of oxidative stress signaling. Surprisingly, the thioredoxin reductase system (the putative target of auranofin) was dispensable in producing synergy with Cyst(e)inase. Moreover, auranofin and Cyst(e)inase combination led to inhibition of mitophagy and accumulation of defective mitochondria. Supplementation with the antioxidant glutathione ethyl ester rescued the combination's cytotoxic effects. Biweekly intraperitoneal Cyst(e)inase treatment of nude mice harboring pancreatic cancer xenografts roughly recapitulated the in vitro sensitivity profile with Panc1 showing the most growth inhibiti