Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than non-malignant cells due to genetic alterations and abnormal growth and as a result, maintenance of the anti-oxidant glutathione (GSH) is essential for their survival and proliferation1–3. Under elevated ROS conditions endogenous l-Cysteine (l-Cys) production is insufficient for GSH synthesis, necessitating l-Cys uptake, predominantly in its disulfide form l-Cystine (CSSC) via the xCT(−) transporter. Here we show that administration of an engineered, pharmacologically optimized, human Cyst(e)inase enzyme mediates sustained depletion of the extracellular l-Cys and CSSC pool in mice and non-human primates, selectively causes cell cycle arrest and death (PI and Annexin-V staining) in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS, yet results in no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in prostate and breast cancer xenografts and doubled the median survival time of TCL1-Tg:p53−/− mice that develop disease resembling human chronic lymphocytic leukemia. The observation that enzyme-mediated depletion of the serum l-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods suggests that Cyst(e)inase represents a safe and effective therapeutic modality for inactivating anti-oxidant cellular responses in a wide range of malignancies4,5.

SummaryCancer cells reprogram their metabolism, altering both uptake and utilization of extracellular nutrients. We individually depleted amino acid nutrients from isogenic cells expressing commonly activated oncogenes to identify correspondences between nutrient supply and viability. In HME (human mammary epithelial) cells, deprivation of cystine led to increased cell death in cells expressing an activated epidermal growth factor receptor (EGFR) mutant. Cell death occurred via synchronous ferroptosis, with generation of reactive oxygen species (ROS). Hydrogen peroxide promoted cell death, as both catalase and inhibition of NADPH oxidase 4 (NOX4) blocked ferroptosis. Blockade of EGFR or mitogen-activated protein kinase (MAPK) signaling similarly protected cells from ferroptosis, whereas treatment of xenografts derived from EGFR mutant non-small-cell lung cancer (NSCLC) with a cystine-depleting enzyme inhibited tumor growth in mice. Collectively, our results identify a potentially exploitable sensitization of some EGFR/MAPK-driven tumors to ferroptosis following cystine depletion.

Prostate cancer (PCa) is the most common non-skin neoplasm and second leading cause of cancer death in men in the USA. The major drawbacks of PCa treatment is the development of resistance to androgen ablation therapy. Due to abnormal growth and genetic alterations, cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than the normal cells. The L-cysteine (Cys) containing tripeptide, glutathione (GSH) is the major intracellular antioxidant and is essential for the survival and proliferation of cancer cells. Under conditions of elevated ROS, endogenous Cys production is insufficient for GSH synthesis. This necessitates uptake of Cys that is predominantly in its disulfide form, L-cystine (CSSC), via the xCT(-) transporter. Cys is a non-essential amino acid in animals; therefore, eliminating Cys and CSSC uptake should selectively impact tumors that display increased ROS production, without causing an adverse effect on normal physiology. However, inhibition of xCT(-) alone is insufficient because free Cys is still imported via other transporters. A superior approach is the elimination of Cys and CSSC through the action of an enzyme that converts these amino acids into non-toxic products. Based on the idea that enzyme mediated systemic depletion of the serum Cys/CSSC pool would constitute a powerful and completely novel therapeutic approach, we developed a genetically engineered and pharmacologically optimized human enzyme called cyst(e)inase. We show that administration of cyst(e)inase mediates sustained depletion of the extracellular Cys and CSSC pool in mice. Treatment with this enzyme selectively causes cell cycle arrest and death in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS; yet no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts and reduced tumor growth in PCa xenografts. Mechanistically, cyst(e)inase treatment increased AMPK phosphorylation, reduced mTORC1 activity, formation of LC3 II as well as modulation of several cell cycle proteins including p27, c-Myc, CDK2, CDK4, pRB, E2F4 and cyclins A, D1 and E1. Further studies showed cyst(e)inase produced synergistic effects with a GSH synthesis inhibitor, buthionine sulfoximine and the natural compound, curcumin for cell growth inhibition and ROS production in vitro. Cyst(e)inase also showed synergistic tumor growth inhibition with curcumin in a xenograft model of human castrate resistant PCa cells. Collectively, enzyme-mediated depletion of serum Cys and CSSC pool suppresses the growth of prostate tumors, and is very well tolerated. These results suggest that cyst(e)inase represents a potentially safe and effective therapeutic modality as a single agent or in combination for the treatment of prostate and possibly other cancers. Citation Format: Achinto Saha, Shira L. Cramer, Sabin Kshattry, Stefano Tiziani, Everett Stone, George Georgiou, John DiGiovanni. Novel therapeutic approach through systemic depletion of L-cyst(e)ine with engineered cyst(e)inase enzyme for suppression of prostate tumor growth [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 2132. doi:10.1158/1538-7445.AM2017-2132

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>MIA-PaCa2>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

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.