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Page 2 of 38 J Cancer Metastasis Treat 2020;6:5 I http://dx.doi.org/10.20517/2394-4722.2020.13

1. Amino acid depleting enzymes alone or in combinations as a therapeutic strategy for
cancer treatment

John DiGiovanni

Division of Pharmacology & Toxicology, College of Pharmacy and Livestrong Cancer Institutes, Dell Medical
School, University of Texas at Austin, Austin, TX 78712, USA.

Background and aim: Significant differences exist between the metabolism and antioxidant requirements
of normal and malignant cells. Tumor cells depend on exogenous nutrients in their microenvironment to
fulfill the elevated energy requirements and for maintaining appropriate intracellular antioxidant levels.
Deprivation of amino acids results in growth inhibition or death of tumor cells by the modulation of
various signaling cascades and in some cases redox balance. We have been evaluating potential therapeutic
enzymes that degrade critical amino acids required for tumor growth. These engineered human enzymes
include one that degrades either L-cysteine and one that degrades methionine.

Experimental procedures: (1) in vitro cell culture experiments to evaluate cell survival using 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet assays; (2) metabolomics analyses of amino
acids and metabolites; (3) analyses of oncogenic cell signaling, reactive oxygen species (ROS) levels, and
DNA damage as well as cell cycle changes using flow cytometry; (4) in vivo allograft and xenograft tumor
experiments with various cancer cell lines.

Results: Depletion of extracellular L-cys/cystine led to depletion of intracellular L-cys, decreased levels
of intracellular glutathione (GSH), and increases in intracellular ROS leading to activation of cellular
signaling pathways, oxidative DNA damage, and ultimately cancer cell death. Cyst(e)inase, given i.p.,
significantly reduced serum levels of L-cys and significantly inhibited tumor growth in vivo of both prostate
and pancreatic cancer xenograft and allograft tumor models. Notably, targeting a second antioxidant
pathway together with cyst(e)inase (i.e., the thioredoxin pathway) using a thioredoxin reductase inhibitor
led to synergistic cancer cell killing and also sensitized tumor cells found to be more resistant to cyst(e)
inase alone. These and other studies on the mechanisms associated with the potential anticancer activity
of Cyst(e)inase are presented. In addition, we have also studied the potential therapeutic application of a
human engineered methionine (L-met) degrading enzyme called methionine gamma lyase (hMGL). Both
mechanistic studies as well as in vivo preclinical therapeutic studies demonstrate significant efficacy against
several cancers with hMGL.

Conclusion: Depletion of amino acids such as L-cys and L-met using human engineered enzymes offer
novel approaches for treating cancer either given alone or more likely in combination with other agents.

2. Investigating metabolic cancer vulnerabilities by high-content metabolomic screening

Stefano Tiziani

Department of Nutritional Sciences, University of Texas at Austin, Austin, TX 78712, USA.

Background and aim: The important role of cell metabolism in furthering cancer development and growth
[1,2]
is increasingly recognized . Recent advances in high-throughput metabolomics technology are leading to