Cysteine and Cystine Depletion Targets Leukemia Stem Cells
Authors: Jones Courtney L , Stevens Brett M , D'Alessandro Angelo , Culp-Hill Rachel , Reisz Julie , Ye Haobin , Khan Nabilah , Minhajuddin Mohd , DeGregori James , Pollyea Daniel A , Jordan Craig T 
Published: October 2019

Abstract
The goal of this project was to identify and target metabolic vulnerabilities of leukemia stem cells (LSCs) to improve therapeutic outcomes for patients with AML. We have previously shown that primary human LSCs reside in a unique metabolic condition characterized by a relatively low oxidative state (termed "ROS-low") and increased levels of glutathione (Lagadinou et al. Cell Stem Cell, 2013). Cells in this condition are highly dependent on oxidative phosphorylation for survival, in striking contrast to many tumor cells which often rely on glycolysis; indicating that LSCs are governed by distinct metabolic properties. To further elucidate key metabolic properties of LSCs, we measured differences in the global metabolome of ROS-Low LSCs in comparison to ROS-high AML blasts. Our preliminary data demonstrated that ROS-low LSCs have higher levels of amino acids and require amino acid catabolism for survival. We hypothesized that certain individual amino acids may be more important for LSC survival. If true, then targeting specific amino acids may be an avenue towards improved AML therapy.
To determine if any individual amino acid is essential for LSC survival, we analyzed AML cells from five patients that were systematically cultured in media lacking one of the twenty amino acids. Cysteine depletion was consistently the most cytotoxic, showing decreased cell viability and colony forming potential of LSCs. We next determined the effect of an engineered human enzyme that selectively degrades cysteine and cystine (AEB3103, Aeglea BioTherapeutics, Inc.) on LSC viability and colony forming potential. We found that AEB3103 treatment decreased viability of LSCs in all AML specimens tested and significantly decreased colony formation (p<0.05). Importantly, AEB3103 treatment did not decrease the frequency or colony forming ability of HSCs in normal mobilized peripheral blood or normal bone marrow.
To determine how AEB3103 decreased LSC viability we investigated the metabolic changes that occur upon AEB3103 treatment by mass spectroscopy. We found that AEB3103 treatment decreased the abundance of metabolites involved in glutathione synthesis (cysteine, cystine, glutathione, taurine, and Cys-Gly) in ROS-low LSCs. Tracing of cysteine13C315N in LSCs demonstrated that all detectable heavy cysteine was metabolized to cystine and glutathione. Furthermore, pretreatment with cell permeable glutathione rescued cell viability and colony-forming potential upon AEB3103 treatment. These data suggest that modulation of glutathione is central to the mechanism by which AEB3103 kills LSCs. Glutathione is a well-characterized antioxidant, therefore, we measured ROS levels and the expression of genes known to be expressed upon ROS induction in LSCs. Surprisingly, we did not observe changes in ROS induction or the expression of ROS induced genes upon AEB3103 treatment.
Next, we interrogated cellular functions of glutathione that are independent from the role of glutathione as an antioxidant. Glutathione has previously been shown to mediate electron transport chain complex II function via posttranslational glutathione modification, glutathionylation. Therefore, we hypothesized that decreased glutathione levels upon AEB3103 treatment could result in decreased complex II activity and therefore decreased levels of OXPHOS and ATP production. To test this hypothesis, we measured complex II activity, complex II glutathionylation, oxidative phosphorylation, and ATP levels upon AEB3103 treatment. We observed AEB3103 treatment significantly decreased glutathionylation, complex II activity, oxidative phosphorylation, and ATP levels in AML cells (p<0.05). Furthermore, pretreatment with cell permeable glutathione completely reversed the metabolic consequences of AEB3103 treatment. Overall, our data demonstrates that cysteine depletion selectively targets LSCs by decreasing glutathione levels resulting in decreased electron transport chain complex II activity. Furthermore, targeting cysteine and cystine metabolism may be a promising approach to eradicate LSCs.

Disclosures
Pollyea: AbbVie: Consultancy, Research Funding; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy; Curis: Membership on an entity's Board of Directors or advisory committees.