Cysteine depletion targets leukemia cells in a glutathione-dependent manner. (A) Viability of ROS-low LSCs and AML ROS-high blasts cultured without cysteine (-C), arginine (-R), or glutamine (-Q) for 24 hours normalized to cell cultured in complete media. (B) Colony number of primary AML ROS-low LSCs and AML ROS-high blasts after a 24-hour culture with (CM) or without cysteine (-C), arginine (-R), or glutamine (-Q). The methylcellulose contained all amino acids. Data were normalized to colony number in CM ROS-low LSCs. (C) Stable isotope-labeled cysteine and glutathione in ROS-low LSCs after incubation with cysteine (¹³C₃¹⁵N) for 1, 6, 12, or 24 hours. Y-axis represents intensity of metabolite detected by mass spectroscopy in arbitrary units (AU). The experiment was run in replicates of 5. (D) Cysteine and glutathione levels in ROS-low LSCs 8 hours postincubation with 125 nM cyst(e)inase. (E) Representative data of levels of cellular (measured by CellROX and DCF) and mitochondrial (measured by MitoSOX and MitoPY) reactive oxygen species in vehicle and cyst(e)inase-treated cells for 8 hours. Statistical significance was determined by a 2-way analysis of variance analysis. *P < .05; **P < .01; ***P < .005; ****P < .001.