AbstractGlucocorticoids (GCs) are metabolic hormones with immunosuppressive effects that have proven effective drugs against childhood acute lymphoblastic leukemia (ALL). Yet, the role of metabolic reprogramming in GC-induced ALL cell death is poorly understood. GCs efficiently block glucose uptake and metabolism in ALL cells, but this does not fully explain the observed induction of autophagy and cell death. Here, we have performed parallel time-course proteomics, metabolomics, and isotope-tracing studies to examine in detail the metabolic effects of GCs on ALL cells. We observed metabolic events associated with growth arrest, autophagy, and catabolism prior to onset of apoptosis: nucleotide de novo synthesis was reduced, while certain nucleobases accumulated; polyamine synthesis was inhibited; and phosphatidylcholine synthesis was induced. GCs suppressed not only glycolysis but also entry of both glucose and glutamine into the TCA cycle. In contrast, expression of glutamine-ammonia ligase (GLUL) and cellular glutamine content was robustly increased by GC treatment, suggesting induction of glutamine synthesis, similar to nutrient-starved muscle. Modulating medium glutamine and dimethyl-α-ketoglutarate (dm-αkg) to favor glutamine synthesis reduced autophagosome content of ALL cells, and dm-αkg also rescued cell viability. These data suggest that glutamine synthesis affects autophagy and possibly onset of cell death in response to GCs, which should be further explored to understand mechanism of action and possible sources of resistance.