Depletion of GATOR1 complex or TSC components decreases B-ALL cell dexamethasone sensitivity. (A) Schematic representation of the mTORC1-mediated signaling pathway. Both GATOR1 and TSC complexes negatively regulate mTORC1 signals depending on cellular metabolic status, reducing de novo protein synthesis and increasing autophagic activity. (B) Competition assay using NALM-6 cells stably expressing Cas9 (NALM-6-Cas9). Cells were transduced with a lentivirus vector encoding an sgRNA and a Crimson cassette and cultured with or without dexamethasone (10 nM). Crimson-positive cell fractions were assayed by fluorescence-activated cell sorting (FACS) at indicated times, and proportions were normalized to the number of Crimson-positive cells present at day 0 (2 days after transduction). Empty vector served as control. Each condition was assessed in triplicate, and data are represented as means ± standard deviation (SD). (C) NPRL2-WT or -KO NALM-6 cells were treated with either DMSO or dexamethasone, and proportions of apoptotic cells were assessed by annexin V staining 48 hour later. Conditions were assessed in triplicate, and data are represented as means ± SD. P values were calculated by Student t test. (D) NPRL2 WT, NPRL2 KO only, or NPRL2-KO cells overexpressing WT NPRL2 were treated for 96 hours with either dexamethasone at various doses (0.32 nM, 1 nM, 3.2 nM, 10 nM, or 32 nM) or DMSO. Viability of dexamethasone-treated cells was analyzed based on luminescence signal values, and values at each dose were normalized to values seen in DMSO-treated controls. Each condition was assessed in triplicate, and data are represented as means ± SD. (E) NPRL2-WT or -KO NALM-6 cells were treated for 72 hours with either DMSO, dexamethasone, RMC-5552, or a combination of dexamethasone and RMC-5552, and cell viability was assessed as in panel D. P values were calculated by analysis of variance (ANOVA) with Dunnett multiple-comparison test. The data presented are representative of multiple independent experiments.