Fig. 5: GATOR1 deficient lymphoma cells are highly sensitive to mTORC1 inhibition in vitro and in vivo.

A, B Response curves of sgControl, sgNprl3 or sgDepdc5 Eµ-Myc;Cas9 lymphoma cell lines to treatment in vitro with increasing doses of rapamycin (A) or Torin1 (B). Lymphoma cell viability was measured after 24 h of treatment with drug or vehicle by staining with Annexin V plus PI followed by flow cytometric analysis. Annexin V/PI double negative cells were deemed viable. Flow cytometry gating strategy represented in Supplementary Fig. S8B. n = 3 sgControl and 6 of each sgNprl3, sgDepdc5 or sgNprl2 lymphoma cell lines per genotype across 3 technical replicates. Data are presented as mean ± SEM, log transformed and fitting to non-linear regression. IC50 values are shown in brackets. Two-way ANOVA with Dunnett’s multiple comparison statistical test used to compare dose response to sgControl, significant P values displayed in corresponding color per genotype. C Schematic of in vivo rapamycin treatment experiments. One million sgControl, sgNprl3 or sgDepdc5 Eµ-Myc;Cas9 lymphoma cells were transplanted i.v. into the tail vein of RAG1-deficient mice, which lack B and T cells, to prevent lymphoma rejection due to an immune response against Cas9 and/or eGFP. Two days later, mice were randomly assigned into treatment arms, receiving either rapamycin at a dose of 8 mg/kg of body weight for 5 consecutive days by i.p. injection or vehicle as a control. Mice were monitored for lymphoma growth. Schematic created in BioRender. Potts, M. (https://BioRender.com/16f33zs). D Tumor-free survival curve of RAG1-deficient mice that had been transplanted with 1 × 10⁶ negative control (sgControl), sgNprl3 or sgDepdc5 Eµ-Myc;Cas9 lymphoma cell lines. n = 9 mice per treatment arm, with three cell lines per genotype, each injected into three recipient mice. Two-sided log-rank (Mantel-Cox) statistical test for survival curve comparison. Source data are provided as a Source Data file.