Fig. 1. Functional genomics identifies metabolic determinants of proliferation under ASNase treatment.

(A) Right: Schematic outlining cell line competition assay. Left: Log₂ fold change in abundance from initial pool, of barcodes (n = 3) representing indicated cell lines in the competition assay, for ASNase-treated tumors (n = 10) relative to mean of vehicle-treated tumors (n = 10). Boxes represent the median and first and third quartiles, and whiskers represent the minimum and maximum of all data points. Statistics: false discovery rate (FDR)–adjusted P, by two-tailed unpaired t test for unequal variances, of ASNase tumor group versus vehicle tumor group. Individual CCLE RNA-seq ASNS expression levels of cell lines are also shown (x indicates no data available). (B) Schematic depicting pooled CRISPR screen under ASNase treatment (0.25 U/ml) using a metabolism-focused single-guide RNA (sgRNA) library. (C) Left: The top 25 genes differentially required under ASNase treatment are shown. Right: Gene scores for Jurkat cells grown in untreated versus ASNase-treated vessels. Most genes, as well as nontargeting control sgRNAs, gave similar scores in untreated and treated vessels. AA, amino acid. (D) Log₂ fold change in the abundance of individual sgRNAs in untreated (black) or ASNase-treated (gray) for top-scoring genes, ASNS, TPK1, GOT2, and MDH2. (E) Schematic demonstrating that top-scoring genes in the CRISPR screen highlight a specific route from glutamine to asparagine as essential under ASNase treatment.