Metabolic landscape provides the biological rationale for amino acid–depletion therapies in MM. (A) Heat map showing expression levels for the probe sets corresponding to ASNase signature defined by Sugimoto et al36 in plasma cells from patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), active disease (MM), and plasma cell leukemia (PCL), as well as in HMCLs compared with normal plasma cells (accession numbers GSE66293 and GSE47552). The color scale spans the relative gene expression changes standardized on the variance. (B) Bar graph showing the frequency of primary patient MM samples included in GSE66293 with t(11;14) (left panel) and t(4;14) (right panel) displaying ASNase high vs low signature. The P value was calculated using Fisher’s exact test. (C) Kaplan-Meier survival curves on OS (left panel) and PFS (right panel) data for low and high z-scores of ASNase signature MM groups from CoMMpass study. Log-rank test P values and MM patients are stratified in each group, according to their risk at the time of diagnosis. (D) Waterfall plots showing ASNS and GLS2 mRNA levels related to their DNA methylation in MM patients.27 Each bar represents a tumor sample. The P values were calculated based on the Spearman correlations test (2-sided). Red indicates expression above the median, and blue indicates expression below the median. (E) Asn (upper panel) and Gln (lower panel) concentration, as measured in conditioned media collected at each time point from RPMI-8226 cells treated with 0.3 U/mL E coli ASNase or Erw-ASNase. Amino acid concentration was normalized to relative control. (F) Drug effects on the indicated MM cell lines treated with increasing doses of Erw-ASNase (0.1-3 U/mL for 48 hours). (G) MM.1S cells were treated with 0.5 U/mL Erw-ASNase at indicated time. Immunoblots for cMyc, CDK4, CDK6, and p21 are shown. γ-tubulin was used as loading control. (H) Cell cycle analysis of MM.1S cells at the indicated time points following Erw-ASNase treatment (0.5 U/mL), by flow cytometry. **P < .01, unpaired Student t test.