Functional characterization of relapse-specific mutations. (A) Top, relapse-specific NR3C1 mutation locations within the NR3C1 protein, with the x-axis indicating amino acid position. Bottom, their effects on NR3C1 transcriptional activator activity (left) and ALL sensitivity to glucocorticoids (right). Left, mutant, wild-type (WT), and empty vector (EV) transcription activator activity was measured in HEK293T cells by using the reporter gene assay. Mutations are grouped and color-coded according to their locations in protein domains. Right, glucocorticoid sensitivity (ie, dose–response curve) was measured by using cell viability after treatment with prednisolone for 72 hours in ALL cell line REH expressing WT or mutant NR3C1 (color-coded according to protein domain), using the MTT assay. The y-axis represents percent cell viability compared with untreated control. Error bars indicate standard error. (B) Top, FPGS relapse-specific mutations. Bottom, their effects on polyglutamation enzymatic activity using MTX as the substrate. Purified mutant and WT FPGS were tested at 3 different amounts (5 ng, 2.5 ng, or 1.25 ng) in triplicate, and the enzymatic activity was relative to the activity of the WT at 5 ng. The first 2 columns represent controls with no MTX or FPGS. Error bars represent standard error. (C) Top, NT5C2 relapse-specific mutations. Bottom, cell viability of REH or Nalm6 cells treated with 6-thioguanine (6-TG, left) or 6-mercaptopurine (6-MP, right) in cells expressing indicated NT5C2 mutations (or WT NT5C2 or uninfected control [-]). Error bars represent standard deviation. (D) Top, TP53 relapse-specific mutations. Bottom, functional validation of TP53 R248Q and R196G. Nalm6 cells underwent TP53 knockout (KO) by CRISPR and were reintroduced with TP53 WT, R196G, or the known hotspot R248Q mutant. Left, cell viability in response to idarubicin and vincristine treatment. Right, fold change in proportion of Annexin V–positive apoptotic cells (top) and in proportion of EdU+ cells in S phase (bottom; values <1.0 indicate G1/S checkpoint arrest induced by functional p53 in response to treatment), compared with untreated controls, after treatment with 1 µM (top) or 0.01 µM (bottom) idarubicin for 24 hours. Error bars represent standard deviation. Dotted line in right panels represents 1.0. Protein domains are as reported on pecan.stjude.cloud (A,D), National Center for Biotechnology Information (B), or Dieck et al³²  (C).