Relapse-specific mutations in NT5C2 in childhood acute lymphoblastic leukemia

Abstract

Relapsed childhood acute lymphoblastic leukemia (ALL) carries a poor prognosis, despite intensive retreatment, owing to intrinsic drug resistance. The biological pathways that mediate resistance are unknown. Here, we report the transcriptome profiles of matched diagnosis and relapse bone marrow specimens from ten individuals with pediatric B-lymphoblastic leukemia using RNA sequencing. Transcriptome sequencing identified 20 newly acquired, novel nonsynonymous mutations not present at initial diagnosis, with 2 individuals harboring relapse-specific mutations in the same gene, NT5C2, encoding a 5'-nucleotidase. Full-exon sequencing of NT5C2 was completed in 61 further relapse specimens, identifying additional mutations in 5 cases. Enzymatic analysis of mutant proteins showed that base substitutions conferred increased enzymatic activity and resistance to treatment with nucleoside analog therapies. Clinically, all individuals who harbored NT5C2 mutations relapsed early, within 36 months of initial diagnosis (P = 0.03). These results suggest that mutations in NT5C2 are associated with the outgrowth of drug-resistant clones in ALL.

Figure 1. Relapse Specific Mutations in NT5C2 Alter Enzymatic Activity

a, Dimer of human cytosolic 5′-nucleotidase II subunits. There are two such dimers, linked by a different interface to form the tetrameric active form of this enzyme. The backbone traces of the structures are displayed as ribbon diagrams. The bottom monomer ribbon is colored in a gradient from its N-terminus (purple) to its C-terminus (red). The location of the active site is indicated with an asterisk (*). Note that the C-terminus of one monomer extends into a groove in the other monomer in order to form the dimer. The upper monomer ribbon is colored green for contrast. The location of the disordered loop at positions 400-417 is indicated as an orange dashed line in the bottom monomer and as a transparent green U-shaped arrow in the top monomer to show its expected area of interaction. Mutations p.Arg238Trp, p.Arg367Gln and p.Ser445Phe are displayed as space-filling spheres colored red for oxygen, blue for nitrogen and white for carbon and are also labeled. The projected locations of the insertion mutation (p.Lys404LysAsp) and point mutation (p.Ser408Arg) in the disordered loop, which is not visible in the crystal structure, are indicated by dashed circles and labeled. A straight transparent green arrow indicates the expected trajectory of the acidic C-terminal tail of the upper monomer, which is not present in the crystal structure, as it lies across the bottom monomer. b, NT5C2 coding region diagram with mutations. Three mutations were found at the same site in exon 9 encoding amino acid 238. c, Western blot analysis of cN-II protein induction by IPTG in BL21 cells. 10ug of each protein lysate run per lane and blotted with antibody against NT5C2. d, Equivalent volumes of BL21 protein lysate were subjected to the 5′-nucleotidase assay (Diazyme, Inc) according to protocol. Mean activity levels were normalized per protein concentration for each sample. Columns show the mean of three independent experiments ± s.d. P values calculated using two-sided unpaired Student's t-test (*p ≤ 0.01).

Figure 2. NT5C2 Mutants Confer Chemoresistance to Purine Nucleoside Analogue Treatment

Reh cells transiently infected with WT, mutant or control GFP lentivirus were treated with increasing concentrations of a, 6-TG, b, 6-MP, c, cytarabine, d, gemcitabine, e, doxorubicin, f, prednisolone and assayed for apoptosis. Columns show a mean of three independent determinations ± s.d. from a representative experiment repeated 3 times with similar results. P values calculated using two-sided unpaired Student's t-test (*p < 0.001). g, Western blot of infected Reh cells demonstrated presence of flag tagged NT5C2 constructs compared to GFP control and Reh cells alone. Actin shown as loading control.