Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL

Abstract

Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric patients and over 50% of adult patients with ALL do not achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most substantial challenge in the treatment of this disease. Using whole-exome sequencing, we identify mutations in the cytosolic 5'-nucleotidase II gene (NT5C2), which encodes a 5'-nucleotidase enzyme that is responsible for the inactivation of nucleoside-analog chemotherapy drugs, in 20/103 (19%) relapse T cell ALLs and 1/35 (3%) relapse B-precursor ALLs. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside-analog metabolism in disease progression and chemotherapy resistance in ALL.

Figure 1. NT5C2 mutations in relapsed pediatric T-ALL

(a) Schematic representation of the structure of the NT5C2 protein. The haloacid dehalogenase (HAD) and the substrate binding domains (SB) are indicated. NT5C2 mutations identified in relapsed pediatric samples are shown. Filled circles represent heterozygous mutations. Multiple circles in the same amino acid position account for multiple patients with the same variant. (b) DNA sequencing chromatograms of paired diagnosis and relapse genomic T-ALL DNA samples showing representative examples of relapse specific heterozygous NT5C2 mutations, with the mutant allele sequence highlighted in red.

Figure 2. Structure-function analysis of the NT5C2 K359Q mutant protein

(a) Molecular surface representation of NT5C2 protein structure. The position of the NT5C2 K359Q mutation found is highlighted in red. The substrate inosine monophosphate (IMP) is depicted in purple; the ATP allosteric activator is shown in yellow. (b) Structure representation of the NT5C2 catalytic center and allosteric regulatory site devoid of substrate or ligands (PDB 2XCX). (c) Structure representation of the NT5C2 catalytic center and allosteric regulatory site bound to IMP and ATP, respectively (PDB 2XCW). (d) Structure representation of the NT5C2 K359Q mutant model corresponding to the catalytic center and allosteric regulatory sites. (e) Overlay of the structures shown in b–d. The white arrow indicates the repositioning of Phe354 from the inactive NT5C2 configuration to the active –ATP-bound NT5C2 and NT5C2 K359Q– structures. Mg²⁺ ions are depicted as green spheres.

Figure 3. Increased 5'-IMP nucleotidase activity in NT5C2 mutant proteins

5'-Nucleotidase activity levels of recombinant mutant proteins relative to wild type NT5C2 control are shown. Data are means ± s.d.

Figure 4. Expression of NT5C2 mutations in ALL cells induces resistance to chemotherapy with 6-MP and 6-TG

(a) Viability assays in CCRF-CEM and CUTLL1 T-ALL cells expressing wild type NT5C2, relapse-associated mutant NT5C2 alleles or a red fluorescent protein control (RFP), treated with increased concentrations of 6-mercaptopurine (6-MP). (b) 6-Thioguanine (6-TG) dose response cell viability curves. Data is shown as means ± s.d.