A Novel Aberrantly Spliced Gain-of-Function NT5C2 Isoform Contributes to Thiopurine Resistance in Acute Lymphoblastic Leukemia

Abstract

AbstractRelapse-specific mutations do not account for all chemotherapy failures in patients with B-cell acute lymphoblastic leukemia (B-ALL). By mining RNA-seq datasets of paired diagnostic/relapse pediatric B-ALL samples, we discovered pervasive aberrant splicing (AS) patterns linked with relapse. They affected drivers of resistance to glucocorticoids, anti-folates, and thiopurines. Most splicing variations represent exon skipping, “poison” exon inclusion, and intron retention, phenocopying well-documented loss-of-function mutations. In addition, relapse-associated AS ofNT5C2mRNA yields an isoform with a cryptic 24-nt in-frame exon 6a. Inclusion of the extra 8 amino acids into this enzyme results in elevated nucleosidase activity, a known consequence of gain-of-function mutations in NT5C2 and a common determinant of 6-mercaptopurine resistance. Furthermore, in B-ALL cells NT5C2ex6a and the R238W hotspot variant confers comparable levels of resistance to 6-mercaptopurinein vitroandin vivo. These results support a role for alternative splicing as a prevalent mechanism driving chemotherapy resistance in relapsed B-ALL.Statement of significanceMutations in chemoresistance genes are found in relapsed/refractory acute lymphoblastic leukemia. However, in this low-mutational-burden disease, up to 30% of cases have no known relapse-specific genetic alterations. Our identification of aberrant splicing as an alternative mechanism of acquired drug resistance fills this gap and suggests new opportunities for therapeutic interventions.