Non-Canonical Exon Usage in Pediatric Leukemia: Implications for Chemoresistance

B-lymphoblastic leukemia (B-ALL) is a heterogeneous, chromosome translocation-driven disease where the prevalence of somatic mutations and copy number variations is relatively low. Previous B-ALL whole exome sequencing efforts by other groups have focused upon mutations acquired under therapeutic pressure, but they have not identified universal resistance gene(s). Instead, relapse-specific mutations occurred in multiple genetic loci often involved in resistance to either glucocorticoids or purine analogs (e.g., 6-mercaptopurine, or 6-MP). The most prevalent target, NT5C2, encodes the enzyme 5'-nucleotidase/cytosolic II involved in 6-MP catabolism, but these gain-of-function mutations were found in a minority of relapsed/refractory (r/r) B-ALL samples (~25%). This lack of concordance between the genotype and the phenotype suggested a possibility that instead of mutations, NT5C2 is predominantly affected by post-transcriptional events, such as aberrant mRNA splicing (AS).

To address this possibility, we have analyzed several richly annotated RNA-Seq datasets, including NCI TARGET, which presently includes several hundred baseline childhood B-ALL samples as well as 48 paired diagnostic and relapse samples. In baseline B-ALL samples, we indeed discovered an abnormally spliced NT5C2 mRNA isoform containing the cryptic in-frame exon 4a (NT5ex4a). Of note, NT5ex4a levels were further increased in relapses compared to diagnostic samples, consistent with its putative role in chemoresistance. Furthermore, NT5ex4a mapped to full-length protein-coding transcripts and resulted in inclusion of 8 extra amino acids near the ATP-binding effector site 2.

Using bacterially produced protein, we demonstrated that at low concentrations of ATP NT5ex4a exhibited elevated enzymatic activity compared to the canonical isoform. Consistent with this biochemical finding, in reconstituted NT5C2low B-ALL cells NT5ex4a conferred the same level of resistance to 6-MP as the variant with the R238W hotspot mutation (2-log difference in IC50). Conversely, NT5ex4a-KO CRISPR/Cas9 engineered cells reduced cell survival in the presence of 6-MP. The role of NT5ex4a in chemoresistance in vivo was further confirmed in B-ALL cells expressing this alternative isoform. Therefore, inclusion of NT5C2 exon 4a phenocopies relapse-specific mutations and could serve as both a valuable predictive biomarker in B-ALL and potentially chronic myelogenous (CML) and acute myeloid leukemia (AML). Additionally, at least in vitro, expression of this non-canonical isoform conferred collateral sensitivity to the purine biosynthesis inhibitor Mizoribine, suggesting the existence of a therapeutic window to treat leukemias with dysregulated splicing.