ABSTRACT
Background Mutations in splicing factor (SF) genes cause myelodysplastic syndromes and chronic lymphocytic leukemia (CLL). While such mutations had not been found in B-cell acute lymphoblastic leukemia (B-ALL), we previously reported that alternative splicing of the CD19 transcript is a robust mechanism of resistance to CD19-directed immunotherapy in children with B-ALL. We thus hypothesized that additional mRNAs may be alternatively spliced in these leukemias.
Results Using flow cytometry-based cell purification protocols, deep RNA sequencing (RNA-seq), and the MAJIQ algorithm, we compared transcriptomes of CD19+/CD34+ pro-B cells from normal bone marrow donors to 18 primary pediatric B-ALL samples. We found 4,000-5,000 differential local splice variations (LSV) per leukemia sample, with 279 LSVs in 241 genes differentially spliced in every B-ALL sample. The consistently mis-spliced genes were significantly enriched in the RNA splicing pathway components and encoded ~100 different SFs, many from the SRSF and hnRNP families. Since aberrant LSVs in hnRNPA1 mRNA were present in 100% of B-ALL samples, we knocked down this transcript in a B-lymphoblastoid cell line using siRNA and defined 213 robust hnRNPA1-dependent events. Nearly 30% of the hnRNPA1-dependent LSVs were detectable in B-ALL samples, with one of the affected genes being DICER1, which is commonly mutated or down-regulated in many hematologic malignancies and included in the COSMIC dataset. We next asked how many other COSMIC genes are affected by aberrant splicing. We discovered 81 LSVs (mainly hnRNPA1-independent) in 41 COSMIC genes, including FBXW7, which was alternatively spliced in all 18 primary B-ALL samples. We were able to confirm 77 out of 81 of these LSVs in at least one of the two large independent RNA-seq B-ALL datasets generated by the TARGET Consortium and St Jude Children's Research Hospital. In fact, the twenty most common B-ALL drivers showed much higher prevalence of aberrant splicing than of somatic mutations.
Conclusions B-ALL has widespread changes in splicing, likely due to the aberrant exon usage by SFencoding transcripts. Aberrant splicing also affects most known B-ALL drivers, suggesting that this type of post-transcriptional regulation contributes to disease pathogenesis.