Characterization of aberrant splicing in pediatric central nervous system tumors reveals CLK1 as a candidate...

Pediatric brain cancer is the leading cause of disease-related mortality in children, yet many aggressive tumors lack effective therapies. Aberrant RNA splicing is a hallmark of cancer, but its role in pediatric central nervous system (CNS) tumors remains underexplored.

We analyzed 752 primary pediatric brain tumors spanning histologies and molecular subtypes to characterize aberrant splicing. We developed the Splicing Burden Index (SBI) as a sample-level metric and performed hierarchical clustering of variable splice events. Cluster-specific events were annotated for overlap with functional protein domains. We conducted in vitro experiments to assess the role of the splicing regulator CDC-like kinase 1 (CLK1).

Clustering identified 11 splicing-defined groups, several enriched for distinct tumor types, though heterogeneity was observed across histologies. Cluster 6, with the poorest event-free survival, comprised multiple histologies and contained 39,528 aberrant splice events across 10,412 genes, including 8,369 single exon (SE) events overlapping known functional protein domains. CLK1 expression was most positively correlated with SE SBI in Cluster 6, and inclusion of its exon 4 (active kinase isoform) was widespread and associated with inferior event-free survival. Pharmacologic inhibition of CLK1 with cirtuvivint suppressed viability and proliferation in KNS-42 cells, while morpholinos targeting exon 4 reduced CLK1 RNA and protein abundance, impaired growth, and induced splicing and expression changes in cancer-related genes.

This study maps splicing dysregulation in pediatric brain tumors, defines biologically- and clinically-relevant splicing-defined subgroups, and identifies CLK1 as a regulator of oncogenic splicing programs. These findings support therapeutic targeting of splicing kinases in high-risk pediatric CNS tumors.