# Alternative splicing in pediatric central nervous system tumors highlights oncofetal candidate CLK1 exon 4

## TL;DR

This study explores RNA splicing in pediatric brain tumors, identifying CLK1 exon 4 as a potential oncofetal target for new therapies.

## Contribution

The study introduces the Splicing Burden Index and identifies CLK1 exon 4 as a novel oncofetal target in pediatric brain tumors.

## Key findings

- SBI reveals significant splicing heterogeneity across and within tumor types.
- CLK1 exon 4 inclusion follows an oncofetal pattern and is functionally important for tumor viability.
- Pharmacologic inhibition of CLK1 reduces tumor cell viability and disrupts cancer-related processes.

## Abstract

Pediatric brain tumors are the leading cause of disease-related mortality in children, yet many aggressive tumors lack effective therapies. RNA splicing is a hallmark of cancer, but it has not yet been systematically studied in pediatric brain tumors.

We analyzed 729 pediatric brain tumors spanning histologies and molecular subtypes to quantify differential tumor splicing. We developed the Splicing Burden Index (SBI) to enable cross-sample comparisons and performed hierarchical clustering of highly variable splice events to define splicing-informed tumor groups. These were integrated with clinical outcomes, pathway activity, and proteogenomic data. Recurrent splice events were prioritized for predicted functional impact, and in vitro perturbation studies were performed targeting the splicing kinase CDC-like kinase 1 (CLK1).

SBI revealed substantial interhistology and intrahistology heterogeneity. Clusters were enriched for histologies and molecular subtypes, several of which were independently associated with survival beyond histology and clinical covariates. Spliceosome pathway activity varied across clusters and was associated with worse survival, yet was not correlated with SBI, indicating distinct dimensions of splicing dysregulation. Functional prioritization identified a recurrent in CLK1 exon 4, required for canonical kinase activity. CLK1 exon 4 inclusion followed an oncofetal pattern and showed context-dependent associations with outcome distinct from total CLK1 expression. Pharmacologic inhibition and exon 4-specific perturbation of CLK1 reduced tumor cell viability and disrupted cancer-relevant splicing and transcriptional programs.

This study systematically characterizes splicing in pediatric brain tumors, identifies splicing-informed subgroups, and prioritizes CLK1 exon 4 as an oncofetal tumor-specific event, motivating further preclinical exploration.