New insights into RNA splicing across pediatric brain tumors - Children's National

Pediatric brain and other central nervous system tumors are the leading cause of cancer-related death in children. Over the past decade, DNA sequencing has helped doctors better classify these tumors and search for new treatments. However, gene mutations are only part of the story. A new study from researchers at Children's National Hospital and Children's Hospital of Philadelphia (CHOP) looks at another powerful layer of biology: how RNA is "spliced" inside tumor cells.

Splicing is the process cells use to cut and rearrange RNA before it becomes a protein. This allows one gene to create multiple versions of a protein. In healthy development, splicing is tightly controlled. In cancer, it can shift in ways that change how cells grow and survive.

A deeper dive

In a large-scale analysis of 729 pediatric central nervous system tumors, researchers mapped splicing patterns across many tumor types. They found that splicing patterns varied widely not only between tumor types, but also within them.

To better understand these differences, the team created a new measurement called the Splicing Burden Index. This tool measures how much a tumor's splicing patterns differ from others in the group. Using this approach, the researchers identified tumor groups based on shared splicing patterns. Some of these splicing-defined groups were linked to patient outcomes, even after accounting for tumor type and other clinical factors. That means RNA splicing may provide important information beyond what we currently detect in the clinic.

"Splicing adds an entirely new layer of biology to how we understand these tumors," said Jo Lynne Rokita, PhD, principal investigator of the Rokita Lab in the Center for Cancer and Immunology Research at Children's National and senior author of the study. "When we look at tumors at the exon level, we see patterns that are not visible through DNA mutations or even overall gene expression alone."

The team also found that overall splicing activity and spliceosome pathway signaling are not the same thing. Some tumors showed high activity in splicing-related pathways that were linked to worse survival, even if their overall splicing burden was not high. This suggests that splicing adds additional complexity to tumor biology.

A closer look

After mapping these patterns, the researchers focused on specific splicing events that might affect how proteins function. One event stood out in a gene called CDC-like kinase 1, or CLK1.

CLK1 helps regulate other splicing proteins. For CLK1 to work normally as a kinase, it must include a specific section called exon 4. When exon 4 is skipped, the protein loses its full activity.

The team found that exon 4 inclusion in CLK1 followed what is known as an oncofetal pattern. It is common in early brain development, usually decreases as the brain matures, and then appears again in many pediatric brain tumors. They found that higher levels of exon 4 inclusion were linked to improved patient outcomes in certain types of tumors, and these effects were different from simply measuring how much of the overall CLK1 gene was present. These findings highlight that looking at specific pieces of a gene can provide more meaningful insight than measuring total gene activity alone.

"CLK1 exon 4 is a clear example of why exon-level analysis matters," said Ammar Naqvi, PhD, Principal Bioinformatics Scientist at CHOP and lead author of the study. "It is not just whether the gene is turned on or off. It is which version of the gene the tumor is using."

What this means

To test whether this splicing event might influence tumor cells, researchers performed laboratory experiments in a pediatric high-grade glioma model. They used both a drug that targets CLK1 activity and a targeted approach to reduce inclusion of exon 4. In both cases, tumor cell growth decreased, and important cancer-related gene programs were disrupted. More work in additional models and in vivo systems will be needed to further confirm these findings. Still, the results suggest that exon-level splicing changes may help shape tumor behavior.

What comes next

This study represents one of the first systematic efforts to examine alternative splicing across many types of pediatric CNS tumors. By introducing the Splicing Burden Index and identifying splicing-defined tumor groups, the research offers a new framework for understanding how RNA regulation influences childhood brain cancer. For clinicians and families, the message is clear - DNA mutations are only one piece of tumor biology. RNA splicing adds another layer that may affect prognosis and, in the future, treatment strategies.

Read the full study "Alternative splicing in pediatric central nervous system tumors highlights oncofetal candidate CLK1 exon 4" in Neuro-Oncology Pediatrics.

This project was funded in part by the National Institutes of Health (R03OD036498), the anonymous private investors to the Children's National Hospital Brain Tumor Institute, Children's Brain Tumor Network, Chad Tough Foundation, CHOP Division of Neurosurgery, Mildred L. Roeckle Endowed Chair in Pathology at CHOP, and the Cure Search for Children's Cancer Foundation. This research utilized the Common Fund Cloud Workspace Pilot (1OT2OD030162-01) and the Kids First Data Research Center Cloud Credit Program (U2C HD109731 - 08S1).