Abstract B031: Uncovering genetic and regulatory drivers of tumor-specific splicing in pediatric brain tumors

Abstract Introduction: Aberrant pre-mRNA splicing is a hallmark of many cancers, yet the underlying genetic mechanisms driving these alterations remain incompletely understood. While splice-altering variants can explain a subset of events, many tumor-specific splicing changes arise in the absence of nearby sequence variants. This study aims to systematically dissect the contribution of proximal splice-altering variants to tumor-specific splicing in pediatric brain tumors and to define the residual cohort likely driven by regulatory mechanisms such as mutations affecting splicing factors or the spliceosome machinery. We hypothesized that some fraction of tumor-specific splice events in pediatric brain tumors are not associated with nearby genetic variants, and that these may highlight broader disruptions to splicing regulation. Methods: We applied replicate Multivariate Analysis of Transcript Splicing (rMATS-turbo) to stranded total RNA-seq data (N=635) from the Pediatric Brain Tumor Atlas (PBTA). Tumor-specific single exon (SE) splicing events were defined as a change in percent spliced in (| ΔPSI |) > 0.3 between tumors and healthy brain samples sourced from GTEx (<40 years, n=2,642) and pediatric normals (n=7). Tumor-normal matched whole genome sequencing data were used to identify somatic and rare (<0.1% Allele Frequency in gnomad) germline SNVs/Indels proximal (<250nt) to rMATS-defined splice junctions. Results: Across the cohort (N=635), we identified 107,827 tumor-specific SE alternative splicing events. Only 44 tumors harbored a variant proximal to the affected splice site (11 germline, 39 somatic), leaving most events without a clear cause. We are now investigating this variant-negative subgroup as a preliminary cohort. Current efforts focus on excluding structural drivers before turning to potential regulatory mechanisms, including the disruption of spliceosome components. Preliminary findings suggest some tumors show altered expression or mutations in spliceosome components, though further validation is needed. Conclusion: This dual-ended analysis of tumor-specific splicing events reveals that while a subset can be attributed to local genetic variation, the majority likely arise from broader disruptions in splicing regulation. These findings underscore the importance of integrating RNA- and DNA-based analyses to fully capture the molecular basis of splicing dysregulation in cancer. Moreover, the variant-negative cohort may serve as a rich resource to uncover novel regulatory mechanisms and potential therapeutic targets in splicing-driven pediatric brain tumors. Citation Format: Patricia J Sullivan, Ryan J Corbett, Ammar S Naqvi, Alexander Sickler, Rebecca Kaufman, Bo Zhang, Chuwei Zhong, Sharon J Diskin, Jo Lynne Rokita. Uncovering genetic and regulatory drivers of tumor-specific splicing in pediatric brain tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr B031.