Abstract 2725: Molecular mechanisms of aberrant splicing in pediatric high grade gliomas

Abstract

Abstract Pediatric brain cancer is the number one cause of disease-related death in children. While much of the somatic mutation variation in these tumors have been profiled, there have been few studies exploring their transcriptional splicing programs. Here, we characterize aberrant alternative splicing across pediatric brain tumors. We identified three splicing-based clusters that spanned across seven broad histological tumor classifications. Patients in clusters 1 and 2 had significantly lower overall survival (p < 0.001) than those in cluster 3. High-grade gliomas (HGGs) displayed the greatest splicing heterogeneity, spanning all three clusters. We focused on midline HGGs (n = 84), identifying 19,275 recurrent and significant (20% change from control, P < 0.05, FDR < 0.05) aberrant splicing events in 8,587 genes compared to non-diseased brainstem controls. We also identified several splicing factor genes, including SRSF11, that were differentially expressed (p < 0.05), suggesting a potential tumor-specific novel mechanism of splicing dysregulation. Strikingly, 27% (5,157) of these splicing events resulted in either a gain or loss of a functional site in 3,294 genes: disulfide bonding (n = 391), localization signaling (n = 216), modifications, such as phosphorylation sites (n = 1,670), and other, such as ion-binding sites (n = 4,245). We prioritized splice variants affecting targetable kinases and found that mRNAs encoding CDC-like kinase 1 (CLK1), a known modulator of the SR family of master splicing regulators, exhibit significantly increased exon 4 inclusion. This leads to a gain of two known phosphorylation sites in CLK1, increased protein expression, and hyper-phosphorylation of SR proteins, inducing cooperative binding of SR proteins to RNA. To assess the impact of this CLK1 event, we compared splicing in tumors with high exon 4 inclusion (n = 5) to those with low exon 4 inclusion (n = 5) and discovered 862 differential splicing events (DSE). Samples with high CLK1 exon 4 inclusion had significantly lower percent spliced in events, suggesting that increased activity of CLK1 resulting from the gain of the exon 4 promotes overall exon skipping. Additionally, more than 80% of the DSE (n = 695), including those in known brain tumor oncogenes such as CDC27 and E2H2, were recurrently mis-spliced in midline HGG tumors, suggesting that CLK1 splicing dysregulation leads to a global aberrant splicing phenotype in HGGs. Finally, we determined a subset of events significantly associated with prolonged survival, suggesting that stratification of patients based on splicing phenotypes can be utilized as a prognostic indicator. In summary, we demonstrate aberrant splicing in pediatric HGGs as an alternative mechanism driving tumorigenesis and future work will focus on urgently needed therapeutic targeting of these pathways toward our overall goal of improving survival outcomes in children with brain tumors. Citation Format: Ammar S. Naqvi, Brian Ennis, Run Jin, Karina L. Conkrite, Komal S. Rathi, Adam A. Kraya, Poonam Sonawane, Phillip B. Storm, Adam C. Resnick, Jo Lynne Rokita. Molecular mechanisms of aberrant splicing in pediatric high grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2725.