Abstract B005A: Tumor-enriched alternative splicing in pediatric central nervous system tumors reveals amyloid precursor protein exon 8 inclusion as a candidate therapeutic target

Abstract

Alternative splicing (AS) is dysregulated in pediatric CNS tumors, including high-grade gliomas (HGGs) and atypical teratoid/rhabdoid tumors (ATRTs) which have poor survival outcomes and urgently need new therapeutic strategies. AS is increasingly being explored as a source of tumor-specific antigens for immunotherapy, yet the tumor-specific splicing landscape in pediatric CNS cancers remains underexplored. We leveraged RNA-sequencing data from the Pediatric Brain Tumor Atlas (PBTA) to identify tumor-enriched splice junctions (TEJs). Using rMATS-turbo, we quantified junction expression in 1,894 tumors (N=1,285 primary), 82 derived cell lines, and reference cohorts of normal brains spanning fetal to young adult stages (N=250). TEJs were defined as junctions that exhibit 1) counts per million fold-change and signal-to-noise ratio >5 in tumors relative to reference samples, and 2) delta percent spliced in (|dPSI|) >0.1 in tumor versus normal brains. We identified 10,676 recurrent TEJs across CNS tumor histologies, with ATRTs harboring the greatest number (N=3,570). Across histologies, 16% of TEJs involve unannotated junctions or novel splice sites and 9.4% (1,006) produce alterations to protein extracellular domains; among these, TEJs supporting inclusion of Amyloid Precursor Protein (APP) exon 8, generating the APP770 isoform, was associated with significantly worse event-free survival in ATRT. APP695, the predominant neuronal isoform, lacks exons 7–8; therefore, we hypothesized that APP770 promotes ATRT tumor cell fitness. APP770 was the dominant APP isoform across patient-derived ATRT models confirmed by orthogonal transcriptomic and proteomic analyses. We used a morpholino to induce skipping of APP exon 8 in three ATRT cell lines, and observed APP770 expression was reduced by 39% and 74% at 24 and 48 hours, respectively, without altering total APP expression. Across all three models, treatment produced dose-dependent reductions in viability and proliferation and altered cellular morphology, consistent with an APP770 isoform-specific requirement in ATRT. In summary, we characterize the tumor-specific AS landscape of pediatric CNS tumors and identify APP exon 8 inclusion as a recurrent, tumor-enriched proteoform that supports ATRT cell fitness in vitro. Notably, APP exon 8 encodes a portion of the extracellular domain making APP770 a candidate for antibody- and cellular-based immunotherapy. Ongoing work includes development of monoclonal antibodies against the exon 8–specific epitope to characterize surface expression and kinetics of APP770 in primary ATRT tumors, supporting preclinical therapeutic study in pediatric CNS malignancies.