Abstract 462: Therapy and malignant progression reshape the splicing landscape to generate shared, tumor-wide neoantigens in IDH-mutant gliomas.

Abstract Background: The low mutational burden and high heterogeneity of gliomas limit the efficacy of immunotherapy. Aberrant RNA splicing can generate targetable neojunctions (NJs) (Kwok et al., 2025), yet the impact of therapy and malignant transformation on this landscape remains unclear. We investigate these effects in IDH-mutant gliomas to identify conserved, upregulated, and potentially targetable splice-derived neoantigens. Methods: Paired bulk RNA-seq data from primary and recurrent IDH-mutant gliomas were obtained from the UCSF Brain Tumor Center patient data and AG-120-treated glioma cell line data (Wu et al., Science 2025). NJs and their subsequent peptide sequences were characterized using the SSNIP pipeline (Kwok et al., Nature 2025). High confidence n-mer sequences were prioritized using HLAthena, MHCflurry, NetMHCpan, MUNIS, and HLApollo across five HLA-I alleles (A0101, A0201, A0301, A1101, A2402). Peptides scoring within the top first percentile on ≥ 3 platforms were filtered by FPKM × splice read frequency. DESeq2 and GSEA were used for differential gene expression analyses. Publicly available eCLIP sequencing near NJ sites identified potential cis-binding RNA-binding regulators. Results: In patients treated with standard chemoradiation (n = 90), 1,806 of 57,400 NJs (3.1%) were significantly upregulated (log2JPM 1; p 0.05). In AG120-treated IDH-mutant glioma cell lines, 90 of 5,520 NJs (1.6%) were significantly upregulated versus non-treated controls. Across paired samples, NJ expression increased at recurrence (p = 0.033), a trend absent with AG120-treated cell lines (p = 0.7). NJ elevation was primarily associated with grade progression and differential gene expression analysis showed enrichment of intron-recognition/exclusion pathways. Further analysis of the chemoradiation cohort identified BCAN, PTPRZ1 and EEF1A1 NJ-derived targets as top HLA-A*11:01, HLA-A*24:02, HLA-A*11:01 candidates, respectively. Notably, the PTPRZ1 and EEF1A1 NJs were intratumorally conserved, and were more abundant than the immunogenic GNAS NJ reported by Kwok et al. Two upregulated AG120 NJs overlapped with those from the chemoradiation cohort and generated proteins. These two NJs generated distinct cancer 9-mers from S100A13 and RANBP2, which were recurrently expressed and strongly predicted to bind HLA-A*02:01. Leading-edge analysis showed IGF2BP3 upregulation, and eCLIP confirmed binding sites near the S100A13 and RANBP2 NJ sites. IGF2BP3 knockdown in HepG2 cells caused distinct splicing alterations without global loss, while S100A13 NJ expression decreased significantly post-KD. Conclusion: We show that therapy and malignant progression remodel the splicing landscape of IDH-mutant gliomas via altered splicing regulator expression. Moreover, this remodeling produces conserved, tumor-wide, and putatively immunogenic NJs. Citation Format: Tim Wu, Darwin Kwok, Joseph Costello, Hideho Okada. Therapy and malignant progression reshape the splicing landscape to generate shared, tumor-wide neoantigens in IDH-mutant gliomas abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 462.