Abstract 4250: Tumor-wide RNA splicing aberrations generate immunogenic public neoantigens across cancers

Abstract Background: High tumor heterogeneity and low mutational burden in cancers pose significant challenges for immunotherapy. To address this, we developed a novel in silico pipeline to characterize cancer-specific splicing events (neojunctions; NJs) expressed ubiquitously across ten cancer types. This approach successfully identified tumor-wide, public, alternatively splicing neoantigens (ASNs) that elicit CD8+ T-cell-mediated cytotoxicity across multiple cancers. Methods: Our SSNIP pipeline identified recurring public NJs expressed in TCGA RNA-seq data (positive sample rate (PSR) > 10%) but not in GTEx normal tissue RNA-seq data (PSR < 1%) across 10 cancer types. We utilized multi-site RNA sequencing data to characterize intratumorally conserved NJs. Our in-house glioma dataset included 56 patients with approximately 10 spatially distinct intratumoral biopsy sites per patient (n=535). In vitro sensitization (IVS) of CD8+ T-cells from healthy donors against high-confidence ASN candidates, followed by 10x VDJ scRNA-seq, identified ASN-specific TCR sequences. TCR-transduced donor-derived CD8+ T-cells were cultured against NJ-expressing glioma and melanoma cell lines to evaluate tumor-specific killing. CRISPRi KD of disease-specific splicing factors was performed to assess NJ modulation. Results: Our pipeline identified 789 public NJs, with 32 NJs concurrently identified in transcriptomic and proteomic data and predicted to be presented by HLA-A*02:01 with high confidence. We captured TCR clonotypes reactive against NJs in RPL22 (n=7) and GNAS (n=1), the latter being highly intratumorally-conserved (detected in > 90% of spatially-mapped biopsies across 17/56 patients (26.78%)). Mutant GNAS-derived ASN-specific CD8+ T-cell clones were detectable from glioma patient PBMC by IVS. TCR-transduced T-cells demonstrated recognition and tumor-specific killing against endogenously processed and presented ASNs in multiple glioblastoma and melanoma cell lines. Furthermore, IDH1-mutant oligodendroglioma samples demonstrated significantly elevated expression of NJs over IDH1-mutant astrocytoma and IDH1wt subtypes. Differential gene expression identified decreased expression of splicing factors due to oligodendroglioma-specific co-deletion of Chromosomes 1p/19q. CRISPRi KD of these splicing factors (e.g. SF3A3, SNRPD2) in IDH1wt glioma cells resulted in significantly increased expression of corresponding NJs. Performing DESeq2 on the remaining cancer types revealed similar splicing gene set-associated differences. Conclusions: SSNIP identified novel public tumor-wide splice-derived neoantigen candidates and ASN-specific TCRs, offering a promising off-the-shelf immunotherapy approach for diverse cancer types. Characterization of intratumorally conserved neoantigens addresses the critical challenge of intratumoral heterogeneity in immunotherapy resistance. Citation Format: Darwin Kwok, Nicholas Stevers, Inaki Etxeberria, Takahide Nejo, Maggie Colton Cove, Lee Chen, Jangham Jung, Kaori Okada, Senthilnath Lakshmanachetty, Marco Gallus, Abhilash Barpanda, Chibo Hong, Gary Chan, Jerry Liu, Samuel Wu, Emilio Ramos, Akane Yamamichi, Payal Watchmaker, Hirokazu Ogino, Atsuro Saijo, Aidan Du, Nadia Grishanina, James Woo, Aaron Diaz, Susan Chang, Joanna Phillips, Arun Wiita, Christopher Klebanoff, Joseph Costello, Hideho Okada. Tumor-wide RNA splicing aberrations generate immunogenic public neoantigens across cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr XX.