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Abstract Splicing dysregulation is a major hallmark of cancer, affecting tumor progression, metastasis, and therapy resistance. Aberrant alternative splicing (AS) plays a critical role in generating tumor-specific neoantigens targetable through an arsenal of immunotherapeutic modalities. Such neoantigens cannot be discovered using conventional DNA-based tools; they require specialized software for AS analysis using RNA-seq data. SpliceIO is Envisagenics’ AI platform for AS-derived neoantigen discovery. SpliceIO uses a “predictive ensemble” approach to uncover neoantigens that are both stably expressed and located on the extracellular membrane of cancer cells. Using SpliceIO, we have analyzed more than 10, 000 RNA-seq samples from 5 patient cohorts covering 5 breast cancer subtypes as well as 2, 647 normal RNA-seq samples from GTEx. Our analysis has identified tumor-specific neoantigens present in AS transmembrane proteins specific for Triple Negative Breast Cancer. SpliceIO target candidates were validated experimentally using our well-established pipeline in which isoform specific transcripts are tested in cancer cell lines by PCR and primary tumor tissues by qPCR to confirm RNA expression. Tumor heterogeneity and prevalence is confirmed by RNA in-situ hybridization in Tissue Microarrays. Extracellular localization of the novel epitope is confirmed by clone generation with GFP and c-Myc tagging followed by microscopy, Western blotting, and proteomic analysis. Our top validated target presents high TNBC tumor specificity and heterogeneous distribution, and contains a novel extracellular N-terminal domain. These results strongly support the role of aberrant AS as a major source of tumor-specific neoantigens. Our combined computational and experimental approach promotes novel target discovery, enabling the development of novel immunotherapeutic for TNBC patients who are in critical need of better treatment options. Citation Format: Nicole Williams, Miguel A. Manzanares, Kendall Anderson, Hasan Zunrut, Sakshi Gera, Nayab Habib, Shaleigh Stanton, Taylor Floyd, Sanjana Shah, Devon Trahan, Martin Akerman, Gayatri Arun. Novel splicing-derived neoantigen in triple negative breast cancer uncovered by SpliceIO™ a robust platform for the discovery and validation of disease-specific immunotherapies abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 5894.
Williams et al. (Fri,) studied this question.