SpliceIO™ a novel AI platform for the discovery of splicing-derived immunotherapeutic targets

Alternative splicing (AS) plays a critical role in generating tumor-specific neoepitopes targetable through an arsenal of immunotherapeutic modalities. Previous studies have demonstrated tumor-promoting activity of deregulated AS isoforms and mutated splicing factors, both of which increase the yield of neoepitope-encoding AS isoforms. Therefore, the identification of AS isoforms has the potential to produce novel therapeutic targets, especially in tumor types where splicing deregulation is frequent, like breast cancer. SpliceIOTM is Envisagenics’ AI platform for AS-derived neoepitope discovery. SpliceIO uses a “predictive ensemble” approach to uncover neoepitopes that are both stably expressed and located on the extracellular membrane of cancer cells. Using SpliceIO, we have analyzed >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 to identify tumor-specific neoepitopes present in AS transmembrane proteins. Here, we present experimental evidence for SpliceIO-identified neoepitopes in breast cancer. We have validated RNA expression of the neoepitope-expressing transcripts in breast cancer cell lines, primary cells and breast tumor FFPE tissues using a combination of RT-PCR as well as RNA-FISH. Further, we have confirmed the expression of neoepitope-containing isoforms at the protein level using western blotting as well as proteomics. Interestingly, one such candidate is an endoplasmic reticulum protein overexpressed in TNBC tumors. The TNBC specific isoform of the protein is localized to plasma membrane in TNBC cells, suggesting that different splice isoforms of a protein can alter protein localization. This demonstrates the potential of utilizing RNA splicing for identifying many more membrane associated proteins for tumor specific targeting. The combination of computational and experimental approaches to neoepitope identification using SpliceIO allows for novel target discovery amenable for targeted immunotherapeutic development for cancer treatment and clinical care. Conflict of interest: Other Substantive Relationships: All the authors are salaried employees of Envisagenics.