Project Summary/Abstract (limited 30 lines of text): In humans, cellular diversity and phenotypic variation stem not only from differential gene expression but also from the expression of different gene isoforms across distinct cellular contexts. Across various cancer types, including both liquid and solid tumors, mutations and expression imbalances in splicing factors contribute significantly to disease progression and heterogeneity. The lack of comprehensive understanding of splicing aberrations and their impact on individual cell phenotypes in cancers like myelodysplastic syndromes (MDS) and high-grade serous ovarian cancer (HGSOC) presents a critical gap in knowledge. The proposed research aims to explore the role of aberrant splicing in cancer cell heterogeneity and its potential as targets for novel immunotherapeutic strategies. Aim 1 will utilize GoT-Splice to study mutations in splicing factors such as SRSF2, U2AF1, and ZRSR2 in MDS, enhancing the characterization of retained introns and identify tumor-specific isoforms with translational potential. Aim 2 will explore the cell-to-cell variability of isoform usage in HGSOC, leveraging single-cell long-read sequencing data to elucidate the relationship between splicing regulation, genomic instability, and immune interactions within the tumor microenvironment. Finally, Aim 3 seeks to identify splicing-derived neoantigens for immunotherapy, utilizing multi-omic characterization of single-cell splicing variation to develop models that distinguish between tumor-specific and normal isoforms predicting their immunological potential. The proposal emphasizes the importance of integrating advanced computational frameworks with multimodal single-cell approaches to comprehensively characterize splicing aberrations and their functional implications in cancer. It also highlights the potential of splicing-derived neoantigens as promising targets for precision immunotherapies, while addressing challenges related to neoantigen off-target effects and HLA variability. Overall, this proposal seeks to provide a comprehensive understanding of isoform regulation in cancer, offering insights into potential targets for precision medicine approaches in cancer immunotherapy.

PROJECT NARRATIVE Human phenotypic variation is not only driven by differences in gene expression but also in variability of isoform usage. In this proposal, I hypothesize that alternative splicing isoforms play a pivotal role in driving cell-to-cell variation, particularly in hematologic malignancies and solid tumors. Using cutting-edge single-cell technologies and long-read sequencing, I identify altered RNA products unique to cancer cells, in the context of myelodysplastic syndrome (MDS) and high-grade serous ovarian cancer (HGSOC), aiming to identify potential targets for more precise immunotherapies.