Alternate splicing as a source of shared neoantigens in a non-small cell lung cancer

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PROJECT SUMMARY/ABSTRACT Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality in the United States. Immune checkpoint inhibitors (ICIs) like anti-PD-1 have increased overall survival in NSCLC, but most patients still do not respond to treatment. Cancer vaccines that target tumor-specific antigens, known as neoantigens, may increase the efficacy of ICIs and other immunotherapies by expanding neoantigen-reactive CD8+ T cells that can recognize and destroy tumor cells. Alternative splicing is a ubiquitous post-transcriptional regulatory process that allows cells to produce different mRNA and protein sequences from the same gene. Alternative splicing is broadly dysregulated in many cancer types including NSCLC and may generate novel peptide sequences absent from normal tissue that can be recognized as neoantigens by CD8+ T cells. To identify alternative splicing- derived neoantigens in NSCLC, we used long-read RNA sequencing to comprehensively map full-length mRNA isoforms in NSCLC tumors and predict the proteins they encode with high accuracy. We found 145,914 predicted peptides that were specific to tumors and shared by up to 70% of NSCLC patients. To identify which of these peptides might be immunogenic, we used immunopeptidomics to directly sequence peptides bound to MHC Class I in three NSCLC cell lines. We identified 21 peptides that are bound to MHC Class I on NSCLC cells and are encoded by tumor-specific alternatively spliced mRNA isoforms. These splicing-derived peptides are potentially shared neoantigens that might represent vaccine targets for NSCLC. Therefore, Aim 1 will test whether any of these 21 splicing-derived peptides can be recognized by CD8+ T cells from NSCLC patients. We will examine whether patient CD8+ T cells can proliferate, secrete cytokines like interferon-gamma, and lyse target cells in response to these peptides. The experiments proposed in Aim 1 will provide crucial insight into the frequency and immunogenicity of alternative splicing-derived neoantigens in NSCLC. Aim 2 will examine which regulators of alternative splicing are driving production of these peptides. To this end, we will leverage publicly available databases to identify splicing factors whose expression in tumors or target binding sites suggest an association with the mRNA isoforms that code for the 21 splicing-derived peptides. We will use targeted genetic approaches to study whether candidate splicing factors directly regulate peptide-coding isoform splicing in vitro. This work will highlight mechanisms that can drive the production of tumor-specific splicing-derived peptides and may reveal novel targets that can be exploited to enhance NSCLC immunogenicity. Altogether, these studies may identify candidates for new immunotherapies, including personalized NSCLC cancer vaccines that can be used to treat multiple patients who share expression of immunogenic splicing-derived neoantigens. This proposal will provide me excellent training that will facilitate my career goals as a physician-scientist who leverages advances in genomics and immunology to improve care for patients with cancer.