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

项目总结/摘要 非小细胞肺癌（NSCLC）是美国癌症死亡率的主要原因。免疫 检查点抑制剂（ICI），如抗PD-1，增加了NSCLC的总生存期，但大多数患者仍然这样做 对治疗没有反应。靶向肿瘤特异性抗原（称为新抗原）的癌症疫苗可能 通过扩增新抗原反应性CD 8 + T细胞来增加ICI和其他免疫疗法的功效， 可以识别并摧毁肿瘤细胞。选择性剪接是一种普遍存在的转录后调控过程 它允许细胞从同一基因产生不同的mRNA和蛋白质序列。选择性剪接是 在包括NSCLC在内的许多癌症类型中广泛失调，并且可能产生缺失的新肽序列 来自正常组织，可以被CD 8 + T细胞识别为新抗原。去识别选择性剪接- 我们使用长读段RNA测序来全面绘制全长mRNA 在NSCLC肿瘤中的亚型，并预测其编码的蛋白质具有高准确性。我们发现有145,914人预测 这些肽对肿瘤具有特异性，并为高达70%的NSCLC患者所共有。为了确定这些 肽可能是免疫原性的，我们使用免疫肽组学直接测序与MHC结合的肽 三种NSCLC细胞系中的I类。我们鉴定了21种与NSCLC细胞上的I类MHC结合的肽， 由肿瘤特异性可变剪接mRNA同种型编码。这些剪接衍生的肽是 可能代表NSCLC疫苗靶点的潜在共享新抗原。因此，目标1将测试 这21种剪接衍生肽中的任何一种是否可以被来自NSCLC患者的CD 8 + T细胞识别。我们 将检查患者的CD 8 + T细胞是否可以增殖，分泌细胞因子，如干扰素-γ，并溶解 靶细胞对这些肽的反应。目标1中提出的实验将提供关键的洞察力， NSCLC中可变剪接衍生的新抗原的频率和免疫原性。目标2将研究 选择性剪接的调节因子驱动这些肽的产生。为此，我们将公开 可用的数据库来鉴定在肿瘤或靶结合位点中表达的剪接因子， 与编码21个剪接衍生肽的mRNA同种型的关联。我们将使用靶向基因 研究候选剪接因子是否在体外直接调节肽编码同种型剪接的方法。 这项工作将突出机制，可以驱动生产的肿瘤特异性剪接衍生肽， 可能揭示了新的靶点，可用于增强NSCLC免疫原性。总之，这些研究 可以确定新的免疫疗法的候选者，包括个性化的NSCLC癌症疫苗， 用于治疗共同表达免疫原性剪接衍生的新抗原的多个患者。这项建议 将为我提供优秀的培训，这将有助于我的职业目标，作为一个医生，科学家谁利用 基因组学和免疫学的进步，以改善对癌症患者的护理。

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.