Effect of APOBEC3 on Bladder Cancer Biology and Response to Immunotherapy

APOBEC3 对膀胱癌生物学和免疫治疗反应的影响

• 批准号: 9911447
• 负责人: Andrew Truong
• 金额: $ 4.55万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9911447
• 关键词: Adenoviruses; Alleles; Allografting; Anchorage-Independent Growth; Antigens; Automobile Driving; Bladder; Bladder Neoplasm; Cancer Biology; Cancer Model; Cancer Patient; Cancer cell line; Cisplatin; Code; Cytidine; DNA biosynthesis; Development; Ear; Enzymes; Evolution; Experimental Models; Family; Fibroblasts; Genes; Genetic Transcription; Genetically Engineered Mouse; Immune; Immune checkpoint inhibitor; Immunocompetent; Immunotherapy; Impairment; In Vitro; Individual; Interferons; Intuition; Knock-in; Knock-in Mouse; Knowledge; Malignant Neoplasms; Malignant neoplasm of liver; Malignant neoplasm of urinary bladder; Mammalian Cell; Mediating; Missense Mutation; Modeling; Mus; Mutagenesis; Mutation; Natural Immunity; Nature; Nonsense Mutation; Nucleic Acids; PD-1 blockade; Patients; Pattern; Peptides; Play; Protein Family; Protein Overexpression; Proteins; Refractory; Resistance; Role; Shapes; Signal Transduction; Single Nucleotide Polymorphism; Single-Stranded DNA; Survival Rate; Testing; Transgenic Mice; Transitional Cell Carcinoma; UPK3 gene; United States; Uracil; Urothelium; Viral; Virus Diseases; Virus Replication; anti-PD-1; anti-PD1 therapy; anti-tumor immune response; apolipoprotein B mRNA editing enzyme; base; cancer cell; cancer type; effective therapy; exome sequencing; immune checkpoint blockade; improved; inhibitor/antagonist; member; mouse model; neoantigens; novel; overexpression; polypeptide; prevent; response; tool; tumor; tumor heterogeneity; tumor progression

PROJECT SUMMARY The apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3) family of proteins play an important role in innate immunity by restricting retroviral replication. During a viral infection, these enzymes are upregulated by interferon signaling. They catalyze the conversion of cytidine to uracil within reverse-transcribed, single-stranded DNA, resulting in the degradation or integration of defective viral nucleic acid, and therefore, impaired viral replication. During DNA replication and transcription in mammalian cells, single-stranded DNA can also serve as the substrate for APOBEC3 enzymes. Given the highly proliferative nature of cancer cells, and a high level of interferon signaling in certain cancer types, APOBEC-induced mutagenesis can contribute significantly to their mutational spectrum. In cancer, high APOBEC expression correlates with an increased tumor mutational burden (TMB) and predicted neoantigen load. Studies in vitro and in liver cancer transgenic mouse models have shown that APOBEC overexpression can promote tumor progression. In urothelial carcinoma, the expression of APOBEC3B, a particular member of this protein family, is significantly upregulated. Strikingly, upwards of 70% of mutations can be attributed to APOBEC-induced mutagenesis. I have generated mouse bladder cancer cell lines with inducible murine Apobec3. I confirmed the induction of Apobec3 protein, induction of an APOBEC mutational pattern, and the ability of Apobec3 to promote anchorage-independent growth. In addition, we have generated a novel Rosa26-Apobec3 knock-in allele in which the expression of Apobec3 is Cre-inducible, allowing for spatial and temporal control of Apobec3 expression. Currently, no studies have examined APOBEC's role in mediating intratumoral heterogeneity, or in response to immune checkpoint blockade. High intratumoral heterogeneity has been shown to negatively correlate with response to immunotherapy. Additionally, because of the increased mutational load (and therefore, neoantigens) imparted by APOBEC-induced mutagenesis, one would expect a robust response to immune checkpoint blockade. However, many of the neoantigens may be subclonal and persistent APOBEC mutagenesis, in the context of a neoantigen driven anti-tumor immune response, may allow for antigen escape (i.e. due to nonsense mutations in the gene encoding the neoantigen). I will utilize the tools we have developed to answer these important questions. Specifically, in Aim 1, I will cross our novel Apobec3 knock-in allele to our established Pten and Trp53-deficient mouse model of bladder cancer and characterize the intratumoral heterogeneity by multiregional whole-exome sequencing. In Aim 2, I will utilize an allograft bladder cancer model with inducible Apobec3 to test whether Apobec3 mutagenic activity can dampen the efficacy of immunotherapy. Results of the proposed aims will contribute to a greater understanding of the role of APOBEC3 mutagenesis in fueling the evolution of bladder cancer. This expansion of knowledge will provide the rationale to develop more effective treatments for bladder cancer such as an APOBEC3 inhibitor.

项目总结 载脂蛋白B信使核糖核酸编辑酶催化多肽3(APOBEC3)家族蛋白发挥重要作用 通过限制逆转录病毒复制，在先天免疫中发挥重要作用。在病毒感染期间，这些酶是 干扰素信号上调。它们催化胞苷在逆转录过程中转化为尿嘧啶， 单链DNA，导致有缺陷的病毒核酸降解或整合，因此， 病毒复制受损。在哺乳动物细胞的DNA复制和转录过程中，单链DNA可以 也是APOBEC3酶的底物。鉴于癌细胞的高度增殖特性，以及一种 在某些癌症类型中高水平的干扰素信号，APOBEC诱导的突变可能起到作用 对它们的突变谱有显著影响。在癌症中，APOBEC高表达与肿瘤增加相关 突变负荷(TMB)和预测的新抗原负荷。肝癌转基因小鼠的体外和体内研究 模型显示，APOBEC过表达可促进肿瘤进展。在尿路上皮癌中， APOBEC3B是该蛋白家族中的一个特殊成员，其表达显著上调。令人惊讶的是， 超过70%的突变可归因于APOBEC诱导的突变。我已经生成了鼠标 含可诱导小鼠载脂蛋白3的膀胱癌细胞系。我证实了APOBEC3蛋白的诱导，诱导 APOBEC突变模式，以及APOBEC3促进非锚定生长的能力。在……里面 此外，我们还产生了一个新的rosa26-APOBEC3敲入等位基因，其中APOBEC3的表达是 Cre可诱导，允许APOBEC3表达的空间和时间控制。目前，还没有研究表明 研究APOBEC在调节肿瘤内异质性或对免疫检查点的反应中的作用 封锁。高度的肿瘤内异质性已被证明与对 免疫疗法。此外，由于增加的突变负荷(因此，新的抗原) 通过APOBEC诱导的突变，人们可以预期对免疫检查点阻断的强烈反应。 然而，许多新抗原可能是亚克隆的和持久的apobec突变，在 新抗原驱动的抗肿瘤免疫反应，可能允许抗原逃逸(即由于无义突变 编码新抗原的基因中)。我将利用我们开发的工具来回答这些重要的问题 问题。具体地说，在目标1中，我将把我们新的APOBEC3敲入等位基因与我们已经建立的Pten和 TrP53基因缺陷小鼠膀胱癌模型的建立及肿瘤内异质性的多区域表征 全外显子组测序。在目标2中，我将利用带有诱导性载脂蛋白BEC3的同种异体膀胱癌模型来测试 APOBEC3诱变活性是否会抑制免疫治疗的疗效。拟议目标的结果 将有助于更好地理解APOBEC3突变在促进膀胱进化中的作用 癌症。这种知识的扩展将为开发更有效的膀胱治疗方法提供理论基础。 癌症，如APOBEC3抑制剂。