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: 10092808
• 负责人: Andrew Truong
• 金额: $ 4.6万
• 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/10092808
• 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.

项目摘要 载脂蛋白B mRNA编辑酶催化多肽3（APOBEC3）蛋白质蛋白质发挥作用 通过限制逆转录病毒复制，在先天免疫中的重要作用。在病毒感染期间，这些酶是 由干扰素信号上调。它们催化了胞丁在反向转录中的尿嘧啶的转化， 单链DNA，导致有缺陷的病毒核酸的降解或整合，因此 病毒复制受损。在哺乳动物细胞中的DNA复制和转录过程中，单链DNA可以 还用作APOBEC3酶的底物。考虑到癌细胞的高度增殖性质，A 在某些癌症类型中，高水平的干扰素信号传导，APOBEC诱导的诱变可能有助于 重要的是它们的突变频谱。在癌症中，高APOBEC表达与肿瘤增加相关 突变负担（TMB）和预测新抗原负荷。体外和肝癌转基因小鼠的研究 模型表明，APOBEC过表达可以促进肿瘤进展。在尿路上皮癌中 该蛋白质家族的特定成员Apobec3b的表达显着上调。令人惊讶的是， 超过70％的突变可以归因于APOBEC诱导的诱变。我已经产生了鼠标 膀胱癌细胞系带有诱导鼠APOBEC3。我证实了Apobec3蛋白的诱导，诱导 APOBEC突变模式，以及APOBEC3促进独立于锚固的生长的能力。在 此外，我们已经产生了一种新颖的Rosa26-Apobec3敲入等位基因，其中Apobec3的表达为 CRE诱导，允许对APOBEC3表达的空间和时间控制。目前，没有研究 检查了APOBEC在介导肿瘤内异质性或对免疫检查点的作用 封锁。高肿瘤内异质性已被证明与对响应的反应负相关 免疫疗法。此外，由于突变负荷增加（因此，新抗原）赋予了 通过APOBEC诱导的诱变，人们会期望对免疫检查点阻滞产生强大的反应。 但是，许多新抗原可能是亚克隆和持续的apobec诱变，在一个背景下 新抗原驱动的抗肿瘤免疫反应可能允许抗原逃脱（即由于胡说八道的突变 在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。具体而言，在AIM 1中，我将把我们的小说Apobec3敲入等位基因跨到我们已建立的PTEN和 TRP53缺乏膀胱癌的小鼠模型，并通过多区域的肿瘤内异质性来表征 全外观测序。在AIM 2中，我将使用具有诱导APOBEC3的同种异体移植膀胱癌模型进行测试 APOBEC3诱变活性是否会抑制免疫疗法的功效。拟议目的的结果 将有助于更深入地了解APOBEC3诱变在加剧膀胱进化中的作用 癌症。知识的扩展将为开发膀胱开发更有效治疗的理由 癌症，例如APOBEC3抑制剂。