Effect of APOBEC3 on Bladder Cancer Biology and Response to Immunotherapy

项目摘要 载脂蛋白B mRNA编辑酶催化多肽3（APOBEC 3）蛋白家族在人的肝细胞中起着重要的作用。 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 被干扰素信号上调。它们催化胞苷在逆转录， 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。在哺乳动物细胞的DNA复制和转录过程中，单链DNA可以 也用作APOBEC 3酶的底物。鉴于癌细胞的高度增殖性， 在某些癌症类型中，高水平的干扰素信号传导，APOBEC诱导的诱变可以有助于 显著地改变了它们的突变谱。在癌症中，高APOBEC表达与肿瘤生长相关。 突变负荷（TMB）和预测的新抗原负荷。体外及肝癌转基因小鼠的研究 模型显示APOBEC过表达可促进肿瘤进展。在尿路上皮癌中， APOBEC 3B（该蛋白质家族的特定成员）的表达显著上调。引人注目的是， 超过70%的突变可归因于APOBEC诱导的诱变。我已经生成了鼠标 膀胱癌细胞系与诱导型鼠Apobec 3.我证实了Apobec 3蛋白的诱导， 的APOBEC突变模式，和Apobec 3的能力，以促进锚定非依赖性生长。在 此外，我们已经产生了一种新的Rosa 26-Apobec 3敲入等位基因，其中Apobec 3的表达是 Cre诱导型，允许Apobec 3表达的空间和时间控制。目前，没有研究表明 研究了APOBEC在介导肿瘤内异质性或响应免疫检查点中的作用 封锁已显示高肿瘤内异质性与对以下治疗的反应呈负相关： 免疫疗法此外，由于增加的突变负荷（并且因此，新抗原）赋予了 通过APOBEC诱导的诱变，人们将预期对免疫检查点阻断的强烈应答。 然而，许多新抗原可能是亚克隆的和持续的APOBEC诱变，在一个特定的背景下， 新抗原驱动的抗肿瘤免疫应答可允许抗原逃逸（即由于无义突变 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， 问题.具体地说，在目标1中，我将我们新的Apobec 3敲入等位基因与我们建立的Pten杂交， Trp 53缺陷型小鼠膀胱癌模型的建立及多区域异质性分析 全外显子组测序在目标2中，我将利用具有诱导型Apobec 3的同种异体膀胱癌模型来测试 Apobec 3诱变活性是否会抑制免疫疗法的功效。拟议目标的成果 将有助于更好地理解APOBEC 3突变在促进膀胱癌进化中的作用， 癌这种知识的扩展将为开发更有效的膀胱癌治疗方法提供理论基础。 例如APOBEC 3抑制剂。

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.