Investigation of human DNA polymerase epsilon variants

摘要 了解细胞如何未能保护自己免受 所有癌症都是最终治疗这种疾病的关键。突变的复制DNA聚合酶在一些 在已知的高度突变的肿瘤中。这些肿瘤也有非常独特的突变谱。在我们的 我们发表了一项工作，并开始描述这些肿瘤的小鼠和人类细胞模型。而当 最初认为这种突变对肿瘤的发展既是必要的，也是充分的，我们的 已发表的工作和初步数据有力地暗示了极部肿瘤所必需的其他因素 发展。 这个项目的主要目标是检验我们的中心假设，虽然极部肿瘤是最常见的 观察到高度突变的肿瘤，极点突变的基本致癌特征是不稳定 停滞/折叠的复制叉子通过某些同源定向的失活来响应受损的DNA 修复BRCA2等基因和/或失去核PTEN活性，这两种基因通常都能稳定停滞的复制 分叉，从而抑制基因组的不稳定性。极点突变同时通过促进 在这些途径中获得突变这一假设是基于我们已发表的工作和未发表的工作 初步证据。具体地说，这个项目将1)定义极点突变的机制起源 签名；2)表征极点突变和同源定向修复基因之间的合作，在 以及3)描述了非规范PTEN突变体与极点之间的相互作用 突变的等位基因。 这项拟议的工作意义重大，因为这些研究将极点功能障碍与其他调节失调联系在一起 这些途径将为基因组不稳定的基本机制提供新的见解，但也提供了新的可能性 治疗复制缺陷癌症的治疗方法。这项工作也很重要，因为它将 促进我们对免疫检查点治疗中的合成致命性和新表位的理解。这个 所提出的工作具有创新性，因为1)首次发现了极地突变体之间的遗传互作 肿瘤中的等位基因和其他途径，然后在哺乳动物系统中描述这些相互作用；2)我们 将首先检查这些相互作用对基因组稳定性的影响，然后比较 从可公开获得的大型癌症基因组数据库中测序信息；3)建议的研究包括 在使用基于CRISPR的多种系统(小鼠、人类细胞、 有机物)，用于比较对表型和诱变的影响。

SUMMARY Understanding how cells fail to protect against the mutations and structural rearrangements that are found in all cancers is critical to ultimately treating the disease. Mutant replication DNA polymerases are found in some of the most highly mutated tumors known. These tumors also have a very unique mutation spectrum. In our published work we generated and began to characterize mouse and human cell models of these tumors. While it was originally thought that this mutagenesis was both necessary and sufficient for tumor development, our published work and preliminary data strongly implicates additional factors as being necessary for POLE tumor development. The main goal of this project is to test our central hypothesis that while POLE tumors are some of the most highly mutagenic tumors observed, the essential tumorigenic feature of POLE mutants is a failure to stabilize stalled/collapsed replication forks in response to damaged DNA via inactivation of certain homology-directed repair genes like BRCA2 and/or loss of a nuclear PTEN activity that both normally stabilize stalled replication forks and thus suppress genome instability. POLE mutagenesis is concurrently enhanced by promoting acquisition of mutations in these pathways This hypothesis is based on our published work and unpublished preliminary evidence. Specifically, this project will 1) Define the mechanistic origins of POLE mutational signatures; 2) Characterize the cooperation between POLE mutations and homology-directed repair genes, in particular BRCA2; and 3) Characterize the interactions between a noncanonical PTEN mutant and POLE mutant alleles. The proposed work is significant because these studies linking POLE dysfunction to other dysregulated pathways will provide novel insight into both basic mechanisms of genome instability, but also new possible therapeutic approaches to treating replication-defective cancers. The work is also significant because it will advance our understanding of synthetic lethality and neo-epitopes in immune checkpoint therapies. The proposed work is innovative because 1) it is the first to identify genetic interactions between POLE mutant alleles and other pathways in tumors and then characterize these interactions in a mammalian system; 2) we will be the first to examine the effects of these interactions on genome stability and then compare to sequencing information from large, publicly available cancer genome databases; 3) the proposed studies are innovative in their use of CRISPR-based genetic engineering of multiple systems (mice, human cells, organoids) for comparing effects on phenotype and mutagenesis.