Investigation of human DNA polymerase epsilon variants

总结 了解细胞如何无法保护细胞免受突变和结构重排的影响， 所有的癌症都是最终治疗疾病的关键。突变复制DNA聚合酶被发现在一些 已知最高度变异的肿瘤之一这些肿瘤也具有非常独特的突变谱。在我们 发表的工作，我们产生并开始表征这些肿瘤的小鼠和人类细胞模型。而 最初认为这种诱变对于肿瘤的发展是必要的和充分的，我们 已发表的工作和初步数据强烈暗示其他因素是必要的极肿瘤 发展 这个项目的主要目标是测试我们的中心假设，虽然POLE肿瘤是一些最重要的肿瘤， 观察到高度致突变性肿瘤，POLE突变体的基本致瘤特征是不能稳定 停滞/崩溃的复制叉响应于受损的DNA，通过某些同源定向的失活， 修复基因如BRCA 2和/或核PTEN活性的丧失，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。POLE诱变同时通过促进 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 初步证据具体来说，这个项目将1）定义POLE突变的机制起源 2）表征POLE突变和同源定向修复基因之间的合作， 3）表征非典型PTEN突变体和POLE之间的相互作用 突变等位基因 这项工作是重要的，因为这些研究将POLE功能障碍与其他失调联系起来。 通路将提供新的见解，既基因组不稳定的基本机制，但也有新的可能， 治疗复制缺陷型癌症的治疗方法。这项工作也很重要，因为它将 推进我们对免疫检查点疗法中的合成致死性和新表位的理解。的 提出的工作是创新的，因为1）它是第一个确定POLE突变体之间的遗传相互作用 等位基因和其他途径，然后在哺乳动物系统中表征这些相互作用; 2）我们 将首先研究这些相互作用对基因组稳定性的影响，然后与 来自大型公开的癌症基因组数据库的测序信息; 3）拟议的研究是 创新地使用基于CRISPR的多系统基因工程（小鼠，人类细胞， 类器官）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enhanced by promoting acquisition of mutations in these pathways This hypothesis is based on our published work and unpublished preliminary evidence.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.