Mechanisms of Genome Instability in Tumors with DNA Polymerase Epsilon Mutations

编码DNA聚合酶e(POLE)的POL基因突变与先天性心脏病的发生有关 具有极高水平超突变的结直肠癌和子宫内膜癌。这一机制通过它 极点变异导致的超变异性仍然令人费解。因为核酸外切酶中的氨基酸残基 结构域优先受到影响，极点突变最初被认为是通过灭活核酸外切作用 校对。然而，我们实验室随后的研究对这一概念提出了挑战。的诱变子效应 模型系统中的癌症相关变异大大超过了校对缺陷的影响(在这种情况下 在最常见的变异中，P286R--下降了两个数量级)。PoleP286R小鼠也被发现有很多 与校对缺陷的波兰小鼠相比，它们更容易患上癌症。因此，除核酸外切酶丢失以外的其他因素必须 对突变和癌症风险的增加负责。这个应用程序的目标是破译 在极突变肿瘤中驱动基因组不稳定性的机制。初步研究使我们提出， 对于许多极地核酸外切酶结构域变体来说，主要后果不仅是核酸外切酶的丢失，而且是一种 DNA聚合酶活性显著增加，这影响了POLE对复制和 干扰其他突变避免机制的功能。这是一个高度创新的假说， 因为它将肿瘤中的极变异体定义为功能获得变异体，而不是原来的功能丧失变异体 之前的想法是。我们将通过追求两个具体目标来检验这一假设。在目标1中，我们将使用纯化的 四个亚单位的极点，以表征癌症相关突变的生化效应。在目标2中，我们将 明确POLE变异体在增殖细胞中诱变效应的机制。这些研究将会有所帮助 了解相当大比例的结直肠癌和子宫内膜癌的病因，并可能确定新的 为极点突变携带者开发个性化治疗的目标。生化和细胞- 我们将在这项工作过程中建立的基于分析的方法也将有助于评估功能 在肿瘤中检测到多个知之甚少的极变异体的意义。

Mutations in the POLE gene encoding DNA polymerase e (Pole) have been linked to the development of colorectal and endometrial cancers with extremely high levels of hypermutation. The mechanism through which Pole variants cause hypermutability remains puzzling. Because amino acid residues in the exonuclease domain are preferentially affected, POLE mutations were originally thought to act by inactivating exonucleolytic proofreading. However, subsequent research by our laboratory challenged this concept. Mutator effects of cancer-associated variants in model systems greatly exceed the effect of proofreading deficiency (in the case of the most recurrent variant, P286R - by two orders of magnitude). PoleP286R mice were also found to be much more cancer-prone than proofreading-deficient Pole mice. Thus, factors other than loss of exonuclease must be responsible for the elevated mutagenesis and cancer risk. The goal of this application is to decipher the mechanisms that drive genomic instability in POLE-mutant tumors. Preliminary studies led us to propose that, for many Pole exonuclease domain variants, the main consequence is not merely the loss of exonuclease but a dramatically increased DNA polymerase activity, which affects the overall contribution of Pole to replication and interferes with the function of other mutation avoidance mechanisms. This is a highly innovative hypothesis, since it defines POLE variants in tumors as gain-of-function variants rather than loss-of-function as was previously thought. We will test this hypothesis by pursuing two Specific Aims. In Aim 1, we will use purified four-subunit Pole to characterize the biochemical effects of cancer-associated mutations. In Aim 2, we will define the mechanisms of the mutator effects of Pole variants in proliferating cells. These studies will help understand the etiology of a significant proportion of colorectal and endometrial cancers and may pinpoint new targets for the development of personalized therapies for POLE mutation carriers. The biochemical and cell- based assays we will establish in the course of this work will also be instrumental for assessing the functional significance of multiple poorly understood POLE variants detected in tumors.