Mechanisms of Genome Instability in Tumors with DNA Polymerase Epsilon Mutations

编码DNA聚合酶e（Pole）的POLE基因突变与以下疾病的发生有关： 大肠癌和子宫内膜癌，具有极高的超突变水平。的机制 极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 结构域优先受影响，POLE突变最初被认为是通过失活外切核酸溶解作用 校对然而，我们实验室随后的研究挑战了这一概念。突变体效应 模型系统中与癌症相关的变异大大超过了校对缺陷的影响（在这种情况下， 最经常发生的变异，P286 R-两个数量级）。PoleP 286 R小鼠也被发现有很多 比缺乏校对能力的北极鼠更容易患癌症。因此，除了核酸外切酶的损失以外的因素必须 是导致突变和癌症风险升高的原因。这个应用程序的目标是破译 导致POLE突变型肿瘤基因组不稳定的机制。初步研究使我们提出， 对于许多Pole核酸外切酶结构域变体，主要后果不仅是核酸外切酶的丢失， DNA聚合酶活性显著增加，这影响了Pole对复制的总体贡献， 干扰其他突变避免机制的功能。这是一个高度创新的假设， 因为它将肿瘤中的POLE变体定义为功能获得变体，而不是像以前那样的功能丧失变体。 以前认为。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 四亚基极点来表征癌症相关突变的生化效应。在目标2中，我们将 定义极变异体在增殖细胞中的增变效应的机制。这些研究将有助于 了解结直肠癌和子宫内膜癌的重要比例的病因，并可能查明新的 为POLE突变携带者开发个性化治疗的目标。生物化学和细胞- 我们将在这项工作的过程中建立的基于测定也将有助于评估功能性 在肿瘤中检测到的多个知之甚少的POLE变体的意义。

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.