The mechanism and consequences of MCM degradation induced by CDK4/6 inhibition

项目摘要 癌症是由细胞周期进程失调和细胞分裂失控引起的。大多数肿瘤改变了 CDK 4，6/RB/E2 F通路促进肿瘤发生，使其成为有希望的治疗靶点。再加上 抗激素治疗、CDK 4/6抑制剂显著改善雌激素受体患者的预后 （ER）阳性/Her 2阴性乳腺癌。然而，CDK 4/6抑制剂的临床使用受到以下因素的限制： 剂量限制性毒性和耐药性。因此，更好地了解CDK 4/6的作用机制 需要抑制剂来最大化它们的治疗功效。这项提案的核心目标是审查 微染色体维持（MCM）复合物蛋白稳定性的变化 CDK 4/6抑制（CDK 4/6 i）。MCM是一种必需的DNA复制蛋白，其失调可导致 复制压力DNA损伤和癌症为了避免这种情况，MCM的细胞定位受到高度调节 然而，在整个细胞周期中，蛋白质丰度保持恒定。令人惊讶的是，我们发现， CDK 4/6抑制剂导致未转化上皮细胞中MCM的蛋白酶体依赖性降解 和乳腺癌细胞中。据我们所知，这是第一个已知的调节MCM的机制 通过活性蛋白质降解而获得丰度。因此，仍然不清楚为什么CDK 4/6 i诱导的细胞停滞， 而不是其它形式的停滞，例如静止，导致活性MCM降解。在本提案的目标1中， 我们将确定CDK 4/6 i诱导的MCM降解的机制和后果， E3泛素连接酶，其标记MCM用于降解并通过定义泛素化的精确靶标。在我们 最初发现，我们将CDK 4/6 i诱导的MCM降解与复制应激和DNA损伤相关联， 但没有直接暗示MCM降解为主要来源。我们将检验MCM 降解是CDK 4/6 i诱导的复制应激和DNA损伤的关键来源， 降解，并确定这是否阻止了这些表型在从CDK 4/6释放时的积累 抑制作用这些实验的结果将提供深入了解是否可以利用MCM降解 增加CDK 4/6抑制剂诱导细胞死亡的能力。在目标2中，我们将检验以下假设： CDK 4/6 i诱导的MCM降解由RB/E2 F介导的基因表达改变引起。我们已经 发现MCM降解是RB依赖性的，然而，仍然不清楚它是否是E2 F依赖性的。测试 这将首先确定CDK 4/6 i诱导的MCM降解是否直接由受抑制的E2 F活性引起。如果是这样的话， 我们将操纵下游E2 F调控基因的表达，以阐明调控途径。 这导致MCM退化。如果MCM降解是E2 F独立的，我们将探索替代机制 RB介导这种表型。综合起来，这些目标将为小说提供机械的见解 决定复制能力的途径。我们预计，我们的研究结果将用于提高 通过增加抗肿瘤活性和最小化有害的副作用来提高CDK 4/6抑制剂的临床功效。

Project Summary Cancer results from dysregulated cell cycle progression and uncontrolled cell division. Most tumors alter the CDK4,6/RB/E2F pathway to promote oncogenesis, making it a promising therapeutic target. When coupled with anti-hormone therapy, CDK4/6 inhibitors significantly improve the prognosis of patients with estrogen receptor (ER)-positive/Her2-negative breast cancer. Nevertheless, the clinical use of CDK4/6 inhibitors is restricted by dose-limiting toxicities and resistance. Thus, a better understanding of the mechanism of action of CDK4/6 inhibitors is required to maximize their therapeutic efficacy. The central goal of this proposal is to examine changes in the stability of the minichromosome maintenance (MCM) complex proteins induced by CDK4/6 inhibition (CDK4/6i). MCM is an essential DNA replication protein and its dysregulation can result in replication stress, DNA damage, and cancer. To avoid this, the cellular localization of MCM is highly regulated throughout the cell cycle, however, the protein abundance remains constant. Surprisingly, we discovered that CDK4/6 inhibitors result in proteasome-dependent degradation of MCM in both untransformed epithelial cells and in breast cancer cells. To our knowledge, this represents the first known mechanism of regulating MCM abundance through active protein degradation. It thus remains unclear why a CDK4/6i-induced cellular arrest, but not other forms of arrest such as quiescence, leads to active MCM degradation. In Aim 1 of this proposal, we will determine the mechanism and consequences of CDK4/6i-induced MCM degradation by identifying the E3 ubiquitin ligase(s) that tags MCM for degradation and by defining the precise target of ubiquitination. In our initial discovery, we associated CDK4/6i-induced MCM degradation with replication stress and DNA damage, but did not directly implicate MCM degradation as the primary source. We will test the hypothesis that MCM degradation is a key source of CDK4/6i-induced replication stress and DNA damage by preventing its degradation and determining if this prevents the accumulation of these phenotypes upon release from CDK4/6 inhibition. The results of these experiments will provide insight into whether MCM degradation can be exploited to increase the cellular death-inducing capabilities of CDK4/6 inhibitors. In Aim 2, we will test the hypothesis that CDK4/6i-induded MCM degradation results from altered RB/E2F-mediated gene expression. We have already discovered that MCM degradation is RB-dependent, however, it is still unclear if it is E2F-dependent. To test this, will first determine if CDK4/6i-induced MCM degradation results directly from repressed E2F activity. If so, we will manipulate the expression of downstream E2F-regulated genes to elucidate the regulatory pathway(s) that results in MCM degradation. If MCM degradation is E2F-independent, we will probe alternative mechanisms by which RB mediates this phenotype. Taken together, these aims will provide mechanistic insights into a novel pathway that determines replication competence. We anticipate that our findings will be used to enhance the clinical efficacy of CDK4/6 inhibitors by increasing anti-tumor activity and minimizing harmful side effects.