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

项目摘要 癌症是由于细胞周期进程失调和细胞分裂失控造成的。大多数肿瘤会改变 CDK4、6/Rb/E2F通路促进肿瘤发生，使其成为一个有前景的治疗靶点。当与 抗激素治疗，CDK4/6抑制剂显著改善雌激素受体患者的预后 (ER)-阳性/Her2-阴性乳腺癌。然而，CDK4/6抑制剂的临床应用受到以下因素的限制 剂量限制毒性和耐药性。从而对CDK4/6的作用机制有了更好的理解 需要使用抑制剂来最大限度地发挥其治疗效果。这项提案的中心目标是审查 小染色体维持(MCM)复合体蛋白稳定性的变化 CDK4/6抑制(CDK4/6i)。MCM是一种重要的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控 然而，在整个细胞周期中，蛋白质的丰度保持不变。令人惊讶的是，我们发现 CDK4/6抑制剂导致两种未转化上皮细胞蛋白酶体依赖的MCM降解 以及在乳腺癌细胞中。据我们所知，这是已知的第一个调节MCM的机制 通过活性蛋白质降解获得丰富的营养。因此，目前尚不清楚为什么CDK4/6i诱导细胞停滞， 但其他形式的抑制，如静止，不会导致活跃的MCM降解。在本提案的目标1中， 我们将通过鉴定CDK4/6I诱导的MCM降解的机制和后果来确定 E3泛素连接酶(S)，标记MCM进行降解，并通过定义泛素化的精确目标。在我们的 最初的发现，我们将CDK4/6I诱导的MCM降解与复制应激和DNA损伤联系起来， 但并没有直接表明MCM的降解是主要来源。我们将检验MCM的假设 降解是CDK4/6I诱导的复制应激和DNA损伤的关键来源，它通过阻止其 降解，并确定这是否阻止这些表型在从CDK4/6释放时积累 抑制力。这些实验的结果将为MCM的降解是否可以被利用提供洞察 提高CDK4/6抑制剂诱导细胞死亡的能力。在目标2中，我们将检验假设 CDK4/6I介导的MCM降解是由于Rb/E2F介导的基因表达改变所致。我们已经这么做了 发现MCM的降解是RB依赖的，然而，它是否依赖E2F仍然不清楚。为了测试 这将首先确定CDK4/6i诱导的MCM降解是否直接源于抑制的E2F活性。如果是的话， 我们将操纵E2F下游调控基因的表达来阐明调控途径(S) 这会导致MCM的降解。如果MCM降解与E2F无关，我们将探索替代机制 Rb通过它来调节这种表型。综上所述，这些目标将为一部小说提供机械性的见解 决定复制能力的途径。我们预计我们的发现将被用来加强 CDK4/6抑制剂通过提高抗肿瘤活性和最大限度减少不良反应的临床疗效。

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.