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

CDK4/6抑制引起MCM降解的机制和后果

基本信息

批准号：
10668948
负责人：
Brandon Lee Mouery
金额：
$ 3.46万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10668948
关键词：
Address Alleles Binding Biological Assay Breast Cancer Cell CDK4 gene CRISPR library Candidate Disease Gene Cell Cycle Cell Cycle Inhibition Cell Cycle Progression Cell Proliferation Cell division Cells Cessation of life Chimeric Proteins Chromosomes Clinical Combined Modality Therapy Competence Complex Coupled Cytostatics DNA Damage DNA biosynthesis DNA replication origin Data Development Dose Limiting E2F transcription factors Ensure Epithelial Cells Estrogen receptor positive G1 Phase Gene Expression Genes Genome Genomic Instability Global Change Goals Human Kinetics Knowledge Ligase Maintenance Malignant Neoplasms Maps Mediating Messenger RNA Molecular Pathway interactions Patients Pharmaceutical Preparations Phenotype Phosphorylation Process Proliferating Proteins Regulation Regulatory Pathway Replication Initiation Replication Licensing Reporting Repression Resistance Retinoblastoma Retinoblastoma Protein Role S phase Side Site Source Testing Therapeutic Toxic effect Treatment Efficacy Ubiquitin Ubiquitination antitumor effect cancer cell candidate identification clinical efficacy drug efficacy experimental study helicase hormone therapy improved in silico inhibitor insight knock-down malignant breast neoplasm multicatalytic endopeptidase complex mutant novel patient prognosis patient subsets prevent protein complex protein degradation proteostasis replication stress response side effect therapeutic target treatment response tumor tumorigenesis tumorigenic ubiquitin-protein ligase

项目摘要

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.

项目概要癌症是由细胞周期进程失调和不受控制的细胞分裂引起的。大多数肿瘤会改变 CDK4,6/RB/E2F通路促进肿瘤发生，使其成为一个有前途的治疗靶点。当与抗激素治疗、CDK4/6抑制剂显着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：剂量限制性毒性和耐药性。从而更好地了解CDK4/6的作用机制抑制剂需要最大限度地发挥其治疗功效。该提案的中心目标是审查微染色体维持（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 的细胞定位受到严格监管然而，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 CDK4/6 抑制剂导致两种未转化上皮细胞中 MCM 的蛋白酶体依赖性降解和乳腺癌细胞中。据我们所知，这代表了第一个已知的 MCM 调节机制通过活性蛋白质降解获得丰度。因此，尚不清楚为什么 CDK4/6i 诱导细胞停滞，但其他形式的停滞（例如静止）不会导致活跃的 MCM 降解。在本提案的目标 1 中，我们将通过确定 CDK4/6i 诱导的 MCM 降解的机制和后果 E3 泛素连接酶通过定义泛素化的精确目标来标记 MCM 进行降解。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 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，通过阻止其降解并确定这是否会阻止这些表型在从 CDK4/6 释放后积累抑制。这些实验的结果将有助于深入了解 MCM 降解是否可以被利用增加 CDK4/6 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： CDK4/6i 诱导的 MCM 降解是由 RB/E2F 介导的基因表达改变引起的。我们已经发现MCM降解是RB依赖性的，然而，目前还不清楚它是否是E2F依赖性的。测试首先确定 CDK4/6i 诱导的 MCM 降解是否直接由 E2F 活性抑制所致。如果是这样，我们将操纵下游 E2F 调节基因的表达来阐明调节途径这会导致 MCM 降解。如果 MCM 退化与 E2F 无关，我们将探讨替代机制 RB 通过它介导这种表型。总而言之，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增强 CDK4/6 抑制剂通过增加抗肿瘤活性并最大限度地减少有害副作用的临床疗效。