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：
10387685
负责人：
Brandon Lee Mouery
金额：
$ 3.36万
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/10387685
关键词：
Address Alleles Binding Biological Assay Breast Cancer Cell CDK4 gene CRISPR library Candidate Disease Gene Cell Cycle Cell Cycle Progression Cell Proliferation Cell division Cells Cessation of life 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 Gene Proteins Genes Genome Genomic Instability Global Change Goals Human Kinetics Knowledge Lead Ligase Maintenance Malignant Neoplasms Maps Mediating Messenger RNA Molecular Pathway interactions Patients Pharmaceutical Preparations Phenotype Phosphorylation Process Proteins Regulation Regulatory Pathway Reporting Resistance Retinoblastoma Retinoblastoma Protein Role S phase Side Site Source Testing Toxic effect Treatment Efficacy Ubiquitin Ubiquitination antitumor effect cancer cell clinical efficacy drug efficacy experimental study helicase 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.

项目摘要癌症来自失调的细胞周期进展和不受控制的细胞分裂。大多数肿瘤改变了 CDK4,6/RB/E2F促进肿瘤发生的途径，使其成为有前途的治疗靶标。与抗激素治疗，CDK4/6抑制剂可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抑制剂的临床使用受到限制剂量限制毒性和抗性。因此，更好地理解CDK4/6的作用机理需要抑制剂来最大化其治疗功效。该提议的核心目标是检查微型纯体维持（MCM）复合蛋白的稳定性变化 CDK4/6抑制（CDK4/6i）。 MCM是必不可少的DNA复制蛋白，其失调可能导致复制应力，DNA损伤和癌症。为避免这种情况，高度调节了MCM的细胞定位但是，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抑制剂在两个未转化的上皮细胞中导致MCM的蛋白酶体依赖性降解在乳腺癌细胞中。据我们所知，这代表了调节MCM的第一个已知机制通过活性蛋白质降解的丰度。因此，尚不清楚为什么CDK4/6I诱导的细胞停滞，但没有其他形式的停滞，例如静止，会导致积极的MCM退化。在本提案的目标1中，我们将通过识别CDK4/6-6-6I诱导的MCM降解的机制和后果 E3泛素连接酶（S）标记MCM进行降解并定义泛素化的精确靶标。在我们的最初的发现，我们将CDK4/6I-6-I诱导的MCM降解与复制应力和DNA损伤相关联，但并没有直接将MCM降解作为主要来源。我们将测试MCM的假设降解是CDK4/6I-6-6-诱导的复制应力和DNA损伤的关键来源降解并确定从CDK4/6释放时是否可以防止这些表型的积累抑制。这些实验的结果将提供有关是否可以利用MCM降解的洞察力增加CDK4/6抑制剂的细胞死亡诱导能力。在AIM 2中，我们将检验以下假设。 RB/E2F介导的基因表达改变的CDK4/6-IND降解引起。我们已经发现MCM降解是RB依赖性的，但是，仍然不清楚它是否依赖于E2F。测试这将首先确定CDK4/6-i诱导的MCM降解是否直接来自抑制E2F活性。如果是这样，我们将操纵下游E2F调节的基因的表达，以阐明调节途径这导致MCM降解。如果MCM降解不依赖于E2F，我们将探测替代机制 RB介导了这种表型。综上所述，这些目标将为小说提供机械洞察力决定复制能力的途径。我们预计我们的发现将用于增强 CDK4/6抑制剂的临床功效通过增加抗肿瘤活性并最大程度地减少有害副作用。