Molecular basis of MED12 in the pathogenesis of uterine fibroids

MED12在子宫肌瘤发病机制中的分子基础

基本信息

批准号：
10539362
负责人：
THOMAS G BOYER
金额：
$ 36.37万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2027-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10539362
关键词：
Accounting Address Benign Binding Biochemical Cells Chemicals Chromosomal Breaks Chromosomal Instability Cryoelectron Microscopy Defect Development Drug Targeting Drug usage Enhancers Enzyme Kinetics Enzymes Etiology Fibroid Tumor Functional disorder Gene Expression Genetic Genetic Transcription Genetic Transformation Genomic Instability Habitual Abortion Health Care Costs Impairment Induced Mutation Infertility Leiomyoma Link Mass Spectrum Analysis Measures Mediator of activation protein Medical Menorrhagia Mitotic Molecular Molecular Conformation Monitor Morbidity - disease rate Muscle Mutation Myometrial Operative Surgical Procedures Output Pathogenesis Pathogenicity Pathway interactions Pelvic Pain Pharmaceutical Preparations Phenotype Phosphotransferases Polymerase Pre-Clinical Model Premature Labor RNA Polymerase II Recurrence Role Route SOX8 gene Seeds Somatic Mutation Structural Biochemistry Structure Testing Therapeutic Therapeutic Intervention Transcription Initiation Transcriptional Regulation Treatment Efficacy Uterine Fibroids Women&apos s Health atomic interactions biological adaptation to stress cell growth cell transformation clinically relevant crosslink dosage driver mutation effective therapy efficacy evaluation genome-wide in vivo inhibitor insight mouse model mutant neoplastic novel novel therapeutics pre-clinical programs promoter replication stress segregation self-renewal small molecule stem cell differentiation stem cells stress kinase structural biology transcription factor tumor tumor growth tumor initiation tumor progression tumorigenic

项目摘要

PROJECT SUMMARY/ABSTRACT Uterine fibroids (UFs) are the most important benign neoplastic threat to women’s health worldwide. As no long- term non-invasive treatment option exists for UFs, deeper insight into tumor etiology is key to develop more effective therapies. Accordingly, this proposal is impactful as it suggests a novel etiological basis for the predominant UF subtype and further offers proof of concept for therapeutic intervention in this specific genetic setting. UFs arise from the genetic transformation of a single myometrial stem cell (MM SC) into a tumor initiating cell (UF SC) that seeds monoclonal tumor growth. Notably, recurrent somatic mutations in the RNA polymerase II (RNAPII) Mediator subunit MED12 account for ~70% of UFs, but how these mutations drive cell transformation and tumor formation is unclear. Previously, we showed that MED12 mutations disrupt CycC- CDK8 kinase activity in Mediator, revealing the first and heretofore only known biochemical defect arising from these pathogenic mutations and further implying a new etiological role for CDK8 in UF pathogenesis. This breakthrough discovery was the basis for our original application which spawned major advances that justify studies in this renewal application to clarify the molecular basis and therapeutic implications of Mediator kinase dysfunction in the pathogenesis of MED12-mutant UFs. Herein, we show that MED12 mutations impair CDK8 activity through T-loop destabilization, leading to a profoundly altered phosphoproteome and dysregulation of cell growth and myogenic gene expression programs that dictate MM SC fate. Further, we show that MED12 mutation-induced CDK8 inactivation triggers R-loop-dependent replication stress, suggesting a possible basis for genomic instability and a new therapeutic vulnerability in this dominant UF subclass. Accordingly, we hypothesize that MED12 mutation-induced Mediator kinase disruption drives tumor initiation and progression through aberrant MM SC reprogramming and replication stress-dependent chromosomal instability. We further propose that clinically relevant ATR axis inhibitors will provide therapeutic benefit in a preclinical model of MED12-mutant UFs. To test this, we will: (1) Elucidate the biochemical basis by which MED12 mutations disrupt Mediator kinase activity. Using structural biology and biochemistry, we will determine the impact of mutant MED12 on CDK8 T-loop stability and conformational dynamics as well as CycC-CDK8 substrate binding and catalytic efficiency; (2) Elucidate the molecular basis by which Mediator kinase disruption drives UF initiation. We will link Mediator kinase-dependent changes in MM SC self-renewal and differentiation with genome-wide enhancer reprogramming and altered transcriptional output and further ask if Mediator kinase disruption can reprogram MM SCs to form UF tumors vivo; (3) Elucidate the molecular basis by which Mediator kinase disruption drives UF progression. We will investigate RNAPII promoter pausing defects as a basis for aberrant R-loop accrual, determine if R-loop-induced replication stress triggers mitotic chromosomal breaks, and evaluate the efficacy of ATR axis inhibitors in a preclinical mouse model of MED12-mutant UFs.

项目摘要/摘要子宫纤维（UFS）是对全球妇女健康的最重要的良性肿瘤威胁。没有长期术语非侵入性治疗选项存在于UFS，更深入地了解肿瘤病因是发展更多的关键有效的疗法。根据该提议的影响很大，因为它暗示了一个新颖的病因学基础主要的UF亚型和进一步的概念证明了这种特定的治疗干预措施遗传环境。 UFS是由单个子宫内膜干细胞（MM SC）转化为肿瘤的遗传转化引发单克隆肿瘤生长的细胞（UF SC）。值得注意的是，RNA中的复发体突变聚合酶II（RNAPII）介体亚基Med12占UFS的70％，但这些突变如何驱动细胞转化和肿瘤的形成尚不清楚。以前，我们表明Med12突变破坏了Cycc- 介体中的CDK8激酶活性，揭示了第一和迄今为止仅由已知的生化缺陷引起的这些致病突变，进一步暗示了CDK8在UF发病机理中的新病因作用。这突破性发现是我们原始申请的基础，该申请产生了重大进步以证明合理的在此更新应用中的研究以阐明介体激酶的分子基础和治疗意义 Med12突变UFS发病机理的功能障碍。在此，我们表明Med12突变会损害CDK8 通过T环的不稳定活性，导致磷光蛋白质组和失调症的深刻变化决定MM SC命运的细胞生长和肌原基因表达程序。此外，我们表明 Med12突变诱导的CDK8失活触发R-loop依赖性复制应力，表明A 基因组不稳定性的可能基础和该主要UF子类中新的治疗脆弱性。据此，我们假设Med12突变诱导的介质激酶破坏驱动肿瘤的启动以及通过异常MM SC重新编程和复制应力依赖性染色体的进展不稳定。我们进一步建议，临床上相关的ATR轴抑制剂将在 Med12突变UFS的临床前模型。为了进行测试，我们将：（1）阐明生化基础 MED12突变破坏介体激酶活性。使用结构生物学和生物化学，我们将确定突变Med12对CDK8 T环稳定性和构象动力学以及CYCC-CDK8的影响底物结合和催化效率；（2）阐明介体激酶破坏的分子基础推动UF启动。我们将在MM SC自我更新和差异化中链接介体激酶依赖性变化随着全基因组增强子的重新编程和转录输出的改变，并进一步询问介质激酶是否破坏可以对MM SC进行重新编程以形成UF肿瘤体内；（3）阐明介体的分子基础激酶破坏驱动UF进展。我们将调查RNAPII启动子暂停缺陷作为基础异常的R环核，确定R环诱导的复制应力是否触发有丝分裂染色体断裂，并且评估ATR轴抑制剂在Med12突变UFS的临床前小鼠模型中的效率。