Molecular basis of MED12 in the pathogenesis of uterine fibroids

项目摘要/摘要 子宫肌瘤(UF)是威胁全球妇女健康的最重要的良性肿瘤。不会再有了- UF存在长期非侵入性治疗选择，对肿瘤病因的更深入了解是开发更多的关键 有效的治疗方法。因此，这一建议是有影响的，因为它提出了一种新的病因基础 主要的UF亚型，进一步为本病的治疗干预提供了概念证明 遗传背景。UFS源于单个子宫肌层干细胞(MM SC)向肿瘤的遗传转化 种子单克隆性肿瘤生长的起始细胞(UF SC)。值得注意的是，RNA中反复出现的体细胞突变 聚合酶II(RNAPII)介体亚单位MED12约占UF的70%，但这些突变如何驱动细胞 转化和肿瘤形成尚不清楚。此前，我们发现MED12突变会破坏CycC- 介体中CDK8激酶的活性，揭示了第一例也是迄今为止唯一已知的由 这些致病突变进一步暗示了CDK8在UF发病机制中的新病因学作用。这 突破性的发现是我们最初应用程序的基础，它催生了重大进展，证明 在这一新的应用中阐明介体蛋白激酶的分子基础和治疗意义的研究 MED12突变尿路综合征发病机制中的功能障碍。在这里，我们发现MED12突变会损害CDK8 通过T环失稳的活性，导致磷蛋白质组的深刻变化和调节失调 决定多发性骨髓间充质干细胞命运的细胞生长和肌源性基因表达程序。此外，我们还证明了 MED12突变诱导的CDK8失活触发R环依赖的复制应激，提示 在这一主要的UF亚类中，基因组不稳定和新的治疗脆弱性的可能基础。 因此，我们假设MED12突变诱导的介体激酶破坏驱动肿瘤的启动 通过异常MM SC重新编程和复制应激依赖的染色体而进展 不稳定。我们进一步提出，临床上相关的ATR轴抑制剂将在 MED12突变型尿失禁的临床前模型。为了验证这一点，我们将：(1)阐明生物化学基础 MED12突变破坏了介体激活酶的活性。利用结构生物学和生物化学，我们将确定 突变体MED12对CDK8 T环稳定性和构象动力学及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 驱动UF启动。我们将把多发性骨髓间充质干细胞自我更新和分化过程中介体蛋白依赖的变化联系起来。 全基因组的增强子重新编程和改变的转录输出，并进一步询问介体蛋白激酶 破坏可以重新编程MM SCs以在体内形成UF肿瘤；(3)阐明介体的分子基础 激酶的破坏推动了UF的进展。我们将调查RNAPII启动子暂停缺陷作为基础 R-环异常增加，确定R-环诱导的复制应激是否触发有丝分裂染色体断裂，以及 评估ATR轴抑制剂在MED12突变UF临床前小鼠模型中的疗效。

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.