Molecular basis of MED12 in the pathogenesis of uterine fibroids

项目总结/摘要 子宫肌瘤（UFs）是世界范围内对妇女健康最重要的良性肿瘤威胁。因为不再- 长期非侵入性治疗方案存在于UFs中，深入了解肿瘤病因是开发更多 有效的治疗。因此，这一提议是有影响力的，因为它为这种疾病提出了一种新的病因学基础。 主要的UF亚型，并进一步提供了在这种特定的治疗干预的概念证明 遗传背景UFs是由单个子宫肌层干细胞（MM SC）遗传转化为肿瘤引起的 启动细胞（UF SC），种子单克隆肿瘤生长。值得注意的是，RNA中的复发性体细胞突变 聚合酶II（RNAPII）介体亚基MED 12占UF的约70%，但这些突变如何驱动细胞 转化和肿瘤形成尚不清楚。以前，我们发现MED 12突变会破坏CycC- 介体中的CDK 8激酶活性，揭示了第一个也是迄今为止唯一已知的生物化学缺陷， 这些致病性突变，并进一步暗示了CDK 8在UF发病机制中的新的病因学作用。这 突破性发现是我们最初应用的基础，它产生了重大进展，证明了 本更新申请中的研究旨在阐明介体激酶的分子基础和治疗意义 MED 12突变型UF发病机制中的功能障碍。在此，我们发现MED 12突变损害CDK 8 活性通过T环不稳定，导致深刻改变磷酸化蛋白质组和失调 决定MM SC命运的细胞生长和肌源性基因表达程序。此外，我们表明， MED 12突变诱导的CDK 8失活触发了R环依赖性复制应激，这表明 基因组不稳定性和新的治疗漏洞在这个占主导地位的UF亚类的可能基础。 因此，我们假设MED 12突变诱导的介体激酶破坏驱动肿瘤的发生。 通过异常MM SC重编程和复制应激依赖性染色体 不稳定我们进一步提出，临床相关的ATR轴抑制剂将提供治疗效益， MED 12突变型UF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， MED 12突变破坏介体激酶活性。利用结构生物学和生物化学，我们将确定 突变MED 12对CDK 8 T环稳定性和构象动力学以及CycC-CDK 8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MM SC自我更新和分化中的介导激酶依赖性变化与MM SC的自我更新和分化联系起来。 与全基因组增强子重编程和改变转录输出，并进一步询问是否介导激酶 破坏可以重新编程MM SC以形成体内UF肿瘤;（3）阐明介导剂 激酶破坏驱动UF进展。我们将研究RNAPII启动子暂停缺陷作为基础， 异常的R环增加，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 评价ATR轴抑制剂在MED 12突变型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.