Epigenome, MED12 and Progesterone Action in Uterine Leiomyomas

子宫平滑肌瘤（LM，纤维瘤）是妇女最常见的肿瘤。没有长期的治疗 available.每个LM似乎起源于单个突变的LM干细胞（LSC）在细胞中的克隆扩增。 子宫肌层（MYO）。LSC占肿瘤质量的5%，并分化为中间细胞群 (LIC，7%），其然后变成占肿瘤块的88%的终末分化细胞（LDC）。司机 介体复合物亚基12（mut-MED 12）突变发生在所有LM的70%中。P4及其 受体PR是LM生长所必需的。PR富集的LIC LIP 4通过旁分泌向PR缺陷的LSC信号传导 激活其增殖的因素。醋酸乌利司他（UPA），一种PR选择性P4拮抗剂， 减轻了症状，但由于有肝损伤的风险而停止使用。我们的总体目标是定义 全基因组P4作用在mut-MED 12 β相关LM肿瘤发生的病因学中的作用， 治疗目标我们发现mut-MED 12与PR和全基因组PR染色质发生物理相互作用， 与正常MYO组织相比，表达mut-MED 12的LM中的相互作用景观显著失调 携带野生型MED 12 Mut-MED 12增强PR募集至P4靶基因的顺式调控元件 编码对LSC增殖至关重要的旁分泌生长因子、细胞因子和细胞外基质蛋白， LM肿瘤发生。此外，在Med 12中具有人类等效功能获得性突变的小鼠的子宫 发展LM响应P4，而Med 12敲除阻断小鼠子宫中的P4/PR信号传导。我们 假设mut-MED 12改变PR-染色质相互作用特征并增强LIC中P4作用， 为LSC存活和增殖提供支持生态位。使用ChIP-seq、RNA-seq、STARR-seq和 CRISPR/Cas9基因编辑策略，以及体内PDX和mut-Med 12敲入小鼠模型，我们提出了 （1）确定PR-染色质相互作用位点是否与mut-1基因特异性相关， MED 12刺激P4在不同的干细胞支持细胞群（LIC）中的作用，然后将致瘤性 旁分泌信号增加LSC活性和肿瘤生长。我们将检验mut-MED 12 与PR相互作用并改变其与染色质的相互作用，从而增强LIC对P4的反应性， 激活支持邻近LSC功能的基因转录和旁分泌信号。(2)定义 mut-Med 12的致瘤活性是否是通过改变PR-染色质相互作用的景观介导的， 人等效mut-Med 12小鼠模型。我们将检验mut-Med 12破坏染色质的假设， PR结合位点周围的特征，从而支持LM关键的基因转录和途径 肿瘤发生，而UPA通过改变异常PR-染色质-表观基因组相互作用收缩LM， 逆转疾病相关基因的不适当表达。破译基因组机制， 确定的细胞群体水平将帮助我们识别与mut-MED 12相关的基因型特异性新靶点 用于治疗LM的药物基因组学和精准医学。

Uterine leiomyoma (LM, fibroid) is the most common tumor in women. No long-term medical treatment is available. Each LM seems to originate from the clonal expansion of a single mutated LM stem cell (LSC) in the myometrium (MYO). LSC comprise 5% of tumor mass and differentiate into an intermediate cell population (LIC, 7%), which then become terminally differentiated cells (LDC) comprising 88% of the tumor bulk. Driver mutations of mediator complex subunit 12 (mut-MED12) occur in 70% of all LM. Progesterone (P4) and its receptor PR are essential for LM growth. PR-rich LIC transduce P4 signaling to PR-deficient LSC via paracrine factors to activate their proliferation. Ulipristal acetate (UPA), a PR-selective P4 antagonist, shrank LM and reduced its symptoms, but its use was halted because of risk of liver injury. Our overall goal is to define the role of genomewide P4 action in the etiology of mut-MED12‒associated LM tumorigenesis and identify novel therapeutic targets. We found that mut-MED12 physically interacts with PR and genomewide PR-chromatin interaction landscapes are dramatically dysregulated in LM expressing mut-MED12 vs. normal MYO tissue carrying wild type MED12. Mut-MED12 enhances PR recruitment to cis-regulatory elements of P4 target genes encoding paracrine growth factors, cytokines and extracellular matrix proteins critical for LSC proliferation and LM tumorigenesis. Furthermore, the uteri of mice with a human-equivalent gain-of-function mutation in Med12 develop LM in response to P4, whereas Med12 knockout blocks P4/PR signaling in mouse uterus. We hypothesize that mut-MED12 alters PR-chromatin interaction signatures and enhances P4 action in LIC, providing a support niche for LSC survival and proliferation. Using ChIP-seq, RNA-seq, STARR-seq, and CRISPR/Cas9-gene editing strategies, and in vivo PDX and mut-Med12 knock-in mouse models, we propose the following Aims: (1) Determine whether PR-chromatin interaction loci specifically associated with mut- MED12 stimulate P4 action in a distinct stem-support cell population (LIC), which then send tumorigenic paracrine signals to increase LSC activity and tumor growth. We will test the hypothesis that mut-MED12 interacts with PR and alters its interaction with chromatin, thereby enhancing P4 responsiveness of LICs to activate gene transcription and paracrine signaling that support the function of adjacent LSC. (2) Define whether tumorigenic activity of mut-Med12 is mediated via altering PR-chromatin interaction landscapes in a human-equivalent mut-Med12 mouse model. We will test the hypothesis that mut-Med12 disrupts chromatin features surrounding PR-binding sites, thereby supporting gene transcription and pathways critical for LM tumorigenesis, whereas UPA shrinks LM via altering the aberrant PR-chromatin-epigenomic interactions and reversing inappropriate expression of disease-associated genes. Deciphering the genomewide mechanisms at a defined cell population level will help us identify genotype-specific novel targets associated with mut-MED12 for pharmacogenomics and precision medicine in the treatment of LM.