New Epigenetic Targets in Ovarian Cancer Stem Cells

摘要 化疗耐药性的发展与癌症干细胞（CSC）的持续存在有关。csc被 其特征在于在免疫缺陷的细胞中自我更新、生长为球体、分化和产生肿瘤的能力。 小鼠他们对传统的治疗方法，包括化疗和放疗都有抗药性。充分发挥两国 先前的研究表明，用铂治疗后残留的卵巢CSC显示DNA增加， 甲基化，我们假设发生了其他表观遗传修饰，促进了干细胞样表型， 并使CSC易受表观基因组修饰剂的影响。为了开始解决这个问题，我们使用 在纳米级水平上可视化染色质的分波光谱， 染色质与高通量测序（ATAC-seq）映射可访问的启动子区域，和蛋白质组 分析以测量CSC与非CSC中的组蛋白标记。我们发现， 与非CSC相比，CSC中的抑制性组蛋白标记，并鉴定了H379组蛋白甲基化的表达增加 CSCs与非CSCs中的Dot 1 L转移酶。我们的初步数据表明，Dot 1 L抑制剂阻断了干性 功能. FOXK 2是通过ATAC-Seq在CSCs vs. non-CSC被证明是Dot 1 L靶基因，并且在卵巢CSC中高度表达。这些初步 这些观察使我们提出了剖析Dot 1 L诱导的H3 K79甲基化、癌症 干性和耐铂性，解决三个主要目标。 目的1：确定Dot 1 L调控的H3 K79甲基化是否是OCSC的标志物和靶点。H3K79 单、二和三甲基化将通过使用 染色质免疫沉淀（ChIP）测序。Dot-1 L抑制剂和Dot-1 L敲低对茎的影响 将在体外和体内评估细胞特性和对铂的反应。 目的2：确定H3 K79甲基化是否在铂耐药OC模型和肿瘤中改变。 配对的亲本和铂耐药细胞系、异种移植物和患者来源的异种移植物模型（PDX） 本课题组开发的新方法将用于检测DOT 1 L表达和H3 K79甲基化。表达及 Dot 1 L的功能将在基于铂的化疗后敏感或持续的人卵巢肿瘤中测量。 化疗遗传和药理学抑制策略对铂类药物反应的影响将在 在体外和体内研究。 目的3：确定FOXK 2，一个Dot 1 L靶点在卵巢CSCs中的功能。FOXK 2表达将是 在铂耐药与铂初治模型和人类卵巢肿瘤中测量。敲门敲门 我们的实验将确定其在卵巢CSC中的转录调控功能。 总之，拟议的研究将解决一个具有直接临床应用的重要生物学问题。 我们在癌症干细胞生物学和铂耐药性方面的初步数据和广泛专业知识强烈支持 支持项目的可行性和影响力。我们将使用最先进的技术来推动发展 新的表观基因组定向策略，如Dot 1 L抑制剂，靶向卵巢CSC。我们久经考验的记录 实验室到诊所的干预措施将促进我们的研究结果转化为临床应用。的结果 该项目将立即适用于卵巢癌，但可能对其他铂- 耐药实体瘤。总之，该项目将弥合铂耐药恶性肿瘤的知识差距， 影响了大量的美国退伍军人和美国军队。

Abstract Development of resistance to chemotherapy was linked to persistence of cancer stem cells (CSCs). CSCs are characterized by the ability to self-renew, grow as spheres, differentiate and generate tumors in immunodeficient mice. They are resistant to traditional forms of treatment, including chemo and radio-therapy. Building on our previous work showing that ovarian CSCs residual after treatment with platinum display increased DNA methylation, we hypothesized that other epigenetic modifications occur, promote a stem-like phenotype, and render CSCs vulnerable to epigenome modifying agents. To begin to address this question, we used partial wave spectroscopy to visualize chromatin at the nanoscale level, the Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) to map accessible promoter regions, and proteomic analysis to measure histone marks in CSCs vs. non-CSCs. We found less open chromatin associated with repressive histone marks in CSCs vs. non-CSC and identified increased expression of the H379 histone methyl transferase Dot1L in CSCs vs. non-CSCs. Our preliminary data show that Dot1L inhibitors blocked stemness features. FOXK2, one of the transcription factors found to have an open promoter by ATAC-Seq in CSCs vs. non-CSCs was shown to be a Dot1L target gene and was highly expressed in ovarian CSCs. These preliminary observations led us to propose dissecting the link between Dot1L-induced H3K79 methylation, cancer stemness, and platinum-resistance by addressing three main objectives. Aim 1: Determine whether H3K79 methylation regulated by Dot1L is a mark and a target in OCSCs. H3K79 mono-, di-, and trimethylation will be mapped to chromatin extracted from OCSCs vs. non-CSCs by using chromatin-immunoprecipitation (ChIP)-sequencing. Effects of Dot-1L inhibitors and Dot-1L knock-down on stem cell characteristics and response to platinum will be assessed in vitro and in vivo. Aim 2: Determine whether H3K79 methylation is altered in platinum-resistant OC models and tumors. Paired parental and platinum resistant cell lines, xenografts and patient-derived xenograft models (PDX) developed by our group will be used to measure DOT1L expression and H3K79methylation. Expression and function of Dot1L will be measured in human ovarian tumors sensitive or persistent after platinum-based chemotherapy. Effects of genetic and pharmacological inhibitory strategies on response to platinum will be investigated in vitro and in vivo. Aim 3: Determine the function of FOXK2, a Dot1L target in ovarian CSCs. FOXK2 expression will be measured in platinum-resistant vs. platinum-naïve models and in human ovarian tumors. Knock-in and knock- out experiments will determine its function on transcription regulation in ovarian CSCs. In summary, the proposed studies will address an important biological question with direct clinical applications. Our preliminary data and extensive expertise on cancer stem cell biology and platinum resistance strongly support the project’s feasibility and impact. We will use state of the art technologies to propel the development of new epigenome-directed strategies, such as Dot1L inhibitors, to target ovarian CSCs. Our proven track record of bench-to-clinic interventions will facilitate translation of our findings to clinical applications. The results of this project will be immediately applicable to ovarian cancer, but may have broader implications for other platinum- resistant solid tumors. In all, this project will bridge a gap of knowledge in platinum-resistant malignancies that affect a significant number of US Veterans and US military.