New Epigenetic Targets in Ovarian Cancer Stem Cells

摘要 化疗耐药性的产生与肿瘤干细胞(CSCs)的持续存在有关。CSC是 以免疫缺陷患者的自我更新、球状生长、分化和生成肿瘤为特征 老鼠。他们对包括化疗和放射治疗在内的传统治疗方式具有抵抗力。建立在我们的 先前的工作表明，用铂治疗后残留的卵巢干细胞显示DNA增加 甲基化，我们假设发生了其他表观遗传修饰，促进了茎状表型， 并使CSCs易受表观基因组修饰剂的影响。为了开始解决这个问题，我们使用了 部分波谱学在纳米水平上显示染色质，转座酶的检测是可访问的 染色质高通量测序(ATAC-SEQ)，以绘制可访问的启动子区域和蛋白质组 分析以测量CSCs与非CSCs中的组蛋白标记。我们发现较少的开放染色质与 CSCs与非CSC的抑制性组蛋白标记及H379组蛋白甲基表达增加 CSCs与非CSCs中的转移酶DOT1L。我们的初步数据显示DOT1L抑制剂阻断了茎的生长 功能。FOXK2是ATAC-Seq发现的具有开放启动子的转录因子之一，在CSCs vs. Non-CSCs是DOT1L靶基因，在卵巢CSCs中高表达。这些初步的 观察结果使我们建议解剖DOT1L诱导的H3K79甲基化与癌症之间的联系 通过解决三个主要目标来解决茎和铂的抗性问题。 目的1：确定DOT1L调控的H3K79甲基化是否是口腔鳞状细胞癌的标志和靶点。H3K79 单甲基化、双甲基化和三甲基化将被映射到从OCSCs和非CSCs中提取的染色质 染色质免疫沉淀(CHIP)-测序。Dot-1L抑制剂和Dot-1L击倒对茎的影响 细胞特性和对铂的反应将在体外和体内进行评估。 目的2：确定H3K79甲基化在铂耐药卵巢癌模型和肿瘤中是否发生改变。 成对的亲代和铂耐药细胞系、异种移植和患者来源的异种移植模型(PDX) 本课题组开发的将用于检测DOT1L表达和H3K79甲基化。表达式和 人卵巢肿瘤对铂类药物敏感或持久后DOT1L功能的测定 化疗。遗传和药物抑制策略对铂反应的影响将是 体外和体内研究。 目的3：确定DOT1L靶基因FOXK2在卵巢CSCs中的功能。FOXK2表达式将为 在铂耐药模型与铂天真模型以及在人类卵巢肿瘤中进行测量。敲门敲门- OUT实验将确定其在卵巢CSCs转录调控中的作用。 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方面的初步数据和广泛的专业知识 支持项目的可行性和影响力。我们将用最先进的技术来推动发展 新的表观基因组导向策略，如DOT1L抑制剂，靶向卵巢CSCs。我们久经考验的业绩记录 从工作台到诊所的干预措施将有助于将我们的研究成果转化为临床应用。这样做的结果 该项目将立即适用于卵巢癌，但可能对其他铂类- 耐药实体瘤。总而言之，该项目将弥合对铂耐药恶性肿瘤的知识空白 影响到相当数量的美国退伍军人和美国军人。

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.