Characterizing ETV6 as a regulator of EWS-FLI oncoprotein in Ewing Sarcoma

将 ETV6 表征为尤文肉瘤中 EWS-FLI 癌蛋白的调节因子

• 批准号: 9905767
• 负责人: Diana Ye Lu
• 金额: $ 3.7万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9905767
• 关键词: Address; Adult; Apoptosis; Binding; Binding Sites; Biological Assay; Biology; Bone neoplasms; CRISPR screen; CRISPR/Cas technology; Cancer cell line; Cell Cycle Arrest; Cell Line; Cell Proliferation; Cell Survival; Cells; ChIP-seq; Characteristics; Chemicals; Childhood; Chimeric Proteins; Chromatin; Chromosomal translocation; Clinical; Data; Dependence; Development; Disease; ETV6 gene; EWSR1 gene; Enhancers; Enzymes; Epigenetic Process; Essential Genes; Ewings sarcoma; Exhibits; FLI1 gene; Family; Frequencies; Fusion Oncogene Proteins; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomic approach; Genomics; Growth; HDAC3 gene; Histone Deacetylase; Histones; Impairment; In Vitro; Knock-out; Malignant Childhood Neoplasm; Malignant Neoplasms; Mediating; Modeling; Molecular; Mus; Mutation; Oncogenic; Oncoproteins; Patients; Pediatric Neoplasm; Process; Recurrent disease; Regulation; Site; Survival Rate; System; Testing; Therapeutic; Work; bone; cancer cell; cancer type; cell growth; drug discovery; experimental study; gene repression; genome-wide; in vivo; inhibitor/antagonist; member; novel; novel therapeutic intervention; novel therapeutics; overexpression; programs; recruit; subcutaneous; targeted treatment; transcription factor; transcriptome sequencing; tumor; tumor growth; tumorigenesis

PROJECT SUMMARY/ABSTRACT Ewing sarcoma (ES) is the second most common pediatric tumor involving bone. Survival rates for patients with metastatic disease remain dismal at 20-30%, presenting an unmet clinical need. Pediatric cancers, on average, exhibit lower mutational burdens compared to adult cancers, concealing disease biology and potential targets for treatment, posing a major barrier to novel drug discovery. Indeed, ES tumors exhibit extremely few oncogenic mutations, with the exception of the characteristic 11;22 chromosomal translocation, which creates the oncogenic EWS-FLI fusion transcription factor in 85-90% of cases. The EWS-FLI oncoprotein closes and opens chromatin at specific motifs in the genome, inducing profound epigenetic dysregulation. To investigate ES biology, a genome-scale CRISPR-Cas9 screen was performed, which identified the transcription factor, ETV6, as a gene essential for ES cell survival. ETV6 is a repressive transcription factor that has previously been shown to mediate gene repression through the direct recruitment of the histone deacetylating (HDAC) enzyme, HDAC3. Preliminary experiments have demonstrated that ETV6 shares thousands of genomic binding sites with EWS- FLI in ES cells, including binding at EWS-FLI-up-regulated genes. Furthermore, ETV6 over-expression reduces abundance of the promotive histone mark, H3K27ac, consistent with its previously described repressive function. Thus, this proposal will address the hypothesis that ETV6 is essential for ES cell survival by repressing a subset of EWS-FLI-induced target genes via the recruitment of HDAC3. Aim 1 will assess the essentiality of this gene in models of ES in vitro and in vivo and elucidate the molecular mechanism of decreased survival of ES cells when ETV6 is suppressed. Aim 2 will define the transcriptional programs of ETV6 and EWS-FLI to determine whether ETV6 represses a subset of EWS-FLI target genes that are deleterious for cell survival and growth. Finally, Aim 3 will investigate whether ETV6 represses EWS-FLI target genes by recruiting HDAC3 and whether ES cells are sensitive to the selective HDAC3 inhibitor, RGFP966. Altogether, this work will characterize a novel essential gene in ES and highlight a potential novel therapeutic approach for ES.

项目总结/摘要 尤因肉瘤（ES）是第二常见的儿科骨肿瘤。患者的生存率 转移性疾病仍令人沮丧地保持在20- 30%，这表示临床需求未得到满足。儿童癌症，平均来说， 与成人癌症相比，表现出较低的突变负担，隐藏了疾病生物学和潜在靶点 这是新药研发的主要障碍。事实上，ES肿瘤几乎没有致癌性 突变，除了特征性的11;22染色体易位，这导致了 致癌EWS-FLI融合转录因子在85-90%的情况下。EWS-FLI癌蛋白关闭和打开 在基因组中的特定基序的染色质，诱导深刻的表观遗传失调。研究ES 在生物学方面，进行了基因组规模的CRISPR-Cas9筛选，其鉴定了转录因子ETV 6， 作为胚胎干细胞生存所必需的基因。ETV 6是一种抑制性转录因子， 通过组蛋白去乙酰化（HDAC）酶HDAC 3的直接募集来介导基因抑制。 初步实验表明，ETV 6与EWS共享数千个基因组结合位点， ES细胞中的FLI，包括结合EWS-FLI上调基因。此外，ETV 6过表达降低了 丰富的促进性组蛋白标记，H3 K27 ac，与其先前描述的抑制功能一致。 因此，该提议将解决ETV 6通过抑制一个亚群而对ES细胞存活至关重要的假设 EWS-FLI诱导的靶基因通过HDAC 3的募集。目的1将评估该基因的重要性 在体外和体内ES模型中，阐明ES细胞存活率降低的分子机制 当ETV 6被抑制时。目的2将定义ETV 6和EWS-FLI的转录程序，以确定 ETV 6是否抑制对细胞存活和生长有害的EWS-FLI靶基因的子集。 最后，目的3将研究ETV 6是否通过募集HDAC 3来抑制EWS-FLI靶基因，以及是否 ES细胞对选择性HDAC 3抑制剂RGFP 966敏感。总而言之，这部作品将成为一部 ES的必需基因，并强调了ES的潜在新治疗方法。