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: 10088329
• 负责人: Diana Ye Lu
• 金额: $ 5.1万
• 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/10088329
• 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.

项目概要/摘要 尤文肉瘤 (ES) 是第二常见的儿童骨肿瘤。患者的生存率 转移性疾病的比例仍然很低，为 20-30%，临床需求尚未得到满足。平均而言，儿童癌症 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目标 治疗，这对新药发现构成了主要障碍。事实上，ES 肿瘤表现出极少的致癌性 突变，但特征性 11;22 染色体易位除外，该易位会产生 85-90% 的病例中存在致癌 EWS-FLI 融合转录因子。 EWS-FLI 癌蛋白关闭和打开 染色质位于基因组中的特定基序，引起严重的表观遗传失调。调查ES 生物学上，进行了基因组规模的 CRISPR-Cas9 筛选，鉴定出了转录因子 ETV6， 作为 ES 细胞生存所必需的基因。 ETV6 是一种抑制性转录因子，之前已被证明 通过直接招募组蛋白脱乙酰 (HDAC) 酶 HDAC3 介导基因抑制。 初步实验表明 ETV6 与 EWS- 共享数千个基因组结合位点 ES 细胞中的 FLI，包括与 EWS-FLI 上调基因的结合。此外，ETV6 过度表达会降低 促进组蛋白标记 H3K27ac 的丰度与其之前描述的抑制功能一致。 因此，该提案将解决以下假设：ETV6 通过抑制子集对 ES 细胞存活至关重要 通过招募 HDAC3 来表达 EWS-FLI 诱导的靶基因。目标 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ETV6被抑制时。目标 2 将定义 ETV6 和 EWS-FLI 的转录程序以确定 ETV6 是否抑制对细胞存活和生长有害的 EWS-FLI 靶基因子集。 最后，目标 3 将研究 ETV6 是否通过招募 HDAC3 来抑制 EWS-FLI 靶基因，以及是否 ES 细胞对选择性 HDAC3 抑制剂 RGFP966 敏感。总而言之，这部作品将描绘一部小说 ES 中的重要基因，并强调了 ES 的潜在新治疗方法。

项目成果

The ETS transcription factor ETV6 constrains the transcriptional activity of EWS-FLI to promote Ewing sarcoma.

• DOI: 10.1038/s41556-022-01059-8
• 发表时间: 2023-02
• 期刊: NATURE CELL BIOLOGY
• 影响因子: 21.3
• 作者: Lu, Diana Y. Y.;Ellegast, Jana M.;Ross, Kenneth N.;Malone, Clare F.;Lin, Shan;Mabe, Nathaniel W.;Dharia, Neekesh V.;Meyer, Ashleigh;Conway, Amy;Su, Angela H. H.;Selich-Anderson, Julia;Taslim, Cenny;Byrum, Andrea K.;Seong, Bo Kyung A.;Adane, Biniam;Gray, Nathanael S.;Rivera, Miguel N.;Lessnick, Stephen L.;Stegmaier, Kimberly
• 通讯作者: Stegmaier, Kimberly