A collaborative approach to analyze and target the EWS-FLI1 transcription factor in patients with Ewing sarcoma

分析和靶向尤文肉瘤患者 EWS-FLI1 转录因子的协作方法

• 批准号: 10219991
• 负责人: Patrick J Grohar
• 金额: $ 35.73万
• 依托单位: SARC
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10219991
• 关键词: Avidity; Biological; Biological Markers; Biology; Bone Tissue; Cell Nucleolus; Cell Nucleus; Cell Survival; Cells; Chromosomal translocation; Clinic; Clinical; Correlative Study; Data; Dependence; Disease model; Dose; Drug resistance; EWS-FLI1 fusion protein; Evaluation; Ewings sarcoma; FLI1 Transcription Factor; Future; Gene Expression; Gene Expression Profiling; Gene set enrichment analysis; Genes; Genetic; Genetic Transcription; Goals; Hour; In Vitro; Infusion procedures; Localized Disease; Manuscripts; Mediating; Messenger RNA; Mutation; NR0B1 gene; Neoplasm Metastasis; Oncogenes; Patients; Pharmaceutical Preparations; Positron-Emission Tomography; Pre-Clinical Model; Preparation; Property; Proteins; Publishing; Recurrence; Recurrent disease; Research Design; Safety; Sampling; Schedule; Series; Serum; Soft tissue sarcoma; TK1 gene; Testing; Therapeutic; Time; Translating; Work; Xenograft Model; actionable mutation; chemotherapy; collaborative approach; genetic signature; imaging biomarker; improved; in vivo; inhibitor/antagonist; insight; irinotecan; multimodality; novel; patient derived xenograft model; phase 1 designs; pre-clinical; preclinical study; programs; promoter; protein expression; response; t(11; 22)(q24; q12); therapeutic target; transcription factor; transcriptome; transcriptome sequencing; tumor; tumor growth; tumorigenesis

Project Abstract It has been known for more than 25 years that Ewing sarcoma cells are absolutely dependent on the EWS- FLI1 transcription factor for cell survival. EWS-FLI1 is a dysregulated transcription factor formed by the t(11;22)(q24;q12) chromosomal translocation. This fusion oncogene alters the expression of over 500 genes to establish a transcriptional program responsible for tumorigenesis and progression. Despite this known dependence, the clinical realization of an EWS-FLI1 inhibitor has not been achieved. We have previously shown that trabectedin interferes with EWS-FLI1 activity. We showed that the drug blocks expression of EWS- FLI1 downstream targets, including the very specific target gene NR0B1. We demonstrated reversal of expression at the promoter, mRNA, and protein levels both in vitro and in vivo in xenograft models of the disease. In addition, we showed that the drug reverses the gene signature of EWS-FLI1 using gene expression profiling and Gene Set Enrichment Analysis (GSEA). We subsequently determined the mechanism of EWS- FLI1 suppression. Treatment of Ewing sarcoma cells with trabectedin redistributes EWS-FLI1 within the nucleus to the nucleolus. Importantly, this redistribution of EWS-FLI1 is extremely concentration dependent, requiring relatively high, but clinically achievable concentrations of the drug. In addition, irinotecan can both amplify and sustain the trabectedin mediated block of EWS-FLI1 activity. Therefore, the goal of this study is to use the combination of trabectedin and irinotecan to achieve the therapeutic suppression of EWS-FLI1. In order to accomplish this, in aim 1, we will establish the optimal dose and schedule of administration of trabectedin in combination with low dose irinotecan. We will use a traditional 3 + 3 phase I design and administer trabectedin as a 1-hour infusion in order to achieve the highest serum concentration of drug possible. We will then perform a limited dose escalation of irinotecan to both amplify and sustain suppression of EWS-FLI1. In aim 2, we will determine if we can use 18F-FLT as a biomarker of EWS-FLI1 suppression. We have demonstrated that silencing of EWS-FLI1 suppresses expression of the proteins responsible for 18F-FLT activity, ENT1, ENT2 and TK1. This results in a loss of PET avidity of Ewing sarcoma cells with EWS-FLI1 suppression in preclinical models of the disease. In this study, we will determine if these effects translate to patients. Finally, in aim 3, we will do a series of correlative studies to characterize the EWS-FLI1 transcriptome for the first time in patients. We will perform both single cell and bulk tumor RNA sequencing to identify the putative EWS-FLI1 targets for the first time in the clinic. We will organize the gene signatures into transcriptional networks, compare the results to preclinical data and establish a series of Patient Derived Xenografts (PDXs). This will provide insight into mechanisms of tumorigenesis and drug resistance. In summary, if the goals of this study are achieved, we will achieve the therapeutic suppression of EWS-FLI1, clinical evidence of suppression and the first evaluation of the EWS-FLI1 transcriptome in the clinic.

项目摘要 25年前就知道尤文肉瘤细胞绝对依赖于EWS- FLI 1转录因子对细胞存活的影响。EWS-FLI 1是一种失调的转录因子， t（11;22）（q24;q12）染色体易位。这种融合癌基因改变了500多个基因的表达 建立一个负责肿瘤发生和发展的转录程序。尽管这是已知的 由于EWS-FLI 1抑制剂的依赖性，尚未实现EWS-FLI 1抑制剂的临床实现。我们先前已经 显示曲贝替丁干扰EWS-FLI 1活性。我们发现，药物阻断EWS的表达- FLI 1下游靶点，包括非常特异的靶基因NR 0 B1。我们证明了 在体外和体内在启动子、mRNA和蛋白质水平的表达， 疾病此外，我们发现，药物逆转EWS-FLI 1的基因签名使用基因表达 分析和基因集富集分析（GSEA）。我们随后确定了EWS的机制- FLI 1抑制。用Trabectedin处理尤文肉瘤细胞使EWS-FLI 1在肿瘤细胞内重新分布。 核到核仁。重要的是，EWS-FLI 1的这种重新分布是高度浓度依赖性的， 需要相对高但临床上可达到的药物浓度。此外，伊立替康可以同时 放大并维持Trabectedin介导的EWS-FLI 1活性阻断。因此，本研究的目的是 使用曲贝替丁和伊立替康的组合来实现EWS-FLI 1的治疗性抑制。在 为了实现这一点，在目标1中，我们将建立最佳剂量和给药方案， 曲贝替定与低剂量伊立替康组合。我们将采用传统的3 + 3一期设计， 以1小时输注的形式施用曲贝替定以达到药物的最高血清浓度 可能然后，我们将进行伊立替康的有限剂量递增，以放大和维持抑制 EWS-FLI1。在目标2中，我们将确定是否可以使用18F-FLT作为EWS-FLI 1抑制的生物标志物。我们 已经证明EWS-FLI 1的沉默抑制了负责18F-FLT的蛋白质的表达 活动，ENT 1，ENT 2和TK 1。这导致尤文肉瘤细胞与EWS-FLI 1的PET亲合力丧失 该疾病的临床前模型中的抑制。在这项研究中，我们将确定这些影响是否转化为 患者最后，在目标3中，我们将对EWS-FLI 1进行一系列相关的研究 这是第一次在患者身上进行转录组。我们将进行单细胞和大量肿瘤RNA测序， 在临床上首次确定推定的EWS-FLI 1靶点。我们将把这些基因特征组织成 转录网络，将结果与临床前数据进行比较，并建立一系列患者来源的 异种移植物（PDX）。这将为深入了解肿瘤发生和耐药性的机制提供帮助。在 总之，如果本研究的目标得以实现，我们将实现EWS-FLI 1的治疗性抑制， 抑制的临床证据和EWS-FLI 1转录组在临床上的首次评估。