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

项目摘要 人们已经知道尤文肉瘤细胞绝对依赖于EWS-25年。 FLI1转录因子对细胞存活的影响。EWS-FLI1是由EWS-FLI1形成的一种转录因子。 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。尽管如此，众所周知 依赖，EWS-FLI1抑制剂的临床实现尚未实现。我们之前已经 结果表明，Trabectedin干扰EWS-FLI1的活性。我们发现该药物可阻断EWS-1的表达。 FLI1下游靶基因，包括非常特异的靶基因NR0B1。我们演示了反转 在体外和体内异种移植模型中启动子、mRNA和蛋白水平的表达 疾病。此外，我们还表明，该药物通过基因表达逆转了EWS-FLI1的基因签名 基因图谱和基因集浓缩分析(GSEA)。我们随后确定了EWS的机制- FLI1抑制。小梁蛋白治疗尤文肉瘤细胞后EWS-FLI1在细胞内的重新分布 从核到核仁。重要的是，EWS-FLI1的这种重新分布非常依赖于浓度， 需要相对较高的药物浓度，但临床上可以达到。此外，伊立替康既可以 扩增并维持Trabectedin介导的EWS-FLI1活性的阻断。因此，这项研究的目标是 联合应用曲贝替丁和伊立替康实现对EWS-FLI1的治疗抑制。在……里面 为了实现这一目标，在目标1中，我们将制定最佳剂量和给药时间表 曲贝替丁联合小剂量伊立替康。我们将使用传统的3+3阶段I设计， 静脉滴注曲贝替丁1小时，以达到最高血药浓度 有可能。然后，我们将对伊立替康进行有限剂量的升级，以扩大和维持抑制 EWS-FLI1。在目标2中，我们将确定是否可以使用18F-Flt作为EWS-FLI1抑制的生物标志物。我们 已经证明沉默EWS-FLI1可以抑制18F-Flt相关蛋白的表达 Activity、ENT1、ENT2和TK1。这导致EWS-FLI1对尤文肉瘤细胞的PET亲和力丧失 在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会转化为 病人。最后，在目标3中，我们将做一系列相关研究来表征EWS-FLI1 转录组在患者中的首次应用。我们将同时进行单细胞和整体肿瘤RNA测序 首次在临床上鉴定可能的EWS-FLI1靶点。我们将把基因签名组织成 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(PDX)。这将为深入了解肿瘤发生和耐药的机制提供依据。在……里面 综上所述，如果本研究的目标得以实现，我们将实现对EWS-FLI1的治疗抑制， 抑制EWS-FLI1转录组的临床证据和临床首次评估。

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.