Gene expression signature based screening in Ewing sarcoma

项目总结 尤文肉瘤是一种由EWS-FLI1融合引起的高度侵袭性的骨和软组织癌症。 蛋白。EWS-FLI1癌蛋白的部分功能是作为一种异常转录因子，是 肿瘤的生长和存活率。然而，直接以药物靶向EWS-FLI1一直是具有挑战性的，并且作为一种 因此，迫切需要确定EWS-FLI1的下游目标和引发的独特漏洞 由癌蛋白引起的。为了确定EWS-FLI1的下游靶点，我们使用了人类胚胎干细胞 表达可诱导的EWS-FLI1来模拟尤文肉瘤的发生和发展 定义的系统。然后，我们使用这个模型系统和基于基因表达的方法来鉴定尤因 肉瘤细胞对核糖核苷酸还原酶(RNR)抑制剂特别脆弱，包括吉西他滨， 它通过阻止脱氧核糖核苷酸的合成来损害DNA复制。此外，我们还 证实RNR在尤文肉瘤细胞中的抑制导致复制应激，激活未折叠 蛋白质反应，以及对受损的DNA反应所需的蛋白质合成受阻 复制。值得注意的是，吉西他滨与检查点激酶1抑制剂(CHK1)的组合，主要是 对DNA复制受损的反应的调节，导致体外协同作用和显著延长 小鼠在异种移植实验中的存活率。在强劲的初步数据指引下，我们现在将继续 具体目标如下：1)剖析未折叠蛋白激活和调控的分子基础 核糖核苷酸还原酶抑制剂对尤文肉瘤细胞的影响2)体内疗效检测 吉西他滨和第二代CHK1抑制剂prexasertib，使用尤文肉瘤异种移植模型； 以及3)使用高通量筛选来测试使用我们的基因表达确定的候选疗法 基于抗尤文肉瘤细胞活性的方法。这些目标将使用以下组合进行测试 癌细胞系、细胞系异种移植、患者来源的异种移植以及我们的同基因异种移植的方法 已定义的细胞系。这项工作将具有重要意义，因为预计它将与 儿童和成人尤文肉瘤的治疗，以及导致更广泛的理解 尤文肉瘤肿瘤发生的基本机制。这项拟议的研究具有创新性，因为它 将尤文肉瘤的新型干细胞模型与基于基因表达特征的筛查相结合 寻找新的治疗靶点的方法。

PROJECT SUMMARY Ewing sarcoma is a highly aggressive bone and soft tissue cancer that is caused by the EWS-FLI1 fusion protein. The EWS-FLI1 oncoprotein functions, in part, as an aberrant transcription factor and is required for tumor growth and survival. However, directly targeting EWS-FLI1 with drugs has been challenging and, as a result, there is a critical need to identify downstream targets of EWS-FLI1 and unique vulnerabilities incurred by the oncoprotein. In order to identify downstream targets of EWS-FLI1, we used human embryonic stem cells that express inducible EWS-FLI1 to model the initiation and development of Ewing sarcoma in a genetically defined system. We then used this model system and a gene expression based approach to identify that Ewing sarcoma cells are uniquely vulnerable to inhibitors of ribonucleotide reductase (RNR), including gemcitabine, which impair DNA replication by blocking the synthesis of deoxyribonucleotides. Moreover, we have also identified that the inhibition of RNR in Ewing sarcoma cells results replication stress, activation of the unfolded protein response, and a block in the synthesis of proteins required for the response to impaired DNA replication. Notably, the combination of gemcitabine with an inhibitor of checkpoint kinase 1 (CHK1), the major regulator of the response to impaired DNA replication, results in synergy in vitro and a significant prolongation of mouse survival in xenograft experiments. Guided by strong preliminary data, we will now pursue the following specific aims: 1) dissect the molecular basis of the activation and regulation of the unfolded protein response in Ewing sarcoma cells treated with inhibitors of ribonucleotide reductase; 2) test the in vivo efficacy of gemcitabine and a second-generation CHK1 inhibitor, prexasertib, using Ewing sarcoma xenograft models; and 3) use high-throughput screening to test candidate therapeutics identified using our gene expression based approach for activity against Ewing sarcoma cells. These aims will be tested using a combination of approaches in cancer cell lines, cell line xenografts, patient-derived xenografts, and our isogenic, genetically defined cell lines. This work will be significant because it is expected to have translational relevance for the treatment of children and adults with Ewing sarcoma tumors, as well as lead to a broader understanding of the basic mechanisms of tumorigenesis in Ewing sarcoma tumors. The proposed research is innovative because it integrates a novel, stem cell model of Ewing sarcoma with a gene expression signature based screening approach to identify new therapeutic targets.