Mechanisms of Toxicity Induced by EWS/FLI1 Overdose in Ewing Sarcoma

项目摘要/摘要 EWS融合癌蛋白是许多恶性肿瘤的关键驱动因素，包括儿童实体瘤 尤文肉瘤，第二常见的儿童癌症，累及骨骼。就尤因肉瘤而言， EWS与ETS转录因子FLI1的融合产生致癌先锋转录因子 一组独特的调控元件来控制致癌基因的表达。镇压对人民的影响 EWS/FLI1与shRNA、CRISPR或靶向蛋白质降解等方法的融合现在很好 被描述和已知对尤文肉瘤细胞有害。相比之下，EWS/FLI1过量服药的可能性 是新描述的，而且还未得到充分的研究。我们最近发现了一种强大而新颖的选择性 尤文肉瘤对E3泛素连接酶TRIM8的依赖值得注意的是，TRIM8控制着 EWS/FLI1蛋白，其失活导致EWS/FLI1蛋白水平增加，这是 肿瘤细胞。尤文肉瘤细胞因此必须在一个狭窄的“恰到好处”的范围内仔细调节EWS/FLI1的水平。 “金发女孩”系列。太少或太多的融合蛋白都是致命的。控制最优的机制 EWS/FLI1的水平以及高水平的融合蛋白介导的细胞杀伤力尚不清楚。我们预计 定义这些机制将揭示尤因肉瘤生物学的重要原理，这些原理也可能是 适用于其他EWS融合蛋白，这一知识可能指向新的治疗策略。这个 因此，这项建议的总体目标是确定EWS/FLI1在TRIM8后诱导的毒性机制 尤文肉瘤通过三个目标的损失。EWS/FLI1的基因调控特性高度依赖 关于生物分子凝聚体的形成，这反过来又需要EWS的相变性质 无序的普恩样结构域。同样，TRIM8也被描述为在凝析油的形成中发挥作用。因此， 在目标1中，我们将确定TRIM8和EWS/FLI1生物分子凝聚体之间的关系 假设TRIM8对EWS/FLI1调节依赖于凝析油的形成以及反过来的损失 TRIM8的含量会导致凝析油的变化。在目标2中，我们将确定TRIM8缺失对表观遗传学的影响 尤文肉瘤的状态，包括3D相互作用。在目标3中，我们将确定EWS/FLI1 过量服药是有毒的。基于基因表达和ORF筛选数据，我们将通过假设来引导这一目标 谷氨酸受体GRM2介导EWS/FLI1过量给药的下游毒性。新疗法 患者需要不同于传统细胞毒化疗的方法和局部控制措施 与尤因肉瘤一样，几十年来在治疗高危患者方面进展甚微。这些 探索癌基因过量的创新策略的拟议研究具有未来翻译的潜力 除了揭示疾病的基本潜在机制外，还将产生重大影响。

PROJECT SUMMARY/ABSTRACT EWS fusion oncoproteins are critical drivers in a number of malignancies, including the pediatric solid tumor Ewing sarcoma, the second most common pediatric cancer involving bone. In the case of Ewing sarcoma, the fusion of EWS to the ETS transcription factor FLI1 generates an oncogenic pioneer transcription factor that alters a distinctive set of regulatory elements to control oncogenic gene expression. The impact of repression of the EWS/FLI1 fusion with approaches such as shRNA, CRISPR, or targeted protein degradation is now well described and known to be deleterious to Ewing sarcoma cells. In contrast, the potential of EWS/FLI1 overdose is just newly described and has been understudied. We recently discovered a powerful and novel selective dependency in Ewing sarcoma on TRIM8, an E3 ubiquitin ligase. Remarkably, TRIM8 controls the levels of EWS/FLI1 protein, and its inactivation leads to an increase in EWS/FLI1 protein levels that is not tolerated by tumor cells. Ewing sarcoma cells must thus carefully regulate the levels of EWS/FLI1 within a narrow “just right,” “Goldilocks” range. Either too little or too much fusion protein is lethal. The mechanisms that control optimal levels of EWS/FLI1 and that mediate cell lethality from high levels of the fusion protein are unknown. We expect that defining these mechanisms will reveal important principles of Ewing sarcoma biology that may also be applicable to other EWS fusion proteins, and that this knowledge may point to new therapeutic strategies. The overall goal of this proposal is thus to determine the mechanisms of EWS/FLI1 induced toxicity following TRIM8 loss in Ewing sarcoma through three aims. The gene regulation properties of EWS/FLI1 are highly dependent on the formation of biomolecular condensates, which in turn require the phase transition properties of the EWS disordered prion-like domain. Similarly, TRIM8 has been described to have a role in condensate formation. Thus, in Aim 1, we will determine the relationship between TRIM8 and EWS/FLI1 biomolecular condensates with the hypothesis that that EWS/FLI1 regulation by TRIM8 is dependent on condensate formation and that, in turn, loss of TRIM8 leads to changes in condensates. In Aim 2, we will determine the impact of TRIM8 loss on epigenetic states in Ewing sarcoma including 3D interactions. In Aim 3, we will identify the mechanisms by which EWS/FLI1 overdose is toxic. Based on gene expression and ORF screening data, we will lead this aim with the hypothesis that the glutamate receptor GRM2 mediates toxicity downstream of EWS/FLI1 overdose. New therapeutic approaches distinct from traditional cytotoxic chemotherapy and local control measures are needed for patients with Ewing sarcoma as little progress has been made in treating high-risk patients over several decades. These proposed studies exploring an innovative strategy of oncogene overdose have potential for future translational impact in addition to revealing basic underlying mechanisms of disease.