Genomics and epigenomics approaches to characterize novel therapy targets in Ewing sarcoma

尤文肉瘤是一种高度恶性的骨和软组织肿瘤，主要发生在儿童和年轻人的骨盆和长骨，早期转移到肺和骨。近年来，儿科恶性肿瘤的预期存活率有所提高。然而，目前只有三分之二的无转移的ES患者通过多模式治疗方法实现了长期持续缓解。此外，晚期ES的预后更差。在遗传学上，ES是由平衡的染色体EWS/ETS易位定义的，这些易位导致了目前还不能靶向的致癌嵌合转录因子(EWS-ETS)。其他与疾病发展有关的体细胞突变只在很低的频率下被观察到。最近，我们发现EZH2是ES转移发展的重要驱动因素。此外，我们还对初级胚胎干细胞进行了完整的外显子组和基因组测序。我们发现了几个低频率的重复突变基因。值得注意的是，激活FGFR1的点突变，该基因在原发肿瘤中的频繁扩增(31.7%)，以及广泛的表达增加，表明FGFR1的显著活性是该病的重要属性。FGFR1激活的各种机制和与EZH2相关的表观遗传机制指向ES的表观遗传和遗传驱动改变的组合。因此，这项建议的主要目的是描述ES中相关驱动机制的治疗机会。我们将在致死性筛选中将通过基因编辑产生的特定基因敲除与CRISP/Cas9系统相结合，并在ES细胞系中使用不同亚致死剂量的波纳替尼。此外，我们将结合先前确定的ES转移的关键角色的基因敲除，在这样的人工致命性基因缺陷的筛查中。有希望的候选组合将与基因组、表观遗传学和临床数据一起进行评估，其潜在的可药性将在临床前模型系统中进行测试。我们期待对关键的恶性机制有更好的理解，这有助于改善ES的治疗，并开发新的癌症治疗方法。

Ewing sarcoma (ES) is a highly malignant bone and soft tissue neoplasm that arises predominantly in the pelvis and long bones in children and young adults with early metastasis to lung and bone. Survival expectancy increased for pediatric malignancies in recent years. However, only two thirds of ES patients without metastases currently achieve long lasting remissions by multimodal therapy approaches. Moreover, advanced ES has an even worse prognosis. Genetically, ES is defined by balanced chromosomal EWS/ETS translocations, which give rise to oncogenic chimeric transcription factors (EWS-ETS) that are currently not targetable. Other contributing somatic mutations involved in disease development have only been observed at low frequency. Recently, we identified EZH2 as an important driver for metastasis development in ES. Also, we have performed whole exome and genome sequencing in primary ES. We identified several recurrently mutated genes at low frequency. Of interest, activating point mutations of FGFR1, frequent amplification of this locus (31.7%) in primary tumors and widespread increased expression suggested significant FGFR1 activity as an important attribute of this disease. The various mechanisms of FGFR1 activation and the epigenetic mechanisms associated with EZH2 point towards a combination of epigenetic and genetic driver alterations in ES. The main aim of this proposal is therefore to characterize the therapeutic opportunities of relevant driver mechanisms in ES. We will combine specific gene knock outs generated via gene editing with a CRISP/Cas9-based system in a lethality screen together with different sublethal doses of Ponatinib in ES cell lines. Furthermore, we will combine gene knock-outs of previously identified key players of ES metastasis in such a screen for synthetic lethality gene defects. Promising candidate combinations will be evaluated together with genomic, epigenetic and clinical data and their potential druggability will be tested in preclinical model systems. We anticipate a better comprehension of key malignant mechanisms that help to improve therapy for ES, and to develop new therapeutic treatment modalities for cancer in general.