Defining and targeting the molecular vulnerabilities of the PAX3-FOXO1 protein in rhabdomyosarcoma

摘要--总体(修订) 融合阳性的肺泡型横纹肌肉瘤(ARM)仍然是最致命但最不为人所知的儿童癌症之一。ARM中的驱动癌蛋白是PAX3-FOXO1融合蛋白，这是一种嵌合转录因子，可以劫持正常的基因表达和染色质状态。患有PAX3-FOXO1阳性手臂的儿童的五年存活率在所有患者中约为30%，在转移时为10%。尽管1993年发现了PAX3-FOXO1，但对受影响儿童的治疗策略仍然没有改变。这一缺陷同样源于对疾病的基本生物学缺乏了解，以及无法直接针对融合蛋白。目前还没有系统或全面的方法来确定支持PAX3-FOXO1介导的肿瘤发生所需的蛋白质和调控元件。因此，该领域一直局限于拼凑而成的数据，没有统一的科学战略。为了克服这一问题，FusonC2中心拥有一个创新的团队和动态的环境，在这个环境中，数据解释通过互补的技术方法以及生物学和临床知识来提供信息。这一综合方法将改变对PAX3-FOXO1介导的肿瘤发生的理解，并为治疗干预创造机会。该中心的首要目标是通过全面鉴定PAX3-FOXO1的可药物共调节因子、调节剂和内在活性，提高PAX3-FOXO1融合蛋白在ARM中的治疗可操作性。为了实现这一目标，该中心包括两个互补的项目，每个项目都由RMS专家生物学家领导，并与从事癌症生物学、基因组学、蛋白质组学、结构生物学和药物化学方面最尖端研究的开创性实验方法专家配对。这些项目将得到每个项目中RMS研究人员的支持，他们将提供经过策划的RMS细胞系、独特的人类原代RMS肿瘤细胞和小鼠模型，以实现目标的快速体外和体内验证和交叉优先排序。行政核心将整合和协调中心各组成部分，提供领导和监督，并促进思想和资源的交叉授粉。总的具体目标是：(1)定义并靶向PAX3-FOXO1相互作用组；以及(2)为PAX3-FOXO1进行化学探针发现，以创造更多的工具来研究PAX3-FOXO1和融合阳性RMS的基础生物学和可操作性。使用的方法包括邻近标记、饱和突变、单一和组合CRISPR筛选、高通量表型分析，以及使用新型高通量结合分析发现化学探针的机械无偏方法。我们将使用收集到的信息来确定验证的目标和试剂的优先顺序，并为化合物优化和PROTAC准备提供信息。该中心的优势和资源将与FusOnC2联盟协同作用，加快可推广到儿童癌症多种融合癌蛋白生物学的知识的发展，加速临床护理的进步。

ABSTRACT – Overall (revised) Fusion-positive alveolar rhabdomyosarcoma (ARMS) remains one of the most fatal but least understood cancers of childhood. The driving oncoprotein in ARMS is the PAX3-FOXO1 fusion protein, a chimeric transcription factor that hijacks normal gene expression and chromatin state. Five-year survival for children with PAX3-FOXO1- positive ARMS is ~30% for all-comers, and <10% when metastatic. Despite the discovery of PAX3-FOXO1 in 1993, treatment strategies for affected children remain unchanged. This deficiency stems equally from a lack of understanding of the basic biology of the disease and an inability to directly target the fusion protein. No systematic or comprehensive approach has been undertaken to identify the proteins and regulatory elements required to support PAX3-FOXO1-mediated tumorigenesis. As a result, the field has been limited to a patchwork of data with no unified scientific strategy. To overcome this, this FusOnC2 Center has an innovative team and dynamic environment in which data interpretation is informed by complementary technological approaches and by biological and clinical knowledge. This comprehensive approach will transform understanding of PAX3-FOXO1-mediated oncogenesis and create opportunities for therapeutic intervention. The Center’s overarching goal is to advance the therapeutic tractability of the PAX3-FOXO1 fusion protein in ARMS by comprehensively identifying the druggable co-regulators, modulators, and intrinsic activities of PAX3-FOXO1. To accomplish this goal, the Center includes two complementary Projects, each led by expert RMS biologists paired with specialists in pioneering experimental approaches undertaking the most cutting-edge research in cancer biology, genomics, proteomics, structural biology, and medicinal chemistry. The Projects will be supported by RMS investigators within each project who will provide curated RMS cell lines, unique human primary RMS tumor cells, and murine models to enable rapid in vitro and in vivo validation and cross-prioritization of targets. An Administrative Core will integrate and coordinate the Center components, providing leadership and oversight, and promoting cross-pollination of ideas and resources. The Overall Specific Aims are to: (1) define and target the PAX3-FOXO1 interactome; and (2) perform chemical probe discovery for PAX3-FOXO1 to create additional tools for investigating the fundamental biology and tractability of PAX3-FOXO1 and fusion-positive RMS. Approaches used include proximity labeling, saturation mutagenesis, single and combinatorial CRISPR screens, high-throughput phenotypic assays, and mechanistically unbiased approaches to chemical probe discovery using novel high-throughput binding assays. We will use information gleaned to prioritize targets and agents for validation and to inform compound optimization and PROTAC preparation. This Center’s strengths and resources will synergize with the FusOnC2 Consortium, speeding development of knowledge generalizable to the biology of multiple fusion oncoproteins in childhood cancers, accelerating advances in clinical care.