Pilot Project Investigating the PAX3-FOXO1 Protein in the Rare Disease Rhabdomyosarcoma

摘要 罕见的儿童肌肉癌泡状横纹肌肉瘤(ARM)通常在以下情况下无法存活 转移性的。然而，ARMS通常最初在临床上对化疗有反应。关于该事业 对于复发，临床医生观察到易位介导的PAX3：Foxo1嵌合蛋白存在于大多数 ARM病例调解了治疗耐药性，导致10年生存率下降45%--这不是临床结果 与不太常见的PAX7：Foxo1蛋白有关。Pax3、PAX7和FOXO1都是单独的密钥 正常肌肉、大脑和心脏发育中的转录因子蛋白--尚未完全研究。这个 Pax3/7：Foxo1融合蛋白的特性更差。我们假设有选择性的小的 这些嵌合转录因子的分子抑制物可以被设计成探针来揭示这些 蛋白质的分子功能、表观遗传功能、细胞功能和体内功能。因此，我们的目标是：(1)发展小企业 PAX3-FOXO1和PAX7：Foxo1的分子抑制剂作为工具化合物。我们的方法是建立 Pax同源结构域和配对结构域转录报告在各种细胞模型系统中的分析 不同的表观遗传环境(骨骼肌成肌细胞、心肌细胞、RMS、对照成纤维细胞和神经元 细胞)；反筛选不相关的转录反应元件靶标(例如，NFkB)；进行表面处理 重组野生型和突变型PAX3：Foxo1或 PAX7：Foxo1蛋白；并进行臂细胞隔室分级以确定PROTAC相容 核E3连接酶用于PAX3/7-FOXO1的PROTAC工具的生成。通过这些研究，我们希望 促进PAX3/7的研究：Foxo1作为转录因子发挥作用，而不一定干扰复合体 (通过小分子抑制剂)，并允许实时研究表观遗传蛋白质功能，而不需要二次 效果(通过最终的PROTAC工具)。

SUMMARY The rare childhood muscle cancer alveolar rhabdomyosarcoma (ARMS) is generally not survivable when metastatic. However, ARMS does usually respond clinically to chemotherapy initially. With respect to the cause of recurrences, clinicians observe that the translocation-mediated PAX3:FOXO1 chimeric protein present in most ARMS cases mediates treatment resistance, causing a 45% drop in 10-year survival – a clinical outcome not associated with the less common PAX7:FOXO1 protein. PAX3, PAX7 and FOXO1 are all individually key transcription factor proteins in normal muscle, brain and heart development – yet incompletely studied. The PAX3/7:FOXO1 fusion proteins are even less well characterized. We hypothesize that selective small molecule inhibitors of these chimeric transcription factor can be designed as probes to uncover these proteins' molecular, epigenetic, cellular and in vivo functions. Thus, our aim is to: (1) Develop small molecule inhibitors of PAX3-FOXO1 and PAX7:FOXO1 as tool compounds. Our approach is to establish PAX homeodomain and paired domain transcriptional reporter assays in a variety of cell model systems with different epigenetic contexts (skeletal muscle myoblasts, cardiomyocytes, RMS, control fibroblasts and neuronal cells); counter-screen for an unrelated transcriptional response element target (e.g., NFkB); perform surface plasmon resonance affinity testing of compound hits with recombinant wildtype and mutant PAX3:FOXO1 or PAX7:FOXO1 proteins; and perform ARMS cell compartment fractionation to define PROTAC-compatible nuclear E3 ligases towards generation of a PROTAC tool for PAX3/7-FOXO1. From these studies, we hope to facilitate the study of PAX3/7:FOXO1 function as transcription factors without necessarily disturbing complexes (via small molecule inhibitors), and to allow study of epigenetic protein function in real time without secondary effects (via eventual PROTAC tools).