Mechanism of regulation for the oncogenic Pax3-FOXO1 in Alveolar Rhabdomyosarcoma

描述（由申请人提供）：腺泡状横纹肌肉瘤（ARMS）是一种侵袭性儿童实体肌肉肿瘤，预后不良，通常以t（2;13）染色体易位为特征，导致两种肌源性转录因子Pax 3和FOXO 1融合。Pax 3与FOXO 1融合导致的Pax 3转录活性的改变被认为有助于Pax 3-FOXO 1依赖性抑制肌源性分化，这对ARMS的发展很重要。该提案的目标是阐明调节致癌Pax 3-FOXO 1融合蛋白的转录活性的分子机制，该融合蛋白可能有助于抑制肌生成和ARMS的发展。我们将“解构”融合蛋白，以了解野生型转录因子的功能调节的分子机制。FOXO 1的调节已被广泛研究，将不再进一步讨论。相比之下，由于很少有人知道的Pax 3的功能的调节，这个建议的重点是建立一个分子机制，在正常的肌肉发生的Pax 3的调节。该模型的后续应用Pax 3-FOXO 1将提供关键信息，以了解融合蛋白的作用，抑制肌生成和发展的ARMS。了解这些机制将允许识别新的分子靶点，如激酶和磷酸化位点，这些靶点可用于合理开发治疗ARMS的潜在疗法。利用一种新的半体外激酶分析、二维磷酸肽分析和磷酸化特异性抗体，我们证明酪蛋白激酶II（CKII）和糖原合成酶激酶32（GSK 32）在体外磷酸化Pax 3和Pax 3-FOXO 1有助于Pax 3 DNA结合能力，Pax 3和Pax 3-FOXO 1在增殖的原代成肌细胞中在Ser 205处磷酸化，并且在诱导肌源性分化时，这种磷酸化在Pax 3上而不是Pax 3-FOXO 1上迅速丧失。我们假设Pax 3在增殖的成肌细胞中被CKII和GSK 32磷酸化，在分化过程中磷酸化的丧失调节其对正常肌生成重要的转录活性，并且在分化过程中Pax 3-FOXO 1的异常磷酸化有助于ARMS的发展。我们将通过以下具体目标来解决这一假设：（1）确定磷酸化对增殖和分化的原代成肌细胞中Pax 3和Pax 3-FOXO 1转录活性调节的后果。(2)在体外分析CKII和GSK 32对Pax 3和Pax 3-FOXO 1的磷酸化，并研究这些激酶对原代成肌细胞中Pax 3和Pax 3-FOXO 1转录活性的影响。(3)确定Pax 3-FOXO 1的磷酸化如何调节其与ARMS相关的活性。公共卫生相关性：腺泡状横纹肌肉瘤（ARMS）是一种侵袭性的儿童实体肌肉肿瘤，预后不良，其特征是致癌融合转录因子Pax 3-FOXO 1。了解调节Pax 3-FOXO 1转录活性的分子机制以及这种调节对ARMS发展的贡献，将确定新的分子靶点，如激酶和磷酸化位点，这些靶点可用于药物的合理设计，以创造治疗ARMS的新疗法。

DESCRIPTION (provided by applicant): Alveolar Rhabdomyosarcoma (ARMS), an aggressive childhood solid muscle tumor with a poor prognosis, is frequently characterized by a t(2;13) chromosomal translocation resulting in the fusion of two myogenic transcription factors, Pax3 and FOXO1. Alterations in Pax3 transcriptional activity resulting from its fusion to FOXO1 are believed to contribute to the Pax3-FOXO1-dependent inhibition of myogenic differentiation important for the development of ARMS. The goal of this proposal is to elucidate the molecular mechanisms regulating the transcriptional activities of the oncogenic Pax3-FOXO1 fusion protein that may contribute to the inhibition of myogenesis and the development of ARMS. We will "deconstruct" the fusion protein to understand the molecular mechanisms regulating the functions of the wild-type transcription factors. The regulation of FOXO1 has been extensively studied and will not be discussed further. In contrast, since very little is known about the regulation of the functions of Pax3, this proposal focuses on establishing a molecular mechanism for the regulation of Pax3 in normal myogenesis. The subsequent application of this model to Pax3-FOXO1 will provide critical information to understand the role of the fusion protein in the inhibition of myogenesis and the development of ARMS. Understanding these mechanisms will allow the identification of novel molecular targets, such as kinases and sites of phosphorylation, which can be exploited for the rational development of potential therapies for the treatment of ARMS. Using a novel semi-in vitro kinase assay, two-dimensional phosphopeptide analysis, and a phospho-specific antibody, we demonstrate that casein kinase II (CKII) and glycogen synthase kinase 32 (GSK32) phosphorylate Pax3 and Pax3-FOXO1 in vitro contributing to Pax3 DNA binding ability, Pax3 and Pax3-FOXO1 are phosphorylated at Ser205 in proliferating primary myoblasts, and that this phosphorylation is rapidly lost on Pax3 but not Pax3-FOXO1 upon the induction of myogenic differentiation. We hypothesize that Pax3 is phosphorylated by CKII and GSK32 in proliferating myoblasts that the loss of phosphorylation during differentiation regulates its transcriptional activity important for normal myogenesis, and the aberrant phosphorylation of Pax3-FOXO1 during differentiation contributes to the development of ARMS. We will address this hypothesis through the following specific aims: (1) Determine the consequences of phosphorylation on the regulation of the transcriptional activities of Pax3 and Pax3-FOXO1 in proliferating and differentiating primary myoblasts. (2) Analyze the phosphorylation of Pax3 and Pax3-FOXO1 by CKII and GSK32 in vitro and investigate the effects of these kinases on Pax3 and Pax3-FOXO1 transcriptional activities in primary myoblasts. (3) Determine how phosphorylation of Pax3-FOXO1 regulates its activity as it relates to ARMS. PUBLIC HEALTH RELEVANCE: Alveolar Rhabdomyosarcoma (ARMS) is an aggressive childhood solid muscle tumor with a poor prognosis that is characterized by the oncogenic fusion transcription factor Pax3-FOXO1. Understanding the molecular mechanisms regulating the transcriptional activities of Pax3-FOXO1 and the contributions this regulation makes to the development of ARMS will identify novel molecular targets, such as kinases and sites of phosphorylation, which can be exploited for the rational design of drugs to create novel therapies for the treatment of ARMS.