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

描述（由申请人提供）：肺泡横纹肌肉瘤（Alveolar Rhabdomyosarcoma， ARMS）是一种侵袭性儿童期实体肌肉肿瘤，预后较差，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Pax3和FOXO1融合。Pax3与FOXO1融合导致的转录活性改变被认为有助于Pax3-FOXO1依赖性的肌源分化抑制，这对ARMS的发展很重要。本研究的目的是阐明调控致癌Pax3-FOXO1融合蛋白转录活性的分子机制，该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，以了解调节野生型转录因子功能的分子机制。fox01的调控已被广泛研究，不再进一步讨论。相比之下，由于对Pax3的功能调控知之甚少，因此本提案的重点是建立Pax3在正常肌生成中的调控分子机制。该模型在Pax3-FOXO1中的后续应用将为了解融合蛋白在抑制肌生成和ARMS发展中的作用提供关键信息。了解这些机制将有助于识别新的分子靶点，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。利用一种新型的半体外激酶试验、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 （GSK32）在体外磷酸化Pax3和Pax3- foxo1，有助于Pax3的DNA结合能力，Pax3和Pax3- foxo1在增殖的原代成肌细胞中被磷酸化在205位点，并且这种磷酸化在Pax3上迅速丢失，而在诱导成肌分化时Pax3- foxo1则不会。我们假设Pax3在增殖的成肌细胞中被CKII和GSK32磷酸化，分化过程中磷酸化的缺失调节了其对正常肌肉形成至关重要的转录活性，而Pax3- foxo1在分化过程中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Pax3和Pax3- foxo1在原代成肌细胞增殖和分化中的转录活性调控的影响。(2)体外分析CKII和GSK32对Pax3和Pax3- foxo1的磷酸化作用，探讨这些激酶对原代成肌细胞中Pax3和Pax3- foxo1转录活性的影响。(3)确定Pax3-FOXO1磷酸化如何调节其与ARMS相关的活性。公共卫生相关性：肺泡横纹肌肉瘤（Alveolar Rhabdomyosarcoma， ARMS）是一种侵袭性儿童实体肌肉肿瘤，预后较差，其特征是致癌融合转录因子Pax3-FOXO1。了解调控Pax3-FOXO1转录活性的分子机制，以及这种调控对ARMS发展的贡献，将有助于发现新的分子靶点，如激酶和磷酸化位点，这些靶点可用于合理设计药物，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.