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

描述(申请人提供)：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，常以t(2；13)染色体易位为特征，导致两个肌源性转录因子Pax3和FOXO1融合。由于其与FOXO1融合而导致的Pax3转录活性的改变被认为有助于Pax3-FOXO1依赖于对手臂发育至关重要的肌源性分化的抑制。这项建议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，这些机制可能有助于抑制肌肉发生和手臂的发育。我们将对融合蛋白进行解构，以了解野生型转录因子功能的分子调控机制。FOXO1的调控已经得到了广泛的研究，不会进一步讨论。相反，由于对Pax3功能的调节知之甚少，本建议侧重于建立Pax3在正常肌肉发生中调节的分子机制。随后将该模型应用于Pax3-FOXO1，将为理解融合蛋白在抑制肌肉发生和手臂发育中的作用提供关键信息。了解这些机制将有助于确定新的分子靶点，如激酶和磷酸化位点，这些靶点可用于合理开发治疗武器的潜在疗法。利用一种新的半体外激酶分析、二维磷酸肽分析和磷酸特异性抗体，我们证明了酪蛋白激酶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 DNA结合能力，在增殖的原代成肌细胞中Pax3和Pax3-FOXO1在Ser205处被磷酸化，并且这种磷酸化在Pax3上迅速丢失，而不是Pax3-FOXO1。我们假设，在增殖的成肌细胞中，Pax3被CKII和GSK32磷酸化，分化过程中磷酸化的丧失调节其转录活性，对正常的肌肉发生至关重要，分化过程中Pax3-FOXO1的异常磷酸化有助于ARM的发育。我们将通过以下具体目标解决这一假说：(1)确定在原代成肌细胞增殖和分化过程中，磷酸化对Pax3和Pax3-FOXO1转录活性调节的后果。(2)分析CKII和GSK32在体外对Pax3和Pax3-FOXO1的磷酸化作用，并探讨其对原代成肌细胞Pax3和Pax3-FOXO1转录活性的影响。(3)确定Pax3-FOXO1的磷酸化如何调节其与武器相关的活性。公共卫生相关性：肺泡型横纹肌肉瘤(ARMS)是一种儿童侵袭性实体肌肉肿瘤，以致癌融合转录因子Pax3-FOXO1为特征，预后差。了解调控Pax3-FOXO1转录活性的分子机制以及这一调控对ARM发育的贡献将识别新的分子靶点，如激酶和磷酸化位点，这些可用于合理设计药物以创造治疗ARM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.