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.
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.
|Loupe, Jacob M; Miller, Patrick J; Crabtree, Judy S et al. (2017) Acquisition of an oncogenic fusion protein is sufficient to globally alter the landscape of miRNA expression to inhibit myogenic differentiation. Oncotarget 8:87054-87072|
|Loupe, Jacob M; Miller, Patrick J; Bonner, Benjamin P et al. (2016) Acquisition of an oncogenic fusion protein serves as an initial driving mutation by inducing aneuploidy and overriding proliferative defects. Oncotarget 7:62814-62835|
|Iyengar, Aditi S; Miller, Patrick J; Loupe, Jacob M et al. (2014) Phosphorylation of PAX3 contributes to melanoma phenotypes by affecting proliferation, invasion, and transformation. Pigment Cell Melanoma Res 27:846-8|
|Iyengar, Aditi S; Loupe, Jacob M; Miller, Patrick J et al. (2012) Identification of CK2 as the kinase that phosphorylates Pax3 at Ser209 in early myogenic differentiation. Biochem Biophys Res Commun 428:24-30|
|Dietz, Kevin N; Miller, Patrick J; Iyengar, Aditi S et al. (2011) Identification of serines 201 and 209 as sites of Pax3 phosphorylation and the altered phosphorylation status of Pax3-FOXO1 during early myogenic differentiation. Int J Biochem Cell Biol 43:936-45|
|Sidhu, Alpa; Miller, Patrick J; Hollenbach, Andrew D (2010) Isolation of putative FOXO1 genomic elements using an improved in vitro technique to isolate genomic regulatory sequences. Gene 458:45-53|
|Dietz, Kevin N; Miller, Patrick J; Hollenbach, Andrew D (2009) Phosphorylation of serine 205 by the protein kinase CK2 persists on Pax3-FOXO1, but not Pax3, throughout early myogenic differentiation. Biochemistry 48:11786-95|
|Miller, Patrick J; Dietz, Kevin N; Hollenbach, Andrew D (2008) Identification of serine 205 as a site of phosphorylation on Pax3 in proliferating but not differentiating primary myoblasts. Protein Sci 17:1979-86|
|Miller, Patrick J; Hollenbach, Andrew D (2007) The oncogenic fusion protein Pax3-FKHR has a greater post-translational stability relative to Pax3 during early myogenesis. Biochim Biophys Acta 1770:1450-8|