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

Summary

Principal Investigator: ANDREW DURRELL HOLLENBACH
Abstract: 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.
Funding Period: 2009-06-01 - 2015-03-31
more information: NIH RePORT

Top Publications

  1. pmc The oncogenic fusion protein Pax3-FKHR has a greater post-translational stability relative to Pax3 during early myogenesis
    Patrick J Miller
    Department of Genetics, Louisiana State University Health Sciences Center, 533 Bolivar Street, New Orleans, LA 70112, USA
    Biochim Biophys Acta 1770:1450-8. 2007
  2. pmc Identification of serine 205 as a site of phosphorylation on Pax3 in proliferating but not differentiating primary myoblasts
    Patrick J Miller
    Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA
    Protein Sci 17:1979-86. 2008
  3. pmc Phosphorylation of serine 205 by the protein kinase CK2 persists on Pax3-FOXO1, but not Pax3, throughout early myogenic differentiation
    Kevin N Dietz
    Department of Genetics, Louisiana State University Health Sciences Center, 533 Bolivar Street, New Orleans, Louisiana 70112, USA
    Biochemistry 48:11786-95. 2009
  4. pmc Isolation of putative FOXO1 genomic elements using an improved in vitro technique to isolate genomic regulatory sequences
    Alpa Sidhu
    Department of Genetics, Louisiana State University Health Sciences Center, 533 Bolivar Street, CSRB 6th floor, New Orleans, LA 70112, USA
    Gene 458:45-53. 2010
  5. pmc Identification of serines 201 and 209 as sites of Pax3 phosphorylation and the altered phosphorylation status of Pax3-FOXO1 during early myogenic differentiation
    Kevin N Dietz
    Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA
    Int J Biochem Cell Biol 43:936-45. 2011
  6. pmc Identification of CK2 as the kinase that phosphorylates Pax3 at Ser209 in early myogenic differentiation
    Aditi S Iyengar
    Department of Genetics, Louisiana State University Health Sciences Center, 533 Bolivar Street, CSRB 6th floor, New Orleans, LA 70112, USA
    Biochem Biophys Res Commun 428:24-30. 2012

Research Grants

  1. Novel Compounds to Inactivate Oncogenic Fusion Proteins
    Jeffrey A Toretsky; Fiscal Year: 2013
  2. Elucidating the role of NF-kB signaling in rhabdomyocarcoma
    Charles Keller; Fiscal Year: 2013
  3. Signaling in Inflammation, Stress, and Tumorigenesis
    GEORGE ROBERT STARK; Fiscal Year: 2013
  4. PAPOVA VIRUS TRANSFORMING MECHANISMS
    DAVID MORSE LIVINGSTON; Fiscal Year: 2013

Detail Information

Publications7

  1. pmc The oncogenic fusion protein Pax3-FKHR has a greater post-translational stability relative to Pax3 during early myogenesis
    Patrick J Miller
    Department of Genetics, Louisiana State University Health Sciences Center, 533 Bolivar Street, New Orleans, LA 70112, USA
    Biochim Biophys Acta 1770:1450-8. 2007
    ..Finally, the persistence of expression of Pax3-FKHR prevents the terminal differentiation of primary myoblasts demonstrating a biological consequence of its aberrant expression...
  2. pmc Identification of serine 205 as a site of phosphorylation on Pax3 in proliferating but not differentiating primary myoblasts
    Patrick J Miller
    Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA
    Protein Sci 17:1979-86. 2008
    ....
  3. pmc Phosphorylation of serine 205 by the protein kinase CK2 persists on Pax3-FOXO1, but not Pax3, throughout early myogenic differentiation
    Kevin N Dietz
    Department of Genetics, Louisiana State University Health Sciences Center, 533 Bolivar Street, New Orleans, Louisiana 70112, USA
    Biochemistry 48:11786-95. 2009
    ....
  4. pmc Isolation of putative FOXO1 genomic elements using an improved in vitro technique to isolate genomic regulatory sequences
    Alpa Sidhu
    Department of Genetics, Louisiana State University Health Sciences Center, 533 Bolivar Street, CSRB 6th floor, New Orleans, LA 70112, USA
    Gene 458:45-53. 2010
    ....
  5. pmc Identification of serines 201 and 209 as sites of Pax3 phosphorylation and the altered phosphorylation status of Pax3-FOXO1 during early myogenic differentiation
    Kevin N Dietz
    Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA
    Int J Biochem Cell Biol 43:936-45. 2011
    ..Taken together, our results allow us to propose a molecular model to describe the changing pattern of phosphorylation for Pax3 and the altered phosphorylation for Pax3-FOXO1 during early myogenic differentiation...
  6. pmc Identification of CK2 as the kinase that phosphorylates Pax3 at Ser209 in early myogenic differentiation
    Aditi S Iyengar
    Department of Genetics, Louisiana State University Health Sciences Center, 533 Bolivar Street, CSRB 6th floor, New Orleans, LA 70112, USA
    Biochem Biophys Res Commun 428:24-30. 2012
    ..Taken together, our results allow us to propose a mechanism to describe the ordered phosphorylation of Pax3 throughout early myogenesis...

Research Grants30

  1. Novel Compounds to Inactivate Oncogenic Fusion Proteins
    Jeffrey A Toretsky; Fiscal Year: 2013
    ..This project will specifically address the need for new targeted therapies for ESFT and a large number of related malignancies that have chromosomal translocations. ..
  2. Elucidating the role of NF-kB signaling in rhabdomyocarcoma
    Charles Keller; Fiscal Year: 2013
    ..Results from these studies are likely to yield significant mechanistic insight into the role of NF-kappaB signaling in alveolar rhabdomyosarcoma, which may prove useful for treatment of this lethal childhood cancer. ..
  3. Signaling in Inflammation, Stress, and Tumorigenesis
    GEORGE ROBERT STARK; Fiscal Year: 2013
    ..abstract_text> ..
  4. PAPOVA VIRUS TRANSFORMING MECHANISMS
    DAVID MORSE LIVINGSTON; Fiscal Year: 2013
    ..The goal of this Program is to continue to shed new light on cellular transformation events that also underpin human cancer development and generate insights that lead to new cancer therapeutic strategies. ..