Alveolar Rhabdomyosarcoma (ARMS) patients carrying the t (2;13) (q35;q14) chromosomal translocation have the lowest survival rate of all rhabdomyosarcomas;however, the reason for this fact is not clear. The translocation encodes an oncogenic fusion protein Pax3-FOXO1, and the strong correlation between the presence of the translocation and increased mortality indicates that this fusion protein directly participates n the mechanism of ARMS development. The contributions of the Pax3 portion of the fusion protein to oncogenesis have been extensively studied. By contrast little is known about the role the FOXO1 portion makes to ARMS development. Despite a lack of experimental evidence, the truncated FOXO1 DNA binding domain has been assumed to be nonfunctional. However, sequence analysis reveals that key amino acids that mediate FOXO1-dependent DNA interactions are present within the fusion protein. Therefore, this evidence emphasizes the need to examine the role of the truncated FOXO1 DBD in targeting promoter regions to aid in identifying novel Pax3-FOXO1 transcriptional targets that contribute to the development of ARMS. The long-term goal is to determine the mechanism by which Pax3-FOXO1 binds to and activates regulatory elements, and how activation of these specific genes leads to the development of ARMS. The initial step in achieving this goal is to determine the contribution made by the truncated FOXO1 DBD to Pax3-FOXO1 DNA binding by examining both the DNA and amino acid contacts necessary for this interaction with the following two specific aims:
Specific Aim 1 : Determine structural elements necessary for Pax3-FOXO1 DNA binding and transcriptional activation in vitro. Using synthetic promoter constructs with both Pax3 and FOXO1 recognition sequences, we will examine the effect of mutating the FOXO1 portion of the sequence and amino acids necessary for FOXO1 binding on DNA binding and transcriptional activation.
Specific Aim 2 : Identify the contribution made by the FOXO1 element to the activation of known Pax3-FOXO1 direct transcriptional targets. We will test the importance of putative FOXO1 DNA sequence elements in the ability of Pax3-FOXO1 to bind and activate CNR1, TFAP2B, PRRX1, and MEOX2 in a rhabdomyosarcoma cell line. The significance of the proposed project is that it will provide both the data and the appropriate mentoring required to position the PI to undertake subsequent definitive studies on the role of the truncated FOXO1 DNA- binding domain in mediating DNA binding by Pax3-FOXO1 at the SC1 level. The research proposed in this application is innovative because it focuses on the contributions that the truncated FOXO1 DNA-binding domain makes to Pax3-FOXO1 DNA binding and gene expression. These data will provide basic information about the molecular mechanism by which Pax3-FOXO1 recognizes and regulates the expression of genes involved in ARMS development.
Alveolar Rhabdomyosarcoma (ARMS) is a malignant, skeletal muscle tumor that occurs mainly in older children and adolescents. The most lethal cases are caused by a chromosomal translocation that results in the fusion of two proteins that regulate gene expression. This proposed pilot project will provide key information as the first step in understanding the mechanism underlying the oncogenic nature of this fusion protein. Such data will enhance the ability of researchers to develop new therapies to stop the development and progression of this cancer.