Role of Myc Family Proteins in Fusion-Positive Rhabdomyosarcoma

Authors

• MJ Koretsky
• C Alvarado
• MB Makarious
• D Vitale
• K Levine
• S Bandres-Ciga
• A Dadu
• SW Scholz
• L Sargent
• F Faghri
• H Iwaki
• C Blauwendraat
• A Singleton
• M Nalls
• H Leonard

Abstract

Fusion-positive rhabdomyosarcoma is a pediatric cancer characterized by a PAX3-FOXO1 (P3F) fusion gene and high-level expression of MYCN and/or MYC. To model interaction of P3F with Myc family proteins, we generated human myoblasts with doxycycline-inducible P3F (iP3F) with or without constitutively expressed MYCN. Myoblasts expressing both MYCN and P3F (MYCN-iP3F) form foci in vitro and tumors in vivo, while myoblasts expressing only P3F (iP3F) do not form foci and form tumors more slowly. Protein expression analysis revealed very high MYCN and low MYC levels in parental and tumor-derived (TD) MYCN-iP3F lines, moderate MYC and very low MYCN levels in parental iP3F lines, and high MYC and moderate MYCN levels in TD iP3F lines. Using CRISPR-Cas9 technology to knockdown MYCN or MYC, oncogenic activity in parental and TD MYCN-iP3F lines is primarily dependent on MYCN whereas oncogenicity in TD iP3F lines is primarily dependent upon MYC. To elucidate whether Myc proteins regulate P3F target genes, we measured RNA expression of multiple targets in parental and TD lines. Several targets (such as FGFR4) showed comparable upregulation by P3F in parental and TD lines with or without high MYCN. In contrast, a few targets (such as FGF8) were stimulated by P3F at low levels in parental iP3F lines and at much higher levels in TD iP3F lines and both parental and TD MYCN-iP3F lines. We postulate that the dependence of fusion-positive rhabdomyosarcoma on high-level Myc family expression is at least partly due to the need to stimulate P3F targets in this latter category.

Scientific Focus Area: Cancer Biology

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