An engineered PAX3-KRAB transcriptional repressor inhibits the malignant phenotype of alveolar rhabdomyosarcoma cells harboring the endogenous PAX3-FKHR oncogene

The t(2;13) chromosomal translocation in alveolar rhabdomyosarcoma tumors ( ARMS) creates an onco genic transcriptional activator by fusion of PAX3 DNA binding motifs to a COOH-terminal activation domain derived from the FKHR gene. The dominant oncogenic potential of the PAX3-FKHR fusion protein is d ependent on the FKHR activation domain. We have fused the KRAB repression m odule to the PAX3 DNA binding domain as a strategy to suppress the activity of the PAX3-FKHR oncogene. The PAX3-KRAB protein bound PAX3 target DNA seq uences and repressed PAX3-dependent reporter plasmids. Stable expression of the PAX3-KRAB protein in ARMS cell lines resulted in loss of the ability o f the cells to grow in low-serum or soft agar and to form tumors in SCID mi ce. Stable expression of a PAX3-KRAB mutant, which lacks repression functio n, or a KRAB protein alone, lacking a PAX3 DNA binding domain, failed to su ppress the ARMS malignant phenotype. These data suggest that the PAX3-KRAB repressor functions as a DNA-binding-dependent suppressor of the transforme d phenotype of ARMS cells, probably via competition with the endogenous PAX 3-FKHR oncogene and repression of target genes required for ARMS tumorigene sis. The engineered repressor approach that directs a transcriptional repre ssion domain to target genes deregulated by the PAX3-FKHR oncogene may be a useful strategy to identify the target genes critical for ARMS tumorigenes is.