An Engineered PAX3-KRAB Transcriptional Repressor Inhibits the Malignant Phenotype of Alveolar Rhabdomyosarcoma Cells Harboring the Endogenous PAX3-FKHR Oncogene

Abstract

The t(2;13) chromosomal translocation in alveolar rhabdomyosarcoma tumors (ARMS) creates an oncogenic 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 dependent on the FKHR activation domain. We have fused the KRAB repression module 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 sequences and repressed PAX3-dependent reporter plasmids. Stable expression of the PAX3-KRAB protein in ARMS cell lines resulted in loss of the ability of the cells to grow in low-serum or soft agar and to form tumors in SCID mice. Stable expression of a PAX3-KRAB mutant, which lacks repression function, or a KRAB protein alone, lacking a PAX3 DNA binding domain, failed to suppress the ARMS malignant phenotype. These data suggest that the PAX3-KRAB repressor functions as a DNA-binding-dependent suppressor of the transformed phenotype of ARMS cells, probably via competition with the endogenous PAX3-FKHR oncogene and repression of target genes required for ARMS tumorigenesis. The engineered repressor approach that directs a transcriptional repression domain to target genes deregulated by the PAX3-FKHR oncogene may be a useful strategy to identify the target genes critical for ARMS tumorigenesis.

ACKNOWLEDGMENTS

We thank Giovanni Rovera, Naomi Galili, and Sunil Mukopadhyay for the anti-FKHR antiserum and the PAX3-FKHR mouse-human hybrid plasmid. We thank J. Biegel for the RD cell line. We also thank Beat Schafer for the 6x-CD19-2(A-ins)LUC reporter plasmid and Claude Desplan for the P3-pD33ADH-CAT reporter plasmid. Peter Sobaille of the Dr. Meenhard Herlyn laboratory at the Wistar Institute provided assistance with tumorigenicity studies in SCID mice. George Prendergast, Jennifer Morris, and Laura Benjamin provided helpful suggestions. We are grateful to Thanos Halazonetis, Mark Lechner, Hongzhuang Peng, and David Schultz for their editorial comments.

W.J.F. was supported by the Wistar Basic Cancer Research Training Grant CA 09171. J.R.F. was supported by the Medical Science Training Program, University of Pennsylvania School of Medicine. F.J.R. is supported in part by National Institutes of Health grants CA 52009, Core grant CA 10815, DK 49210, GM 54220, DAMD 17-96-1-6141, and ACS NP-954; the Irving A. Hansen Memorial Foundation; the Mary A. Rumsey Memorial Foundation; and the Pew Scholars Program in the Biomedical Sciences.