[CANCER RESEARCH (SUPPL.)59, 1711s-1715s, April 1, 1999] The Role of Chimeric Paired Box Transcription Factors in the Pathogenesis of Pediatric Rhabdomyosarcomal Frederic G. Barr 2 Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6082 Abstract for RMS and other pediatric solid tumors to present as collections of poorly differentiated cells. Evidence of myogenic differentiation can Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric soft be detected by immunohistochemical assays for muscle proteins as tissue tumor with striated muscle differentiation. Chromosomal studies of well as electron microscopic examination for myofilaments. these tumors identified 2;13 and 1;13 translocations. Using physical map- ping and cloning strategies, we determined that t(2;13) and t(1;13) rear- Although no consistent structural chromosomal changes have been range PAX3 and PAX7, which encode members of the paired box tran- found in ERMS, cytogenetic studies have identified nonrandom chro- scription factor family, and juxtapose these genes with FKHR, which mosomal translocations in ARMS (2). The most prevalent finding is encodes a novel member of the fork head transcription factor family. a translocation involving chromosomes 2 and 13, t(2;13)(q35-37; These translocations result in chimeric transcripts consisting of 5' PAX3 q14), which was detected in 70% of published ARMS cases. There or PAX7 exons fused to 3' FKHR exons, which encode fusion proteins have also been several reports of a t(1;13)(p36;q14) variant translo- containing the PAX3 or PAX7 DNA-binding domain and the COOH- cation. The 2; 13 and 1; 13 translocations have not been associated with terminal FKHR transcriptional activation domain. In transfection studies, any other tumor type and appear to be specific markers for ARMS. the PAX3-FKHR fusion activates transcription of reporter genes contain- ing PAX DNA-binding sites, and is 10-100-fold more potent as a tran- Mapping and Cloning of Loci Involved in t(2;13) scriptional activator than is wild-type PAX3. This increased function results from the insensitivity of the COOH-terminal FKHR activation Using a physical mapping approach, PAX3 was found to be the domain to the inhibitory effects of NH2-terminal PAX3 domains. In chromosome 2 locus rearranged by the t(2;13) (Ref. 3; Fig. 1). This addition to functional alterations, our studies demonstrated PAX3-FKHR and PAX7-FKHR overexpression resulting from two distinct mechanisms, gene is a member of the paired box family and encodes a transcription increased transcription of PAX3-FKHR by a copy number-independent factor with an NHz-terminal DNA-binding domain containing paired mechanism, and gene amplification of PAX7-FKHR. These findings indi- box and homeobox motifs (4). Furthermore, PAX3 was found to be cate that the genetic changes in these tumors result in high levels of split by the translocation such that the 5' PAX3 region is located on chimeric transcription factors that are hypothesized to inappropriately the derivative chromosome 13 and the 3' PAX3 region is on the activate transcription of genes with PAX DNA-binding sites and thereby derivative chromosome 2 (3). Structural alterations of PAX3 were induce tumorigenic behavior. The differences in overexpression strategies confirmed by Southern analysis of genomic DNA from ARMS cell suggest important differences between the mechanisms for regulating lines and were localized to the 3' end of the PAX3 gene. PAX3 and PAX7 expression. These differences extend to the phenotypic Northern analysis with a PAX3 probe demonstrated a novel 7.2-kb level, at which clinical differences have been found between the two transcript in t(2;13)-containing cell lines (3). The corresponding ARMS subtypes: PAX7-FKHR tumors more often occur as localized lesions in the extremities of younger patients and are associated with cDNA was cloned and revealed a fusion of the PAX3 sequence 5' to longer event-free survival as compared to PAX3-FKHR tumors. There- the t(2;13) breakpoint to a novel sequence from chromosomal region fore, the clinical heterogeneity within the ARMS category is associated 13q14. These findings indicate that the t(2;13) results in a chimeric with genetic heterogeneity. Further analysis of the transcriptional func- transcript composed of 5' PAX3 and sequences from a chromosome tion, regulation of expression, and phenotypic effects will help to elucidate 13 gene. A full-length cDNA corresponding to the wild-type chromo- the action of these fusion products and the biological basis of the clinical some 13 gene was isolated and found to contain a 1965-bp open heterogeneity. reading frame encoding a 655-amino acid protein (5, 6). Sequence analysis revealed a region of homology to the conserved DNA- Introduction binding motif characteristic of the fork head or winged-helix tran- scription factor family (7), and thus this gene has been named FKHR RMS 3 is a family of soft tissue tumors that generally occur in (fork head in rhabdomyosarcoma). pediatric patients and are related to the skeletal muscle lineage (1). On the basis of histopathological criteria, RMS can be divided into two Chimeric Products Generated by t(2;13) principle subtypes, ARMS and ERMS, which are associated with distinct clinical behaviors. ARMS presents mainly in adolescents and In the PAX3-FKHR cDNA, the 5' PAX3 and 3' FKHR coding young adults, often occurs in the extremities and trunk, and is asso- sequences are fused in-frame to generate a 2508-nt open reading ciated with an unfavorable prognosis. ERMS mainly presents in frame, encoding an 836-amino acid fusion protein (Refs. 5 and 6; Fig. children who are <10 years old; occurs in the head and neck, 1). The FKHR breakpoint occurs within the fork head DNA-binding genitourinary tract, and retroperitoneum; and is associated with a domain, whereas the PAX3 breakpoint occms distal to the PAX3 favorable prognosis. Diagnosis of RMS is often complicated by a DNA-binding domain. Therefore, this fusion protein contains an paucity of features of striated muscle differentiation, the subtle his- intact PAX3 DNA-binding domain, the COOH-terminal half of the tological criteria for distinguishing RMS subtypes, and the tendency fork head domain, and the COOH-terminal FKHR region. The con- sistency of this chimeric transcript was confirmed by RT-PCR assays Received 9/18/98; accepted 2/1/99. of ARMS cell lines with oligonucleotide primers specific for the 5' 1 Presented at the "General Motors Cancer Research Foundation Twentieth Annual PAX3 and 3' FKHR sequences. The fusion protein product was Scientific Conference: Developmental Biology and Cancer," June 9-10, 1998, Bethesda, MD. This work was supported in part by NIH Grants CA64202 and CA71838. subsequently detected with antisera specific for the PAX3 and FKHR 2 To whom requests for reprints should be addressed. proteins (5, 8). In contrast, the reciprocal translocation product con- 3 The abbreviations used are: RMS, rhabdomyosarcoma; ARMS, alveolar RMS; ERMS, embryonal RMS; RT-PCR, reverse transcriptase-PCR; FISH, fluorescence in situ sisting of 5' FKHR and 3' PAX3 exons was not detected in ARMS hybridization. lines by northern blot or immunoprecipitation analysis and was only 171 ls Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1999 American Association for Cancer Research. CHIMERIC PAX PROTEINS IN RHABDOMYOSARCOMA lkb 100 a.a. m DNA Binding Transactivation Domain Domain w,,<,.,,,. HMN l HH _._._,.. !i!~ PB H iilii iii~iii~iiiiiil PAX3 gene // I/// I "l Breakpoint Inhibitory Domains I distribution I Ch,,,,.,,o m HN !I I gene // Chimeric FKHR-PAX3 1 // 5' i:i~~:::~ gene I I I WIld'type 1 IllL__ ..._...p~ ,, FKHR gene //Z. l~ I I Breakpoint DNA Binding Transactivation distribution Domain Domain Fig. 1. Chimericgenes and products generatedby the 2;13 translocationin ARMS. Left, the exons of the wild-typeand fusion genes are shown as boxes above each map, and the translocation breakpoint distributions are shown as line segments below the map of the wild-typegenes. Right, the protein products of the wild-typeand chimericgenes are shown. [~, the paired box (PB), homeobox(HD), and fork head (FD) domains; l, octapeptide;I, transcriptional domains. Vertical dashed line, the translocation fusion point. detected in a subset of ARMS lines with a sensitive RT-PCR assay (3, combined with a reverse 3' FKHR primer to amplify PAX3-FKHR or 5). The findings of higher and more consistent expression of the 5' PAX7-FKHR in a single RT-PCR assay (13). A two-step assay PAX3-3' FKHR fusion suggest that PAX3-FKHR is the product procedure thus consists of a consensus PAX3-/PAX7-FKHR RT-PCR involved in ARMS pathogenesis. assay to identify whether either fusion is present and then hybridiza- The consistent structure of the PAX3-FKHR product is clarified by tion of the RT-PCR product with PAX3- and PAX7-specific oligo- a consideration of the genomic organization of the wild-type genes nucleotide probes to type the specific fusion. (Fig. 1). PAX3 consists of eight exons dispersed over 100 kb; exons 2, A detection methodology that directly visualizes the genomic DNA 3, and 4 encode the paired box, whereas the homeodomain is encoded fusion in interphase or metaphase cells is FISH. PAX3-FKHR is by exons 5 and 6 and the transactivation domain is encoded by exons assayed by labeling 5' PAX3- and 3' FKHR-containing cosmids with 6, 7, and 8 (9). The t(2;13) breakpoints consistently disrupt the 20-kb biotin- and digoxigenin-modifieddUTP and hybridizing these probes intron separating exons 7 and 8 (10), and thus the translocation to cells plated on glass slides (14). Following detection of the hybrid- maintains the integrity of the NH2-terminal DNA-binding domain and ized probe with fluorescent affinity reagents, the genomic fusion is separates it from an essential part of the transactivation domain.