Gene Fusions Involving PAX and FOX Family Members in Alveolar Rhabdomyosarcoma

Gene fusions involving PAX and FOX family members in alveolar rhabdomyosarcoma

Frederic G Barr*,1

1Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 36th Street and Hamilton Walk, Philadelphia, Pennsylvania, PA 19104-6082, USA

The chromosomal translocations t(2;13)(q35;q14) and Clinical, pathologic and cytogenetic features of alveolar t(1;13)(p36;q14) are characteristic of alveolar rhabdo- rhabdomyosarcoma myosarcoma, a pediatric soft tissue cancer related to the striated muscle lineage. These translocations rearrange Alveolar rhabdomyosarcoma (ARMS) is one subtype PAX3 and PAX7, members of the paired box transcrip- of rhabdomyosarcoma (RMS), a family of pediatric tion factor family, and juxtapose these genes with soft tissue tumors that are related to the skeletal FKHR, a member of the fork head transcription factor muscle lineage (Wexler and Helman, 1997). ARMS was family. This juxtaposition generates PAX3 ± FKHR and initially distinguished from the other major RMS PAX7 ± FKHR chimeric genes that are expressed as subtype, embryonal rhabdomyosarcoma (ERMS), by chimeric transcripts that encode chimeric proteins. The histologic criteria. As the criteria were re®ned, the fusion proteins, which contain the PAX3/PAX7 DNA subtypes were found to be associated with distinct binding domain and the FKHR transcriptional activation clinical behaviors (Tsokos et al., 1992). ARMS presents domain, activate transcription from PAX-binding sites mainly in adolescents and young adults, often in the with higher potency than the corresponding wild-type vicinity of skeletal muscle in the extremities and trunk, PAX proteins. This increased function results from the and is associated with an unfavorable prognosis. This insensitivity of the FKHR activation domain to inhibitory unfavorable prognosis is related to the propensity for eects of N-terminal PAX3/PAX7 domains. In addition early and wide dissemination, often involving bone to altered function, the fusion products are expressed in marrow, and to poor response to chemotherapy. In ARMS tumors at higher levels than the corresponding contrast, ERMS mainly presents in children less than wild-type PAX products due to two distinct mechanisms. 10 years old in regions without abundant skeletal The PAX7 ± FKHR fusion is overexpressed as a result of muscle such as the head and neck, genitourinary tract, in vivo ampli®cation while the PAX3 ± FKHR fusion is and retroperitoneum, and has a more favorable overexpressed due to a copy number-independent prognosis. increase in transcriptional rate. Finally, though FKHR Diagnosis of ARMS is often complicated by scant subcellular localization is regulated by an AKT- evidence of striated muscle dierentiation and rela- dependent pathway, the fusion proteins are resistant to tively subtle histologic criteria for distinguishing these signals and show exclusively nuclear localization. ERMS and ARMS (Tsokos, 1994). The problem is Therefore, these translocations alter biological activity at compounded by the fact that other pediatric solid the levels of protein function, gene expression, and tumors including neuroblastoma, Ewing's sarcoma and subcellular localization with the cumulative outcome lymphoma can present as collections of poorly postulated to be aberrant regulation of PAX3/PAX7 dierentiated cells. Subtle evidence of myogenic target genes. This aberrant gene expression program is dierentiation can be detected with immunohistochem- then hypothesized to contribute to tumorigenic behavior ical reagents speci®c for muscle proteins (such as by impacting on the control of growth, apoptosis, MyoD, desmin, or muscle-speci®c actin) and electron dierentiation and motility. Oncogene (2001) 20, microscopic analysis of myo®laments. However, there 5736 ± 5746. is no well-established immunohistochemical or ultra- structural marker that distinguishes ARMS and Keywords: rhabdomyosarcoma; transcription factor; ERMS. translocation; paired box; fork head Chromosomal studies identi®ed nonrandom translocations that distinguish the majority of ARMS tumors from ERMS and other pediatric solid tumors. The most prevalent ®nding in ARMS is a translocation, t(2;13)(q35-37;q14), which was detected in 70% of ARMS cases (Douglass et al., 1987; Turc-Carel et al., 1986; Wang-Wuu et al., 1988) (Figure 1). In addition, a variant translocation, t(1;13)(p36;q14), was identi®ed in *Correspondence: FG Barr; E-mail: [email protected] a smaller subset of ARMS cases (Biegel et al., 1991; Alveolar rhabdomyosarcoma gene fusions FG Barr 5737 Douglass et al., 1991). The 2;13 and 1;13 translocations have not been detected in any other tumor type and appear to be speci®c and sensitive markers of ARMS.

Mapping and cloning of loci involved in 2;13 and 1;13 translocations

Based on physical mapping studies, PAX3 was localized to the vicinity of the t(2;13) breakpoint on chromosome 2 (Barr et al., 1993). Analysis of somatic cell hybrids derived from ARMS cells indicated that PAX3 is split such that the 5' PAX3 region is located on the derivative chromosome 13 [der(13)] and the 3' PAX3 region is on the der(2) (Figure 2). In conjunction Figure 3 Comparison of wild-type and fusion products asso- with Southern blot ®ndings of PAX3 structural ciated with the 2;13 and 1;13 translocations. The paired box, alterations in ARMS cell lines, these studies established octapeptide, homeobox and fork head domain are indicated as open boxes, and transcriptional domains (DNA binding domain, that PAX3 is the chromosome 2 locus rearranged by DBD; transcriptional activiation domain, TAD; transcriptional the t(2;13). Northern analysis with a 5' PAX3 probe inhibitory domain, TID) are shown as solid bars. The sites demonstrated a novel 7.2 kb transcript unique to cell phosphorylated by Akt are indicated by stars, and the alternative lines with the t(2;13). Clones containing 5' PAX3 splice in the paired box is shown by an arrowhead. The vertical sequences fused to a novel sequence from chromosomal dash line indicates the translocation fusion point region 13q14 were then isolated from cDNA libraries constructed from ARMS cell lines (Galili et al., 1993; Shapiro et al., 1993) (Figure 3). The corresponding acid protein. A BLAST database search showed wild-type chromosome 13 gene generates a 6.5 kb homology to the fork head transcription factor family, widely expressed transcript that encodes a 655 amino and thus this chromosome 13 gene was named FKHR (fork head in rhabdomyosarcoma) (Galili et al., 1993); this gene was also termed ALV (Shapiro et al., 1993), and was recently renamed FOXO1 (Kaestner et al., 2000). These ®ndings indicate that the t(2;13) results in a chimeric transcript composed of 5' PAX3 sequences fused to 3' FKHR sequences. Analysis of the PAX3 ± FKHR cDNA revealed that the 5' PAX3 and 3' FKHR coding sequences are fused in-frame to encode an 836 amino acid fusion protein (Galili et al., 1993; Shapiro et al., 1993) (Figure 3). The consistency of this chimeric transcript was con®rmed by reverse transcriptase (RT) ± PCR experiments, and sequence analysis of these RT ± PCR products revealed an invariant fusion point. The reciprocal translocation Figure 1 Diagram of t(2;13)(q35;q14) and t(1;13)(p36;q14) product, the der(2) chimeric transcript consisting of 5' chromosomal translocations FKHR and 3' PAX3 exons, was not detected in ARMS lines by Northern blot analysis (Barr et al., 1993), but low level expression was detected by RT ± PCR analysis in a subset of ARMS cell lines and specimens (Galili et al., 1993). The ®ndings of higher, more consistent expression of the 5' PAX3-3' FKHR fusion indicate that the der(13) encodes the product involved in the pathogenesis of ARMS. In ARMS cases with the 1;13 translocation, attention focused on PAX7, another member of the paired box- containing transcription factor family located in chromosomal region 1p36 (Davis et al., 1994). RT ± PCR and Southern blot studies demonstrated that PAX7 is rearranged and fused to FKHR in tumors containing this translocation. This fusion results in a Figure 2 Generation of chimeric genes by the t(2;13)(q35;q14) chimeric transcript consisting of 5' PAX7 and 3' translocation in ARMS. The exons of the wild-type and fusion genes are shown as boxes above each map and the translocation FKHR regions (Figure 3), which is nearly identical in breakpoint distributions are shown as line segments below the structure and organization to the 5' PAX3 ± 3' FKHR map of the wild-type genes transcript formed by the 2;13 translocation.

Oncogene Alveolar rhabdomyosarcoma gene fusions FG Barr 5738 Paired box family of transcription factors disrupted Pax7 gene demonstrate abnormalities in neural crest-derived structures and musculature (Man- PAX3 and PAX7 encode members of the paired box or souri et al., 1996; Seale et al., 2000). These studies thus PAX transcription factor family that is characterized provide functional evidence of an important role for by a conserved paired box DNA binding domain ®rst Pax3 and Pax7 in myogenic development. identi®ed in Drosophila segmentation genes (Tremblay and Gruss, 1994). There are nine human members of this family. Several family members also contain a Fork head family of transcription factors complete or truncated version of a homeobox DNA binding domain and a short conserved octapeptide The fork head domain was ®rst identi®ed as a 100 motif distal to the paired box. PAX3 and PAX7 amino acid region of sequence similarity between the constitute a subfamily within the PAX family and Drosophila fork head and the rat HNF-3a proteins, and encode very similar proteins containing an N-terminal was subsequently identi®ed in a large family of genes in DNA binding domain consisting of a paired box, species ranging from yeast to human (Kaufmann and octapeptide and complete homeodomain, and a pro- Knochel, 1996). Genetic, expression and functional line-, serine- and threonine-rich C-terminal domain analyses of the various fork head (or FOX) genes (Figure 3). indicate diverse roles including control of embryonic PAX family members function in the transcriptional development and adult tissue-speci®c gene expression. control of pattern formation during embryogenesis The FOX gene products function as transcription (Tremblay and Gruss, 1994). Each gene has a unique factors in which the fork head domain constitutes a temporal and spatial expression pattern during early functional unit that is necessary and sucient for DNA development, and some are also expressed with a binding, and cannot be further subdivided without loss restricted distribution in the adult. In situ hybridization of the DNA binding function. In addition, other analysis of embryos revealed that Pax3 and Pax7 are divergent regions of the proteins confer transcriptional both expressed in the developing nervous system and in regulatory function. somite compartments that give rise to skeletal muscle Within the FOX family, FKHR is the prototype of a progenitors (Goulding et al., 1991; Jostes et al., 1990). subfamily with highly similar sequence in the fork head Pax7 expression is activated later and persists longer domain and additional regions of sequence similarity than that of Pax3. In addition, Pax3 becomes localized outside the fork head domain (Anderson et al., 1998; to the lateral dermomyotome while Pax7 becomes more Borkhardt et al., 1997; Hillion et al., 1997). Other prominent in the medial dermomyotome, and Pax3 but subfamily members include AFX (also called FOXO4) not Pax7 is expressed by myogenic progenitors that and FKHRL1 (also called FOXO3a). A subfamily migrate to the limbs (Bober et al., 1994; Goulding et member named DAF-16 was also identi®ed in C. al., 1994; Williams and Ordahl, 1994). Finally, while elegans (Lin et al., 1997; Ogg et al., 1997). Analysis of Pax3 expression was not detected in the adult mouse, adult and embryonic murine tissues demonstrated that Pax7 expression was detected in muscle satellite cells of the three mammalian subfamily members are widely the adult mouse (Seale et al., 2000). Therefore, PAX3 expressed, with quantitative dierences in expression and PAX7 are expressed with overlapping but distinct that are somewhat complementary (Furuyama et al., patterns in myogenic precursors that are the presumed 2000). Following the ®nding that DAF-16 functions in progenitors of ARMS. insulin-like signaling pathways and longevity control in The important developmental role of the paired box C. elegans (Lin et al., 1997; Ogg et al., 1997), genes is further highlighted by phenotypic analyses of subsequent studies of FKHR, AFX, and FKHRL1 germline mutations (Dahl et al., 1997; Tremblay and demonstrated roles in insulin-response and growth Gruss, 1994). Mutations of these genes were identi®ed arrest/apoptosis pathways (Brunet et al., 1999; Guo in several spontaneous murine and human heritable et al., 1999; Medema et al., 2000). A further striking developmental disorders. Point mutations and deletions role of this subfamily in tumorigenesis is indicated by aecting functional domains of the Pax3 gene were the ®nding that AFX and FKHRL1 are both fused to identi®ed in the splotch mouse (Epstein et al., 1991), the MLL gene by chromosomal translocations in acute which is characterized by abnormalities of the neural leukemias (Borkhardt et al., 1997; Hillion et al., 1997). tube, neural crest-derived structures and peripheral musculature. The human disease Waardenburg syndrome, characterized by deafness and pigmentary Structural analyses of translocation breakpoints disturbances, is caused by mutations in the PAX3 gene (Baldwin et al., 1992; Tassabehji et al., 1992). The structure of the chimeric PAX3 ± FKHR and Functional studies showed that splotch and Waarden- PAX7 ± FKHR products is clari®ed by a consideration burg mutations alter or abolish transcriptional activity of the genomic organization of the wild-type and (Chalepakis et al., 1994a), and result in a loss of chimeric genes (Figure 2). In addition to the high function. In addition to these spontaneous mutations, degree of sequence similarity between the PAX3 and gene targeting technology was used to generate murine PAX7 products, the organization of the PAX3 and strains in which other PAX genes were inactivated PAX7 loci is very similar, with highly comparable (Dahl et al., 1997). Animals that are homozygous for a exon-intron boundaries and exon distributions (Burri

Oncogene Alveolar rhabdomyosarcoma gene fusions FG Barr 5739 et al., 1989; Macina et al., 1995; Vorobyov et al., 1997). ARMS cases (Barr et al., 1996). These ®ndings were Each locus consists of nine exons dispersed over con®rmed and extended by quantitative Southern blot 100 kb; exons 2, 3 and 4 encode the paired box assays of the relative copy number of wild-type and whereas the homeodomain is encoded by exons 5 and 6 rearranged alleles (Barr et al., 1996; Davis and Barr, and the transactivation domain is encoded by exons 6, 1997). In a series of ARMS cases, fusion gene 7 and 8 (Bennicelli et al., 1995, 1999). A recently ampli®cation was detected in one of 24 PAX3 ± FKHR discovered exon 9 in each gene encodes a C-terminal cases and 10 of 11 PAX7 ± FKHR cases (Fitzgerald et extension with unknown function (Barr et al., 1999). al., 2000). Therefore, in PAX7 ± FKHR tumors, The t(2;13) and t(1;13) breakpoints consistently disrupt translocation and ampli®cation often occur sequen- the seventh introns, which span 17.5 and 32 kb in the tially to alter both gene structure and copy number and PAX3 and PAX7 loci, respectively; the distribution of thereby activate oncogenic activity by complementary breakpoints within these introns appears random (Barr strategies. et al., 1998; Fitzgerald et al., 2000). Therefore, these The mechanism of overexpression in PAX3 ± FKHR- breakpoints are situated to maintain the integrity of expressing tumors was elucidated by analyses of the N-terminal DNA binding domain and separate it mRNA stability and transcription rate (Davis and from an essential part of the transactivation domain. Barr, 1997). Ribonuclease protection analysis of RNA The FKHR locus consists of three exons spanning from actinomycin D-treated ARMS cell lines showed 140 kb (Davis et al., 1995) (Figure 2). The fork head that PAX3 ± FKHR and PAX3 transcripts have domain is encoded by portions of exons 1 and 2 and comparable stabilities. However, nuclear runo analy- the transcriptional activation domain is encoded by sis using hybridization targets ¯anking the t(2;13) exon 2; the last exon consists entirely of 3' untranslated breakpoint revealed that PAX3 ± FKHR is more region. The translocation breakpoints occur within the actively transcribed than PAX3. Therefore, PAX3 ± ®rst intron, which spans 130 kb. This intron provides a FKHR overexpression results from a copy number- large target for rearrangements and allows disruption independent increase in transcriptional rate that is of the fork head DNA binding domain and fusion of postulated to result from synergism of PAX3 and the C-terminal FKHR transactivation domain to the FKHR regulatory elements. In contrast, PAX7 ± N-terminal PAX DNA binding domain. Furthermore, FKHR overexpression results from a second genetic the translational reading frame is maintained from event, fusion gene ampli®cation. These ®ndings in- PAX3/PAX7 exon 7 to FKHR exon 2. A similar fusion dicate biological dierences between the two fusion cannot be created by any other combination of PAX3/ genes that are probably related to dierences in the PAX7 and FKHR exons, because of incompatible regulation of their expression. reading frames or loss of functional domains. These ®ndings support the premise that rearrangements of PAX3/PAX7 intron 7 and FKHR intron 2 are selected Regulation of subcellular localization of wild-type and due to functional constraints related to the genomic chimeric FOX proteins organization of these loci. Genetic studies in C. elegans revealed that DAF-16 is involved in a signaling pathway involving the homo- Expression characteristics of wild-type and chimeric logues of the insulin receptor (DAF-2), phosphoinosi- PAX genes tide 3-kinase (AGE-1), and AKT serine/threonine kinases (Lin et al., 1997; Ogg et al., 1997). In Ribonuclease protection assays showed several-fold mammalian cells, various growth/survival factors higher levels of PAX3 ± FKHR mRNA relative to (including insulin and IGF-1) activate phosphoinositide wild-type PAX3 mRNA in most PAX3 ± FKHR- 3-kinase and subsequently activate AKT. Based on the expressing ARMS specimens (Davis and Barr, 1997). C. elegans ®ndings, AKT was shown to phosphorylate Immunoprecipitation analysis con®rmed that PAX3 ± FKHR, FKHRL1 and AFX at three conserved sites FKHR is overexpressed at the protein level. Similar that conform to the AKT consensus target sequence studies of PAX7 ± FKHR-expressing tumors revealed (Brunet et al., 1999; Takaishi et al., 1999; Tang et al., that this fusion is consistently overexpressed relative to 1999) (Figure 3). Following phosphorylation, these wild-type PAX7. These ®ndings indicate that over- proteins localize within the cytoplasm, and thus this expression of PAX3 ± FKHR and PAX7 ± FKHR signaling pathway inactivates transcriptional function relative to the corresponding wild-type PAX genes is by preventing these proteins from reaching their site of characteristic of ARMS tumors, and suggest that action. The mechanism for this change in subcellular overexpression is required to generate a level of fusion localization appears to involve interaction with 14-3-3 product above a critical threshold for oncogenic scaolding proteins and cytoplasmic sequestration. activity. Of the three AKT phosphorylation sites, two are Genomic studies revealed a striking dierence in the retained in the PAX3 ± FKHR and PAX7 ± FKHR basis of PAX3 ± FKHR and PAX7 ± FKHR over- proteins in the region distal to the fork head domain expression. Fluorescence in situ hybridization assays (Figure 3). In that ARMS cells produce high levels of showed in vivo ampli®cation of the fusion gene on IGF2 (Toretsky and Helman, 1996), the fusion proteins extrachromosomal elements in PAX7 ± FKHR-positive would be sequestered in the cytoplasm if these proteins

Oncogene Alveolar rhabdomyosarcoma gene fusions FG Barr 5740 are regulated by this pathway. However, transfection removes a single glutamine residue in the paired box of experiments showed that PAX3 ± FKHR has a nuclear PAX3 and increases the anity for class I sites (Vogan localization and is transcriptionally active in the et al., 1996) (Figure 3). An analysis of PAX7 showed presence of AKT (del Peso et al., 1999). Therefore, the that the analogous glutamine-containing isoform inter- fusion protein is resistant to upstream regulatory signals acts with e5 and related class II sites with a binding and is constitutively retained in the nucleus. Possible anity similar to that of PAX3 (Schafer et al., 1994). explanations include a necessary role for the N-terminal Furthermore, a comparable alternative splice that FKHR phosphorylation site that is not retained by the removes a glutamine from the paired box was also fusion protein or antagonism by N-terminal PAX identi®ed in PAX7, though binding activity was not domains, such as the nuclear localization signal. assayed (Vogan et al., 1996). Since both splice forms of PAX3 and PAX7 are co-expressed, the diering binding activity of the two isoforms will contribute to DNA binding properties of wild-type and chimeric PAX a complex pattern of overall binding function. proteins Electrophoretic mobility shift assays of the PAX3- FKHR protein showed binding to e5 and related sites In PAX3 ± FKHR and PAX7 ± FKHR, the paired box (Fredericks et al., 1995; Sublett et al., 1995). Whereas and homeobox are intact whereas the fork head mutations within the paired box or homeodomain of domain is truncated (Davis et al., 1994; Galili et al., PAX3 ± FKHR abolished interaction with these sites, 1993; Shapiro et al., 1993) (Figure 3). Since mutations deletion of the truncated fork head domain did not aect of other fork head domains inactivate DNA binding binding. Comparison of the autoradiographic intensities function (Kaufmann and Knochel, 1996), the truncated of the protein ± DNA complexes formed with PAX3 and fork head domain in the fusion proteins is probably PAX3 ± FKHR indicated that the binding anity of inactive or modi®es DNA binding by the PAX3/PAX7 isolated wild-type PAX3 protein for e5 was greater than domains. Therefore, the PAX3 and PAX7 DNA that of PAX3 ± FKHR. Therefore, even though the wild- binding domains are postulated to provide the DNA type and fusion proteins contain the same PAX3 DNA binding speci®city for the fusion proteins. binding domain, in vitro binding function is partly Initial studies focused on binding to the e5 sequence, dependent on the larger protein context. which was identi®ed in the Drosophila even-skipped gene PAX3 binding sites were also isolated from random as a binding site for the paired gene product, which also oligonucleotide pools by a PCR-based selection strategy. contains a paired box and homeodomain (Goulding et A bacterially synthesized protein containing the PAX3 al., 1991; Treisman et al., 1991). Footprinting experi- paired box identi®ed consensus binding sequences ments with e5 and its derivatives showed that the paired of TCGTCAC(G/A)C(T/C/A)(T/C)(C/A/T)A and and PAX3 proteins protect an 18 ± 27 bp region, which CGTCACG(G/C)TT (Chalepakis and Gruss, 1995; can be subdivided into paired box and homeodomain Epstein et al., 1996). Similar experiments with a bacterial binding sites that include GTTCC and ATTA motifs, synthetic protein containing the PAX3 paired box and respectively (Chalepakis et al., 1994a; Treisman et al., homeodomain identi®ed ATTA and GTNNN motifs in 1991). Optimal binding of PAX3 to e5 or its derivatives the majority of isolated binding sites, and the sequence requires both sites (Chalepakis et al., 1994a; Goulding ATTA-(N)n-GTTAT in 20% of the binding sites (Phelan et al., 1991). Comparison of full-length PAX3 to a and Loeken, 1998). This ®nding is consistent with the truncated form lacking the homeodomain demonstrates hypothesis of paired box and homeobox binding motifs a qualitative dierence in binding anity. Subsequent connected by a variable spacer. studies of PAX3 proteins with paired box or home- Several studies also demonstrated protein ± protein odomain mutations (from splotch and Waardenburg interactions involving PAX3 and/or PAX7 that may syndrome cases) revealed altered binding activity of in¯uence DNA binding activity. PAX3 and PAX7 are both the unmutated and mutated domains, and thus capable of forming homodimers (PAX3/PAX3 or indicated a functional interaction between the paired PAX7/PAX7) and heterodimers (PAX3/PAX7) that box and homeodomain within the wild-type PAX3 can bind to a palindromic homeodomain-binding site protein (Fortin et al., 1997; Underhill and Gros, 1997; (P2-TAATCAATTA) with comparable or enhanced Underhill et al., 1995). binding compared to the monomers (Schafer et al., Additional studies of PAX family members indicated 1994). In addition, the homeobox-containing protein that the paired box is comprised of two subdomains Msx1, which is coexpressed with Pax3 in migrating (Czerny et al., 1993). PAX5 targets can be divided into limb muscle precursors, can interact with the Pax3 two sets of binding sites, the longer class I sites that protein and inhibit Pax3 DNA binding activity require both N-terminal and C-terminal subdomains, (Bendall et al., 1999). and the shorter class II sites (which includes e5 and related sequences) that only require the N-terminal subdomain. A comparison of PAX3 and PAX5 DNA Transcriptional properties of wild-type and chimeric binding initially indicated that only PAX5 can bind PAX proteins class I sites whereas both PAX3 and PAX5 can bind class II sites (Schafer et al., 1994). However, sub- To analyse sequence-speci®c transcriptional regulatory sequent studies identi®ed an alternative splice that function of the wild-type and fusion proteins, con-

Oncogene Alveolar rhabdomyosarcoma gene fusions FG Barr 5741 structs expressing the full-length proteins were trans- to two orders of magnitude less activity than the C- fected into mammalian cells with a reporter construct terminal PAX3 or PAX7 construct. These ®ndings containing a minimal adenoviral E1b promoter and indicate that an inhibitory domain is present in the N- PAX3/PAX7 binding sites (Bennicelli et al., 1996, 1999; terminal PAX3 and PAX7 regions that eectively Fredericks et al., 1995). In experiments using various inhibits the activity of the C-terminal PAX3 and cell lines and binding sites, wild-type PAX3 expression PAX7 domains but only has a modest eect on the resulted in a low but detectable level of transcriptional C-terminal FKHR domain (Figure 3). Therefore the activation whereas wild-type PAX7 expression did not translocations create potent transcriptional activators result in detectable transcriptional activity. In contrast, by introducing a C-terminal transactivation domain the PAX3 ± FKHR and PAX7 ± FKHR proteins simi- that is relatively insensitive to the inhibitory eects of larly induced much higher levels of transcriptional the N-terminal PAX3 and PAX7 domains. Further- activity, greater than 10-fold more activity than wild- more, diering interactions between the N-terminal type PAX3. In separate experiments using reporter inhibitory and C-terminal activation domains in PAX3 constructs with a herpesvirus thymidine kinase pro- and PAX7 may explain the diering transcriptional moter and PAX3 binding sites, transcriptional activa- activities of these wild-type proteins. tion by PAX3 ± FKHR was only twofold higher than Additional studies suggest that the N-terminal PAX3 that by PAX3 (Sublett et al., 1995). These experiments and PAX7 domains function as transcriptional in- demonstrate that the PAX3 ± FKHR and PAX7 ± hibitory modules in intermolecular and intramolecular FKHR proteins can function as transcription factors, regulation by binding co-repressors. Deletion analysis speci®cally activating expression of genes containing localized the inhibitory activity to two N-terminal PAX3/PAX7 DNA binding sites. Furthermore, the domains, the homeodomain and the N-terminus fusion proteins are more potent transcriptional activa- including the ®rst half of the paired box (Bennicelli tors than the wild-type proteins, though the increased et al., 1996, 1999) (Figure 3). These same two regions potency may be promoter-dependent. were also shown to have independent transcriptional To investigate the diering transcriptional potencies repression activity when fused to the GAL4 DNA of the wild-type and fusion proteins, initial studies binding domain (Chalepakis et al., 1994b). Protein focused on the activities of the C-terminal PAX3, interaction studies subsequently determined that the PAX7, and FKHR regions (Bennicelli et al., 1995, putative co-repressors DAXX, RB1, and HIRA bind 1999; Chalepakis et al., 1994b; Sublett et al., 1995). to PAX3, and for DAXX and HIRA, interactions with These C-terminal regions were examined independent PAX7 were also noted (Hollenbach et al., 1999; of the respective DNA binding domains by generating Magnaghi et al., 1998; Wiggan et al., 1998). Deletion fusions with the GAL4 DNA binding domain. In studies suggest that these proteins interact with the these GAL4 fusion constructs, the C-terminal PAX3, PAX3 homeodomain, though the DAXX interactions PAX7, and FKHR regions acted as highly potent also appear to involve the octapeptide and other N- activation domains with essential transactivation terminal regions. When co-expressed with PAX3, both domains located at the 3' end of each coding region DAXX and RB1 result in titratable inhibition of (Figure 3). The PAX3 and PAX7 transactivation PAX3-mediated transcriptional activation, thus sup- domains are serine- and threonine-rich, whereas the porting their function as transcriptional co-repressors. FKHR transactivation domain contains both acidic- In addition, DAXX can also bind PAX3 ± FKHR but and serine-, threonine-rich regions. In addition to is unable to repress transcriptional activation by the these domains, positive modifying elements were found fusion transcription factor. These ®ndings support the in adjacent regions. These data demonstrate that the transcriptional gain of function model and suggest that wild-type and fusion proteins contain comparably increased transcriptional potency of the fusion proteins potent, yet structurally distinct transcriptional activa- results from unresponsiveness to the repressive activity tion domains which are switched by the translocations of bound co-repressors. in ARMS. The ®nding of similar transcriptional potencies of the C-terminal domains and contrasting potencies of Transcriptional targets of wild-type and chimeric PAX the full-length proteins suggested that the activity of proteins the C-terminal activation domains is modulated by other portions of the full-length protein. To explore Using electrophoretic mobility shift assays in conjunc- this hypothesis, the GAL4 DNA binding domain was tion with partial or full-length protein constructs, joined to the full-length wild-type and fusion proteins putative PAX3 binding sites were identi®ed in several (Bennicelli et al., 1996, 1999). When fused to GAL4, mammalian genes. These sites were located within the full-length PAX3 ± FKHR or PAX7 ± FKHR activated 5' promoters of the MET, MITF, TYRP1, PDGFRA, transcription of a GAL4-dependent reporter gene with BCL2L1 (BCL ± XL), and RET genes as well as in the an activity 10-fold higher than the corresponding full- 3' untranslated region of the NF1 gene and the ®rst length wild-type protein. In addition, the full-length intron of the NRCAM gene (Epstein et al., 1995, 1996, PAX ± FKHR constructs had several-fold lower activ- 1998; Galibert et al., 1999; Kallunki et al., 1995; Lang ity than the C-terminal FKHR construct whereas the et al., 2000; Margue et al., 2000; Watanabe et al., full-length wild-type PAX3 or PAX7 construct had one 1998). These putative binding sites contain sequences

Oncogene Alveolar rhabdomyosarcoma gene fusions FG Barr 5742 resembling a paired box binding motif (NF1, MET and Phenotypic roles of wild-type PAX proteins: gene RET), a homeobox binding motif (PDGFRA and knockout studies BCL ± XL), or both motifs (MITF, TYRP1 and NRCAM). In one other study, a fragment from the The transcriptional studies of PAX3 ± FKHR and NCAM promoter was shown to bind to cellular PAX7 ± FKHR are consistent with the hypothesis that extracts expressing the paired box but not extracts the translocations activate the oncogenic potential of expressing the entire PAX3 protein and thus the PAX3 and PAX7 by dysregulating or exaggerating their signi®cance of this binding site is unclear (Chalepakis normal function in the myogenic lineage. Clues for this et al., 1994b). normal function may be deduced from the skeletal These binding sites were further investigated in muscle phenotype of splotch and Pax7 knockout mice. In assays in which a PAX3 or PAX3 ± FKHR expression the homozygous splotch animals, limb musculature fails construct was co-transfected with a reporter construct to develop whereas the axial musculature shows varying containing the binding sites. In these assays, PAX3 degrees of malformation (Franz et al., 1993). The defects activated transcription from MITF, TYRP1 and RET in muscle development appears to result from increased binding sites, whereas PAX3 repressed transcription apoptosis within the myogenic precursor pool of the from the NCAM element (Chalepakis et al., 1994b; lateral dermomyotome (Borycki et al., 1999) and from Galibert et al., 1999; Lang et al., 2000; Watanabe et al., failure of surviving myogenic precursors to migrate from 1998). In the case of the PDGFRA site, the ®nding of the somites into the limb buds (Bober et al., 1994; Daston activation by PAX3 ± FKHR but not PAX3 supports et al., 1996; Goulding et al., 1994). This limb musculature the gain of transcriptional function model described defect in splotch mice is associated with reduced previously (Epstein et al., 1998). In contrast, both expression of the Met receptor in myogenic progenitors PAX3 and PAX3 ± FKHR activated transcription from (Daston et al., 1996; Epstein et al., 1995; Yang et al., NF1 and BCL ± XL sites without substantial dier- 1996), and is also found in murine strains in which the ences in transcriptional activity between the wild-type gene encoding Met was mutated by gene targeting (Bladt and fusion proteins (Epstein et al., 1995; Margue et al., et al., 1995). The established role of Met in cell motility 2000). signaling and the ®nding that the gene encoding Met is a Additional studies investigated endogenous gene potential transcriptional target of the Pax3 protein expression changes following manipulations of PAX3 suggest that the migration problem is due to defective or PAX3 ± FKHR expression. Two widely used regulation of Met expression by the mutant Pax3 strategies are introduction of a PAX3 or PAX3 ± transcription factor. Therefore, these studies of splotch FKHR expression construct into speci®c cell types, and mice suggest possible roles for PAX3 in the stimulation analysis of homozygous splotch mice. The transfection of motility and maintenance of a viable population by approach demonstrated enhanced expression of MET, facilitating growth or inhibiting apoptosis. BCL ± XL, and RET and thus provides further support Additional studies of splotch mice and Pax7 knockout that these genes are direct transcriptional targets of mice revealed roles for Pax3 and Pax7 in facilitating PAX3 or PAX3 ± FKHR (Epstein et al., 1996; Lang et myogenic dierentiation. In particular, when the homo- al., 2000; Margue et al., 2000). A more complicated zygous Pax3 mutation is combined with a homozygous situation exists for PDGFRA in which transient Myf5 mutation, almost all trunk (as well as limb) transfection of PAX3 ± FKHR but not PAX3 into musculature fails to form (Tajbakhsh et al., 1997). This P19 cells activates PDGFRA expression whereas stable defect in myogenic development involves a failure to integration of either PAX3 or PAX3 ± FKHR in activate MyoD expression and subsequent myogenic NIH3T3 cells represses expression (Epstein et al., events. Furthermore, though mice with a homozygous 1998; Khan et al., 1998). These transfection studies inactivating Pax7 mutation generally have normal also identi®ed additional candidate targets, as exem- muscle development, these animals have a postnatal pli®ed by the cDNA array analysis of NIH3T3 cells muscular de®ciency evidenced by smaller muscle ®bres that stably express PAX3 ± FKHR (Khan et al., 1998). and decreased size of muscles such as the diaphragm This transcriptional pro®ling study showed increased (Seale et al., 2000). This muscle de®ciency is attributed to expression of IGF2, IGFBP5 and MERTK; and the absence of satellite cells, and implicates Pax7 in the decreased expression of FISP12, PMX1 and DAF. speci®cation of myogenic satellite cells from uncom- The homozygous splotch expression studies also mitted progenitors. Therefore, in addition to motility identi®ed genes whose expression is increased (e.g., and viability, PAX3 and PAX7 are proposed to regulate versican) or decreased (e.g., lbx1) in speci®c tissues in downstream myogenic events. Exaggeration of these the absence of functional Pax3 protein (Henderson et activities by PAX3 ± FKHR and PAX7 ± FKHR suggests al., 1997; Mennerich et al., 1998). Though these various several hypotheses relevant to ARMS pathogenesis. studies suggest possible target genes, these approaches do not speci®cally determine whether these genes are directly bound and regulated by PAX3 or PAX3 ± Phenotypic roles of wild-type and chimeric PAX FKHR. Some of these altered expression events may proteins: gene transfer studies occur further downstream such that additional steps or signals intervene between the PAX transcription factor Gene transfer studies in several model systems further and the assayed gene. investigated speci®c functions of these proteins and

Oncogene Alveolar rhabdomyosarcoma gene fusions FG Barr 5743 suggest that the fusion proteins may exert an oncogenic including the transcription factors MyoD, myogenin, eect through multiple pathways that exaggerate the Six1, and Slug (but not Myf5) as well as downstream normal role of the wild-type PAX proteins. In studies myogenic products such as various troponins and investigating growth control, under conditions in which myosin light chain (Khan et al., 1998). The ®nding wild-type PAX3 does not transform NIH3T3 cells or that PAX3 was not able to induce this myogenic chicken embryo ®broblasts, introduction of a PAX3 ± transcription program in NIH3T3 cells suggests that FKHR expression construct resulted in potent trans- PAX3 ± FKHR has greater transcriptional potency in forming activity (Lam et al., 1999; Scheidler et al., this less permissive cell environment. A ®nal study 1996). As a complementary experiment, introduction of focused on the ability of these proteins to inhibit a fusion of PAX3 to the KRAB transcriptional terminal dierentiation in two myogenic cell lines, repression domain into ARMS cells resulted in loss C2C12 myoblasts and MyoD-expressing 10T1/2 cells of transforming activity in culture and tumor suppres- (Epstein et al., 1995). Whereas dierentiation is sion in mice (Fredericks et al., 2000). Mutation analysis typically induced in most cells (86 ± 87%) by growth demonstrated that the homeodomain but not the factor withdrawal, PAX3 cDNA transfection reduced paired box is required for transformation by PAX3 ± the percentage of myosin-expressing colonies to 40 ± FKHR (Lam et al., 1999; Scheidler et al., 1996), and 43%, and PAX3 ± FKHR transfection further reduced thus altered expression of target genes whose binding this percentage to 17 ± 27%. In contrast to the sites require paired box function may not be required transformation studies, both the paired box and for cellular transformation. Mutation analysis also homeodomain were required to inhibit terminal indicated that the C-terminal FKHR activation domain dierentiation. The apparent contrast of these ®ndings is required for transformation. Furthermore, substitu- from those of the preceding two studies may be tion of the VP16 activation domain for the C-terminal explained by the hypothesis that these PAX proteins domain of PAX3 activated transforming activity, facilitate entry into the myogenic pathway but inhibit whereas substitution of the PAX3 activation domain the ®nal steps of the pathway. Alternatively, the for the C-terminal domain of PAX3 ± FKHR ablated myogenic consequences of aberrant PAX3 and transforming activity (Cao and Wang, 2000). These PAX3 ± FKHR expression may be dependent on the swapping studies support the gain of transcriptional speci®c cell type and other environmental features. function model, and suggest that the eect of the C- Several recent studies investigated the eect of terminal FKHR domain can be mimicked by other PAX3 ± FKHR on mammalian development, and have activation domains. found a diversity of phenotypic eects. Transgenic In addition to stimulating transforming activity, gene mice expressing PAX3 ± FKHR from a cloned murine transfer studies also indicated that PAX3 ± FKHR Pax3 promoter demonstrate pigmentary and neurolo- functions in the maintenance of cell viability by gical alterations reminiscent of the splotch phenotype inhibiting apoptosis. Treatment of ARMS cells with (Anderson et al., 2001). In a second study that used a an antisense oligonucleotide directed against the PAX3 gene targeting strategy to introduce the 3' FKHR translational start site resulted in a transient decrease in cDNA into the endogenous murine Pax3 locus, mice PAX3 ± FKHR protein expression (Bernasconi et al., with the heterozygous knock-in allele were not viable, 1996). This expression change was associated with a probably as a result of heart defects and a small signi®cant decrease in cell number as well as morpho- disorganized diaphragm (Lagutina et al., 2000). logical characteristics and a DNA fragmentation Finally, in a third study, ES cells expressing the pattern characteristic of apoptosis. In a parallel set of PAX3 ± FKHR cDNA from CMV expression elements studies, introduction of a tamoxifen-inducible PAX3 ± were used to generate chimeric mice that demonstrated KRAB construct into ARMS cells revealed evidence of an overgrowth phenotype reminiscent of Beckwith ± apoptosis when the construct is induced in low serum Wiedemann syndrome (Xie et al., 2001). Though the conditions or in tumor xenografts (Ayyanathan et al., phenotypic features varied in these animal systems, 2000). A role for BCL ± XL is supported by the ®nding tumor susceptibility was not found in any of these that BCL-XL was downregulated in association with studies. Therefore, the tumorigenic eects of the gene apoptosis in these PAX3 ± KRAB studies. fusion gene may require cooperating events or other A ®nal set of studies addressed the roles of PAX3 features that are not present in these animal models. and PAX3 ± FKHR in regulating myogenic pathways. The ability of PAX3 to activate the myogenic program in vitro was revealed by studies in which Pax3- Molecular diagnostic and therapeutic studies of ARMS expressing retroviruses were introduced into explanted gene fusions paraxial mesoderm and other embryonic tissues (Maroto et al., 1997). In this setting, Pax3 induced As the genetics and biology of the gene fusions in expression of the myogenic transcription factors ARMS were elucidated, studies were initiated to MyoD, Myf5, and myogenin as well as myogenic explore clinical applications of these gene fusions dierentiation products. Similarly, when a PAX3 ± (Arden et al., 1996; Barr et al., 1995; de Alava et al., FKHR expression construct was introduced into 1995; Downing et al., 1995; Frascella et al., 1998; NIH3T3 cells, cDNA array analysis revealed induction Reichmuth et al., 1996). Using RT ± PCR assays for of a large set of genes involved in myogenesis, the PAX3 ± FKHR and PAX7 ± FKHR transcripts,

Oncogene Alveolar rhabdomyosarcoma gene fusions FG Barr 5744 multiple tumors were assayed to determine the PAX3 or PAX7 with FKHR to generate PAX3 ± FKHR frequency of these fusions. Approximately 80% of and PAX7 ± FKHR chimeric genes. A consideration of ARMS cases expressed one of the two fusions, with the the biological and clinical data on these two fusion frequency of PAX3 ± FKHR being 5 ± 10 times that of genes indicates important similarities that point to a PAX7 ± FKHR. Molecular diagnostic studies also common fundamental mechanism in the pathogenesis revealed a small subset of ARMS cases that do not of this tumor. However, the biological and clinical data express either gene fusion. Though these negative also reveal several striking dierences between the two results may be explained by variable application of fusions that highlight the heterogeneity within this histopathologic diagnostic criteria or the suboptimal tumor category and distinctions between two highly quality of some samples, there is clearly a small subset related members of the paired box family. of well-characterized ARMS cases in which these gene The genetic changes resulting from the 2;13 fusions are not detectable by standard RT ± PCR translocation alter biological activity at the levels of assays. In these cases, the possibilities of rare variant protein function, gene expression, and protein localiza- fusions, other genetic events that can substitute for the tion. The result is high level, constitutively nuclear characteristic fusions, or a biologically distinct entity expression of a potent fusion transcription factor. with a similar histological appearance should also be These biological changes are postulated to promote considered. aberrant expression of genes that are transcriptional Comparison of the clinical characteristics of PAX3 ± targets of wild-type PAX3 and that normally function FKHR and PAX7 ± FKHR-expressing tumors showed in the control of early myogenic development. The that PAX7 ± FKHR tumors tend to occur in younger aberrant or inappropriate expression of these gene patients, more often present in extremities, are more products is postulated to contribute to oncogenic often localized lesions, and are associated with longer initiation or progression by stimulating cell behaviors event-free survival (Kelly et al., 1997). In contrast, such as growth and motility or inhibiting cell behaviors PAX3 ± FKHR tumors tend to occur in adolescents as such as apoptosis and terminal dierentiation. higher stage lesions in a wider variety of sites. The t(1;13) bears a strong similarity to the t(2;13) in Therefore, clinical heterogeneity in ARMS is associated that the PAX7 ± FKHR fusion product joins the PAX7 with genetic heterogeneity in the involved fusion genes, DNA binding domain and FKHR activation domain. and thus identi®cation of the speci®c gene fusion may Functional studies indicate that the PAX7 ± FKHR provide valuable information for management and protein functions an aberrant transcription factor outcome assessment. similar to PAX3 ± FKHR. In contrast to PAX3 ± In addition to providing diagnostic information, FKHR, the juxtaposition of PAX7 and FKHR studies are also being conducted to investigate whether regulatory elements is apparently not sucient for the fusions can be targeted by therapeutic strategies. As PAX7 ± FKHR overexpression, and instead an ampli- described, strategies were developed to antagonize ®cation event ensues to increase PAX7 ± FKHR gene PAX3 ± FKHR expression with antisense oligonucleo- copy number. This ®nding indicates a biological tides or to antagonize PAX3 ± FKHR function with an distinction between the two fusions, the potential engineered PAX3 ± KRAB transcriptional repressor signi®cance of which is indicated by the ®nding of (Ayyanathan et al., 2000; Bernasconi et al., 1996; clinical dierences between PAX3 ± FKHR and Fredericks et al., 2000). Another therapeutic strategy PAX7 ± FKHR-expressing tumors. involves transfer of a construct consisting of PAX Future investigations will integrate the gene expres- binding sites that regulate expression of a toxin- sion, target gene, and functional studies to develop a encoding gene (Massuda et al., 1997). Cell culture comprehensive understanding of the aberrant genetic studies demonstrated that this construct is selectively program induced by these translocations. Furthermore, toxic for cells expressing the PAX3 ± FKHR fusion. In increased eorts must be directed at identifying the a ®nal strategy, the immunogenicity of peptides collaborating events in ARMS tumorigenesis and spanning the fusion point is being tested (Mackall et developing appropriate model systems to recapitulate al., 2000). Animal studies provided evidence that these oncogenic pathways in a controlled fashion. In cytotoxic T lymphocytes directed against the fusion addition to further elucidating how the PAX3 ± FKHR peptide can be generated and will recognize and kill and PAX7 ± FKHR fusions contribute to tumorigen- PAX3 ± FKHR-expressing tumor cells. esis, these studies need to address the dierences between the two fusions in tumorigenic pathways. These studies will ultimately enhance the utility of Conclusions molecular genetics in the diagnosis and monitoring of this cancer, and will indicate directions in which In ARMS, the 2;13 and 1;13 translocations are possible therapeutic strategies can be developed to consistent and speci®c events that provide opportu- interrupt these tumorigenic pathways. nities to investigate the molecular etiology of this cancer, identify novel markers for diagnosis and monitoring, and develop therapeutic approaches Acknowledgments against tumor-speci®c targets. These translocations This work was supported in part by NIH grants CA64202, juxtapose the transcription factor-encoding genes CA71838 and CA89461.

Oncogene Alveolar rhabdomyosarcoma gene fusions FG Barr 5745 References

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Oncogene