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[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 mapping 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 containing 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 transcription 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

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. visualized by fluorescence microscopy as juxtaposed or hybrid sig- FKHR consists of three exons spanning 140 kb; the fork head domain nals. Comparison of this assay with an RT-PCR assay for the PAX3- is encoded by portions of exons 1 and 2, and the transcriptional FKHR fusion demonstrated excellent concordance in a series of RMS activation domain is encoded by the COOH-terminal portion of exon cases (15). A similar FISH assay was developed to detect the PAX7- 2 (11). The t(2;13) breakpoints occur within the 130-kb intron be- FKHR fusion (16). tween FKHR exons 1 and 2; this intron provides a large target for Multiple tumors have been assayed to determine the frequency rearrangements and allows disruption of the fork head DNA-binding of PAX3-FKHR and PAX7-FKHR fusions. In published studies, domain and in-frame fusion of the COOH-terminal FKHR transacti- the frequencies of PAX3-FKHR and PAX7-FKHR fusions in vation domain to the NHz-terminal PAX3 DNA-binding domain. A ARMS were 54-87% and 8-15%, respectively (15, 17-19). These similar fusion cannot be created by any other combination of PAX3 studies found a small subset of cases of histologically diagnosed and FKHR exons because of incompatible reading frames or loss of ARMS tumors (13-31%) that do not express PAX3-FKHR or needed functional domains. These findings support the premise that PAX7-FKHR fusions. Although these negative results may be rearrangements of PAX3 intron 7 and FKHR intron 2 are selected due explained by variable application of histopathological criteria or to functional constraints related to the genomic organization of PAX3 suboptimal samples, the possibilities of variant fusions or other and FKHR. genetic events that can substitute for the fusions should also be Chimeric Products Generated by t(1;13) considered. Although the majority, by far, of ERMS cases do not contain either fusion, PAX3-FKHR and PAX7-FKHR fusions were For the variant t(1;13), Southern blot and RT-PCR analyses indi- detected in a few cases diagnosed as ERMS. These findings cated that PAX7, another member of the paired box-containing tran- indicate that there is substantial but not perfect overlap between scription factor family, is rearranged in the t(1 ;13)-containing tumors molecular and histopathological analyses. and fused to FKHR (12). The PAX3 and PAX7 genes have very similar Analysis of clinical characteristics indicated that PAX3-FKHR and organization and highly homologous coding sequences. Therefore, the PAX7-FKHR are associated with different presentations and tumor t(1;13) results in a chimeric transcript consisting of 5' PAX7 and 3' behavior (20). Tumors with the PAX7-FKHR fusion occur more often FKHR regions, which is nearly identical in structure and organization in younger patients and in extremity sites than tumors with the to the PAX3-FKHR product of the t(2;13). PAX3-FKHR fusion. Furthermore, these PAX7-FKHR tumors more Molecular Diagnostic Evaluation of Chimeric Products often present as localized lesions without metastatic disease at diagnosis. Kaplan-Meier analysis showed significantly longer event-free The consistent structure of the chimeric transcripts permits detec- survival in the PAX7-FKHR group. These findings indicate that there tion of these RNA species by RT-PCR. A single consensus primer was is a close association between the genetic and clinical heterogeneity identified in the homologous 5' PAX3 and 5' PAX7 regions and within the ARMS tumor category. 1712s

Expression Characteristics of Wild-Type and Chimeric gested that the wild-type PAX3 protein binding affinity is 3.5-fold Products greater than that of the PAX3-FKHR fusion (8). Despite this difference in binding function, the PAX3-FKHR fusion protein is a more Use of RNase protection assays for the wild-type PAX3 and PAX3- potent transcriptional activator than the wild-type PAX3 protein (8, FKHR transcripts permitted detection of several-fold greater expres- 27). The regulatory function was analyzed by transfecting expression sion of PAX3-FKHR relative to wild-type PAX3 in 18 of 19 tumor constructs into mammalian cells along with a reporter construct con- specimens (21). Immunoprecipitation analysis with PAX3-specific taining a minimal adenoviral Elb promoter and PAX3 binding sites. antiserum confirmed that PAX3-FKHR is also overexpressed at the In studies with a variety of cell lines and binding sites, the wild-type protein level. In addition, using similar assays for wild-type PAX7 PAX3 protein induced a low but detectable level of transcriptional and PAX7-FKHR transcripts, analysis of eight tumor specimens re- activation, whereas the PAX3-FKHR fusion protein induced up to vealed that PAX7-FKHR is consistently overexpressed relative to 10-100-fold more activity. wild-type PAX7. These findings indicate that overexpression of PAX3-FKHR and PAX7-FKHR relative to wild-type PAX3 and To investigate the increased transcriptional potency of PAX3- PAX7 is characteristic of ARMS tumors and suggest that overexpres- FKHR, initial studies focused on the transcriptional regulatory activ- sion generates a level of fusion product above a critical threshold for ities of the COOH-terminal PAX3 and FKHR regions. These COOH- oncogenic activity. terminal regions were examined independent of their respective DNA A striking difference in the basis of PAX3-FKHR and PAX7- binding domains by generating fusions with the GALA DNA-binding FKHR overexpression was revealed by FISH and quantitative South- domain (28). In these GAL4 constructs, both the COOH-terminal ern blot studies. In the FISH assays, hybridization patterns indicative PAX3 and FKHR regions acted as highly potent activation domains of in vivo amplification on extrachromosomal elements were identi- (Fig. 1). Deletion mapping demonstrated that these comparably potent fied in several PAX7-FKHR-positive ARMS cases (16). These find- domains are structurally distinct. The finding of similar transcriptional ings were confirmed and extended by quantitative Southern blot potencies of the COOH-terminal PAX3 and FKHR domains and studies that determined the relative copy number of wild-type and contrasting potencies of the full-length PAX3 and PAX3-FKHR pro- rearranged alleles (16, 21). Using a combination of the two method- teins suggested that the activity of the COOH-terminal activation ologies, fusion gene amplification was detected in 1 of 24 PAX3- domains is modulated by other portions of the full-length protein. To FKHR cases and 8 of 9 PAX7-FKHR cases. These findings indicate explore this hypothesis, additional fusions with the GAL4 DNA- that PAX7-FKHR overexpression is directly related to PAX7-FKHR binding domain were analyzed and revealed that a negative modula- fusion gene amplification, whereas PAX3-FKHR overexpression is tory domain is present in the NHz-terminal PAX3 region and that this generally copy number independent. In the PAX7-FKHR subset, domain effectively inhibits the activity of the COOH-terminal PAX3 translocation and amplification occur sequentially to alter both gene activation domain (27). Sublocalization studies indicated that this structure and copy number and, thereby, activate oncogenic activity negative modulatory domain is bipartite and overlaps the DNA- by complementary strategies. binding domain. In contrast to the effect on the COOH-terminal PAX3 The mechanism of overexpression in PAX3-FKHR-expressing tu- activation domain, the NH2-terlrtinal PAX3 negative modulatory do- mors was further analyzed by mRNA stability and transcription level main has a very modest effect on the COOH-terminal FKHR activa- assays (21). RNase protection analysis of RNA isolated from actino- tion domain. Therefore, the t(2;13) appears to create a potent tran- mycin D-treated ARMS cell lines demonstrated that PAX3-FKHR scriptional activator by introducing a COOH-terminal transactivation and PAX3 transcripts have comparable stabilities. However, nuclear domain that is relatively insensitive to the negative modulatory effects runoff analysis using hybridization targets flanking the t(2; 13) break- of the NH2-terminal PAX3 domain. point revealed that PAX3-FKHR is more actively transcribed than PAX3. Therefore, despite the common feature of fusion gene over- Phenotypic Roles of Wild-Type and Chimeric Proteins expression in ARMS tumors, the mechanism of overexpression is The transcriptional studies indicate that the t(2; 13) results in a gain fusion gene specific. PAX3-FKHR overexpression is the result of a of function and are consistent with the hypothesis that the fusion copy number-independent increase in transcriptional rate that is pos- activates the oncogenic potential of PAX3 by exaggerating its normal tulated to result from a favorable juxtaposition of PAX3 and FKHR function in the myogenic lineage. Clues for this normal function may regulatory elements in the fusion gene. In contrast, PAX7-FKHR be deduced from the skeletal muscle phenotype of splotch mice in overexpression results from a second genetic alteration, amplification which the murine Pax3 gene is homozygously mutated (29-32). In of the PAX7-FKHR fusion gene. These findings indicate important these animals, the limb musculature fails to develop, whereas the axial biological differences between the PAX3-FKHR and PAX7-FKHR musculature is reduced but develops relatively normally. The defect in fusion genes and suggest significant differences in the regulation of limb musculature appears to result from the failure of myogenic expression of these fusion genes. precursors to migrate from the somites into the limb buds (33). This Transcriptional Properties of Wild-Type and Chimeric Proteins limb musculature defect in splotch mice is associated with reduced expression of the c-met receptor in myogenic progenitors (34, 35). In the fusion products, the PAX3/PAX7 DNA-binding domain is The established role of c-met in cell motility signaling and the finding intact, whereas the FKHR DNA-binding domain is split (Fig. t). that c-met is a potential transcriptional target of the Pax3 protein Because mutations of other fork head domains inactivate DNA- suggests that the migration problem is due to defective regulation of binding function (22, 23), the PAX3/PAX7 domains are postulated to c-met expression by the mutant Pax3 transcription factor (34). provide the DNA binding specificity for the fusion transcription Gene transfer studies have investigated the activity of PAX3-FKHR factors. Due to the paucity of known mammalian gene targets for in several model systems. One study indicated a role for PAX3-FKHR PAX3 and PAX7, initial transcriptional studies focused on their in the control of cell growth (36). Under conditions in which wild-type ability to bind e5, a sequence from the region upstream of the PAX3 does not transform chicken embryo fibroblasts, the PAX3- Drosophila even-skipped gene that binds the Drosophila paired gene FKHR fusion has been shown to have potent transforming activity. product (24-26). Electrophoretic mobility shift experiments revealed Another study focused on the ability to inhibit the myogenic differ- specific binding of the PAX3-FKHR protein to these sites and sug- entiation of C2C12 myoblasts or MyoD-expressing 10T1/2 cells (37). 1713s

Differentiation is typically induced by the withdrawal of growth 39, 1998. factors and results in myosin expression in the majority of cells. 11. Davis, R. J., Bennicclli, J. L., Macina, R. A., Nycum, L. M., Biegel, J. A., and Barr, F. G. Structural characterization of the FKHR gene and its rearrangement in alveolar Transfection of PAX3 cDNA reduced the percentage of myosin- rhabdomyosarcolna. Hum. Mol. Genet., 4: 2355-2362, 1995. expressing colonies from 86-87% to 40-43%, and PAX3-FKHR 12. Davis, R. J., D'Cruz, C. M., Lovell, M. A., Biegel, J. A., and Barr, F. G. Fusion of transfection further reduced this percentage to 17-27%. A third study PAX7 to FKHR by the variant t(1;13)(p36;q14) translocation in alveolar rhabdomyosarcoma. Cancer Res., 54: 2869-2872, 1994. indicated that PAX3-FKHR functions in the maintenance of ARMS 13. Barr, F. G., Xiong, Q-B., and Kelly, K. A consensus polymerase chain reaction- cellular viability by inhibiting apoptotic pathways (38). Using an oligonucleotide hybridization approach for the detection of chromosomal transloca- antisense oligonucleotide directed against the PAX3 translational start tions in pediatric bone and soft tissue cancers. Am. J. Clin. Pathol., 104: 627-633, 1995. site, a transient decrease in PAX3-FKHR protein was induced in 14. Biegel, J. A., Nycum, L. M., Valentine, V., Barr, F. G., and Shapiro, D. N. Detection ARMS cells that was associated with a significant drop in cell num- of the t(2;13)(q35;q14) chromosomal translocation and PAX3-FKHR fusion in alve- ber. On the basis of the morphological appearance of the cells as well olar rhabdomyosarcoma by fluorescence in situ hybridization. Genes Chromosomes Cancer, 12: 186-192, 1995. as the finding of DNA fragmentation, this reduced viability was 15. Barr, F. G., Chatten, J., D'Cruz, C. M., Wilson, A. E., Nanta, L. E., Nycum, L. M., concluded to be the result of apoptosis. The findings of these various Biegel, J. A., and Womer, R. B. Molecular assays for chromosomal translocations in the diagnosis of pediatric soft tissue sarcomas. J. Am. Med. Assoc., 27: 553-557, phenotypic studies indicate that PAX3-FKHR can influence cellular 1995. growth, differentiation, and apoptosis and thus may exert an onco- 16. Barr, F. G., Nauta, L. E., Davis, R. J., Schafer, B. W., Nycum, L. M., and Biegel, J. A. genic effect through multiple pathways that exaggerate the normal In vivo amplification of the PAX3-FKHR and PAX7-FKHR fusion genes in alveolar rhabdomyosarcoma. Hum. Mol. Genet., 5: 15-21, 1996. role of the wild-type PAX3 protein. 17. Downing, J. R., Khandekar, A., Shurtleff, S. A., Head, D. R., Parham, D. M., Webber, B. L., Pappo, A. S., Hulshof, M. G., Conn, W. P., and Shapiro, D. N. Multiplex Conclusions RT-PCR assay for the differential diagnosis of alveolar rhabdomyosarcoma and Ewing's sarcoma. Am. J. Pathol., 146: 626-634, 1995. 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Kelly, K. M., Womer, R. B., Sorensen, P. H., Xiong, Q-B., and Barr, F. G. Common and variant gene fusions predict distinct clinical phenotypes in rhabdomyosarcoma. mechanism in the pathogenesis of this tumor. However, the biological J. Clin. Oncol., 15: 1831-1836, 1997. and clinical data also reveal several striking differences between the 21. Davis, R. J., and Barr, F. G. Fusion genes resulting from alternative chromosomal two fusions that highlight the heterogeneity within this tumor category translocations are overexpressed by gene-specific mechanisms in alveolar rhabdomyosarcoma. Proc. Natl. Acad. Sci. USA, 94: 8047-8051, 1997. and distinctions between two highly related members of the paired 22. Lai, E., Prezioso, V. R., Smith, E., Litvin, O., Costa, R. H., and Darnell, J. E., Jr. box family. Future investigations will refine the alterations in gene HNF-3A, a hepatocyte-enriched transcription factor of novel structure is regulated transcriptionally. 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Discussion

Speaker: Can you tell us anything about the normal functions of genesis and, if not, is there a compelling reason to think it is a gain of FKHR and whether FKHR function is, in a sense, deregulated in the function that leads to these tumors, as opposed to some loss of translocations? function associated with the translocation? Dr. Barr: FKHR appears to be a widely, if not ubiquitously, Dr. Barr: A number of model cell culture studies have shown that expressed gene, although its pattern of expression during development cells such as NIH 3T3 and chicken embryo fibroblasts can be trans- has not yet been established. Recently, a subfamily of genes related to formed by PAX3-FKHR. Similarly, in C2C12 myoblasts, their dif- FKHR has emerged that includes genes on chromosome 6 and the X ferentiation can be inhibited by PAX3-FKHR. Finally, Beat Schafer chromosome. This subfamily shows homology to Daf-16, a Caeno- showed that down regulation of PAX3-FKHR expression in an alve- rhabditis elegans gene that seems to be involved in insulin signaling. olar rhabdomyosarcoma cell line causes those cells to go through A number of groups are currently studying whether FKHR or its two apoptosis. At least at those three levels, there seems to be a gain of cousins, AF6q21 and AFX, may also be involved in insulin-regulated function such that PAX3-FKHR may be able, in certain contexts, to pathways. This information would suggest that FKHR may be in- transform cells, inhibit differentiation, and inhibit apoptosis, which volved in signaling pathways and may, in fact, be a regulated tran- would all be desirable oncogenic functions. scription factor. Whether those regulatory signals are still being re- Speaker: Do you have any data about whether MET is overex- ceived by PAX3-FKHR remains to be seen. One of the major pressed in rhabdomyosarcomas? regulatory regions may be in the NH 2 terminus of FKHR, which Dr. Barr: For the alveolar rhabdomyosarcomas, there is a relation- would be removed in the fusion and would then change FKHR from ship between PAX3-FKHR expression and MET expression. Exactly being a regulated transcription factor to a constitutive activator in the how one determines whether increased expression is true overexpres- context of the PAX3-FKHR fusion. sion is somewhat dependent on the system to which you compare Dr. Mina Bissell: In the model system that you have created in these expression levels. In embryonal rhabdomyosarcomas, for in- culture, where you put in PAX3-FKHR and find increased MET stance, we find low expression levels in some cases, but there actually expression, what are the physiological consequences of increasing is a subset of embryonal rhabdomyosarcomas that express relatively MET? Can you actually get the same phenotype by just overexpress- high levels of MET. Therefore, I do not know whether these levels ing MET? represent overexpression or just what is typical for these sublineages. Dr. Barr: The PAX3-FKHR-transfected RD subclones are not In addition, aside from PAX3-FKHR-induced expression, there are phenotypically different in culture. The problem is that RD, like most certainly other mechanisms for inducing MET expression, as shown tumor cell lines, is a highly evolved tumor cell line that has already by the embryonal rhabdomyosarcomas as well as all of other tumor gone through many events to acquire transformed behavior in culture; types arising from epithelial lineages. I do not think adding yet another additional insult is going to cause them to be any more transformed. If anything, they may be a little less Dr. Phillip Sharp: The phenotype associated with this fusion on- transformed, basically because we have perturbed the steady state. cogene is that of a generic dominant oncogene. Where is the speci- The question of whether MET expression can reproduce any of the ficity that causes this fusion to confer phenotype on this cell type? phenotypes associated with PAX3-FKHR expression is a set of ex- Dr. Barr: The question is why is this fusion only found in this periments that we are planning to conduct in the future. tumor of the myogenic lineage. In fact for most cancer-associated Dr. George Vande Woude: Did you look to see if the MET receptor translocations, these translocations are specific for very narrow lin- was activated either in the original tumor or in the embryonal rhab- eages. The basis of this specificity is unclear at this point. For PAX3 domyosarcoma cells into which you introduced PAX3-FKHR? The and PAX7, part of the specificity arises because these genes are MET receptor in the tumor may be activated through an autocrine expressed with such limited distribution in the neural and the myo- process because often the cells of mesenchymal origin endogenously genic lineages. However, I cannot explain why these or similar express the factor. fusions are not found in tumors of neural cells. I think that the Dr. Barr: We have not examined that issue. I think other groups specificity is probably associated with constraints either at the level of have looked and shown hepatocyte growth factor expression within expression, at the level of rearrangement, or at the level of function the environment of these tumors, so the hypothesis currently stands that somehow pins specific fusions to particular lineages. The specif- that there is activated signaling through MET, but that issue still needs ics of these constraints are unknown for most of these translocations. to be formally addressed. To explore these issues, it will be important to examine phenotypic Dr. Mark Israel: Have you shown in either culture or mouse models consequences not only in generic model systems but in experimental that the fusion products are actually oncogenic or contribute to onco- systems more closely related to these specific lineages.

1715s

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1999 American Association for Cancer Research. The Role of Chimeric Paired Box Transcription Factors in the Pathogenesis of Pediatric Rhabdomyosarcoma

Frederic G. Barr

Cancer Res 1999;59:1711s-1715s.

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