Frederic G. Barr, M.D., Ph.D.
Associate Professor, Dept of Pathology and Laboratory Medicine

Cancer Biology Program

Address

505C Stellar Chance
422 Curie Boulevard
Philadelphia, PA 19104-6069

Office tel.: 215 898-0884
Lab tel.: 215 898-0883
Fax: 215 898-4227
E-mail: barrfg@mail.med.upenn.edu

Williams College: BA (Chemistry), 1980.

Washington University: PhD (Molecular Biology), 1987.

Washington University: MD, 1987.

Research Interests

• rhabdomyosarcoma (a cancer of the muscle lineage), chromosomal translocations, paired box transcription factors

Key words: rhabdomyosarcoma (a cancer of the muscle lineage), chromosomal translocations, paired box transcription factors, gene amplification.

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Description of Research

The major focal points of my research program are chromosomal translocations, paired box transcription factors, and the pediatric malignancy rhabdomyosarcoma. These issues are unified by a major research focus on the 2;13 and 1;13 chromosomal translocations in the pediatric cancer alveolar rhabdomyosarcoma (ARMS). My laboratory's previous studies identified PAX3 and PAX7, which encode members of the paired box transcription factor family, as the chromosome 2 and chromosome 1 loci disrupted by these translocations. We subsequently cloned FKHR, which encodes a novel member of the fork head transcription factor family, as the chromosome 13 locus joined to these genes to generate PAX3-FKHR and PAX7-FKHR chimeric genes. These chimeric genes encode fusion proteins containing the PAX3/PAX7 DNA binding domain and the FKHR transcriptional activation domain. Our studies demonstrated that these gene fusion events result in alterations at the level of protein function, gene expression, and subcellular localization. The end result of these alterations is high levels of exclusively nuclear localized chimeric transcription factors that inappropriately activate transcription of genes with PAX3/PAX7 DNA binding sites.

More recent studies in my laboratory examined the downstream effects of these chimeric transcription factors. Though PAX3-FKHR is capable of exerting transforming activity in immortalized cell lines such as NIH3T3 murine fibroblasts, this oncogenic activity is mostly demonstrable at low expression levels. In contrast, at higher expression levels comparable to the levels in human ARMS tumors, PAX3-FKHR causes cell death and/or growth suppression in various murine and human cell lines. Therefore, these higher "physiologic" levels are not tolerated by non-ARMS cells whereas human ARMS cells can tolerate these high expression levels. We hypothesize that additional genetic alterations are necessary in ARMS to attenuate the toxic/growth suppressive effects of the fusion protein. In current studies, we are studying the effect of the fusion protein in human myogenic cells. We identified an immortalized non-transformed human myogenic cell line for these studies and found conditions under which PAX3-FKHR will transform these cells in culture and cause tumors in mice.

In my laboratory's current studies, we are investigating several questions:

1. What are the phenotypic effects of these fusion products in various cell types?
2. What are the downstream target genes of these fusion transcription factors?
3. How do phenotype and target genes change with varying fusion gene expression levels?
4. What are the frequent secondary genetic alterations in ARMS?
5. Do these secondary alterations collaborate with PAX3-FKHR cell culture & animal systems?

In an effort to identify genetic events that may collaborate with the gene fusions in the molecular pathogenesis of ARMS, we initiated a genome-wide screen for amplifications, deletions, and other genomic copy number changes. In particular, we screened 59 ARMS cases (33 PAX3-FKHR, 10 PAX7-FKHR, and 16 fusion-negative) on a 50K Affymetrix GeneChip copy number array. Based on previous data, we focused on amplification events and found evidence of amplification in 56% of cases, ranging from 48% of PAX3-FKHR cases to 90% of PAX7-FKHR cases. Multiple regions of the genome were found to have high recurrent amplicons, including 12q14, 13q14, and 13q31. The candidate critical genes in these amplicons are being localized to the minimal common region of amplification and then the level of expression is being compared between amplified and non-amplified cases using data from accompanying Affymetrix GeneChip expression arrays.

Recent Publications

Xia S.J., Barr F.G. (2004). Analysis of the transforming and growth suppressive activities of the PAX3-FKHR oncoprotein. Oncogene, 9, 6864-71

Tomescu O, Xia, SJ, Strezlecki, D., Bennicelli, J.L., Ginsberg, J., Pawel, B., Barr, F.G. (2004). Inducible short-term and stable long-term cell culture systems reveal that the PAX3-FKHR fusion oncoprotein regulates CXCR4, PAX3, and PAX7 expression. Lab Invest. 84, 1060-70.

Du, S., Lawrence, E.J., Strzelecki, D., Rajput, P., Xia, S.J., Gottesman, D.M., Barr, F.G. (2005). Co-expression of alternatively spliced forms of PAX3, PAX7, PAX3-FKHR, and PAX7-FKHR with distinct DNA binding and transactivation properties in rhabdomyosarcoma. Int J Cancer. 115, 85-92.

Barr, F.G., Smith, L.M., Lynch, J.C., Strzelecki, D., Parham, D.M., Qualman, S.J., Breitfeld, P.B. (2006). Examination of gene fusion status in archival samples of alveolar rhabdomyosarcoma entered on the IRS-III trial: a report from the Children's Oncology Group, J Mol Diag, 8, 202-8.

Xia, S.J., Rajput, P., Strzelecki, D.M., Barr, F.G. (2006). Analysis of genetic events that modulate the oncogenic and growth suppressive activities of the PAX3-FKHR fusion oncoprotein. Lab Invest. 87, 318-325.

Lab

Rotation Projects

1. Phenotypic consequences of PAX3-FKHR and PAX7-FKHR in human myoblasts - We propose that these fusion proteins may affect expression of genes controlling the processes of proliferation, apoptosis, differentiation, or motility. We are using inducible and constitutive retroviral constructs to introduce these fusions into immortalized human myoblast cell lines. The studies that are now ready to be performed are….
   
   
   a. Assay for various alterations in cellular phenotype using appropriate cell culture assays
   b. Use in vitro mutagenesis to investigate role of specific domains of transcription factor.
   c. Compare PAX3-FKHR vs. PAX7-FKHR and various splice forms of paired box.
   
   
2. Expression consequences of PAX3-FKHR and PAX7-FKHR in human myoblasts - Using cells constitutively expressing PAX3-FKHR, we performed an initial set of expression profiling experiments and identified a set of downstream genes in cells that are transformed by PAX3-FKHR (and collaborating oncogenes). The studies that are ready to be performed are….
   
   
   a. Validate selected downstream genes in both constitutive & inducible expression systems
   b. For selected genes, test promoter constructs in inducible system
   c. For selected genes, test expression constructs for functional activity in myoblasts
   
   
3. Defining critical genes in recurrent ARMS amplicons - Using the copy number data from the ARMS panel, we identified multiple recurrent amplicons, which we hypothesize to represent the site of overexpressed genes that collaborate with the gene fusions in ARMS pathogenesis. For each amplicon, comparison of the copy number data from the various amplified cases will permit designation of a minimal region of amplification. Comparison of this mapping data with human genome databases will then permit identification of the involved genes. The gene expression consequences of these copy number changes will then be investigated by corresponding analysis of data from Affymetrix expression arrays and determination which amplified genes are also significantly overexpressed.
   
   
4. Testing the role of ARMS collaborating events. - Several candidate genes with varying but interesting functions will be identified in the analysis of the ARMS amplicons. Selected genes will be introduced into human PAX3-FKHR-expressing myoblast and ARMS cell lines using retroviral constructs. Various phenotypic assays in cell culture as well as animal assays will be considered to investigate the function of these amplified genes.
   

Lab Personnel:

Jenny Xia, Ph.D. - Senior Research Investigator
Jennifer Reichek, M.D. - Postdoctoral Researcher
Karen Chagin, M.D. - Postdoctoral Researcher
Donna Strzelecki, B.S. - Research Technician
Chune Zhang, B.S. - Research Technician
Dara Holder - Undergraduate Student
Lucas Cocia - Undergraduate Student