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Supplementary Figure 1 from Credentialing a Preclinical Mouse Model of Alveolar Rhabdomyosarcoma

Supplementary Table 10 from Credentialing a Preclinical Mouse Model of Alveolar Rhabdomyosarcoma

The highly aggressive muscle cancer alveolar rhabdomyosarcoma (ARMS) is one of the most common soft tissue sarcoma of childhood, yet the outcome for the unresectable and metastatic disease is dismal and unchanged for nearly three decades. To better understand the pathogenesis of this disease and to facilitate novel preclinical approaches, we previously developed a conditional mouse model of ARMS by faithfully recapitulating the genetic mutations observed in the human disease, i.e., activation of Pax3:Fkhr fusion gene with either p53 or Cdkn2a inactivation. In this report, we show that this model recapitulates the immunohistochemical profile and the rapid progression of the human disease. We show that Pax3:Fkhr expression increases during late preneoplasia but tumor cells undergoing metastasis are under apparent selection for Pax3:Fkhr expression. At a whole-genome level, a cross-species gene set enrichment analysis and metagene projection study showed that our mouse model is most similar to human ARMS when compared with other pediatric cancers. We have defined an expression profile conserved between mouse and human ARMS, as well as a Pax3:Fkhr signature, including the target gene, SKP2. We further identified 7 “druggable” kinases overexpressed across species. The data affirm the accuracy of this genetically engineered mouse model. [Cancer Res 2009;69(7):2902–11]

Supplementary Figure 2 from Credentialing a Preclinical Mouse Model of Alveolar Rhabdomyosarcoma

Supplementary Methods and Materials from Credentialing a Preclinical Mouse Model of Alveolar Rhabdomyosarcoma

Supplementary Figure 5 from Credentialing a Preclinical Mouse Model of Alveolar Rhabdomyosarcoma

Supplementary Tables 1-7 from Credentialing a Preclinical Mouse Model of Alveolar Rhabdomyosarcoma

Supplementary Figure 3 from Credentialing a Preclinical Mouse Model of Alveolar Rhabdomyosarcoma

Supplementary Table 8 from Credentialing a Preclinical Mouse Model of Alveolar Rhabdomyosarcoma

Supplementary Table 9 from Credentialing a Preclinical Mouse Model of Alveolar Rhabdomyosarcoma

Supplementary Figure 4 from Credentialing a Preclinical Mouse Model of Alveolar Rhabdomyosarcoma

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Supplementary Figure Legends 1-5 from Credentialing a Preclinical Mouse Model of Alveolar Rhabdomyosarcoma

The highly aggressive muscle cancer alveolar rhabdomyosarcoma (ARMS) is one of the most common soft tissue sarcoma of childhood, yet the outcome for the unresectable and metastatic disease is dismal and unchanged for nearly three decades. To better understand the pathogenesis of this disease and to facilitate novel preclinical approaches, we previously developed a conditional mouse model of ARMS by faithfully recapitulating the genetic mutations observed in the human disease, i.e., activation of Pax3:Fkhr fusion gene with either p53 or Cdkn2a inactivation. In this report, we show that this model recapitulates the immunohistochemical profile and the rapid progression of the human disease. We show that Pax3:Fkhr expression increases during late preneoplasia but tumor cells undergoing metastasis are under apparent selection for Pax3:Fkhr expression. At a whole-genome level, a cross-species gene set enrichment analysis and metagene projection study showed that our mouse model is most similar to human ARMS when compared with other pediatric cancers. We have defined an expression profile conserved between mouse and human ARMS, as well as a Pax3:Fkhr signature, including the target gene, SKP2. We further identified 7 “druggable” kinases overexpressed across species. The data affirm the accuracy of this genetically engineered mouse model. [Cancer Res 2009;69(7):2902–11]

We previously identified increased copies of CEBPB in a subset of Ewing sarcoma (ES) tumors, and this gain was associated with worse clinical outcome. Due to CEBPB's role in cell growth and differentiation, as well as the gene's ability to transform normal mammary epithelial cells, we hypothesized that CEBPB acts as an oncogene in ES by increasing cell proliferation and transformation. To address this hypothesis, we altered gene expression using viral gene delivery systems in ES cell lines. First, we tested if CEBPB is a target of the EWS-FLI1 fusion protein using a FLI1 shRNA to knockdown EWS-FLI1 in an ES cell line (A673), followed by re-expression of the fusion gene. We then measured expression of the three C/EBPβ (protein) isoforms by western blot. Knockdown of EWS-FLI1 expression led to decreased protein expression of all three C/EBPβ isoforms. Re-expression of EWS-FLI1 rescued C/EBPβ protein expression, suggesting that C/EBPβ is a target of EWS-FLI1. To explore the functional consequence of altered expression of CEBPB, we transduced ES cell lines to knockdown, overexpress, and rescue C/EBPβ. Changes in protein expression were confirmed by western blot. Following transduction and antibiotic selection, cell proliferation and colony formation were measured by quantification of cellular ATP (Cell Titer Glo, Promega). Overexpression of C/EBPβ-1, the largest of the three C/EBPβ isoforms, led to a significant (p Citation Format: Lisa M. Abegglen, Jamie D. Gardiner, Clinton C. Mason, Bryce E. Carter, Elizabeth A. Schackmann, Marcus Stucki, Angelica R. Putnam, R Lor Randall, Heinrich Kovar, Stephen L. Lessnick, Joshua D. Schiffman. Functional validation of CEBPB as an oncogenic target of EWS-FLI1 in Ewing sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 483. doi:10.1158/1538-7445.AM2015-483

Purpose: The purpose of this study is to investigate whether individuals with Ewing's sarcoma are at increased risk of developing other cancers. The presence of excess cancers in first-, second-, and third-degree relatives of ES cases is also investigated. Background: Ewing's sarcoma (ES) is a rare, aggressive bone cancer that affects children and young adults. In 85% of ES cases, a chromosomal translocation between the EWSR1 gene on chromosome 22 and the FLI1 gene on chromosome 7 is observed in somatic cells. In the remainder of cases, the EWSR1 gene fuses to other ETS family gene members. The resulting EWS/ETS fusion protein formed in ES cases dysregulates target genes–thereby promoting oncogenesis. Methods: The Utah Population Database (UPDB) is a population-based genealogy linked to both the Utah Cancer Registry and to hospital data from the University of Utah Health Sciences Center. We used the UPDB to calculate the relative risks (RR) of 35 cancer sites in ES cases and in their relatives. RR was calculated using age-, birth-state- (Utah vs. non-Utah), birth county- (urban vs. rural) and birth-year-specific cancer rates. Results: We identified 133 individuals with ES who had ancestors in the UPDB. Brain cancer and complex genotype/karyotype sarcomas were observed at significant excess in more than one ES case (RR = 10.26; 95% CI: 1.24, 37.05; RR = 28.33, 95%CI: 3.43, 102.33, respectively). Individuals with ES had 695 first-degree, 2129 second-degree, and 6285 third-degree relatives in the UPDB. RRs for four cancer sites were significantly elevated in first-degree relatives: prostate (1.75; 1.06, 2.94), female genital (6.38; 1.13, 23.06), brain (3.68; 1.26, 9.42), and lung (2.31; 1.01, 5.04). In second-degree relatives, there was a significantly increased risk of four cancers: Non-Hodgkins lymphoma (2.07; 1.16, 3.42), breast (1.47; 1.11, 2.01), malignant peripheral nerve sheath (15.63; 1.89, 56.45), and tongue (4.02; 1.10, 11.75). Lastly, third-degree relatives had a significantly increased risk for five cancer sites: acute lymphocytic leukemia (3.74; 1.75, 7.70), stomach (1.87; 1.17, 3.03), prostate (1.23; 1.04, 1.49), brain (1.86; 1.07, 3.24), and gallbladder (2.73; 1.08, 6.37). Conclusion: The increased risk of several cancers other than ES in ES cases and in their relatives suggests the possibility of an inherent genetic predisposition to mutagenesis that predisposes both to observed mutations and chromosomal rearrangements seen in ES cases and to additional cancers (primarily brain and prostate) in individuals who do not have ES. Citation Format: Diana Abbott, R. Lor Randall, Joshua Schiffman, Stephen Lessnick, Lisa A. Cannon-Albright. A population-based survey of excess cancers observed in Ewing's sarcoma and in their first-, second-, and third-degree relatives. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2748. doi:10.1158/1538-7445.AM2015-2748

Study Rationale: Unlike other cancer models, the risk factors predisposing patients to the development of Ewing's sarcoma and predicting subsequent disease behavior are largely unknown. Interestingly, the incidence of Ewing's sarcoma is highest in populations of European descent, which is 10X higher than the incidence observed in those of African descent. Furthermore, cure rates and overall survival are significantly worse in patients of African descent. Recently, it has been shown that the EWS/FLI fusion product selectively binds to a (GGAA)n microsatellite response element embedded within the promoter region of key EWS/FLI-target oncogenes. An increasing number of these repeats also appears to positively affect DNA binding and gene activation. We hypothesize that differences in polymorphic GGAA microsatellites within the promoter regions of key EWS/FLI-target genes may exist in African and European populations, contributing to observed differences in Ewing's sarcoma incidence and outcomes. Methods: Genomic DNA samples were obtained from 100 subjects each of African and European descent. The microsatellite regions within the promoter elements of two key EWS/FLI target genes (NR0B1 and CAV1) were PCR amplified, cloned and sequenced. Microsatellite sequences were analyzed and the overall length of the microsatellite, the total number of GGAA repeats and the maximal number of consecutive repeats were quantified. Data was statistically interpreted using a comparative two-tailed t-test. Results: Measurements of the CAV1 microsatellite response element were quite similar for both African and European subjects. In sharp contrast, marked inter-ethnic variation was observed for the NR0B1 GGAA microsatellite response element. African samples had a significantly longer GGAA microsatellite, a greater number of total (GGAA)n repeats and longer consecutive (GGAA)n repeats (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3979. doi:1538-7445.AM2012-3979