Credentialing a preclinical mouse model of alveolar rhabdomyosarcoma

Abstract

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.

Figure 1

High penetrance of conditional mouse model of ARMS. Bi-allelic activation of Pax3:Fkhr expression concurrent with p53 or Cdkn2a inactivation is critical for ARMS development. A and B, Disease-free survival of Pax3:Fkhr, p53 mice (A) and Pax3:Fkhr, Cdkn2a mice (B). P denotes conditional Pax3:Fkhr allele, F denotes floxed conditional knockout allele, WT denotes wildtype allele. C, Comparison of Myf6^ICNm/WTPax3^P3Fm/P3Fmp53^F2-10/F2-10 (abbreviated as Pax3(P/P) p53(F/F)) and Myf6^ICNm/WTPax3^P3Fm/P3FmCdkn2a^F2-3/F2-3 mice (Pax3(P/P) CDK(F/F)). For cohort size, see Supplementary Table S6). D, H&E staining (top row), Trichrome staining (2^nd row). Immunohistochemistry positive for Myogenin (3^rd row), and MyoD (bottom row) in Pax3:Fkhr, p53 and Pax3:Fkhr, Cdkn2a mouse tumors.

Figure 2

The conditional mouse model of ARMS reflects the aggressiveness of the human disease. A, Limb tumor on at diagnosis (A-left) and 6 days later (A-right). B, MicroCT scan of a right chest tumor (T); yellow arrow, lymphatic metastasis. PA; posterior-anterior. C, Pulmonary metastases can be quantified using microCT-based Virtual Histology. 8µm resolution scan of normal lung (left) and lung with metastasis (right). White arrows denote gross metastases (0.8×0.92×0.74 mm). D, Comparison of scan (D, top left) and histology (D, top right). Histological verification was performed after epoxy embedding. Higher magnification (D-bottom). Yellow arrows point to macrophages found in locations of black-appearing dots on microCT. Macrophages are completely surrounded by tumor cells.

Figure 3

Stepwise increments in Pax3:Fkhr expression with disease progression. A, quantitative PCR of Pax3:Fkhr expression in 4 week-old wildtype skeletal muscle, 4 week-old preneoplastic skeletal muscle, and tumors. Pax3:Fkhr was not detected in wildtype muscle (WT-SKM). Primary rhabdomyosarcoma tissue from Myf6^ICNm/WTPax3^P3Fm/P3Fmp53^F2-10/F2-10 (M6-P3F-p53 Prim-ARMS) mice expressed Pax3:Fkhr at a significantly higher level than the preneoplastic muscle of the same genotype. Pax3:Fkhr expression in metastatic tumor tissues was significantly higher than primary tumors. Pax3:Fkhr expression was normalized relative to Gapdh expression. Ct values of Pax3:Fkhr PCR were 25–26 in mouse tumors. B, Pax3:Fkhr protein in mouse tumors using anti-Fkhr antibody. NIH3T3 cell was loaded as a positive control for wildtype Fkhr. C, immunofluolescence of Pax3:Fkhr during disease progression (400x). A surrogate marker for Pax3:Fkhr expression was assayed using anti-GFP antibody (green) since both genes are expressed on the same mRNA by means of the Pax3:Fkhr-ires-eYFP allele. Dystrophin (red), and DAPI (blue). Metastatic tumors taken from the liver contained a higher number of eYFP-expressing tumor cells than the primary tumor from the same animal.

Figure 4

Expression of human ARMS-specific genes were studied by quantitative RT-PCR in mouse ARMS tumors (n=10) and skeletal muscles (SKM, n=6). * represents significant difference between mouse tumors and skeletal muscle (p-value <0.05). Nine genes out of 12 were significantly overexpressed in mouse tumors compared to skeletal muscle.

Figure 5

The mouse model phenocopies the human disease, allowing Pax3:Fkhr target identification. A, Northern blotting showed induction of SKP2 expression by Pax3:Fkhr was maintained even after cycloheximide treatment. B, reporter assay showing a genomic fragment 3723bp upstream to SKP2 gene did not respond to Pax3:Fkhr. C, a putative Pax3:Fkhr binding site 49kb downstream (3’) to SKP2, showed response to Pax3:Fkhr in NIH3T3 cells and p53-deficient MEFs. PDGFRA promoter was used as a positive control. (Asterisks;p<0.01, N.S.; no significant difference). D, SKP2 downregulation represses cell proliferation in human rhabdomyosarcoma cells. Rh30 was stably transfected with SKP2-specific or control shRNA vector. Western blot analysis confirmed reduced steady state level of SKP2 protein without affecting PAX3-FKHR protein level. Concomitant to reduced SKP2 expression was an increase in p27 protein. Rhabdomyosarcoma cells transfected with SKP2-specific shRNA showed substantially decreased cell growth compared to control non-specific shRNA (analysis of covariance, p<0.001). Population doubling time for SKP2-shRNA and non-targetting shRNA was 46.2 and 36.2 hours, respectively.

Figure 6

Cross-species identification of overexpressed protein kinases as potential therapeutic targets. A, Expression of 19 protein kinases overexpressed in mouse ARMS. 16 kinases were significantly upregulated in human ARMS. Red-highlighted genes are potentially druggable targets. B, Quantitative RT-PCR confirming overexpression of the protein kinases for which kinase inhibitors are available.