Abstract A65: Unraveling the rhabdomyosarcoma genome using mouse mosaicism
Authors: Timothy McKinnon, Rosemarie Venier, Manon Alkema, Leah Kabaroff, Javed Khan, Brendan Dickson, Rebecca Gladdy
Journal: Cancer Research (2014)

Abstract Introduction: Rhabdomyosarcoma (RMS) represents the most common pediatric soft tissue sarcoma. Despite advances in multimodality therapy, outcomes in intermediate and high-risk RMS groups have plateaued. To develop more efficacious therapies, improved biologic models of RMS are required to better characterize the molecular pathogenesis of RMS. Results: We have developed a mouse model of RMS that targets maturing myoblasts, the likely cell-of-origin for RMS. In this study, myoblasts were isolated from neonatal skeletal muscle harvested from p53-/- mice (Trp53tm1Tyj), as loss of function in the p53 pathway is a common mutation in RMS. Lentiviral particles encoding a bicistronic construct of a known RMS oncogene (Kras) and a green fluorescent protein (GFP) reporter (KrasG12DIRES-Emerald) were used to transduce two different p53-/- primary myoblast cell lines. GFP+ cells were purified using fluorescence activated cell sorting (FACS) and expanded. In parallel, myoblasts expressing empty vector (IRES-Emerald) were also selected using FACS. Kras overexpression was confirmed by immunoblot and resulted in a striking transformation of p53-/- myoblasts, demonstrated by increased colony formation in anchorage-independent growth assays when compared to parental cell lines and empty vector controls. Additionally, a concomitant increase in proliferation and decrease in differentiation potential was observed in myoblasts expressing oncogenic Kras compared to controls. Intramuscular injection of KrasG12D-overexpressing myoblasts into the hind limbs of neonatal p53+/- hosts resulted in rapid tumor formation with a median latency of 4.2 weeks (range 2.7 to 5.9 weeks) and a penetrance of 85%. Live mouse imaging demonstrated that the hind limb tumors expressed GFP. Empty vector control constructs did not form when host animals were aged for at least 35 weeks. All tumors (n=21) were examined using histological staining and immunohistochemistry with a panel of sarcoma specific markers. Histopathologic analysis revealed that the tumors represented high-grade sarcomas with myogenic differentiation based on their expression of multiple muscle markers, including desmin, MyoD1 and myogenin. Metastases were not identified in any mice. Transcriptome analysis using gene expression arrays will assist in further characterization of the tumors and determine whether this murine model of RMS recapitulates aberrant gene expression described in human RMS. Conclusions: Our novel mosaic mouse model supports previous studies describing the development of RMS following transformation of maturing myoblasts. Moreover, we observed that dysregulation of p53 and RAS signalling are synergistic events in the molecular pathogenesis of RMS. Using a lentiviral system to deliver genes and transform key cell populations, followed by engraftment into syngeneic hosts, represents a powerful method to experimentally generate RMS and, potentially, other solid tumors. This flexible functional genomics platform is highly amenable to the study of candidate drivers of RMS, and as a preclinical tumor model to test novel therapeutic agents. Citation Format: Timothy McKinnon, Rosemarie Venier, Manon Alkema, Leah Kabaroff, Javed Khan, Brendan Dickson, Rebecca Gladdy. Unraveling the rhabdomyosarcoma genome using mouse mosaicism. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A65.