Abstract C3: Candidate osteosarcoma metastatic drivers identified in highly clonal metastases using transposon-mediated insertional mutagenesis

Abstract

Abstract Osteosarcoma (OS) is the most common primary bone tumor and the third leading pediatric cancer. Despite combined treatments, over 30% of patients with localized OS and 75% of patients with metastases at presentation experience treatment failure within 5 years of diagnosis. Identification of OS initiation, progression, and metastatic driver genes and pathways has been difficult due to the genomic instability and subsequent complexity of OS. To address this, we created transgenic mice that develop OS via Sleeping Beauty transposon-mediated mutagenesis. Using a Trp53 pathway deficient background (Trp53R270H/+) to accelerate tumorigenesis and metastasis, the T2/Onc transposon system is activated in osteoblasts via an osteoblast specific osterix- cre recombinase transgene (Osx-Cre). Our quadruple transgenic mice (Osx-Cre; R26-LSL-SB11; LSL-Trp53R270H; T2/Onc) develop OS with an average latency of 10.5 months and a penetrance of 75% (n=96), as compared to 60% (n=49) in Trp53R270H/+ controls. Importantly, the SB-accelerated OS predilection sites are more similar to human OS as compared to other models; >40% arose in the hind or front legs, as most human OS is appendicular in origin. SB tumors also resemble human OS in gross anatomy, histological appearance, and presence of collagen. Over 100 metastatic nodules were collected from 16 quadruple transgenic mice with OS. Analysis of recurrent SB integration sites revealed the well-known OS genes RB1 and CMYC, validating the screen, and novel genes not previously reported, including 10 genes common among metastatic nodules. Non-supervised hierarchical clustering analysis was performed on metastatic and primary tumor sets using transposon integration sites as molecular tags. Metastases from the same mice were found to be clonal derivatives from the primary tumor and generally more related to each other than to the primary tumor, even if they were collected from different tissues. Additionally, in each set of metastases from a given mouse, there was a set of transposon insertion mutations found only in the metastatic cells and not in the primary tumor. PCR confirmation experiments of metastasis-specific insertions are underway. Thus far, it is clear that some mutations identified in only metastatic nodules are present in the indicated samples and not detectable in the primary OS tumors from the same mouse. Functional validation using published in vitro assays for migration and invasion are being performed on cell lines derived from several lung and primary tumor pairs collected from the quadruple transgenic SB mice and in well-characterized human and murine OS cell lines: U2OS, HOS, MNNG/HOS, 143B, K12, and K7M2. Gene expression will be increased using stably integrated, over expression vectors containing cDNA and/ or silenced using transcription activator-like effector nuclease (TALENs) mediated gene knockout. The SB transposon screen has revealed high clonality among metastases and identified several candidate metastatic genes for further study. Citation Format: Tracy A. Powell, Branden S. Moriarity, Aaron L. Sarver, David A. Largaespada. Candidate osteosarcoma metastatic drivers identified in highly clonal metastases using transposon-mediated insertional mutagenesis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C3.