Abstract A18: Comprehensive identification of bone cancer driver genes by using Li-Fraumeni syndrome iPSCs

Osteosarcoma, the primary malignant tumor of bone, is the most frequent primary non-hematologic malignancy in children and adolescents. Despite the advances in surgery and multiagent chemotherapy, the survival rate of osteosarcoma has not improved as much as for other malignancies, with 5-year survival of 60-65% for localized osteosarcoma and far worse for metastatic disease. Osteosarcoma is also the second leading cause of cancer-related death in children and adolescents. Therefore, identifying novel driver genes involved in osteosarcoma initiation, development, and progression is of great translational importance for osteosarcoma detection, prevention and treatment. Li-Fraumeni syndrome (LFS) is an autosomal dominant disease caused by germline mutations in p53 gene, which predispose individuals to a wide range of malignancies, especially osteosarcoma; thus it provides an ideal model system to study this malignancy. Utilizing induced pluripotent stem cells (iPSCs) established from LFS patients, our group successfully recapitulated tumorigenic characteristics of osteosarcoma by differentiating LFS iPSC-derived mesenchymal stem cells (MSCs) to osteoblasts. This first human LFS disease model holds great potential in identifying and characterizing novel driver genes during osteosarcoma development. In this study, we plan to extend our previous findings by applying deep sequencing and trackable Sleeping Beauty forward genetic screen to identify cancer drivers that contribute to LFS osteosarcomagenesis. Succinctly, whole-genome sequencing will be performed in LFS samples with different tumorigenic potential (MSCs, osteoblasts, osteoblasts grown in soft agar and in nude mice), followed by systematic analyses of whole-genome alterations during this process. We will also model clonal evolution in LFS osteosarcoma utilizing deep sequencing results acquired from different stages of LFS osteosarcoma. We expect to sequentially determine genomic driver events and define clonal architecture during LFS-associated osteosarcoma development. On the other hand, a cancer forward genetic screening will be performed by transducing hybrid viral-transposon system (Lentihop) into LFS iPSC-derived MSCs, followed by differentiation into osteoblasts. These differentiated osteoblasts will be inoculated into immune-deficient mice to allow de novo tumor development. The developed tumors will be used to identify gain-of-function and loss-of-function common insertion sites. By either knoc