System analysis of p53 mutant associated cancer development with LFS patient- specific iPSCs

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Project Summary/Abstract Human induced pluripotent stem cells (iPSCs) not only represent a promising resource for regenerative medicine, but are also rising in their utility as a tool for studying human disease. Specifically, modeling cancer using patient specific iPSCs provides an opportunity to study human cancer pathogenesis. In our previous work in Cell 2015, `Modeling Familial Cancer with Induced Pluripotent Stem Cells', we demonstrated that iPSCs derived from human familial cancer patients of Li-Fraumeni Syndrome (LFS) mimic cancer pathogenesis in a dish. Additionally, systems analysis of LFS patient osteosarcoma development revealed that the gene H19 and its imprinted gene network, which is required for normal osteoblast differentiation, become dysregulated during LFS patient osteoblast differentiation. Although we reported the abnormal regulation of H19 and its imprinted gene network in LFS iPSC-derived osteoblasts, there is no known clinically feasible strategy to target abnormal H19 and its non-coding RNA regulation. In order to uncover clinically relevant and therapeutically targetable molecules, I continued researching our LFS iPSC-derived osteosarcoma model and identified secreted frizzled-related protein 2 (sFRP2) as an important oncogenic factor of osteosarcoma development. I further demonstrated that sFRP2 increases the phosphorylation of AXL receptor in osteoblasts, a known oncogene associated with colon cancer, melanoma, and chronic myelogenous leukemia. Additionally, using CRISPR/Cas9 gene editing technology I corrected the p53 mutation in our LFS iPSC lines. In this proposal, I will use functional and comparative analysis to identify the molecular differences of LFS- isogenic controls to wild type and the original LFS iPSCs. From this analysis, I anticipate showing that correcting the causative p53 mutation in osteosarcoma using CRISPR/Cas9 can reverse pathogenesis of osteosarcoma in an iPSC platform. Additionally, I will demonstrate the efficacy of targeting sFRP2 and the AXL receptor as potential therapeutic strategies for LFS-associated osteosarcoma. Finally, to check the clinical relevance of my approach, I will analyze the expression level of sFRP2 in human osteosarcoma samples using osteosarcoma tissue microarrays. Overall, the proposed studies will provide corrected iPSC clones that will serve as perfect isogenic controls to study the role of p53 mutation, as well as the resulting downstream changes in sFRP2 expression and AXL receptor phosphorylation, in LFS-associated osteosarcoma development. This work will not only expand our knowledge of p53 mutation mediated osteosarcoma progression, but also describe multiple potential therapeutic methods to improve clinical outcomes for p53 mutation osteosarcoma patients.