Abstract 1611: Temporal recapitulation of genome instability in an iPSC model of osteosarcoma

Abstract

Abstract Osteosarcoma (OS) is characterized by widespread genomic instability, with cellular transformation associated with extensive structural (SV) and copy number variation (CNV). TP53 and RB1 mutations contribute to the initiation and maintenance of genome instability, which serves as a key driver of relapse. Significant effort has been made to model OS genome instability; however, current models have limited capacity to recapitulate genomic instability in real time. To model OS initiation and development, TP53 and RB1 mutations were installed in human iPSC using CRISPR/Cas9, followed by differentiation to mesenchymal (iMSC) and osteoblast (iOB) lineages in vitro. Functional assays of transformation were then conducted at intermediate stages of development for each genotype. Compared to isogenic WT controls, TP53-/- and TP53-/-/RB1-/- iOB exhibited increased proliferation, whereas anchorage independent growth was only observed in TP53-/-/RB1-/- iOB. Overexpression of cMYC and hRAS significantly increased the magnitude of these results in iOB. In contrast to iOB, TP53-/-/RB1-/- iMSC did not form colonies without cMYC and hRAS overexpression. To evaluate this phenomenon in vivo, iMSC and iOB were injected into the calcaneal bone of immunodeficient mice. Consistent with our in vitro observations, TP53-/-/RB1-/- and TP53-/-/RB1-/-+cMYC/hRAS iOB, and TP53-/-/RB1-/-+cMYC/hRAS iMSC gave rise to tumors. Histological evaluation of MSC-derived tumors revealed a largely undifferentiated pleomorphic phenotype, with a progressive increase in complexity in tumors derived from later stage iOB. Global gene expression signatures of xenografted tumors bore a striking similarity to OS patient samples compared to other pediatric tumors. In follow-on experiments, a progressive increase in cytogenetic abnormalities were observed in mutant but not WT iMSC, which became more pronounced in mutant iOB and correlated with a transformed phenotype. We are currently evaluating the temporal kinetics of this process, including through the use of low-coverage whole genome sequencing to elucidate the existence of recurrent patterns. These data highlight the utility of our iPSC approach as a novel model of OS, allowing for the first time the ability to study the evolution of genome instability in real time. We envision that this approach may uncover heretofore undiscovered common patterns underlying the seemingly chaotic nature of OS genomes, providing new opportunities for therapeutic intervention. Citation Format: Kelsie Lynn Becklin, Rebecca Madden, Lauren Mills, Nicole Thue, Nathan Carroll, Anthony DeFeo, Branden Moriarity, Beau Webber. Temporal recapitulation of genome instability in an iPSC model of osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1611.