Single cell RNA analysis of murine osteosarcoma uncovers Skp2 function in metastasis, genomic instability and immune activation and reveals additional target pathway

Osteosarcoma (OS) is the most common primary pediatric bone malignancy. One promising new target is SKP2, encoding a substrate recognition factor of the SCF E3 ubiquitin ligase that targets p27 for proteasomal degradation, driving cellular proliferation. Knockout of Skp2 in an immunocompetent transgenic mouse model of OS improved survival, drove apoptosis, and induced anti-tumor immunity. Here, we applied single-cell RNA-sequencing (scRNA-seq) to primary OS tumors from Osx-Cre conditional Rb1/Trp53 knockout mice. We further compared with models of Skp2 disruption: Skp2 knockout or disruption of the Skp2-p27 interaction (resulting in p27 overexpression). We report that murine OS models recapitulate the tumor heterogeneity and microenvironment complexity observed in patient tumors. Skp2 disruption led to reduction of T cell exhaustion and upregulation of interferon signaling, as well as induction of cell-type specific replicative and endoplasmic reticulum stress, which we validated with proteomics analysis. Further, we showed that interferon induction was correlated with improved survival in OS patients. Additionally, our scRNA-seq analysis uncovered decreased expression of metastasis-related gene signatures in Skp2-disrupted OS, which we validated by a strong reduction in lung metastasis in the Skp2 knockout mice. Finally, we report several mechanisms potentially used by OS to escape from Skp2 targeting, including upregulation of Myc targets, induction of genomic instability, overexpression of alternative E3 ligases, and lineage plasticity. These mechanistic insights into OS tumor biology and Skp2 function suggest novel targets for new, synergistic therapies, while the data and our comprehensive analysis may serve as a public resource for further big data-driven OS research.