SKP2 knockout in Rb1/p53 deficient mouse models of osteosarcoma induces immune infiltration and drives a transcriptional program with a favorable prognosis

Abstract

PurposeOsteosarcoma (OS) is an aggressive bone malignancy with a poor prognosis. One putative proto-oncogene in OS is SKP2, encoding a substrate recognition factor of the SCF E3 ubiquitin ligase. We previously demonstrated that SKP2 knockout in murine OS improved survival and delayed tumorigenesis. Here we aim to define the SKP2 drives transcriptional program and its clinical implication in OS. Experimental DesignWe performed RNA-sequencing (RNA-seq) on tumors from a transgenic OS mouse model with conditional Trp53 and Rb1 knockouts in the osteoblast lineage ("DKO": Osx1-Cre;Rb1lox/lox;p53lox/lox) and a triple-knockout model with additional Skp2 germline knockout ("TKO": Osx1-Cre;Rb1lox/lox;p53lox/lox;SKP2-/-). We validated our RNA-seq findings using qPCR and immunohistochemistry. To investigate the clinical implications of our results, we analyzed a human OS patient cohort ("NCI-TARGET OS") with RNA-seq and clinical data. ResultsWe found large differences in gene expression after SKP2 knockout. Strikingly, we observed increased expression of genes related to immune microenvironment infiltration in TKO tumors. We observed significant increases in signature genes for macrophages and to a lesser extent, T cells, B cells and vascular cells. We also uncovered a set of relevant transcription factors that may mediate the changes. In OS patient cohorts, high expression of genes upregulated in TKO was correlated with favorable overall survival, which was largely explained by the macrophage gene signatures. This relationship was further supported by our finding that SKP2 expression was negatively correlated with macrophage infiltration in the NCI-TARGET OS and the TCGA Sarcoma cohort. ConclusionOur findings indicate that SKP2 may mediate immune exclusion from the OS tumor microenvironment, suggesting that SKP2 modulation in OS may induce anti-tumor immune activation. Translational RelevanceOsteosarcoma (OS) is an aggressive bone malignancy. Standard care treatment involving chemotherapy and surgery remains unchanged for decades. OS prognosis remains poor and targeted therapies are critically needed. Immunotherapy failed in clinical trials despite extensive genomic instability, suggesting OS tumors employ active immune exclusion. One putative oncogene in OS is SKP2, which activates cell proliferation. SKP2 is correlated with worse prognosis in patients and knockout of SKP2 improved survival in murine OS. Here, we performed comparative transcriptomic analysis between transgenic OS murine models with SKP2 knockout and controls. We showed that SKP2 knockout dramatic increased immune gene expression largely due to macrophage, and to a lesser extent lymphocytes, infiltration. Interestingly, we found that the increased gene program uncovered from our SKP2 knockout model was correlated with improved survival in OS patients. Our findings indicate a completely new function for SKP2 in mediating immune exclusion in sarcoma and suggest that SKP2 inhibition may lead to improved immune activation and potential treatment.