Abstract 553: SKP2 knockout induces macrophage infiltration in p53/Rb1 null transgenic mouse models of osteosarcoma and drives gene expression correlated with improved survival in patients

Abstract

Abstract Background: Osteosarcoma (OS) is an aggressive cancer of the bone with a poor prognosis. OS often involve mutations of tumor suppressors RB1 and TP53. One putative proto-oncogene in OS is SKP2, which codes for a substrate-recognition factor of the SCF E3 ubiquitin ligase. We have demonstrated that germline knockout of SKP2 in a pre-clinical transgenic mouse model of OS doubly deficient in Rb1 and Trp53 improved survival, delayed tumorigenesis, reduced malignant cell proliferation, slowed tumor growth, diminished stemness, and induced apoptosis. However, the role that SKP2 plays in OS progression remains unclear. Method: We performed bulk RNA-sequencing on pre-clinical transgenic mouse models of OS. To model OS in mice, we generated a conditional double-knockout line of Trp53 and Rb1 mutations induced in cells of the bone-forming osteoblast lineage via Cre-Lox recombination (“DKO”: Osx1-Cre;Rb1lox/lox;p53lox/lox). We compared this with a triple-knockout model, which additionally features a germline Skp2 knockout (“TKO”: Osx1-Cre;Rb1lox/lox;p53lox/lox;SKP2-/-). We compared tumors from the TKO and DKO models via bulk RNA-sequencing (n=3 per group) to study differential expression and immune infiltration. We used immunohistochemistry (IHC) and quantification to validate differences in immune markers between genotypes. Using the transcriptomic results from the mouse models, we analyzed a human OS patient cohort of 83 participants (NCI TARGET OS) with RNA-seq and clinical data via survival analysis to infer the impact of modulating SKP2 in OS patients. Result: We found a large difference in gene expression between the TKO and DKO models. Surprisingly, we observed a dramatic increase in the expression of genes related to immune microenvironment infiltration in TKO tumors compared to DKO. Macrophage transcriptomic signatures were the strongest signal detected (P<0.001), but significant increases in B cells, endothelial cells and T cell signatures were also observed. IHC also showed macrophage markers were higher in TKO (P = 0.08). In the NCI TARGET OS cohort, high expression of genes up-regulated in TKO was significantly correlated with 5-year overall survival (p=0.02). This effect was driven by macrophage transcriptomic signatures. This finding was supported by an immune microenvironment deconvolution analysis, which showed that macrophages and T cells were associated with improved overall survival in the patient cohort (p=0.01). We are currently performing single-cell RNA-sequencing analysis to study immune infiltration and cellular heterogeneity in more detail. Conclusion: Taken together, our new findings indicate that SKP2 modulation in OS may induce a vigorous anti-tumor immune activation especially in the form of macrophage and lymphocyte infiltration into the tumor microenvironment. Citation Format: Alexander Ferrena, Jichuan Wang, Ranxin Zhang, Rui Yang, David Geller, Bang Hoang, Deyou Zheng. SKP2 knockout induces macrophage infiltration in p53/Rb1 null transgenic mouse models of osteosarcoma and drives gene expression correlated with improved survival in patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 553.