Development and characterization of a c-Myc-driven preclinical mouse model of osteosarcoma to investigate the tumor immune microenvironment

Abstract Osteosarcoma (OS) is the most common bone tumor in pediatric patients, particularly in adolescents and young adults. Currently, no effective molecular targeted therapies are available for the OS, however, many OS patients possess genetically defined somatic DNA copy number alterations, including amplification of chromosome 8q24, which harbors the oncogene Myc, and correlates with a worse prognosis. To better understand the molecular pathogenesis, and identify targeted therapy for OS, a robust and reliable preclinical model is needed both for the primary and metastatic disease. We have developed a novel conditional, osteoblast-specific Myc knock-in murine model to understand the mechanisms that underlie the ability of Myc to drive the development and progression of OS and utilize it for the identification of Myc-dependent intrinsic and extrinsic molecular vulnerabilities. A tissue-specific Myc-in genetically engineered murine model was generated by crossing Col2.3-Cre; TP53Flox/+ mice with LSL-MycT58A mice and characterized integrating RNA-seq and Mass Spectrometry. Syngeneic murine OS cell lines were also generated and characterized. The molecular characterization of the GEMM model was compared with high and low Myc expressing human tumors using the OS-TARGET dataset. Differential gene expression in Myc-driven and non-driven-tumor tissue samples were quantified by RNA sequencing. The immune landscape of OS was evaluated using SymphonyFACS and IHC staining. Murine OS tumor type was confirmed by H&E staining. OS tumor was highly aggressive and metastatic in more than 60% of the mice. Myc expression was significantly higher both at the transcriptional and protein levels in the Myc-in tissue/cells compared to the non-Myc-driven model. Expression of 1479 genes (out of 13781 genes) were significantly altered in which 543 were upregulated and 936 were downregulated at transcriptional levels in Myc-in tumor tissue samples as compared to the low-Myc-driven OS tumor model analyzed by RNA sequencing. Myc-target-genes, splicing factor-related genes, E2F, G2M check-point, and DNA repair associated genes were significantly upregulated whereas the immune-related genes, oxidative phosphorylation, adipogenesis, and E2M transition genes were significantly downregulated when compared between two groups, which correlates to differential gene ontologies noted in high Myc human tumors. The total immune cell population, specifically the myeloid and macrophage populations were significantly diminished in the Myc-in tumor models, which could contribute to the poor prognosis in OS.Our murine model closely resembles human OS and will provide new opportunities for dissecting the molecular pathogenesis, identification of novel therapeutic strategies, and pre-clinical modeling for direct translational applications and targeting of Myc-driven OS. Citation Format: Bikesh K. Nirala, Lyazat Kurenbekova, Ryan L. Shuck, Tajhal Patel, Kimal Rajapakshe, Jason T. Yustein. Development and characterization of a c-Myc-driven preclinical mouse model of osteosarcoma to investigate the tumor immune microenvironment [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 1668.