Objectives/Goals: Osteosarcoma (OS) is the most common primary bone malignancy in humans and dogs. >40% of children and >90% of dogs succumb to metastatic disease. We hypothesize MYC overexpression in metastatic canine and human OS contributes to an immunosuppressive tumor environment by driving tumor-associated macrophage influx and T lymphocyte exclusion. Methods/Study Population: To characterize the role of oncogenic MYC signaling in the canine metastatic tumor immune microenvironment (TIME), 42 archived FFPE lung metastatic canine OS samples were evaluated for MYC copy number variation (CNV), mRNA, and protein expression via ddPCR, nanostring analysis, and immunohistochemistry (IHC). Seven samples also underwent GeoMX spatial profiling to more specifically evaluate T cell and macrophage transcriptional profiles based on MYC status. To determine the role of MYC target modulation as a potential therapeutic option, canine and human OS cell lines were treated with a novel MYC inhibitor (MYCi975) and assessed for effects on survival, proliferation, and cytokine profiles. Results/Anticipated Results: We demonstrate that copy number gains are not a key driver of MYC hyperactivity in canine metastatic OS. However, stratification based on MYC protein expression demonstrates that “MYC-high” tumors are associated with downregulation of cytotoxic effector T-cell associated transcripts and upregulation of tumor-associated macrophage (TAM) and extracellular matrix remodeling transcripts. We also report that MYCi975 treatment of canine and human OS cell lines results in significant inhibition of OS cell survival and proliferation at concentrations that are pharmacologically achievable in mice. Furthermore, we demonstrate MYC inhibition by MYCi975 is associated with reduced pro-inflammatory cytokine secretion in OS cell culture models. Discussion/Significance of Impact: While MYC overactivity in metastatic canine OS may not be genomically driven, other mechanisms that lead to increased MYC protein expression are associated with transcriptomic profiles supportive of local immunosuppression. Pharmacologic targeting of MYC may serve as a strategy to bolster immunotherapeutic options in metastatic OS treatment.