(P 259) PRECLINICAL MODELING OF CHEMOTHERAPY RELEASE IDENTIFIES MITOCHONDRIAL ADAPTATIONS IN DOXORUBICIN TREATED OSTEOSARCOMA

Objective: Osteosarcoma (OS), the most common bone tumor in children, is often associated with poor outcomes due to chemotherapy resistance. Studies of therapy-resistant OS have focused on mechanisms of resistance but have largely neglected the temporal dynamics of chemotherapy delivery and release that occur in real-world settings. To address this, we have developed models of chemotherapy-released OS to determine how cellular adaptations to the stress of chemotherapy may enhance fitness after chemotherapy exposure. Our preliminary data identified alterations in mitochondrial Complex I (CI) activity and transcriptional enrichment in mitochondrial genes supporting CI after long-term doxorubicin (DOX) exposure in OS cell lines. The objective of this study is to determine how mitochondrial function in OS is impacted after short-term DOX exposure with an overall goal to identify and target mechanisms of mitochondrial remodeling in chemotherapy-resistant OS.

Methods: DOX was administered to canine OS patient-derived organoids (PDOs) for 48 hours and then removed from the culture to begin DOX release. DOX-naïve and -released PDOs were rechallenged with DOX to assess phenotypic resistance and growth. Respiratory flux assays and RNA sequencing were performed to explore adaptations in respiratory capacity and expression of mitochondrially-supporting genes.

Results: Canine OS PDOs demonstrate heterogeneity in cell viability in response to DOX and rate of growth, reflecting a biologically diverse cohort. After DOX-release, PDOs demonstrate increased DOX-resistance (Release/Naïve DOX IC50 fold change 1.5-72.8). Relative to DOX-naive PDOs, DOX-treated PDOs demonstrate transcriptional enrichment in numerous mitochondrial and CI pathways and preserve or increase maximal respiratory capacity.

Conclusion: OS PDOs demonstrate enhancement in mitochondrial function and gene expression following DOX-exposure. Ongoing work aims to further characterize the mitochondrial stress response in OS after DOX treatment and release to determine how mitochondrial adaptations contribute to DOX-resistance and how to target mitochondrial remodeling during DOX-release.