Targeting high risk osteosarcoma: MYC modulation alters metastasis

Osteosarcoma (OS) is a bone tumor that affects human and canine patients. Standard of care is neoadjuvant chemotherapy and surgery resulting in a 5 year survival rate for patients with localized disease of ~70%. However, patients with metastatic disease and relapsed disease have a 5 year overall survival of less than 30%. Therefore, there is a critical need for improved therapies and a better understanding of the biological underpinnings of high risk disease. A subset of patients with particularly poor outcomes are known to have copy number amplification of MYC. However, it is not known if MYC contributes to the high risk phenotype by driving metastatic progression or drug resistance. Importantly, 20 compounds have been described as MYC inhibitors and perturb different steps of MYC driven transcription. In this report, we found that MYC drives cell migration and outgrowth but does not appear to contribute to drug resistance in OS cells. More precisely, MYC silencing reversed the metastatic phenotypes of migration and outgrowth of OS cells. Further, MYC downstream targets play an important role in metastatic progression. Silencing of MYC in 5 different cell lines revealed 45 common induced targets, many of which are known to modulate different steps in the metastatic cascade. We screened all 20 compounds previously shown to interfere with MYC transcription using an approach designed to capture the compound that modulates both MYC activity and the metastatic phenotype. Fourteen compounds modulated expression of MYC and/or downstream targets in 4 different OS models. Of those,10 compounds had a profound impact in cell viability in both 2D and 3D assays. Five of these showed selective toxicity in 3D relative to 2D; a phenotype linked to metastatic progression. Importantly, not all compounds that modulated MYC showed therapeutically favorable effects on migration or metastatic organization and outgrowth with at least 2 compounds driving a dramatic increase in migration despite suppressing expression of MYC. Nevertheless, 2 compounds, samuraciclib and THZ531, blocked MYC expression, downstream target expression, cell migration, metastatic organization and outgrowth. We confirmed these results and showed reversal of metastatic competence and complete reversal of metastatic outgrowth using the in vivo/ex vivo pulmonary metastasis assay (PuMA). We are now working to integrate CUT&Tag with BRUseq, an assay of nascent transcription, to determine if modulation of different steps in MYC transcription drives diverse cellular phenotypes as we hypothesized. Nevertheless, the top hit of the screen, samuraciclib, convincingly reverses MYC activity and the associated metastatic phenotype and is undergoing additional testing in metastatic OS mouse models and a canine clinical trial is under development. Correlative biology such as spatial transcriptomics will be used to guide the translation of samuraciclib to patients with high risk osteosarcoma.