Epigenetic and transcriptional programs define osteosarcoma subtypes and establish targetable vulnerabilities.

Osteosarcoma is a genomically complex tumor characterized by widespread structural rearrangements. This complexity has limited development of therapeutic strategies informed by molecular mechanisms of oncogenesis. We hypothesized that epigenetic mechanisms could drive distinct subtypes of osteosarcoma. Through analysis of chromatin accessibility, we identified an "early osteoblast-derived" (EOD) cell state characterized by upregulation of transcription factors associated with early bone development, and a "late osteoblast-derived" state (LOD), characterized by upregulation of genes involved in late bone development. We then defined core regulatory circuitries governing the underlying gene expression programs in these two cell states. Multiomic single-cell analysis indicates that these cell states co-exist in a single tumor. Finally, using a panel of patient-derived xenograft models, we identified differential drug responses dependent on these cellular states. These findings create opportunities for developing new combination therapy strategies for osteosarcoma treatment and underscore the value of defining epigenetic subtypes in highly genomically complex cancers.