Osteosarcoma is a genomically complex tumor characterized by widespread structural rearrangements. This complexity has limited the 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” cell state, characterized by upregulation of transcription factors associated with early bone development, and a “late osteoblast–derived” state, 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 coexist 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. Significance: This study identifies two distinct cellular states in osteosarcoma, driven by specific transcription factor circuitries linked to normal bone development. These epigenetically defined states demonstrate differential drug responses, are identifiable in patient samples, and are correlated with survival.

Accurate DNA replication and segregation is key to reproduction and cell viability in all organisms. Autonomously replicating sequence-binding factor 1 (Abf1) is a multifunctional protein that has essential roles in replication, transcription, and regional silencing in the model yeast Saccharomyces cerevisiae. In the opportunistic pathogenic fungus Candida glabrata, which is closely related to S. cerevisiae, these processes are important for survival within the host, for example, the regulation of transcription of virulence-related genes like those involved in adherence. Here, we describe that CgABF1 is an essential gene required for cell viability and silencing near the telomeres, where many adhesin-encoding genes reside. CgAbf1 mediated subtelomeric silencing depends on the 43 C-terminal amino acids. We also found that abnormal expression, depletion, or overexpression of Abf1, results in defects in nuclear morphology, nuclear segregation, and transit through the cell cycle. In the absence of ABF1, cells are arrested in G2 but start cycling again after 9 h, coinciding with the loss of cell viability and the appearance of cells with higher DNA content. Overexpression of CgABF1 causes defects in nuclear segregation and cell cycle progression. We suggest that these effects could be due to the deregulation of DNA replication.

Supplementary Table S5. Primers and data from qRT-PCR used to generate networks related to Figure 4E-4F.

Supplementary Figure S7 shows the Multiomics for single nucleus ATAC-seq + RNA-seq from cell lines.

Supplementary Table S2. Differential accessible regions (DAR) related to Figure 1B.