Abstract 1721: Epigenetic heterogeneity of osteosarcoma reveals distinct cellular states driven by enhancer regulation

Abstract Osteosarcoma (OS) is characterized by structural rearrangements and copy number changes which drive considerable heterogeneity. What is less clear is how these genetic rearrangements alter are in turn regulated at the epigenetic level to drive gene expression. Defining the epigenetic circuitry of osteosarcoma could help to understand the role of chromatin accessibility in determining cell plasticity. Chromatin accessibility can prime gene expression and define a cellular state. As chromatin accessibility is a major determinant of epigenetic transcriptional regulation, understanding the variability of chromatin accessibility could identify clinically-relevant subtypes and biomarkers of therapy response in Osteosarcoma, a disease that has seen few advances in therapy for over 40 years. Using ATAC-seq, we identified two epigenetic subtypes (EC1 and EC2) of OS with distinct chromatin accessibility. These studies were done in a panel of 11 PDX-derived cell lines and 9 established OS cell lines. We also performed ATACseq on the matching PDX for the 11 PDX-derived lines as well as 8 primary tumor samples. Analysis of transcription factor (TF) motifs and footprints of ATAC-seq peaks identified transcription factors distinctly activated in these two subtypes. For example, EC1 is characterized by high level of activity of developmental transcription factors including RUNX2, MEF2C and homeobox TFs. In contrast, EC2 is characterized by high activity of AP1 transcription factors such as FOSL1 and FOSL2. In order to understand the underlying transcriptional circuitry of these cell states, we performed CUT&RUN assay for the complete panel of PDX-derived cell lines. We found that the subtypes are driven by enhancer regulation (H3K27ac). We defined the specific Core Regulatory Circuitry (CRC) per subtype by defining the superenhancers by ROSE. We overlapped ATAC-seq data in the acetylated regions to demarcate TF binding regions dramatically and reduce the search space for TF cis-regulatory sites. In parallel, we used our PDX-derived cell line panel to screen cluster-specific targeted therapies and found differential responses dependent on cellular states defined by chromatin accessibility. For example, cell lines in the EC2 cluster are marked by upregulation of AP1 transcription factors, have evidence of ERK activation and are highly responsive to ERK inhibitors. In contrast cell lines in the EC1 cluster are sensitive to AURKA and AURKB inhibitors. Single-cell RNAseq analysis indicates that these subtypes may coexist within a single tumor. In summary, we discovered two epigenetically distinct cell states that are controlled by a state-specific collection of transcription factors, creating a gene signature that may aid in distinguishing osteosarcoma subclasses and predicting treatment response. Citation Format: Eunice Lopez Fuentes, Andrew Clugston, Alex Lee, Leanne Sayles, Maria Pons, Alejandro Sweet-Cordero. Epigenetic heterogeneity of osteosarcoma reveals distinct cellular states driven by enhancer regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1721.