Abstract 4763: Two epigenetically distinct cellular states in osteosarcoma are regulated by a state-specific set of transcription factors driving differential drug response
Authors: Fuentes Eunice Lopez , Clugston Andrew , Sayles Leanne , Ventura Maria Pons , Lee Alex , Ramani Vijay , Sweet-Cordero E. Alejandro 
Published: April 2023

Osteosarcoma is a highly aggressive cancer seen mostly in children and young adults which is characterized by aneuploidy and dramatic structural rearrangements associated with large copy number gain and widespread chromosomal loss. Osteosarcoma therapy has not change in over forty years, highlighting the need for deeper molecular understanding of this disease which could in turn identify new therapeutic opportunities. While DNA-level heterogeneity between tumors has been well-described, not much is known regarding epigenetic heterogeneity within osteosarcoma and how this may impact critical phenotypes such as therapy response and metastasis. To characterize the osteosarcoma epigenome, we integrated ATAC-seq and RNA-seq across a unique set of patient samples, PDXs and PDX-derived cell lines obtained across the disease continuum including biopsies, resections, and metastases. ATAC-seq analysis revealed the presence of at least two distinct epigenetic cell states, epigenetic cluster 1 (EC1) and epigenetic cluster 2 (EC2). We used the GREAT algorithm to identify enriched pathways for the genes linked to these differential open regions (~2000 unique peaks per EC). EC1 is defined by genes related to mesenchymal cell proliferation and osteoblast differentiation, whereas EC2 is enriched in genes related to extracellular matrix organization and regulation of kinase activity. Accessible chromatin peaks in EC1 showed transcription factor binding motifs related to development including RUNX2/3, MEOX2, HOXA2/5 and DLX; whereas members of the AP1 complex including FOSL1/2, c-FOS, JUN were enriched in open regions in EC2. This is correlated with high protein expression of these transcription factors in a cluster-specific manner and differential binding to chromatin. We defined a gene-signature of 343 genes by integrating differential open chromatin with differential gene expression of EC1 and EC2. This gene-signature is able to separate gene expression data of patient samples into 2 clusters, suggesting these epigenetic cell states are found in patient samples. We assigned the patient samples to one EC and performed a survival analysis using only primary patient samples. EC1 shows a lower survival probability than EC2. To identify possible therapeutic implications of these EC, we evaluated a drug response dataset generated with ~40 targeted agents for the same panel of PDX-cell lines. Whereas EC1 cell lines were highly sensitive to a selective inhibitor of Aurora B, cell lines in EC2 were inhibited by a MEK inhibitor. PDX-cell lines injected in a subcutaneous model recapitulated the differential drug response in vivo. In summary, we have identified two epigenetically cell states regulated by a state-specific set of transcription factors defining a gene signature that could help to define osteosarcoma subclasses and predict drug response.

Citation Format: Eunice Lopez Fuentes, Andrew Clugston, Leanne Sayles, Maria Pons Ventura, Alex Lee, Vijay Ramani, E. Alejandro Sweet-Cordero. Two epigenetically distinct cellular states in osteosarcoma are regulated by a state-specific set of transcription factors driving differential drug response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4763.