(P 379) THE ROLE OF EZHIP IN DRIVING AGGRESSIVE OSTEOSARCOMA

Objective: Osteosarcoma (OS) is the most common bone cancer and accounts for 20% of all bone tumours. OS is a disease of genomic instability characterized by extensive structural variants and somatic copy number alteration that frequently lead to gain in oncogene expression and loss of tumor suppressors. While oncogenic mutations in histone proteins (oncohistones) have also been found in osteosarcoma tumors, very little is known about the role of epigenetics and chromatin remodeling in these malignancies. We hypothesized that histone mutations and aberrant expression of the onco-histone mimic, EZHIP, play a role in osteosarcoma pathogenesis.

Methods: The expression of oncohistones (H3K27M and H3G34W), and the oncohistone-mimic EZHIP was evaluated by immunohistochemistry in a cohort of 55 pediatric and adult osteosarcomas. We investigated the presence of co-occurring partner mutations by molecular profiling (WES and WGS) and clinical correlates with EZHIP expression.To study the role of EZHIP in OS pathogenesis, we used patient derived OS cell models of different genetic backgrounds. Using gain of function (lentiviral mediated overexpression) and loss of function modalities (CRISPR-Cas9) we investigated the oncogenic role of EZHIP and the consequences of its loss on the epigenome, transcriptome and on OS cells tumorigenic capacity in-vivo..

Results: EZHIP is highly expressed in a subset of osteosarcomas in both our discovery cohort and an external validation cohort of 28 additionalOS cases. EZHIP overexpression in the osteosarcoma cell of origin (human mesenchymal stroma cells) stalls its differentiation capacity into mature bone tissue and rather enhances its proclivity to differentiate towards the myogenic lineage and upregulates of oncogenic pathways. In high-grade OS cell lines, overexpression of EZHIP promotes tumorigenesis and enhances aggressiveness resulting in shorter survival time in orthotopically implanted mouse models. Similarly, the loss of EZHIP in lines endogenously expressing this oncogenes results in a drastic loss in tumorigenic capacity that can be rescued upon EZHIP re-expression. EZHIP phenocopies H3K27M and results in K27me3 restriction onto CPG-islands resulting in increased KRAS signaling and an epigenomic profile reminiscent of human embryonic stem cells.

Conclusion: The study of epigenetic modifiers in the pathogenesis and progression of bone tumors such as OS has been largely neglected influenced by the view that that these tumors are mostly driven by genetic mechanisms. In addition to the previously reported oncogenic mutations of histone proteins, we describe a potential role for EZHIP in a subset of Osteosarcomas. In osteosarcoma cell lines, EZHIP induction caused aggressive tumor behavior and significantly shortened animal survival. We conclude that altered levels of epigenetic players such as EZHIP may constitute a novel mechanism of OS pathogenesis and progression and strategies targeting such mechanisms should be considered in the future as treatment options for osteosarcoma patients.