(P 022) EZHIP ECTOPIC EXPRESSION AND ITS ROLE IN OSTEOSARCOMA PATHOGENESIS

Objective: Osteosarcoma (OS) is the most common bone cancer and accounts for 20% of all bone tumours; malignant and benign. In North America, around 450 new OS cases are diagnosed annually. Comprehensive molecular profiling revealed that OS is a disease of genomic instability due to frequent mutations in mitotic checkpoints and impaired DNA repair pathways. This instability results in complex OS genomes with extensive structural variants and somatic copy number alterations 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 remodelling in these malignancies. We hypothesized that histone mutations and aberrant expression of the onco-histone mimic, EZHIP, play a role in osteosarcoma tumor formation and progression.

Methods: The expression of oncohistones (H3K27M and H3G34W), and oncohistone-mimic EZHIP was evaluated by immunohistochemistry in a cohort of pediatric and adult osteosarcomas. We correlated their expression with histone methylation status (H3K27me3) and investigated the presence of co-occurring partner mutations by molecular profiling. Next, we studied the role of EZHIP in OS pathogenesis using patient derived OS cell models, by in vitro and in vivo gain of function (lentiviral mediated overexpression) and loss of function experiments (CRISPR-Cas9).

Results: EZHIP is highly expressed in a subset of osteosarcomas and its expression is correlated to the relative loss of the heterochromatin mark H3K37me3. EZHIP overexpression in the osteosarcoma cell of origin (human mesenchymal stromal cells) alters its differentiation propensity and leads to the upregulation 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.

Conclusion: Conclusion: The study of epigenetic modifiers in the pathogenesis and progression of bone tumors such as OS has been largely neglected under the assumption that these tumors are exclusively caused by genetic, copy number driven 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.