(P 299) EXPLORING THE POTENTIAL OF PHOSPHOPROTEOMIC PROFILING FOR TARGET DISCOVERY IN HIGH-RISK OSTEOSARCOMA: A PATHWAY TO NOVEL THERAPIES?

Objective: Osteosarcoma (OS) is the most common primary malignant tumour of the bone in children and adolescents and young adults (AYAs). Currently there are no effective targeted treatments for advanced disease, and these patients rarely harbour actionable genomics alterations. There is evidence to suggest that activated (phosphorylated) protein kinase signalling is a key driver event in subsets of human osteosarcoma. Phosphoproteomics is an emerging technique that can be used to determine the abundance of phosphorylated proteins in an unbiased way and detect aberrantly activated proteins that may drive tumour growth. This study aims to utilise a phosphoproteomic approach to identify aberrantly activated protein targets (kinases) in human OS samples, which, if targeted, could indicate novel, effective, patient-specific osteosarcoma therapies.

Methods: Eighteen unique patient-derived xenograft (PDX) models from high-risk (expected chance of survival < 30%) sarcoma patients enrolled in Australia’s national Precision Medicine Trial for children with cancer (ZERO), including four OS (one with matched disease samples), were subjected to Mass Spectrometry-based phosphoproteomic profiling. Selected activation signatures in OS were validated by Western Blot (WB) and linked to molecular genomics/transcriptomics. Eight drug targets were selected for functional validation in vitro in short- and long-term drug viability assays across standard OS (n=3) and newly generated high-risk OS PDX cell lines derived from patients in ZERO (n=6). Two top-performing novel agents were selected for further in vitro combination testing using ZERO’s clinically applicable 150-compound drug library and in vivo exploration. Drug mechanism-of-action was investigated by WB and FACS cell cycle analysis.

Results: In OS, we identified ~4900 phosphosites, corresponding to 1843 unique proteins. Eight potential novel drug targets with elevated phosphorylation across OS ZERO PDX models, with no apparent link to molecular genomics/transcriptomics aberrations in the respective genes, were selected for further validation with ten kinase inhibitors. Two clinically applicable inhibitors demonstrated remarkably low IC50 values of < 0.05uM or < 0.1uM in standard OS and OS PDX cell lines in short-term in vitro assays and were selected for further validation. On-target drug activity and drug mechanism-of-action was confirmed by WB and cell cycle analysis. Long-term viability assays further confirmed the remarkable sensitivity of all 9 tested OS cell lines to these drugs in vitro. In vitro combination testing revealed synergy with at least 3 different targeted agents. Studies exploring the in vivo efficacies of these drugs as single agents and in combinations are currently underway.

Conclusion: Our study shows significant anticancer activity of two novel kinase inhibitors in all tested in vitro OS cell lines, including 6 newly established high-risk ZERO PDX cell line models. Combinatorial drug approaches to maximise anti-tumour responses and in vivo drug efficacies are being further explored. As these drugs were selected based on drug target protein activation signatures in the absence of apparent genomics/transcriptomics aberrations, this underlines the potential of phosphoproteomics for the discovery of actionable targets in high-risk OS, where the prognosis is particularly poor.