Abstract 7049: Harnessing phosphoproteomic profiling for target discovery in high-risk osteosarcoma: A pathway to novel therapies?

Abstract Background and Aims: Osteosarcoma (OS) is the most common primary malignant tumor of the bone in children and adolescents and young adults (AYAs). Currently there are no effective targeted treatments for advanced disease, and tumours rarely possess actionable, genomic alterations. Activated (phosphorylated) protein kinase signaling has been identified as a key driver event in subsets of human osteosarcoma. This study aims to utilize a phosphoproteomic approach to identify aberrantly activated kinases in OS, which novel targeted therapies can be directed against. Hypothesis: Activated kinase signaling is a key driver event in subsets of human OS. Phosphoproteomic profiling of OS tumours will identify aberrantly activated kinases, which if targeted by matched drugs could indicate novel, effective, patient-specific OS therapies. Methods: Eighteen unique patient-derived xenograft (PDX) models from high-risk (<30% chance of survival) sarcoma patients enrolled in the ZERO Childhood Cancer Precision Medicine Trial (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 established OS PDX cell lines from ZERO patients (n=6). Two top-performing novel agents underwent 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 cell cycle analysis. Results: In OS, we identified ∼4900 phosphosites, corresponding to 1843 unique proteins. Eight novel drug targets with elevated phosphorylation across OS patient models were selected for further validation with ten kinase inhibitors. Two clinically applicable inhibitors demonstrated remarkably low IC50 values of <0.05uM or <0.1uM across standard OS and OS PDX cell lines in short-term in vitro assays and were selected for further validation. On-target drug activity and mechanism-of-action was confirmed by WB, where reduced expression of CDK downstream targets was observed. Clonogenic assays further confirmed the remarkable sensitivity of tested cell lines to these drugs. In vitro combination testing revealed synergy with at least three targeted agents. Conclusion: Our study demonstrates significant anticancer activity of two novel kinase inhibitors in vitro in OS and newly established high-risk ZERO PDX cell lines. Studies exploring the in vivo efficacies of these drugs as single agents and in combinations are underway. This study demonstrates the potential application of phosphoproteomics for the discovery of actionable targets in high-risk OS. Citation Format: Callum M. Perkins, Zara Barger, Ashleigh Fordham, Alice Salib, Terry Lim Kam Sian, Roger J. Daly, Paul Ekert, Emmy Fleuren. Harnessing phosphoproteomic profiling for target discovery in high-risk osteosarcoma: A pathway to novel therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7049.