Poster Presentation 36th Lorne Cancer Conference 2024

Phosphoproteomic profiling for novel target identification in childhood sarcomas: Paving the road towards improved outcomes for relapsed and metastatic osteosarcoma (#249)

Callum Perkins 1 2 , Zara Barger 1 2 , Ashleigh Fordham 1 2 , Alice Salib 1 2 , Terry Lim Kam Sian 3 4 , Roger J Daly 3 4 , Paul Ekert 1 2 5 6 7 , Emmy Fleuren 1 2 5
  1. Sarcoma Biology and Therapeutics, Children's Cancer Institute, Sydney, NSW, Australia
  2. School of Clinical Medicine, UNSW Medicine & Health, Sydney, NSW, Australia
  3. Cancer Program, Monash Biomedicine Discovery Institute, Melbourne, VIC, Australia
  4. Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, Australia
  5. University of New South Wales Centre for Childhood Cancer Research, Sydney, NSW, Australia
  6. Murdoch Children's Research Institute, Melbourne, VIC, Australia
  7. Peter MacCallum Cancer Centre, Melbourne, VIC, Australia

Background & Aims: 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. This study aims to utilise a phosphoproteomic approach to identify aberrantly activated kinases in OS, which novel therapies can be directed against.

Hypothesis: Activated kinase signalling is a key driver event in subsets of human osteosarcoma. Phosphoproteomic profiling of OS tumours will identify aberrantly activated kinases, which if targeted by matched drugs could indicate novel, effective, patient-specific osteosarcoma therapies.

Methods: Eighteen unique patient-derived xenograft (PDX) models from sarcoma patients enrolled in the ZERO Childhood Cancer Precision Medicine Trial (ZERO), including four OS (one with matched disease samples), were subjected to MS-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 and newly established and validated OS PDX cell lines from patients enrolled in ZERO (n=6). Drug mechanism-of-action was investigated by WB.

Results: In OS, we identified ~4900 phosphosites, corresponding to 1843 unique proteins. Eight potential novel targets with enriched phosphorylation across OS patient models were selected for further validation with ten kinase inhibitors, of which two clinically applicable inhibitors demonstrated profound sensitivities, with IC50 values <0.05uM or <0.1uM in OS and OS PDX cell lines in short-term in vitro assays. These kinase inhibitors were selected for further validation, where on-target activity was confirmed by WB and drug mechanism-of-action determined. Long-term viability assays further confirmed a remarkable sensitivity of OS and OS PDX cells to these drugs in vitro.

Conclusion: These findings demonstrate significant anticancer activity of two novel kinase inhibitors in OS and OS PDX cell lines. Future studies will validate the efficacy of these in combination with other drugs, including standard chemotherapy agents, and in in vivo models.