Poster Presentation 27th Annual Lorne Proteomics Symposium 2022

Phosphoproteomic profiling to complement genomics approaches to guide treatment selection for paediatric high-grade gliomas. (#116)

Izac Findlay 1 2 , Dilana Staurdt 1 2 , Padraic Kearny 1 2 , Ryan J. Duchatel 1 2 , Evangeline R. Jackson 1 2 , Nathan D. Smith 3 , Nicholas A. Vitanza 4 5 , Jason E. Cain 6 7 , Sebastian M. Waszak 8 9 , Mitchell Hansen 10 , Frank Alvaro 1 2 11 , Matthew D. Dun 1 2
  1. Precision Medicine Program, Hunter Medical Research Institute, Newcastle, NSW, Australia
  2. Cancer Signalling Research Group, School of Biomedical Sciences and Pharmacy, College of Health, Medicine & Wellbeing, University of Newcastle, Newcastle, NSW, Australia
  3. Analytical and Biomolecular Research Facility, University of Newcastle, Newcastle, NSW, Australia
  4. Ben Towne Center for Childhood Cancer Research, Seattle Children's Research Institute , Seattle, WA, USA
  5. Division of Pediatric Hematology/Oncology, Department of Pediatrics, Seattle Children's Hospital, Seattle, WA, USA
  6. Hudson Institute of Medical Research, Melbourne, VIC, Australia
  7. Department of Paediatrics, Monash University, Melbourne, VIC, Australia
  8. Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, Oslo, Norway
  9. Department of Pediatric Research, Division of Pediatric and Adolescent Medicine, Rikshospitalet, Oslo University Hospital, Oslo, Norway
  10. Surgical Department, John Hunter Hospital, Newcastle, NSW, Australia
  11. John Hunter Children's Hospital, Newcastle, NSW, Australia

Paediatric high-grade glioma (pHGG) is the leading cause of cancer-related death in children. Current treatment is centred on maximal safe resection, followed by radiation therapy, systemic conventional chemotherapies, or even precision therapies targeting somatic tumour mutations. For most children with pHGG, this treatment approach has failed to increase overall survival, which remains an unacceptable 15-months. To improve outcomes, we have investigated the proteomic heterogeneity of pHGG to better understand the functional consequences of somatic alterations. Utilising a novel ‘pharmaco-phospho-proteo-genomics’ pipeline in real-time, we have analysed tumour tissue (partial resection) from a 19-month-old female with relapsed congenital glioblastoma (cGBM). Genomic (TruSight Oncology 500 (TSO500) next-generation sequencing (NGS)), proteomic and phosphoproteomic (‘pHASED’ - phospho Heavy-labelled-spiketide FAIMS StEpped-CV DDA) signatures were identified and compared from the same primary tumour tissue and compared to control tissues and cell types. Our pHASED protocol employed the use of phosphorylated and non-phosphorylated heavy-labelled spiketides spanning the chromatography and enriched with tumour-associated phosphopeptides using titanium dioxide. Phosphoenriched and non-modified peptides were separated by nRPLC, stepped-CV-FAIMS high and low field mobility separation, prior to high-resolution MS/MS using an Orbitrap Exploris 480. TSO500-NGS identified 11 somatic alterations of unknown clinical significance, with pHASED identifying hyperactivated MAPK, HSP90 and PRKCB signalling, and increased expression and phosphorylation of SRC-family kinases relative to normal brain tissues. This strategy identified several TGA/FDA approved therapies; trametinib (MAPKs), dasatinib (SRC) and enzastaurin (PRKCB), as well as onalespib (HSP90) – targets invisible to NGS. We established a cell line (UON-IONA2) from the resected tumour tissue and confirmed the utility of the treatment approaches predicted by studying the proteomic and phosphoproteomic heterogeneity of this lethal tumour. These preliminary data provide vital evidence to support the inclusion of clinically relevant phosphoproteomic profiling to complement and extend treatment strategies revealed by genomic approaches.