Poster Presentation Hunter Cell Biology Meeting 2022

Optimising a real-time pharmaco-phospho-proteo-genomic pipeline to improve precision medicine based treatments for paediatric high-grade glioma patients (#88)

Izac J. Findlay 1 2 , Dilana Staudt 1 2 , Padraic Kearny 1 2 , Holly McEwan 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. Cancer Signalling Research Group, School of Biomedical Sciences and Pharmacy, College of Health, Medicine & Wellbeing, University of Newcastle, Newcastle, NSW, Australia
  2. Precision Medicine Program, Hunter Medical Research Institute, 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

Background/ Motivation

Paediatric high-grade gliomas (pHGG) are the leading cause of cancer-related death in children. Current treatment centres on maximal safe resection, followed by radiation therapy, systemic conventional chemotherapies, or even precision therapies targeting somatic tumour mutations identified through genomic approaches using biopsy or resected tissues. Unfortunately, we are yet to see an increase in clinical outcomes for pHGG patients with overall survival remaining 15-months.

Objective

To develop a real-time pipeline that provides accurate proteomics, phosphoproteomics and genomics data to be used to design effective treatment strategies validated preclinically using a congenital glioblastoma (cGBM) tissue sample and cell line we established from the resection.

Approach/ Method

Through the use of a novel ‘pharmaco-phospho-proteo-genomics’ pipeline in real-time, we have analysed the partial resection from a relapsed cGBM patient (UON-IONA2). Genomic characterisation was performed using TruSight Oncology 500 (TSO500) next-generation sequencing (NGS). Whole proteome and phosphoproteome analysis was then performed using our high-fidelity global phosphoproteomics profiling technique, pHASED (phospho Heavy-labelled-spiketide FAIMS StEpped-CV DDA). Genomic, proteomic and phosphoproteomic signatures from UON-IONA2 were compared with diffuse midline glioma (DMG) and normal brain control signatures to identify aberrantly enriched proteins / pathways unique to UON-IONA2. In vitro colony formation assays and cytotoxicity assays were performed to test the preclinical utility of the predicted targets.

Findings/ Results

TSO500 sequencing of UON-IONA2 tissue identified 18 somatic mutations, 8 of which possessed a high predicted impact severity, however, are of currently unknown clinical significance. Notable copy number losses were observed on two chromosome 22 genes; SMARCB1 and EP300, the loss of which acts as a prognostic biomarker in GBMs. Proteomic and phosphoproteomic characterisation of UON-IONA2 revealed distinct proteome and phosphoproteome profiles relative to the other pHGGs and normal brain tissues analysed. Downstream analysis of these profiles identified a high abundance of MAPK1, MAPK3 and PRKCB phosphoproteins, compared to normal brain controls with pathway analysis confirming the enrichment of MAPK and PRKCB-related pathways. This strategy identified several TGA/FDA approved therapies such as trametinib (MAPKs), and enzastaurin (PRKCB) that, when tested using in vitro colony formation assays and cytotoxicity assays, confirmed their preclinical utility as treatment regimens. Additional in vivo patient derived xenograft mouse models are underway to provide the necessary preclinical proof of this optimised precision medicine pipeline.

Conclusion

This real-time data provides critical evidence to support the use of systems-wide, multi-omics approaches to inform treatment selection/development for these highly aggressive, and poorly survived paediatric cancers.