Poster Presentation NSW State Cancer Conference 2023

Identification of drivers of resistance in BRAFV600E paediatric high-grade glioma and novel therapeutic targets (#129)

Rebecca Lehmann 1 2 , Joshua Zhu 1 3 , Chelsea Mayoh 2 4 , Maria Tsoli 1 2 , Benjamin Rayner 1 2 , David Ziegler 1 2 5
  1. Brain Tumours Research Group, Children's Cancer Institute, Sydney, NSW, Australia
  2. School of Clinical Medicine, UNSW Medicine and Health, Sydney, NSW, Australia
  3. School of Biomedical Sciences, UNSW Medicine and Health, Sydney, NSW, Australia
  4. Bioinformatics Network , Children's Cancer Insitute, Sydney, NSW, Australia
  5. Kids Cancer Centre, Sydney Children’s Hospital, Randwick, NSW, Australia

Paediatric high-grade glioma (pHGG) is a devastating form of brain cancer with extremely poor prognosis. The BRAFV600E mutation has recently been discovered as a driver mutation in a subset of pHGG, with tumours driven by this mutation responding to targeted BRAF and MEK inhibitors. Unfortunately, resistance inevitability develops, resulting in disease progression. There is currently limited understanding of the mechanisms underlying such drug resistance in BRAFV600E pHGG and no effective counter-therapies exist. Therefore, this study aimed to identify driver pathways of resistance in BRAFV600E pHGG to subsequently identify new therapeutic targets.

 

BRAF inhibitor-resistant, MEK inhibitor-resistant and BRAF+MEK inhibitor-resistant BRAFV600E pHGG cultures were derived through chronic exposure of a BRAFV600E patient-derived culture to either vemurafenib (BRAF inhibitor), trametinib (MEK inhibitor) or a combination of both drugs, respectively. Cytotoxicity assays confirmed the resistance profile of the three cell lines, displaying acquired resistance to both vemurafenib and trametinib. All three resistant cell lines spontaneously changed from a spheroid phenotype to adherent growth, indicating profound changes in tumour characteristics. RNAseq analyses identified more than 1500 genes of interest, with subsequent Gene Set Enrichment Analysis (GSEA) identifying key pathways as novel drivers of resistance, the top-ranked gene sets being neural development and plasma membrane/cell adhesion. Approximately 45% of identified gene sets were enriched across all three drug-resistant lines, indicating overlapping mechanisms of resistance to current therapeutic regimes. Interrogation of the receptor tyrosine kinase (RTK) GSEA gene sets identified potent upregulation of several RTKs in all three drug-resistant cell lines. Notably, overexpression of EGFR, a RTK upstream of the RAF/BRAF/MEK pathway, was confirmed through RNAseq and western blot as a potential key mediator of the acquired resistance. Combined treatment with vemurafenib and the brain-penetrant EGFR inhibitor, dacomitinib, resulted in synergistic activity against both vemurafenib-resistant BRAFV600E pHGG cells and matched parental cells, providing a viable therapeutic option for drug-resistant BRAFV600E pHGG.

 

Overall, this study has identified novel drivers of resistance in BRAFV600E pHGG. EGFR overexpression was pinpointed as a key resistance mediator and dual BRAF/EGFR inhibition displayed promising efficacy as a therapeutic option. Future studies will continue to elucidate the mechanisms of resistance using multi-omic approaches and test the in vivo efficacy of dual BRAF/EGFR inhibition.