Poster Presentation 35th Lorne Cancer Conference 2023

Mutation of the E3 ubiquitin ligase CBL as a mechanism of kinase activation and a potential therapeutic target in paediatric cancer (#117)

Lauren M Brown 1 2 , Teresa Sadras 3 4 , Chelsea Mayoh 1 2 , Paul G Ekert 1 2 3 4 5
  1. Children's Cancer Institute, UNSW Sydney, NSW, Australia
  2. School of Clinical Medicine, UNSW Sydney, Sydney, NSW, Australia
  3. Peter MacCallum Cancer Centre, Parkville, VIC, Australia
  4. The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC, Australia
  5. University of New South Wales Centre for Childhood Cancer Research, UNSW Sydney, Sydney, NSW, Australia

The molecular analysis of individual patient tumours by personalised medicine programs like the Zero Childhood Cancer Program (ZERO) enables the detection of potentially targetable lesions, unlocking new therapeutic opportunities for patients. Somatic mutations in the E3-ubiquitin ligase, CBL, are known to activate receptor tyrosine kinases (RTKs) in cancer and have been exclusively characterised in haematological malignancies. This includes acute myeloid leukaemia, where CBL mutation leads to FLT3 activation. Using molecular data from ZERO we have identified known and novel CBL variants in novel tumour contexts. Our findings raise the possibility that CBL mutation may be a marker of RTK activation in a range of paediatric cancer types, which may identify additional patients that will benefit from tyrosine kinase inhibitor (TKI) therapy.

We analysed whole genome sequencing and RNA sequencing data from the ZERO cohort and identified 20 somatic CBL variants in 17 individual patients, spanning haematological (5 patients), brain and CNS tumours (10 patients), and individual cases in neuroblastoma and germ cell tumour. Interestingly, we identified an established oncogenic CBL exon 8/9 deletion variant in novel tumour types - neuroblastoma and germ cell tumour. In addition, we identified 3 novel variants/transcripts located in the linker region, which is critical for E3 ubiquitin ligase function, of CBL in high-grade glioma patients.

To assess the functional and signalling impacts of these CBL variants we have generated overexpression models in cancer cell lines reflecting the tumour types in which the variants were identified. We first used CRISPR/Cas9 to knockout CBL, followed by overexpression of either FLAG-tagged CBL variants or a wild-type control. These models will be subjected to proliferation assays to assess transforming capacity and phosphoproteomics and FLAG-immunoprecipitation to understand the impact of CBL variant expression on RTK activation and binding by CBL.

This study will establish which RTKs are activated by CBL variant expression in specific cancer types, which may enable the use of clinically available TKIs in high-risk paediatric cancer patients.