E-Poster Presentation 33rd Lorne Cancer Conference 2021

Therapeutic targeting of ubiquitin-specific protease 5 in MYCN-driven neuroblastoma (#119)

Ane Kleynhans 1 , Ritu Mittra 1 2 , Zsuzsanna Nagy 1 2 , Patrick Kim 1 , Olivia OC Ciampa 1 , Shizhen Zhu 3 , Daniel R Carter 1 4 , Belamy B Cheung 1 2 , Glenn M Marshall 1 5
  1. Children Cancer Institute, Sydney, NSW, Australia
  2. School of Women’s and Children’s Health, University Of New South Whales , Sydney, NSW, Australia
  3. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, America
  4. School of Biomedical Engineering, University of Technology Sydney, Sydney, NSW, Australia
  5. Kids Cancer Centre, Sydney Children's Hospital, Sydney, NSW, Australia

Histone deacetylase (HDAC) inhibitors are effective in preclinical models of the childhood cancer, neuroblastoma. HDAC inhibitor, suberanoyl hydroxamic acid (SAHA), has shown clinical success in the treatment of cutaneous T-cell lymphoma but lacks efficacy as a single agent in many solid tumours, including neuroblastoma.

First, we aimed to identify new compounds that potentiate SAHA activity and characterize their molecular mechanism. We identified a small enhancer molecule, SE486-11, that was able to synergistically enhance the cytopathic effects of SAHA in neuroblastoma cells. Treatment with the combination led to a decrease in cell viability, an increase in MYCN ubiquitination and inhibited tumour growth in TH-MYCN+/+ homozygous mice and zebrafish. A SILAC analysis of protein from neuroblastoma cells treated with combination therapy, revealed that Ubiquitin-Specific Protease 5 (USP5) expression was significantly reduced. USP5 is responsible for regulating the relative cellular levels of unanchored mono- and polyubiquitin. We found that the combination treatment caused an increase in Lys48-linked polyubiquitination and that overexpression of USP5 reduced polyubiquitin levels following combination treatment.

Next, we aimed to determine the oncogenic role of USP5 in neuroblastoma. An online data base revealed that high USP5 expression predicted poor prognosis in MYCN amplified neuroblastoma patients. This implicates USP5 as a potential oncogenic driver in neuroblastoma. To determine the oncogenic role of USP5 in MYCN-driven neuroblastoma, siRNA knockdown of USP5 in MYCN-amplified neuroblastoma cell lines, caused a significant decrease in MYCN protein expression and half-life along with a significant reduction in cell viability, cell proliferation and colony formation. Chromatin immunoprecipitation assays demonstrated an enrichment of MYCN protein near the USP5 transcription start site. Repression of MYCN expression correlated with decreased USP5 protein and mRNA levels. Co-immunoprecipitation assays showed that USP5 and MYCN formed a protein complex.

Taken together, our findings suggest that USP5 and MYCN participate in a forward feedback expression loop and that inhibition of USP5 function in neuroblastoma could be a novel therapeutic approach for MYCN-amplified tumours.