Flash Talk & E-Poster Presentation 33rd Lorne Cancer Conference 2021

MYCN interacts with SNRPD3 and alters spliceosome activity in neuroblastoma to promote cell viability and proliferation (#50)

Alice Salib 1 2 , Chelsea Mayoh 1 2 , Nisitha Jayatilleke 1 , Andrew J Gifford 1 2 3 , Zsuzsanna Nagy 1 2 , Belamy B Cheung 1 2 , Daniel R Carter 1 2 4 , Glenn M Marshall 1 5
  1. Children's Cancer Institute Australia, University of New South Wales, Randwick, NSW, Australia
  2. School of Women's & Children's Health, UNSW Sydney, Kensington, NSW, Australia
  3. Department of Anatomical Pathology, Prince of Wales Hospital, Randwick, NSW, Australia
  4. School of Biomedical Engineering, University of Technology Sydney, Sydney, NSW, Australia
  5. Kids Cancer Centre, Sydney Children's Hospital, Sydney, NSW, Australia

MYCN amplification is the most important predictor of poor prognosis for neuroblastoma. Pro-tumorigenic functions of MYCN are attributed to its ability to regulate global gene expression programs. Alternative splicing is an important component of genetic regulation and has been implicated in neuroblastoma tumorigenesis. In this study, we examined the hypothesis that MYCN drives neuroblastoma tumorigenesis by promoting alternative splicing in neuroblastoma cells.

Gene expression analysis on ganglia cells and tumour tissue collected during tumour initiation in transgenic TH-MYCN+/+ homozygous and wildtype mice revealed a significant increase in the mRNA expression of SNRPD3, a core component of the spliceosome. siRNA knockdown of SNRPD3 reduced cell viability, proliferation and colony numbers in MYCN-amplified neuroblastoma cells. Further investigation using the MYCN-inducible, SHEP.tet21N cells, showed that SNRPD3 knockdown exhibited a more pronounced lethality only in the presence of MYCN. Furthermore, RNA-sequencing revealed a genome-wide increase in the number of genes being differentially spliced when MYCN is expressed and depletion of SNRPD3 in the presence of MYCN resulted in a further 2.5-fold increase in the number of genes being differentially spliced. SNRPD3 knockdown alone showed little effect on global differential splicing. Gene ontology and pathway enrichment analysis of MYCN-on/SNRPD3-off gene splicing changes identified enrichment in cell cycle and DNA damage pathways. Further analysis by co-immunoprecipitation identified SNRPD3 as a novel MYCN binding protein. In addition, we showed that protein expression of SNRPD3, SNRPD3 methylation and the protein methyltransferase, PRMT5 decreased with MYCN knockdown. Utilizing PRMT5 inhibitors, currently in clinical trials, showed MYCN-amplified cell lines have greater sensitivity to the inhibitors compared to MYCN non-amplified cells.

We conclude that SNRPD3 contributes to changes in alternative splicing that perpetuate MYCN-driven oncogenesis. Disruption of SNRPD3 in the presence of MYCN was sufficient to induce widespread mis-splicing that impact the cell cycle resulting in cell death. Our data suggests a role for PRMT5 inhibitors in the treatment of neuroblastoma and highlights SNRPD3 as a novel therapeutic vulnerability.