Oral Presentation Advances in Neuroblastoma Research Congress 2016

Identification of spliceosomal components as novel therapeutic targets for the treatment of high-risk, MYCN-driven neuroblastoma (#46)

Melinda Halasz 1 , Tapesh Santra 1 , Simon Furney 2 , Kristiina Iljin 3 , Dirk Fey 1 , Matthias Fischer 4 , Frank Westermann 5 , Walter Kolch 1
  1. Systems Biology Ireland, University College Dublin, Dublin, Ireland
  2. Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland
  3. VTT Technical Research Centre of Finland, Espoo, Finland
  4. Department of Paediatric Haematology and Oncology and Center for Molecular Medicine Cologne, University Hospital Cologne, Cologne, Germany
  5. German Cancer Research Center, Heidelberg, Germany

Background: Despite extensive research, the five-year survival rate for children with high-risk neuroblastoma (NB) is still as low as 40-50%; thus, better therapies are desperately needed. Amplification of the MYCN oncogene drives about half of the aggressive forms. Unfortunately, MYCN is considered undruggable. Based on the hypothesis that we can target MYCN through its protein-protein interactions/interactors (PPI), we aim to identify novel therapeutic targets to treat children with MYCN-amplified neuroblastoma.

Methods: We mapped the MYCN interactome across a panel of neuroblastoma cell lines with differential MYCN status and paediatric tumour samples by using label-free quantitative interaction proteomics. We inhibited selected MYCN PPIs by RNAi or drugs (splicing inhibitors); and measured cell viability as well as anchorage-independent growth of NB cells. We inferred the differential exon usage from mRNA-Seq data by using DexSeq; and analysed alternative splicing of selected genes in the presence of splicing inhibitors by qPCR. To further validate the effect of splicing inhibition on NB growth in vivo, we treated Tg[dbh:MYCN-EGFP] zebrafish (a generous gift from A. T. Look, Dana-Farber Cancer Institute) developing tumours in the fish-equivalent of adrenal medulla with splicing inhibitors.

Results: By comparing proteomics data obtained from MYCN-amplified tumours and NB cell lines, we shortlisted MYCN PPIs that are relevant in MYCN-amplified tumours. Ingenuity Pathway Analysis of MYCN PPIs revealed that pre-mRNA splicing is among the top altered pathways. Knocking down spliceosomal MYCN PPIs decreased cell viability of MYCN-amplified NB cells. Moreover, selected splicing inhibitors reduced transformation of MYCN-amplified NB cells in soft agar assays. Furthermore, we identified differentially spliced genes in MYCN-amplified NB; many of them are tumour suppressors, and regulate invasion and apoptosis. In addition, the splicing inhibitor reduced tumour size in the zebrafish model of MYCN-driven neuroblastoma.

Conclusions: We confirmed a novel strategy to target the pharmacologically undruggable MYCN. We pinpointed spliceosomal components as novel MYCN PPIs, and confirmed that we can inhibit MYCN-amplified NB growth by altering mRNA splicing.