Oral Presentation Advances in Neuroblastoma Research Congress 2016

Identifying mechanisms of neuroblastoma tumorigenesis using single cell transcriptomics (#52)

Daniel R Carter 1 , Belamy B Cheung 1 , Dominik Beck 2 , Anneleen Beckers 3 , Katleen De Preter 3 , Frank Speleman 3 , Murray Norris 1 , Michelle Haber 1 , Alexander Swarbrick 4 , Glenn Marshall 1
  1. Children's Cancer Institute, Randwick, NSW, Australia
  2. Adult Cancer Program, Lowy Cancer Research Centre, University of New South Wales, Randwick, NSW, Australia
  3. Center for Medical Genetics (CMGG), Ghent University, Ghent, Belgium
  4. The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia

Neuroblastoma is a pediatric cancer of the sympathetic nervous system. MYCN oncogene amplification occurs in approximately 20% of patients and is associated with poor patient prognosis. MYCN overexpression has been confirmed to recapitulate neuroblastoma tumorigenesis in TH-MYCN transgenic mice. In this model, sympathetic ganglia exhibit hyperplasia of a premalignant neuroblast population, which are clonally selected prior to malignant transformation. The genetic determinants of clonal selection in this heterogenous population of premalignant cells is unknown.

To decipher early changes in gene expression during neuroblastoma tumor initiation, we conducted single cell transcriptomics on sympathetic ganglia derived from 10 day old pre-tumor TH-MYCN+/+ mice using the Fluidigm C1 Single-Cell Auto Prep System. We successfully profiled the expression of 152 single cells according to 69 custom-derived genes (a 62 gene tumorigenesis signature, 5 lineage markers and 2 housekeeping genes). Hierarchical clustering of the profiled cells identified that hyperplastic neuroblasts (Phox2b+,Dbh+,PCNAHigh) fell within two dominant clusters by gene expression, with only one of the clusters showing exclusive expression of a 11 member subgroup of genes involved in regulation of mitosis (ASPM, BUB1, BUB1B, NUF2, KIF23, PRR11, CCNB2, KIFC1, DEPDC1, SGOL1, CCNB1). Interrogation of a 649 patient neuroblastoma tumor dataset, showed that the same 11 mitotic genes were strongly correlated with MYCN target gene expression, stage 4 disease and MYCN amplification. Moreover in a panel of 30 neuroblastoma cell lines, high expression of the 11 mitotic genes sensitized cells to anti-mitotic compounds such as preclinical agents that target Aurora Kinase B, PLK1 and EG5, as well as clinically used microtubule inhibitors. Together this supports a hypothetical model: that deregulation of mitotic genes occurs as a secondary event to MYCN amplification in neuroblastoma tumor initiation, and high mitotic gene expression is associated with clonal selection, malignant transformation and tumor progression. These findings also support that mitotic deregulation may impart a therapeutic targeting vulnerability that could be exploited as a novel targeted strategy for MYCN-amplified neuroblastoma patients.