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Neuroblastoma is an aggressive childhood cancer arising from sympatho-adrenergic neuronal progenitors. The low survival rates for high-risk disease point to an urgent need for novel targeted therapeutic approaches. Detailed molecular characterization of the neuroblastoma genomic landscape indicates that ALK-activating mutations are present in 10% of primary tumours. Together with other mutations causing RAS/MAPK pathway activation, ALK mutations are also enriched in relapsed cases and ALK activation was shown to accelerate MYCN-driven tumour formation through hitherto unknown ALK-driven target genes. To gain further insight into how ALK contributes to neuroblastoma aggressiveness, we searched for known oncogenes in our previously reported ALK-driven gene signature. We identified ETV5, a bona fide oncogene in prostate cancer, as robustly upregulated in neuroblastoma cells harbouring ALK mutations, and show high ETV5 levels downstream of the RAS/MAPK axis. Increased ETV5 expression significantly impacted migration, invasion and colony formation in vitro, and ETV5 knockdown reduced proliferation in a murine xenograft model. We also established a gene signature associated with ETV5 knockdown that correlates with poor patient survival. Taken together, our data highlight ETV5 as an intrinsic component of oncogenic ALK-driven signalling through the MAPK axis and propose that ETV5 upregulation in neuroblastoma may contribute to tumour aggressiveness.

Long intergenic non-coding RNAs (lincRNAs) are emerging as integral components of signaling pathways in various cancer types. In neuroblastoma, only a handful of lincRNAs are known as upstream regulators or downstream effectors of oncogenes. Here, we exploit RNA sequencing data of primary neuroblastoma tumors, neuroblast precursor cells, neuroblastoma cell lines and various cellular perturbation model systems to define the neuroblastoma lincRNome and map lincRNAs up- and downstream of neuroblastoma driver genes MYCN, ALK and PHOX2B. Each of these driver genes controls the expression of a particular subset of lincRNAs, several of which are associated with poor survival and are differentially expressed in neuroblastoma tumors compared to neuroblasts. By integrating RNA sequencing data from both primary tumor tissue and cancer cell lines, we demonstrate that several of these lincRNAs are expressed in stromal cells. Deconvolution of primary tumor gene expression data revealed a strong association between stromal cell composition and driver gene status, resulting in differential expression of these lincRNAs. We also explored lincRNAs that putatively act upstream of neuroblastoma driver genes, either as presumed modulators of driver gene activity, or as modulators of effectors regulating driver gene expression. This analysis revealed strong associations between the neuroblastoma lincRNAs MIAT and MEG3 and MYCN and PHOX2B activity or expression. Together, our results provide a comprehensive catalogue of the neuroblastoma lincRNome, highlighting lincRNAs up- and downstream of key neuroblastoma driver genes. This catalogue forms a solid basis for further functional validation of candidate neuroblastoma lincRNAs.

Chemotherapy resistance is responsible for high mortality rates in neuroblastoma. MYCN, an oncogenic driver in neuroblastoma, controls pluripotency genes including LIN28B. We hypothesized that enhanced embryonic stem cell (ESC) gene regulatory programs could mark tumors with high pluripotency capacity and subsequently increased risk for therapy failure. An ESC miRNA signature was established based on publicly available data. In addition, an ESC mRNA signature was generated including the 500 protein coding genes with the highest positive expression correlation with the ESC miRNA signature score in 200 neuroblastomas. High ESC m(i)RNA expression signature scores were significantly correlated with poor neuroblastoma patient outcome specifically in the subgroup of MYCN amplified tumors and stage 4 nonamplified tumors. Further data-mining identified FOXM1, as the major predicted driver of this ESC signature, controlling a large set of genes implicated in cell cycle control and DNA damage response. Of further interest, re-analysis of published data showed that MYCN transcriptionally activates FOXM1 in neuroblastoma cells. In conclusion, a novel ESC m(i)RNA signature stratifies neuroblastomas with poor prognosis, enabling the identification of therapy-resistant tumors. The finding that this signature is strongly FOXM1 driven, warrants for drug design targeted at FOXM1 or key components controlling this pathway.