The ETV5 oncogene is a target of activated ALK signaling in neuroblastoma

AIMS: Neuroblastoma (NB) is the most common solid childhood cancer, arising in the symptho-adrenergic neural crest cells, for which the overall survival rates remain unsatisfactory. Molecular characterization of the NB genomic landscape identified activating ALK receptor mutations in 10% of primary NBs, marking patients for treatment with small-molecule inhibitors. However, recent studies show that multiple mechanisms drive resistance to these molecular therapies resulting in relapse upon exposure to a single compound. Importantly, ALK mutations emerged as an important event in relapsed cases. In order to design effective novel targeted therapeutic approaches, gaining detailed insights into downstream ALK signaling is crucial. Therefor, we performed detailed mapping of the oncogenic ALK-driven signaling in NB to identify potential fragile nodes as additional targets for combination therapies. We previously identified PI3K/AKT and RAS/MAPK as major downstream signaling axes where oncogenes RET and ETV5 came forward as major ALK regulated targets. Here, we further established ETV5 as a major RAS/MAPK downstream target regulated through ALK and its functional implications in the NB setting. METHODS: ALK activity was modulated in a panel of ALK mutated and non-ALK mutated NB cell lines to investigate downstream ALK targets. To unravel the ETV5 functionality within the NB setting, proliferation, invasion and colony formation assays following short term knock down experiments of ETV5 in different NB cell lines were performed. Xenografting experiments were used for in vivo confirmation. RNA-sequencing on ETV5 knock down cell lines and tumors upon xenografting was performed to shed a light upon ETV5 downstream targets in NB. RESULTS: Multiple ALK activating and inhibiting cell line models firmly establish ETV5 as a major RAS/MAPK downstream target regulated through ALK in both ALK addicted and ALK WT NB cells. In literature, this RAS/MAPK pathway and ETV5 are known to mediate in the process of metastasis and neuronal cell fate decisions, respectively. Here, knock down of ETV5 reduced the clonogenic potential and growth of NB cells in vitro. Xenografting of ETV5 stable knock down cell lines similarly pointed towards a reduction in growth capacity in vivo. Next, RNAseq transcriptome profiling, following ETV5 knock down in vitro and in vivo, provided us an ETV5 signature score which identifies patients with poor overall survival. Interestingly, the ETV5 knock down gene expression profile was enriched for a Ewing Sarcoma (EWS) signature, another member of the undifferentiated small-blue-round cell pediatric tumors, suggesting a possible common pertubated gene regulatory pathway in both tumor entities. Further, we identified CXCR4 and TRIM67 as possible ETV5 downstream targets in the NB setting, with CXCR4 known as an important mediator of invasion and metastasis and TRIM67 known as a negative regulator of the RAS-pathway. CONCLUSION: Our data highlight ETV5, CXCR4 and TRIM67 as intrinsic components of ALK - RAS/MAPK signaling in NB with immediate potential relevance given that deregulation of this axis by drugging can impede NB cells of their migratory capacity and therapy resistance