Characterization of ALK driven molecular networks identifies perturbed RET and cholinergic signaling in neuroblastoma

Purpose: Activating ALK mutations are present in nearly 10% of primary neuroblastomas and mark patients for treatment with ALK inhibitors. Clinical trials for small molecule ALK inhibitors have been initiated for neuroblastoma and other ALK-driven tumor entities. Recent studies have shown that multiple mechanisms drive resistance to molecular therapies targeting receptor tyrosine kinases including oncogene switching and modulation of various regulatory loops. We anticipated that detailed mapping of the oncogenic ALK-driven signaling in neuroblastoma can pave the way to identify fragile nodes that may serve as additional targets for combination therapies. Experimental design: To achieve this aim, we analysed the transcriptional consequences of mutant ALK signaling in neuroblastoma. As such, we established a 79-gene signature recapitulating the transcriptional response upon ALK inhibition based on transcriptome profiling of ALKwt, ALKF1174L and ALKR1275Q mutant, as well as ALKamplified NB cell lines following ALK inhibition by either NVP-TAE-684, LDK-378, X-396 or Crizotinib. Further, the 79-gene signature was validated in an ALK-inducible NB cell line and primary tumor samples. Results: We established a 79-gene signature marking ALK activity in neuroblastoma cells, which was also preserved to a large extent in other ALK driven tumor entities and across ALKF1174L- and MYCN-driven human and murine NB tumors. Further data mining confirmed that MEK/MAPK, AKT/mTOR and MYC/MYCN pathways are important downstream branches of ALK signaling and highlight a disabled MAPK negative feedback loop. Moreover, using cross-species genomics analysis we show that mutant ALK induces RET and RET driven cholinergic sympathetic neuronal markers such as VIP, VGF, ChAT and VAChT, pointing out to a perturbed RET and cholinergic signaling in neuroblastoma. Conclusions: These novel insights are important leads for further studies exploring novel combination therapy strategies in ALK mutant neuroblastomas and can also pave the way for deeper understanding of the role of ALK signaling in normal sympathetic nervous system development. Moreover, our data emphasize RET as a target for further molecular combination therapies