Phosphoproteome and gene expression profiling of ALK inhibition in neuroblastoma cell lines reveals conserved oncogenic pathways

Background: Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that is a clinical target of major interest in cancer. Mutations and rearrangements in ALK trigger the activation of the encoded receptor and its downstream signaling pathways. ALK mutations have been identified in both familial and sporadic neuroblastoma cases as well as in 30 to 40% of relapses, which makes ALK a bona fide target in neuroblastoma therapy. Tyrosine kinase inhibitors (TKIs) that target ALK are currently in clinical use for the treatment of patients with ALK-positive non–small cell lung cancer. However, monotherapy with the ALK inhibitor crizotinib has been less encouraging in neuroblastoma patients with ALK alterations, raising the question of whether combinatorial therapy would be more effective. Methods: In this study, we established both phosphoproteomic and gene expression profiles of ALK activity in neuroblastoma cells exposed to first- and third-generation ALK TKIs, to identify the underlying molecular mechanisms and identify relevant biomarkers, signaling networks, and new therapeutic targets. Results: The phosphoproteomic analysis identified several conserved oncogenic downstream signaling pathways of ALK, similar to those involved in insulin receptor (INSR)/tropomyosin receptor kinase (TRK) and fibroblast growth factor receptor (FGFR) signaling. In addition, signaling events involved in feedback and cross-talk were identified, including modulation of DUSP (dual-specificity phosphatase) family phosphatases. Furthermore, from analysis of the RNA-seq data, several transcription factors were predicted and validated as responsive to ALK signaling, including members of the FOXO (forkhead box O) and ETS (E26 transformation-specific or E-twenty-six) transcription factor families. Conclusions: Although neuroblastoma is a complex heterogeneous disease, this in-depth investigation of downstream targets of ALK signaling offers future avenues to pursue to inhibit ALK-driven neuroblastoma. Legal entity responsible for the study: Bengt Hallberg. Funding: Barncancerfonden. Disclosure: All authors have declared no conflicts of interest.