Summary Genetic studies have established anaplastic lymphoma kinase (ALK), a cell surface receptor tyrosine kinase, as a tractable molecular target in neuroblastoma. We describe comprehensive genomic, biochemical, and computational analyses of ALK mutations across 1596 diagnostic neuroblastoma samples. ALK tyrosine kinase domain mutations occurred in 8% of samples; at three hotspots plus 13 minor sites – and correlated significantly with poorer survival in high- and intermediate-risk neuroblastoma. Biochemical and computational studies distinguished oncogenic (constitutively activating) from non-oncogenic mutations and allowed robust computational prediction of their effects. We also established differential in vitro crizotinib sensitivity of mutated variants. Our studies identify ALK genomic status as a clinically important therapeutic stratification tool in neuroblastoma, and will allow tailoring of ALK-targeted therapy to specific mutations.

Erlotinib and gefitinib, tyrosine kinase inhibitors used to block EGFR (epidermal growth factor receptor) signalling in cancer, are thought to bind only the active conformation of the EGFR-TKD (tyrosine kinase domain). Through parallel computational and crystallographic studies, we show in the present study that erlotinib also binds the inactive EGFR-TKD conformation, which may have significant implications for its use in EGFR-mutated cancers.

Neuroblastomas (NBs) harboring activating point mutations in Anaplastic Lymphoma Kinase (ALK) are differentially sensitive to the ALK inhibitor crizotinib, with certain mutations conferring intrinsic crizotinib resistance. To overcome this clinical obstacle, our goal was to identify inhibitors with improved potency that can target intractable ALK variants such as F1174L. We find that PF-06463922 has high potency across ALK variants, and inhibits ALK more effectively than crizotinib in vitro. Most importantly, PF-06463922 induces complete tumor regression in both crizotinib-resistant and sensitive xenograft mouse models of NB, as well as in PDXs harboring the crizotinib-resistant F1174L or F1245C mutations. These studies demonstrate that PF-06463922 has the potential to overcome crizotinib resistance, and exerts unprecedented activity as a single targeted agent against F1174L and F1245C ALK-mutated xenograft tumors, while also inducing responses in a R1275Q xenograft model. Taken together, these results provide the rationale to move PF-06463922 into clinical trials for treatment of patients with ALK-mutated NB.

As with other groups of protein kinases, approximately 10% of the receptor tyrosine kinases (RTKs) in the human proteome contain intracellular pseudokinases that lack one or more conserved catalytically important residues. These include ErbB3, a member of the epidermal growth factor receptor (EGFR) family, and a series of unconventional Wnt receptors. We recently showed that, despite its reputation as a pseudokinase, the ErbB3 tyrosine kinase domain (TKD) does retain significant – albeit weak – kinase activity. This led us to suggest that a subgroup of RTKs may be able to signal even with very inefficient kinases. Recent work suggests that this is not the case, however. Other pseudokinase RTKs have not revealed significant kinase activity, and mutations that impair ErbB3’s weak kinase activity have not so far been found to exhibit signaling defects. These findings therefore point to models in which the TKDs of pseudokinase RTKs participate in receptor signaling by allosterically regulating associated kinases (such as ErbB3 regulation of ErbB2) and/or function as regulated ‘scaffolds’ for other intermolecular interactions central to signal propagation. Further structural and functional studies – particularly of the pseudokinase RTKs involved in Wnt signaling – are required to shed new light on these intriguing signaling mechanisms.

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