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Simple Summary Neuroblastoma is a pediatric cancer that arises in the sympathetic nervous system. High-risk neuroblastoma is clinically challenging and identification of novel therapies, particularly those that offer a reduction in morbidity for these patients, is a high priority. Combining genetic analyses with investigation of molecular mechanisms, while considering recent advances in our understanding of key developmental events, provides avenues for future treatment. Here we review and highlight several recently published articles that address novel molecular mechanisms arising from chromosome 1p, 2p, and 11q aberrations, which likely contribute to high-risk neuroblastoma, and discusses their potential impact on treatment options. Abstract Neuroblastoma is the most common extracranial solid pediatric tumor, with around 15% childhood cancer-related mortality. High-risk neuroblastomas exhibit a range of genetic, morphological, and clinical heterogeneities, which add complexity to diagnosis and treatment with existing modalities. Identification of novel therapies is a high priority in high-risk neuroblastoma, and the combination of genetic analysis with increased mechanistic understanding—including identification of key signaling and developmental events—provides optimism for the future. This focused review highlights several recent findings concerning chromosomes 1p, 2p, and 11q, which link genetic aberrations with aberrant molecular signaling output. These novel molecular insights contribute important knowledge towards more effective treatment strategies for neuroblastoma.

Simple Summary The anaplastic lymphoma kinase (ALK) and rearranged during transfection (RET) receptor tyrosine kinases (RTKs) are expressed in both the developing neural crest and the pediatric cancer neuroblastoma. Moreover, ALK is mutated in approximately 10% of neuroblastomas. Here, we investigated ALK and RET in neuroblastoma, with the aim of better understanding their respective contributions. Using neuroblastoma cell lines, we show that ALK modulates RET signaling at the level of RET phosphorylation, as well as at the level of transcription. Using CRISPR/Cas9, we generated RET knockout neuroblastoma cell lines and performed a multi-omics approach, combining RNA-Seq and proteomics to characterize the effect of deleting RET in a neuroblastoma context. Remarkably, we could show that loss of RET results in a striking epithelial-to-mesenchymal transition (EMT) phenotype, and we provide evidence that RET activity suppresses the mesenchymal phenotype in neuroblastoma. Abstract Aberrant activation of anaplastic lymphoma kinase (ALK) drives neuroblastoma (NB). Previous work identified the RET receptor tyrosine kinase (RTK) as a downstream target of ALK activity in NB models. We show here that ALK activation in response to ALKAL2 ligand results in the rapid phosphorylation of RET in NB cells, providing additional insight into the contribution of RET to the ALK-driven gene signature in NB. To further address the role of RET in NB, RET knockout (KO) SK-N-AS cells were generated by CRISPR/Cas9 genome engineering. Gene expression analysis of RET KO NB cells identified a reprogramming of NB cells to a mesenchymal (MES) phenotype that was characterized by increased migration and upregulation of the AXL and MNNG HOS transforming gene (MET) RTKs, as well as integrins and extracellular matrix components. Strikingly, the upregulation of AXL in the absence of RET reflects the development timeline observed in the neural crest as progenitor cells undergo differentiation during embryonic development. Together, these findings suggest that a MES phenotype is promoted in mesenchymal NB cells in the absence of RET, reflective of a less differentiated developmental status.

High‐risk neuroblastoma (NB) is responsible for a disproportionate number of childhood deaths due to cancer. One indicator of high‐risk NB is amplification of the neural MYC (MYCN) oncogene, which is currently therapeutically intractable. Identification of anaplastic lymphoma kinase (ALK) as an NB oncogene raised the possibility of using ALK tyrosine kinase inhibitors (TKIs) in treatment of patients with activating ALK mutations. 8–10% of primary NB patients are ALK‐positive, a figure that increases in the relapsed population. ALK is activated by the ALKAL2 ligand located on chromosome 2p, along with ALK and MYCN, in the “2p‐gain” region associated with NB. Dysregulation of ALK ligand in NB has not been addressed, although one of the first oncogenes described was v‐sis that shares > 90% homology with PDGF. Therefore, we tested whether ALKAL2 ligand could potentiate NB progression in the absence of ALK mutation. We show that ALKAL2 overexpression in mice drives ALK TKI‐sensitive NB in the absence of ALK mutation, suggesting that additional NB patients, such as those exhibiting 2p‐gain, may benefit from ALK TKI‐based therapeutic intervention., ALKAL2 misregulation facilitates ALK receptor tyrosine kinase signaling and drives ALK‐dependent cancer irrespective of oncogenic mutations.

SUMMARY Although activating mutations of the anaplastic lymphoma kinase (ALK) membrane receptor occur in ~10% of neuroblastoma (NB) tumors, the role of the wild-type (WT) receptor, which is aberrantly expressed in most non-mutated cases, is unclear. Both WT and mutant proteins undergo extracellular domain (ECD) cleavage. Here, we map the cleavage site to Asn654-Leu655 and demonstrate that cleavage inhibition of WT ALK significantly impedes NB cell migration with subsequent prolongation of survival in mouse models. Cleavage inhibition results in the downregulation of an epithelial-to-mesenchymal transition (EMT) gene signature, with decreased nuclear localization and occupancy of β-catenin at EMT gene promoters. We further show that cleavage is mediated by matrix metalloproteinase 9, whose genetic and pharmacologic inactivation inhibits cleavage and decreases NB cell migration. Together, our results indicate a pivotal role for WT ALK ECD cleavage in NB pathogenesis, which may be harnessed for therapeutic benefit., Graphical abstract, In brief Huang et al. show that extracellular domain (ECD) cleavage of the ALK cell surface tyrosine kinase receptor mediates neuroblastoma cell migration through induction of an EMT phenotype. ECD cleavage is caused by MMP-9 whose inhibition leads to decreased cell migration.

Significance Neuroblastoma is a pediatric tumor arising from the neural crest. Dysregulation of the receptor tyrosine kinase ALK has been linked to neuroblastoma, making it important to understand its function in native conditions. In zebrafish, a related receptor—Ltk—is also expressed in neural crest and regulates development of specific pigment cells—iridophores. Ligands activating human ALK were recently identified as the ALKAL proteins (FAM150, AUG) by biochemical means. Our data show that this ligand–receptor pair functions in vivo in the neural crest of zebrafish to drive development of iridophores. Removal of Ltk or all three zebrafish ALKALs results in larvae completely lacking these cells. Using Drosophila and human cell lines, we show evolutionary conservation of this important interaction., Mutations in anaplastic lymphoma kinase (ALK) are implicated in somatic and familial neuroblastoma, a pediatric tumor of neural crest-derived tissues. Recently, biochemical analyses have identified secreted small ALKAL proteins (FAM150, AUG) as potential ligands for human ALK and the related leukocyte tyrosine kinase (LTK). In the zebrafish Danio rerio, DrLtk, which is similar to human ALK in sequence and domain structure, controls the development of iridophores, neural crest-derived pigment cells. Hence, the zebrafish system allows studying Alk/Ltk and Alkals involvement in neural crest regulation in vivo. Using zebrafish pigment pattern formation, Drosophila eye patterning, and cell culture-based assays, we show that zebrafish Alkals potently activate zebrafish Ltk and human ALK driving downstream signaling events. Overexpression of the three DrAlkals cause ectopic iridophore development, whereas loss-of-function alleles lead to spatially distinct patterns of iridophore loss in zebrafish larvae and adults. alkal loss-of-function triple mutants completely lack iridophores and are larval lethal as is the case for ltk null mutants. Our results provide in vivo evidence of (i) activation of ALK/LTK family receptors by ALKALs and (ii) an involvement of these ligand–receptor complexes in neural crest development.

Numerous mutations have been observed in the Anaplastic Lymphoma Kinase (ALK) receptor tyrosine kinase (RTK) in both germline and sporadic neuroblastoma. Here, we have investigated the Y1278S mutation, observed in four patient cases, and its potential importance in the activation of the full length ALK receptor. Y1278S is located in the 1278-YRASYY-1283 motif of the ALK activation loop, which has previously been reported to be important in the activation of the ALK kinase domain. In this study, we have characterized activation loop mutations within the context of the full length ALK employing cell culture and Drosophila melanogaster model systems. Our results show that the Y1278S mutant observed in patients with neuroblastoma harbors gain-of-function activity. Secondly, we show that the suggested interaction between Y1278 and other amino acids might be of less importance in the activation process of the ALK kinase than previously proposed. Thirdly, of the three individual tyrosines in the 1278-YRASYY-1283 activation loop, we find that Y1283 is the critical tyrosine in the activation process. Taken together, our observations employing different model systems reveal new mechanistic insights on how the full length ALK receptor is activated and highlight differences with earlier described activation mechanisms observed in the NPM-ALK fusion protein, supporting a mechanism of activation more in line with those observed for the Insulin Receptor (InR).

The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori), has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo. In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALKF1174L/MYCN. Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients., Summary: Our results suggest that PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients.

Aberrant activation of anaplastic lymphoma kinase (ALK) has been described in a range of human cancers, including non-small cell lung cancer and neuroblastoma (Hallberg and Palmer, 2013). Vertebrate ALK has been considered to be an orphan receptor and the identity of the ALK ligand(s) is a critical issue. Here we show that FAM150A and FAM150B are potent ligands for human ALK that bind to the extracellular domain of ALK and in addition to activation of wild-type ALK are able to drive 'superactivation' of activated ALK mutants from neuroblastoma. In conclusion, our data show that ALK is robustly activated by the FAM150A/B ligands and provide an opportunity to develop ALK-targeted therapies in situations where ALK is overexpressed/activated or mutated in the context of the full length receptor. DOI: http://dx.doi.org/10.7554/eLife.09811.001, eLife digest Cells have receptor proteins on their surface that enable them to detect changes in their environment and communicate with other cells. Signal molecules bind to a segment of the receptor called the extracellular domain that faces out from the cell. This can result in the activation of another domain in the receptor that is just inside the cell, which, in turn, activates signaling pathways that relay the information around the cell. However, these communication systems are often disrupted in cancer cells. This helps the cells to override the strict growth controls imposed upon them by other (healthy) cells in the body. The gene that encodes a receptor protein called Anaplastic Lymphoma Kinase (or ALK for short) is often mutated in some types of human cancer so that the protein is always active. However, we still do not know what signal molecules bind to the ALK protein to activate it in normal cells. Guan, Umapathy et al. used a variety of cell biology and biochemical techniques to study the role of ALK. The experiments show that when either of two proteins called FAM150A and FAM150B are produced in rat nerve cells alongside ALK, the nerve cells rapidly respond and form outgrowths. Experiments using cancer cells derived from human nerve cells also yielded similar results. Guan, Umapathy et al. found that the extracellular domain of ALK can physically interact with FAM150A and FAM150B. The eyes of fruit flies that had been genetically modified to produce the human ALK protein alongside either FAM150A or FAM150B grew more than normal, giving the eyes an abnormal "rough" appearance. Further experiments showed that FAM150A and FAM150B are also able to increase the level of activation of an ALK mutant protein that is already active. Therefore, in future, the development of drugs that stop FAM150A and FAM150B from binding to ALK may be useful for treating cancers that are driven by high levels of ALK activity. Many challenging questions lie ahead to better understand how FAM150A and FAM150B interact with ALK. DOI: http://dx.doi.org/10.7554/eLife.09811.002