ALK signaling primes the DNA damage response sensitizing ALK-driven neuroblastoma to therapeutic ATR inhibition.

High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and Anaplastic Lymphoma Kinase (ALK), which are often involved in high-risk NB, lead to increased replication stress in cancer cells, suggesting therapeutic strategies. We previously identified an ATR (ataxia telangiectasia and Rad3-related)/ALK inhibitor (ATRi/ALKi) combination as such a strategy in two independent genetically modified mouse NB models. Here, we identify an underlying molecular mechanism, in which ALK signaling leads to phosphorylation of ATR and CHK1, supporting an effective DNA damage response. The importance of ALK inhibition is supported by mouse data, in which ATRi monotreatment resulted in a robust initial response, but subsequent relapse, in contrast to a 14-d ALKi/ATRi combination treatment that resulted in a robust and sustained response. Finally, we show that the remarkable response to the 14-d combined ATR/ALK inhibition protocol reflects a robust differentiation response, reprogramming tumor cells to a neuronal/Schwann cell lineage identity. Our results identify an ability of ATR inhibition to promote NB differentiation and underscore the importance of further exploring combined ALK/ATR inhibition in NB, particularly in high-risk patient groups with oncogene-induced replication stress.

IGF1R Contributes to Cell Proliferation in ALK-Mutated Neuroblastoma with Preference for Activating the PI3K-AKT Signaling Pathway.

Aberrant activation of anaplastic lymphoma kinase (ALK) by activating point mutation or amplification drives 5-12% of neuroblastoma (NB). Previous work has identified the involvement of the insulin-like growth factor 1 receptor (IGF1R) receptor tyrosine kinase (RTK) in a wide range of cancers. We show here that many NB cell lines exhibit IGF1R activity, and that IGF1R inhibition led to decreased cell proliferation to varying degrees in ALK-driven NB cells. Furthermore, combined inhibition of ALK and IGF1R resulted in synergistic anti-proliferation effects, in particular in ALK-mutated NB cells. Mechanistically, both ALK and IGF1R contribute significantly to the activation of downstream PI3K-AKT and RAS-MAPK signaling pathways in ALK-mutated NB cells. However, these two RTKs employ a differential repertoire of adaptor proteins to mediate downstream signaling effects. We show here that ALK signaling led to activation of the RAS-MAPK pathway by preferentially phosphorylating the adaptor proteins GAB1, GAB2, and FRS2, while IGF1R signaling preferentially phosphorylated IRS2, promoting activation of the PI3K-AKT pathway. Together, these findings reveal a potentially important role of the IGF1R RTK in ALK-mutated NB and that co-targeting of ALK and IGF1R may be advantageous in clinical treatment of ALK-mutated NB patients.

Integrating scRNA and bulk-RNA sequencing develops a cell senescence signature for analyzing tumor heterogeneity in clear cell renal cell carcinoma.

Introduction: Cellular senescence (CS) plays a critical role in cancer development, including clear cell renal cell carcinoma (ccRCC). Traditional RNA sequencing cannot detect precise molecular composition changes within tumors. This study aimed to analyze cellular senescence's biochemical characteristics in ccRCC using single RNA sequencing (ScRNA-seq) and traditional RNA sequencing (Bulk RNA-seq). Methods: Researchers analyzed the biochemical characteristics of cellular senescence in ccRCC using ScRNA-seq and Bulk RNA-seq. They combined these approaches to identify differences between malignant and non-malignant phenotypes in ccRCC across three senescence-related pathways. Genes from these pathways were used to identify molecular subtypes associated with senescence, and a new risk model was constructed. The function of the gene DUSP1 in ccRCC was validated through biological experiments. Results: The combined analysis of ScRNA-seq and Bulk RNA-seq revealed significant differences between malignant and non-malignant phenotypes in ccRCC across three senescence-related pathways. Researchers identified genes from these pathways to identify molecular subtypes associated with senescence, constructing a new risk model. Different subgroups showed significant differences in prognosis level, clinical stage and grade, immune infiltration, immunotherapy, and drug sensitivity. Discussion: Senescence signature markers are practical biomarkers and predictors of molecular typing in ccRCC. Differences in prognosis level, clinical stage and grade, immune infiltration, immunotherapy, and drug sensitivity between different subgroups indicate that this approach could provide valuable insights into senescence-related treatment options and prognostic assessment for patients with ccRCC. The function of the gene DUSP1 in ccRCC was validated through biological experiments, confirming its feasibility as a novel biomarker for ccRCC. These findings suggest that targeted therapies based on senescence-related mechanisms could be an effective treatment option for ccRCC.

ALK F1174S mutation impairs ALK kinase activity in EML4-ALK variant 1 and sensitizes EML4-ALK variant 3 to crizotinib.

Objective: To assess the influence of F1174S mutation on kinase activity and drug sensitivity of the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) fusion (EML4-ALK) variants 1 and 3. Methods: We constructed mammalian expression plasmids of both wildtype and F1174 mutant EML4-ALK variants 1 and 3, and then characterized them with cell models by performing immunoblotting, neurite outgrowth assay, focus formation assay as well as protein stability assay. Drug sensitivity to ALK tyrosine kinase inhibitors was also compared between wildtype and F1174 mutant EML4-ALK fusions. In addition, we characterized the effect of different F1174 kinase domain mutations in the context of EML4-ALK fusions. Results: In contrast to the oncogenic ALK-F1174S mutation that has been reported to be activating in the context of full-length ALK in neuroblastoma, EML4-ALK (F1174S) variant 1 exhibits impaired kinase activity leading to loss of oncogenicity. Furthermore, unlike the previously reported F1174C/L/V mutations, mutation of F1174 to S sensitizes EML4-ALK variants 3a and 3b to crizotinib. Conclusion: These findings highlight the complexity of drug selection when treating patients harboring resistance mutations and suggest that the F1174S mutation in EML4-ALK variant 1 is likely not a potent oncogenic driver. Additional oncogenic driver or other resistance mechanisms should be considered in the case of EML4-ALK variant 1 with F1174S mutation.

Chromosome Imbalances in Neuroblastoma-Recent Molecular Insight into Chromosome 1p-deletion, 2p-gain, and 11q-deletion Identifies New Friends and Foes for the Future.

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.

Mapping the Phospho-dependent ALK Interactome to Identify Novel Components in ALK Signaling.

Protein-protein interactions (PPIs) play essential roles in Anaplastic Lymphoma Kinase (ALK) signaling. Systematic characterization of ALK interactors helps elucidate novel ALK signaling mechanisms and may aid in the identification of novel therapeutics targeting related diseases. In this study, we used the Mammalian Membrane Two-Hybrid (MaMTH) system to map the phospho-dependent ALK interactome. By screening a library of 86 SH2 domain-containing full length proteins, 30 novel ALK interactors were identified. Many of their interactions are correlated to ALK phosphorylation activity: oncogenic ALK mutations potentiate the interactions and ALK inhibitors attenuate the interactions. Among the novel interactors, NCK2 was further verified in neuroblastoma cells using co-immunoprecipitation. Modulation of ALK activity by addition of inhibitors lead to concomitant changes in the tyrosine phosphorylation status of NCK2 in neuroblastoma cells, strongly supporting the functionality of the ALK/NCK2 interaction. Our study provides a resource list of potential novel ALK signaling components for further study.

Extracellular domain shedding of the ALK receptor mediates neuroblastoma cell migration.

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 ß-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.

BioID-Screening Identifies PEAK1 and SHP2 as Components of the ALK Proximitome in Neuroblastoma Cells.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is mutated in approximately 10% of pediatric neuroblastoma (NB). To shed light on ALK-driven signaling processes, we employed BioID-based in vivo proximity labeling to identify molecules that interact intracellularly with ALK. NB-derived SK-N-AS and SK-N-BE(2) cells expressing inducible ALK-BirA* fusion proteins were generated and stimulated with ALKAL ligands in the presence and absence of the ALK tyrosine kinase inhibitor (TKI) lorlatinib. LC/MS-MS analysis identified multiple proteins, including PEAK1 and SHP2, which were validated as ALK interactors in NB cells. Further analysis of the ALK-SHP2 interaction confirmed that the ALK-SHP2 interaction as well as SHP2-Y542 phosphorylation was dependent on ALK activation. Use of the SHP2 inhibitors, SHP099 and RMC-4550, resulted in inhibition of cell growth in ALK-driven NB cells. In addition, we noted a strong synergistic effect of combined ALK and SHP2 inhibition that was specific to ALK-driven NB cells, suggesting a potential therapeutic option for ALK-driven NB.

Loss of RET Promotes Mesenchymal Identity in Neuroblastoma Cells.

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.

ALK ligand ALKAL2 potentiates MYCN-driven neuroblastoma in the absence of ALK mutation.

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.

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

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. 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. This analysis has unveiled various important leads for novel combinatorial treatment strategies for patients with neuroblastoma and an increased understanding of ALK signaling involved in this disease.

Clinical response of the novel activating ALK-I1171T mutation in neuroblastoma to the ALK inhibitor ceritinib.

Tumors with anaplastic lymphoma kinase (ALK) fusion rearrangements, including non-small-cell lung cancer and anaplastic large cell lymphoma, are highly sensitive to ALK tyrosine kinase inhibitors (TKIs), underscoring the notion that such cancers are addicted to ALK activity. Although mutations in ALK are heavily implicated in childhood neuroblastoma, response to the ALK TKI crizotinib has been disappointing. Embryonal tumors in patients with DNA repair defects such as Fanconi anemia (FA) often have a poor prognosis, because of lack of therapeutic options. Here we report a child with underlying FA and ALK mutant high-risk neuroblastoma responding strongly to precision therapy with the ALK TKI ceritinib. Conventional chemotherapy treatment caused severe, life-threatening toxicity. Genomic analysis of the initial biopsy identified germline FANCA mutations as well as a novel ALK-I1171T variant. ALK-I1171T generates a potent gain-of-function mutant, as measured in PC12 cell neurite outgrowth and NIH3T3 transformation. Pharmacological inhibition profiling of ALK-I1171T in response to various ALK TKIs identified an 11-fold improved inhibition of ALK-I1171T with ceritinib when compared with crizotinib. Immunoaffinity-coupled LC-MS/MS phosphoproteomics analysis indicated a decrease in ALK signaling in response to ceritinib. Ceritinib was therefore selected for treatment in this child. Monotherapy with ceritinib was well tolerated and resulted in normalized catecholamine markers and tumor shrinkage. After 7.5 mo treatment, the residual primary tumor shrunk, was surgically removed, and exhibited hallmarks of differentiation together with reduced Ki67 levels. Clinical follow-up after 21 mo treatment revealed complete clinical remission including all metastatic sites. Therefore, ceritinib presents a viable therapeutic option for ALK-positive neuroblastoma.

ALKALs are in vivo ligands for ALK family receptor tyrosine kinases in the neural crest and derived cells.

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.

MEK inhibitor trametinib does not prevent the growth of anaplastic lymphoma kinase (ALK)-addicted neuroblastomas.

Activation of the RAS-RAF-MEK-ERK signaling pathway is implicated in driving the initiation and progression of multiple cancers. Several inhibitors targeting the RAS-MAPK pathway are clinically approved as single- or polyagent therapies for patients with specific types of cancer. One example is the MEK inhibitor trametinib, which is included as a rational polytherapy strategy for treating EML4-ALK-positive, EGFR-activated, or KRAS-mutant lung cancers and neuroblastomas that also contain activating mutations in the RAS-MAPK pathway. In addition, in neuroblastoma, a heterogeneous disease, relapse cases display an increased rate of mutations in ALK, NRAS, and NF1, leading to increased activation of RAS-MAPK signaling. Co-targeting ALK and the RAS-MAPK pathway is an attractive option, because monotherapies have not yet produced effective results in ALK-addicted neuroblastoma patients. We evaluated the response of neuroblastoma cell lines to MEK-ERK pathway inhibition by trametinib. In contrast to RAS-MAPK pathway-mutated neuroblastoma cell lines, ALK-addicted neuroblastoma cells treated with trametinib showed increased activation (inferred by phosphorylation) of the kinases AKT and ERK5. This feedback response was mediated by the mammalian target of rapamycin complex 2-associated protein SIN1, resulting in increased survival and proliferation that depended on AKT signaling. In xenografts in mice, trametinib inhibited the growth of EML4-ALK-positive non-small cell lung cancer and RAS-mutant neuroblastoma but not ALK-addicted neuroblastoma. Thus, our results advise against the seemingly rational option of using MEK inhibitors to treat ALK-addicted neuroblastoma.

Novel Mechanisms of ALK Activation Revealed by Analysis of the Y1278S Neuroblastoma Mutation.

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).

Anaplastic lymphoma kinase L1198F and G1201E mutations identified in anaplastic thyroid cancer patients are not ligand-independent.

Activating mutations in full length anaplastic lymphoma kinase (ALK) have been reported in neuroblastoma and in anaplastic thyroid cancer. ALK-L1198F and ALK-G1201E mutations were originally identified in anaplastic thyroid cancer (ATC) and characterized as constitutively activating mutations. In this study, we employed in vitro cell culture assays together with biochemical and in vivo Drosophila analyses to characterize their sensitivity to either activation by the FAM150A (AUG-ß) and FAM150B (AUG-α) ALK ligands or inhibition by ALK inhibitors. Here we report that neither ALK-L1198F nor ALK-G1201E mutations result in ligand independent gain-of-function (GOF) activity in either in vitro biochemical analysis or the various model systems employed. ALK-L1198F is activated by the FAM150 (AUG) ligands and its ligand-dependant activity is similar to the wild type full length ALK receptor. ALK-G1201E is only very weakly activated by the FAM150 (AUG) ligands, most likely due to impaired protein stability. We conclude that neither ALK-L1198F nor ALK-G1201E displays ligand independent kinase activity, with ALK-L1198F belonging to the class of ligand dependent ALK mutations which are not constitutively active but that responds to ligand activation, while the ALK-G1201E mutation generates an unstable receptor with very low levels of kinase activity.

Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK-positive neuroblastoma cells, Drosophila and mice.

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor which has been implicated in numerous solid and hematologic cancers. ALK mutations are reported in about 5-7% of neuroblastoma cases but the ALK-positive percentage increases significantly in the relapsed patient population. Crizotinib, the first clinically approved ALK inhibitor for the treatment of ALK-positive lung cancer has had less dramatic responses in neuroblastoma. Here we investigate the efficacy of a second-generation ALK inhibitor, brigatinib, in a neuroblastoma setting. Employing neuroblastoma cell lines, mouse xenograft and Drosophila melanogaster model systems expressing different constitutively active ALK variants, we show clear and efficient inhibition of ALK activity by brigatinib. Similar abrogation of ALK activity was observed in vitro employing a set of different constitutively active ALK variants in biochemical assays. These results suggest that brigatinib is an effective inhibitor of ALK kinase activity in ALK addicted neuroblastoma that should be considered as a potential future therapeutic option for ALK-positive neuroblastoma patients alone or in combination with other treatments.

FAM150A and FAM150B are activating ligands for anaplastic lymphoma kinase.

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.