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Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder caused by pathogenic variants in FBN1, with a hitherto unknown association with cancer. Here, we present two females with MFS who developed pediatric neuroblastoma. Patient 1 presented with neonatal MFS and developed an adrenal neuroblastoma with unfavorable tumor genetics at 10 months of age. Whole genome sequencing revealed a germline de novo missense FBN1 variant (NP_000129.3:p.(Asp1322Asn)), resulting in intron 32 inclusion and exon 32 retention. Patient 2 was diagnosed with classic MFS, caused by a germline de novo frameshift variant in FBN1 (NP_000129.3:p.(Cys805Ter)). At 18 years, she developed high-risk neuroblastoma with a somatic ALK pathogenic variant (NP_004295.2:p.(Arg1275Gln)). We identified 32 reported cases of MFS with cancer in PubMed, yet none with neuroblastoma. Among patients, we observed an early cancer onset and high frequency of MFS complications. We also queried cancer databases for somatic FBN1 variants, finding 49 alterations reported in PeCan, and variants in 2% of patients in cBioPortal. In conclusion, we report the first two patients with MFS and neuroblastoma and highlight an early age at cancer diagnosis in reported patients with MFS. Further epidemiological and functional studies are needed to clarify the growing evidence linking MFS and cancer.
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Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila, including functions in the central nervous system (CNS), however the molecular details are poorly understood. To gain mechanistic insight, we employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output. The resulting TaDa data were analyzed together with larval CNS scRNA-seq datasets performed under similar conditions, identifying a role for Alk in the transcriptional regulation of neuroendocrine gene expression. Further integration with bulk and scRNA-seq datasets from larval brains in which Alk signaling was manipulated identified a previously uncharacterized Drosophila neuropeptide precursor encoded by CG4577 as an Alk signaling transcriptional target. CG4577, which we named Sparkly (Spar), is expressed in a subset of Alk-positive neuroendocrine cells in the developing larval CNS, including circadian clock neurons. In agreement with our TaDa analysis, overexpression of the Drosophila Alk ligand Jeb resulted in increased levels of Spar protein in the larval CNS. We show that Spar protein is expressed in circadian (clock) neurons, and flies lacking Spar exhibit defects in sleep and circadian activity control. In summary, we report a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.
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Quinase do Linfoma Anaplásico , Sistema Nervoso Central , Proteínas de Drosophila , Animais , Sistema Nervoso Central/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Quinase do Linfoma Anaplásico/metabolismo , Quinase do Linfoma Anaplásico/genética , Larva/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Neuropeptídeos/metabolismo , Neuropeptídeos/genética , Transdução de Sinais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Drosophila/genética , Drosophila/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão GênicaRESUMO
Solute Carrier Family 3, Member 2 (SLC3A2 or 4F2hc) is a multifunctional glycoprotein that mediates integrin-dependent signaling, acts as a trafficking chaperone for amino acid transporters, and is involved in polyamine transportation. We identified SLC3A2 as a potential Anaplastic Lymphoma Kinase (ALK) interacting partner in a BioID-proximity labeling screen in neuroblastoma (NB) cells. In this work we show that endogenous SLC3A2 and ALK interact in NB cells and that this SLC3A2:ALK interaction was abrogated upon treatment with the ALK inhibitor lorlatinib. We show here that loss of ALK activity leads to decreased SLC3A2 expression and reduced SLC3A2 protein stability in a panel of NB cell lines, while stimulation of ALK with ALKAL2 ligand resulted in increased SLC3A2 protein levels. We further identified MARCH11, an E3 ligase, as a regulator of SLC3A2 ubiquitination downstream of ALK. Further, knockdown of SLC3A2 resulted in inhibition of NB cell growth. To investigate the therapeutic potential of SLC3A2 targeting, we performed monotreatment of NB cells with AMXT-1501 (a polyamine transport inhibitor), which showed only moderate effects in NB cells. In contrast, a combination lorlatinib/AMXT-1501 treatment resulted in synergistic inhibition of cell growth in ALK-driven NB cell lines. Taken together, our results identify a novel role for the ALK receptor tyrosine kinase (RTK), working in concert with the MARCH11 E3 ligase, in regulating SLC3A2 protein stability and function in NB cells. The synergistic effect of combined ALK and polyamine transport inhibition shows that ALK/MARCH11/SLC3A2 regulation of amino acid transport is important for oncogenic growth and survival in NB cells.
Assuntos
Quinase do Linfoma Anaplásico , Proliferação de Células , Cadeia Pesada da Proteína-1 Reguladora de Fusão , Neuroblastoma , Transdução de Sinais , Humanos , Quinase do Linfoma Anaplásico/metabolismo , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Quinase do Linfoma Anaplásico/genética , Neuroblastoma/patologia , Neuroblastoma/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Lactamas/farmacologia , Aminopiridinas/farmacologia , Ubiquitinação/efeitos dos fármacos , Pirazóis/farmacologiaRESUMO
Neuroblastoma (NB) is the most common cancer in infancy with an urgent need for more efficient targeted therapies. The development of novel (combinatorial) treatment strategies relies on extensive explorations of signaling perturbations in neuroblastoma cell lines, using RNA-Seq or other high throughput technologies (e.g. phosphoproteomics). This typically requires dedicated bioinformatics support, which is not always available. Additionally, while data from published studies are highly valuable and raw data (e.g. fastq files) are nowadays released in public repositories, data processing is time-consuming and again difficult without bioinformatics support. To facilitate NB research, more user-friendly and immediately accessible platforms are needed to explore newly generated as well as existing high throughput data. To make this possible, we developed an interactive data centralization and visualization web application, called CLEAN (the Cell Line Explorer web Application of Neuroblastoma data; https://ccgg.ugent.be/shiny/clean/). By focusing on the regulation of the DNA damage response, a therapeutic target of major interest in neuroblastoma, we demonstrate how CLEAN can be used to gain novel mechanistic insights and identify putative drug targets in neuroblastoma.
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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.
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Neuroblastoma , Receptores Proteína Tirosina Quinases , Humanos , Camundongos , Animais , Quinase do Linfoma Anaplásico/genética , Receptores Proteína Tirosina Quinases/metabolismo , Proliferação de Células , Linhagem Celular Tumoral , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/patologia , Reparo do DNA , Dano ao DNA , Proteínas Mutadas de Ataxia Telangiectasia/genéticaRESUMO
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.
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Anaplastic lymphoma kinase (ALK) fusion variants in Non-Small Cell Lung Cancer (NSCLC) consist of numerous dimerizing fusion partners. Retrospective investigations suggest that treatment benefit in response to ALK tyrosine kinase inhibitors (TKIs) differs dependent on the fusion variant present in the patient tumor. Therefore, understanding the oncogenic signaling networks driven by different ALK fusion variants is important. To do this, we developed controlled inducible cell models expressing either Echinoderm Microtubule Associated Protein Like 4 (EML4)-ALK-V1, EML4-ALK-V3, Kinesin Family Member 5B (KIF5B)-ALK, or TRK-fused gene (TFG)-ALK and investigated their transcriptomic and proteomic responses to ALK activity modulation together with patient-derived ALK-positive NSCLC cell lines. This allowed identification of both common and isoform-specific responses downstream of these four ALK fusions. An inflammatory signature that included upregulation of the Serpin B4 serine protease inhibitor was observed in both ALK fusion inducible and patient-derived cells. We show that Signal transducer and activator of transcription 3 (STAT3), Nuclear Factor Kappa B (NF-κB) and Activator protein 1 (AP1) are major transcriptional regulators of SERPINB4 downstream of ALK fusions. Upregulation of SERPINB4 promotes survival and inhibits natural killer cell-mediated cytotoxicity, which has potential for therapeutic impact targeting the immune response together with ALK TKIs in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Serpinas , Humanos , Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Oncogenes , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/genética , Proteômica , Estudos Retrospectivos , Serpinas/genéticaRESUMO
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.
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Activating anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK) mutations occur in pediatric neuroblastoma and are associated with poor prognosis. To study ALK-activating mutations in a genetically controllable system, we employed CRIPSR/Cas9, incorporating orthologs of the human oncogenic mutations ALKF1174L and ALKY1278S in the Drosophila Alk locus. AlkF1251L and AlkY1355S mutant Drosophila exhibited enhanced Alk signaling phenotypes, but unexpectedly depended on the Jelly belly (Jeb) ligand for activation. Both AlkF1251L and AlkY1355S mutant larval brains displayed hyperplasia, represented by increased numbers of Alk-positive neurons. Despite this hyperplasic phenotype, no brain tumors were observed in mutant animals. We showed that hyperplasia in Alk mutants was not caused by significantly increased rates of proliferation, but rather by decreased levels of apoptosis in the larval brain. Using single-cell RNA sequencing, we identified perturbations during temporal fate specification in AlkY1355S mutant mushroom body lineages. These findings shed light on the role of Alk in neurodevelopmental processes and highlight the potential of Alk-activating mutations to perturb specification and promote survival in neuronal lineages. This article has an associated First Person interview with the first author of the paper.
Assuntos
Quinase do Linfoma Anaplásico , Diferenciação Celular , Proteínas de Drosophila , Neurônios , Quinase do Linfoma Anaplásico/genética , Animais , Criança , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Humanos , Hiperplasia , Mutação , Neurônios/citologia , Receptores Proteína Tirosina Quinases/genéticaRESUMO
Understanding protein-protein interactions (PPIs) and interactome networks is essential to reveal molecular mechanisms mediating various cellular processes. The most common method to study PPIs in vivo is affinity purification combined with mass spectrometry (AP-MS). Although AP-MS is a powerful method, loss of weak and transient interactions is still a major limitation. Proximity labeling (PL) techniques have been developed as alternatives to overcome these limitations. Proximity-dependent biotin identification (BioID) is one such widely used PL method. The first-generation BioID enzyme BirA*, a promiscuous bacterial biotin ligase, has been effectively used in cultured mammalian cells; however, relatively slow enzyme kinetics make it less effective for temporal analysis of protein interactions. In addition, BirA* exhibits reduced activity at temperatures below 37°C, further restricting its use in intact organisms cultured at lower optimal growth temperatures ( e.g., Drosophila melanogaster ). TurboID, miniTurbo, and BirA*-G3 are next generation BirA* variants with improved catalytic activity, allowing investigators to use this powerful tool in model systems such as flies. Here, we describe a detailed experimental workflow to efficiently identify the proximal proteome (proximitome) of a protein of interest (POI) in the Drosophila brain using CRISPR/Cas9-induced homology-directed repair (HDR) strategies to endogenously tag the POI with next generation BioID enzymes.
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BACKGROUND: In the last years, several efforts have been made to classify colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, known as Consensus Molecular Subtypes (CMSs). METHODS: In this work, we performed a meta-analysis of CRC patients stratified into four CMSs. We identified a negative correlation between a high level of anaplastic lymphoma kinase (ALK) expression and relapse-free survival, exclusively in CMS1 subtype. Stemming from this observation, we tested cell lines, patient-derived organoids and mice with potent ALK inhibitors, already approved for clinical use. RESULTS: ALK interception strongly inhibits cell proliferation already at nanomolar doses, specifically in CMS1 cell lines, while no effect was found in CMS2/3/4 groups. Furthermore, in vivo imaging identified a role for ALK in the dynamic formation of 3D tumor spheroids. Consistently, ALK appeares constitutively phosphorylated in CMS1, and it signals mainly through the AKT axis. Mechanistically, we found that CMS1 cells display several copies of ALKAL2 ligand and ALK-mRNAs, suggesting an autocrine loop mediated by ALKAL2 in the activation of ALK pathway, responsible for the invasive phenotype. Consequently, disruption of ALK axis mediates the pro-apoptotic action of CMS1 cell lines, both in 2D and 3D and enhanced cell-cell adhesion and e-cadherin organization. In agreement with all these findings, the ALK signature encompassing 65 genes statistically associated with worse relapse-free survival in CMS1 subtype. Finally, as a proof of concept, the efficacy of ALK inhibition was demonstrated in both patient-derived organoids and in tumor xenografts in vivo. CONCLUSIONS: Collectively, these findings suggest that ALK targeting may represent an attractive therapy for CRC, and CMS classification may provide a useful tool to identify patients who could benefit from this treatment. These findings offer rationale and pharmacological strategies for the treatment of CMS1 CRC.
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Quinase do Linfoma Anaplásico , Neoplasias do Colo , Citocinas , Quinase do Linfoma Anaplásico/genética , Animais , Neoplasias do Colo/genética , Citocinas/genética , Humanos , Ligantes , Camundongos , Recidiva Local de NeoplasiaAssuntos
Benzamidas/uso terapêutico , Indazóis/uso terapêutico , Neuroblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinase do Linfoma Anaplásico/fisiologia , Cromossomos Humanos Par 2/genética , Citocinas/genética , Variação Genética , Células Germinativas , Humanos , Lactente , Masculino , Neuroblastoma/genética , Neuroblastoma/secundário , Receptor trkA/fisiologiaRESUMO
Development of the Drosophila visceral muscle depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signaling, which specifies founder cells (FCs) in the circular visceral mesoderm (VM). Although Alk activation by its ligand Jelly Belly (Jeb) is well characterized, few target molecules have been identified. Here, we used targeted DamID (TaDa) to identify Alk targets in embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differentially expressed genes, including the Snail/Scratch family transcription factor Kahuli (Kah). We confirmed Kah mRNA and protein expression in the VM, and identified midgut constriction defects in Kah mutants similar to those of pointed (pnt). ChIP and RNA-Seq data analysis defined a Kah target-binding site similar to that of Snail, and identified a set of common target genes putatively regulated by Kah and Pnt during midgut constriction. Taken together, we report a rich dataset of Alk-responsive loci in the embryonic VM and functionally characterize the role of Kah in the regulation of embryonic midgut morphogenesis.
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Quinase do Linfoma Anaplásico , Proteínas de Ligação a DNA , Proteínas de Drosophila , Desenvolvimento Embrionário , Proteínas do Tecido Nervoso , Proteínas Proto-Oncogênicas , Fatores de Transcrição , Animais , Quinase do Linfoma Anaplásico/genética , Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Desenvolvimento Embrionário/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Desenvolvimento Muscular/genética , Músculos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA-Seq , Transdução de Sinais/genética , Análise de Célula Única , Fatores de Transcrição/genéticaRESUMO
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.
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High-risk neuroblastoma (NB) often involves MYCN amplification as well as mutations in ALK. Currently, high-risk NB presents significant clinical challenges, and additional therapeutic options are needed. Oncogenes like MYCN and ALK result in increased replication stress in cancer cells, offering therapeutically exploitable options. We have pursued phosphoproteomic analyses highlighting ATR activity in ALK-driven NB cells, identifying the BAY1895344 ATR inhibitor as a potent inhibitor of NB cell growth and proliferation. Using RNA-Seq, proteomics and phosphoproteomics we characterize NB cell and tumour responses to ATR inhibition, identifying key components of the DNA damage response as ATR targets in NB cells. ATR inhibition also produces robust responses in mouse models. Remarkably, a 2-week combined ATR/ALK inhibition protocol leads to complete tumor regression in two independent genetically modified mouse NB models. These results suggest that NB patients, particularly in high-risk groups with oncogene-induced replication stress, may benefit from ATR inhibition as therapeutic intervention.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Quinase do Linfoma Anaplásico/genética , Quinase do Linfoma Anaplásico/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Neuroblastoma/genética , Neuroblastoma/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/farmacologia , Pirazóis/uso terapêutico , RNA-Seq , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Anaplastic lymphoma kinase (Alk) is an evolutionary conserved receptor tyrosine kinase belonging to the insulin receptor superfamily. In addition to its well-studied role in cancer, numerous studies have revealed that Alk signaling is associated with a variety of complex traits such as: regulation of growth and metabolism, hibernation, regulation of neurotransmitters, synaptic coupling, axon targeting, decision making, memory formation and learning, alcohol use disorder, as well as steroid hormone metabolism. In this study, we used BioID-based in vivo proximity labeling to identify molecules that interact with Alk in the Drosophila central nervous system (CNS). To do this, we used CRISPR/Cas9 induced homology-directed repair (HDR) to modify the endogenous Alk locus to produce first and next generation Alk::BioID chimeras. This approach allowed identification of Alk proximitomes under physiological conditions and without overexpression. Our results show that the next generation of BioID proteins (TurboID and miniTurbo) outperform the first generation BirA* fusion in terms of labeling speed and efficiency. LC-MS3-based BioID screening of AlkTurboID and AlkminiTurbo larval brains revealed an extensive neuronal Alk proximitome identifying numerous potential components of Alk signaling complexes. Validation of Alk proximitome candidates further revealed co-expression of Stardust (Sdt), Discs large 1 (Dlg1), Syntaxin (Syx) and Rugose (Rg) with Alk in the CNS and identified the protein-tyrosine-phosphatase Corkscrew (Csw) as a modulator of Alk signaling.
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Quinase do Linfoma Anaplásico/metabolismo , Encéfalo/embriologia , Encéfalo/metabolismo , Organogênese , Proteoma , Proteômica , Quinase do Linfoma Anaplásico/genética , Animais , Drosophila/embriologia , Drosophila/genética , Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Organogênese/genética , Proteômica/métodos , Transdução de SinaisRESUMO
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.
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Quinase do Linfoma Anaplásico/metabolismo , Proteínas de Transporte/metabolismo , Mapeamento de Interação de Proteínas , Transdução de Sinais , Linhagem Celular Tumoral , Humanos , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/métodosRESUMO
BACKGROUND: Neuroblastoma (NB) is one of the most frequently diagnosed tumors in infants. NB is a neuroendocrine tumor type with various characteristics and features, and with diverse outcome. The most malignant NBs have a 5-year survival rate of only 40-50%, indicating the need for novel and improved treatment options. 177Lu-octreotate is routinely administered for treatment of neuroendocrine tumors overexpressing somatostatin receptors (SSTR). The aim of this study was to examine the biodistribution of 177Lu-octreotate in mice bearing aggressive human NB cell lines, in order to evaluate the potential usefulness of 177Lu-octreotate for treatment of NB. METHODS: BALB/c nude mice bearing CLB-BAR, CLB-GE or IMR-32 tumor xenografts (n = 5-7/group) were i.v. injected with 0.15 MBq, 1.5 MBq or 15 MBq 177Lu-octreotate and sacrificed 1 h, 24 h, 48 h and 168 h after administration. The radioactivity concentration was determined for collected tissue samples, tumor-to-normal-tissue activity concentration ratios (T/N) and mean absorbed dose for each tissue were calculated. Immunohistochemical (IHC) staining for SSTR1-5, and Ki67 were carried out for tumor xenografts from the three cell lines. RESULTS: High 177Lu concentration levels and T/N values were observed in all NB tumors, with the highest for CLB-GE tumor xenografts (72%IA/g 24 h p.i.; 1.5 MBq 177Lu-octreotate). The mean absorbed dose to the tumor was 6.8 Gy, 54 Gy and 29 Gy for CLB-BAR, CLB-GE and IMR-32, respectively, p.i. of 15 MBq 177Lu-octreotate. Receptor saturation was clearly observed in CLB-BAR, resulting in higher concentration levels in the tumor when lower activity levels where administered. IHC staining demonstrated highest expression of SSTR2 in CLB-GE, followed by CLB-BAR and IMR-32. CONCLUSION: T/N values for all three human NB tumor xenograft types investigated were high relative to previously investigated neuroendocrine tumor types. The results indicate a clear potential of 177Lu-octreotate as a therapeutic alternative for metastatic NB.
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Lutécio/uso terapêutico , Neuroblastoma/radioterapia , Octreotida/análogos & derivados , Radioisótopos/uso terapêutico , Animais , Apoptose , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Quinase do Linfoma Anaplásico/metabolismo , Neuroblastoma/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteômica/métodos , Aminopiridinas/farmacologia , Animais , Linhagem Celular Tumoral , Cromatografia Líquida , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Lactamas/farmacologia , Células PC12 , Fosforilação , Piperidinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ratos , Espectrometria de Massas em TandemRESUMO
Although activating mutations of the anaplastic lymphoma kinase (ALK) membrane receptor occur in â¼10% 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.