RESUMEN
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.
Asunto(s)
Quinasa de Linfoma Anaplásico , Diferenciación Celular , Proteínas de Drosophila , Neuronas , Quinasa de Linfoma Anaplásico/genética , Animales , Niño , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Humanos , Hiperplasia , Mutación , Neuronas/citología , Proteínas Tirosina Quinasas Receptoras/genéticaRESUMEN
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.
Asunto(s)
Quinasa de Linfoma Anaplásico/química , Quinasa de Linfoma Anaplásico/metabolismo , Movimiento Celular , Neuroblastoma/patología , Secuencia de Aminoácidos , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico/genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Membrana Celular/metabolismo , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal/genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glicina/química , Células HEK293 , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Células 3T3 NIH , Invasividad Neoplásica , Neuroblastoma/genética , Unión Proteica , Dominios ProteicosRESUMEN
Anaplastic lymphoma kinase (Alk) is a receptor tyrosine kinase of the insulin receptor super-family that functions as oncogenic driver in a range of human cancers such as neuroblastoma. In order to investigate mechanisms underlying Alk oncogenic signaling, we conducted a genetic suppressor screen in Drosophila melanogaster. Our screen identified multiple loci important for Alk signaling, including members of Ras/Raf/ERK-, Pi3K-, and STAT-pathways as well as tailless (tll) and foxo whose orthologues NR2E1/TLX and FOXO3 are transcription factors implicated in human neuroblastoma. Many of the identified suppressors were also able to modulate signaling output from activated oncogenic variants of human ALK, suggesting that our screen identified targets likely relevant in a wide range of contexts. Interestingly, two misexpression alleles of wallenda (wnd, encoding a leucine zipper bearing kinase similar to human DLK and LZK) were among the strongest suppressors. We show that Alk expression leads to a growth advantage and induces cell death in surrounding cells. Our results suggest that Alk activity conveys a competitive advantage to cells, which can be reversed by over-expression of the JNK kinase kinase Wnd.
Asunto(s)
Quinasa de Linfoma Anaplásico/metabolismo , Proteínas de Drosophila/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Transducción de Señal , Quinasa de Linfoma Anaplásico/genética , Animales , Muerte Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Quinasas Quinasa Quinasa PAM/genéticaRESUMEN
Basement membranes (BMs) are specialized layers of extracellular matrix (ECM) mainly composed of Laminin, type IV Collagen, Perlecan and Nidogen/entactin (NDG). Recent in vivo studies challenged the initially proposed role of NDG as a major ECM linker molecule by revealing dispensability for viability and BM formation. Here, we report the characterization of the single Ndg gene in Drosophila. Embryonic Ndg expression was primarily observed in mesodermal tissues and the chordotonal organs, whereas NDG protein localized to all BMs. Although loss of Laminin strongly affected BM localization of NDG, Ndg-null mutants exhibited no overt changes in the distribution of BM components. Although Drosophila Ndg mutants were viable, loss of NDG led to ultrastructural BM defects that compromised barrier function and stability in vivo Moreover, loss of NDG impaired larval crawling behavior and reduced responses to vibrational stimuli. Further morphological analysis revealed accompanying defects in the larval peripheral nervous system, especially in the chordotonal organs and the neuromuscular junction (NMJ). Taken together, our analysis suggests that NDG is not essential for BM assembly but mediates BM stability and ECM-dependent neural plasticity during Drosophila development.
Asunto(s)
Membrana Basal/metabolismo , Tipificación del Cuerpo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Animales , Membrana Basal/ultraestructura , Conducta Animal , Fenómenos Biomecánicos , Proteínas de Unión al Calcio/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/genética , Proteínas de la Matriz Extracelular/genética , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Laminina/metabolismo , Larva/genética , Unión Neuromuscular/patología , Sistema Nervioso Periférico/embriología , Sistema Nervioso Periférico/patología , Permeabilidad , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , VibraciónRESUMEN
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.
Asunto(s)
Citocinas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas de Pez Cebra/metabolismo , Secuencia de Aminoácidos , Quinasa de Linfoma Anaplásico , Animales , Línea Celular Tumoral , Drosophila , Ojo/metabolismo , Humanos , Linfoma/enzimología , Cresta Neural/enzimología , Células PC12 , Pigmentación , Ratas , Pez CebraRESUMEN
In Drosophila melanogaster, the receptor tyrosine kinase (RTK) anaplastic lymphoma kinase (Alk) and its ligand jelly belly (Jeb) are required to specify muscle founder cells in the visceral mesoderm. We identified a critical role for the scaffolding protein Cnk (connector enhancer of kinase suppressor of Ras) in this signaling pathway. Embryos that ectopically expressed the minimal Alk interaction region in the carboxyl terminus of Cnk or lacked maternal and zygotic cnk did not generate visceral founder cells or a functional gut musculature, phenotypes that resemble those of jeb and Alk mutants. Deletion of the entire Alk-interacting region in the cnk locus affected the Alk signaling pathway in the visceral mesoderm and not other RTK signaling pathways in other tissues. In addition, the Cnk-interacting protein Aveugle (Ave) was critical for Alk signaling in the developing visceral mesoderm. Alk signaling stimulates the MAPK/ERK pathway, but the scaffolding protein Ksr, which facilitates activation of this pathway, was not required to promote visceral founder cell specification. Thus, Cnk and Ave represent critical molecules downstream of Alk, and their loss genocopies the lack of visceral founder cell specification of Alk and jeb mutants, indicating their essential roles in Alk signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Diferenciación Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Quinasa de Linfoma Anaplásico , Animales , Bases de Datos de Ácidos Nucleicos , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Humanos , Mesodermo/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Eliminación de Secuencia , CigotoRESUMEN
The Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) plays a critical role in the specification of founder cells (FCs) in the Drosophila visceral mesoderm (VM) during embryogenesis. Reporter gene and CRISPR/Cas9 deletion analysis reveals enhancer regions in and upstream of the Alk locus that influence tissue-specific expression in the amnioserosa (AS), the VM and the epidermis. By performing high throughput yeast one-hybrid screens (Y1H) with a library of Drosophila transcription factors (TFs) we identify Odd-paired (Opa), the Drosophila homologue of the vertebrate Zic family of TFs, as a novel regulator of embryonic Alk expression. Further characterization identifies evolutionarily conserved Opa-binding cis-regulatory motifs in one of the Alk associated enhancer elements. Employing Alk reporter lines as well as CRISPR/Cas9-mediated removal of regulatory elements in the Alk locus, we show modulation of Alk expression by Opa in the embryonic AS, epidermis and VM. In addition, we identify enhancer elements that integrate input from additional TFs, such as Binou (Bin) and Bagpipe (Bap), to regulate VM expression of Alk in a combinatorial manner. Taken together, our data show that the Opa zinc finger TF is a novel regulator of embryonic Alk expression.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas Tirosina Quinasas Receptoras/genética , Factores de Transcripción/genética , Complejo 1 de Proteína Adaptadora/genética , Complejo 1 de Proteína Adaptadora/metabolismo , Subunidades beta de Complejo de Proteína Adaptadora/genética , Subunidades beta de Complejo de Proteína Adaptadora/metabolismo , Quinasa de Linfoma Anaplásico , Animales , Animales Modificados Genéticamente , Sitios de Unión , Sistemas CRISPR-Cas , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Embrión no Mamífero , Elementos de Facilitación Genéticos , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/metabolismo , Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Neuroblastoma/patología , Compuestos Organofosforados/farmacología , Pirimidinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico , Animales , Animales Modificados Genéticamente , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Drosophila , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neuroblastoma/enzimología , Neuroblastoma/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Citocinas/metabolismo , Activación Enzimática , Proteínas Tirosina Quinasas Receptoras/metabolismo , Quinasa de Linfoma Anaplásico , Línea Celular , Humanos , Datos de Secuencia Molecular , Unión Proteica , Análisis de Secuencia de ADNRESUMEN
After spinal cord injury, transected axons fail to regenerate, yet significant, spontaneous functional improvement can be observed over time. Distinct central nervous system regions retain the capacity to generate new neurons and glia from an endogenous pool of progenitor cells and to compensate neural cell loss following certain lesions. The aim of the present study was to investigate whether endogenous cell replacement (neurogenesis or gliogenesis) in the brain (subventricular zone, SVZ; corpus callosum, CC; hippocampus, HC; and motor cortex, MC) or cervical spinal cord might represent a structural correlate for spontaneous locomotor recovery after a thoracic spinal cord injury. Adult Fischer 344 rats received severe contusion injuries (200 kDyn) of the mid-thoracic spinal cord using an Infinite Horizon Impactor. Uninjured rats served as controls. From 4 to 14 days post-injury, both groups received injections of bromodeoxyuridine (BrdU) to label dividing cells. Over the course of six weeks post-injury, spontaneous recovery of locomotor function occurred. Survival of newly generated cells was unaltered in the SVZ, HC, CC, and the MC. Neurogenesis, as determined by identification and quantification of doublecortin immunoreactive neuroblasts or BrdU/neuronal nuclear antigen double positive newly generated neurons, was not present in non-neurogenic regions (MC, CC, and cervical spinal cord) and unaltered in neurogenic regions (dentate gyrus and SVZ) of the brain. The lack of neuronal replacement in the brain and spinal cord after spinal cord injury precludes any relevance for spontaneous recovery of locomotor function. Gliogenesis was increased in the cervical spinal cord remote from the injury site, however, is unlikely to contribute to functional improvement.
Asunto(s)
Neuroglía/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Médula Espinal/fisiopatología , Animales , Encéfalo/fisiopatología , Proliferación Celular , Vértebras Cervicales , Contusiones/complicaciones , Proteína Doblecortina , Actividad Motora/fisiología , Regeneración Nerviosa/fisiología , Neurogénesis , Ratas Endogámicas F344 , Traumatismos de la Médula Espinal/etiología , Vértebras Torácicas , Factores de TiempoRESUMEN
In this study, we aimed to identify molecular mechanisms involved in the specification of the 4d (mesentoblast) lineage in Platynereis dumerilii. We employ RT-PCR and in situ hybridization against the Platynereis dumerilii twist homolog (Pdu-twist) to reveal mesodermal specification within this lineage. We show that Pdu-twist mRNA is already maternally distributed. After fertilization, ooplasmatic segregation leads to relocation of Pdu-twist transcripts into the somatoblast (2d) lineage and 4d, indicating that the maternal component of Pdu-twist might be an important prerequisite for further mesoderm specification but does not represent a defining characteristic of the mesentoblast. However, after the primordial germ cells have separated from the 4d lineage, zygotic transcription of Pdu-twist is exclusively observed in the myogenic progenitors, suggesting that mesodermal specification occurs after the 4d stage. Previous studies on spiral cleaving embryos revealed a spatio-temporal correlation between the 4d lineage and the activity of an embryonic organizer that is capable to induce the developmental fates of certain micromeres. This has raised the question if specification of the 4d lineage could be connected to the organizer activity. Therefore, we aimed to reveal the existence of such a proposed conserved organizer in Platynereis employing antibody staining against dpERK. In contrast to former observations in other spiralian embryos, activation of MAPK signaling during 2d and 4d formation cannot be detected which questions the existence of a conserved connection between organizer function and specification of the 4d lineage. However, our experiments unveil robust MAPK activation in the prospective nephroblasts as well as in the macromeres and some micromeres at the blastopore in gastrulating embryos. Inhibition of MAPK activation leads to larvae with a shortened body axis, defects in trunk muscle spreading and improper nervous system condensation, indicating a critical function for MAPK signaling for the reorganization of embryonic tissues during the gastrulation process.
Asunto(s)
Embrión no Mamífero/embriología , Activación Enzimática , Regulación del Desarrollo de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Poliquetos/embriología , ARN Mensajero/genética , Proteína 1 Relacionada con Twist/genética , Animales , Embrión no Mamífero/metabolismo , Femenino , Poliquetos/genética , ARN Mensajero/análisisRESUMEN
The basic helix-loop-helix transcription factor twist plays a key role during mesoderm development in Bilateria. In this study, we identified a twist ortholog in the polychaete annelid Platynereis dumerilii and analyze its expression during larval development, postlarval growth up to the adult stage, and caudal regeneration after amputation of posterior segments. At late larval stages, Pdu-twist is expressed in the mesodermal anlagen and in developing muscles. During adulthood and caudal regeneration, Pdu-twist is expressed in the posterior growth zone, in mesodermal cells within the newly forming segments and budding parapodia. Our results indicate that Pdu-twist is involved in mesoderm formation during larval development, posterior growth, and caudal regeneration.
Asunto(s)
Poliquetos/embriología , Poliquetos/metabolismo , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Mesodermo/metabolismo , Datos de Secuencia Molecular , Filogenia , Proteína 1 Relacionada con Twist/químicaRESUMEN
The capability of regenerating posterior segments and pygidial structures is ancestral for annelids and has been lost only a few times within this phylum. As one of the three major segmented taxa, annelids enable us to monitor reconstruction of lost tissues and organs. During regeneration, regional identities have to be imprinted onto the newly formed segments. In this study, we show spatial and temporal localization of expression of nine Hox genes during caudal regeneration of the polychaete annelid Platynereis dumerilii. Hox genes are homeodomain genes encoding transcriptional regulators of axial patterning in bilaterian animals during development. We demonstrate that five Platynereis Hox genes belonging to paralog groups (PG) 1, 4, 5, 6, and 9-14 are expressed in domains of the regenerating nervous system consistent with providing positional information along the anteroposterior axis of the regenerate. We report that expression in regenerating neuromeres is limited to varying subsets of perikarya, called gangliosomes. Four of nine genes analyzed do not appear to be involved in axial patterning. Two genes, Pdu-Hox2 and Pdu-Hox3, are predominantly expressed in the growth zone region. For some Hox genes expression in newly formed coelomic epithelia can be observed. Platynereis Hox genes do not exhibit temporal or spatial colinearity. Although there are some similarities to previously reported expression patterns during larval and postlarval development in Nereididae (Kulakova et al. 2007), expression patterns observed during caudal regeneration also show unique patterns.