Ruth H. Palmer

WASP and SCAR have distinct roles in activating the Arp2/3 complex during myoblast fusion

Myoblast fusion takes place in two steps in mammals and in Drosophila. First, founder cells (FCs) and fusion-competent myoblasts (FCMs) fuse to form a trinucleated precursor, which then recruits further FCMs. This process depends on the formation of the fusion-restricted myogenic-adhesive structure (FuRMAS), which contains filamentous actin (F-actin) plugs at the sites of cell contact. Fusion relies on the HEM2 (NAP1) homolog Kette, as well as Blow and WASP, a member of the Wiskott-Aldrich-syndrome protein family. Here, we show the identification and characterization of schwächling – a new Arp3-null allele. Ultrastructural analyses demonstrate that Arp3schwächling mutants can form a fusion pore, but fail to integrate the fusing FCM. Double-mutant experiments revealed that fusion is blocked completely in Arp3 and wasp double mutants, suggesting the involvement of a further F-actin regulator. Indeed, double-mutant analyses with scar/WAVE and with the WASP-interacting partner vrp1 (sltr, wip)/WIP show that the F-actin regulator scar also controls F-actin formation during myoblast fusion. Furthermore, the synergistic phenotype observed in Arp3 wasp and in scar vrp1 double mutants suggests that WASP and SCAR have distinct roles in controlling F-actin formation. From these findings we derived a new model for actin regulation during myoblast fusion.

Myoblast determination in the somatic and visceral mesoderm depends on Notch signalling as well as on milliways(mili(Alk)) as receptor for Jeb signalling.

The visceral muscles of the Drosophila midgut consist of syncytia and arise by fusion of founder and fusion-competent myoblasts, as described for the somatic muscles. A single-step fusion results in the formation of binucleate circular midgut muscles, whereas a multiple-step fusion process produces the longitudinal muscles. A prerequisite for muscle fusion is the establishment of myoblast diversity in the mesoderm prior to the fusion process itself. We provide evidence for a role of Notch signalling during establishment of the different cell types in the visceral mesoderm, demonstrating that the basic mechanism underlying the segregation of somatic muscle founder cells is also conserved during visceral founder cell determination. Searching for genes involved in the determination and differentiation of the different visceral cell types, we identified two independent mutations causing loss of visceral midgut muscles. In both of these mutants visceral muscle founder cells are missing and the visceral mesoderm consists of fusion-competent myoblasts only. Thus, no fusion occurs resulting in a complete disruption of visceral myogenesis. Subsequent characterisation of the mutations revealed that they are novel alleles of jelly belly (jeb) and the Drosophila Alk homologue named milliways (miliAlk). We show that the process of founder cell determination in the visceral mesoderm depends on Jeb signalling via the Milliways/Alk receptor. Moreover, we demonstrate that in the somatic mesoderm determination of the opposite cell type, the fusion-competent myoblasts, also depends on Jeb and Alk, revealing different roles for Jeb signalling in specifying myoblast diversity. This novel mechanism uncovers a crosstalk between somatic and visceral mesoderm leading not only to the determination of different cell types but also maintains the separation of mesodermal tissues, the somatic and splanchnic mesoderm.

Crizotinib : latest champion in the cancer wars?

Three articles in this issue of the Journal report on the therapeutic potential of a new kid on the kinase inhibitor block: crizotinib, an ATP-competitive inhibitor of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase. Kwak et al.1 summarize a study involving patients with non–small-cell lung cancer who were enrolled in a phase 1 trial, starting in 2008, hot on the heels of a study in which cell lines derived from non–small-cell lung tumors were shown to be sensitive to NVP-TAE6842 and crizotinib (PF-02341066).3,4 From a cohort of 1500 patients with non–small-cell lung cancer, 82 (5.5%) were found to .xa0.xa0.

Appearance of the novel activating F1174S ALK mutation in neuroblastoma correlates with aggressive tumour progression and unresponsiveness to therapy

Mutations in the kinase domain of the ALK kinase have emerged recently as important players in the genetics of the childhood tumor neuroblastoma. Here, we report the appearance of a novel ALK mutation in neuroblastoma, correlating with aggressive tumor behavior. Analyses of genomic DNA from biopsy samples initially showed ALK sequence to be wild type. However, during disease progression, mutation of amino acid F1174 to a serine within the ALK kinase domain was observed, which correlated with aggressive neuroblastoma progression in the patient. We show that mutation of F1174 to serine generates a potent gain-of-function mutant, as observed in 2 independent systems. First, PC12 cell lines expressing ALK(F1174S) display ligand-independent activation of ALK and further downstream signaling activation. Second, analysis of ALK(F1174S) in Drosophila models confirms that the mutation mediates a strong, rough eye phenotype upon expression in the developing eye. Thus, we report a novel ALK(F1174S) mutation that displays ligand-independent activity in vivo, correlating with rapid and treatment-resistant tumor growth. The study also shows that initial screening in the first tumor biopsy of a patient may not be sufficient and that further molecular analysis, in particular in tumor progression and/or tumor relapse, is warranted for better understanding of the treatment of neuroblastoma patients.

Anaplastic Lymphoma Kinase (ALK) regulates initiation of transcription of MYCN in neuroblastoma cells

Neuroblastoma is a neural crest-derived embryonal tumour of the postganglionic sympathetic nervous system and a disease with several different chromosomal gains and losses, which include MYCN-amplified neuroblastoma on chromosome 2, deletions of parts of the chromosomes 1p and 11q, gain of parts of 17q and triploidy. Recently, activating mutations of the ALK (Anaplastic Lymphoma Kinase) RTK (Receptor Tyrosine Kinase) gene have been described in neuroblastoma. A meta-analysis of neuroblastoma cases revealed that ALK mutations (49 of 709 cases) in relation to genomic subtype were most frequently observed in MYCN amplified tumours (8.9%), correlating with a poor clinical outcome. MYCN proteins target proliferation and apoptotic pathways, and have an important role in the progression of neuroblastoma. Here, we show that both wild-type and gain-of-function mutants in ALK are able to stimulate transcription at the MYCN promoter and initiate mRNA transcription of the MYCN gene in both neuronal and neuroblastoma cell lines. Further, this stimulation of MYCN gene transcription and de novo MYCN protein expression is abrogated by specific ALK inhibitors, such as crizotinib (PF-2341066), NVP-TAE684, and by small interfering RNA to ALK resulting in a decrease in proliferation rate. Finally, co-transfection of ALK gain-of-function mutations together with MYCN leads to an increase in transformation potential. Taken together, our results indicate that ALK signalling regulates initiation of transcription of the MYCN gene providing a possible explanation for the poor clinical outcome observed when MYCN is amplified together with activated ALK.

The constitutive activity of the ALK mutated at positions F1174 or R1275 impairs receptor trafficking

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK), which is transiently expressed during development of the central and peripheral nervous system. ALK has been recently identified as a major neuroblastoma predisposition gene and activating mutations have also been identified in a subset of sporadic neuroblastoma tumors. Two hot spots of ALK mutations have been observed at positions F1174 and R1275. Here, we studied stably transfected cell lines expressing wild-type or F1174L- or R1275Q-mutated ALK in parallel with a neuroblastoma cell line (CLB-GE) in which the allele mutated at position F1174 is amplified. We observed that the mutated ALK variants were essentially intracellular and were largely retained in the reticulum/Golgi compartments. This localization was corroborated by a defect of N-linked glycosylation. Although the mutated receptors exhibited a constitutive activation, the minor pool of receptor addressed to the plasma membrane was much more tyrosine phosphorylated than the intracellular pool. The use of antagonist monoclonal antibodies suggested that the constitutive activity of the mutated receptors did not require the dimerization of the receptor, whereas adequate dimerization triggered by agonist monoclonal antibodies increased this activity. Finally, kinase inactivation of the mutated receptors restored maturation and cell-surface localization. Our results show that constitutive activation of ALK results in its impaired maturation and intracellular retention. Furthermore, they provide a rationale for the potential use of kinase inhibitors and antibodies in ALK-dependent tumors.

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. DOI: http://dx.doi.org/10.7554/eLife.09811.001

Anaplastic lymphoma kinase activates the small GTPase Rap1 via the Rap1-specific GEF C3G in both neuroblastoma and PC12 cells

Many different types of cancer originate from aberrant signaling from the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), arising through different translocation events and overexpression. Further, activating point mutations in the ALK domain have been recently reported in neuroblastoma. To characterize signaling in the context of the full-length receptor, we have examined whether ALK is able to activate Rap1 and contribute to differentiation/proliferation processes. We show that ALK activates Rap1 via the Rap1-specific guanine-nucleotide exchange factor C3G, which binds in a constitutive complex with CrkL to activated ALK. The activation of the C3G/Rap1 pathway results in neurite outgrowth of PC12 cells, which is inhibited by either overexpression of Rap1GAP or siRNA-mediated knockdown of Rap1 itself or the guanine nucleotide exchange factor C3G. Significantly, this pathway also appears to function in the regulation of proliferation of neuroblastoma cells such as SK-N-SH and SH-SY5Y, because abrogation of Rap1 activity by Rap1-specific siRNA or overexpression of Rap1GAP reduces cellular growth. These results suggest that ALK activation of Rap1 may contribute to cell proliferation and oncogenesis of neuroblastoma driven by gain-of-function mutant ALK receptors.

Godzilla-dependent transcytosis promotes Wingless signalling in Drosophila wing imaginal discs

The apical and basolateral membranes of epithelia are insulated from each other, preventing the transfer of extracellular proteins from one side to the other. Thus, a signalling protein produced apically is not expected to reach basolateral receptors. Evidence suggests that Wingless, the main Drosophila Wnt, is secreted apically in the embryonic epidermis. However, in the wing imaginal disc epithelium, Wingless is mostly seen on the basolateral membrane where it spreads from secreting to receiving cells. Here we examine the apico-basal movement of Wingless in Wingless-producing cells of wing imaginal discs. We find that it is presented first on the apical surface before making its way to the basolateral surface, where it is released and allowed to interact with signalling receptors. We show that Wingless transcytosis involves dynamin-dependent endocytosis from the apical surface. Subsequent trafficking from early apical endosomes to the basolateral surface requires Godzilla, a member of the RNF family of membrane-anchored E3 ubiquitin ligases. Without such transport, Wingless signalling is strongly reduced in thisxa0tissue.

ALK and NSCLC: Targeted therapy with ALK inhibitors

For many years treatment for advanced or metastatic non-small cell lung cancer (NSCLC) has employed chemotherapy regimens for patient care, with limited effect. Five-year survival rates for these patients are not encouraging. However, for a subgroup of these patients, there have been radical changes over recent years. Our understanding of the basic pathology behind NSCLC at the molecular level has offered up a host of new molecularly targeted therapies, which are revolutionizing this area of cancer care. Results from recent clinical trials provide hope for NSCLC patients harboring oncogenic translocations involving the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase. Just as inhibition of the breakpoint cluster region–ABL complex has changed the face of chronic myeloid leukemia diagnosis, oncogenic ALK fusions offer a step forward in the diagnosis and treatment of ALK-positive NSCLC. This article discusses the current knowledge and potential implications concerning ALK inhibitors and NSCLC.