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Activating ALK mutations found in neuroblastoma are inhibited by Crizotinib and NVP-TAE684
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
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2011 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 440, 405-413 p.Article in journal (Refereed) Published
Abstract [en]

Mutations in the kinase domain of ALK (anaplastic lymphoma kinase) have recently been shown to be important for the progression of the childhood tumour neuroblastoma. In the present study we investigate six of the putative reported constitutively active ALK mutations, in positions G1128A, I1171N, F1174L, R1192P, F1245C and R1275Q. Our analyses were performed in cell-culture-based systems with both mouse and human ALK mutant variants and subsequently in a Drosophila melanogaster model system. Our investigation addressed the transforming potential of the putative gain-of-function ALK mutations as well as their signalling potential and the ability of two ATP-competitive inhibitors, Crizotinib (PF-02341066) and NVP-TAE684, to abrogate the activity of ALK. The results of the present study indicate that all mutations tested are of an activating nature and thus are implicated in tumour initiation or progression of neuroblastoma. Importantly for neuroblastoma patients, all ALK mutations used in the present study can be blocked by the inhibitors, although some mutants exhibited higher levels of drug sensitivity than others.

Place, publisher, year, edition, pages
2011. Vol. 440, 405-413 p.
Keyword [en]
anaplastic lymphoma kinase (ALK), cancer, Crizotinib, gain-of-function mutation, neuroblastoma, NVP-TAE684
National Category
Cell and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:umu:diva-51470DOI: 10.1042/BJ20101796ISI: 000298440300012OAI: oai:DiVA.org:umu-51470DiVA: diva2:482609
Available from: 2012-01-24 Created: 2012-01-23 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Anaplastic Lymphoma Kinase mutations and downstream signalling
Open this publication in new window or tab >>Anaplastic Lymphoma Kinase mutations and downstream signalling
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The oncogene Anaplastic Lymphoma Kinase (ALK) is a Receptor Tyrosine Kinase (RTK) and was initially discovered as the fusion protein NPM (nucleophosmin)-ALK in a subset of Anaplastic Large Cell Lymphomas (ALCL). Since then more fusion proteins have been identified in a variety of cancers. Further, overexpression of ALK due to gene amplification has been observed in many malignancies, amongst others neuroblastoma, a pediatric cancer. Lately, activating point mutations in the kinase domain of ALK have been described in neuroblastoma patients and neuroblastoma cell lines. In contrast, the physiological function of ALK is still unclear, but ALK is suggested to play a role in the normal development and function of the nervous system.

By employing cell culture based approaches, including a tetracycline-inducible PC12 cell system and the in vivo D. melanogaster model system, we aimed to analyze the downstream signalling of ALK and its role in neuroblastoma. First, we wished to analyze whether ALK is able to activate the small GTPase Rap1 contributing to differentiation/proliferation processes. Activated ALK recruits a complex of the GEF C3G and CrkL and activates C3G by tyrosine phosphorylation. This activated complex is able to activate Rap1 resulting either in neurite outgrowth in PC12 cells or proliferation of neuroblastoma cells suggesting a potential role in the oncogenesis of neuroblastoma driven by gain-of-function mutant ALK. Next, we could show that seven investigated ALK mutations with a high probability of being oncogenic (G1128A, I1171N, F1174L, F1174S, R1192P, F1245C and R1275Q), are true gain-of-function mutations, respond differently to ALK inhibitors and have different transforming ability. Especially the F1174S mutation correlates with aggressive disease development. However, the assumed active germ line mutation I1250T is in fact a kinase dead mutation and suggested to act as a dominant-negative receptor. Finally, ALK mutations are most frequently observed in MYCN amplified tumours correlating with a poor clinical outcome. Active ALK regulates mainly the initiation of MYCN transcription in human neuroblastoma cell lines. Further, ALK gain-of-function mutants and MYCN synergize in transforming NIH3T3 cells.

Overall, somatic mutations appear to be more aggressive than germ line mutations, implying a different impact on neuroblastoma. Further, successful application of ALK inhibitors suggests a promising future for the development of patient-specific treatments for neuroblastoma patients.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2012. 85 p.
Keyword
Anaplastic Lymphoma Kinase, oncogene, RTK, neuroblastoma, crizotinib, NVP-TAE684, gain-of-function, MYCN, transcription factor, small GTPase, Rap1, C3G, PC12 cells, neurite outgrowth
National Category
Cell Biology
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:umu:diva-54562 (URN)978-91-7459-387-7 (ISBN)
Public defence
2012-08-24, NUS - Norrlands universitetssjukhus, Byggnad 6M, Betula, Umeå Universitet, Umeå, 09:00 (English)
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Available from: 2012-05-16 Created: 2012-04-30 Last updated: 2012-05-09Bibliographically approved

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Schönherr, ChristinaRuuth, KristinaYamazaki, YasuoEriksson, TheresePalmer, Ruth HHallberg, Bengt

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