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Mechanistic Implications and Characterization of Anaplastic Lymphoma Kinase (ALK) mutations in Neuroblastoma
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Professor Bengt Hallberg)
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that was first reported as a fusion partner of nucleophosmin in Anaplastic large cell lymphoma in 1994. ALK is involved in myriad of cancers including neuroblastoma which is the most common extracranial solid tumor affecting young children. It arises in the neural crest cells of sympathetic nervous system origin and is responsible for 12% of all childhood cancer deaths. Several point mutations in ALK have been described in both familial and sporadic neuroblastoma.

With the aim to understand the role of ALK in neuroblastoma further, we investigated the point mutations in ALK reported in patients. Using cell culture based methods and Drosophila as a model organism; we first characterized these mutations under three broad categories: 1) Ligand independent mutations that were constitutively active, 2) Kinase dead mutation and 3) Ligand dependent mutations that behaved as inducible wild type. Further, to understand the activation mechanism of ALK, we constructed mutations that could potentially alter ALK’s conformation based on the available crystal structure. From the data generated, we were able to provide a new perspective to the activation of full length ALK receptor. This was more in line with activation mechanism of insulin receptor and different from that suggested for ALK fusion protein. From a clinical point of view, all the mutations in the study were blocked to different degrees using the ALK inhibitor, crizotinib. Lastly, we identified potential downstream targets of ALK using phosphoproteomics. From the various targets identified, we focused on STAT3 and confirmed its role as a mediator in ALK initiated MYCN transcription. We showed that STAT3 inhibition led to reduction of MYCN levels and thereby identifying it as a potential therapeutic target in neuroblastoma. Overall, our study highlights clinical relevance of ALK mutations in neuroblastoma and from a basic biology viewpoint; it reveals important mechanistic insight into receptor activation.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2015. , 79 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1709
Keyword [en]
neuroblastoma, ALK, crizotinib, receptor tyrosine kinase
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-106663ISBN: 978-91-7601-254-3 (print)OAI: oai:DiVA.org:umu-106663DiVA: diva2:848575
Public defence
2015-10-02, KBC-huset, Stora hörsalen, KKB3B1, Umeå universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2015-09-04 Created: 2015-07-28 Last updated: 2015-09-04Bibliographically approved
List of papers
1. Cell and Drosophila model systems define three classes of anaplastic lymphoma kinase mutations in neuroblastoma
Open this publication in new window or tab >>Cell and Drosophila model systems define three classes of anaplastic lymphoma kinase mutations in neuroblastoma
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2013 (English)In: Disease models & mechanisms, ISSN 1754-8411, Vol. 6, no 2, 373-382 p.Article in journal (Refereed) Published
Abstract [en]

Neuroblastoma is a childhood extracranial solid tumor which is associated with a number of genetic changes. Included in these genetic alterations are mutations in the kinase domain of the Anaplastic Lymphoma Kinase (ALK) receptor tyrosine kinase (RTK), which have been found in both somatic and familial neuroblastoma. In order to treat patients accordingly required characterisation of these mutations in terms of their response to ALK tyrosine kinase inhibitors (TKIs). Here, we report the identification and characterisation of two novel neuroblastoma ALK mutations (A1099T and 1464STOP) which we have investigated together with several previously reported but uncharacterised ALK mutations (T1087I, D1091N, T1151M, M1166R, F1174I and A1234T). In order to understand the potential role of these ALK mutations in neuroblastoma progression we have employed cell culture based systems together with the model organism Drosophila as a readout for ligand-independent activity. Mutation of ALK at position F1174I generates a gain-of-function receptor capable of activating intracellular targets, such as ERK (extracellular signal regulated kinase) and STAT3 (signal transducer and activator of transcription 3) in a ligand independent manner. Analysis of these previously uncharacterised ALK mutants and comparison with ALK(F1174) mutants suggests that ALK mutations observed in neuroblastoma fall into three classes. These are: (i) gain-of-function ligand independent mutations such as ALK(F1174), (ii) kinase-dead ALK mutants, e.g. ALK(I1250T)(Schonherr et al 2011a) or (iii) ALK mutations which are ligand-dependent in nature. Irrespective of the nature of the observed ALK mutants, in every case the activity of the mutant ALK receptors could be abrogated by the ALK inhibitor crizotinib (PF-02341066, Xalkori), albeit with differing levels of sensitivity.

Place, publisher, year, edition, pages
Cambridge, UK: The Company of Biologists Ltd, 2013
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-61265 (URN)10.1242/dmm.010348 (DOI)23104988 (PubMedID)
Available from: 2012-11-07 Created: 2012-11-07 Last updated: 2015-09-01Bibliographically approved
2. Novel mechanisms of ALK activation revealed by analysis of the Y1278S neuroblastoma mutation.
Open this publication in new window or tab >>Novel mechanisms of ALK activation revealed by analysis of the Y1278S neuroblastoma mutation.
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(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-106700 (URN)
Note

Authors Damini Chand and Jikui Guan are shared first authors for this manuscript.

Available from: 2015-08-11 Created: 2015-08-03 Last updated: 2015-09-01Bibliographically approved
3. Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells
Open this publication in new window or tab >>Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells
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2013 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, no 21, Special Issue, 5269-5282 p.Article in journal (Refereed) Published
Abstract [en]

Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin-ALK and echinoderm microtubule-associated protein-like 4-ALK oncoproteins. It is now also appreciated that the full-length ALK receptor can be activated by point mutations and by deletions within the extracellular domain, such as those observed in neuroblastoma. Several studies have employed phosphoproteomics approaches to find substrates of ALK fusion proteins. In this study, we used MS-based phosphotyrosine profiling to characterize phosphotyrosine signaling events associated with the full-length ALK receptor. A number of previously identified and novel targets were identified. One of these, signal transducer and activator of transcription 3 (STAT3), has previously been observed to be activated in response to oncogenic ALK signaling, but the significance of this in signaling from the full-length ALK receptor has not been explored further. We show here that activated ALK robustly activates STAT3 on Tyr705 in a number of independent neuroblastoma cell lines. Furthermore, knockdown of STAT3 by RNA interference resulted in a reduction in myelocytomatosis neuroblastom (MYCN) protein levels downstream of ALK signaling. These observations, together with a decreased level of MYCN and inhibition of neuroblastoma cell growth in the presence of STAT3 inhibitors, suggest that activation of STAT3 is important for ALK signaling activity in neuroblastoma.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2013
Keyword
anaplastic lymphoma kinase, cancer, neuroblastoma, SHP-2, signal transducer and activator of transcription 3 (STAT3)
National Category
Cell and Molecular Biology Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-81092 (URN)10.1111/febs.12453 (DOI)23889739 (PubMedID)
Conference
6th Garvan Signalling Symposium, 2012
Funder
Swedish Cancer Society, 12-0722, 120796Swedish Research Council, 621-2011-5181, 521-2012-2831
Note

Special Issue: Frontiers in Cell Signalling, and Ca2+-Signalling and Transport in Health and Disease

Available from: 2013-10-02 Created: 2013-10-02 Last updated: 2017-12-06Bibliographically approved

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