Constitutive activation of Jak2 contributes to proliferation and resistance to apoptosis in NPM/ALK-transformed cells.
OBJECTIVE
The t(2;5) translocation results in a 80-kDa oncogenic fusion protein consisting of NPM and the kinase domain of the tyrosine kinase ALK and is present in over half the cases of anaplastic large cell lymphoma (ALCL). NPM/ALK exerts its transforming potential via activation of multiple signaling pathways promoting growth factor independence and protection from apoptosis. Jak/Stat signaling is aberrantly activated in several human hematopoietic malignancies. We investigated the role of Jak2 in the context of NPM/ALK-mediated oncogenesis.


METHODS
Constitutive tyrosine phosphorylation of Jak2 was analyzed by Jak2 immunoprecipitation and subsequent anti-phosphotyrosine Western blotting. NPM/ALK-transformed cells were treated with the Jak2 inhibitor AG490 or transfected with wild-type or dominant-negative Jak2 expression constructs to measure 3[H]-thymidine incorporation. Apoptosis was assessed by flow cytometric analysis of annexin V-stained cells. The effect of Jak2 on Stat5-dependent transcriptional activity was measured by beta-casein promoter-dependent luciferase expression.


RESULTS
Jak2 was found to be constitutively tyrosine phosphorylated in ALCL cells and in NPM/ALK-transformed hematopoietic cells. Also, NPM/ALK was present in immunoprecipitates of Jak2. Inhibition of Jak2 led to a reduction of NPM/ALK-mediated proliferation and induced apoptosis. Stat5-dependent transcriptional activity was inhibited by transfection of NPM/ALK-transformed cells with a dominant-negative Jak2 expression construct or treatment with AG490.


CONCLUSIONS
Constitutive activation of Jak2 constitutes a pro-proliferative, anti-apoptotic signaling pathway in NPM/ALK-transformed hematopoietic cells.
Ruchatz
Holger
H
The National Cancer Institute, Oncogenic Fusion Proteins Unit, Department of Experimental Oncology, Milan, Italy.
Coluccia
Addolorata Maria Luce
AM
Stano
Paola
P
Marchesi
Edoardo
E
Gambacorti-Passerini
Carlo
C
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001402:B-Lymphocytes / Q000378:metabolism; D002364:Caseins / Q000235:genetics; D002455:Cell Division; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic; D004268:DNA-Binding Proteins / Q000502:physiology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D007163:Immunosorbent Techniques; D053614:Janus Kinase 2; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008894:Milk Proteins; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D011518:Proto-Oncogene Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators / Q000502:physiology; D014158:Transcription, Genetic; D014162:Transfection; D020032:Tyrphostins / Q000494:pharmacology
D002364:Caseins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; D019000:Phosphotyrosine; D008156:Luciferases; C107023:p80(NPM-ALK) protein; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2