NPM/ALK binds and phosphorylates the RNA/DNA-binding protein PSF in anaplastic large-cell lymphoma.

The oncogenic fusion tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) induces cellular transformation in anaplastic large-cell lymphomas (ALCLs) carrying the t(2;5) chromosomal translocation. Protein-protein interactions involving NPM/ALK are important for the activation of downstream signaling pathways. This study was aimed at identifying novel NPM/ALK-binding proteins that might contribute to its oncogenic transformation. Using a proteomic approach, several RNA/DNA-binding proteins were found to coimmunoprecipitate with NPM/ALK, including the multifunctional polypyrimidine tract binding proteinassociated splicing factor (PSF). The interaction between NPM/ALK and PSF was dependent on an active ALK kinase domain and PSF was found to be tyrosine-phosphorylated in NPM/ALK-expressing cell lines and in primary ALK(+) ALCL samples. Furthermore, PSF was shown to be a direct substrate of purified ALK kinase domain in vitro, and PSF Tyr293 was identified as the site of phosphorylation. Y293F PSF was not phosphorylated by NPM/ALK and was not delocalized in NPM/ALK(+) cells. The expression of ALK fusion proteins induced delocalization of PSF from the nucleus to the cytoplasm and forced overexpression of PSF-inhibited proliferation and induced apoptosis in cells expressing NPM/ALK. PSF phosphorylation also increased its binding to RNA and decreased the PSF-mediated suppression of GAGE6 expression. These results identify PSF as a novel NPM/ALK-binding protein and substrate, and suggest that PSF function may be perturbed in NPM/ALK-transformed cells.

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. MATERIALS AND 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. CONCLUSION: Constitutive activation of Jak2 constitutes a pro-proliferative, anti-apoptotic signaling pathway in NPM/ALK-transformed hematopoietic cells.

Differences between in vivo and in vitro sensitivity to imatinib of Bcr/Abl+ cells obtained from leukemic patients.

Imatinib mesylate (imatinib) inhibits Bcr/Abl, an oncogenic fusion protein. The in vitro effects of imatinib on BCR/ABL+ leukemic cells include inhibition of Bcr/Abl tyrosine phosphorylation, block of proliferation, and induction of apoptosis. The in vivo effects of imatinib were evaluated in 12 CML (chronic myeloid leukemia) patients in blast crisis or accelerated phase who were treated with imatinib. Treatment caused a decrease in spontaneous proliferation of leukemic cells in 10 of 12 evaluable patients and the development of apoptosis in 9 of 11 cases. Imatinib also caused an inhibition of Bcr/Abl autophosphorylation; however, the degree of inhibition obtained in vivo was substantially lower than that achieved in vitro with similar concentrations of imatinib. In seven patients cells could be evaluated at relapse: spontaneous proliferation was no longer inhibited and Bcr/Abl phosphorylation was comparable or superior to that present at the beginning of treatment, before imatinib administration. Plasma imatinib concentrations were not reduced. Leukemic cells obtained at relapse maintained in vitro sensitivity (Bcr/Abl autophosphorylation and proliferation inhibition) to imatinib concentration measured in vivo (3 microM or higher), although a partial resistance to the antiproliferative effects of imatinib was present at low (0.01-0.3 microM) concentrations. In four patients, addition of erythromycin to blood samples obtained at relapse restored imatinib sensitivity in terms of phosphorylation inhibition, indicating that the majority of plasma imatinib was not available to cells and probably bound to alpha1 acid glycoprotein. These data suggest that measurements of Bcr/Abl kinase activity in peripheral blood samples may represent a more reliable indicator of active concentrations than the measurement of imatinib plasma levels.