Nucleophosmin-anaplastic Lymphoma Kinase Fusion Research Articles

Characterization and diagnostic application of genomic NPM-ALK fusion sequences in anaplastic large-cell lymphoma.

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion genes resulting from the translocation t(2;5)(p23;q35) are present in almost 90% of childhood ALK-positive anaplastic large-cell lymphomas (ALCL). Detection and quantification of minimal disseminated disease (MDD) by measuring NPM-ALK fusion transcript levels in the blood provide independent prognostic parameters. Characterization of the genomic breakpoints provides insights into the pathogenesis of the translocation and allows for DNA-based minimal disease monitoring.We designed a nested multiplex PCR assay for identification and characterization of genomic NPM-ALK fusion sequences in 45 pediatric ALCL-patients, and used the sequences for quantitative MDD monitoring. Breakpoint analysis indicates the involvement of inaccurate non-homologous end joining repair mechanisms in the formation of NPM-ALK fusions. Parallel quantification of RNA and DNA levels in the cellular fraction of 45 blood samples from eight patients with NPM-ALK-positive ALCL correlated, as did cell-free circulating NPM-ALK DNA copies in the plasma fraction of 37 blood samples. With genomic NPM-ALK fusion sequence quantification, plasma samples of ALCL patients become an additional source for MRD-assessment. Parallel quantification of NPM-ALK transcripts and fusion genes in ALCL cell lines treated with the ALK kinase inhibitor crizotinib illustrates the potential value of supplementary DNA-based quantification in particular clinical settings.

Crizotinib (PF-2341066) induces apoptosis due to downregulation of pSTAT3 and BCL-2 family proteins in NPM-ALK+ anaplastic large cell lymphoma

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is an aberrant fusion gene product with tyrosine kinase activity and is expressed in substantial subset of anaplastic large cell lymphomas (ALCL). It has been shown that NPM-ALK binds to and activates signal transducer and activator of transcription 3 (STAT3). Although NPM-ALK(+) ALCL overall shows a better prognosis, there is a sub-group of patients who relapses and is resistant to conventional chemotherapeutic regimens. NPM-ALK is a potential target for small molecule kinase inhibitors. Crizotinib (PF-2341066) is a small, orally bioavailable molecule that inhibits growth of tumors with ALK activity as shown in a subgroup of non-small lung cancer patients with EML4-ALK expression. In this study, we have investigated the in vitro effects of Crizotinib in ALCL cell line with NPM-ALK fusion. Crizotinib induced marked downregulation of STAT3 phosphorylation, which was associated with significant apoptotic cell death. Apoptosis induction was attributed to caspase-3 cleavage and marked downregulation of the Bcl-2 family of proteins including MCL-1. These findings implicate that Crizotinib has excellent potential to treat patients with NPM-ALK(+) ALCL through induction of apoptotic cell death and downregulation of major oncogenic proteins in this aggressive lymphoma.

Involvement of Grb2 Adaptor Protein in Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK)-mediated Signaling and Anaplastic Large Cell Lymphoma Growth

Most anaplastic large cell lymphomas (ALCL) express oncogenic fusion proteins derived from chromosomal translocations or inversions of the anaplastic lymphoma kinase (ALK) gene. Frequently ALCL carry the t(2;5) translocation, which fuses the ALK gene to the nucleophosmin (NPM1) gene. The transforming activity mediated by NPM-ALK fusion induces different pathways that control proliferation and survival of lymphoma cells. Grb2 is an adaptor protein thought to play an important role in ALK-mediated transformation, but its interaction with NPM-ALK, as well as its function in regulating ALCL signaling pathways and cell growth, has never been elucidated. Here we show that active NPM-ALK, but not a kinase-dead mutant, bound and induced Grb2 phosphorylation in tyrosine 160. An intact SH3 domain at the C terminus of Grb2 was required for Tyr(160) phosphorylation. Furthermore, Grb2 did not bind to a single region but rather to different regions of NPM-ALK, mainly Tyr(152-156), Tyr(567), and a proline-rich region, Pro(415-417). Finally, shRNA knockdown experiments showed that Grb2 regulates primarily the NPM-ALK-mediated phosphorylation of SHP2 and plays a key role in ALCL cell growth.

Proteome-Wide Identification of Novel Binding Partners to the Oncogenic Fusion Gene Protein, NPM-ALK, using Tandem Affinity Purification and Mass Spectrometry

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), an oncogenic fusion gene protein that is characteristically found in a subset of anaplastic large cell lymphomas, promotes tumorigenesis through its functional and physical interactions with various biologically important proteins. The identification of these interacting proteins has proven to be useful to further our understanding of NPM-ALK-mediated tumorigenesis. For the first time, we performed a proteome-wide identification of NPM-ALK-binding proteins using tandem affinity purification and a highly sensitive mass spectrometric technique. Tandem affinity purification is a recently developed method that carries a lower background and higher sensitivity compared with the conventional immunoprecipitation-based protein purification protocols. The NPM-ALK gene was cloned into an HB-tagged vector and expressed in GP293 cells. Three independent experiments were performed and the reproducibility of the data was 68%. The vast majority of the previously reported NPM-ALK-binding proteins were detected. We also identified proteins that are involved in various cellular processes that were not previously described in association with NPM-ALK, such as MCM6 and MSH2 (DNA repair), Nup98 and importin 8 (subcellular protein transport), Stim1 (calcium signaling), 82Fip (RNA regulation), and BAG2 (proteosome degradation). We believe that these data highlight the functional diversity of NPM-ALK and provide new research directions for the study of the biology of this oncoprotein.

Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell lymphoma

A t(2;5) chromosomal translocation resulting in expression of an oncogenic kinase fusion protein known as nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) has been implicated in the pathogenesis of anaplastic large-cell lymphoma (ALCL). PF-2341066 was recently identified as a p.o. bioavailable, small-molecule inhibitor of the catalytic activity of c-Met kinase and the NPM-ALK fusion protein. PF-2341066 also potently inhibited NPM-ALK phosphorylation in Karpas299 or SU-DHL-1 ALCL cells (mean IC(50) value, 24 nmol/L). In biochemical and cellular screens, PF-2341066 was shown to be selective for c-Met and ALK at pharmacologically relevant concentrations across a panel of >120 diverse kinases. PF-2341066 potently inhibited cell proliferation, which was associated with G(1)-S-phase cell cycle arrest and induction of apoptosis in ALK-positive ALCL cells (IC(50) values, approximately 30 nmol/L) but not ALK-negative lymphoma cells. The induction of apoptosis was confirmed using terminal deoxyribonucleotide transferase-mediated nick-end labeling and Annexin V staining (IC(50) values, 25-50 nmol/L). P.o. administration of PF-2341066 to severe combined immunodeficient-Beige mice bearing Karpas299 ALCL tumor xenografts resulted in dose-dependent antitumor efficacy with complete regression of all tumors at the 100 mg/kg/d dose within 15 days of initial compound administration. A strong correlation was observed between antitumor response and inhibition of NPM-ALK phosphorylation and induction of apoptosis in tumor tissue. In addition, inhibition of key NPM-ALK signaling mediators, including phospholipase C-gamma, signal transducers and activators of transcription 3, extracellular signal-regulated kinases, and Akt by PF-2341066 were observed at concentrations or dose levels, which correlated with inhibition of NPM-ALK phosphorylation and function. Collectively, these data illustrate the potential clinical utility of inhibitors of NPM-ALK in treatment of patients with ALK-positive ALCL.

Identification of Critical Phosporylation Sites within NPM-ALK That Regulate JUNB mRNA Translation.

Anaplastic large cell lymphoma (ALCL) accounts for approximately 30% of childhood lymphomas and 3% of adult non-Hodgkin lymphomas. The nucleophosmin - anaplastic lymphoma kinase (NPM-ALK) fusion which is the product of a t(2;5)(p23;q35) chromosomal translocation is present in about half of nodal ALCL. Expression of this fusion kinase results in induction of the AP-1 transcription factor JunB and IL-3 independent outgrow of murine hematopoietic Ba/F3 cells. We demonstrated that wild type NPM-ALK increases the amount of ribosomes bound to JUNB mRNA resulting in its more effective translation in large polysomes. The NPM-ALK fusion tyrosine kinase has 20 potential tyrosine residues available for autophosphorylation and phosphorylation by other protein tyrosine kinases. Here we used series of Y-to-F-substituted mutants of NPM-ALK to identify tyrosine residues that are required to regulate the segregation of JUNB mRNAs between polysomes and monosomes as well as ribonucleic particles (RNPs). Neither JUNB transcription nor JunB translation was altered in Ba/F3 cells expressing NPM-ALK mutants Y17F/Y29F/Y67F Y138F/Y152F Y156F/Y191F/Y299F Y378F/Y418F/Y445F and Y646F/Y664F compared to NPM-ALK wild type. Conversely, in NPM-ALK Y567F/Y461F/Y644F mutant cells proliferation was markedly decreased. These cells demonstrated active MEK-ERK pathway, while AKT, mTOR, and rpS6 phosphorylation was impaired. Moreover a shift of JUNB mRNA from the polysomic to the monosomic/mRNP fraction could be observed. In conclusion, we identified specific NPM-ALK phosphorylation sites required to mediate the effect of NPM-ALK on the JUNB translational regulation and therefore provide further insights in the transforming mechanisms of the oncoprotein NPM-ALK.

The Role of Forkhead Transcription Factors FoxO1, FoxO3 and FoxO4 in Hematopoiesis and Leukemogenesis.

FoxO is a family of forkhead transcription factors that negatively regulate proliferation and survival signals in hematopoietic cells. We and others have previously shown that inhibition of the three members of this family (FoxO1, FoxO3 and FoxO4) by leukemogenic tyrosine kinase fusion genes results in enhanced proliferative and survival signaling in leukemic cells. For example, the transforming activities of the lymphoma associated NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) fusion, BCR-ABL, or FLT3-ITD, are mediated in part by inactivation of FoxO through phosphorylation and ubiquitin mediated degradation by constitutively active Akt (Gu TL, et al. Blood 2004), with subsequent induction of proliferative and survival signals. Furthermore, inhibition of FoxO is required for efficient transformation of hematopoietic cells. However, the roles of FoxO in adult hematopoiesis are unknown. We have initiated studies to examine the role of FoxO in the context of normal hematopoiesis and leukemogenesis using a triple conditional knockout mouse for each of the FoxO1, FoxO3 and FoxO4 alleles. The FoxO alleles are flanked by lox-P sites and conditional excision is mediated by Cre expression under the control of the interferon inducible Mx1 promoter. Based on the normal function of FoxO family members to repress proliferative and survival signals, we hypothesized that the deletion of FoxO subfamily members would lead to an enhanced proliferation and survival in the hematopoietic compartment, and might contribute to the development of a myeloproliferative and/or lymphoproliferative phenotype in vivo. Triple homozygous conditional FoxO knockout mice were generated in an Mx1-Cre background to allow for excision of the FoxO alleles in the hematopoietic stem cell compartment after treatment with pIpC. Complete excision of each of the three alleles in the hematopoietic compartment was confirmed. However, in contrast with our working hypothesis, we observed that loss of function of FoxO family members was associated with a relatively subtle hematopoietic phenotype with 12 months of follow-up. The phenotype includes a non-fatal mild myeloproliferative phenotype that is progressive over time and characterized by modest splenomegaly, extramedullary hematopoiesis and increased mature myeloid populations in bone marrow and spleen. In addition, there are subtle alterations in both B and T lymphoid cell populations, including a decrease in both immature and mature B cells in the spleen and bone marrow; and abnormalities of CD4+CD8+ double positive and CD4+ and CD8+ T cells in the thymus. Examination of stem and progenitor populations also revealed subtle differences in the HSC and CLP progenitor populations at 4 weeks post pIpC. Thus, these data indicate that complete loss of FoxO function in the adult hematopoietic compartment results in a relatively subtle hematopoietic phenotype. They further demonstrate that although inhibition of FoxO family members is required for efficient transformation of hematopoietic cells by leukemogenic fusion tyrosine kinases, loss of FoxO function alone is not sufficient to induce a leukemic phenotype.

Genes Regulated by NPM-ALK Fusion Kinase Play a Key Role in the Activation of AP-1 Transcription Factors.

Background: About half of nodal anaplastic large cell lymphomas (ALCL) express the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein, which is the product of a t(2;5)(p23;q35) chromosomal translocation. Expression of this protein has been shown to result in neoplastic change. Combining suppression subtractive hybridization (SSH) and cDNA microarray analysis we aimed at elucidating the consequences of NPM-ALK expression.Methods: SSH cDNA libraries were constructed using mRNA from human embryonic kidney (293) cells transfected with active or kinase-dead NPM-ALK constructs, as well as pools of NPM-ALK positive and negative ALCL cell lines. The resulting cDNA clones and genes relevant for cancer pathogenesis were spotted, generating specific cDNA microarrays comprising 4992 genes. mRNA expression patterns were analyzed in individual cell lines. Real time quantitative RT-PCR of 20 selected genes validated the mRNA expression data of the microarrays.Results: Expression of a set of 102 genes distinguishes NPM-ALK-negative (FE-PD, MAC-2A) from NPM-ALK-positive ALCL cell lines (SU-DHL-1, JB-6, SUP-M2, SR-786, DEL and Karpas 299). The majority are involved in regulation of cell cycle and apoptosis. 38 of these genes also discriminate 293 cells with respect to their NPM-ALK expression. Interestingly, AP-1 target genes, such as GM-CSFRA, GM-CSFRB, ARF5, FAS, FASL, and BCL3, are increased in NPM-ALK expressing cell lines. Electrophoretic mobility shift assay (EMSA) verifies NPM-ALK dependent AP-1 DNA binding activity.Conclusion: This study reveals genes specifically regulated by NPM-ALK lymphoma kinase. Further, we demonstrate that AP-1 activation is a critical target of NPM-ALK signalling.

Cell Proliferation Induced by the NPM-ALK Fusion Tyrosine Kinase of Anaplastic Large Cell Lymphoma Is Mediated by mTOR/S6K1 and MEK/ERK Signaling.

The nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion is the product of the t(2;5)(p23;q35) chromosomal translocation found in approximately half of anaplastic large cell lymphomas (ALCL). Moreover, this fusion kinase, as well as other ALK fusion proteins, have been found in large B-cell lymphomas. This fusion kinase has been shown to regulate multiple signal transduction pathways and to induce hematopoietic malignancy in murine models. Nonetheless, the functional role of signaling events caused by NPM-ALK expression is incompletely understood. Here we report that NPM-ALK activates the kinase S6K1 (p70/p85) and the extracellular regulated kinase (ERK1/2). In Ba/F3 murine hematopoietic cells that express NPM-ALK, S6K1 activition by NPM-ALK was sensitive to mTOR inhibition by rapamycin. However, treatment of NPM-ALK-Ba/F3 cells with the MEK inhibitor UO126 did not attenuate the activation of S6K1 by NPM-ALK. Pharmacological inhibition of either mTOR or MEK in Ba/F3 cells expressing NPM-ALK resulted in impaired cytokine-independent outgrowth of these cells. For either inhibitor, suppression of cytokine-independent outgrowth was due to impairment of cell proliferation. In contrast, cell survival signaling was not compromised by either inhibitor alone or in combination. Combined inhibition of both the mTOR/S6K1 and MEK/ERK signaling modules resulted in an additive impairment of cytokine-independent outgrowth. Moreover, rapamycin attenuated the proliferation of the human NPM-ALK-expressing ALCL cell line Karpas299. The mTOR/S6K1 and MEK/ERK signaling modules may serve as effective chemotherapeutic targets in the treatment of ALCL and other malignancies that express the activated ALK protein tyrosine kinase.

NPM-ALK fusion kinase of anaplastic large-cell lymphoma regulates survival and proliferative signaling through modulation of FOXO3a

AbstractBetween 30% and 50% of patients with advanced-stage anaplastic large-cell lymphoma (ALCL) harbor the balanced chromosomal rearrangement t(2;5)(p23;q35), which results in the generation of the fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). To further study survival signaling by NPMALK, we generated Ba/F3 cell lines with either inducible or constitutive expression of NPM-ALK and examined the regulation of the AKT target FOXO3a. We hypothesized that NPM-ALK signaling through phosphoinositol 3-kinase (PI 3-kinase) and AKT would regulate FOXO3a, a member of the forkhead family of transcription factors, thereby stimulating proliferation and blocking programmed cell death in NPM-ALK-transformed cells. In Ba/F3 cells with induced or constitutive expression of NPM-ALK, concomitant AKT activation and phosphorylation of its substrate, FOXO3a, was observed. In addition, transient expression of NPM-ALK in U-20S cells inhibited FOXO3a-mediated transactivation of reporter gene expression. Furthermore, NPM-ALK-induced FOXO3a phosphorylation in Ba/F3 cells resulted in nuclear exclusion of this transcriptional regulator, up-regulation of cyclin D2 expression, and down-regulation of p27kip1 and Bim-1 expression. NPMALK reversal of proliferation arrest and of p27kip1 induction was dependent on the phosphorylation of FOXO3a. Thus, FOXO3a is a barrier to hematopoietic transformation that is overcome by phosphorylation and cytoplasmic relocalization induced by the expression of NPM-ALK. (Blood. 2004;103:4622-4629)

The oncogenic fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) induces two distinct malignant phenotypes in a murine retroviral transplantation model.

A t(2;5) (p23;q35) chromosomal translocation can be found in a high percentage of anaplastic large-cell lymphomas (ALCL). This genetic abnormality leads to the expression of the NPM-ALK fusion protein, which encodes a constitutively active tyrosine kinase that plays a causative role in lymphomagenesis. Employing a modified infection/transplantation protocol utilizing an MSCV-based vector, we were able to reproducibly induce two phenotypically different lymphoma-like diseases dependent on the retroviral titers used. The first phenotype presented as a polyclonal histiocytic malignancy of myeloid/macrophage origin with a short latency period of 3-4 weeks. Clinically, the diseased mice showed rapidly progressive wasting, lymphadenopathy and pancytopenia. Mice displaying the second phenotype developed monoclonal B-lymphoid tumors with a longer latency of approximately 12-16 weeks, primarily involving the spleen and the bone marrow, with less extensive lymph node but also histologically evident extranodal organ infiltration by large immature plasmoblastic cells. The described retroviral mouse model will be useful to analyse the role of NPM-ALK in lymphomagenesis in vivo and may contribute to the development of new treatment options for NPM-ALK induced malignancies.