ALBERT

Description: The NPM-ALK fusion tyrosine kinase generated by the t(2;5)(p23;q35) chromosomal translocation is present in approximately 50% of anaplastic large cell lymphomas. NPM-ALK is known to play a causal role in the generation of hematopoietic neoplasia, and it has multiple potential sites of tyrosine phosphorylation that can bind to signaling effector molecules. We found that expression of NPM-ALK regulates mediators of translational initiation, including mTOR, S6K1, and 4E-BP1. Moreover, other investigators have shown that expression of proteins that mediate the control of translational initiation can be rate-limiting in the pathogenesis of neoplasia. Collectively, these findings stimulated our hypothesis that NPM-ALK promotes neoplasia by altering the partitioning of specific mRNA’s to polysomes and thereby regulating their translation. To evaluate this hypothesis, we assessed the global profile of polysomal mRNA in cells that express the NPM-ALK fusion kinase. In this study, we generated Ba/F3 murine hematopoietic cells that express kinase-active NPM-ALK or a kinase-deficient NPM-ALK mutant (K210A). Cells were maintained in the presence of the cytokine IL-3 to minimize changes secondary to cell cycle redistribution or early apoptosis. We used these cell lines to compare in a global manner the partitioning of mRNA species to polysomes. We isolated polyribosome preparations using conventional sucrose gradient fractionation, and from this purified polysomal material we extracted mRNA. Similarly, we prepared total mRNA from these cell lines. The global profile of gene expression for both total and polysomal RNA was assessed using Affymetrix mouse genome microarrays. The data generated in this effort were analyzed using the GeneChip robust multichip analysis (GCRMA) method. Significance Analysis of Microarrays (SAM) and Linear Models for Microarray Data (Limma) were employed to identify significantly differentially expressed genes. By culturing each cell line in the presence of IL-3, we found little difference in total cellular mRNA levels. However, we found that expression of kinase-active NPM-ALK markedly altered the partitioning of mRNA’s to the polysomal pool. We identified 286 genes whose mRNA’s exhibited altered polysomal partitioning, including 102 decreased in cells with kinase-active NPM-ALK and 184 with increased polysome association. These translationally regulated mRNA’s encode proteins associated with transcriptional control, post-translational modification of proteins, and hematopoietic neoplasia. Our findings support the assertion that NPM-ALK signaling regulates gene expression in part through translational control. We speculate that concerted dysregulation of transcription and translation by oncogenes might broadly mediate the pathogenesis of hematopoietic neoplasia.

Description: 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.

Description: Between 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)

Description: The nucleophosmin (NPM) - anaplastic lymphoma kinase (ALK) fusion protein, which is the product of the balanced chromosomal rearrangement t(2;5)(p23;q35), occurs in about 50% of nodal anaplastic large cell lymphoma (ALCL). Expression of this fusion kinase results in neoplastic transformation by modulating multiple intracellular signaling molecules, such as the PI3-kinase and the ERK1/2 kinases. Here we show high activation of JunB in various human ALCL cell lines. Moreover, using human embryonic kidney (HEK293) and murine hematopoietic Ba/F3 cells ectopically expressing NPM-ALK, we demonstrate that NPM-ALK is the trigger for JunB activation. Knock down of JUNB in NPM-ALK expressing cells using RNA interference results in upregulation of p16INK4a and downregulation of Cyclin D1, causing G1/S cell cycle arrest. Thus, JunB plays a critical oncogenic role in NPM-ALK transformed cells. Using the PI3-kinase inhibitor LY294002 and the MEK-inhibitor UO126, we demonstrate that NPM-ALK-induced JunB activity as well as cellular proliferation are dependent on both PI3-kinase and MEK-ERK signaling. Moreover, we illustrate that NPM-ALK and subsequently PI3-kinase activate AKT and mTOR/S6K1 which are implicated in the translational control of specific mRNA molecules containing a polypyrimidine motif (see pathway diagram). Since JUNB mRNA harbors such a motif we confirmed that JUNB mRNA is translated in large polysomes using ribosomal gradient preparations. Selective block of mTOR with the immunosuppressant rapamycin decreases proliferation and reduces protein but not mRNA levels of JunB in human and murine NPM-ALK positive cell lines. A similar effect is seen when inhibiting the upstream activator of mTOR, PI3-kinase. In contrast, a significant decrease of JUNB mRNA levels is shown in cells treated by the MEK-inhibitor UO126. An analogous effect of NPM-ALK is observed in ALCL patient samples. Hyperphosphorylation of S6K1 at Thr389 indicating activated mTOR/S6K1 is seen in 9/10 NPM-ALK positive ALCL cases in contrast to only 1/15 of NPM-ALK negative ALCL samples (P 〈 0,001). Hence, these findings suggest that the signaling cascade NPM-ALK→PI3K→AKT→mTOR/S6K1 is crucial to induce JUNB translation in human NPM-ALK positive ALCL. In conclusion, we reveal that JunB acts as an oncogene in NPM-ALK positive neoplastic cells and therefore represents a valid therapeutic target. Our data indicate a novel mechanism for regulation of AP-1 activity in general and suggest a new therapeutic approach to specifically modulate translation of an oncogene. Figure Figure

Description: Acute leukemias are classified into myeloid and lymphoid subtypes according to their phenotypic characteristics. Leukemic cells, however, sometimes co-express myeloid and lymphoid phenotypes, or switch their phenotype from myeloid to lymphoid lineages and vice versa. The conventional classification of leukemias is based mainly upon the concept that leukemic phenotype reflects a progenitor stage at which leukemic transformation occurs, or that it represents a stage at which transformed hematopoietic stem cells (HSCs) become incapable of further differentiation. Here we propose another possibility that phenotype of leukemias could be determined by instructive signals from the leukemic transformation mechanism itself. We found that TEL/PDGFβR (T/P), a tyrosine kinase fusion isolated from chronic myelomonocytic leukemia, can instruct myeloid lineage commitment and conversion at stem and progenitor stages of hematopoiesis. The T/P gene was transduced into purified progenitors or HSCs by using a retrovirus carrying a green fluorescent protein reporter. HSCs transduced with T/P (T/P+ HSCs) spontaneously formed GM colonies without cytokines. Furthermore, T/P+ HSCs were incapable of differentiation into B cells on OP9 stromal layer in the presence of IL-7. To test the effect of T/P on lymphoid commitment more precisely, we transduced T/P into purified common lymphoid progenitors (CLPs) that normally differentiate only into T, B and NK lineages. In a limiting dilution assay, 1 in 7 control-GFP transduced CLPs generated B cell progeny in vitro, while only 1 in 500 T/P+ CLPs differentiated into B cells. Instead, surprisingly, the majority of T/P+ CLPs and even T/P+ thymic proT cells quickly differentiated into granulocytes and monocytes in vitro. We then transplanted T/P+ CLPs into lethally irradiated congenic mice. T/P+ CLPs again differentiated into Gr-1+ granulocytes and monocytes in vivo. Gene expression analyses showed that T/P+ CLPs upregulated GM-related molecules including C/EBPα and GM-CSFRα immediately after T/P transduction, while transduction of either C/EBPα or GM-CSFRα also reprogrammed CLPs into the myeloid lineage as we reported previously. Thus, T/P signaling can activate these critical GM-related molecules in lymphoid progenitors to convert them into the myeloid lineage. These data collectively suggest that at least some types of oncogenic tyrosine kinase fusions can specify leukemic phenotypes, through activating critical signals for lineage commitment.

Description: Anaplastic large cell lymphomas (ALCLs) are highly proliferating tumors that commonly express the AP-1 transcription factor JunB. ALK fusions occur in approximately 50% of ALCLs, and among these, 80% have the t(2;5) translocation with NPM-ALK expression. We report greater activity of JunB in NPM-ALK–positive than in NPM-ALK–negative ALCLs. Specific knockdown of JUNB mRNA using small interfering RNA and small hairpin RNA in NPM-ALK–expressing cells decreases cellular proliferation as evidenced by a reduced cell count in the G2/M phase of the cell cycle. Expression of NPM-ALK results in ERK1/2 activation and transcriptional up-regulation of JUNB. Both NPM-ALK–positive and –negative ALCL tumors demonstrate active ERK1/2 signaling. In contrast to NPM-ALK–negative ALCL, the mTOR pathway is active in NPM-ALK–positive lymphomas. Pharmacological inhibition of mTOR in NPM-ALK–positive cells down-regulates JunB protein levels by shifting JUNB mRNA translation from large polysomes to monosomes and ribonucleic particles (RNPs), and decreases cellular proliferation. Thus, JunB is a critical target of mTOR and is translationally regulated in NPM-ALK–positive lymphomas. This is the first study demonstrating translational control of AP-1 transcription factors in human neoplasia. In conjunction with NPM-ALK, JunB enhances cell cycle progression and may therefore represent a therapeutic target.

Description: Significant advances have been made towards understanding the molecular pathogenesis and prognostic determinants in acute myelogenous leukemia of normal karytype (AMLNK). One of these, somatic mutation within exon 12 of the nucleophosmin gene (NPM1), is present in 50–60% of AML-NK and has been associated with favorable response to induction chemotherapy, overall survival, and event-free survival, but only in the absence of FLT3-ITD mutation. In addition to exon 12 mutation, NPM1 is disrupted in hematologic malignancies through fusion to partner proteins such as the anaplastic lymphoma kinase (ALK), myeloid leukemia factor 1 in myelodysplasia (MLF1), and retinoic acid receptor-α (RARα). The NPM1 gene encodes a 37-kDa protein that is predominantly localized to the nucleolus but also shuttles to the nucleoplasm and cytoplasm. A strong association (perhaps a 100% correlation) exists between NPM1 mutation and aberrant localization of the nucleophosmin protein in the cytoplasm. This mislocalization of nucleophosmin has been attributed to the loss of tryptophan residues 288 and 290 (or 290 only) in the carboxy terminus of this protein, and these motifs are required for nucleolar localization of nucleophosmin. Importantly, the NPM1 mutation also creates a de novo nuclear export signal within nucleophosmin. The functional role of wild-type nucleophosmin has been implicated in the regulation of cell growth control through p14ARF and p53 interactions, ribosome biogenesis, centrosome duplication, as well as other functions. Pediatric AML samples with NPM1 mutation were reported to have a distinct gene expression signature, including altered expression of homeobox (HOX) genes, and adult AML specimens carrying mutant NPM1 were reported to have a distinct microRNA expression signature. In addition to alterations in the expression of mRNA and microRNA species, the critical function of nucleophosmin in ribosome biogenesis, as well as its reported association with poly(A)(+) mRNA’s in vivo, suggests that mutant NPM1 could disrupt gene expression through aberrant translational control. Regulators of translational initiation can be rate-limiting for neoplasia in animal models, and we evaluated the hypothesis that cytoplasmic nucleophosmin promotes leukemogenesis by similarly altering the translational control of gene expression. Here, we present data to show that enforced expression of mutant nucleophosmin significantly alters the partitioning of mRNA’s to polyribosomes. Polyribosomal extracts were purified from cells that express wild-type or mutant nucleophosmin, RNA was extracted from this material, and the global profile of mRNA in these fractions was evaluated by gene expression microarray analysis. Enforced expression of cytoplasmic nucleophosmin significantly altered mRNA recruitment to polysomes. Moreover, we found common features in the polysome signature of cells expressing mutant NPM or the NPM-ALK fusion, suggesting that cytoplasmic NPM and the NPM-ALK fusion might disrupt translational initiation through partially overlapping mechanisms. These findings suggest that mutant nucleophosmin can perturb mRNA translational initiation in concert with other molecular mechanisms in the pathogenesis of AML-NK.

Description: Nucleophosmin (NPM) exon 12 mutations are found in 50–60 percent of acute myelogenous leukemia (AML) with normal karyotype, and the altered protein (termed NPMc) is relocalized from the nucleolus to the cytoplasm. Multiple functions have been attributed to NPM, and research by other investigators demonstrated that NPM serves a critical role in nucleolar-to-cytoplasmic shuttling of rRNA’s and in ribosome biogenesis. An early indication of the role of NPM in hematopoietic neoplasia was its fusion to the ALK protein in patients with anaplastic large cell lymphoma (ALCL). We and others have demonstrated that the expression of NPM-ALK regulates mediators of translational initiation, including mTOR, S6K1, and 4E-BP1, and we showed that NPM-ALK expression significantly alters the partitioning of mRNA’s to polyribosomes. Regulators of translational initiation can be rate-limiting for neoplasia in animal models, and we hypothesized that NPMc promotes leukemogenesis in a similar manner by altering the recruitment of specific mRNA’s to polysomes and thereby altering their translation. To evaluate this hypothesis, we assessed the global profile of polysomal mRNA in cells engineered to express NPMc. We generated pTRE2-NPMc(A)-Ba/F3 murine hematopoietic cells which express NPMc under the control of a tetracycline-inducible promoter. These cells carry “variant A” NPMc, which contains an insertion of a TCTG tetranucleotide at positions 956 through 959 and is the most common NPMc variant in AML. Control cells contained the empty vector. We used these cell lines to compare in a global manner the partitioning of mRNA species to polysomes, which were isolated using sucrose gradient fractionation of post-nuclear supernatants. From this enriched polysomal material we purified RNA, and the global profile of mRNA in these fractions was assessed by using Affymetrix mouse genome microarrays. The data generated in this effort was analyzed using the GeneChip robust multichip analysis (GCRMA) method. Significance Analysis of Microarrays (SAM) and Linear Models for Microarray Data (Limma) were employed to identify significant differentially expressed genes. We identified 345 genes whose mRNA’s exhibited altered polysomal partitioning, including 253 increased in cells with NPMc and 192 with decreased polysome association (FDR≤ 0.05). These translationally regulated mRNA’s encode proteins that control transcription, post-translational modification of proteins, and proteins associated with hematopoietic neoplasia. One of these differentially recruited mRNA’s encodes the Dub-1 protein, a ubiquitin peptidase that has been implicated previously in hematopoietic cell growth control, and the mRNA showed a 6-fold increase in polysome recruitment. Interestingly, in our previous evaluation comparing cells with NPM-ALK versus the kinase-deficient variant, there was a 50-fold increase in the partitioning of Dub-1 mRNA to polysomes. These findings suggest that alteration of Dub-1 expression through mRNA partitioning might be commonly utilized by both NPMc as well as NPM-ALK. Collectively, these findings suggest that concerted disruption of gene transcription and translation might broadly mediate the pathogenesis of hemotopoietic neoplasia.

Description: High expression of the tumor necrosis factor receptor CD30 and the AP-1 transcription factor JunB are the hallmark of anaplastic large cell lymphoma (ALCL). In contrast to the prototypic AP-1 factor c-Jun, JunB exerts an antioncogenic function in most cell types. Its functional role in ALCL remains uncertain. In about 50% of nodal ALCL the balanced chromosomal rearrangement t(2;5)(p23;q35), generating the fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), can be detected. Expression of this fusion kinase induces malignancy in mice and also leads to IL-3 independent outgrowth of the murine hematopoietic cell line Ba/F3. Using NPM-ALK transduced Ba/F3 cells, we show that NPM-ALK induces JunB expression and activation as verified by quantitative RT-PCR, immunoblot and electro-mobility supershift assay. Interestingly, NPM-ALK transduced Ba/F3 cells also express CD30, which is undetectable in the corresponding wild type cells. Since NPM-ALK induces JunB and CD30 and also leads to growth factor independent proliferation in Ba/F3 cells, these cells mimic conditions present in ALCL. Knock down of JUNB in NPM-ALK expressing cells using RNA interference leads to downregulation of CD30. Moreover, this partial loss of JunB induces upregulation of p16INK4a and downregulation of CCND1, which directly affect the cell cycle at the G1/S transition. These observations indicate that JunB is an essential factor for CD30 regulation and for neoplastic transformation. To test if JunB by itself is sufficient to induce CD30 expression and IL-3 independence, we stably transduced Ba/F3 cells with JUNB. In Ba/F3 wild type (WT) cells, JunB expression leads to reverse effects compared to that observed in NPM-ALK transduced Ba/F3. Ba/F3 WT cells do not become IL-3 independent. In addition, compared to vector control, JUNB-transduced Ba/F3 cells show a decrease in proliferation. Furthermore, an induction of p16INK4a and a decrease of CCND1 expression are observed. Moreover, aberrant JunB expression does not trigger CD30 expression in this system. Taken together, we show that both NPM-ALK and JunB are essential to induce CD30 expression. Furthermore the opposing effects of JunB on p16INK4a and CCND1 in the presence or absence of NPM-ALK indicate that NPM-ALK converts JUNB from a tumor suppressor to an oncogene.

Description: 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.