Vasiliki Leventaki

Activation of Mammalian Target of Rapamycin Signaling Pathway Contributes to Tumor Cell Survival in Anaplastic Lymphoma Kinase–Positive Anaplastic Large Cell Lymphoma

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in aberrant expression of chimeric nucleophosmin-ALK. Previously, nucleophosmin-ALK has been shown to activate phosphatidylinositol 3-kinase (PI3K) and its downstream effector, the serine/threonine kinase AKT. In this study, we hypothesized that the mammalian target of rapamycin (mTOR) pathway, which functions downstream of AKT, mediates the oncogenic effects of activated PI3K/AKT in ALK+ ALCL. Here, we provide evidence that mTOR signaling phosphoproteins, including mTOR, eukaryotic initiation factor 4E-binding protein-1, p70S6K, and ribosomal protein S6, are highly phosphorylated in ALK+ ALCL cell lines and tumors. We also show that AKT activation contributes to mTOR phosphorylation, at least in part, as forced expression of constitutively active AKT by myristoylated AKT adenovirus results in increased phosphorylation of mTOR and its downstream effectors. Conversely, inhibition of AKT expression or activity results in decreased mTOR phosphorylation. In addition, pharmacologic inhibition of PI3K/AKT down-regulates the activation of the mTOR signaling pathway. We also show that inhibition of mTOR with rapamycin, as well as silencing mTOR gene product expression using mTOR-specific small interfering RNA, decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis in ALK+ ALCL cells. Cell cycle arrest was associated with modulation of G(1)-S-phase regulators, including the cyclin-dependent kinase inhibitors p21(waf1) and p27(kip1). Apoptosis following inhibition of mTOR expression or function was associated with down-regulation of antiapoptotic proteins, including c-FLIP, MCL-1, and BCL-2. These findings suggest that the mTOR pathway contributes to nucleophosmin-ALK/PI3K/AKT-mediated tumorigenesis and that inhibition of mTOR represents a potential therapeutic strategy in ALK+ ALCL.

Selective inhibition of STAT3 induces apoptosis and G1 cell cycle arrest in ALK-positive anaplastic large cell lymphoma

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is an aberrant fusion gene product expressed in a subset of cases of anaplastic large cell lymphoma (ALCL). It has been shown that NPM-ALK binds to and activates signal transducer and activator of transcription 3 (STAT3) in vitro, and that STAT3 is constitutively active in ALK+ ALCL cell lines and tumors. In view of the oncogenic potential of STAT3, we further examined its biological significance in ALCL using two ALK+ ALCL cell lines (Karpas 299 and SU-DHL-1) and an adenoviral vector that carries dominant-negative STAT3 (AdSTAT3DN). Infection by AdSTAT3DN led to the expression of STAT3DN in both ALK+ ALCL cell lines at a similar efficiency. Subcellular fractionation studies showed that a significant proportion of the expressed STAT3DN protein translocated to the nucleus, despite the fact that STAT3DN has a mutation at residue 705tyrosine → phenylalanine, a site that is believed to be crucial for STAT3 activation and nuclear translocation. Introduction of STAT3DN induced apoptosis and G1 cell cycle arrest. Western blot studies showed that expression of STAT3DN resulted in caspase-3 cleavage, downregulation of Bcl-2, Bcl-xL, cyclin D3, survivin, Mcl-1, c-Myc and suppressor of cytokine signaling 3. These results support the concept that STAT3 activation is pathogenetically important in ALCL cells by deregulating the expression of multiple target proteins that are involved in the control of apoptosis and cell cycle progression.

Inhibition of JAK3 induces apoptosis and decreases anaplastic lymphoma kinase activity in anaplastic large cell lymphoma

Signal transducer and activator of transcription 3 (STAT3), normally activated by Janus kinase (JAK) in response to cytokine stimulation, has been shown to have oncogenic potential. In addition to JAK, recent data suggest that STAT3 can also be activated by other proteins such as the aberrant fusion protein, NPM-ALK, which is expressed in a subset of systemic anaplastic large cell lymphoma (ALCL). In this study, we investigated the possible role of JAK in activating STAT3 in ALCL using two ALK-positive ALCL cell lines, Karpas 299 and SU-DHL-1. At the steady state, JAK3 showed detectable tyrosine phosphorylation by immunoprecipitation. Treatment with AG490, a JAK inhibitor, decreased but did not completely abrogate tyrosine phosphorylation of JAK3 and STAT3 in a concentration-dependent manner. Similar results were obtained using two other inhibitors of JAK3, WHI-P131 and WHI-P154. These biochemical changes were associated with apoptosis in both cell lines that was coupled with activation of caspase 3 and decreased bcl-xL and bcl-2. Cell cycle analysis revealed a decrease in the S phase, which may be attributed to cyclin D3 downregulation and p21waf1 upregulation. Importantly, the tyrosine kinase activity of NPM-ALK, as assessed by an in vitro assay, decreased with increasing concentrations of AG490. Our findings highlight the importance of JAK3 in activating STAT3 in ALCL, and that NPM-ALK-mediated activation of STAT3 is influenced by the functional status of JAK3.

Characterization of 4 Mantle Cell Lymphoma Cell Lines Establishment of an In Vitro Study Model

CONTEXT Mantle cell lymphoma (MCL) is a distinct type of B-cell non-Hodgkin lymphoma characterized by t(11;14)(q13;q32) and cyclin D1 overexpression. The pathogenesis of MCL has not been comprehensively studied, which can be attributed in part to the paucity of well-characterized MCL cell lines. OBJECTIVES We collected 4 previously developed MCL cell lines and performed extensive characterization, including the susceptibly of these cell lines to transduction by adenovirus vectors. Our aim was to facilitate the establishment of an in vitro model that can be reliably used to study the pathogenesis of MCL. METHODS Standard techniques were used to compare the morphologic, immunophenotypic, and cytogenetic features of the 4 cell lines. In addition, Western blotting was used to investigate the presence of several cell cycle- and apoptosis-related proteins. TP53 DNA sequencing was also performed on the cell lines. The adenoviral transduction efficiency was assessed using an adenoviral vector carrying the gene encoding for the green fluorescence protein (Ad-GFP). RESULTS All cell lines demonstrated evidence of t(11;14)(q13;q32) and overexpression of cyclin D1. Cyclin D2 was not detectable in all cell lines, whereas cyclin D3 was weakly expressed in JeKo-1 and SP-53. Other abnormalities of the cell cycle G1 phase regulatory pathway were detected, including loss of expression of p53 (JeKo-1) and p16(INK4a) (SP-53 and Granta 519), as well as TP53 mutation (Mino). All cell lines express high levels of cyclin E, c-Myc, Bcl-2, Bax, Bcl-x(L), and Mcl-1. Retinoblastoma protein is hyperphosphorylated in all cell lines. With the exception of Mino, MCL cell lines are highly transducible with adenoviral vectors. CONCLUSION These cell lines are representative of MCL and can be used as an in vitro model to further explore the pathogenesis of this disease. The susceptibility of these cell lines to gene transfer provides opportunities to evaluate the importance of various oncogenes and tumor suppressor genes that may have an impact on developing effective therapeutic regimens for MCL.

Inhibition of p53-Murine Double Minute 2 Interaction by Nutlin-3A Stabilizes p53 and Induces Cell Cycle Arrest and Apoptosis in Hodgkin Lymphoma

Purpose: p53 is frequently expressed but rarely mutated in Hodgkin and Reed-Sternberg (HRS) cells of Hodgkins lymphoma (HL). p53 protein levels are regulated by murine double minute 2 (MDM2) through a well-established autoregulatory feedback loop. In this study, we investigated the effects of nutlin-3A, a recently developed small molecule that antagonizes MDM2 and disrupts the p53-MDM2 interaction, on p53-dependent cell cycle arrest and apoptosis in cultured HRS cells. Experimental Design: HL cell lines carrying wild-type (wt) or mutated p53 gene were treated with the potent MDM2 inhibitor nutlin-3A or a 150-fold less active enantiomer, nutlin-3B. Results: We show that nutlin-3A, but not nutlin-3B, stabilizes p53 in cultured HRS cells carrying wt p53 gene resulting in p53-dependent cell cycle arrest and apoptosis. Cell cycle arrest was associated with up-regulation of the cyclin-dependent kinase inhibitor p21. Nutlin-3A–induced apoptotic cell death was accompanied by Bax and Puma up-regulation and caspase-3 cleavage and was abrogated, in part, by inhibition of caspase-9 and caspase-3 activity. By contrast, no effects on cell cycle or apoptosis were found in HL cell lines harboring mutated p53 gene. Furthermore, combined treatment with nutlin-3A and doxorubicin revealed enhanced cytotoxicity in HRS cells with wt p53 gene. Blocking of nuclear export by leptomycin B, or inhibition of proteasome by MG132, stabilized p53 at a level comparable with that of nutlin-3A treatment in HRS cells with wt p53. Conclusions: These data suggest that nutlin-3A stabilized p53 by preventing MDM2-mediated p53 degradation in HRS cells. wt p53 stabilization and activation by nutlin-3A may be a novel therapeutic approach for patients with HL.

Cuplike nuclei (prominent nuclear invaginations) in acute myeloid leukemia are highly associated with FLT3 internal tandem duplication and NPM1 mutation

A small subset of patients with acute myeloid leukemia (AML) have cuplike nuclei. Other investigators have demonstrated that these neoplasms have distinctive clinicopathologic and molecular features.

Expression of STAT3 and its phosphorylated forms in mantle cell lymphoma cell lines and tumours.

The pathogenesis of mantle cell lymphoma (MCL) is incompletely understood, although cyclin D1 overexpression leading to deregulated cell proliferation is probably important. Recent data suggest that interleukin (IL)‐10 can increase the proliferative activity of MCL cells. STAT3 (signal transducer and activator of transcription 3) is the signal transducer of IL‐10, and STAT3 is activated by phosphorylation. The hypothesis of this study is that STAT3 is activated in MCL. The expression of the two phosphorylated (i.e. active) forms of STAT3, pSTAT3‐tyr (phosphorylated at the tyrosine705 residue) and pSTAT3‐ser (phosphorylated at the serine727 residue), was assessed in four MCL cell lines and 12 MCL tumours using western blots and/or immunofluorescence staining techniques. All MCL cell lines expressed STAT3, but only one had detectable pSTAT3‐tyr and none had pSTAT3‐ser. Addition of IL‐10 rapidly resulted in expression of pSTAT3‐tyr but not pSTAT3‐ser. All eight cases of frozen MCL tumours examined had detectable pSTAT3‐tyr and pSTAT3‐ser. Immunofluorescence studies using four formalin‐fixed, paraffin wax‐embedded MCL tumours demonstrated cytoplasmic localization of STAT3, as opposed to the nuclear localization of the pSTAT3 species. In conclusion, these findings provide evidence that STAT3 is constitutively activated in MCL, supporting the concept that STAT3 signalling may be important in the pathogenesis of these tumours. Copyright

c-Jun expression and activation are restricted to CD30+ lymphoproliferative disorders

Cellular Jun (c-Jun), a member of the JUN family, is an activator protein-1 transcription factor involved in cell differentiation, proliferation, and apoptosis that can be activated by phosphorylation at serine-73 and -63 residues. Using tissue microarrays and immunohistochemistry, we investigated c-Jun expression and serine-73 phosphorylation in 112 CD30+ lymphomas and 232 CD30− lymphomas of B- or T-cell lineage, and 24 cases of lymphomatoid papulosis. c-Jun was expressed exclusively by CD30+ lymphoproliferative disorders including 41/41 (100%) classical Hodgkin lymphoma (cHL), 20/23 (87%) anaplastic lymphoma kinase (ALK)+ anaplastic large cell lymphoma (ALCL), 18/26 (69%) ALK− ALCL, 5/9 (56%) primary cutaneous ALCL, 4/11 (36%) CD30+ diffuse large B-cell lymphoma (DLBCL), and 11/24 (46%) cases of lymphomatoid papulosis. The percentage of c-Jun-positive tumor cells was highest in cHL and ALCL (P=0.002). In contrast, all CD30− lymphomas, including nodular lymphocyte predominant HL and CD30− non-Hodgkin lymphomas of B- or T-cell lineage were negative for c-Jun. Serine-73 phosphorylated c-Jun (Ser73p-c-Jun), the activated form of c-Jun, was expressed more frequently and at a higher level in cHL and ALK+ ALCL than other CD30+ tumors. The percentage of Ser73p-c-Jun-positive tumor cells correlated significantly with the percentage of total c-Jun-positive cells (P<0.0001), suggesting that activated c-Jun positively regulates total c-Jun levels in CD30+ lymphomas through a well-established positive feedback loop. We conclude that CD30+ lymphomas are characterized by common patterns of c-Jun expression and activation suggesting a potential role of c-Jun in the pathogenesis of these tumors.

The oncogenic JUNB/CD30 axis contributes to cell cycle deregulation in ALK+ anaplastic large cell lymphoma

Anaplastic lymphoma kinase (ALK)+ anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in expression of NPM1(NPM)‐ALK oncogenic kinase. The latter is capable of activating ERK kinase, which upregulates JUNB expression through ETS1. JUNB, in turn, interacts with the TNFRSF8 (CD30) gene promoter and induces CD30 (TNFRSF8) overexpression. However, the role of CD30 overexpression in ALK+ ALCL oncogenesis remains unknown. Here we show that the JUNB gene is frequently amplified in ALK+ ALCL, suggesting gene amplification as an additional underlying mechanism for JUNB overexpression. Silencing of JUNB resulted in reduced cell growth and colony formation associated with decreased activator protein‐1 activity and G1/S and G2/M cell cycle arrest. These effects were linked to decreased CD30 levels, downregulation of CCNA2 (Cyclin A), CCND2 (Cyclin D2) and CCND3 (Cyclin D3) and upregulation of cyclin‐dependent kinase inhibitors CDKN2A (p14) and CDKN1A (p21), but not CDKN1B (p27). Similar cell cycle changes were observed following the knock‐down of TNFRSF8 gene or blockade of its function using anti‐CD30 antibodies, which were associated with upregulation of CDKN2A and CDKN1A, but not CDKN1B. These findings indicate that JUNB may partly operate through CD30 signalling. Silencing of JUNB also sensitized NPM1‐ALCL+ cells to standard chemotherapeutic agents. Our findings uncover the oncogenic role of the JUNB/CD30 axis and its potential as therapeutic target in ALK+ ALCL.

Expression of Eukaryotic Initiation Factor 4E Predicts Clinical Outcome in Patients With Mantle Cell Lymphoma Treated With Hyper-CVAD and Rituximab, Alternating With Rituximab, High-Dose Methotrexate, and Cytarabine

Oncogenic AKT/mammalian target of rapamycin (mTOR) signaling has recently been shown to contribute to tumor survival and proliferation in mantle cell lymphoma (MCL) through its downstream effector eukaryotic initiation factor 4E (eIF4E), which may control cyclin D1 protein levels. However, the clinical significance of eIF4E expression in MCL is unknown.