NPM-ALK Promotes Cell Cycle Progression through Activation of JNK and c-Jun in Anaplastic Large Cell Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2057-2057
Author(s):  
Vasiliki Leventaki ◽  
Elias Drakos ◽  
Megan Lim ◽  
Kojo S. Elenitoba-Johnson ◽  
Francois-Xavier Claret ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Western Blot ◽  
Anaplastic Large Cell Lymphoma ◽  
Blot Analysis ◽  
Cell Cycle Progression ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Cycle Progression ◽  
Large Cell Lymphoma

Abstract Anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in aberrant expression of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) chimeric protein. NPM-ALK mediates its oncogenic effects through phosphorylation of a number of proteins involved in known signal transduction pathways including PLC, PI3K-AKT and JAK-STAT. ALK+ ALCL cells also are known to overexpress c-Jun, a member of the activator protein-1 (AP-1) transcription factor family that controls cell proliferation, differentiation, growth and apoptosis. Phosphorylation of c-Jun at serine 73 and serine 63 residues substantially increases AP-1 transcriptional activity and the levels of c-Jun protein through an autoregulatory positive feedback loop. In this study, we hypothesized that NPM-ALK activates JNK which , in turn, phosphorylates and activates c-Jun, resulting in uncontrolled cell cycle progression in ALCL. 293T and Jurkat (T-acute lymphoblastic leukemia) cells were transfected with a vector expressing NPM-ALK with active kinase domain (pDest40-NPM-ALK) or a construct lacking NPM-ALK kinase activity (pDest40-K210R) or empty vector. Cells were harvested at 48 hours and analyzed for protein expression by Western blot analysis and for AP-1 activity by luciferase reporter assay. Two ALK+ ALCL cell lines Karpas 299 and SU-DHL-1, found to express high levels of serine phosphorylated and total c-Jun in immunoblots, were treated with JNK (SP600125), ERK (U0126), or ALK (WHI-P154) inhibitors or were transiently transfected with siRNAs specific for JNK1 and c-Jun. Cell proliferation was assessed by MTS assay, and cell cycle was analyzed by BrdU assay or propidium iodide staining and flow cytometry. Forced expression of NPM-ALK in 293T and Jurkat cells resulted in increased levels of JNK and c-Jun phosphorylation in immunoblots and a dramatic increase in AP-1 activity. Conversely, pharmacologic inhibition of ALK activity in Karpas 299 and SU-DHL1 resulted in a concentration-dependent decrease of JNK and c-Jun phosphorylation levels. Co-immunoprecipitation studies revealed that NPM-ALK physically binds to JNK1 and its upstream activator MKK7 in ALK+ ALCL cells. Selective inhibition of JNK, but not ERK, in Karpas 299 and SU-DHL1 decreased the level of c-Jun phosphorylation in a dose-dependent manner as shown by Western blot analysis and in vitro kinase assays. Inhibition of JNK by SP600125 or silencing of the JNK1 gene by siRNA also resulted in decreased cell proliferation associated with decreased AP-1 activity, cell cycle arrest mostly at G2 phase, and up-regulation of the cyclin-dependent inhibitor p21, a transcriptional target of c-Jun. Similarly, silencing of c-Jun by specific siRNA led to decreased S-phase fraction of cell cycle, which was associated with up-regulation of p21 and downregulation of cyclin D3. These findings reveal a novel function of NPM-ALK oncoprotein, phosphorylation and activation of JNK, which may contribute to uncontrolled cell cycle progression through activation of c-Jun. Modulation of JNK or c-Jun activity may be a target for therapy in patients with ALCL.

cJun Is Phosphorylated at Ser73 and Contributes to Cell Cycle Progression in Anaplastic Large Cell Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2620-2620
Author(s):  
Vassiliki Leventaki ◽  
Elias Drakos ◽  
Francois-Xavier Claret ◽  
L. Jeffrey Medeiros ◽  
George Z. Rassidakis
Keyword(s):  
Cell Cycle ◽  
Tumor Cells ◽  
Cell Lines ◽  
Anaplastic Large Cell Lymphoma ◽  
Cell Cycle Progression ◽  
Cell Lymphoma ◽  
Large Cell ◽  
S Phase ◽  
Cycle Progression ◽  
Large Cell Lymphoma

Abstract Anaplastic Large Cell Lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) or variant translocations resulting in overexpression of anaplastic lymphoma kinase (ALK). cJun is a member of the activator protein-1 (AP-1) family, which is a group of transcription factors that control cell proliferation, differentiation, growth and apoptosis. The activity of cJun can be regulated by phosphorylation at serine 73 (Ser73) and serine 63 (Ser63) residues of the N-terminal domain. It is believed that cJun promotes cell cycle progression, in part, through downregulation of the cyclin-dependent kinase inhibitor p21. Previous studies have shown high AP-1 activity and cJun overexpression in Hodgkin lymphoma and ALCL (Mathas et al, EMBO J2002; 21:4104). In this study, we assessed for expression of cJun and its Ser73- and Ser63-phosphorylated forms in two ALK+ (Karpas 299 and SU-DHL-1) and one ALK- (Mac2A) ALCL cell lines by western blot analysis, and in 31 ALCL tumors (15 ALK+, 16 ALK-) by immunohistochemistry using tissue microarrays and specific antibodies. To examine the role of cJun in cell survival and proliferation in our in vitro system, ALCL cells were transiently transfected with small interfering RNA (siRNA) specific for cJun. Cell viability, proliferation of viable cells and cell cycle progression from G1 to S-phase were assessed by trypan blue exclusion, MTS and BrdU assays, respectively. All three ALCL cell lines expressed total cJun and Ser73-phosphorylated cJun (Ser73p-cJun) at a high level, whereas Ser63-phosphorylated cJun was expressed at a low level. In addition, all 31 ALCL tumors expressed total cJun in most neoplastic cells. Ser73p-cJun was also detected in all ALCL tumors at a variable level with the percentage of Ser73p-cJun-positive tumor cells ranging from 5% to 95%. By contrast, Ser63p-cJun was detected rarely in tumor cells. Transient transfection of ALCL cells with specific siRNA resulted in almost complete silencing of total cJun expression and absence of Ser73p-cJun expression, which was associated with decreased cell viability and a substantial (40%) decrease of cell growth. cJun silencing also resulted in cell cycle arrest as shown by decreased S-phase fraction. These cell cycle changes were associated with a marked increase of p21 levels and downregulation of cyclin D2 and D3. In conclusion, cJun is highly phosphorylated at serine 73 in ALCL cell lines and tumors and may contribute to cell cycle progression. Targeting cJun expression or phosphorylation using gene therapy approaches may represent a novel therapeutic strategy for patients with ALCL.

NPM-ALK oncogenic kinase promotes cell-cycle progression through activation of JNK/cJun signaling in anaplastic large-cell lymphoma

Blood ◽  
2007 ◽  
Vol 110 (5) ◽  
pp. 1621-1630 ◽  
Author(s):  
Vasiliki Leventaki ◽  
Elias Drakos ◽  
L. Jeffrey Medeiros ◽  
Megan S. Lim ◽  
Kojo S. Elenitoba-Johnson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Anaplastic Large Cell Lymphoma ◽  
Cell Cycle Progression ◽  
Transcriptional Activity ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Cyclin D3 ◽  
Phase Cell ◽  
Cycle Progression ◽  
Large Cell Lymphoma

Abstract Anaplastic large-cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35), resulting in aberrant expression of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). We show that in 293T and Jurkat cells, forced expression of active NPM-ALK, but not kinase-dead mutant NPM-ALK (210K>R), induced JNK and cJun phosphorylation, and this was linked to a dramatic increase in AP-1 transcriptional activity. Conversely, inhibition of ALK activity in NPM-ALK+ ALCL cells resulted in a concentration-dependent dephosphorylation of JNK and cJun and decreased AP-1 DNA-binding. In addition, JNK physically binds NPM-ALK and is highly activated in cultured and primary NPM-ALK+ ALCL cells. cJun phosphorylation in NPM-ALK+ ALCL cells is mediated by JNKs, as shown by selective knocking down of JNK1 and JNK2 genes using siRNA. Inhibition of JNK activity using SP600125 decreased cJun phosphorylation and AP-1 transcriptional activity and this was associated with decreased cell proliferation and G2/M cell-cycle arrest in a dose-dependent manner. Silencing of the cJun gene by siRNA led to a decreased S-phase cell-cycle fraction associated with upregulation of p21 and downregulation of cyclin D3 and cyclin A. Taken together, these findings reveal a novel function of NPM-ALK, phosphorylation and activation of JNK and cJun, which may contribute to uncontrolled cell-cycle progression and oncogenesis.

scholarly journals Inhibition of Akt increases p27Kip1 levels and induces cell cycle arrest in anaplastic large cell lymphoma

Blood ◽  
2005 ◽  
Vol 105 (2) ◽  
pp. 827-829 ◽  
Author(s):  
George Z. Rassidakis ◽  
Marianna Feretzaki ◽  
Coralyn Atwell ◽  
Ioannis Grammatikakis ◽  
Quan Lin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Anaplastic Large Cell Lymphoma ◽  
Cell Cycle Progression ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Cycle Progression ◽  
Anaplastic Lymphoma ◽  
Cycle Arrest ◽  
Large Cell Lymphoma

Abstract Anaplastic large cell lymphoma (ALCL) is a highly proliferative neoplasm that frequently carries the t(2;5)(p23;q35) and aberrantly expresses nucleophosmin–anaplastic lymphoma kinase (NPM-ALK). Previously, NPM-ALK had been shown to activate the phosphatidylinositol 3 kinase (PI3K)/Akt pathway. As the cyclin-dependent kinase (CDK) inhibitor p27Kip1 (p27) is usually not expressed in ALCL, we hypothesized that activated Akt (pAkt) phosphorylates p27 resulting in increased p27 proteolysis and cell cycle progression. Here we demonstrate that inhibition of pAkt activity in ALCL decreases p27 phosphorylation and degradation, resulting in increased p27 levels and cell cycle arrest. Using immunohistochemistry, pAkt was detected in 24 (57%) of 42 ALCL tumors, including 8 (44%) of 18 ALK-positive tumors and 16 (67%) of 24 ALK-negative tumors, and was inversely correlated with p27 levels. The mean percentage of p27-positive tumor cells was 5% in the pAkt-positive group compared with 26% in the pAkt-negative group (P = .0076). These findings implicate that Akt activation promotes cell cycle progression through inactivation of p27 in ALCL.

PI3K/AKT-and JAK3-Dependent Regulation of SKP2 Expression Is Mediated by E2F1 in Anaplastic Large Cell Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3742-3742
Author(s):  
Jean-Marc Fontaine ◽  
Kojo S.J. Elenitoba-Johnson ◽  
Megan S Lim
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Cycle Progression ◽  
Gene Promoter ◽  
Large Cell ◽  
Cycle Progression ◽  
F Box Protein

Abstract The majority of anaplastic large cell lymphomas (ALCL) are characterized by the chromosomal translocation t(2;5)(p23;q35) leading to the expression of NPM/ALK. The constitutive activation of the NPM/ALK tyrosine kinase induces downstream mediators such as phosphoinositide 3-kinase (PI3-kinase)/AKT, JAK3 and STAT3 that result in increased cell proliferation and enhanced survival. Although the molecular mechanism by which these pathways deregulate the cell cycle machinery is not fully understood, previous studies have shown that NPM/ALK-mediated PI3K/AKT activation is required for cell cycle progression and that inhibition of PI3K/AKT results in decreased p27Kip1 degradation and cell cycle arrest. The expression of S-phase kinase protein 2 (SKP2), an F-box motif-containing protein which targets cell cycle regulators including cyclin-dependent kinase inhibitor p27Kip1 via ubiquitin-mediated degradation, was evaluated in a panel of ALCL cell lines. Western blot analysis of five t(2;5)-positive ALCL-derived cell lines demonstrated an inverse pattern of expression between F-box protein SKP2 and p27Kip1. We hypothesized that SKP2 deregulation contributes to the oncogenic activity of NPM/ALK by regulating the degradation of p27Kip1. In this study we investigated regulation of SKP2 and p27Kip1 expression as a consequence of inhibition of two well-known pathways downstream of NPM/ALK. Inhibition of PI3K/AKT with Ly294002 (20 mM) or JAK3 with WHI-P154 (10 mM) resulted in a dose and time-dependent decrease in cell viability (50% or 20% respectively at 24h). To determine the mechanism of SKP2 transcriptional regulation by PI3K, we performed quantitative RT-PCR and western blot analysis which demonstrated a decrease in both SKP2 transcript and protein levels after PI3K/AKT and JAK2 inhibition (33% or 47% at 24h respectively), with increase in the levels of p27 transcript and protein (47% or 71% at 24h respectively). Furthermore, the levels of E2F1 (a transcription factor associated with cell cycle progression) also decreased upon PI3K/AKT and JAK3 inhibition. Chromatin immunoprecipitation (ChIP) assays revealed that E2F1 binding to the SKP2 gene promoter was reduced as early as 4 hours after inhibition of PI3K/AKT or JAK3 (80% and 59% respectively) while no binding was detected with the GAPDH gene promoter (control). In conclusion, these results indicate that the expression of the F-box protein SKP2 is regulated by NPM/ALK mediators, PI3K/AKT and JAK3, and that E2F1 mediates the transcriptional control of SKP2 expression. Our data supports the role of SKP2–mediated regulation of p27Kip1 in ALCLs and implicates SKP2 and E2F1 as a potential therapeutic target in ALCLs.

Wiskott-Aldrich Protein Is Phosphorylated by NPM-ALK and Regulates the Proliferation and Invasion of Anaplastic Large Cell Lymphoma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1535-1535
Author(s):  
Carlos Murga-Zamalloa ◽  
Steven R Hwang ◽  
Anagh A Sahasrabuddhe ◽  
Scott RP McDonnell ◽  
Venkatesha Basrur ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Western Blot ◽  
Anaplastic Large Cell Lymphoma ◽  
Blot Analysis ◽  
Colony Formation ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Actin Dynamics ◽  
Large Cell Lymphoma

Abstract Abstract 1535 Introduction: Anaplastic large cell lymphoma (ALCL) is a subtype of mature T cell lymphoma. The NPM-ALK fusion protein is expressed in 80% of pediatric ALCL and arises from a chromosomal translocation t(2;5)(p23;q35). NPM-ALK is a constitutively active oncogenic tyrosine kinase that regulates the function of numerous proteins involved in apoptosis and cell cycle progression. In order to better understand the mechanisms of NPM-ALK mediated oncogenesis, we utilized an unbiased mass spectrometry –based phosphoproteomic approach to identify novel downstream signaling targets of the oncogenic NPM-ALK. Approach: The phosphoproteome was enriched using a two-step procedure including immobilized metal affinity chromatography (IMAC) and phospho-tyrosine immunoaffinity purification and subsequently analyzed by liquid chromatography and high mass accuracy tandem mass spectrometry (LC-MS/MS). Western blot analysis with phospho-specific antibodies and in vitro kinase assays were used to validate the MS results. Stable lentiviral transduction of shRNA vectors was used to evaluate the functional consequences. Cell proliferation was measured using the WST-1 assay. Effect on transformation capacity was assessed with colony formation assay using methyl-cellulose agar. Invasive properties were assessed using collagen based transwell assays and adhesion was evaluated by attachment onto fibronectin substratum. Results: Phosphoproteomic analysis identified several proteins involved in actin dynamics to be regulated by ALK tyrosine kinase activity. These include WASP, CRKL, NCK1, ARP2/3 and ITSN2. Pathway network analysis situated WASP at the center of the actin network. Western blot analysis demonstrated that phosphorylation of WASP at Y290 is regulated by NPM-ALK. Expression of wild type NPM-ALK increased the phosphorylation of WASP Y290 in 293T cells compared to a kinase-defective K210R mutant. In vitro kinase assays demonstrated that NPM-ALK phosphorylates WASP at Y290 as well as a novel residue Y102. The interaction between NPM-ALK and WASP was demonstrated using immunoprecipitation of co-transfected 293T cells as well as in ALCL cell lines. Because Y102 residue of WASP is located in the domain involved in interaction with its chaperone WIP, we evaluated whether its phosphorylation regulates the interaction. The interaction of WASP and WIP was decreased when Y102 was phosphorylated as demonstrated by immunoprecipitation of phospho-mutants of WASP (Y102F and Y102E). Surface plasmon resonance studies with recombinant WT or phosphomimetic (Y102E) WASP and WIP confirmed these observations. Furthermore, cycloheximide chase experiments demonstrated that phosphorylation of WASP at Y102 decreased it protein stability. Stable WASP knockdown in SUDHL1 cells led to reduced adhesion to fibronectin (21% ± SEM 0.4% vs 13% ± SEM 0.6%, p<0.01) and invasion through collagen (68% ± SEM 16% vs 12% ± SEM 3%, p<0.05). Interestingly, stable knock down of WASP resulted in increased proliferation (0.79 RFU ± SEM 0.07 vs 0.54 RFU ± SEM 0.019, p<0.01) and colony formation (58 colonies ± SEM 1.2 vs 26 colonies ± SEM 3.3, p< 0.01) of SUDH1L cells. Conclusions: Our results show that NPM-ALK regulates the phosphorylation of a network of proteins involved in actin dynamics, one of which is WASP. NPM-ALK directly phosphorylates WASP at residues Y290 and Y102 and thereby regulates WASP function in actin dynamics critical for cell adhesion, invasion and proliferation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Usefulness of BATF3 Immunohistochemistry in Diagnosing Classical Hodgkin Lymphoma

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1123
Author(s):  
Julian Benckendorff ◽  
Johanna Kuchar ◽  
Frank Leithäuser ◽  
Malena Zahn ◽  
Peter Möller
Keyword(s):  
Transcription Factor ◽  
Western Blot ◽  
Hodgkin Lymphoma ◽  
Anaplastic Large Cell Lymphoma ◽  
Diagnostic Marker ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Classical Hodgkin Lymphoma ◽  
Highly Sensitive ◽  
Large Cell Lymphoma

It is well recognized that the AP-1 transcription factor BATF3 is constitutively expressed in Hodgkin/Reed-Sternberg (HRS) cells, but its potential as a diagnostic marker for classical Hodgkin lymphoma (cHL) has not yet been addressed. In this study, we performed immunohistochemistry and analyzed the BATF3 expression in lymphoma cells on 218 lymphoma samples belonging to 14 different lymphoma entities. We observed varying degrees of BATF3 expression in nearly half of the cases (n = 100) with BATF3 expression being a constitutive feature of cHL (n = 53) and anaplastic large cell lymphoma (ALCL). By scoring BATF3 expression (BATF3-score) we observed constitutively high BATF3-scores in cHL and ALCL and low to moderate BATF3-scores in all other entities examined. Western blot analysis confirmed BATF3 protein expression in cell lysates from cHL cell lines (n = 7). Thus, BATF3 can be considered a useful IHC marker for the diagnosis of cHL as it is highly sensitive and sufficiently specific when analyzed by BATF3-scoring.

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

Oncogene ◽  
2004 ◽  
Vol 23 (32) ◽  
pp. 5426-5434 ◽  
Author(s):  
Hesham M Amin ◽  
Timothy J McDonnell ◽  
Yupo Ma ◽  
Quan Lin ◽  
Yasushi Fujio ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Anaplastic Large Cell Lymphoma ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Selective Inhibition ◽  
Cycle Arrest ◽  
G1 Cell Cycle Arrest ◽  
Large Cell Lymphoma ◽  
Alk Positive

scholarly journals PTBP3 regulates proliferation of lung squamous cell carcinoma cells via CDC25A‐mediated cell cycle progression

2022 ◽  
Vol 22 (1) ◽  
Author(s):  
Yingji Chen ◽  
Ying Ji ◽  
Suo Liu ◽  
Yicai Liu ◽  
Wei Feng ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Squamous Cell Carcinoma ◽  
Western Blot ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Cell Cycle Progression ◽  
Lung Squamous Cell Carcinoma ◽  
Molecular Therapy ◽  
Cycle Progression

Abstract Background The roles of Polypyrimidine tract-binding protein 3 (PTBP3) in regulating lung squamous cell carcinoma (LUSC) cells progression is unclear. The aim of this study was to investigate the role of PTBP3 in LUSC. Methods Expression and survival analysis of PTBP3 was firstly investigated using TCGA datasets. Quantitative reverse transcription PCR and Western blot were performed to detect PTBP3 expression in clinical samples. Moreover, cell counting kit 8 (CCK-8) assays, colony formation assays and in vivo tumor formation assays were used to examine the effects of PTBP3 on LUSC cell proliferation. RNA-sequence and analysis explores pathways regulated by PTBP3.Flow cytology was used analyzed cell cycle. Cell cycle-related markers were analyzed by Western blot. Results PTBP3 was found to be overexpressed in LUSC tissues compared with normal tissues. High PTBP3 expression was significantly correlated with poor prognosis. In vitro and vivo experiments demonstrated that PTBP3 knockdown caused a significant decrease in the proliferation rate of cells. Bioinformatics analysis showed that PTBP3 involved in cell cycle pathway regulation in LUSC. Furthermore, PTBP3 knockdown arrested cell cycle progression at S phase via decreasing CDK2/Cyclin A2 complex. In addition, downregulation of PTBP3 significantly decreased the expression of CDC25A. Conclusions Our results suggest that PTBP3 regulated LUSC cell proliferation via cell cycle and might be a potential target for molecular therapy of LUSC.

The CALM/AF10 Interactor CATS Is a Substrate of KIS, a Positive Regulator of Cell Cycle Progression in Leukemia Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2549-2549
Author(s):  
Leticia Fröhlich Archangelo ◽  
Fabíola Traina ◽  
Philipp A Greif ◽  
Alexandre Maucuer ◽  
Valérie Manceau ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Cycle Progression ◽  
Phosphorylation Site ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Cycle Progression

Abstract Abstract 2549 The CATS protein (also known as FAM64A and RCS1) was first identified as a novel CALM (PICALM) interactor that interacts with and influences the subcellular localization of CALM/AF10, a leukemic fusion protein found in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and in malignant lymphoma. CATS is highly expressed in leukemia, lymphoma and tumor cell lines but not in non-proliferating T-cells or in peripheral blood lymphocytes (PBLs). The protein levels of CATS are cell cycle-dependent, induced by mitogens (e.g. PHA) and correlate with the proliferative state of the cell. Thus, CATS is as a marker for proliferation. Using CATS as a bait in a yeast two-hybrid screen we identified the Kinase Interacting Stathmin (KIS or UHMK1) as a CATS interacting partner. KIS is a serine/threonine kinase that positively regulates cell cycle progression through phosphorylation of p27KIP in leukemia cell lines. The interaction between CATS and KIS was confirmed by GST pull-down, and co-immunopreciptation. KIS interaction region was mapped to CATS N-terminal portion. Searching through the phosphorylation site databases PhosphoSitePlus™ (http://www.phosphosite.org) and Phosida (http://www.phosida.com/) we identified 9 residues within CATS shown to be subject of post-translational modification. Phosphorylation assay with recombinant KIS demonstrated that this kinase efficiently phosphorylated full length CATS and its N-terminal part, but not the C-terminal of the protein. To map the KIS phosphorylation site of CATS, peptides comprising all known phospho-sites of CATS N-terminal (S16, S129, S131, T133 and S135) and mutations of the putative KIS target motif (S129 and S131) were tested for KIS phosphorylation. Thereby, we identified CATS S131 as the unique target site for KIS phosphorylation. Western blot analysis of U2OS cells, which had undergone cell cycle synchronization by a double thymidine block, revealed that KIS fluctuated throughout the cell cycle and counteracted CATS levels. Furthermore, we analyzed KIS protein expression on bone marrow mononuclear cells (MNCs) of MDS and AML patients. We studied 5 healthy donors, 13 MDS patients (7 low-risk [RA/RARS] and 6 high-risk [RAEB/RAEBt] according to FAB classification) and 10 AML patients (7 de novo and 3 secondary). Western blot analysis revealed elevated levels of KIS in MDS and AML compared to the control samples. We used a reporter gene assay in order to determine the influence of KIS on the CATS-mediated transcriptional repression and to elucidate the role of KIS-dependent phosphorylation of CATS at serine 131 in this context. Coexpression of GAL4-DBD-CATS and KIS enhanced the inhibitory function of CATS on transactivation of the GAL4-tk-luciferase reporter. This effect of KIS was observed for both CATS wild type and CATS phospho-defective mutant (CATS S131A) but not when the kinase dead mutant KISK54R was used. Moreover, CATS phosphomimetic clone (CATSS131D) exerted the same transcriptional activity as the CATS wild type. These results demonstrate that KIS enhances the transcriptional repressor activity of CATS, and this effect is independent of CATS phosphorylation at S131 but dependent on the kinase activity of KIS. Finally, we investigated whether CATS would affect the CALM/AF10 function as an aberrant transcription factor. Coexpression of constant amounts of GAL4-DBD-CALM/AF10 and increasing amounts of CATS lead to reduced transactivation capacity of CALM/AF10 in a dose dependent manner. Our results show that CATS not only interacts with but is also a substrate for KIS, suggesting that CATS function might be modulated through phosphorylation events. The identification of the CATS-KIS interaction further supports the hypothesis that CATS plays an important role in the control of cell proliferation. Moreover the elevated levels of KIS in hematological malignances suggest that KIS could regulate CATS activity and/or function in highly proliferating leukemic cells. Thus our results indicate that CATS function might be important to understand the malignant transformation mediated by CALM/AF10. Disclosures: No relevant conflicts of interest to declare.

scholarly journals FOXM1 and the NPM-ALK/STAT3 Axis Form a Novel Positive Feedback Loop in Promoting the Oncogenesis of ALK-Positive Anaplastic Large Cell Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3921-3921
Author(s):  
Peng Wang ◽  
Moinul Haque ◽  
Jing Li ◽  
Yung-Hsing Huang ◽  
Meaad Almowaled ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Anaplastic Large Cell Lymphoma ◽  
Feedback Loop ◽  
Transcriptional Activity ◽  
Cell Lymphoma ◽  
Large Cell ◽  
University Of Alberta ◽  
Large Cell Lymphoma ◽  
Alk Positive

Abstract Peng Wang1, Moinul Haque2, Jing Li2,3, Yung-Hsing Huang2, Meaad Almowaled2, Carter Bargar4, Adam Karpf4, Will Chen2, Suzanne Turner5 and Raymond Lai2,6 1Division of Hematology, Dept of Medicine, University of Alberta, Edmonton, 2 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton Alberta, Canada; 3Electron Microscopy Center, Basic Medical Science College, Harbin Medical University, Harbin, Heilongjiang, China; 4Eppley Institute and Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, USA; 5Division of Cellular and Molecular Pathology, Department of Pathology, University of Cambridge, Cambridge, UK;6Department of Oncology, University of Alberta, Edmonton, Alberta, Canada 1. Backgrounds and Aims Forkhead Box M1 (FOXM1) is a transcription factor implicated in the pathogenesis of solid tumors, and it has been shown to promote cell-cycle progression, stem cell renewal and chemotherapeutic resistance in cancer cells. Nonetheless, the biological significance of FOXM1 in hematologic malignancies has not been extensively studied. Here, we studied the expression and role of FOXM1 in ALK-positive anaplastic large cell lymphoma (ALK+ALCL). 2 Methods and Results In contrast with normal lymphocytes, FOXM1 was highly expressed in all ALK+ALCL cell lines (5/5), tumors from patients (6/6) and tumors arising from NPM-ALK transgenic mice. Experiments using nuclear/cytoplasmic fractionation, immunocytochemistry and reporter assays had provided evidence that FOXM1 is transcriptionally active in ALK+ALCL. Down-regulation of FOXM1 expression using shRNA and a pharmacologic agent (thiostrepton) resulted in a significant reduction in cell growth, colony formation in soft agar and cell-cycle arrest in ALK+ALCL cells. Further studies revealed that the oncogenic potential of FOXM1 is linked to substantial increases in the phosphorylation/activation status of NPM-ALK and STAT3, and the upregulations of a host of cytokines that have been previously shown to activate the NPM-ALK/STAT3 axis, including IGF-1, IL9 and IL21. Using co-immunoprecipitation, we found that NPM-ALK binds to FOXM1 in the nucleus of ALK+ALCL cells. Importantly, the binding of NPM-ALK to FOXM1 promotes the DNA binding ability and transcriptional activity of FOXM1, and functional inhibition of NPM-ALK using crizotinib or depletion of NPM-ALK using siRNA in ALK+ALCL cells significantly decreased the transcriptional activity of FOXM1.Conclusions: In conclusion, we have identified a novel oncogenic feedback loop involving FOXM1 and the NPM-ALK/STAT3 axis in ALK+ALCL. This study has revealed the first clear example in which NPM-ALK exerts important oncogenic functions in the nuclei of ALK+ALCL cells, by means of its binding to an oncogenic transcription factor so as to promote its DNA binding and transcription activity. Disclosures No relevant conflicts of interest to declare.

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