Leptin-Induce Proliferation Mediated by JAK/STAT and MAPK Activation in Acute Myelogenous Leukemia Cells.

Abstract Leptin, secreted as a product of the ob gene that is mainly produced by adipose tissue, has been involved in the regulation of energy metabolism. Recently, leptin has been suggested to stimulate normal cells as well as various cancer cells including acute myelogenous leukemia (AML). However, the molecular mechanism of leptin as a proliferative effector is not poorly understood in AML. In the present study, we show that leptin-induced cellular proliferation is mediated by JAK/STAT and MAPK activation in AML. Expression of the long form (Ob-Rb) and the short form (Ob-Ra) of leptin receptor (Ob-R) was observed in 10 AML cell lines examined, and up-regulated by treatment of leptin. Leptin induced the proliferation of HEL, which was shown to express the highest Ob-R among AML cell lines, in a dose-dependent manner. Treatment with 100ng/ml of leptin enhanced the expression of Janus kinase 2 (JAK2) phosphorylation, signal transducer and activator of transcription (STAT)-3 phosphorylation and mitogen-activated protein kinases (MAPKs) in HEL. Blocking of STAT3 phosphorylation with a specific inhibitor, AG490 (50 μM), significantly reduced leptin-induced ERK1/2 phosphorylation and cellular proliferation of HEL, whereas blocking of ERK1/2 activation by a specific ERK1/2 kinase inhibitor, PD98059 (25 μM), did not affect the STAT3 phosphorylation and leptin-induced proliferation in HEL. Furthermore, knockdown of Ob-R expression with small interfering RNA (siRNA) reduced leptin-induced proliferation of HEL, and also significantly attenuated leptin-induced STAT3 and ERK1/2 activation. This results provides that leptin promotes AML cell growth by activating JAK/STAT3 and MAPK, although not directly dependent on ERK. Blocking as direct receptor level could be a rational therapeutic strategy of AML.

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Advanced Glycation End Products (AGEs)-Mediated Cell Growth of Human Acute Myelogenous Leukemia Via Mitogen-Activated Protein (MAP) Kinase Pathways.

Blood ◽

10.1182/blood.v106.11.2762.2762 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2762-2762

Author(s):

Ju Young Kim ◽

Hyun Ki Park ◽

Jin Sun Yoon ◽

Eun Shil Kim ◽

Kwang Sung Ahn ◽

...

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Cell Lines ◽

Cellular Proliferation ◽

Myelogenous Leukemia ◽

Dependent Manner ◽

Glycation End Products ◽

Mitogen Activated Protein ◽

Acute Myelogenous ◽

Dose Dependent

Abstract Advanced glycation end products (AGEs) are products of non-enzymatic glycation/oxidation of proteins/lipids that accumulate slowly during natural aging and at a much accelerated rate in a variety of disorders such as diabetes, renal failure, and Alzheimer’s disease. AGE modifications do not only change the physicochemical properties of the afflicted molecules, but also induce cellular signaling, activation of transcription factors and subsequent gene expression in vitro and in vivo. Most of the biologic activities associated with AGEs have been transduced by receptor for AGE (RAGE). Recently, AGEs are known to be in association with diverse cancers in terms of cellular proliferation and metastasis. However, little is known about the role of AGEs in acute myelogenous leukemia (AML). Here we examined the effects of the AGEs-RAGE interaction on the cell proliferation and intracellular signaling of AGEs in human leukemia cell lines. Expression of RAGE was observed in 8 AML cell lines examined, and up-regulated by treatment of AGE. AGE induced the proliferation of AML cell lines, HL60 and HEL, in a dose-dependent manner. Treatment with 5 μM of antisense S-ODN for RAGE did effectively inhibit cell growth of HEL cells. Exposure of HL60 and HEL with AGE induced a significant increase in the numbers of cells in S phase of cell cycle in a dose-dependent manner. AGE enhanced the expression of cell cycle regulatory proteins such as cyclin-dependent kinase (CDK) 2/4/6, cyclin D1/E/B in a dose- and a time-dependent manner. In addition, the protein levels of the cyclin-dependent kinase inhibitor (CDKI), p21 and p27, were decreased by 24 hr exposure of AGE from 10 to 200 μg/ml in HEL. Furthermore, treatment of HEL with 200 μg/ml of AGE triggered activation of mitogen-activated protein (MAP) kinases, Erk, Akt, and p38, pathways and in nuclear translocation of transcription factors NF-kB. These results indicated that AGE induced the cell growth of human AML cells, HL60 and HEL, via augmentation of cell cycle and activation of MAPK kinase pathways. Up-regulation of RAGE by exposure of AGE suggested that cellular proliferation of AML cells might be mediated in autocrine fashion.

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Pulsing with Blast Cell Lysate or Blast-Derived Total RNA Reverses the Dendritic Cell-Mediated Increase in Cytotoxic Activity of Cytokine-Induced Killer Cells Against Human Allogeneic Acute Myelogenous Leukemia Cells.

Blood ◽

10.1182/blood.v104.11.4531.4531 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4531-4531

Author(s):

Bjorn Schottker ◽

Angela Marten ◽

Carsten Ziske ◽

Marcus Gorschluter ◽

Ingo G.H. Schmidt-Wolf

Keyword(s):

Cytotoxic Activity ◽

Cell Lines ◽

Acute Myelogenous Leukemia ◽

Blast Cell ◽

Myelogenous Leukemia ◽

Dependent Manner ◽

Total Rna ◽

Acute Myelogenous ◽

Cik Cell

Abstract Immunotherapeutic strategies may be a treatment option in patients with refractory acute myelogenous leukemia (AML) or, in cases of complete remission after conventional therapy regimens, may help to reduce disease recurrence or delay time to progression. Because evidence suggests a key role of dendritic cells (DCs) in cancer immunotherapy, we examined cytokine induced killer (CIK) cell responses in vitro after coincubation with autologous peripheral blood monocyte-derived DCs against three cell lines and allogeneic blasts from three patients with de novo AML. Although DCs were unable to enhance CIK cell effects against all three cell lines tested, the cytotoxic activity against the AML cells of the patients increased after coculture with mature DCs, which was significant in two of three patients. However, neither prior pulsing of the DCs with blast cell lysates nor with leukaemic cell-derived total RNA further enhanced the lytic capacity of the CIK cells. On the contrary, pulsing reduced the cytotoxic activity of the effector cells in a concentration-dependent manner. Because this decrease of allogeneic cytotoxicity was observed, we conclude that monocyte-derived DCs may be useful in autologous or allogeneic vaccine strategies for the treatment of AML or in priming donor lymphocytes in vitro, but unfractionated antigens as pulsing agents may have inhibitory effects on T cell efficiency and their employment in immunotherapeutic strategies for AML seems questionable.

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Autocrine stimulation of interleukin 1 beta in acute myelogenous leukemia cells.

Journal of Experimental Medicine ◽

10.1084/jem.166.5.1597 ◽

1987 ◽

Vol 166 (5) ◽

pp. 1597-1602 ◽

Cited By ~ 48

Author(s):

K Sakai ◽

T Hattori ◽

M Matsuoka ◽

N Asou ◽

S Yamamoto ◽

...

Keyword(s):

Acute Myelogenous Leukemia ◽

Interleukin 1 ◽

Myelogenous Leukemia ◽

Leukemic Cells ◽

Dependent Manner ◽

Acute Myelogenous ◽

Autocrine Stimulation ◽

Basal Proliferation ◽

Culture Supernatants ◽

Dose Dependent

A significant increase in CD25 antigen-positive cells by IL-1 was observed in cells of a patient with M7 acute myelogenous leukemia. Basal proliferation and expression of CD25 antigen by the M7 leukemic cells were inhibited by addition of anti-IL-1 beta antibody in a dose-dependent manner, but not by rabbit anti-IL-1 alpha antibody. Culture supernatants of these leukemic cells contained IL-1 activity, which was specifically inhibited by addition of anti-IL-1 beta antibody, and Northern blot analysis detected intracellular IL-1 beta mRNA. These results indicated that autocrine secretion of IL-1 beta was involved in proliferation of some myelogenous leukemic cells.

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Microrna Expression Profiling in Acute Myelogenous Leukemia.

Blood ◽

10.1182/blood.v104.11.1131.1131 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1131-1131

Author(s):

Fernando J. Suarez Saiz ◽

Serban San-Marina ◽

Mark D. Minden

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Cell Lines ◽

Acute Myelogenous Leukemia ◽

Myelogenous Leukemia ◽

Erythroid Cell ◽

Normal Bone Marrow ◽

Hematopoietic Differentiation ◽

Normal Bone ◽

Acute Myelogenous

Abstract Acute myelogenous leukemia (AML) arises due to changes in gene expression that block or alter the normal differentiation program of hematopoietic stem cells. A variety of mutations in protein-encoding genes have been shown to contribute to the development of leukemia. Recently a new class of genes called microRNAs (miRNAs) have been identified. miRNAs are a subgroup of highly conserved, non-coding RNAs found only in eukaryotes. They do not encode proteins, and appear to have a significant effect on the proteome of a cell. Their conservation between species suggests their involvement in important biological functions, and in fact been shown to be involved in hematopoietic differentiation. While the function of most miRNAs is still unknown, it is believed that they regulate expression of target mRNAs by using the siRNA machinery either to promote degradation of the mRNA or to block its translation. To begin to understand the role of miRNAs in AML, we used Quantitative Polymerase Chain Reaction (QPCR) to measure the expression level of 20 miRNA precursors in the pro erythroid cell line K562, the pro-myelocytic cell line NB4, the myelomococytic cell line OCI/AML2, AML patients’ blasts and in normal bone marrow (NBM). The investigated miRNAs included some that are known to be specific for hematopoietic tissues or involved in hematopoietic differentiation, as well as all the miRNAs in chromosome 7, a hot spot for gene deletion in AML. Our findings indicate that miRNAs are differentially expressed in patients and cell lines when compared among themselves and against normal bone marrow. For example pre-miR-142 was expressed in NBM and K562 but was found to be elevated in OCI/AML2, NB4 and in all patient samples. Pre-miR-20 was found to be overexpressed in only a subset of patients. Other miRNAs like pre-miR-335 and pre-miR-148a were expressed in NBM and in some patients and not in the cell lines. In an effort to identify possible regulators of miRNA expression, we analyzed the upstream region of pre-miR-142 and found an LMO2 binding site. In AML, the LMO2 gene can be overexpressed relative to normal bone marrow and healthy lymphocytes. This transcription factor is involved in the regulation of genes important in the development of blood cells. To investigate if LMO2 could be involved in the regulation of miR-142 expression, we performed chromatin immunoprecipitation (ChIP) from K562 using an anti-LMO2 antibody. Only the LMO2 immunoprecipitation, and not those from the pre-immune control, were enriched in promoter DNA for pre-miR-142. This is consistent with the observation that miRNAs and coding RNAs can be regulated by the same environmental signals. Based on this observation we propose that oncogenes regulate in part the phenotype and biological behaviour of leukemia by affecting the expression of miRNAs. This further suggests that different forms of leukemia may be recognized based upon the spectrum of miRNAs they express.

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Janus Kinase (JAK)-2 Does Not Play a Role in Bcr-Abl Signaling in Philadelphia Chromosome (Ph)-Positive (p190) Acute Lymphoblastic Leukemia (ALL) Cells.

Blood ◽

10.1182/blood.v110.11.4241.4241 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4241-4241

Author(s):

Stefan H. Faderl ◽

Quin Van ◽

Patricia E. Koch ◽

David M. Harris ◽

Inbal Hallevi ◽

...

Keyword(s):

Cell Lines ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Janus Kinase ◽

Myelogenous Leukemia ◽

Dependent Manner ◽

Western Immunoblotting ◽

Gm Csf ◽

Dose Dependent

Abstract Novel immunochemotherapy regimens combined with imatinib mesylate (IA) have significantly improved treatment outcome of Ph+ ALL. Nevertheless, most adult patients with Ph+ ALL relapse and succumb to their disease. Recent reports suggested that Jak-2 is engaged in the signaling of Bcr-Abl in chronic myelogenous leukemia (CML) cells. Because Jak-2 inhibitory agents are currently investigated in clinical trials, we sought to explore the role of Jak-2 in the signaling of Bcr-Abl in Ph+ ALL assuming that inhibition of Jak-2 might be beneficial in the treatment of Ph+ ALL. To do this, we used our Ph+ (p190) ALL cell lines Z-119 and Z-181 (Estrov et al. J Cell Physiol166: 618, 1996). We chose these cells because in both lines Jak-2 can be activated. Both Z-119 and Z-181 cells express granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors and GM-CSF activates Jak-2 and stimulates the proliferation of both cell lines. Using a clonogenic assay, we found that IA inhibited the proliferation of these cells at concentrations ranging from 50 to 500 nM. Because Bcr-Abl was found to activate the signal transducer and activator of transcription (STAT)-5 in CML cells, we used Western immunoblotting and found that IA inhibited the phosphorylation (p) of STAT5 in a dose-dependent manner in Ph+ ALL cells. To test whether JAk-2 plays a role in Bcr-Abl (p190) signaling we incubated Z-181 cells for 4 hours with or without 50, 100, 250, and 500 nM IA, extracted cellular protein and immunoprecipitated total STAT5 protein. Then, using Western immunoblotting we detected the Bcr-Abl p190 protein in all STAT5 immunoprecipitates and by using specific pSTAT5 antibodies, we demonstrated that IA induced a dose-dependent reduction in the levels of pSTAT5, but not of p190 protein, suggesting that the p190 Bcr-Abl kinase binds to and activates STAT5. Remarkably, neither Jak-2 nor pJak-2 was detected in either immunoprecipitate. To further delineate the role of Jak-2 in Bcr-Abl signaling we extracted protein from Z-181 cells and immunoprecipitated Jak-2. Neither Bcr-Abl nor STAT5 was detected in these immunoprecipitates, confirming that Jak-2 does not bind Bcr-Abl p190 protein and does not participate in the activation of STAT5. Taken together, our data suggest that Bcr-Abl (p190) binds and phosphorylates STAT5 whereas, Jak-2 is not engaged in Bcr-Abl (p190) signaling in Ph+ ALL cells.

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Gamma-interferon regulation of class II antigens on acute myelogenous leukemia cell lines

Human Immunology ◽

10.1016/0198-8859(88)90296-0 ◽

1988 ◽

Vol 23 (2) ◽

pp. 160-161

Author(s):

J.J. Yunis ◽

H. Band ◽

F. Bonneville ◽

E.J. Yunis

Keyword(s):

Cell Lines ◽

Acute Myelogenous Leukemia ◽

Leukemia Cell ◽

Class Ii ◽

Gamma Interferon ◽

Myelogenous Leukemia ◽

Acute Myelogenous Leukemia Cell ◽

Leukemia Cell Lines ◽

Acute Myelogenous ◽

Class Ii Antigens

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Development of a Novel Treatment Strategy Targeting JAK2 in Acute Myelogenous Leukemia.

Blood ◽

10.1182/blood.v114.22.2619.2619 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2619-2619

Author(s):

Shinsuke Kojima ◽

Takayuki Ikezoe ◽

Jing Yang ◽

Chie Nshioka ◽

Asako Takeuchi ◽

...

Keyword(s):

Acute Myelogenous Leukemia ◽

Conflicts Of Interest ◽

Remission Rate ◽

Hematopoietic Cells ◽

Rare Event ◽

Specific Inhibitor ◽

Janus Kinase ◽

Myelogenous Leukemia ◽

Jak2 Inhibitor ◽

Acute Myelogenous

Abstract Abstract 2619 Poster Board II-595 Somatic mutations in Janus kinase 2 (JAK2) gene activate JAK2/signal transducer and activator of transcriptions (STATs) signaling, leading to proliferation of hematopoietic cells. STAT3 and/or STAT5 were constitutively activated in the majority of patients with acute myelogenous leukemia (AML), although mutation of the JAK2 gene was an extremely rare event in AML. This study performed the immunohistochemichal examination to verify whether JAK2 was activated in AML (n=73, excluding acute promyelocytic leukemia) by utilizing the phosphor-specific antibody against JAK2. p-JAK2 was detectable in all cases examined, although its level varied between each patients (+, n=27; +/−, n=28; −/+, n=18). Statistical examination found that levels of p-JAK2 were correlated with leukocytosis (21.4 × 106/L in patients with high p-JAK2 vs 13.8 × 106/L in those with low p-JAK2, p<0.001) and lower complete remission rate (65.5% in patients with high p-JAK2 vs 83.3% in patients with low p-JAK2, p<0.02). In addition, there was a trend that high expression level of p-JAK2 was associated with poor prognosis in AML patients (median survival time 604 days in patients with high p-JAK2 vs 1431 days in those with low p-JAK2). Moreover, we found that the novel and specific inhibitor of the JAK2 kinase AZ960 potently inhibited the clonogenic growth of freshly isolated CD34+ AML cells from patients (n=6) with IC50 ranging from 0.01 to 0.1 mM. On the other hand, levels of p-JAK2 in CD34+ hematopoietic cells from healthy volunteers (n=3) were lower than those in CD34+AML cells, as measured by FACS, and their colony forming ability was not affected by AZ960. Furthermore, exposure of freshly isolated CD34+ AML cells to AZ960 (0.03–0.1 mM) induced apoptosis as assessed by induction of the cleaved forms of PARP as well as caspase 3, and downregulation of anti-apoptotic protein Bcl-xL. Taken together, JAK2 may be a promising molecular target for treatment of AML. Further studies are warranted to evaluate the efficacy of the JAK2 inhibitor in clinical settings. Disclosures: No relevant conflicts of interest to declare.

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A Role for IL1RAP in Acute Myelogenous Leukemia Stem Cells Following Treatment and Progression

Blood ◽

10.1182/blood.v126.23.4266.4266 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4266-4266 ◽

Cited By ~ 1

Author(s):

Tzu-Chieh Ho ◽

Craig T Jordan ◽

Mark W. LaMere ◽

John M. Ashton ◽

Kristen O'Dwyer ◽

...

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Cell Lines ◽

Small Molecule ◽

Acute Myelogenous Leukemia ◽

Research Funding ◽

Myelogenous Leukemia ◽

Molecule Inhibitor ◽

Acute Myelogenous ◽

Pre Treatment

Abstract Background Acute Myelogenous Leukemia (AML) evolves as many patients who are responsive to therapy upfront are resistant to the same agents when applied at relapse. We previously reported the results of our prospective efforts to formally assess the evolution of the leukemia stem cell (LSC) population(s) during patients' clinical courses. We identified a 9-90 fold increase in LSC activity and greatly increased phenotypic diversity of the LSC population. To identify the potential mechanisms underlying these changes we further characterized functionally-defined LSC populations from paired diagnosis and relapse samples. Methods Primary bone marrow and peripheral blood samples were collected on IRB approved protocols from patients with newly diagnosed AML undergoing induction therapy as well as normal donors. Twenty-five patients who relapsed after achieving a complete remission were selected for further study. Screening studies identified seven patients whose pre-therapy samples demonstrated sustained engraftment of NSG mice following transplantation. Transcriptional profiling of highly enriched LSC populations from seven patients was performed using ABI TaqMan® Low Density Array (TLDA) qPCR analyses following pre-amplification using a novel 153 gene expression platform. Protein expression levels of interleukin-1 receptor accessory protein (IL1RAP) on bulk leukemia cells and LSC populations from 25 patients were assessed by flow cytometry. The impact of loss of IL1RAP was assessed using lentiviral based shRNA targeting all IL1RAP isoforms followed by assessment of proliferation, apoptosis, colony forming unit (CFU) activity and NSG engraftment capacity in human cell lines as well as in primary patient samples. Downstream signaling events for IL1RAP were probed using a small molecule inhibitor approach. Results While the majority of the LSC populations' gene expression profile remained stable, twelve genes were differentially expressed between pre-treatment and relapsed LSC populations including IL1RAP. Flow cytometric analyses confirmed that IL1RAP is overexpressed on both bulk leukemia populations as well as LSC populations at diagnosis and relapse in comparison to normal hematopoietic stem cell (HSC) populations. Targeting ILRAP1 using shRNA in both cell lines and primary AML samples resulted in impaired proliferation, increased apoptosis, a marked loss of CFU capacity and impaired NSG engraftment. IL1 signaling is known to involve both the MAPkinase and NFKappB pathways. To determine which pathways are involved in IL1RAP mediated LSC survival, we performed a small molecule inhibitor screen targeting elements in both signaling cascades. Established inhibitors of the NFKappaB pathway resulted in loss in loss of leukemic cell function while MAPK signaling inhibition had minimal to no effect. Conclusions We identified IL1RAP as being overexpressed in both bulk leukemia and functionally defined LSC populations from pre-treatment and relapsed AML samples. Loss of IL1RAP was associated with a marked decline in LSC function. Preliminary studies support a primary role for the NF Kappa B pathway in LSC function. Our findings support a critical role for IL1RAP in LSC function and support its development as a target for AML therapy in both the upfront and relapse setting. Disclosures Wang: Immunogen: Research Funding. Calvi:Fate Therapeutics: Patents & Royalties. Becker:Millenium: Research Funding.

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