scholarly journals B-myb antisense oligonucleotides inhibit proliferation of human hematopoietic cell lines

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2708-2716 ◽  
Author(s):  
M Arsura ◽  
M Introna ◽  
F Passerini ◽  
A Mantovani ◽  
J Golay
Keyword(s):  
Cell Lines ◽  
Antisense Oligonucleotides ◽  
Hematopoietic Cell ◽  
Dependent Manner ◽  
Myb Gene ◽  
Myb Genes ◽  
Dose Dependent ◽  
Lymphoid Cell Lines ◽  
Hematopoietic Cell Lines

Abstract The B-myb gene is highly homologous to the c-myb protooncogene in several domains and also shares some of the functions of c-myb in that it can act as a transcriptional activator. In addition, the expression of both the B-myb and c-myb genes correlates with proliferation of normal hematopoietic cells. We investigated more directly the role of B- myb in proliferation of hematopoietic cell lines using B-myb-specific antisense oligonucleotides. We showed that several anti-B-myb oligonucleotides, complementary to distinct regions of the gene, inhibit significantly and in a dose-dependent manner the proliferation of all myeloid or lymphoid cell lines tested. This block in proliferation was not accompanied by detectable differentiation of U937 or HL60 cells to macrophages or granulocytes either spontaneously or after exposure to chemical agents. These data suggest that the B-myb gene, like c-myb, is necessary for hematopoietic cell proliferation.

scholarly journals B-myb antisense oligonucleotides inhibit proliferation of human hematopoietic cell lines

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2708-2716 ◽  
Author(s):  
M Arsura ◽  
M Introna ◽  
F Passerini ◽  
A Mantovani ◽  
J Golay
Keyword(s):  
Cell Lines ◽  
Antisense Oligonucleotides ◽  
Hematopoietic Cell ◽  
Dependent Manner ◽  
Myb Gene ◽  
Myb Genes ◽  
Dose Dependent ◽  
Lymphoid Cell Lines ◽  
Hematopoietic Cell Lines

The B-myb gene is highly homologous to the c-myb protooncogene in several domains and also shares some of the functions of c-myb in that it can act as a transcriptional activator. In addition, the expression of both the B-myb and c-myb genes correlates with proliferation of normal hematopoietic cells. We investigated more directly the role of B- myb in proliferation of hematopoietic cell lines using B-myb-specific antisense oligonucleotides. We showed that several anti-B-myb oligonucleotides, complementary to distinct regions of the gene, inhibit significantly and in a dose-dependent manner the proliferation of all myeloid or lymphoid cell lines tested. This block in proliferation was not accompanied by detectable differentiation of U937 or HL60 cells to macrophages or granulocytes either spontaneously or after exposure to chemical agents. These data suggest that the B-myb gene, like c-myb, is necessary for hematopoietic cell proliferation.

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 ◽  
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.

Rbm15 Affects Notch Signaling and Myelopoiesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2545-2545
Author(s):  
Matthew J. Renda ◽  
Ee-Chun Cheng ◽  
Lin Wang ◽  
Xian-Young Ma ◽  
Diane S. Krause
Keyword(s):  
Fusion Protein ◽  
Notch Signaling ◽  
Cell Lines ◽  
Promoter Activity ◽  
Hematopoietic Cell ◽  
Chromosome 1 ◽  
Dominant Negative Effect ◽  
Myeloid Differentiation ◽  
Hematopoietic Cell Lines

Abstract Acute Megakaryoblastic Leukemia (AMKL) presents with one of two different genotypic abnormalities; either Down Syndrome (DS) accompanied with GATA-1 mutations, or a non-DS translocation t(1;22)(p13;q13). In AMKL associated with DS, there is trisomy 21 and various partial loss of function mutations in GATA-1. In non-DS AMKL, the translocation t(1;22)(p13;q13) encodes a fusion protein, RBM15-MKL, comprised of the transcriptional co-factors RBM15 (chromosome 1) and MKL (chromosome 22). In order to elucidate the role of the RBM15-MKL fusion protein in AMKL, we must understand the normal functions of RBM15 and MKL. Herein we demonstrate a role for Rbm15 in myeloid differentiation. Previous work in the lab showed that Rbm15 is expressed at highest levels in hematopoietic stem cells, and at more moderate levels during myelopoiesis of murine cell lines and primary murine cells. Therefore, we hypothesized that Rbm15 plays a role in myeloid differentiation. Indeed, we demonstrate that shRNA-mediated knockdown of Rbm15 enhanced the differentiation of 32DWT18 myeloid precursor cells. Recent studies have shown the Rbm15 homolog, Sharp, interacts with RBPJk, a critical transcription factor in the Notch signaling pathway; and since Notch signaling is essential for hematopoiesis, we hypothesized that Rbm15 may also modulate Notch signaling. We demonstrate that Rbm15 alters Notch-induced HES1 promoter activity in a cell-type specific manner. Rbm15 inhibits Notch-induced HES1 activity in non-hematopoietic cell lines, but stimulates this activity in hematopoietic cell lines including 32DWT18 and HEL (human erythroleukemia). In addition, we show that the N-terminus of Rbm15 co-immunoprecipitates with RBPJk and has a dominant negative effect by impairing the activation ability of HES1 promoter activity by full length-Rbm15. Thus, Rbm15 is differentially expressed during hematopoiesis and may act to inhibit myeloid differentiation in hematopoietic cells via a mechanism that is mediated, at least in part, by stimulation of Notch signaling via interaction with RBPJk. Consistent with a potential mechanistic role of Rbm15 and Notch in myelopoiesis, we have shown that shRNA-mediated knockdown of Rbm15 in 32DWT18 cells promotes myeloid differentiation, suggesting that the RBM15 component of the RBM15-MKL fusion protein may act by blocking differentiation. Currently, we are examining the effect of RBM15 overexpression and knockdown in primary mouse bone marrow cells using in vitro and in vivo assays.

MKC-1 Is a Novel Agent That Induces Cell Cycle Arrest and Disrupts Multiple Survival Pathways in Hematologic Cancers.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4390-4390
Author(s):  
Trisha A. Denny ◽  
Xiaoru Chen ◽  
Cassandra L. Waters ◽  
Patricia A. Burke ◽  
Graham C. Fletcher ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Solid Tumor ◽  
Hematopoietic Cell ◽  
Cycle Arrest ◽  
Survival Pathways ◽  
Dose Dependent ◽  
Hematopoietic Cell Lines

Abstract MKC-1 is a novel, orally active cell cycle inhibitor with in vitro and in vivo activity against a wide range of human solid tumor cell lines, including multi-drug resistant cell lines. MKC-1 has been tested in over 270 patients to date and is currently in Phase II clinical trials. The strong pre-clinical activity of MKC-1 towards solid tumor lines and signs of efficacy in the initial clinical evaluation with lack of neuropathy and cardiotoxicity suggests that MKC-1 may also be of clinical benefit in the treatment of hematopoietic cancers. The antiproliferative activity of MKC-1 was examined against a panel of hematopoietic cell lines including HL-60, U937, MV4;11, THP-1, Jurkat, and OCI-AML 1–5. MKC-1 showed potent and dose-dependent activity towards these cell lines, with IC50 values in the range of 20 – 400 nM. MKC-1 also inhibited in vitro growth of primary cells derived from AML and CML patients. Additionally, MKC-1 showed enhanced activity with Ara-C in combination studies in vitro when added either simultaneously or sequentially using the cell line OCI-AML 4. Binding studies have shown that MKC-1 binds to the colchicine binding site of tubulin and to members of the importin beta family of proteins. Consistent with these results, cell cycle arrest in the G2/M phase of the cell cycle followed by apoptosis was observed in cell lines and patient samples treated with MKC-1. Immunofluorescence analysis of cells treated with MKC-1 revealed that the drug induced a disruption of the microtubule network and the formation of aberrant mitotic spindles. Furthermore, MKC-1 was also shown to induce a dose-dependent reduction in the levels of both phospho-Akt and phospho-p70S6K kinases through Western blot analysis of treated THP-1 cells. In conclusion, our results indicate MKC-1 arrests the cell cycle and disrupts multiple survival pathways to induce apoptosis in hematopoietic cell lines and patient samples. These results suggest that MKC-1 may have clinical potential in the treatment of leukemia either alone or in combination with other agents. Phase I trials in hematological cancers are currently being explored.

scholarly journals HDAC8 Maintain Cytoskeleton Integrity Via Homologous Recombination and Represent a Novel Therapeutic Target in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4385-4385
Author(s):  
Zuzana Chyra ◽  
Maria Gkotzamanidou ◽  
Masood A. Shammas ◽  
Vassilis L. Souliotis ◽  
Yan Xu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Repair ◽  
Homologous Recombination ◽  
Cell Lines ◽  
Hdac Inhibitors ◽  
Mass Spectrometry Analysis ◽  
Dependent Manner ◽  
Large Panel ◽  
Dose Dependent

Multiple Myeloma (MM) is a plasma cell malignancy vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets in combination with current anti-myeloma agents. Pan-HDAC inhibitors are effective as therapeutic agents both in preclinical and clinical setting; however, there is an increasing emphasis on understanding the biological and molecular roles of individual HDACs to limit toxicities observed with pan-HDAC inhibitors. Based on correlation with patient outcome in three independent myeloma datasets, we have evaluated the functional role of HDAC8, a member of Class I HDAC isoenzymes, in MM. Unlike other isoforms, there is limited information about molecular and epigenomic functions of HDAC8. We have previously confirmed expression of HDAC8 in a large panel of MM cell lines, where it is localized predominantly to cytoplasm. Moreover, genetic and pharmacological modulation of HDAC8 with RNAi and specific inhibitor PCI-34051 resulted in a significant inhibition of myeloma cell proliferation and decrease in colony formation (p<.001). HDAC8 inhibition led to an increase in the ongoing spontaneous and radiation-induced DNA damage in MM cells by affecting DNA repair via the homologous recombination (HR) pathway, suggesting a novel function of HDAC8 in promoting HR and DNA repair in MM cells. Using laser micro-irradiation in MM1S and U2OS cells, we observed HDAC8 recruitment to DSBs sites and its co-localization with Rad51 and Scm3, a member of cohesin complex. A transcriptomic analysis of HDAC8 knock-down cells also shows perturbation of number of cytoskeleton-related genes confirming significant role of HAD8 in cytoskeleton rearrangement in MM. Mass-spectrometry analysis to identify the HDAC8 substrates in MM cells is currently ongoing. Classical pan-HDACi, such as SAHA (vorinostat), bind to HDAC8 with substantially diminished activity (IC50 = 2 μM), reflecting a unique binding site of this isoform. To discover and validate new small molecules with HDAC8 subtype selectivity, we have explored the efficacy of OJI-1, a novel selective and potent HDAC8 inhibitor (IC50 = 0.8 nM) with modest inhibition of HDAC6 (1200 nM). Treatment with OJI-1 selectively impact cell viability of a large panel of MM cell lines (n=20) in a time and dose dependent manner, while sparing healthy donors PBMC both in resting and activated state (n=3). The significantly higher IC50 observed in PBMCs suggests a favorable therapeutic index. Western blotting analysis confirmed target selectivity with significant time and dose dependent decrease in H3 and H4 acetylation in MM cells treated with OJI-1. Moreover, pharmacological inhibition of HDAC8 specifically inhibited HR but not non-homologous end joining. These data suggest that targeting of HDAC8 using OJI-1 could be effective treatment approach in MM. Based on molecular data combination studies and in vivo evaluation are ongoing. In conclusion, our results provide insight into the role of HDAC8 in DNA stability and cell growth and viability which can be exploited in future for therapeutic application alone and in combination in MM. Disclosures Munshi: Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Celgene: Consultancy.

Proteosome Inhibitor Treatment Down-Regulates S-Phase Kinase-Associated Protein 2 Causing Inhibition of Proliferation and Induces Apoptosis in Primary Effusion Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4616-4616
Author(s):  
Azhar R. Hussain ◽  
Naif A. Al-Jomah ◽  
Mehar Sultana ◽  
Manugaran S. Pulicat ◽  
Khawla S. Al-Kuraya ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Primary Effusion Lymphoma ◽  
S Phase ◽  
Dependent Manner ◽  
Down Regulation ◽  
Inhibition Of Proliferation ◽  
Dose Dependent ◽  
Inhibitor Treatment

Abstract Proteosome inhibition is a novel approach for treating malignancy and has been approved for clinical use. The proteosome is the primary proteolytic mechanism in eukaryotic cells and inhibition of its catalytic activity initiates a cascade of events affecting cell cycle and apoptotic activities. These activities ultimately lead to cell cycle arrest and apoptosis in malignant cells however, the normal counterpart of these cells are spared. In this study, we used a panel of primary effusion lymphoma cell lines (BC1, BC3, BCBL1 and HBL6) to study the effects of proteosome inhibitor, MG132 on cell proliferation and apoptosis. Our data showed that proteosome inhibitor MG132 decreased cell viability as well as induced apoptosis in a dose dependent manner ranging from 0.5–10μM. Furthermore, treatment with 2.5μM MG132 for 24hours induced 41% apoptosis in BC1, 51% in BC3, 41% in BCBL1 and 48% in HBL6 cell lines as detected by annexinV/PI dual staining. S-phase kinase-associated protein 2 (skp-2) is a proto-oncogene and over expressed in various types of tumors. We sought to determine the role of Skp-2 following proteosome inhibition in PELs. MG132 treatment of PEL cell lines resulted in down-regulation of SKP-2 protein in a dose dependent manner whereas the expression of p-27 was up-regulated demonstrating an inverse relationship between these two proteins. Furthermore, MG132 treatment of PELs led to conformational changes in Bax protein and translocation to the mitochondria leading to the loss of mitochondrial membrane potential with subsequent release of cytochrome c from mitochondria into cytosol. Cytochrome c release caused activation of caspase-3 followed by polyadenosin-5′-diphosphate-ribose polymerase (PARP) cleavage. In addition, proteosome inhibitor treatment also caused down-regulation of inhibitor of apoptosis protein, XIAP. Taken together, our findings show that proteosome inhibition causes down-regulation of skp-2, up-regulation of p-27, inhibition of proliferation as well as caspase-dependent apoptosis in primary effusion lymphoma cells suggesting a role of proteosome inhibitors in the treatment of these aggressive cancers.

Activation Of TAK1 By MYD88 L265P Drives Malignant B Cell Growth In Non-Hodgkin Lymphomas

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 245-245
Author(s):  
Stephen M Ansell ◽  
Lucy S. Hodge ◽  
Frank Secreto ◽  
Michelle Manske ◽  
Esteban Braggio ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Signaling Proteins ◽  
Dependent Manner ◽  
Wild Type ◽  
Dose Dependent

Abstract Massively parallel sequencing analyses have revealed a common mutation within the MYD88 gene (MYD88L265P) occurring at high frequencies in many non-Hodgkin lymphomas (NHL) including the rare lymphoplasmacytic lymphoma, Waldenström’s macroglobulinemia (WM). Using whole exome sequencing, Sanger sequencing and allele-specific PCR, we validate the initial studies and detect the MYD88L265P mutation in the tumor genome of 97% of WM patients analyzed (n=39). MYD88L265P was detected at lower frequencies in other indolent lymphomas including LPL (0%), MALT (4%), nodal MZL (5%) and splenic MZL (8%); all but one MYD88L265P was heterozygous. Due to the high frequency of MYD88 mutation in WM and other NHL, and its known effects on malignant B cell survival, therapeutic targeting of MYD88 signaling pathways may be useful clinically. However, while the effects of MYD88L265P on the activity of IRAK1/4 and NF-κB are have been studied previously, we are lacking a thorough characterization of the role of intermediary signaling proteins such as TRAF6 and TAK1 on the biology of MYD88L265P-expressing B cells. A better understanding of the proteins involved in MYD88L265P signaling may lead to the development of more targeted and effective therapeutic approaches. In an attempt to identify MYD88L265P –specific therapeutic targets we first wanted to characterize the role of intermediary signaling proteins that facilitate the downstream activation of NF-κB. Upon activation of TLRs or IL-1b receptors, MYD88 forms a homodimer and recruits IRAK1/4 and TRAF6 into a complex resulting in association and phosphorylation of TAK1 followed by activation of NF-κB. We monitored the formation of a complex comprised of MYD88, IRAK1, IRAK4 and TRAF6 and immunoprecipitation of either endogenous IRAK4 or IRAK1 revealed constitutive association of IRAK with TRAF6 and MYD88L265P. To assess if the formation of a MYD88L265P/IRAK/TRAF6 complex results in downstream activation of TAK1, constitutive TAK1 phosphorylation was measured and detected in all three cell lines that express MYD88L265P. An association between TAK1 and TRAF6, another measure of TAK1 activation, was also detectable. When a similar analysis of TAK1 was performed in DLBCL cells expressing wild-type MYD88, no phosphorylation of TAK1 was detected, nor was TAK1 associated with TRAF6. IRAK1, IRAK4, TAK1, TRAF6, and MYD88 were expressed at similar levels in all cell lines studied and therefore did not contribute the differences in MYD88 complex formation observed between cell lines. These studies were further confirmed using HEK 293T cells that were transduced with either a vector control plasmid or HA-tagged MYD88WT or MYD88L265P expression plasmids. Together, these studies suggest that MYD88L265P forms a complex with IRAK and TRAF6 resulting in constitutive activation of TAK1 and NF-κB. To confirm the significance of TAK1-mediated MYD88L265P signaling on lymphoma cell growth, the effect of the selective TAK1 inhibitor, (5Z)-7-Oxozeaenol, on cell proliferation was tested. All MYD88L265P-expressing cell lines were sensitive to TAK1 inhibition in a dose-dependent manner (0-10 μM). In contrast, NHL cells expressing MYD88WT were found to be insensitive to TAK1 inhibition. We next tested the impact of the TAK1 inhibitor on a MYD88L265P positive WM patient sample. Similar to what was seen in the WM cell lines, the TAK1 inhibitor inhibited WM cell growth and survival in a dose dependent manner. Additionally, the TAK1 inhibitor significantly reduced the level of IL-10 secreted by each of the cell lines. Together, these data suggest that MYD88L265P drives cell proliferation and cytokine secretion through a TAK1-dependent mechanism. In conclusion, we are the first to validate by NGS in a large patient cohort the high prevalence and specificity of MYD88L265P in WM. Cells harboring the L265P mutation but not wild-type MYD88 exhibit constitutive signaling leading to the hyperactivation of NF-κB. We have established the role of TAK1 as an integral component of MYD88L265P signaling in both WM and DLBCL cell. Our data suggest that targeting TAK1 clinically may be an effective strategy for the treatment of WM and other lymphomas driven by MYD88L265P signaling. Disclosures: Fonseca: millennium: Consultancy; amgen: Consultancy; Binding site: Consultancy; onyx: Consultancy; medtronic: Consultancy; Genzyme: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; lilly: Consultancy; Onyx: Research Funding; cylene: Research Funding.

Regulation and Functional Role of Beta2-Adrenergic Receptor in Acute Myelogenous Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2563-2563
Author(s):  
Ningfei An ◽  
Yeong-Bin Im ◽  
Amr Abdallah Moh'D Qudeimat ◽  
Luciano J Costa ◽  
Robert K Stuart ◽  
...  
Keyword(s):  
Cell Lines ◽  
Acute Myelogenous Leukemia ◽  
Leukemia Cell ◽  
Hematopoietic Cell ◽  
Myelogenous Leukemia ◽  
Pim Kinases ◽  
Leukemia Cell Lines ◽  
Acute Myelogenous ◽  
Hematopoietic Cell Lines

Abstract Abstract 2563 Acute myelogenous leukemia (AML) occurs with an incidence of 2.7 per 100,000 population in the year of 2009, and is associated with significant mortality and morbidity. Despite recent advances in molecular and cytogenetic analytic technologies, initial treatment for AML patients has remained essentially the same over the last 30 years; and the treatment outcome is dreadful with a 5-year survival rate of ∼25%. In an effort to gain a better understanding of AML cell biology and to develop more effective treatments for AML, we have been exploring the roles of the b2-adrenergic receptor (B2-AR) in AML. B2-AR is a G-protein- coupled catecholamine receptor and was recently found to play a direct role in the engraftment, proliferation and migration of hematopoietic stem cells [Spiegel, A., et al. Nat Immunol8, 1123–1131 (2007)]. However, very little is known about the roles of B2-AR in AML. We first screened a number of leukemia/hematopoietic cell lines, including K562, NBAL3, Jurkat, RPMI8226, U937, HEL, HL60, NB4, THP-1, and MV4;11, for B2-AR expression using flow cytometry. We found that B2-AR expression was not uniform in the leukemia/hematopoietic cell lines we tested. B2-AR was absent in acute non-myelogenous leukemia/hematopoietic cell lines such as K562, NBAL3, Jurkat or RPMI8226. In contrast, B2-AR was expressed on all acute myelogenous leukemia cell lines tested, with the highest expression on 2 myelomonocytic leukemia cell lines (THP-1 and MV4;11). We next examined the roles of B2-AR in leukemia cell survival, in leukemia cell responses to chemotherapy, and in leukemia cell migration in response to stromal derived factor-1 (SDF-1). We found that treatment with a B2-AR antagonist (ICI115881) modestly inhibited leukemia cell growth. Interestingly, treatment with a B2-AR agonist (i.e., isoproteronol or clenbuterol) inhibited leukemia cell migration to SDF-1. Additionally, combined treatment of MV4-11 cells with Isoproterenol and SDF-1 increased downstream ERK phosphorylation synergistically, suggesting a potential interaction or reciprocal regulation between B2-AR and CXCR4 receptor. To further understand the regulation and functional role of B2-AR in AML cells, we have been focusing on two biphenotypic leukemia cell lines, that is, MV4;11 and RS4;11 cells. Both MV4;11 and RS4;11 cells carry the t(4;11)(q21;q23) chromosomal translocation. While RS4;11 cells bear only the wild-type version of the Flt3 gene, MV4;11 cells express exclusively the mutated Flt3-ITD gene. Interestingly, we found that in contrast to MV4;11 leukemia cells, RS4;11 cells did not express surface B2-AR (panel A), although the total amount of B2-AR in the whole cell lysate was comparable between these two cell lines (panel B). This significantly different B2-AR expression pattern between these 2 cell lines may be related to the difference in the expression level of proviral insertion in murine lymphoma (Pim) kinases (panel B); RS4;11 has reduced/absent expression of Pim kinases compared to MV4;11 cells. Consistent with the potential regulation of B2-AR expression by Pim kinases, we found that Pim 2 and 3 double knockout mice had reduced B2-AR surface expression in peripheral blood mononucleated cells. Additional experiments are currently ongoing to further dissect the interaction between B2-AR and Pim kinases. Taken together, our current studies demonstrated a novel role of B2-AR in AML and a potential functional interaction between B2-AR, CXCR4, Pim kinase, and Flt3 gene. This work is supported by MUSC Hollings Cancer Center Startup Fund, Hollings Cancer Center ACS IRG, and ASCO Conquer Cancer Foundation Career Development Award Disclosures: No relevant conflicts of interest to declare.

Elevated Serum IL-10 Levels in Patients with Diffuse Large B Cell Lymphoma: A Mechanism of Aberrant JAK2 Kinase Activation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 960-960
Author(s):  
Mamta Gupta ◽  
Mary Stenson ◽  
Jing Jing Han ◽  
Matthew J Maurer ◽  
Linda Wellik ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Elevated Serum ◽  
B Cell Lymphoma ◽  
Cytokine Signaling ◽  
Dependent Manner ◽  
Kinase Activation ◽  
Stat3 Phosphorylation ◽  
Dose Dependent

Abstract Abstract 960 Diffuse large B cell lymphoma (DLBCL) is an aggressive lymphoma whose survival depends on various signaling pathways one of which is Signal transducer and activator of transcription 3 (STAT3)/Janus kinase 2 (JAK2). We have demonstrated that JAK2 is constitutively activated at the auto-phosphorylation site in many cases of DLBCL. The most common mechanism causing abnormal JAK2 activation is through dysregulated cytokine signaling. Cytokines are deregulated in several hematological malignancies and may play a role in tumor cell growth through receptor-mediated and ligand dependent activation of the JAK2 kinase. In this study we investigated the role of cytokine signaling in JAK2 activation in DLBCL and use of a novel JAK2 inhibitor (JAK2i) to inhibit IL-10 induced JAK/STAT signaling. We studied serum cytokines from 70 patients with new untreated DLBCL who participated in a clinical trial in the North Central Cancer Treatment Group (Micallef, IN et al Blood 2011). Compared to control sera, patients with DLBCL had higher levels of several JAK2 kinase related cytokines (IL-2, IL-6, IL-10, and EGF). IL-10 and IL-6 were significantly higher in DLBCL patients vs controls with p values >0.03 and >0.001, respectively. We next examined whether DLBCL cell lines produced IL-10. The phospho-JAK2 (pJAK2) positive DLBCL cell lines Ly10, DHL2 and HBL1 produced more IL-10 (40–80 pg/ml) than the pJAK2 negative cell lines Ly1, Ly19 and DHL6 (0–5 pg/ml). Analysis of the surface expression of IL-10 receptors (IL-10R) determined that most p-JAK2 positive DLBCL cells express either IL-10Ra or IL-10Rb or both. IL-10 had no effect on DLBCL cell survival in vitro; however, it did promote their proliferation. Only IL-10 (but not the other elevated cytokines) was specifically able to activate JAK2 and STAT3 phosphorylation in a subgroup of DLBCL cell lines. IL-10 was not able to activate JAK1, STAT1 and STAT5, which suggests a specific role of IL-10 in the JAK2 pathway. Moreover, neutralizing antibody to IL-10 inhibited IL-10-induced JAK2 and STAT3 tyrosine phosphorylation. We studied the effect of SAR302503 (SAR503, Sanofi, Cambridge, MA), a selective JAK2i currently in clinical trial for myelofibrosis on constitutive JAK2 signaling in DLBCL cells. SAR503 was able to inhibit JAK2 and STAT3 phosphorylation in a dose and time dependent manner in a variety of DLBCL cell lines and patient samples. JAK2 inhibition with SAR2503 caused a dose-dependent inhibitory effect on survival of pJAK2 positive DLBCL cells not observed in pJAK2 negative cells. Activation of STAT3 signaling up-regulates the expression of various genes involved in cell survival and proliferation such as bcl-xl, bcl-2, mcl-1 and c-myc. We observed a dose-dependent decrease in c-myc protein and mRNA levels in Ly3 and DHL2 cells with SAR503 treatment; however, SAR503 did not effect expression of bcl2, mcl-1 and bcl-xl proteins in DLBCL cells. Interestingly, JAK2 inhibition with SAR503 inhibited the autocrine secretion of IL-10 in a dose-dependent manner. Next, we determined if higher pre-treatment serum IL-10 correlates with the overall survival of 81 DLBCL patients. IL-10 by ELISA demonstrated that IL-10 levels were high in 51% (41/81) of patients (median, 57.7pg/ml; range, 26.1–503.7), and low in 49% (40/81) (median, 15.9 pg/ml; range, 0–25.9). Using a cut-off value of 26.1pg/ml, patients with a high serum IL-10 level had a significantly worse event-free survival (p=0.01). Clinical correlation of serum IL-10 with disease parameters showed that the IL-10 level was correlated with elevated serum LDH (p= 0.0085) and IPI score (p=0.01); there was no correlation with the number of extranodal sites, age, or B symptoms. DLBCL tumors from 40 patients were classified into germinal center B-cell type (GCB) and non-GCB type using the Hans algorithm. There was a clear trend towards a higher serum IL-10 in non-GCB DLBCL patients (p <0.06). In summary, our data identifies mechanisms of JAK2 kinase activation in DLBCL and supports the hypothesis that the IL-10-JAK2-cmyc pathway is activated in patients with DLBCL. Finally our data provide a mechanistic basis of selecting and targeting DLBCL tumor cells with high IL-10 levels and/or constitutive JAK2 activity with potent and novel JAK2 inhibitors such as SAR503. Disclosures: No relevant conflicts of interest to declare.

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