MO7e Cells Research Articles

Antiplasmodial action of 4-nitrobenzenesulfonamide chalcones: Design, synthesis, characterisation, in vitro and in silico evaluation against blood stages of Plasmodium falciparum 3D7.

Malaria is an intracellular protozoan parasitic disease caused by Plasmodium species with significant morbidity and mortality in endemic regions. The complex lifecycle of the parasite and the emergence of drug-resistant Plasmodium falciparum have hampered the efficacy of current anti-malarial agents. To circumvent this situation, the present study attempts to demonstrate the blood-stage anti-plasmodial action of 26 hybrid compounds containing the three privileged bioactive scaffolds (sulfonamide, chalcone, and nitro group) with synergistic and multitarget action. These three parent scaffolds exhibit divergent activities, such as antibacterial, anti-malarial, anti-fungal, anti-inflammatory, and anticancer. All the synthesised compounds were characterised using various spectroscopic techniques. The in vitro blood-stage inhibitory activity of 26 hybrid compounds was evaluated against mixed-stage culture (asynchronize) of human malarial parasite P. falciparum, Pf 3D7 at different concentrations ranging from 25.0 µg/mL to 0.78 µg/mL using SYBR 1 green assay, with IC50 values determined after 48 h of treatment based on the drug-response curves. Two potent compounds (11 and 10), with 2-Br and 2,6-diCl substitutions, showed pronounced activity with IC50 values of 5.4 µg/mL and 5.6 µg/mL, whereas others displayed varied activity with IC50 values ranging from 7.0 µg/mL to 22.0 µg/mL. Both 11 and 10 showed greater susceptibility towards mature-stage trophozoites than ring-stage parasites. The hemolytic and in vitro cytotoxicity assays revealed that compounds 11 and 10 did not cause any toxic effects on host red blood cells (uninfected), human-derived Mo7e cells, and murine-derived BA/F3 cells. The in vitro observations are consistent with the in silico studies using P. falciparum-dihydrofolate reductase, where 11 and 10 showed a binding affinity of -10.4 Kcal/mol. This is the first report of the hybrid scaffold, 4-nitrobenzenesulfonamide chalcones, demonstrating its potential as an anti-plasmodial agent.

Design and fabrication of dysprosium impregnated polyvinyl alcohol hydrogels. Physiochemical, mechanical, bioimaging and in vitro evaluation

Tissue engineering has gained prominence during the past decade since it offers a key solution to defects associated with the tissue regeneration. The limited healing potential of the cartilage tissue damage has significant clinical implications. Herein, dysprosium (Dy3+) impregnated polyvinyl alcohol (PVA) hydrogels have been developed to enhance the therapeutic efficacy, enabling simultaneous diagnostic imaging and antibacterial drug delivery for potential applications in articular cartilage. Based on the favorable imaging features, Dy3+ impregnated PVA hydrogels with enhanced stability were formed through successive steps of repeated cycles of freezing at – 30 °C for 21 h, thawing at 25 °C for 4 h and lyophilization. The tensile and compression tests of the hydrogels respectively determined a maximum of 3.88 and 1.58 MPa, which reflected better compatibility towards cartilage. The hydrogels fetched a sustained drug release for a period of 12 h with an associated swelling ratio of 80%. The potential of the resultant hydrogels in image diagnosis has been deliberated through their blue and yellow emissions in the visible region. Further, the computed tomography (CT) and magnetic resonance imaging characteristics of the hydrogels respectively accomplished a maximum of 343 Hounsfiled units (HU) and relaxivity of 7.25 mM−1s−1. The cytocompatibility of the hydrogels is also determined through in vitro tests performed in Murine pro B cell line (BA/F3) and human Megakaryocyte cell line (Mo7e) cell lines.

In VitroandIn VivoInteractions between Stromal and Leukemic Cells

Understanding the mechanisms underlying changes in the microenvironment caused by pathological cells is essential to develop new therapeutic strategies. Such modifications allow leukemia cells to modify their microenvironment in order to promote their growth. Here, we studiedin vivoandin vitrointeractions between Primary Myelofibrosis and the microenvironment. This clonal malignant disorder is caused by three major driver mutations: JAK2V617F, MPLW515L and CALR exon 9 mutations. We performed 72h co-cultures of the mesenchymal stromal cell line MS5 and the MO7e human hematopoietic cell line transduced with either the MPLW515L mutation (MO7eMPLW515L) or GFP as a control. As Mo7e cells are dependent on the human growth factor h-GM-CSF, co-cultures were performed with and without it Flow cytometry confirmed that leukemic cells expressing the MPLW515L mutation proliferated actively in the absence of hGM-CSF, unlike the control MO7e GFP cells. In addition, the presence of MS5 stromal cells did not have a major impact on the proliferation of MO7eMPLW515L but promoted MO7e GFP cell survival. Thus the MPLW515L mutation activates signaling pathways that make the cells independent of cytokines and stroma. On the other hand, the number of stromal cells increased upon co-culture with either MO7e GFP or MO7e W515L and in the presence of h-GM-CSF. Thus, either growth factor addition or the MPLW515L mutation allows MO7e cells to enhance the proliferation of stromal cells. To test our observations in primary cells, we also performed 72h co-cultures using isolated murine cKIT+ hematopoietic progenitor cells from JAK2V617F knock-in or wild type mice. The impact of stromal cells was evaluated in the presence or absence of thrombopoietin (TPO). Cells were analyzed by flow cytometry. The results showed that MS5 stromal cells induced proliferation of both primary wild type and JAK2V617F cKIT+ hematopoietic cells independently of TPO. Moreover, the proliferation of MS5 stromal cells was also significantly induced by both wild type and JAK2617F cKIT + primary cells. In vivointeractions were studied using transgenic mice expressing CXCL12HT-dsRED crossed with JAK2V617F mice (CXCL2-HT-JAK2V617F). We followed different parameters, namely the weight and the size of the spleen, the different blood parameters, the number of hematopoietic progenitors in bone marrow, spleen and blood, and the cellularity and the number of fibroblast colonies (CFU-F). Results showed that CXCL12 HT JAK2KI mice developed significant splenomegaly, associated with changes in blood parameters characteristic of JAK2KI mice, i.e. increases in white blood cells, red blood cells, hematocrit, and hemoglobin. There was also an increase in blood, spleenand bone marrow progenitors compared to the CXCL12HT GFP control mice. The number of fibroblast colonies (CFU-F), reflecting the activity of mesenchymal stem cells (MSC) in the microenvironment, was also greatly increased, especially in the marrow of CXCL12HT JAK2KI mice, indicating that the malignant clone modified the microenvironment of the mice. Taken together, our findings show that hematopoietic cells, either normal or leukemic, can modify their own microenvironmentin vitroandin vivo. This offers a new avenue for therapeutic targeting in the treatment of this type of hemopathologies. Disclosures No relevant conflicts of interest to declare.

The Pediatric Acute Leukemia Fusion Oncogene ETO2‐GLIS2 Increases Self‐Renewal and Alters Differentiation in a Human Induced Pluripotent Stem Cells‐Derived Model

Supplemental Digital Content is available in the text.

Pseudomonas aeruginosa infection stimulates mitogen-activated protein kinases signaling pathway in human megakaryocytes.

Pseudomonas aeruginosa is a major cause of nosocomial infections and contributes to higher mortality in hospitalized individuals. Infection by P. aeruginosa triggers host immune response through activation of pathogen recognition receptors, which are present in innate cells. Several studies have reported the mechanism of P. aeruginosa induced innate immunity in multiple cell types. But so far there is no reports on response of megakaryocytes to P. aeruginosa infection. Hence, our aim was to investigate the precise role and signaling mechanism of megakaryocytes during P. aeruginosa infection. In this study, we used Mo7e cells as representatives of human megakaryocyte and found that P. aeruginosa infection induces cytotoxicity in these cells. We further demonstrated that P. aeruginosa infection modulates p38 and extracellular signal regulated kinase pathways in Mo7e cells. Protein expression profiling in P. aeruginosa lipopolysaccharide-treated Mo7e cells revealed upregulation of importin subunit β and downregulation of metabolic enzymes. Our results suggest that P. aeruginosa infection regulates mitogen-activated protein kinases signaling pathway and importin in Mo7e cells and that this is a potential mechanism for nuclear translocation of nuclear factor binding near the κ light-chain gene in B cells and c-Jun N-terminal kinases to induce cell cytotoxicity.

S888 TNF‐ALPHA PROMOTES CLONAL EXPANSION OF KIT D816V+ CELLS IN SYSTEMIC MASTOCYTOSIS

Background:Systemic mastocytosis (SM) is characterized by clonal mast cell (MC) infiltration in the bone marrow (BM). Most patients harbor the activating KIT mutation D816V. Cytokines are substantially involved in the interactions between neoplastic cells and the BM microenvironment in SM and in classical myeloproliferative neoplasms (MPN) and have been recognized as a major trigger of disease evolution. The pro‐apoptotic/anti‐proliferative and pro‐inflammatory cytokine tumor necrosis factor alpha (TNFa) has been identified as an important driver for the expansion of JAK2 V617F+ clones and the establishment of MPN.Aims:Investigation of the potential role of TNFa in disease evolution of SM patients.Methods:In vitro and in vivo experiments with MC lines and primary material of SM patients.Results:In a first step, lentivirally‐driven expression of the KIT D816V mutant gene in the human Mo7e cells suggested an oncogene‐dependent regulation of the production of TNFa. Correspondingly, the human neoplastic KIT D816V+ MC lines HMC‐1.2 and ROSAKIT D816V expressed and secreted substantial amounts of TNFa and inhibition of KIT D816V either by the recently approved multi‐kinase inhibitor midostaurin or by RNAi‐mediated knockdown of KIT significantly reduced expression of this cytokine. Mechanistically, we studied auto‐/paracrine effects of TNFa on the clonal selection of neoplastic MC using KIT D816V negative (ROSAKIT WT) and positive (ROSAKIT D816V) subclones of ROSA cells. TNFa reduced proliferation of ROSAKIT WT cells (84.9% ± 4.6% of control, p<0.01) whereas ROSAKIT D816V cells were resistant to TNFa (102.7% ± 3.7% of control, n.s.). Preliminary data also indicate a relative resistance of primary neoplastic cells obtained from KIT D816V+ SM patients against TNFa in colony‐forming unit (CFU) assay. To determine the mechanism of TNFa resistance in KIT D816V+ cells, we screened for key regulators of apoptosis and identified the anti‐apoptotic protein survivin to be highly upregulated in KIT D816V+ ROSA cells upon TNFa treatment but not in KIT D816V‐ ROSA cells. We hypothesize that KIT D816V+ MC produce substantial amounts of TNFa that induces apoptosis and suppresses proliferation of normal BM cells while MC themselves are relatively resistant via upregulation of survivin. In a next step, we studied the effect of TNFa on growth of neoplastic MC in vivo in a xenotransplant model using ROSAKIT D816V cells with and without knockout of TNFa in NOD‐SCID IL‐2Rγ‐null (NSG) mice. CRISPR/Cas9‐mediated knockout completely abolished expression and secretion of TNFa in ROSAKIT D816V cells and resulted in a significantly longer survival of mice compared to mice injected with TNFa expressing controls (median 49 vs. 58 days, p < 0.05). Finally, the clinical relevance of TNFa expression was studied in a SM patient's cohort (n = 45). TNFa levels in sera of patients were significantly higher (median 1.95, range 0.72–16.64 pg/ml, p = 0.002) compared to age‐ and sex‐matched controls (median 1.49, range 0.51–5.63 pg/ml). Importantly, when we stratified SM patients into those with clearly elevated TNFa levels and those with TNFa levels within the reference range of controls (≤3.63 pg/ml) a significantly lower overall survival was observed for SM patients with high TNFa serum levels (median 6.3 years vs. not reached, p < 0.005).Summary/Conclusion:In summary, we have identified TNFa as a critical, KIT D816V‐dependent, cytokine‐mediator that promotes clonal expansion of KIT D816V+ MC and may thereby be involved in clonal evolution and disease progression in SM.

Stem cell factor and NSC87877 combine to enhance c-Kit mediated proliferation of human megakaryoblastic cells.

Enhancement of hematopoietic stem cells (HSCs) proliferation is a central aim in bone marrow transplantation (BMT). A stem cell factor (SCF) and c-Kit mediated extracellular signaling trigger proliferation of HSCs. This signaling is negatively regulated by protein tyrosine phosphatases (PTPs), SHP-1 and SHP-2. Although NSC87877 (N) is known to inhibit SHP-1/SHP-2, c-Kit-mediated HSCs proliferation by inhibiting SHP-1/SHP-2 has not been reported. This study investigated the combined effect of SCF (S) and N in c-Kit mediated proliferation and underlying mechanisms. The growth of human megakaryoblastic cell line, MO7e and HSCs, upon treatment with S and N alone, and in combination was assessed by PrestoBlue staining. The expression of c-Kit, phosphorylated c-Kit, SHP-1/SHP-2 and HePTP inhibition using S and N treatment were evaluated in the MO7e cells. Megakaryoblast cell proliferation was determined by quantification of Ki-67+, S-phase, BrdU+ and CFDA-SE+ cells using flow cytometry. The combination of S and N leads to enhanced cell growth compared with either S or N alone. Collectively, the results reveal a novel mechanism by which S in combination with N significantly enhances proliferation of human megakaryoblast cells. The pretreatment of N before S enhances proliferation of cells than S alone. This promising combination would likely play an essential role in enhancing the proliferation of cells.

Enhancement of antibody-dependent cellular cytotoxicity of cetuximab by a chimeric protein encompassing interleukin-15

ABSTRACTEnhancement of antibody-dependent cellular cytotoxicity (ADCC) may potentiate the antitumor efficacy of tumor-targeted monoclonal antibodies. Increasing the numbers and antitumor activity of NK cells is a promising strategy to maximize the ADCC of standard-of-care tumor-targeted antibodies. For this purpose, we have preclinically tested a recombinant chimeric protein encompassing the sushi domain of the IL15Rα, IL-15, and apolipoprotein A-I (Sushi-IL15-Apo) as produced in CHO cells. The size-exclusion purified monomeric fraction of this chimeric protein was stable and retained the IL-15 and the sushi domain bioactivity as measured by CTLL-2 and Mo-7e cell proliferation and STAT5 phosphorylation in freshly isolated human NK and CD8+ T cells. On cell cultures, Sushi-IL15-Apo increases NK cell proliferation and survival as well as spontaneous and antibody-mediated cytotoxicity. Scavenger receptor class B type I (SR-B1) is the receptor for ApoA-I and is expressed on the surface of tumor cells. SR-B1 can adsorb the chimeric protein on tumor cells and can transpresent IL-15 to NK and CD8+ T cells. A transient NK-humanized murine model was developed to test the increase of ADCC attained by the chimeric protein in vivo. The EGFR+ human colon cancer cell line HT-29 was intraperitoneally inoculated in immune-deficient Rag2−/−γc−/− mice that were reconstituted with freshly isolated PBMCs and treated with the anti-EGFR mAb cetuximab. The combination of the Sushi-IL15-Apo protein and cetuximab reduced the number of remaining tumor cells in the peritoneal cavity and delayed tumor engraftment in the peritoneum. Furthermore, Sushi-IL15-Apo increased the anti-tumor effect of a murine anti-EGFR mAb in Rag1−/− mice bearing subcutaneous MC38 colon cancer transfected to express EGFR. Thus, Sushi-IL15-Apo is a potent tool to increase the number and the activation of NK cells to promote the ADCC activity of antibodies targeting tumor antigens.

Anti-proliferative effects of T cells expressing a ligand-based chimeric antigen receptor against CD116 on CD34(+) cells of juvenile myelomonocytic leukemia.

BackgroundJuvenile myelomonocytic leukemia (JMML) is a fatal, myelodysplastic/myeloproliferative neoplasm of early childhood. Patients with JMML have mutually exclusive genetic abnormalities in granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR, CD116) signaling pathway. Allogeneic hematopoietic stem cell transplantation is currently the only curative treatment option for JMML; however, disease recurrence is a major cause of treatment failure. We investigated adoptive immunotherapy using GMR-targeted chimeric antigen receptor (CAR) for JMML.MethodsWe constructed a novel CAR capable of binding to GMR via its ligand, GM-CSF, and generated piggyBac transposon-based GMR CAR-modified T cells from three healthy donors and two patients with JMML. We further evaluated the anti-proliferative potential of GMR CAR T cells on leukemic CD34+ cells from six patients with JMML (two NRAS mutations, three PTPN11 mutations, and one monosomy 7), and normal CD34+ cells.ResultsGMR CAR T cells from healthy donors suppressed the cytokine-dependent growth of MO7e cells, but not the growth of K562 and Daudi cells. Co-culture of healthy GMR CAR T cells with CD34+ cells of five patients with JMML at effector to target ratios of 1:1 and 1:4 for 2 days significantly decreased total colony growth, regardless of genetic abnormality. Furthermore, GMR CAR T cells from a non-transplanted patient and a transplanted patient inhibited the proliferation of respective JMML CD34+ cells at onset to a degree comparable to healthy GMR CAR T cells. Seven-day co-culture of GMR CAR T cells resulted in a marked suppression of JMML CD34+ cell proliferation, particularly CD34+CD38− cell proliferation stimulated with stem cell factor and thrombopoietin on AGM-S3 cells. Meanwhile, GMR CAR T cells exerted no effects on normal CD34+ cell colony growth.ConclusionsLigand-based GMR CAR T cells may have anti-proliferative effects on stem and progenitor cells in JMML.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0256-3) contains supplementary material, which is available to authorized users.

Dragon's blood extracts reduce radiation-induced peripheral blood injury and protects human megakaryocyte cells from GM-CSF withdraw-induced apoptosis

PurposeDragon's blood (DB), a Chinese traditional herb, was shown to have certain protective effects on radiation-induced bone marrow injury due to the presence of several phenolic compounds. The 50% ethanol extracts (DBE) were separated from DB by the methods of alcohol extracting-water precipitating. The protective effects of DBE on hematopoiesis were studied, particularly on megakaryocytes. Materials and methodsIn this study, we investigated the in vivo radioprotective effects of DBE on hematopoiesis and pathological changes using an irradiated-mouse model. Moreover, the protective effects and potential molecular mechanisms of DBE on megakaryocytopoiesis in vitro were explored in GM-CSF depletion-induced Mo7e cell model. ResultsDBE significantly promoted the recovery of peripheral blood cells in irradiated mice. Histology bone marrow confirmed the protective effect of DBE, as shown by an increased number of hematopoietic cells and a reduction of apoptosis. In a megakaryocytic apoptotic model, DBE (50 µg/mL) markedly alleviated GM-CSF withdrawal-induced apoptosis and cell-cycle arrest of Mo7e cells. DBE (50 µg/mL) also significantly decreased the ratio of Bax to Bcl-2 expression, inhibited the active caspase-3 expression. In addition, DBE could induce ERK1/2 phosphorylation in GM-CSF-depleted Mo7e cell, but not Akt. ConclusionsOur data demonstrated that DBE could effectively accelerate the recovery of peripheral blood cells, especially platelet. DBE attenuated cell apoptosis and cell cycle arrest through the decrease of Bax/Bcl-2 ratio and the reduction of active caspase-3 expression. The effect of DBE on Mo7e cells survival and proliferation is likely associated with the activation of ERK, but not Akt.

Abstract 2690: DCC-2618 is a potent inhibitor of wild-type and mutant KIT, including refractory Exon 17 D816 KIT mutations, and exhibits efficacy in refractory GIST and AML xenograft models

Abstract Introduction: KIT kinase mutations are causative of a number of human cancers, including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), mast cell leukemia (MCL), and subtypes of melanoma and acute myeloid leukemia (AML). DCC-2618 is a robust Type II switch pocket control inhibitor which potently inhibits exon 17 KIT mutations that are resistant to conventional TKIs. Experimental procedures: DCC-2618 was tested for inhibition of KIT isoforms using a standard PK/LDH coupled spectrophotometric assay. CHO cells were transiently transfected to express mutant KIT or PDGFRα constructs. Transfected cells were treated with a range of DCC-2618 and levels of phosphorylated KIT or PDGFRα in cell lysates were determined by ELISA or western blot. Cell proliferation of several cell lines was measured using the fluorescent dye resazurin. Experiments were performed in triplicate. In vivo xenograft models were performed at Molecular Imaging, Inc. (Ann Arbor, MI) or Molecular Response, LLC (San Diego, CA). Summary of results: DCC-2618 inhibited various forms of KIT with nanomolar potency: WT (IC50 4 nM), V654A (8 nM), T670I (18 nM), D816H (5 nM), D816V (14 nM). In CHO cells transiently transfected with both single and double (primary/secondary) KIT mutants, DCC-2618 robustly inhibited exon 17, exon 9/13, exon 9/14, and exon 9/17 KIT mutants, as well as exon 11/17 KIT mutants, including exon 17 D816V, D816G, D820A, D820E, D820Y, N822K, N822Y, N822H, and Y823D primary or secondary mutations. DCC-2618 inhibited wild type KIT phosphorylation in the MO7e cell line (IC50 36 nM). DCC-2618 potently inhibited KIT activation in human GIST cell lines, including GIST T1 (exon 11 deletion, IC50 2 nM), GIST 430 (exon 11 deletion/exon 13 V654A, IC50 7 nM), and GIST 48 (exon 11 V560D/exon 17 D820A, IC50 53 nM). In the murine mastocytosis P815 cell line expressing the exon 17 D816Y mutation, DCC-2618 potently inhibited cell proliferation (IC50 2 nM). In vivo, DCC-2618 administration at 50 mg/kg afforded an ED90 for inhibition of KIT phosphorylation in the GIST T1 xenograft model, corresponding to an EC90 concentration of ∼ 470 ng/mL. When give twice daily, this oral dose resulted in almost complete tumor stasis. This dose of DCC-2618 produced tumor regressions in a patient derived xenograft (PDX) GIST expressing KIT exon 11 delW557K558/exon 17 Y823D, and also in a KIT exon 17 N822K AML xenograft model. Conclusion: DCC-2618 is a potent inhibitor of singly and doubly mutated KIT characterized by primary exon 9 or exon 11 mutations paired with secondary mutations in exons 13, 14 or 17. DCC-2618 inhibits exon 17 mutations, including the D816V mutation refractory to currently marketed KIT inhibitors. DCC-2618 has the potential to treat KIT mutant-driven cancers including GIST, systemic mastocytosis, AML, or melanoma. DCC-2618 has been selected for formal IND-enabling clinical development. Citation Format: Bryan D. Smith, Molly M. Hood, Scott C. Wise, Michael D. Kaufman, Wei-Ping Lu, Thomas Rutkoski, Daniel L. Flynn, Michael C. Heinrich. DCC-2618 is a potent inhibitor of wild-type and mutant KIT, including refractory Exon 17 D816 KIT mutations, and exhibits efficacy in refractory GIST and AML xenograft models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2690. doi:10.1158/1538-7445.AM2015-2690

Effects of Resveratrol and Vitamin C on the Self-Renewal Capacity of Blast Progenitors in Hematologic Malignancies

Although a large number of phytochemicals has been shown to inhibit the growth of leukemic and cancer cells, their effects on the self-renewal (SR) capacity of the malignant stem/progenitor cells were not examined. In seven hematologic malignant cell lines including U-937, Mo7e, K-562, HL-60, U-266, Raji and Daudi, the SR capacity of blast progenitors was assessed by measuring cumulative clonogenic cells in suspension, whereas the terminal division of blast progenitors was assessed by blast colony formation (BCF) in methylcellulose. Resveratrol and vitamin C (Vit-C) with mean IC50 values of 41 and 514 μM for inhibiting BCF were added twice per week to cells in long-term suspension at 10 and 300 μM, respectively. Vit-C induced slight increase in senescence or necrosis rates, marked G2/M arrest and significant inhibition of telomerase activity in two, one and five cell lines, respectively. Resveratrol, but not Vit-C, induced cell cycle arrest and/or cellular stress responses such as senescence, apoptosis, and necrosis and inhibited SR in all seven cell lines. Resveratrol coordinately induced S-phase arrest and cellular stress responses, inhibited telomerase activity and abolished SR in U-937, Mo7e and K-562 cells. Resveratrol induced marked increase in p21 and/or p27 transcript levels and senescence rates in U-937, K-562 and U-266 cells. Taken together, resveratrol has been shown to exert multifactorial actions and inhibit SR, whereas Vit-C by inhibition of telomerase activity alone did not affect SR.

Mild Hyperthermia Treatment Of CD34+ Human Cord Blood Cells Increases Lipid Raft Aggregation Resulting In An Earlier Engraftment In NSG Mice Following Transplantation

Umbilical cord blood (UCB) is a viable source of hematopoietic stem cells (HSC) for transplantation to treat patients with malignant and non-malignant disease. Although the limitation of low stem cell dose in single UCB transplantation has been improved upon by the use of double UCB units, this has not resulted in a shortened time to engraftment. Therefore efforts are needed to enhance the speed of engraftment of these cells. Mild heating (39.5°C) of mature hematopoietic cells increases membrane fluidity, resulting in increased lipid raft aggregation, and priming cells for increased sensitization of responsiveness to cytokine signaling. We hypothesized that exposing CD34+ UCB cells to 39.5°C would enhance their capacity to respond to SDF-1/CXCL12, a chemokine secreted in the bone marrow microenvironment that strongly attracts HSCs and progenitor cells, by increasing lipid raft aggregation and CXCR4 expression, thus leading to better engraftment following transplantation. To determine if this mild heating had any effect on engraftment in vivo, NSG mice received 4Gy TBI followed one day later with 20,000 or 80,000 CD34+ UCB cells incubated for 4 hours at 37°C or 39.5°C prior to i.v. injection. Percent chimerism in the blood was determined through the detection of human CD45 by flow cytometry at 1, 2, 4 and 6 months following transplantation. Mild heat treatment significantly increased the percent chimerism following transplantation by 1.7 fold at 1 month (when 80,000 cells were used) and by 1.8 fold at 2 months (when 20,000 cells were used), without significant increases in percent chimerism at 4 or 6 months. This suggests that the effects of heat treatment are most likely occurring early following transplantation, potentially due to an increase in homing of the heated CD34+ UCB cells to the bone marrow microenvironment. To begin to test this hypothesis, CD34+ UCB and Mo7e cells (a human megakaryoblastic leukemia cell line) were exposed to 39.5°C for 4 hours followed by a chemotaxis assay. Exposure of the CD34+ UCB cells to 39.5°C manifested in a significant increase in the percent of cells that migrated towards 12.5 (1.8 fold increase), 25 (1.9 fold increase), 50 (2.2 fold increase), 100 (2.3 fold increase) and 200 ng/mL SDF-1 (2.5 fold increase). Mild heating also significantly increased the percent of Mo7e cells that migrated towards 12.5 (1.8 fold increase), 25 (1.4 fold increase), 50 (1.5 fold increase), 100 (1.3 fold increase), and 200 ng/mL SDF-1 (1.5 fold increase). Increases in chemotaxis towards SDF-1 following mild heating was associated with a significant, but modest, increase (1.3 fold as determined by MFI) in expression of the SDF-1 receptor, CXCR4, on the surface of CD34+ UCB cells as detected by flow cytometry. In order to optimize SDF-1 signaling, CXCR4 must be incorporated into the cholesterol-enriched lipid rafts of the cellular membrane followed by lipid raft aggregation. Mild heating increased the percent of Mo7e cells with aggregated lipid rafts from 13 percent for cells incubated for 4 hours at 37°C to 52% for cells incubated for 4 hours at 39.5C. By using a dose of MbCD (1.25mM), an agent that can deplete cholesterol in the cellular membrane thus blocking lipid raft aggregation, it was determined that the enhancement in chemotaxis seen following mild heating was dependent upon lipid raft aggregation in both CD34+ UCB and Mo7e cells. Overall, mild heating may be a simple and inexpensive novel adjuvant therapy to enhance engraftment following UCB transplantation in patients. Disclosures: No relevant conflicts of interest to declare.

Preclinical Characterization of KB003, a Novel Humaneered™ Monoclonal Anti-GM-CSF Antibody, Demonstrates That the GM-CSF Signaling Axis Is a Therapeutic Target in Chronic Myelomonocytic Leukemia (CMML)

AbstractAbstract 3793 Introduction:Chronic Myelomonocytic Leukemia (CMML) is a genetically diverse hematologic malignancy characterized by cytopenias with or without leukocytosis, marrow dysplasia, monocytosis, splenomegaly, and a propensity to transform to Acute Myeloid Leukemia (AML). It is among the most aggressive chronic myeloid malignancies with a three year overall survival approximating twenty percent. The current treatment armamentarium is generally ineffective and mostly derived from studies that focused on Myelodysplastic Syndromes (MDS). As has been well defined in Juvenile Myelomonocytic Leukemia (JMML), we previously reported that primary CMML bone marrow mononuclear cells exhibit GM-CSF-dependent pSTAT5 hypersensitivity. To determine whether this signaling pathway is a viable therapeutic target, we present the preclinical characterization of KB003 activity on primary CMML bone marrow mononuclear cells. Methods:KB003 was engineered and provided by KaloBios Pharmaceuticals. The ability of KB003 to neutralize GM-CSF was tested in two ways using doses that ranged from .01 to 10,000 ng/ml of KB003. First, 10 ng/ml of GM-CSF was used to induce IL-8 production by the U937 cell line as measured by ELISA. Second, the GM-CSF-dependent cell line, MO7e, was cultured in the absence and in the presence of 0.1, 1, and 10 ng/ml of GM-CSF with or without KB003. Apoptosis and viability was assessed by annexin and 4′,6-diamidino-2-phenylindole (DAPI) staining by flow cytometry. To determine anti-GM-CSF sensitivity in CMML, primary cryoperserved bone marrow samples from 10 patients were reconstituted in prewarmed StemSpan H3000 with 10% FBS and microcultured in a 96 well plate with increasing doses of GM-CSF and KB003. After a 48-hour incubation period, cells were stained with myeloid-specific antibodies (CD34, CD38, CD14, CD33) and measured by multi-color flow cytometry. DAPI was used to measure cell viability. MethoCult hematopoietic progenitor assays were used to quantify the expansion and differentiation of CMML mononuclear cells in the presence of GM-CSF and escalating concentrations of KB003 in those samples with sufficient cell number for analysis (n=7). Results:KB003 effectively neutralized GM-CSF-induced IL-8 secretion in U937 cells with an IC50 of 48.2 ng/ml, as shown in Figure 1a. A dose of 0.1, 1, and 10 ng/ml of GM-CSF was used to protect MO7e cells from apoptosis and a survival benefit was achieved at each dose tested (Fig 1b). In this assay, KB003 neutralized 0.1 ng/ml of GM-CSF, but was unable to neutralize GM-CSF at higher doses (10 ng/ml). Using CMML samples, doses ranging from 10 to 100 μg/ml of KB003 were tested in apoptosis and in colony formation assays. Using the gating strategy shown in Figure 2a, the percentage of mature (CD33+/CD14+) and immature (CD33+/CD14-) myeloid subpopulations within the viable gate after KB003 treatment was decreased (Figure 2b). As expected, KB003 had no effect on CD3+ T-cells in the mixed culture populations. In addition to myeloid subpopulations identified by CD14, the CD33+/CD38+ cells were also more sensitive than CD33+/CD38- or CD38+/CD34+ inhibition by KB003 (Figure 2c, p<0.05). As shown in Figure 2d, hematopoetic colony formation assays confirmed the viability results. Using a one way ANOVA, there was a dose dependent decrease in the number of hematopoietic colonies, coupled with less organized and more diffuse appearance of colonies after KB003 treatment (Figure 2d). Conclusion:GM-CSF neutralization using KB003 suppresses CMML progenitor survival in vitro. These preclinical data suggest that KB003 and other GM-CSF signaling axis inhibitors merit further investigation in CMML. A more committed myeloid precursor expressing CD38 may represent the progenitor population with enhanced GM-CSF dependence in CMML. This is consistent with the results showing CD38 to be a marker of GM-CSF-dependent pSTAT5 sensitivity in JMML and may prove to be predictive biomarker for anti-GM-CSF therapy in CMML. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

An Antibody Fusion Protein for Cancer Immunotherapy Mimicking IL-15 trans-Presentation at the Tumor Site

Cytokines driving the immune response are powerful tools for cancer immunotherapy, but their application is generally limited by severe systemic toxicity. Targeted approaches by means of antibody-cytokine fusion proteins might enable focus on the cytokine activity to the tumor site, thereby reducing unwanted side effects. Here, we investigated the possibility to improve the efficiency of interleukin (IL)-15 presentation in a targeted approach by the incorporation of an IL-15Rα chain fragment, mimicking physiologic trans-presentation. Therefore, an antibody cytokine fusion protein (scFv_RD_IL-15) composed of an antibody moiety targeting the tumor stromal fibroblast activation protein (FAP), an extended IL-15Rαsushi domain (RD) and IL-15 was generated, exhibiting antibody-mediated specific binding and cytokine activity in soluble and targeted form. Comparative analysis with a corresponding antibody fusion protein devoid of RD (scFv_IL-15) showed for scFv_RD_IL-15 in solution enhanced stimulatory activity on Mo7e (IL-15Rβγ) cells and reduced proliferation response on CTLL-2 (IL-15Rαβγ) cells, while in FAP-targeted, that is, membrane-bound form, comparable proliferation of CTLL-2 (IL-15Rαβγ) cells was obtained. In addition, scFv_RD_IL-15 achieved in its soluble and target-bound form stronger proliferation and cytotoxicity on unstimulated and activated T cells, respectively. Furthermore, in vivo analysis in a lung metastasis tumor mouse model revealed a superior antitumor effect for scFv_RD_IL-15 in comparison with that obtained by an untargeted or RD missing version of IL-15 fusion protein. Thus, tumor-directed trans-presentation of IL-15 in association with RD in form of an antibody fusion protein seems to be a promising approach to further improve the antitumor effect of IL-15.

HDLG Associated Protein 1 (DLGAP1) Supports Mpl-Driven Cell Proliferation and Differentiation Through Its Centrosomal Function,

Abstract 3402The Mpl proto-oncogene functions as a basic factor in megakaryocytic development and platelet production as well as contributing to hematopoietic stem cell (HSC) homeostasis and self-renewal. Several mutations in Mpl have been shown to be associated with the myeloproliferative neoplasms. We used retroviral insertional mutagenesis to screen for factors that might give a proliferative and/or survival advantage for cells dependent on Mpl signaling. We developed a retroviral construct expressing a dimerizable form of Mpl that can interrupt genes through insertion, and can activate adjacent genes with its long terminal repeats. We transduced the human leukemia cell line K562 with this construct and blocked the endogenous transforming BCR-ABL kinase using Imatinib. Cells dependent on Mpl signaling were selected by the addition of the dimerizing drug, AP20187. In the absence of Mpl signaling the cells underwent erythroid differentiation and died. Cells that acquired a proliferative advantage and were dependent on Mpl function were expanded. Cloning of retroviral integration sites (RIS) from the selected populations allowed the identification of recurrent RIS that co-localized in the genome. We have identified 668 RIS from 36 independent transductions with a presumed ∼ 1.9 × 106 independent insertions in the initial non-selected, cell population. Among these, 203 RIS represented independent insertion events. Three independent RIS were located in the fourth intron of the Discs large homolog-associated protein 1 (DLGAP1). This protein is a member of the Discs-large/Scribble/Lethal Giant Larvae pathway. DLGAP1, in cooperation with DLG1 and CDC42, has recently been shown to control centrosome positioning and cell polarity in astrocytes (Manneville J-B et al. 2010, JBC, v.191, no 3, pp. 585–598). We investigated the role of DLGAP1 in MPL signaling.Overexpression of full length DLGAP1 had minimal effect on plain K562 cells, but it significantly slowed down the proliferation of K562 cells switched to Mpl signaling. The identified insertion sites would predict the increased expression of an amino-end truncated isoform. When this truncated form was overexpressed in K562 cells and Mpl dependent K562 cells, the cells proliferated at an increased rate. Immunofluorescent studies revealed that the full length DLGAP1 colocalized with major centrosomal markers including gamma-tubulin, PCM1, and APC in K562, HEL, UT7/TPO and Mo7e cell lines. We did not observe colocalization with centrosomes when the truncated isoform of DLGAP1 was overexpressed. Immunoflourescent microscopy revealed that endogenous DLGAP1 in myeloid cell lines was localized in the centriolar satellites, a cellular structure essential for centrosome integrity and function. The immunoflourescent pattern using an anti DLGAP1 antibody showed cell cycle dependent assembly and disassembly. Protein sequence analyses of DLGAP1 indicated consensus sites for presumptive Jak2 phosphorylation as well as consensus sites for SRC tyrosine kinases. To test the possible influence of the two types of kinases on DLGAP1 we treated K562, HEL and UT7/TPO cell lines with an inhibitor of Jak2 (AG490) and independently with an inhibitor of SRC (SU6656). In each case the centrosomal staining of DLGAP1 was diminished with minimal effect on the cytoplasmic fraction of DLGAP1. Interestingly, treatment with SU6656 of the K562 cells switched to Mpl signaling and overexpressing full length GFP-DLGAP1 fusion resulted in a significant increase in the number of polyploid cells. The changes correlated with upregulation of the megakaryocytic marker CD41a and downregulation of the erythroid marker Glycophorin A as assessed by FACS analyses. As centrosomal aberrations are frequent markers of the myeloproliferative neoplasms, we intend to study the role of DLGAP1 in these bone marrow disorders. Disclosures:No relevant conflicts of interest to declare.

Inhibition of TET2 Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine Disturbs Myelopoiesis and Granulo-Monocytic Differentiation.

AbstractAbstract 1584TET2, a gene frequently mutated in myeloid malignancies, encodes an oxygenase that may convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in hematopoietic cells. Using high performance liquid chromatography coupled to tandem mass sprectrometry (HPLC-MS-MS), we found lower levels of 5hmC in erythroblasts from patients with myeloproliferative neoplasms and TET2 mutations than in erythroblasts from non mutated patients. To study the function of TET2 during myelopoiesis, we used lentiviruses expressing short hairpin RNA (shRNA) designed to inhibit TET2 expression in CD34+ cells from normal bone marrow or umbilical cord blood. TET2 mRNA was knocked down by 56 to 74% in several human leukemia cell lines expressing shRNA-TET2, compared to cell lines expressing scramble shRNA. TET2 expression was then assessed in these cell lines by western blot, which showed a 49% reduction of TET2 protein in TF1 cell line, and 80 to 90% reduction in Kasumi, Uke1, and Mo7E cell lines. In addition, knock down of TET2 led to a 73% decrease in 5hmC in Mo7E cell line DNA whereas 5mC and cytosine contents remained unchanged as measured by HPLC-MS-MS. These results indicate that TET2 has a role in the hydroxylation of 5mC in human cells of hematopoietic origin. We then studied the consequences of TET2 knock-down in umbilical cord blood CD34+ cells in vitro. We observed a skewing of CD34+CD38- progenitor differentiation toward the myeloid lineage (52 +/− 4% of the TET2 knock down cells versus 35 +/− 3% of the control cells, n=3, p=0.001) at the expense of lympho-myeloid development and B cell and natural killer (NK) lymphoid lineages in a B/myeloid/NK culture condition. Methylcellulose in the presence of EPO, IL3, SCF, G-CSF showed greater numbers of CFU-G/GM (66 +/− 4 versus 58 +/− 3 colonies per 1000 input CD34+ cells) and lower numbers of BFU-E (60 +/−19 versus 78 +/− 19) in TET2 knock-down samples than in control samples (n= 4, p<0.05) indicating that there was also a skewing toward the granulo-monocytic differentiation at the expense of the erythroid lineage. In addition, in presence of G-CSF, IL3, and FLT3-ligand granulocytic differentiation was delayed in TET2 knock-down cells with a relative excess of monocytic cells at day 10 of culture as assessed by the analysis of cell morphology (47 +/− 4% monocytic cells versus 37 +/− 3%, n=5, p=0.006) and immunophenotype (52 +/− 3% of CD14+ cells versus 39+/−8%, n=5, p=0.002). We then analyzed the expression of a set of transcription factors at the mRNA level that confirmed that some transcription factors specific to granulocytic differentiation were under-expressed in TET2 knocked down cells between day 5 and day 10 of culture. Together, our results show that TET2 participates to the conversion of 5mC to 5hmC in hematopoietic cells, and suggest that TET2 inactivation may have a role in the pathogenesis of myeloid malignancies through the disturbance of myeloid differentiation. Disclosures:No relevant conflicts of interest to declare.

NON-Peptidyl Small Molecule EPO Receptor Agonists Are Potent Pathway Selective Inducers of Erythropoiesis.

AbstractAbstract 1565Erythropoietin (EPO) acts on the homodimeric EPO receptor (EPOR) to stimulate proliferation of erythroid progenitor cells and induce their survival and differentiation into red blood cells. Various recombinant human EPO derivatives, also known as erythropoiesis-stimulating agents (ESAs), are marketed or in clinical development for the treatment of anemia due to renal failure or cancer chemotherapy. However, ESA treatment is associated with an increased risk of adverse cardiovascular complications in patients with kidney disease, and may be related to an increase in mortality in cancer patients, when it is used to increase hemoglobin levels above 13.0 g/dl. We have identified a series of novel non-peptidyl small molecules that selectively activate EPOR function, which may provide a unique therapeutic opportunity in the treatment of anemia. In CD34 positive human bone marrow hematopoietic cells (BM-HCs), a representative analog, LG5640, potently (2 nM EC50) increased the percentage of cells positive for the erythrocyte-specific marker CD235a (glycophorin A) with an efficacy partial (42%) to the maximal effect of EPO (3 U/ml), but greater than the efficacy of the normal serum EPO concentration (∼0.01 U/ml). The erythropoietic effect of LG5640 in BM-HCs was additive to the effect of EPO. LG5640 stimulated the expression of several EPO responsive genes in CD34 positive BM-HCs, including hemoglobin α, EPOR and the anti-apoptotic protein BCL2L1. In addition, LG5640 stimulated the formation of BFUe colonies with partial efficacy (30%) when incubated with BM-HCs for 14 days. The effect of LG5640 on BM-HCs was specific for the erythroid lineage. LG5640 did not increase the percentage of BM-HCs positive for the megakaryocyte marker CD41 or the granulocyte marker CD15. Using human cell lines, we have determined that the action of LG5640 is dependent on EPOR and involves the selective activation of the PI3K/AKT-GATA1 signaling pathway. In the human EPO-dependent cell line UT7EPO, LG5640 blocked apoptosis induced by EPO withdrawal (10 nM EC50), and stimulated the expression of BCL2L1 with an efficacy comparable to EPO. LG5640 stimulated the phosphorylation of EPOR, PI3K, and GATA1, and induced the binding of GATA1 to DNA. Incubation of UT7EPO cells with the PI3K inhibitor LY294002 blocked the effect of LG5640 on cell survival. However, LG5640 did not stimulate phosphorylation of STAT5 or ERK/MAPK, or induce STAT5 DNA binding. LG5640 did not block apoptosis or stimulate BCL2L1 expression in the GM-CSF- and TPO-responsive human leukemia Mo7e cells that lack EPOR. Furthermore, transfection of UT7EPO cells with EPOR- and GATA1-specific siRNAs blocked the activity of LG5640. These data demonstrate that LG5640 is a novel small molecule selective EPOR agonist that unlike other ESAs selectively activates the EPOR/PI3K/GATA1 signal transduction pathway resulting in survival and differentiation of BM-HCs into erythrocytes, possibly through uniquely altering the conformation of the homodimeric EPOR. The selective agonists display an efficacy partial to the maximal effect induced by EPO, and lack excessive erythropoietic stimulation that may possibly contribute to the adverse effects of ESAs. Based on the novel profile of the series, several lead compounds that increase the percentage of CD235a positive BM-HCs with nanomolar potency, and display oral bioavailability in the rat and monkey, have been identified as potential preclinical development candidates. Disclosures:Bissonnette:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Rungta:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Hudson:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Roach:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Hong:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Sun:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Hu:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Ward:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Luo:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Sanders:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Syka:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Slavin:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Zhi:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Marschke:Ligand Pharmaceuticals Inc: Employment, Equity Ownership.

Dasatinib Inhibits the Growth of Molecularly Heterogeneous Myeloid Leukemias

Dasatinib is a dual Src/Abl inhibitor recently approved for Bcr-Abl+ leukemias with resistance or intolerance to prior therapy. Because Src kinases contribute to multiple blood cell functions by triggering a variety of signaling pathways, we hypothesized that their molecular targeting might lead to growth inhibition in acute myeloid leukemia (AML). We studied growth factor-dependent and growth factor-independent leukemic cell lines, including three cell lines expressing mutants of receptor tyrosine kinases (Flt3 or c-Kit) as well as primary AML blasts for responsiveness to dasatinib. Dasatinib resulted in the inhibition of Src family kinases in all cell lines and blast cells at approximately 1 x 10(-9) mol/L. It also inhibited mutant Flt3 or Kit tyrosine phosphorylation at approximately 1 x 10(-6) mol/L. Mo7e cells expressing the activating mutation (codon 816) of c-Kit were most sensitive to growth inhibition with a GI(50) of 5 x 10(-9) mol/L. Primary AML blast cells exhibited a growth inhibition of <1 x 10(-6) mol/L. Cell lines that showed growth inhibition at approximately 1 x 10(-6) mol/L showed a G(1) cell cycle arrest and correlated with accumulation of p21 and p27 protein. The addition of rapamycin or cytotoxic agents enhanced growth inhibition. Dasatinib also caused the apoptosis of Mo7e cells expressing oncogenic Kit. Although all of the precise targets for dasatinib are not known, this multikinase inhibitor causes either growth arrest or apoptosis in molecularly heterogeneous AML. The addition of cytotoxic or targeted agents can enhance its effects.

Soluble GM-CSF Receptor α Is Cytotoxic to Myeloid Cells in a Manner That Is Independent of GM-CSF Binding Inhibition.

Abstract 3618Poster Board III-554The potential influence over cytokine activity by soluble receptors and the precise cytokine specificity they display suggests that soluble cytokine receptors may play a significant role in the response of cells to cytokine-mediated signaling. GM-CSF biology is mediated by binding to a heterodimeric receptor consisting of a major binding subunit (GMRα) and a major signaling subunit (β). GM-CSF Receptor can signal an astonishing variety of cellular functions, including protection from apoptosis, differentiation, maturation, activation and homing. GM-CSF Receptor α (GMRα) exists in both trans-membrane (tmGMRα) and soluble isoforms (sGMRα). sGMRα is believed to arise exclusively through alternative splicing of the GMRα gene product which removes the exon encoding the transmembrane domain. The splicing is such that the amino terminal 317 residues of sGMRα are homologous to the extracellular domain of tmGMRα, however, the deletion and subsequent frameshift replaces the transmembrane and cytoplasmic domains of tmGMRα with a unique 16 amino acid tail on sGMRα. It has been demonstrated that normal human monocytes constitutively secrete sGMRα, and sGMRα has been implicated in the biology of acute myeloid leukemias and the tumor-nerve interactions evoking bone cancer pain.sGMRα has been shown to bind directly to GM-CSF. We hypothesized that recombinant sGMRα(r-sGMRα) competitively inhibits the biological properties of GM-CSF. Using a baculoviral expression system, we produced N-terminal-His-tagged r-sGMRα. We exposed the GM-CSF and IL-3 dependent myeloid cell line, Mo7e, to varying concentrations of r-sGMRα and found that r-sGMRα directly inhibits GM-CSF dependent cell survival and proliferation in a dose-dependent manner. We found that r-sGMRα was toxic to Mo7e cells at concentrations as low as the Kd of r-sGMRα for GM-CSF (3nM). The finding that lower concentrations of GM-CSF resulted in lower Mo7e stimulation, but not toxicity, indicated that the mechanism of r-sGMRα toxicity was unlikely to simply be a reduction in effective GM-CSF concentration.To test whether the r-sGMRα toxicity seen in Mo7e cells was a result of GM-CSF neutralization or a result of signaling inhibition, we demonstrated that GM-CSF neutralizing antibody is cytotoxic during GM-CSF withdrawal, but not cytotoxic in the presence of IL-3, whereas r-sGMRα was cytotoxic under both conditions. Previous kinetic studies have demonstrated a time-dependent strengthening of the GMRα : β : GM-CSF complex; forming discrete “loose” and “tight” binding forms over minutes. Our studies indicate that neither pre-incubation of r-sGMRα with GM-CSF (6 hours) nor pre-incubation of cells with GM-CSF (prior to r-sGMRα addition) diminishes r-sGMRα's ability to inhibit GM-CSF-dependent cell survival and proliferation. We also determined that both r-sGMRα and GM-CSF degradation is independent of the other in vitro.To determine if r-sGMRα mediated cell death resulted from mechanisms other than GM-CSF signaling inhibition, we also tested additional myeloid cell lines that do not require exogenous cytokines for survival, including K562, (which does not express GMRα) as well as KG-1 and HL-60 cells (which both co-express GMRα and GMRb). While r-sGMRα does not inhibit survival or proliferation of KG-1 cells, r-sGMRα does inhibit proliferation of both HL-60 and K562 cells, but only at concentrations tenfold higher than the Kd (30nM). This inhibition was independent of GM-CSF's presence. These data indicate that soluble receptor may have biological effects beyond competition for ligand with the membrane bound receptor, and beyond the modification of GM-CSF half-life.The ability of r-sGMRα to inhibit IL-3 dependent survival in Mo7e cells, but not K562 cells, supports the notion that r-sGMRα may be directly inducing cytotoxicity by interacting with surface components that are likely part of the GM-CSF Receptor complex. Collectively, our data represent the first evidence demonstrating the modulation of GM-CSF signaling in human myeloid neoplasms by r-sGMRα and further illustrates the potentially significant role of soluble receptors in the response of cells to cytokine-mediated signaling. Disclosures:No relevant conflicts of interest to declare.