Vitamin K2 Induces Autophagy and Apoptosis Simultaneously in Leukemia Cells and Bcl-2 Expression Level Determines the Phenotype of Their Cell Death.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3478-3478
Author(s):  
Keisuke Miyazawa ◽  
Tomohisa Yokoyama ◽  
Munekazu Naito ◽  
Juri Toyotake ◽  
Testuzo Tauchi ◽  
...  
Keyword(s):  
Cell Death ◽  
Leukemia Cell ◽  
Vitamin K2 ◽  
Expression Level ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Potent Inducer ◽  
Acridine Orange Staining ◽  
Induced Apoptosis

Abstract Vitamin K2 (menaquinone-2: VK2) is now known to be a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of human bcl-2 gene into HL-60 leukemia cell line, show 5-fold greater expression of Bcl-2 protein compared with that in HL-60neo cells, a control clone transfected with vector alone. Although HL-60neo cells are induced apoptosis in response to VK2, HL-60bcl-2 cells are resistant against apoptosis induction but still show cell growth inhibition along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed autophagosomes and autolysosomes formation in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVO) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunonoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported enhanced autophagy induction in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, autophagosome formation was rather prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells were protected from rapid apoptotic death by higher expression level of Bcl-2.

Cytotoxic T-Lymphocyte Harnessing CML66 Can Kill Human Myeloid Leukemia Cells, In Vitro.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2746-2746
Author(s):  
Koichiro Suemori ◽  
Hiroshi Fujiwara ◽  
Toshiki Ochi ◽  
Masaki Yasukawa
Keyword(s):  
Cell Line ◽  
Myeloid Leukemia ◽  
Cytotoxic T Lymphocyte ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Expression Level ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Myeloid Leukemia Cell

Abstract [Purpose & background] CML66 is a newly identified cancer-testis antigen by SEREX method in post-transplant CML patient who had a second remission by DLI for relapse. Thus CML66 is initially considered to be implicated in graft-versus-leukemia (GvL) effect against CML, while its’ physiological function remains unknown. The identification by SEREX means its’ immunogenicity to produce antibody, however the T-cell response specific for CML66, particularly its’ ability to generate cytotoxic T-lymphocyte (CTL) against leukemia still remains to be verified. Thus we explored a CTL-epitope of CML66 to induce epitope-specific CTL which can kill human leukemia cells, because of the exploration of its’ clinical applicability as an anticancer vaccine for the immunotherapy. [Methods] At first, we synthesized a variety of CML66-derived 9 aminoacid peptides (9 mer) that had computedly-predicted high binding affinity to HLA-A*2402 molecule. CD8+ T lymphocytes from an HLA-A*2402+healthy donor were co-cultured with autologous monocyte-derived mature dendritic cells (mDCs). CD8+T lymphocytes were repeatedly stimulated with peptide-loaded mDCs. Thereafter, the target epitope-specificity of growing cells was examined by a standard 51Cr-release assay. Additionally, the blocking tests by using anti-HLA class I and anti-class II monoclonal antibody (mo.ab.) were conducted to confirm its’ HLA-A*2402-restricted fashion. Next, CML66 mRNA expression level of target cells including myeloid leukemia cell line cells and primary leukemia cells was examined by real-time semi-quantitative PCR (RQ-PCR). The relative expression level of CML66 mRNA was determined by comparative Ct method. [Result] We identified two CML66-derived 9 mer epitopes with high binding affinity to HLA-A*2402 measured by using HLA-A*2402 gene transfected T2 (T2-A24) cell. One of 2 epitopes, the epitope of CML66; aa70–78: WIQDSVYYI generated the epitope-specific CTL, in vitro, and those CTL exerted anti-leukemia activity against human myeloid leukemia cell line cells in an HLA-A*2402-restricted fashion, but not any cytotoxicity against normal cells. Furthermore, the HLA-A*2402 restriction was confirmed by blocking test by HLA-class I and II mo.ab. Next CML66 mRNA expression level was revealed high in myeloid leukemia cell line cells but low in normal cells, which were compared to that of K562 cell line cell. In primay leukemia cells, acute myelogenous leukemia(AML) cells and acute lymphoblastic leukemia(ALL) cells showed the high expression level of CML66 mRNA. Regarding to the FAB classification of AML, the expression level of CML66 mRNA tended to be higher in subsets ranging from M1 to M4, particularly M2 cells. Even by small number, it was of interest that the expression level of CML66 mRNA in primary chronic myelogenous leukemia (CML) cells was high in cells from blastic phase, but low in cells from chronic phase. This finding may suggest the correlation between CML66 and growth activity of tumor cells. [Conclusion] We identified the novel HLA-A*2402 restricted CTL-epitope derived from CML66; aa70–78: WIQDSVYYI, which may be a promising and secure target for immunotherapy against acute leukemias and aggressive CML.

Ubenimex Enhances the Gemtuzumab Ozogamicin-Induced Cytotoxicity Against Myeloid Leukemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4370-4370
Author(s):  
Yasushi Takamatsu ◽  
Shiro Jimi ◽  
Yuka Yoshimura ◽  
Junji Suzumiya ◽  
Kazuo Tamura
Keyword(s):  
Cell Death ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Myeloid Cell ◽  
Quantitative Relationship ◽  
K562 Cells ◽  
Gemtuzumab Ozogamicin ◽  
Leukemia Cells ◽  
Induced Apoptosis

Abstract Gemtuzumab ozogamicin (GO) is a humanized anti-CD33 antibody conjugated to a derivative of a tumoricidal antibiotic calicheamicin. GO alone induces complete remission in 26% of AML patients in first relapse. One way to improve the response rate is to combine GO with other agents. A quantitative relationship has been shown between rates and degrees of CD33 expression and GO-induced cytotoxicity in vitro by using gene transfer to manipulate CD33 expression in myeloid cell lines. We first studied CD33 expression on myeloid leukemia cells by stimulating with several agents in vitro, and found that ubenimex, but not G-CSF, M-CSF, or ATRA, increased CD33 expression on both HL-60 and K562 cells. Ubenimex is an aminopeptidase inhibitor isolated from Streptomyces olivoreticuli and commercially available as an oral agent for clinical use for AML treatment. To investigate whether ubenimex enhances the cytotoxicity of GO, HL-60 and K562 cells were cultured with GO and/or ubenimex for 3 days. When GO was administered, cell viabilities of HL-60 and K562 were reduced to 31.5% and 90.3% as compared with control, respectively. Treatment with ubenimex alone did not influence viabilities of either HL-60 or K562. However, when cells were preincubated with ubenimex and then cultured with GO, cell viabilities decreased to 18.5% and 81.4% for HL-60 and K562, respectively, indicating that pretreatment with ubenimex enhanced GO-induced myeloid leukemia cell death in vitro. We next assessed the mechanism of cell death. The treatment with GO alone and ubenimex alone induced apoptosis in 39.2% and 2.9% of HL-60 cells, respectively. When HL-60 cells were preincubated with ubenimex and then cultured with GO, the number of apoptotic cells increased to 62.9%, demonstrating that ubenimex augments GO-induced apoptosis. Our data suggest that the priming of AML cells with ubenimex should improve the clinical efficacy of GO.

The Human Leukemic HL-60 Cell Line: An in Vitro Model for Cell Death Endpoint Identification

1996 ◽  
Vol 24 (4) ◽  
pp. 581-587
Author(s):  
Cristiana Zanetti ◽  
Arrnalaura Stammati ◽  
Orazio Sapora ◽  
Flavia Zucco
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
In Vitro Model ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Leukemia Cell Line ◽  
Cell Type ◽  
Vitro Model ◽  
Promyelocytic Leukemia Cell Line

The aim of this study was to investigate the endpoints related to cell death, either necrosis or apoptosis, induced by four chemicals in the promyelocytic leukemia cell line, HL-60. Cell morphology, DNA fragmentation, cytofluorimetric analysis and oxygen consumption were used to classify the type of cell death observed. In our analysis, we found that not all the selected parameters reproduced the differences observed in the cell death caused by the four chemicals tested. As cell death is a very complex phenomenon, several factors should be taken into account (cell type, exposure time and chemical concentration), if chemicals are to be classified according to differences in the mechanisms more directly involved in cell death.

scholarly journals Curcumin and Its Analogue Induce Apoptosis in Leukemia Cells and Have Additive Effects with Bortezomib in Cellular and Xenograft Models

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
L. I. Nagy ◽  
L. Z. Fehér ◽  
G. J. Szebeni ◽  
M. Gyuris ◽  
P. Sipos ◽  
...  
Keyword(s):  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Great Promise ◽  
Human Leukemia ◽  
Human Leukemia Cell Line ◽  
Apoptotic Effects

Combination therapy of bortezomib with other chemotherapeutics is an emerging treatment strategy. Since both curcumin and bortezomib inhibit NF-κB, we tested the effects of their combination on leukemia cells. To improve potency, a novel Mannich-type curcumin derivative, C-150, was synthesized. Curcumin and its analogue showed potent antiproliferative and apoptotic effects on the human leukemia cell line, HL60, with different potency but similar additive properties with bortezomib. Additive antiproliferative effects were correlated well with LPS-induced NF-κB inhibition results. Gene expression data on cell cycle and apoptosis related genes, obtained by high-throughput QPCR, showed that curcumin and its analogue act through similar signaling pathways. In correlation with in vitro results similar additive effect could be obsereved in SCID mice inoculated systemically with HL60 cells. C-150 in a liposomal formulation given intravenously in combination with bortezomib was more efficient than either of the drugs alone. As our novel curcumin analogue exerted anticancer effects in leukemic cells at submicromolar concentration in vitro and at 3 mg/kg dose in vivo, which was potentiated by bortezomib, it holds a great promise as a future therapeutic agent in the treatment of leukemia alone or in combination.

ARC Is Regulated by MAPK and PI3K and Confers Drug Resistance and Survival Advantage to AML in Vitro and in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 893-893
Author(s):  
Po Yee Mak ◽  
Duncan H Mak ◽  
Yuexi Shi ◽  
Vivian Ruvolo ◽  
Rodrigo Jacamo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Apoptosis Resistance ◽  
Control Mscs ◽  
Extrinsic Apoptosis ◽  
Induced Apoptosis ◽  
And Control

Abstract Abstract 893 ARC (Apoptosis repressor with caspase recruitment domain) is a unique antiapoptotic protein that has been shown to suppress the activation of both intrinsic and extrinsic apoptosis. We previously reported that ARC is one of the most potent adverse prognostic factors in AML and that high ARC protein expression predicted shorter survival and poor clinical outcome in patients with AML (Carter BZ et al., Blood 2011). Here we report how ARC is regulated and its role in inhibition of AML apoptosis and in cell survival. We provide evidence that ARC expression is regulated by MAPK and PI3K signaling. Inhibition of MAPK and PI3K pathways decreased ARC mRNA and protein levels in AML cells. ARC expression in AML cells is upregulated in co-cultures with bone marrow-derived mesenchymal stromal cells (MSCs) and the upregulation is suppressed in the presence of MAPK or PI3K inhibitors. To investigate the role of ARC in apoptosis resistance in AML, we generated stable ARC overexpressing (O/E) KG-1 and stable ARC knock down (K/D) OCI-AML3 and Molm13 cells and treated them with Ara-C and agents selectively inducing intrinsic (ABT-737) or extrinsic (TRAIL) apoptosis. We found that ARC O/E cells are more resistant and ARC K/D cells more sensitive to Ara-C, ABT-737, and TRAIL-induced apoptosis: EC50s of Ara-C, ABT-737, or TRAIL treatment at 48 hours for ARC O/E KG-1 and control cells were 1.5 ± 0.1 μM vs. 83.5 ± 4.6 nM, 2.2 ± 0.2 μM vs. 60.2 ± 3.1 nM, or 0.97 ± 0.03 μg/mL vs. 0.17 ± 0.08 μg/mL, respectively and for ARC K/D OCI-AML3 and control cells were 0.33 ± 0.02 μM vs. 3.4 ± 0.2 μM, 0.24 ± 0.01 μM vs. 1.3 ± 0.1 μM, or 0.13 ± 0.09 μg/mL vs. 0.36 ± 0.03 μg/mL, respectively. Bone marrow microenvironment is known to play critical roles in AML disease progression and in protecting leukemia cells from various therapeutic agent-induced apoptosis. Leukemia cells were co-cultured with MSCs in vitro study to mimic the in vivo condition. ARC was found to be highly expressed in MSCs and stable ARC K/D MSCs were generated. AML cell lines and primary patient samples were co-cultured with ARC K/D or control MSCs and treated with Ara-C, ABT-737, or TRAIL. Interestingly, ARC K/D MSCs lost their protective activity for leukemia cells treated with these agents. EC50s for OCI-AML3 cells co-cultured with ARC K/D or control MSCs for 48 hours treated with Ara-C, ABT-737, or TRAIL were 1.0 ± 0.04 μM vs. 4.5 ± 0.2 μM, 0.15 ± 0.06 μM vs. 0.53 ± 0.02 μM, or 1.4 ± 0.8 μg/mL vs. 8.1 ± 0.3 μg/mL, respectively. In addition, ARC O/E KG-1 cells grew faster and ARC K/D OCI-AML3 and Molm13 cells and ARC K/D MSCs grew slower than their respective controls. We then injected KG-1 cells into mice and found that NOD-SCID mice harboring ARC O/E KG-1 had significantly shorter survival than mice injected with the vector control KG-1 (median 84 vs. 111 days) as shown in the figure. Collectively, results demonstrate that ARC plays critical roles in AML. ARC is regulated by MSCs through various signaling pathways in AML cells, protects leukemia cells from apoptosis induced by chemotherapy and by agents selectively inducing intrinsic and extrinsic apoptosis. ARC regulates leukemia cell growth in vitro and in vivo. The results suggest that ARC is a potential target for AML therapy. In addition, targeting ARC in MSCs suppresses microenvironmental protection of AML cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Proliferation and differentiation of human myeloid leukemic cells in immunodeficient mice: electron microscopy and cytochemistry

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 1015-1022 ◽  
Author(s):  
EA Machado ◽  
DA Gerard ◽  
CB Lozzio ◽  
BB Lozzio ◽  
JR Mitchell ◽  
...  
Keyword(s):  
Nude Mice ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Immunodeficient Mice ◽  
Human Leukemia Cells

Abstract To study the influence of a biologic environment on cultured human leukemia cells, KG-1, KG-1a, and HL-60 cells were inoculated subcutaneously into newborn nude mice. The cells developed myelosarcomas at the site of inoculation and in lungs and kidneys. KG-1 and HL-60 myelosarcomas were successfully passaged through adult nude mice, whereas KG-1a tumors proliferated only after transplantation into newborn hosts. The human nature of the cells forming myelosarcomas in mice was assessed by chromosomal analyses and detection of cross- reactivity with an antibody to the human leukemia cell line K562. We undertook electron microscopic and cytochemical examinations of the cells proliferating in vitro and in the mice. The granules of KG-1 cells in vivo did not react for acid phosphatase, as observed in vitro, and the HL-60 cells proliferating in mice lost the perinuclear myeloperoxidase (MPO) demonstrated in cultured cells. Although the influence of an in vivo selection of cell subpopulations cannot be ruled out, the enzymatic changes are compatible with induced cell differentiation. Conclusive evidence of differentiation in vivo was observed in the KG-1a cell subline. The undifferentiated KG-1a blasts developed cytoplasmic granules and synthesized MPO during proliferation in vivo. These observations indicate that human leukemia cells from established cell lines proliferate in nude mice and may acquire new differentiated properties in response to the in vivo environment.

scholarly journals In Vitro Inhibitory Effect of Recombinant Human Calprotectin on Nalm6 Leukemia Cell Line

2020 ◽  
Vol 20 (8) ◽  
pp. 951-962
Author(s):  
Samira Charkhizadeh ◽  
Mehdi Imani ◽  
Nematollah Gheibi ◽  
Fateme Shabaani ◽  
Akbar Nikpajouh ◽  
...  
Keyword(s):  
Cell Line ◽  
Leukemia Cell ◽  
Annexin V ◽  
Inhibitory Effect ◽  
New Drugs ◽  
Leukemia Cell Line ◽  
Cytotoxic Effects ◽  
Tumor Expression ◽  
Induced Apoptosis

Background & Purpose: In evaluating new drugs for the treatment of various types of cancer, investigations have been made to discover a variety of anti-tumor compounds with less side effects on normal cells. Investigations have shown that the heterodimers S100A8 and S100A9 inhibit the enzyme casein kinase 2 and then prevent the activation of the E7 oncoprotein. Therefore, the aim of this study was to evaluate the effect of calprotectin as an antitumor compound on the Nalm6 (B cell precursor leukemia cell line). Material & Methods: Transformation of genes encoding S100A8 and S100A9 human, designed in the pQE32 plasmid, was performed by the thermal shock method into E. coli M15 bacteria. After bacterial growth in LB medium, the expression of two S100A8 and S100A9 subunits, the solubility of the protein by SDS-PAGE method was determined. Finally, the S100A8 / A9 complex was equally placed in the microtube. In the next step, the cytotoxic effects of calprotectin produced on the Nalm6 cell line were evaluated using the wst1 test. Then, the apoptosis in these cells was measured using flow cytometry methods with Annexin-V coloration. Results: In the current study, the results showed that the cytotoxic effects of Calprotectin are time and concentration- dependent. Therefore, it can reduce the tumor expression and had a beneficial effect by induced apoptosis in Nalm6 cell line. Conclusion: Calprotectin has an anti-tumor effect on the Nalm6 cell line by increasing apoptosis.

NADPH Oxidase Regulates Cytarabine-Induced Apoptotic Death in Acute Myeloid Leukemia Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4258-4258
Author(s):  
Nazmul H Khan ◽  
Kevin J Sexton ◽  
Melissa J Grimm ◽  
Brahm H Segal ◽  
Carlos E Vigil
Keyword(s):  
Cell Death ◽  
Nadph Oxidase ◽  
Myeloid Leukemia ◽  
Apoptotic Cell ◽  
Leukemia Cell ◽  
Apoptotic Cell Death ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Myelomonocytic Leukemia

Abstract Abstract 4258 Background: Cellular metabolism and oxidative stress are important in the biology and pathophysiology of malignancies. Both increased reactive oxygen species [ROS] levels and induction of anti-oxidative pathways have been described in several malignancies, and may be modulate tumor biology and susceptibility to chemotherapy. Limited studies point to metabolic pathways, including ROS production, influencing pathogenesis and chemo-sensitivity of leukemia. NADPH oxidase is a critical enzyme in antimicrobial host defense and its activation results in ROS generation in myeloid leukemia cells. Our prior studies show that NADPH oxidase can activate Nrf2, a transcriptional factor that induces anti-oxidant and cytoprotective pathways. However the role of NADPH oxidase in chemotherapy-mediated apoptosis induction in leukemic cells is not well-known. Hypothesis: NADPH oxidase-derived ROS will increase sensitivity of AML cells to chemotherapy, whereas Nrf2 will be associated with chemotherapy resistance Methods: We evaluated the role of NADPH oxidase and Nrf2 in regulating cytarabine-induced cell death in wild-type [WT] and engineered PLB-985 cells, a human acute myelomonocytic leukemia cell line derivative. NADPH oxidase-deficient PLB-985 cells were generated by recombination with mutant gp91phox, a necessary component of NADPH oxidase. Nrf2-deficient cells were generated by shRNA (Nrf2shRNA) and depletion (>70%) of Nrf2 mRNA was confirmed by quantitative-PCR. WT and engineered PLB-985 cells were treated with cytarabine (12.5 to 750ng/ml for 24 – 48 hours) and cell death was determined by trypan blue exclusion and Annexin V/7-AAD staining. Results: NADPH oxidase-deficient PLB-985 cells were significantly more resistant to cytarabine compared to WT cells. Cytarabine (500 ng/ml for 48h) induced apoptotic cell death in 25% of NADPH oxidase-deficient vs. 53% of WT PLB-985 cells. Additional dose-response studies confirmed a significant effect of NADPH oxidase in potentiating cytarabine-induced cell death. Nrf2shRNA PLB-985 cells had either similar or modestly increased susceptibility to cytarabine-induced cell death compared to WT PLB-985 cells with empty vectors. NADPH-deficient/Nrf2shRNA PLB-985 cells had similar susceptibility to cytarabine as NADPH-deficient cells with empty vector. Conclusions: Our results show that NADPH oxidase potentiates apoptotic cell death by cytarabine in a myelomonocytic leukemia cell line. However, we did not observe a consistent effect of Nrf2 depletion on apoptotic cell death by cytarabine. These studies suggest that modulation of redox-stress may be a potential therapeutic approach in AML that merits further study. Disclosures: No relevant conflicts of interest to declare.

BMS-936564 (MDX1338): A Fully Human Anti-CXCR4 Antibody Induces Apoptosis in an in Vitro Model of Stromal – Leukemia Cell Interaction for Chronic Lymphocytic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2887-2887
Author(s):  
Manoj Kumar Kashyap ◽  
Deepak Kumar ◽  
Harrison Jones Jones ◽  
Michael Y. Choi ◽  
Johanna Melo-Cardenas ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Leukemia Cell ◽  
Cell Interaction ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Drug Induced ◽  
Induced Apoptosis ◽  
Cells Cultured

Abstract Abstract 2887 Chronic lymphocytic leukemia (CLL) remains incurable despite advances in the biology and treatment of this disease. Current data support the notion that resistance to therapy is promoted by a “protective” tumor microenvironment in which non-leukemia cells produce factors that enhance the resistance of CLL cells to spontaneous or drug-induced apoptosis. One such factor is the chemokine CXCL12, which interacts with its receptor CXCR4 on CLL cells to promote cancer cell survival. To examine the therapeutic potential of blocking CXCL12-CXCR4 interactions, we studied the effect of BMS-936564, a fully human IgG4 anti-CXCR4 antibody, using an in vitro co-culture model of human bone marrow derived stomal-NKter cells – leukemia cell interaction. Such stromal-NKter cells secrete CXCL12 and enhance the resistance of CLL cells to apoptosis in vitro. We observed that primary CLL cells co-cultured with stromal-NKter cells had significantly greater viability than CLL cells cultured alone (20–60% above baseline at 48 hours). Moreover, CLL cells co-cultured with stromal cells had enhanced resistance to drug-induced apoptosis. We found that BMS-936564 antibody at concentrations of 2–200nM could enhance the rate of apoptosis of CLL cells cultured alone or in the presence of stromal cells. CLL cells that expressed unmutated IgVH genes or ZAP-70 appeared equally susceptible to treatment with BMS-936564 as did CLL cells that lack these adverse prognostic markers, as did CLL cells that harbored deletions in 17p13.2 and that were resistant to chemotherapeutic agents, such a fludarabine monophosphate. BMS-936564 antibody inhibited CXCL12 mediated F-Actin polymerization in CLL cells at lower concentrations (20–200nM) compared to AMD-3100 (Mozobil), a small molecule CXCR4 inhibitor (50–150μM). In addition, AMD-3100 did not induce apoptosis in CLL cells (10–300μM). In summary, we observed that the anti-CXCR4 antibody BMS-936564 inhibited CXCL12 mediated activation of the CXCR4 receptor in CLL cells and induced apoptosis in leukemia cells. The pro-apoptotic activity of BMS-936564 was observed in cells cultured alone or together with stromal cells suggesting that this antibody had direct cytotoxic effect on leukemia cells and that it can overcome the protective tumor microenvironment. More over, the activity of BMS-936564 was independent of the presence of poor prognostic factors such as del(17p) suggesting that its mechanism of action is P53 independent. These findings show evidence that the CXCR4-CXCL12 pathway is a valid therapeutic target in CLL and provide additional biological rationale for ongoing clinical trials in CLL and other hematological malignancies using BMS-936564. Disclosures: Kuhne: Bristol-Myers Squibb: Employment. Sabbatini:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Shelat:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Kipps:Abbott: Consultancy, Research Funding.

scholarly journals The Glutaminase Activity of L-Asparaginase Mediates Suppression of Asns Upregulation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3959-3959 ◽  
Author(s):  
Wai Kin Chan ◽  
Lin Tan ◽  
Karine Harutyunyan ◽  
Di Du ◽  
Leona Martin ◽  
...  
Keyword(s):  
Cell Death ◽  
Amino Acid ◽  
Anticancer Activity ◽  
Single Agent ◽  
Leukemia Cell ◽  
Cell Types ◽  
Leukemia Cells ◽  
E Coli ◽  
Glutaminase Activity

Abstract L-asparaginase (ASNase) is a standard component of treatment regimens used for acute lymphoblastic leukemia and is being tested against other cancer types, including acute myeloid leukemia, lymphoma, and pancreatic cancer. We and others have reported that the anticancer activity of ASNase requires the enzyme's glutaminase activity, but the underlying glutaminase-mediated mechanism(s) that lead to leukemia cell death are unknown. Glutamine, the most abundant amino acid in the blood, is known for pleiotropic roles in numerous biological pathways, including energy metabolism, redox metabolism, nucleotide anabolism, and amino acid anabolism. Many cancer cells have been found to reprogram their metabolic pathways to become highly dependent on glutamine for survival and proliferation. Glutaminase (GLS/GLS2)-mediated conversion of glutamine to glutamic acid provides the latter as a substrate for conversion to α‐ketoglutarate by transaminases or glutamate dehydrogenases (GLUD1/GLUD2) to fuel the TCA cycle. Consequently, targeting glutamine metabolism has become an attractive strategy for anticancer therapy. The enzyme asparagine synthetase (ASNS) mediates resistance to ASNase through synthesis of asparagine. ASNS is expressed in most cell types, and its expression is upregulated in response to a wide variety of cell stresses, including amino acid limitation and endoplasmic reticulum stress. We and others have shown that ASNS-positive leukemia cells capable of synthesizing asparagine de novo are less responsive than ASNS-negative leukemia cells to ASNase therapy (Chan et al., Blood, 2014). Moreover, ASNase resistance has been associated with elevated ASNS expression. In fact, we have shown that ASNS expression is a predictive marker of the in vitro response of leukemia cell lines and some solid tumor cell types to ASNase. The expression of ASNS in most cells in the body poses a serious challenge for successful therapy with ASNase; for example, production of asparagine by the liver and cells (e.g., mesenchymal stem cells and adipocytes) of the tumor microenvironment may contribute significantly to ASNase resistance in vivo. Here we used the high-glutaminase E. chrysanthemi ASNase (Erwinaze®), wild-type E. coli ASNase (ASNaseWT), and the glutaminase-deficient E. coli mutant, ASNaseQ59L, as models of high, medium-, and low-glutaminase, respectively, to explore ASNase glutaminase activity-mediated mechanisms of leukemia cell death. Unexpectedly, we found that increasing glutaminase activity caused an increase in the suppression of ASNS upregulation in vitro (Figure 1A). In NSG mice injected with luciferase-labeled Sup-B15 cells, single-agent ASNaseWT yielded a durable response approximating cure, whereas glutaminase-deficient ASNaseQ59L yielded a complete response but with recurrence. Together, the results suggest that ASNase glutaminase activity is associated with suppression of ASNS upregulation, making durable, single-agent anticancer activity easier to achieve. Overall, the results provide new insight into the mechanism of action of ASNase. Disclosures Konopleva: Stemline Therapeutics: Research Funding. Weinstein:NIH: Patents & Royalties: L-asparaginase. Lorenzi:Erytech Pharma: Consultancy; NIH: Patents & Royalties.

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