scholarly journals Lymphocyte Function-Associated Antigen 3 (LFA-3): Key Factor of the Interactions Between Nurse-like-Cells and B Leukemic Cells from Chronic Lymphocytic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1955-1955
Author(s):  
Frederic Boissard ◽  
Jean Jacques Fournie ◽  
Loic Ysebaert ◽  
Mary Poupot
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Lymph Nodes ◽  
B Lymphocytes ◽  
Adhesion Molecule ◽  
Actin Polymerization ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Lymphocyte Function ◽  
Blocking Antibody

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western Countries. This pathology is characterized by an accumulation of monoclonal, non-functional and mature CD5+ CD19+ leukemic B-cells (CLL cells) in lymph nodes, peripheral blood and bone marrow. Despite a high resistance to the in vivo apoptosis, CLL cells die spontaneously in vitro due to a lack in ex vivo conditions of sustaining cells and factors from their microenvironment such as stroma cells (Lagneaux L et al, Blood. 1998; 91:2387-2396), follicular dendritic cells or Nurse-Like-Cells (NLC) (Burger JA et al, Blood. 2000; 96:2655-2663). NLC are derived from CD14+ cells in contact with CLL cells in vitro (Tsukada N et al, Blood. 2002; 99:1030-1037) and were found in lymph nodes of CLL patients (Ysebaert L et al, Leuk Lymphoma. 2011; 52:1404-1406). NLC were shown to have a Tumour Associated Macrophages phenotype and gene expression profile. These cells have been first described to be essential for in vitro CLL cells survival partially through the production of soluble factors such as CXCL12 (Burger JA et al, Blood. 2000; 96:2655-2663), CCL3 and CCL4 (Burger JA et al, Blood. 2009; 113:3050-3058). Thus, other mechanisms are required for CLL cells survival. Indeed, we showed that contact of CLL cells with NLC was necessary to protect CLL cells from the in vitro apoptosis. We then investigated the mechanism of these interactions at a molecular level. We also determined their influences on the in vitro CLL cells survival and on the NLC-induced chemoresistance. We observed close and strong interactions evaluated by the measurement of trogocytosis from NLC to CLL cells. Trogocytosis is an active phenomenon with transfer of membrane fragments from one cell to another. We showed that NLC/CLL cells trogocytosis is dependant to actin polymerization and SRC phosphorylation. To find possible couples of molecules involved in this contact, we compared different transcriptomic data from NLC, monocyte, CLL cells and B lymphocytes. We highlighted potential couples of molecules and confirmed their expression on CLL cells and NLC by flow cytometry analysis. Finally, we obtained 3 couples probably implicated: Lymphocyte Function-Associated Antigen 3 (LFA-3)/CD2, Platelet/Endothelial Cell Adhesion Molecule 1 (PECAM1)/CD38 and Intercellular Adhesion Molecule 1 (ICAM-1)/LFA-1. Antibody blocking strategies revealed that LFA-3, which is up-regulated in CLL cells compared to healthy donors B lymphocytes, was necessary for the interaction between CLL cells/NLC when PECAM1, ICAM-1 and their co-partners were not essential (figure 1). Furthermore, this contact, through LFA-3, induced Akt phosphorylation but not ERK1/2 phosphorylation in CLL cells. Finally, we showed that LFA-3 and its receptor CD2 are necessary to the rescue of CLL cells by NLC (figure 2). To go further, we tested the chemoprotective effect of NLC on CLL cells. We showed that NLC slightly protect CLL cells against bendamustin but not against rituximab, dasatinib or ibrutinib. We hypothesized that the contact through LFA-3 could be involved in this chemoresistance. However, we did not observed a significant effect of the combination of bendamustin and LFA-3-blocking compared to bendamustin alone suggesting that this chemoprotection of CLL cells by NLC involved another pathway. Altogether, our results indicate that overexpression of LFA-3 by CLL cells and its critical implication in the interaction with NLC might be a new therapeutic target in CLL to disturb the interaction of CLL cells with their microenvironment. Figure 1: LFA-3 blocking but not ICAM-1 and PECAM1 decrease trogocytosis from NLC to CLL cells. a) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-LFA-3. b) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-ICAM-1. c) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-PECAM1. Figure 1:. LFA-3 blocking but not ICAM-1 and PECAM1 decrease trogocytosis from NLC to CLL cells. a) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-LFA-3. b) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-ICAM-1. c) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-PECAM1. Figure 2: LFA-3 is critical for the survival of CLL cells in contact with NLC. Figure 2:. LFA-3 is critical for the survival of CLL cells in contact with NLC. Disclosures No relevant conflicts of interest to declare.

Nicotinamide Promotes Apoptosis In Chronic Lymphocytic Leukemia through Activation of the p53/Mir-34a/SIRT1 Tumor Suppressor Network

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4627-4627
Author(s):  
Valentina Audrito ◽  
Tiziana Vaisitti ◽  
Sara Serra ◽  
Davide Rossi ◽  
Daniela Gottardi ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Lymphocytes ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Critical Role ◽  
Disease Model ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Intrinsic Resistance

Abstract Abstract 4627 Nicotinamide (Nam), is the main precursor of nicotinamide adenine dinucleotide (NAD+). It regulates intracellular levels of NAD+ and consequently activities of four classes of NAD+-consuming enzymes, including NADases, mono-ADP-ribosyl transferases (ARTs), poly-ADP-ribose polymerases (PARPs) and sirtuins. Pharmacological doses of Nam inhibit the physiological activation and proliferation of mouse B lymphocytes, suggesting that this agent might affect also human B cell homeostasis. We approaches this issue by comparing the effects of Nam on normal vs. leukemic B lymphocytes. Chronic lymphocytic leukemia (CLL) was selected as disease model, for testing in vitro the therapeutic potential of Nam, due its intrinsic resistance to apoptosis, mediated by an imbalance in the mechanisms regulating cell death, mainly regulated through the activities of NAD+-dependent enzymes. This study shows that pharmacological doses of Nam (5-10 mM) significantly inhibit proliferation and induce apoptosis of CLL cells. At earlier time points, Nam markedly reduces phosphorylation of multiple intracellular substrates, including ERK1/2. Normal B lymphocytes, used as control, were significantly less sensitive to the action of Nam. We hypothesized that these effects could be explained at least in part as a consequence of the inhibitory effects of Nam on NAD+-consuming enzymes. Attention was focused on SIRT1, a deacetylase that plays a critical role in cancer and that acts as a longevity factor. The results demonstrate that Nam exposure inhibits the activity, and also the expression of SIRT1. This effect is apparent only in leukemic cells, where SIRT1 protein levels are significantly higher than in normal B lymphocytes, obtained from spleen or tonsils, markedly less sensitive to Nam effects. The functional block of SIRT1 induced by Nam is followed by activation of p53, transcription of miR-34a and translational repression of SIRT1 mRNA (p53/miR-34a/SIRT1 functional loop). The endpoint is the activation of apoptosis. The same loop is the target of conventional DNA-damaging drugs, such as etoposide. Thus, addition of Nam to conventional DNA-damaging chemotherapeutics agents, leads to an inhibition of SIRT1 through two independent and synergic pathways, resulting in additive effects on apoptosis. In conclusion this work suggests that Nam represents a potentially useful non-chemotherapeutic agent, characterized by a known and established safety profile, to be associated to conventional cytotoxic drugs in the treatment of selected forms of CLL. Disclosures: No relevant conflicts of interest to declare.

Monocytes/Macrophages Are the Major Targets of the CCL3 Chemokine Produced by CD38+CD49d+ Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2350-2350
Author(s):  
Antonella Zucchetto ◽  
Dania Benedetti ◽  
Claudio Tripodo ◽  
Riccardo Bomben ◽  
Fleur Bossi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Chronic Lymphocytic Leukemia ◽  
T Lymphocytes ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Lymphocytic Leukemia ◽  
Bone Marrow Biopsies ◽  
Tnf Α ◽  
Significant Difference

Abstract Abstract 2350 Poster Board II-327 Introduction: CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Recent gene expression profiling studies comparing CLL cases expressing low versus high levels of CD38 and CD49d, identified CCL3 as a gene upregulated by CD38+CD49d+ CLL. The release of CCL3 by cultured CLL cells was also demonstrated upon CD38 triggering, and CCL3 protein was found in CLL cells from bone marrow biopsies (BMB) of CD38+ cases (Zucchetto et al., Cancer Res, 2009; 69:4001-9). Given the role of CCL3 as potent chemoattractant for different cell types, we aimed at identifying the major targets of CCL3, as produced by CD38+CD49d+ CLL cells. Methods: CLL infiltrates of BMB were characterized by immunohistochemistry (IHC). Expression of the CCL3 receptors CCR1 and CCR5 by PB CLL subpopulations was evaluated by flow cytometry. T lymphocyte and monocyte migrations were performed by in-vitro transwell chemotaxis assays. Results: IHC analysis of BMB from 16 CLL cases revealed a higher number of infiltrating CD68+ cells in the context of CLL-involved areas of BMB from CD38+CD49d+CCL3+, compared to CD38−CD49d−CCL3− cases (p=0.01). CD3+ lymphocytes were interspersed in the CLL aggregates, but with no significant difference between the two subgroups. Evaluation of CCR1 and CCR5 in PB cell subpopulations from 40 CLL cases expressing or not surface CD38 and CD49d, showed the highest mean fluorescence intensity (MFI) levels for both CCR1 (624±60) and CCR5 (64±9) in the monocytic component, irrespective of CD38 and CD49d expression by CLL cells. Conversely, both CLL cells and residual T lymphocytes showed low MFI levels for CCR1 (19±4 and 14±3) and CCR5 (21±2 and 20±2). High CCR1 and CCR5 expression levels were detected in in-vitro differentiated monocytes from purified PB cells of four CD38+CD49d+ CLL. Accordingly, CCR1 expression was documented in macrophage-like cells in BMB from CD38+CD49d+ CLL. Next, we evaluated the capability of purified monocytes and T lymphocytes from 10 CLL cases to migrate in response to CCL3. In keeping with the strong expression of CCR1, monocytes migrated toward CCL3 at a concentration of 3 ng/mL (migration index, MI= 8.8±0.9, p=0.03), whereas T lymphocytes required a higher CCL3 concentration (100 ng/mL) to display slight migration capability (MI= 1.6±0.2, p=ns). The increased infiltration of macrophages in BMB from CCL3-producing CD38+CD49d+ CLL, prompted us to verify the capability of CCL3-stimulated macrophages to induce the expression by endothelial cells (EC) of the CD49d specific ligand VCAM-1. By using two different EC models (HUVEC and ADMEC), we documented a significant up-regulation of VCAM-1 by EC exposed to conditioned media (CM) collected from cultures of macrophages challenged in-vitro with CCL3 (p=0.002). Notably, increased levels of the pro-inflammatory cytokine TNF-α were detected in CCL3-CM (p=0.006), and neutralization of TNF-α by specific antibodies reverted the capability of CCL3-CM to induce VCAM-1 by EC models. In agreement with these in-vitro data, we found a more prominent meshwork of VCAM-1+ stromal/endothelial cells in lymphoid infiltrates from CD38+CD49d+ CLL compared to CD38−CD49d− cases (p=0.002), and engagement of CD49d by VCAM-1 was able to significantly delay the spontaneous apoptosis observed in cultured CLL cells. Conclusions: CD68+ monocytes/macrophages are likely the main targets for the CLL3 chemokine produced by CD38+CD49d+ CLL cells, and are active in determining, through the release of TNF-α and other yet unidentified cytokines, the overexpression of VCAM-1 by endothelial cells. Experiments aimed at investigating further roles of CD68+ monocytes/macrophage in CLL are currently matter of study. Disclosures: No relevant conflicts of interest to declare.

Increased CD39 Expression on CD4+ T-Lymphocytes Has Clinical and Prognostic Significance in Chronic Lymphocytic Leukemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3895-3895
Author(s):  
Yair Herishanu ◽  
Inbal Hazan-Hallevi ◽  
Sigi Kay ◽  
Varda Deutsch ◽  
Aaron Polliack ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Prognostic Significance ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Anti Inflammatory

Abstract Abstract 3895 Chronic lymphocytic leukemia (CLL) cells depend on their microenvironment for proliferation and survival. Ectonucleotidase CD39 has anti-inflammatory properties as it hydrolyzes pro-inflammatory extra-cellular ATP, generates anti-inflammatory adenosine and also protects regulatory T cells from ATP-induced cell death. In this study we investigated the clinical significance of CD39 expression on CD4+T-cells in 45 patients with CLL as well as its compartmental regulation and explored the possible mechanisms for its induction. Compared to healthy individuals, CD4+CD39+ lymphocytes were increased in the peripheral blood of patients with CLL (4.6%±2.28 vs. 17.3%±12.49, respectively, p=0.004), and correlated with advanced stage of disease (9.72%±5.76, 18.15%±12.03 and 25.90%±16.34, of CD4+ lymphocytes, in patients with Rai stages 0, 1+2 and 3+4, respectively, p=0.019). CD4+CD39+ cells were also higher in patients with CLL who needed therapeutic intervention (untreated; 12.99%±10.63 vs treated; 22.21%±12.88, p=0.01) and in those who were ZAP70+ or had b2-microglobulin levels>3g/L. There were more CD4+CD39+ lymphocytes in the bone marrow compartment (22.25%±16.16) than in the peripheral blood (16.60%±15.84, p=0.009). In-vitro studies showed that CD39 can be induced on CD4+cells by exposure to ATP or indirectly, following B-cell receptor (BCR) engagement (CD4+CD39+ lymphocytes increased by 1.56 fold, in the BCR engaged samples compared to their paired controls; 20.27%±11.3 vs. 13%±9.42, respectively, p=0.0006). Conclusions: Increased CD39 expression on CD4+ T-lymphocytes in CLL associates with an aggressive disease. This may reflect the ability of the leukemic cells to suppress the surrounding immune environment, and contribute to a poorer prognosis. CD39+ may also serve as a future target for the development of novel therapies with immune modulating anti–tumor agents in CLL. Disclosures: No relevant conflicts of interest to declare.

Mapping the Targets of Dasatinib in Chronic Lymphocytic Leukemia Reveals Distinct Roles for Abl and Btk in Drug Resistance and Adhesion, and Explains Clinical Effects On Lymph Node Reduction

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3900-3900
Author(s):  
Eric Eldering ◽  
Christian R Geest ◽  
Martin FM de Rooij ◽  
Nora Liu ◽  
Bogdan I Florea ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Lymph Node ◽  
Chronic Lymphocytic Leukemia ◽  
Broad Spectrum ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Clinical Effects ◽  
Open Label

Abstract Abstract 3900 In the lymph node (LN) microenvironment, chronic lymphocytic leukemia (CLL) cells are protected from apoptosis by upregulation of anti-apoptotic proteins. In vitro, this can be mimicked via CD40-stimulation of CLL cells, which also provides resistance to various chemotherapeutics. Novel drugs that target kinases involved in B cell signalling, including the broad spectrum kinase inhibitor dasatinib, are currently in clinical development for CLL. We have shown previously that dasatinib prevents CD40-mediated anti-apoptotic changes in CLL (Hallaert et Blood 2008). However, the kinase(s) involved remain unidentified. Here, we coupled dasatinib to an affinity matrix and pulled down its targets from CD40-stimulated CLL cells. By mass-spectrometry and Western blotting, Abl and Btk were identified as dominant targets of dasatinib. Functional analysis revealed that CD40-mediated anti-apoptotic signals and drug-resistance could be overcome both by dasatinib and the Abl inhibitor imatinib, but not by the novel Btk inhibitor PCI-32765 (ibrutinib), whereas BCR- and chemokine-controlled adhesion could be abolished by dasatinib and ibrutinib, but not by imatinib. Thus, dasatinib combines two key aspects that are clinically relevant: inhibition of Abl overrides chemoprotective survival signals, whereas inhibition of Btk impairs integrin-mediated adhesion of CLL cells in the microenvironmental niche. This combined inhibition of Abl and Btk was put to an initial test in an open-label phase 2 trial of dasatinib combined with fludarabine in twenty refractory CLL patients. As might be expected based on the in vitro data, reductions in lymph node size were observed in most patients. A LN reduction of ≥20% provided a significant improved PFS (256 days) and OS (510 days) as compared to non-responders (80 days and 158 days respectively). Details of the clinical study will be presented separately. In conclusion, in agreement with in vitro molecular studies, dasatinib seems to have clinical efficacy in heavily pretreated refractory CLL patients. Combined, these data encourage further studies on a broad-spectrum kinase inhibitor like dasatinib in combination with other classes of drugs in relapsed and refractory CLL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Production of autoantibodies by CD5-expressing B lymphocytes from patients with chronic lymphocytic leukemia.

1989 ◽  
Vol 169 (1) ◽  
pp. 255-268 ◽  
Author(s):  
Z M Sthoeger ◽  
M Wakai ◽  
D B Tse ◽  
V P Vinciguerra ◽  
S L Allen ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Lymphocytes ◽  
Autoimmune Disorders ◽  
Lymphoproliferative Disorders ◽  
Lymphocytic Leukemia ◽  
Antigenic Specificity ◽  
Human B Cells ◽  
Well Differentiated

CD5-expressing B lymphocytes from patients with selected chronic lymphoproliferative disorders were used to determine whether monoclonal populations of CD5+ human B cells produce autoantibodies. CD5+ B cells from 19 patients with chronic lymphocytic leukemia (CLL) and one with diffuse well-differentiated lymphocytic lymphoma (DWDL) were cultured, with and without mitogenic stimulation, to obtain Ig from these cells. 17 of the 20 samples produced Ig in vitro. mAb from nine of the 17 patients were reactive with either IgG, ssDNA, or dsDNA. In every instance, the autoantibodies displayed monotypic L chain usage that correlated precisely with the L chain expressed on the CD5+ leukemic B cell surface. These monoclonal autoantibodies varied in their degree of antigenic specificity; some were quite specific, reacting with only one antigen, whereas others were polyspecific, reacting with two or all three autoantigens tested. Three features distinguish these autoantibodies from those observed in prior studies of CD5+ B cells. First, they are clearly the products of monoclonal populations of CD5+ cells; second, several react with dsDNA, a specificity not previously reported and often seen in association with significant autoimmune disorders; and third, two of the monoclonal autoantibodies secreted by the CD5+ clones were of the IgG class. Although not all of the Ig-producing, CD5-expressing clones elaborated mAbs reactive with the autoantigens tested, greater than 50% did. It is possible that with a broader autoantigenic panel or with larger quantities of CLL/DWDL-derived Ig, even more autoantibody-producing clones might be identified. These studies may have important implications for the antigenic specificity of subsets of human B lymphocytes as well as for lymphoproliferative and autoimmune disorders in general.

Concurrent up-regulation of BCL-XL and BCL2A1 induces approximately 1000-fold resistance to ABT-737 in chronic lymphocytic leukemia

Blood ◽  
2009 ◽  
Vol 113 (18) ◽  
pp. 4403-4413 ◽  
Author(s):  
Meike Vogler ◽  
Michael Butterworth ◽  
Aneela Majid ◽  
Renata J. Walewska ◽  
Xiao-Ming Sun ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Lymph Nodes ◽  
Peripheral Blood ◽  
De Novo ◽  
Phase 1 ◽  
Lymphocytic Leukemia ◽  
Interleukin 4 ◽  
Synergistic Activity

Abstract ABT-737 and its orally active analog, ABT-263, are rationally designed inhibitors of BCL2 and BCL-XL. ABT-263 shows promising activity in early phase 1 clinical trials in B-cell malignancies, particularly chronic lymphocytic leukemia (CLL). In vitro, peripheral blood CLL cells are extremely sensitive to ABT-737 (EC50 ∼7 nM), with rapid induction of apoptosis in all 60 patients tested, independent of parameters associated with disease progression and chemotherapy resistance. In contrast to data from cell lines, ABT-737–induced apoptosis in CLL cells was largely MCL1-independent. Because CLL cells within lymph nodes are more resistant to apoptosis than those in peripheral blood, CLL cells were cultured on CD154-expressing fibroblasts in the presence of interleukin-4 (IL-4) to mimic the lymph node microenvironment. CLL cells thus cultured developed an approximately 1000-fold resistance to ABT-737 within 24 hours. Investigations of the underlying mechanism revealed that this resistance occurred upstream of mitochondrial perturbation and involved de novo synthesis of the antiapoptotic proteins BCL-XL and BCL2A1, which were responsible for resistance to low and high ABT-737 concentrations, respectively. Our data indicate that after therapy with ABT-737–related inhibitors, resistant CLL cells might develop in lymph nodes in vivo and that treatment strategies targeting multiple BCL2 antiapoptotic members simultaneously may have synergistic activity.

Comparison of in Vitro Apoptotic Response of Chronic Lymphocytic Leukemia (CLL) Cells to Bcl-2 Antagonist ABT-737 and IAP Antagonist BV6.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4386-4386
Author(s):  
Shinichi Kitada ◽  
Ryuji Yamaguchi ◽  
Laura Z. Rassenti ◽  
Thomas J. Kipps ◽  
John C. Reed
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Patient Treatment ◽  
Altered Expression ◽  
Over Expression ◽  
Iap Antagonist ◽  
Smac Mimetics

Abstract Abstract 4386 Altered expression of Bcl-2-family and IAP-family proteins has been considered to play central roles in apoptosis dysregulation in cancer and leukemia, promoting malignant cell expansion and contributing to chemoresistance. Anti apoptotic Bcl-2-family proteins Bcl-2 and Bcl-XL as well as IAP proteins, including XIAP, have been thoroughly validated as drug discovery targets for cancer and strategies for inhibiting these proteins have been devised based on mimicking their endogenous antagonists, as represented by ABT-737, a fully synthetic Bcl-2/Bcl-XL antagonist developed at Abbott Laboratories, and BV6, bivalent SMAC mimetics developed at Genentech Inc. Chronic lymphocytic leukemia (CLL) is a quintessential example of a human malignancy caused by defective programmed cell death. Over-expression of the Bcl-2 protein is one of the most consistent and prominent etiological factors associated with this disease, whereas over-expression of XIAP protein has been demonstrated in a majority of CLL patients. In this study, we evaluated the pro-apoptotic effects of ABT-737 and BV6 on CLL cells in side-by-side comparisons. We found that the CLL cells of 31 of 42 patients were highly sensitive to ABT-737, inducing potent dose-dependent killing of cells with an IC50 = 52.5 ± 22.5 (mean ± std error). In contrast, the CLL cells of 11 of 42 patients were relatively resistant to ABT-737, requiring higher concentrations to induce apoptosis (IC50 >1 μM). Preliminary statistical analysis was performed to correlate ABT737-sensitive and -resistant CLL with various other characteristics, including patient treatment status, ZAP-70 expression, and VH mutation status, but no significant correlation was observed. In contrast, the CLL cells from all 42 patients examined were relatively resistant to the IAP antagonist BV6, which required high concentrations to induce apoptosis (IC50 > 1 μM). Similar results were obtained using other SMAC mimetic compounds. Analysis of CLL cells for endogenous SMAC protein expression and for the subcellular location of SMAC protein demonstrated that the leukemia cells of 18 of 23 patients expressed high-levels of SMAC, as assessed by immunoblotting. Furthermore, SMAC was located in the cytosolic fraction of these leukemia cells in 7 of 18 cases examined. However, CLL cells with high-levels of cytosolic SMAC had little evidence of concomitant release of cytochrome c from mitochondria and demonstrated little spontaneous apoptosis in vitro. These results suggest that the leukemia cells of some CLL patients may already express cytosolic SMAC, which might explain the relative insensitivity of CLL cells to SMAC mimetics such as BV6. Taken together, these studies suggest that Bcl-2 antagonists such as ABT-737, but not SMAC mimetics such as BV6, may have promising therapeutic activity for most CLL patients. (NIH CA-081534) Disclosures: No relevant conflicts of interest to declare.

AICAR Induces Apoptosis in Chronic Lymphocytic Leukemia Cells through the Mitochondrial Pathway and Independently of p53 Pathway and AMPK Activation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4819-4819
Author(s):  
Joan Gil ◽  
Antonio F Santidrián ◽  
Diana M González-Gironès ◽  
Daniel Iglesias-Serret ◽  
Llorenç Coll-Mulet ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Lymphocytes ◽  
Mitochondrial Pathway ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Ampk Activation ◽  
Cytochrome C Release ◽  
Cell Technologies ◽  
Induced Apoptosis

Abstract Abstract 4819 AICAR (5-aminoimidazole-4-carboxamide riboside or acadesine) induces apoptosis in different cell types including chronic lymphocytic leukemia (CLL) cells. Here, we have analyzed the mechanisms involved in AICAR-induced apoptosis in CLL cells. AICAR induced apoptosis through the mitochondrial pathway, since inhibition of caspase-8 did not protect CLL cells from AICAR-induced apoptosis and caspase inhibition did not alter cytochrome c release induced by AICAR. AICAR induced a significant increase in the mRNA levels of the proapoptotic BH3-only genes BIM, BNIP3, BNIP3L, HRK, MOAP1, and NOXA. These changes were AICA ribotide (ZMP) accumulation-dependent and adenosine monophosphate-activated protein kinase (AMPK) activation-independent in CLL cells. Furthermore, AICAR induced the accumulation of NOXA protein in all CLL samples and BIM protein in about half of these samples, without modifying the levels of other BCL-2 family proteins analyzed. Importantly, AICAR induced apoptosis irrespective of the tumor suppressor TP53 and ataxia telangiectasia mutated status in CLL cells. AMPK activation with phenformin or A-769662 did not induce apoptosis in CLL cells. Finally, AICAR induced apoptosis in B lymphocytes from AMPKa1−/− mice. Taken together, our results demonstrate that AICAR induces apoptosis in B lymphocytes through the mitochondrial pathway by an AMPK- and p53-independent mechanism. Disclosures: Gil: Advancell-In Vitro Cell Technologies S.L.: Patents & Royalties, Research Funding. Campàs:Advancell-In Vitro Cell Technologies : Employment, Patents & Royalties.

Surface IgM of Chronic Lymphocytic Leukemia Cells Displays Unusual Glycans Indicative of Antigen Engagement In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 55-55
Author(s):  
Graham Packham ◽  
Serge Krysov ◽  
Christopher Ian Mockridge ◽  
Kathy N Potter ◽  
Freda K Stevenson
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Variable Region ◽  
Immunoglobulin M ◽  
Lymphocytic Leukemia ◽  
Glycosylation Pattern ◽  
Mature Form

Abstract Abstract 55 Several lines of evidence support the idea that surface immunoglobulin M (sIgM) plays a key role in determining the clinical behavior of chronic lymphocytic leukemia (CLL). For example, the presence of somatic mutations in immunoglobulin variable region genes is a strong prognostic marker with unmutated CLL (U-CLL) associated with a poor outcome relative to mutated CLL (M-CLL). U-CLL also generally express higher levels of sIgM and retain the ability to signal via this receptor. In this study, we used surface biotinylation to analyse sIgM in CLL and discovered that it exists in two forms with differing mobility on SDS-PAGE. Treatment with glycosidases revealed that these forms were due to different N-glycosylation patterns in the μ constant region. One form is similar to that of normal B cells in bearing mature complex glycans common to most cell surface glycoproteins. The other is an immature mannosylated form more characteristic of endoplasmic reticulum (ER)-located μ chains. CLL cells expressed variable proportions of the immature mannosylated form and quantitative analysis demonstrated that, on average, the proportion of mannosylated sIgM was approximately 2-fold higher (p=0.006) in U-CLL compared to M-CLL. Although normal B cells isolated from blood expressed only the mature form of sIgM, in vitro treatment with anti-μ resulted in upregulation of the immature form, suggesting that glycan modification is a consequence of antigen exposure. Consistent with this, in vitro incubation of CLL cells was associated with increased expression of the mature form of sIgM. Phosphotyrosine analysis demonstrated that both forms of sIgM were able to signal following sIgM engagement in vitro. Taken together, these findings support the concept that CLL cells are continuously exposed to antigen in vivo. This process leads to a change in the N-glycosylation pattern of the re-expressed sIgM so that a mannosylated form predominates, especially in U-CLL. Strikingly, expression of mannosylated sIgM is also characteristic of follicular lymphoma, where it is constitutively displayed via N-glycosylation sites in the Ig variable region (Radcliffe et al. J Biol Chem. 2007; 282, 7405-15). Persistent mannosylation of sIgM appears to be a feature common to several B-cell malignancies, suggesting a role in pathogenesis. Disclosures: No relevant conflicts of interest to declare.

CXCL12 Enhances Survival of Chronic Lymphocytic Leukemia Cells Via a RAF-Dependent Pathway That Can Be Inhibited by Sorafenib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2445-2445
Author(s):  
Davorka Messmer ◽  
Jessie-F Fecteau ◽  
Morgan O'Hayre ◽  
Tracy Handel ◽  
Thomas J. Kipps
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Survival ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Growth Factor Receptor ◽  
Lymphocytic Leukemia ◽  
Increased Risk ◽  
Dependent Pathway

Abstract Abstract 2445 The cellular microenvironment is critical for the survival of Chronic Lymphocytic Leukemia (CLL) cells. CLL cells die rapidly in vitro unless they receive survival signals from stromal cells or “nurse-like” cells (NLCs). CLL cell survival is in part mediated by the stromal cell-derived factor-1 (SDF-1alpha, designated as CXCL12), which is expressed by NLCs. CXCL12 is a highly conserved chemokine that can promote CLL-cell survival through its receptor CXCR4. Prior studies showed that treatment of CLL cells with CXCL12 induced activation of Extracellular Signal-Regulated Kinase (ERK). In this study, we examined CXCL12 signaling in CLL cells to characterize the mechanism (s) accounting for its ability to enhance CLL-cell survival. For this we examined CLL cells with high- or low- level expression of the zeta-associated protein of 70 kD (ZAP-70), a tyrosine kinase that is expressed by CLL cells of patients who have an increased risk for early disease progression and short survival. We found that CXCL12 induced a robust intracellular Ca2+ flux in ZAP-70+ CLL cells but only modest-to-poor Ca2+ flux in ZAP-70-negative CLL cells. Furthermore, ZAP-70+ CLL cells (n=10) responded to CXCL12 stimulation with increased and prolonged phosphorylation of ERK and MEK compared to ZAP-70-negative CLL cells (n=9). To investigate the underlying mechanism for MEK activation in ZAP-70+ CLL, we used small molecule inhibitors and found that CXCL12-induced phosphorylation of ERK and MEK could be blocked by sorafenib, a small molecule inhibitor of RAF. The role of RAF was further supported using KG5, a kinase inhibitor of RAF signaling through B-RAF and C-RAF in addition to platelet-derived-growth-factor-receptor (PDGFR) alpha and beta, Flt3, and Kit. As a control, we used a kinase inhibitor that targets all of these kinases except B- and C-RAF (KG1) and found it could not inhibit MEK activation. The involvement of Raf was further substantiated using GW5074, an inhibitor of B-RAF and C-RAF. Both KG5 and GW5074 could inhibit CXCL12-induced MEK activation in ZAP-70+ CLL samples. CXCL12-induced activation of MEK/ERK was not affected by sunitinib, an inhibitor of non-RAF kinases that also are inhibited by sorafenib, including VEGFR, PDGFR, Flt3, and c-Kit. Sorafenib not only inhibited MEK/ERK activation but also caused apoptosis of CLL cells whereby ZAP-70+ CLL cells showed incresed sensitivity to lower doses of sorafenib. Consistent with these results we found that ZAP-70+ CLL cells had a greater responsiveness to CXCL12 for survival in vitro than did ZAP-70-negative CLL cells. We conclude that CXCL12 can enhance survival particularly of ZAP-70+ CLL cells via a RAF dependent pathway, which can be targeted by the kinase inhibitor sorafenib. As such, sorafenib might be effective in blocking the protective influence of the microenvironment on CLL cells, suggesting that this drug could have activity in the treatment of patients with this disease. Disclosures: No relevant conflicts of interest to declare.

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