Protein Expression Profiling of Cytokines and Cytokine Receptors on Purified Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4709-4709
Author(s):  
Zhifeng Yu ◽  
Baohua Sun ◽  
Hagop M. Kantarjian ◽  
Hesham M. Amin ◽  
Xiaoping Sun
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Conditioned Medium ◽  
Mononuclear Cells ◽  
Lymphocytic Leukemia ◽  
Cytokine Receptors ◽  
Cellular Interactions ◽  
Serum Free

Abstract Chronic lymphocytic leukemia (CLL) B-cells rapidly undergo apoptosis when cultured in vitro, which contrasts with their prolonged survival in vivo. Multiple cytokines and cytokine receptors are believed to work together to regulate the survival of CLL cells. The literature is conflicting as to whether the CLL cells themselves produce significant amounts of cytokines compared with normal B-cells and how the CLL cells respond to these cytokines. This discrepancy is largely due to the different experimental conditions that have been used whereby various amounts of exogenous cytokines were introduced into the experimental system from, for example, the serum used to supplement the culture medium and the lysate or conditioned medium of CLL cells where other types of mononuclear cells were not removed. The aim of the current study is to reveal the intrinsic production and secretion of cytokines and cytokine receptors in CLL cells when exogenous sources are minimized. We purified CD19+ cells by magnetic beads from peripheral blood mononuclear cells of five CLL patients who had stage I or II disease and had not received any therapy. CD19+ cells from healthy donors were used as control. We used a cytokine antibody array approach that simultaneously measured 174 cytokines and cytokine receptors. We determined both intracellular levels in purified CLL cells and secreted levels in serum-free conditioned medium. The intracellular levels of cytokines and cytokine receptors of the purified CLL cells and the normal B-cells were not significantly different. However, the secretion of interleukin-6 (IL-6) was 3.0 times lower (p = 0.038) and that of eotaxin was 2.2 times higher (p = 0.028) in CLL-conditioned medium than in normal B-cell-conditioned medium. We further studied the effect of IL-6 and anti-IL-6 antibody on the apoptosis of purified CLL B-cells in serum-free culture, but no significant change was found in the presence or absence of IL-6 or IL-6 antibody. Except for IL-6 and eotaxin, our results suggest that CLL cells and their normal counterparts produce and secrete similar amounts of cytokines and cytokine receptors in vitro and that the in vivo longevity of CLL cells may be due to the concerted effects of various molecules and cellular interactions in the microenvironment.

IL4 and CD40L Prevent Apoptosis of Chronic Lymphocytic Leukemia Cells Via Intracellular pSTAT6 and NFkB Signaling and Not Via Receptor Kinetics

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3893-3893
Author(s):  
Daniel Mertens ◽  
Nupur Bhattacharya ◽  
Sarah Häbe ◽  
Hartmut Döhner ◽  
Stephan Stilgenbauer
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Conditioned Medium ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Malignant Cells ◽  
Downstream Signaling ◽  
Receptor Kinetics

Abstract Abstract 3893 Chronic lymphocytic leukemia (CLL) cells are highly dependent on microenvironmental input for their extended survival in vivo, but the underlying molecular mechanism is still unclear. Compared to non-malignant B-cells, CLL cells are more responsive to contact dependent complex stimuli like coculture on bone marrow derived stromal cell lines of both human (p<0.0001) and murine origin (p<0.01), but also to soluble factors (human conditioned medium p<0.0001, murine conditioned medium p<0.001, all student′s t-test). In order to understand the intrinsic difference of the anti-apoptotic phenotype of CLL cells, the signalling circuitry of the malignant cells was modelled. Compared to candidate ligands like SDF-1 (at concentrations between 10–1000ng/ml), BAFF (250–1000ng/ml), APRIL (250–1000ng/ml) and soluble anti-IgM (1–25μg/ml), the factors CD40L (10–2000ng/ml) and IL4 (0.1–10ng/ml) were the most efficient ligands in rescuing CLL cells from spontaneous death in vitro. The dose response of IL4 and CD40L displayed different saturation and cooperativity between CLL cells and non-malignant B-cells. Using IL4, saturation was reached both for CLL cells and B-cells at 0.2pM, but at 52% survival (+/− 8%) for CLL cells and 28% (+/−7%) for B-cells, and the estimated dissociation constant Kd was 0.01pM for both ligands. For CD40L, CLL cell survival reached saturation at 40nM, while no saturation was reached for B-cells. Intriguingly, B-cells showed cooperativity in their response to CD40L, with a cooperativity coefficient of 2.0 and a Kd of 70pM, while cooperativity for CD40L was lost in CLL cells (Kd of only 2.6pM). This pointed towards distinct differences in ligand-receptor interactions or in downstream signaling between CLL cells and non-malignant B-cells. However, high-throughput spatial analysis with a microscope-coupled cytometer did not show differences of receptor quantity or receptor distribution between malignant and non-malignant cells. In contrast, quantity and phosphorylation levels of downstream signalling nodes like STAT6 (measured by flow cytometry and validated by Western-blot) and the activity of NF-kB (p65 binding to DNA measured by oligonucleotide-coupled ELISA) were higher in CLL cells compared to B-cells from healthy donors. Therefore, the defect in IL4 and CD40L signalling that leads to an enhanced survival in CLL cells is likely caused by changes in the intracellular circuitry. Disclosures: No relevant conflicts of interest to declare.

BAFF Accelerates Development of Chronic Lymphocytic Leukemia in TCL1 Transgenic Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1117-1117
Author(s):  
Thomas Enzler ◽  
George F. Widhopf ◽  
Jason Lee ◽  
Weizhou Zhang ◽  
Carlo M. Croce ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mononuclear Cells ◽  
Lymphoproliferative Disease ◽  
Lymphocytic Leukemia ◽  
Brdu Incorporation

Abstract The B cell- activating factor of the tumor necrosis factor family (BAFF) is a potent regulator of normal B cells. We recently showed that BAFF supports chronic lymphocytic leukemia (CLL) B cell survival in vitro through activation of the canonical NF-kB pathway. To study the influence of BAFF on CLL development, we crossed BAFF transgenic (Tg) mice with mice that express human TCL1 under a B cell specific promoter/enhancer, and that are known to develop a lymphoproliferative disease resembling human B-CLL. BAFF/TCL1-Tg mice had a shorter mean survival than either TCL1-Tg or BAFF-Tg mice (12 mice each; BAFF/TCL1-Tg mice 9.6±3.4 months; TCL1-Tg 17.2±3.9; BAFF-Tg 17.9±3.6; B6 wildtype (wt) >19.2). To monitor for the development of CLL, mice were bled at 6-week intervals starting at 3 months of age, and blood mononuclear cells (PBMC) were analyzed via flow cytometry using fluorochrome-conjugated antibodies for murine CD5, CD3, CD45R, and human TCL1. Whereas all BAFF/TCL1-Tg mice began to develop a pathological CD5+CD3−CD45Rlo cell population at 3 months of age, such a population was not observed in TCL1-Tg mice before 6 months of age. BAFF-Tg or wt mice did not develop CD5+CD3−CD45Rlo cells over the entire observation period (26 months). CD5+CD3−CD45Rlo B cells expressed the TCL1 transgene. Over time, the CD5+CD3−CD45Rlo population increased in BAFF/TCL1-Tg mice, coming to represent >99% of the total PBMC of 9-month-old animals. To examine the capacity of these cells to propagate, 1x106 CD5+CD3−CD45Rlo B cells were transferred i.v. into either BAFF-Tg or wt mice that previously were irradiated with 600 rad. Ten days after transfer, CD5+TCL1+ cells were detected in BAFF-Tg, but not in wt recipients. Most CLL cells were located in the liver and spleen, as assessed by bioluminescent-based imaging of mice that received luciferase expressing CLL cells. Subsequent examination upon autopsy at 6 months of age, however, revealed that the majority of CLL cells populated the spleens of the recipient mice, which were massively enlarged. At this age, CLL cells also were found in wt recipient mice, although tumor burden was less than 20% of that of BAFF-Tg recipients (n=3 per group). We found that BAFF did not promote CLL cell proliferation in vitro or in vivo using assays to measure BrdU incorporation and flow cytometry to evaluate for enhanced intracellular expression of Ki67. However, BAFF induced CLL cells to express high levels of several anti-apoptotic proteins (e.g. Bcl-XL, Bcl-2, Bim, and A1/Bfl1). Also, while death-associated protein kinase 1 was repressed in CLL cells of TCL1-Tg mice, CLL cells of BAFF/TCL1-Tg mice expressed high-levels. Because of this, we examined whether treatment with BAFF-neutralizing BR3-Fc could influence the survival of CLL cells that were adoptively transferred into BAFF-Tg mice. We found that i.p. injection of 200 ug BR3-Fc into the recipient animals reduced the numbers of circulating CLL cells by nearly 20% (18.2%±5.3%; n=3) within 6 days. These data indicate that BAFF can accelerate the development of CLL cells in TCL1-Tg mice by promoting their survival. Because BAFF can similarly promote survival of human CLL cells, BAFF, and the signaling pathways it activates in neoplastic B cells, could be targeted for the development of novel therapies for this disease.

Distinctive features of “nurselike” cells that differentiate in the context of chronic lymphocytic leukemia

Blood ◽  
2002 ◽  
Vol 99 (3) ◽  
pp. 1030-1037 ◽  
Author(s):  
Nobuhiro Tsukada ◽  
Jan A. Burger ◽  
Nathan J. Zvaifler ◽  
Thomas J. Kipps
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Mononuclear Cells ◽  
Cell Types ◽  
Lymphocytic Leukemia ◽  
Blood Monocytes ◽  
Hla Dq ◽  
Blood Mononuclear Cells

Abstract A subset of blood mononuclear cells from patients with chronic lymphocytic leukemia (CLL) can differentiate in vitro into “nurselike” cells (NLCs) that can protect CLL cells from apoptosis. NLCs express cytoplasmic vimentin and stromal-derived factor 1 (SDF-1). NLCs also express CD14, as well as CD11b, CD33, CD40, CD45RO, CD68, CD80, CD86, HLA-DQ, and HLA-DR, but not CD1a, CD2, CD3, CD11c, CD19, CD45RA, CD83, CD106, or CD154. Consistent with this phenotype, NLCs failed to differentiate from blood mononuclear cells that were depleted of CD14+ cells or from isolated CD19+cells. CD14+ blood cells of healthy donors could differentiate into cells with the morphology and phenotype of NLCs when cultured in direct contact with CLL B cells, but not with normal B cells. Despite expressing antigens in common with blood monocytes, monocyte-derived dendritic cells, and macrophages, NLCs expressed significantly higher levels of CD68 than these other cell types. Consistent with the notion that NLCs are present in vivo, CD14+ splenocytes from CLL patients have NLC morphology and express significantly higher levels of CD68 than CD14+splenocytes from persons without known B-cell malignancy. These findings indicate that although NLCs may differentiate from blood monocytes, they probably represent a distinctive hematopoietic cell type that exists in vivo, differentiates from hematopoietic CD14+ cells in the context of CLL, and in turn protect CLL cells from apoptosis via a mechanism that is independent of CD106 (vascular cell adhesion molecule-1). The interaction between CLL cells and NLCs may represent a novel target for therapy of patients with this disease.

scholarly journals Chronic Lymphocytic Leukemia B Cells Can Undergo Somatic Hypermutation and Intraclonal Immunoglobulin VHDJH Gene Diversification

2002 ◽  
Vol 196 (5) ◽  
pp. 629-639 ◽  
Author(s):  
Carmela Gurrieri ◽  
Peter McGuire ◽  
Hong Zan ◽  
Xiao-Jie Yan ◽  
Andrea Cerutti ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Lymphocyte ◽  
Somatic Hypermutation ◽  
Point Mutations ◽  
Single Strand Conformation Polymorphism ◽  
Lymphocytic Leukemia ◽  
Gene Diversification

Chronic lymphocytic leukemia (CLL) arises from the clonal expansion of a CD5+ B lymphocyte that is thought not to undergo intraclonal diversification. Using VHDJH cDNA single strand conformation polymorphism analyses, we detected intraclonal mobility variants in 11 of 18 CLL cases. cDNA sequence analyses indicated that these variants represented unique point-mutations (1–35/patient). In nine cases, these mutations were unique to individual submembers of the CLL clone, although in two cases they occurred in a large percentage of the clonal submembers and genealogical trees could be identified. The diversification process responsible for these changes led to single nucleotide changes that favored transitions over transversions, but did not target A nucleotides and did not have the replacement/silent nucleotide change characteristics of antigen-selected B cells. Intraclonal diversification did not correlate with the original mutational load of an individual CLL case in that diversification was as frequent in CLL cells with little or no somatic mutations as in those with considerable mutations. Finally, CLL B cells that did not exhibit intraclonal diversification in vivo could be induced to mutate their VHDJH genes in vitro after stimulation. These data indicate that a somatic mutation mechanism remains functional in CLL cells and could play a role in the evolution of the clone.

Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2777-2783 ◽  
Author(s):  
Luisa Granziero ◽  
Paolo Ghia ◽  
Paola Circosta ◽  
Daniela Gottardi ◽  
Giuliana Strola ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Survivin Expression ◽  
Lymphocytic Leukemia ◽  
Cd40 Stimulation ◽  
Cell Chronic Lymphocytic Leukemia

Abstract In B-cell chronic lymphocytic leukemia (B-CLL), defective apoptosis causes the accumulation of mature CD5+ B cells in lymphoid organs, bone marrow (BM), and peripheral blood (PB). These cells are the progeny of a proliferating pool that feeds the accumulating compartment. The authors sought to determine which molecular mechanisms govern the proliferating pool, how they relate to apoptosis, and what the role is of the microenvironment. To begin to resolve these problems, the expression and modulation of the family of inhibitor of apoptosis proteins (IAPs) were investigated, with consideration given to the possibility that physiological stimuli, such as CD40 ligand (CD40L), available to B cells in the microenvironment, might modulate IAP expression. The in vitro data on mononuclear cells from PB or BM of 30 patients demonstrate that B-CLL cells on CD40 stimulation express Survivin and that Survivin is the only IAP whose expression is induced by CD40L. Through immunohistochemistry, in vivo Survivin expression in lymph node (LN) and BM biopsies was evaluated. In reactive LN, Survivin was detected only in highly proliferating germinal center cells. In LN from patients with B-CLL, Survivin was detected only in pseudofollicles. Pseudofollicle Survivin+ cells were actively proliferating and, in contrast to Survivin+ B cells found in normal GC, were Bcl-2+. In B-CLL BM biopsies, CD5+, Survivin+ cells were observed in clusters interspersed with T cells. These findings establish that Survivin controls the B-CLL proliferative pool interfacing apoptosis and that its expression may be modulated by microenvironmental stimuli.

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.

Chronic Lymphocytic Leukemia-Derived Exosomes Stimulate Cells From The Microenvironment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3683-3683
Author(s):  
Jerome Paggetti ◽  
Guy J. Berchem ◽  
Etienne Moussay
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Bone Marrow Microenvironment ◽  
Lymphocytic Leukemia ◽  
Target Cells ◽  
And Migration

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in the blood and the primary lymphoid organs of long-lasting, mature, but non-functional B lymphocytes. Although CLL B cells can survive for long time periods in vivo, cells are undergoing apoptosis relatively quickly in vitro. This spontaneous apoptosis and their sensitivity to drugs is strongly reduced in presence of bone marrow mesenchymal stem cells (MSC) and endothelial cells (EC), which provide anti-apoptotic stimuli to CLL cells via direct contact or secretion of soluble factors. We recently reported the first profiling of circulating miRNA obtained from plasma of CLL patients (Moussay et al., PNAS, 2011). Specific miRNAs were found at higher level in the plasma of CLL patients compared to healthy donors. Exosomes, which are small extracellular vesicles of 50-150 nm originating from endosomes, are now known to efficiently transport nucleic acids and transfer mRNA, microRNA and proteins to target cells. Therefore, exosomes constitute a new component of intercellular communication and their role in CLL remains totally unknown. The specific miRNA signature from plasma of CLL patients combined with our observations that primary CLL B cells can transfer vesicles to MSC through 0.4 µm culture inserts in vitro prompted us to investigate whether CLL B cells secrete exosomes that could modify cells of the bone marrow microenvironment to produce tumor growth promoting factors locally in order to favor their own survival. We isolated, purified and characterized exosomes derived from CLL cell lines, primary cells culture supernatants and plasma from CLL patients. Proteins, mRNA and microRNAs contents were evaluated by high-throughput methods (LC-MS, microarrays) revealing in particular the presence of oncogenic molecules. In vitro, purified CLL-exosomes were found to rapidly enter target cells (already after 1h in MSC and endothelial cells) and to transfer proteins and miRNA. Flow cytometry showed that transferred proteins were expressed at cell surface. Luciferase reporter assay confirmed that miRNAs were efficient in targeting cellular mRNA. Exosomes could also be taken up ex vivo and in vivo by mouse bone marrow cells. Functionally, CLL-exosomes activated key signaling pathways (PI3K, AKT, and MAPK) Immunoblotting indicated the rapid phosphorylation of kinases after 5 min of incubation with CLL-exosomes and the subsequent activation of the canonical NF-kB pathway. We also observed that CLL-exosomes modulated gene expression in target cells among which cytokines (BAFF, IL-6, and IL-8), chemokines (CCL2/MCP-1, CCL5/RANTES, and CXCL1), and other factors involved in cell adhesion and migration (ICAM-1 and MMP-1). These factors were also secreted in the supernatants of MSC and EC as detected by antibody arrays. Exosomes were also shown to increase MSC and EC proliferation, to stimulate actin remodeling, cell migration and to enhance EC angiogenic capabilities (tube formation and aortic ring assays). In conclusion, CLL-exosomes contain pro-oncogenic molecules and strongly affect key functions of MSC and EC which are critical component of the bone marrow microenvironment. Activation of these cells by CLL-exosomes led to release of cytokines/chemokines and oncogenic factors that could promote angiogenesis and also favor leukemic cells survival and migration. Our findings may lead to applications in both diagnosis and therapy development. Molecules identified at the surface or inside CLL-exosomes may be further used as cancer biomarkers. Finally, the description of cell-to-cell communication mechanisms will generate opportunities of innovative therapeutic strategies and confirms the crucial role of exosomes in the development of CLL. Disclosures: No relevant conflicts of interest to declare.

CLL Cells from Ibrutinib-Induced Lymphocytosis of Relapsed/Refractory Chronic Lymphocytic Leukemia Patients Are Responsive to Obinutuzumab, but Not Rituximab, Ex Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4157-4157 ◽  
Author(s):  
Loïc Ysebaert ◽  
Christian Klein ◽  
Anne Quillet-Mary
Keyword(s):  
Lymph Node ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Peripheral Blood ◽  
Absolute Number ◽  
Lymphocytic Leukemia ◽  
Viable Cell Number ◽  
Cluster 2

Abstract Introduction: Ibrutinib is an irreversible first-in-class inhibitor of BTK (Bruton tyrosine kinase) approved for the therapy of relapsed/refractory chronic lymphocytic leukemia (R/R CLL). The drug mediates a transient increase in circulating CLL cells together with reduction in spleen and lymph node size, by both cellular mobilization and apoptosis of resident CLL cells (Herman SE, et al. Blood 2014;123:3286-95). These events occur with important patients' inter-variability (Herman SE, et al. Leukemia 2014;28:2188-96), one cluster of patients presents with greater peak lymphocytosis (resolving between 1 to more than 6 months), while another cluster presents with rapid resolution of lymphocytosis and lymph node/spleen size within 2 months. Upon such dramatic shifts in disease distribution the first 2 months of therapy (and sometimes lasting >6-12 months), the question of phenotypic changes, sensitivity to monoclonal antibodies (MoAbs), and subclonal diversity of circulating cells remains central for further combination studies. In this study, we evaluated changes in CD5, CD19, and CD20 expression in vitro/in vivo, and peripheral blood side population (SP) cells (a fraction highly enriched in chemorefractory cells, Gross E, et al. Leukemia 2010;24:1885-92) upon ibrutinib therapy. We also investigated whether patterns of lymphocytosis may predict for response to rituximab (RTX) or obinutuzumab (GA101). Methods: R/R CLL patients (n=25) median prior lines=4, range=2-8), PBMCs were collected before ibrutinib initiation and after 1 and 2 months of therapy. PBMC were seeded at 10 x 106 cells/mL in culture medium and treated for 7 days with 10µg/mL control IgG1 (trastuzumab), RTX or obinutuzumab. The specific percentage of remaining B cells in MoAbs-treated samples was calculated as (absolute number in treated samples/absolute number in control samples) x 100. For each condition, absolute number of remaining B cells =total viable cell number (trypan blue exclusion determination) x % of viable CD19+/CD5+ lymphocytes (flow cytometry determination). For statistical analyses, Student's test (paired, two-sided) was used (*p<0.05;**p<0.01;***p<0.001). Results: We firstanalyzed patterns ofabsolute lymphocytes count ( ALC) across 23 patients receiving ibrutinib (Fig 1a) to classify them into two clusters as previously published (Fig 1b): Cluster 1 and cluster 2 did not differ significantly in terms of initial lymphocytosis, line of therapy, gender, karyotype, IgHV. Interestingly, the SP fraction in peripheral blood was significantly increased (median: 5/microL before ibrutinib, 10/microL at peak lymphocytosis), suggesting mobilization of resident SP cells, although no apoptosis was detected (in vitro or in vivo) with ibrutinib. We next assessed CD5, CD19 and CD20 levels in vitro (n=22) and in vivo (n=15) upon ibrutinib therapy. In vitro, ibrutinib significantly reduced CD20 (Fig 2a) and CD19 surface expression, but not CD5; nevertheless anti-CD20 MoAbs still had activity in vitro (Fig 2b). Expression levels were not linked to clusters 1 or 2. Finally we compared RTX- and obinutuzumab-induced B-cell depletion before administration of ibrutinib, and at various sampling time points (1 to 6 months). Obinutuzumab induced significantly superior depletion at various timepoints than RTX. More interestingly, when analysis was performed from paired samples before/during ibrutinib therapy from the same ibrutinib-exposed patients, only obinutuzumab-induced depletion was increased in cluster 2 (Fig 3). Conclusions: Ongoing and planned clinical studies evaluate the combination of ibrutinib and obinutuzumab in CLL (first-line and relapsed). Some concerns have emerged due to published preclinical data showing that ibrutinib can interfere with efficacy of therapeutic antibodies. Here, we suggest that ibrutinib-exposed CLL cells, despite wide inter-patient heterogeneity, are targetable with obinutuzumab. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Klein: Roche: Employment.

scholarly journals Fas-ligand (CD178) and TRAIL synergistically induce apoptosis of CD40-activated chronic lymphocytic leukemia B cells

Blood ◽  
2005 ◽  
Vol 105 (8) ◽  
pp. 3193-3198 ◽  
Author(s):  
Frank Dicker ◽  
Arnon P. Kater ◽  
Tetsuya Fukuda ◽  
Thomas J. Kipps
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Fas Ligand ◽  
Chinese Hamster ◽  
Death Receptors ◽  
Lymphocytic Leukemia ◽  
Death Receptor 5 ◽  
Cd40 Ligation

AbstractChronic lymphocytic leukemia (CLL) B cells become sensitive to Fas (CD95)–mediated apoptosis 3 to 5 days after CD40 ligation. However, CD4+ cytotoxic T lymphocytes (CTLs) can kill CLL B cells via a Fas-ligand (CD178)–dependent process within 24 hours after CD40 cross-linking, when ligation of CD95 alone is insufficient to induce apoptosis. In addition to CD95, CD40-activated CLL cells also express DR5, a receptor for tumor-necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) that is expressed by CD4+ CTL. In addition, CD40 ligation in vitro and in vivo induces CLL cells to express the proapoptotic protein, BH3 interacting domain death agonist (Bid), which can facilitate crosstalk between mitochondrial-dependent, apoptosis-inducing pathways and death receptors, such as death receptor 5 (DR5). To evaluate whether ligation of CD95 and/or DR5 can induce apoptosis of CD40-activated CLL cells, we generated artificial cytotoxic effector cells that express both human TRAIL and CD178 (Chinese hamster ovary [CHO]–CD178/TRAIL) or only TRAIL (CHO-TRAIL) or CD178 (CHO-CD178). CHO-CD178/TRAIL cells were significantly more effective in killing CD40-activated CLL cells than either CHO-TRAIL or CHO-CD178 and, unlike the latter, could kill CLL cells 24 hours after CD40 ligation. We conclude that CD40 ligation induces CLL cells to express the proapoptotic molecule Bid and the death receptors CD95 and DR5, the latter of which can act synergistically to induce caspase-dependent apoptosis of CD40-activated CLL B cells.

scholarly journals Potent Inhibition of the Growth and Induction of Apoptosis in Lymphoma By the Anthelminthic Drug Niclosamide: In Vitro Data

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5131-5131
Author(s):  
Junaid Ansari ◽  
Paula Polk ◽  
Jeffrey Aufman ◽  
Guillermo A Herrera ◽  
James Cardelli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Lymphocytic Leukemia ◽  
Lymphoma Cell ◽  
B Lymphoma

Abstract Background and Purpose: Niclosamide is an anthelminthic drug which has been used for the treatment of human parasitic infections for many years. Niclosamide interacts with lysosomes and induces autophagy. In recent years, it has demonstrated anti-cancer potential in leukemia, breast cancer, colon cancer, myeloma, ovarian, prostate and lung cancer models. Multiple pathways like Wnt/β-catenin, mTORC1, STAT3, NF-κB and notch signaling were reported to be involved. Only limited studies were done in lymphoma models. We hypothesized that niclosamide may also have in vitro and in vivo activities in lymphomas. Non-Hodgkin lymphomas generally respond well to chemotherapy and/ or immunotherapy, however many patients relapse and ultimately become refractory. Relapses are often caused by tumor stem cells not eliminated by cytostatic drugs. Therefore new treatment approaches and new targets are desirable. Materials and Methods: Established B lymphoma cell lines were exposed to different concentrations of niclosamide (0.1-4µM) and IC50 was calculated at 24, 48 and 72 hours. The cell concentration, viability and proliferation were assessed by CellTiter-Blue viability and trypan blue exclusion assays. Apoptosis was assessed by a combined annexin-V/ propidium iodide stain. Gene expression changes were studied using GeneChip Human Transcriptome Array 2.0 (Affymetrix) with 44 699 annotated genes. Colony forming assays were performed in methylcellulose. Ultrastructural changes were studied using a Hitachi electron microscope. As normal controls, peripheral blood mononuclear cells from individuals without active cancer were incubated with niclosamide for up to 72 hours. Samples from patients with chronic lymphocytic leukemia were also treated under the same conditions. Results: Treatment with niclosamide at doses as low as 0.1 μM resulted in time-and dose- dependent apoptosis, cytotoxicity and inhibition of proliferation in aggressive lymphoma cell lines. The 50% inhibitory concentration in a proliferation assay (mean of data at 24, 48 and 72 hours) is shown in the Table below. Niclosamide also inhibited clonal growth in semi-solid media. Electron microscopy showed that filopodia increased and lipid vacuoles developed whereas mitochondria were less numerous and had fewer cristae (when KOPN-8 was treated with 0.5 μM for 48 hours). The viability of mononuclear cells from 8 individuals without lymphoma was unchanged (or minimally decreased) when incubated with niclosamide. As far as cells from two patients with untreated chronic lymphocytic leukemia are concerned, no cytotoxicity was observed at doses between 0.5 and 5 μM. Gene expression changes were studied the cell lines Daudi and KOPN-8 treated with 2.5 μM for 3 and 6 h. 96 genes were consistently overexpressed , 59 down-regulated. Ten out of the 96 overexpressed genes involved the TNF pathway and immunoregulation including CD95. Thirteen out of the 59 down-regulated genes are involved in mitochondrial function. Table.Cell lineDescription of Cell TypeIC 50STDDaudiBurkitt lymphoma cell line0.37 μM± 0.12HBL-2Diffuse large B cell lymphoma cell line0.68 μM± 0.15KOPN-8B precursor ALL cell line0.6 μM± 0.08RamosBurkitt lymphoma cell line0.58 μM± 0.04RajiBurkitt lymphoma cell line0.65 μM± 0.10SU-DHL4-VRVincristine resistant lymphoma cell line0.5 μM± 0.02 Conclusion: Niclosamide effectively inhibits the proliferation of B lymphoma cell lines and induces apoptosis. Preliminary data show that Niclosamide targets genes involved in the TNF pathway and interferes with mitochondrial function. Normal lymphocytes are not sensitive to niclosamide. The in-vitro activity of niclosamide is at least comparable or superior to the activity seen in other malignancies. Niclosamide may target drug-resistant lymphoma stem cells and has clinical potential. We plan to study combination treatments and perform in vivo studies. Acknowledgments: The authors thank Drs. Borje Andersson, Shile Huang, Nakle Saba, Ben Valdez and Ellen Vitetta for their kind gift of cell lines. Disclosures No relevant conflicts of interest to declare.

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