Overexpression of Lipases Enables Specific Cytotoxicity by the Lipase Inhibitor Orlistat in Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2800-2800
Author(s):  
Christian P. Pallasch ◽  
Janine Schwamb ◽  
Alexandra Schulz ◽  
Svenja Debey ◽  
David Kofler ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Therapeutic Potential ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Lipase Inhibitor ◽  
In Vivo Experiments ◽  
Induction Of Apoptosis

Abstract Genome-wide gene expression profiling of chronic lymphocytic leukemia (CLL) cells in comparison to healthy donor CD5-positive B-cells revealed deregulated expression of lipase-associated genes. A set of 19 lipase activity defined genes, e.g. LPL, phospholipases A1, −A2, −C and −D2 family members and other lipase-associated genes were overexpressed in CLL. Recently lipoprotein lipase (LPL) was identified as prognostic factor in CLL. Here we show that the expression of LPL in CLL is induced by B-cell receptor (BCR) stimulus both in mutated and unmutated CLL samples. In native and BCR-stimulated CD5+ healthy B-cells no LPL-expression was detected. Antigenic stimulation via the BCR is thought to be functionally and prognostically relevant in CLL pathogenesis, LPL expression in CLL cells could reflect a permanent BCR-stimulus due to autoantigens including also unmutated IgVH cases. We hypothesized that the overexpression of lipases and especially of LPL reveals a putative therapeutic target by lipase inhibition through the the FDA-approved obesity drug and lipase inhibitor orlistat (tetrahydrolipstatin), which is known to inhibit LPL as well. Treatment of CLL cells with orlistat in vitro revealed significant cytotoxicity and induction of apoptosis in primary CLL cells with an IC50 of 5.48 μM (n=18). In comparison, no significant cytotoxicity was seen with healthy PBMC’s (n=12; p<0.001), even using high doses of orlistat up to 100 μM. Induction of apoptosis induction was observed both in low-risk (ZAP70 negative, n=15) and high-risk (ZAP70-positive, n=10) CLL samples. Orlistat mediated cytotoxicity was slightly decreased by BCR stimulus while additive cytotoxic effects where observed in combination with fludarabine treatment. Susceptibility to orlistat treatment was not dependent on Binet stage. In summary, we provide in vitro data for a criticial role of fatty acid metabolism in CLL pathogenesis and suggest a therapeutic potential of the lipase inhibitor orlistat. No severe systemic side effects of orlistat have been observed in several animal models and in the current clinical application as anti-obesity drug. Orlistat seems to be a promising candidate for an anti-leukemic therapy in CLL, ongoing in vivo experiments applying the TCL1 mouse model will further elucidate the therapeutic potential of orlistat.

Chronic lymphocytic leukemia: revelations from the B-cell receptor

Blood ◽  
2004 ◽  
Vol 103 (12) ◽  
pp. 4389-4395 ◽  
Author(s):  
Freda K. Stevenson ◽  
Federico Caligaris-Cappio
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Cell Of Origin ◽  
Regulatory B Cell ◽  
V Genes

Abstract The finding that chronic lymphocytic leukemia (CLL) consists of 2 clinical subsets, distinguished by the incidence of somatic mutations in the immunoglobulin (Ig) variable region (V) genes, has clearly linked prognosis to biology. Antigen encounter by the cell of origin is indicated in both subsets by selective but distinct expression of V genes, with evidence for continuing stimulation after transformation. The key to distinctive tumor behavior likely relates to the differential ability of the B-cell receptor (BCR) to respond. Both subsets may be undergoing low-level signaling in vivo, although analysis of blood cells limits knowledge of critical events in the tissue microenvironment. Analysis of signal competence in vitro reveals that unmutated CLL generally continues to respond, whereas mutated CLL is anergized. Differential responsiveness may reflect the increased ability of post-germinal center B cells to be triggered by antigen, leading to long-term anergy. This could minimize cell division in mutated CLL and account for prognostic differences. Unifying features of CLL include low responsiveness, expression of CD25, and production of immunosuppressive cytokines. These properties are reminiscent of regulatory T cells and suggest that the cell of origin of CLL might be a regulatory B cell. Continuing regulatory activity, mediated via autoantigen, could suppress Ig production and lead to disease-associated hypogammaglobulinemia. (Blood. 2004;103:4389-4395)

Nurture versus Nature: The Microenvironment in Chronic Lymphocytic Leukemia

Hematology ◽  
2011 ◽  
Vol 2011 (1) ◽  
pp. 96-103 ◽  
Author(s):  
Jan A. Burger
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Disease Progression ◽  
Drug Testing ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
In Vivo Models

Abstract Intrinsic factors such as genetic lesions, anti-apoptotic proteins, and aberrant signaling networks within leukemia cells have long been the main focus of chronic lymphocytic leukemia (CLL) research. However, over the past decade, it became increasingly clear that external signals from the leukemia microenvironment make pivotal contributions to disease progression in CLL and other B-cell malignancies. Consequently, increasing emphasis is now placed on exploring and targeting the CLL microenvironment. This review highlights critical cellular and molecular pathways of CLL-microenvironment cross-talk. In vitro and in vivo models for studying the CLL microenvironment are discussed, along with their use in searching for therapeutic targets and in drug testing. Clinically, CXCR4 antagonists and small-molecule antagonists of B cell receptor (BCR)-associated kinases (spleen tyrosine kinase [Syk], Bruton's tyrosine kinase [Btk], and PI3Kδ) are the most advanced drugs for targeting specific interactions between CLL cells and the miocroenvironment. Preclinical and first clinical evidence suggests that high-risk CLL patients can particularly benefit from these alternative agents. These findings indicate that interplay between leukemia-inherent and environmental factors, nature and nurture determines disease progression in CLL.

scholarly journals Functional and clinical relevance of VLA-4 (CD49d/CD29) in ibrutinib-treated chronic lymphocytic leukemia

2018 ◽  
Vol 215 (2) ◽  
pp. 681-697 ◽  
Author(s):  
Erika Tissino ◽  
Dania Benedetti ◽  
Sarah E.M. Herman ◽  
Elisa ten Hacken ◽  
Inhye E. Ahn ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Progression Free Survival ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Clinical Activity ◽  
Independent Manner ◽  
Btk Inhibitor

The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, which antagonizes B cell receptor (BCR) signals, demonstrates remarkable clinical activity in chronic lymphocytic leukemia (CLL). The lymphocytosis experienced by most patients under ibrutinib has previously been attributed to inhibition of BTK-dependent integrin and chemokine cues operating to retain the tumor cells in nodal compartments. Here, we show that the VLA-4 integrin, as expressed by CD49d-positive CLL, can be inside-out activated upon BCR triggering, thus reinforcing the adhesive capacities of CLL cells. In vitro and in vivo ibrutinib treatment, although reducing the constitutive VLA-4 activation and cell adhesion, can be overcome by exogenous BCR triggering in a BTK-independent manner involving PI3K. Clinically, in three independent ibrutinib-treated CLL cohorts, CD49d expression identifies cases with reduced lymphocytosis and inferior nodal response and behaves as independent predictor of shorter progression-free survival, suggesting the retention of CD49d-expressing CLL cells in tissue sites via activated VLA-4. Evaluation of CD49d expression should be incorporated in the characterization of CLL undergoing therapy with BCR inhibitors.

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.

Anergic Response to B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia: The Differing Roles of IgD and IgM in the Microenvironment and Peripheral Blood.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2889-2889
Author(s):  
Tom Butler ◽  
Alexander Montoya ◽  
Andrew James Clear ◽  
Rita Coutinho ◽  
David C Taussig ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling ◽  
B Cell Receptor Signaling ◽  
Cell Receptor Signaling

Abstract Abstract 2889 Chronic Lymphocytic Leukemia (CLL) cells depend on B cell receptor signaling as well as other microenvironmental survival signals (1). Drugs targeting the BCR signaling pathways are showing exciting results in CLL clinical trials. A peculiarity of CLL is that IgD signaling is generally preserved, whilst IgM signaling is decreased and it has been suggested that this pattern mimics anergic B-cells, and might be consistent with chronic autoantigen exposure. We examined the differing roles of IgM and IgD signaling in CLL using a theoretical framework of anergy. Peripheral blood (PB) CLL cells exhibited higher IgD expression, as compared to IgM (n=204, p<0.0001), but this did not have prognostic impact. When we examined IgM and IgD expression in LN biopsies compared to paired PB (n=10) expression, IgM expression was lower in LN (p=0.002) whilst IgD expression was unchanged. Although the number of these paired samples is small, cases with lower LN IgM levels had poorer prognosis, and we are investigating this further with a larger cohort. We hypothesize that reduced LN IgM expression reflects antigen engagement and an anergic response in the microenvironment. We sought to replicate Mockridge et al' s model of reversible anergy (2) by monitoring the dynamic changes in IgM/D expression after in vitro incubation. Most (18/20) PB CLL samples underwent calcium (Ca) flux after IgD crosslinking, whereas only 13/20 cases underwent IgM Ca flux, and the level of Ca flux was less than with IgD, a well recognized anergic pattern. Incubation for 24h in vitro led to partial restoration of IgM Ca flux and some improvement in IgD Ca flux. This was impaired by treatment with anti-IgD or IgM F(ab)2 fragments, mimicking antigen exposure, and in keeping with a model of CLL cells engaging autoantigen in vivo. Further support for the pro-survival role of the BCR in CLL was demonstrated by the finding that both IgD and IgM ligation was associated with reduced apoptosis in vitro, with a significant decrease in apoptosis with IgD ligation as compared to IgM. To examine the mechanistic differences of signaling via IgM and IgD further, we used high-throughput mass-spectrometry based phosphoproteomics. This allows analysis of multiple active signaling pathways without a priori knowledge of which pathways to investigate. 6 CLL samples were compared to 5 tonsil controls. 4,575 unique phosphopeptides were identified using MASCOT proteomics software and quantified using a label-free technique based on extracted ion currents. 174 phosphoproteins (p<0.001, fold change up to >4000-fold) were over-expressed in CLL relative to healthy B-cells. These included components of RNA processing complexes, cytoskeletal regulators and MAPK signaling pathway components. Kinase prediction based on phosphoprotein substrates confirmed activation of kinases known to be active in CLL (such as AKT1, ERK1/2, CK2), but several novel kinases (such as CaMK1, CRIK, ROCK1 and BCKDK) were also active in CLL relative to healthy controls. Evaluation of differentially expressed phosphoproteins after BCR ligation included components of the spliceosome, regulators of the cytoskeleton, as well as known BCR signaling components. BCR-induced kinase activities included mTOR, CDK family members, MAPKs, BCKDK and others. There was much overlap between kinases active after IgM and IgD ligation, but also marked differences in CLL and tonsil BCR signaling. CONCLUSIONS Anergic IgM signaling is contrasted with IgD as a dynamic and plastic process that appears different in the LN and PB compartments in CLL. Mass-spectrometry based phosphoproteomics offers a powerful tool for interrogating intracellular signaling, with networks of phosphorylation characterizing the topology of pathways. BCR signaling in healthy B-cells has not previously been studied using this approach and comparisons with CLL highlight known pathways as well as suggesting novel treatment targets. The ultimate goal is to identify kinases active in CLL that will provide rational and effective drug combinations. Disclosures: Gribben: Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

scholarly journals CD4+ T cells sustain aggressive chronic lymphocytic leukemia in Eμ-TCL1 mice through a CD40L-independent mechanism

2021 ◽  
Vol 5 (14) ◽  
pp. 2817-2828
Author(s):  
Matteo Grioni ◽  
Arianna Brevi ◽  
Elena Cattaneo ◽  
Alessandra Rovida ◽  
Jessica Bordini ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Antigen Specificity

Abstract Chronic lymphocytic leukemia (CLL) is caused by the progressive accumulation of mature CD5+ B cells in secondary lymphoid organs. In vitro data suggest that CD4+ T lymphocytes also sustain survival and proliferation of CLL clones through CD40L/CD40 interactions. In vivo data in animal models are conflicting. To clarify this clinically relevant biological issue, we generated genetically modified Eμ-TCL1 mice lacking CD4+ T cells (TCL1+/+AB0), CD40 (TCL1+/+CD40−/−), or CD8+ T cells (TCL1+/+TAP−/−), and we monitored the appearance and progression of a disease that mimics aggressive human CLL by flow cytometry and immunohistochemical analyses. Findings were confirmed by adoptive transfer of leukemic cells into mice lacking CD4+ T cells or CD40L or mice treated with antibodies depleting CD4 T cells or blocking CD40L/CD40 interactions. CLL clones did not proliferate in mice lacking or depleted of CD4+ T cells, thus confirming that CD4+ T cells are essential for CLL development. By contrast, CD8+ T cells exerted an antitumor activity, as indicated by the accelerated disease progression in TCL1+/+TAP−/− mice. Antigen specificity of CD4+ T cells was marginal for CLL development, because CLL clones efficiently proliferated in transgenic mice whose CD4 T cells had a T-cell receptor with CLL-unrelated specificities. Leukemic clones also proliferated when transferred into wild-type mice treated with monoclonal antibodies blocking CD40 or into CD40L−/− mice, and TCL1+/+CD40−/− mice developed frank CLL. Our data demonstrate that CD8+ T cells restrain CLL progression, whereas CD4+ T cells support the growth of leukemic clones in TCL1 mice through CD40-independent and apparently noncognate mechanisms.

scholarly journals Emerging role of kinase-targeted strategies in chronic lymphocytic leukemia

Hematology ◽  
2012 ◽  
Vol 2012 (1) ◽  
pp. 88-96 ◽  
Author(s):  
Adrian Wiestner
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Absolute Lymphocyte Count ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Cell Trafficking

Abstract Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells that depend on host factors in the tissue microenvironment for survival and proliferation. In vitro, CLL cells rapidly undergo apoptosis unless microenvironmental factors are provided that support their survival. Signaling pathways activated in the microenvironment in vivo include the B-cell receptor (BCR) and NF-κB pathways. Thus, CLL is a disease “addicted to the host” and is dependent on pathways that promote normal B-cell development, expansion, and survival; this is particularly true in the case of the BCR signaling cascade. Small-molecule inhibitors of kinases that are essential for BCR signal transduction abrogate the stimulating effects of the microenvironment on CLL cells. The orally administered tyrosine kinase inhibitors fostamatinib and ibrutinib and the phosphatidylinositol 3-kinase inhibitor GS-1101 have induced impressive responses in relapsed and refractory CLL patients, mostly with moderate side effects. Reductions in lymphadenopathy and splenomegaly are seen within weeks and are frequently accompanied by a transient rise in absolute lymphocyte count that is asymptomatic and probably the result of changes in CLL cell trafficking. This review discusses the biologic basis for kinase inhibitors as targeted therapy of CLL and summarizes the exciting early clinical experience with these agents.

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.

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.

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.

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