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.

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.

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.

CCR4:CCL17 Interaction Influences TLR-9 Mediated Cell Survival and Proliferation In Chronic Lymphocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3593-3593
Author(s):  
Sonal C. Temburni ◽  
Ryon M. Andersen ◽  
Luke Janson ◽  
Xiao-Jie Yan ◽  
Barbara Sherry ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Dose Range ◽  
Serum Levels ◽  
Lymphocytic Leukemia ◽  
Novel Therapy

Abstract Abstract 3593 Unlike other hematologic disorders, chronic lymphocytic leukemia(CLL) exhibits remarkable heterogeneity in the rates of disease progression among cases. CLL cells survive by receiving signals from the microenvironment via various receptors: B-cell antigen receptor (BCR), Toll-like receptors (TLRs) and cytokine and chemokine receptors. We previously reported that CLL clones with somatically mutated IGHVs and high (≥30%) percentage of CD38 expressing cells have the highest percentage of CCR4-expressing cells. To further explore the functional contribution of the CCR4:CCL17 axis in CLL, we studied CCL17-induced chemotactic behavior in 16 CLL cases. In transwell cultures we observed a bimodal migratory response to CCL17 at 2 doses in a dose range of 0.78– 25ng/ml, in ~60% of cases; the remaining cases showed maximal migration at a single dose (1.56 or 3.12ng/ml). A comparison of phenotypes of the migrated and non-migrated cell populations was undertaken in 10 cases, analyzing CXCR3, CXCR4, CCR4 and CCR7 that are involved in homing of cells to sites favoring growth, and CD31, CD38 and CD69, activation related molecules. The migrated cells consistently showed significantly higher percentages and densities of CD38 expression than the non-migrated cells suggesting a role for CD38 in the CCR4-mediated downstream pathway. CCR4 ligand, CCL17, is constitutively expressed in the thymus and is produced by dendritic cells, endothelial cells, keratinocytes and fibroblasts, whereas CCL22 is produced by tumor cells and the tumor microenvironment. Serum levels of both these ligands in untreated patients were quantified by ELISA. CCL17 levels ranged between 45-1, 229 pg/ml in U-CLL cases (n=23) and between 43-1, 418 pg/ml in M-CLL cases (n=30). CCL22 levels ranged between 121-5, 497 pg/ml in U-CLL cases (n=23) and 409-5, 502 pg/ml in M-CLL cases (n=30). The percentages of CCR4- expressing B cells directly correlated with percentages of T cells expressing CCR4 in individual cases, whereas they inversely correlated with both, serum levels of CCL17 (p< 0.01) and CCL22 (p< 0.05). CCL17 produced by DCs in peripheral organs may exert an accessory role in BCR- and TLR-9-mediated immune responses in B cells. We therefore tested if CCL17 supported BCR- and TLR-mediated proliferative responses in a cohort of 31 (16 U-CLL and 15M-CLL) CLL cases. CCL17 augmented BCR-mediated B-cell proliferation in 9/16 (56%) U-CLL cases, but only in 3/15 (20%) M-CLL cases. On the other hand, CCL17 showed an additive effect in promoting TLR-9-mediated cell proliferation in 13/15 (87%) M-CLL cases at a dose of 2ng/nl (approximating that detected in serum); it also augmented TLR-9 mediated B cell proliferation in 6/16 U-CLL cases but at a 5-fold or higher dose (10-25 ng/ml). In a subset of this cohort (8 cases) CCL17-induced modulation of molecules involved in the apoptotic process was studied. We found upregulation of anti-apoptotic proteins Mcl-1 and Bcl2 and down-regulation of pro-apoptotic molecules Bim, PUMA, and Bid in 5 of these cases. The pro-survival effects of CCL17 were partially abrogated by the blocking anti-CCR4 mAb (1G1). Taken together, these findings suggest that CCL17 plays a role in modulating TLR-9-mediated signaling and migration in CLL. Therefore, inhibition of CCR4:CCL17 interaction in vivo represents a novel therapy by preventing migration of CLL cells towards an environment that promotes their survival. Disclosures: No relevant conflicts of interest to declare.

Role Of Lyn Kinase In The Pathogenesis Of Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 668-668
Author(s):  
Phuong-Hien Nguyen ◽  
Nina Reinart ◽  
Michael Hallek
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Lymphoid Tissues ◽  
Malignant Cells ◽  
Deficient Mice

Abstract The Src family kinase Lyn is predominantly expressed in B cells and plays a central role in initiating B cell receptor (BCR) signaling. Lyn is associated with BCR complexes and is renowned for its role in B cell activation and proliferation. Active Lyn contributes to positive regulation of signalling through tyrosine phosphorylation of components of the BCR. Intriguingly, Lyn was also shown as a negative regulator of BCR signal transduction. Lyn plays an essential role in negative regulation of signalling through its unique ability to phosphorylate immunoreceptor tyrosine based inhibition motifs (ITIM) in inhibitory cell surface receptors. ITIM phosphorylation induces the recruitment of inhibitory phosphatases such as SHP-1/2 and SHIP-1, which attenuate BCR signalling. Lyn-deficient mice have reduced number of B cells and increased numbers of myeloid progenitors. It was reported that expression and activity of Lyn in human chronic lymphocytic leukemia (CLL) is elevated compared to healthy B cells. Besides, higher levels of Lyn are associated with a shorter treatment-free survival of CLL patients. This rises up a hypothesis about Lyn’s significant role in B cell tumorigenesis, malignant transformation of B cells, and the balance between myeloid cells and B lymphocytes. We generated Eµ-TCL1 transgenic LYN-deficient mice (TCL1+/wtLYN-/-) and monitored them in order to identify the population of malignant B cells and to characterize the development of malignant cells in these mice in comparison with Eµ-TCL1 transgenic mice (TCL1+/wtLYNwt/wt). In comparison to TCL1+/wtLYNwt/wt mice, TCL1+/wtLYN-/- mice show a significantly reduced number of malignant B cells in the peripheral blood, as well as a reduced leukocyte count. Besides, TCL1+/wtLYN-/- mice have significantly decreased infiltration of malignant B cells in lymphoid tissues such as spleen, liver, lymph node and bone marrow. This result is also resembled in a hepato-splenomegaly in the TCL1+/wtLYNwt/wt mice. These mice develop severe splenomegaly and hepatomegaly due to infiltration of malignant cells, while TCL1+/wtLYN-/- mice do not develop hepatomegaly. The non-transgenic LYN-/- control mice develop splenomegaly due to infiltration of myeloid cells. Although TCL1+/wtLYN-/- mice have hindered development of TCL1-induced CLL, preliminary data suggest it is not only due to LYN-deficiency in B cell compartment of these mice. Indeed, B cell of TCL1+/wtLYN-/- mice show enhanced proliferation and better survival ex vivo compared to TCL1+/wtLYNwt/wt mice. Notably, TCL1+/wtLYN-/- mice developed a skewed microenvironment which might contribute to CLL down regulation. LYN-/- microenvironment, particularly in aged mice, does not support engraftment of TCL1-induced leukemic B cell as well as LYNwt/wt mice in our transplantation model. These results point to a complex regulation of Lyn signalling in CLL involving not only leukemic cells but also cells of the micromillieu, that needs further investigation. Disclosures: No relevant conflicts of interest to declare.

Reversible anergy of sIgM-mediated signaling in the two subsets of CLL defined by VH-gene mutational status

Blood ◽  
2007 ◽  
Vol 109 (10) ◽  
pp. 4424-4431 ◽  
Author(s):  
C. Ian Mockridge ◽  
Kathleen N. Potter ◽  
Isla Wheatley ◽  
Louise A. Neville ◽  
Graham Packham ◽  
...  
Keyword(s):  
B Cells ◽  
Direct Evidence ◽  
Lymphocytic Leukemia ◽  
Downstream Signaling ◽  
Specific Effects ◽  
Mutational Status ◽  
Differential Signaling ◽  
Vh Gene

Abstract The 2 subsets of chronic lymphocytic leukemia (CLL), of worse or better prognosis, likely derive from pre-GC unmutated B cells, or post-GC mutated B cells, respectively. Different clinical behavior could relate to the ability of tumor cells to respond to surface (sIg)–mediated signals. Unmutated cases (U-CLL) have an increased ability to phosphorylate p72Syk in response to sIgM ligation compared to mutated cases (M-CLL). We now confirm and further investigate this differential signaling in a large cohort by [Ca2+]i mobilization. Cases responding to sIgM ligation express higher levels of CD38, ZAP-70, and sIgM. However, CD38 does not influence signaling in vitro or associate with response in bimodal CD38-expressing cases. Similarly, ZAP-70 expression is not required for response in either U-CLL or M-CLL. Strikingly, partially or completely anergized sIgM responses from each subset can recover both sIgM expression and signal capacity spontaneously in vitro or following capping/endocytosis. This provides direct evidence for engagement of putative antigen in vivo. Signaling via sIgD differs markedly being almost universally positive in both U-CLL and M-CLL, with no association with CD38 or ZAP-70 expression. Downstream signaling pathways, therefore, appear intact in CLL, locating anergy to sIgM, mainly in M-CLL. Integration of differential isotype-specific effects mediated by (auto)antigen may determine tumor behavior.

Lenalidomide Reduces Survival of Chronic Lymphocytic Leukemia Cells in Primary Co-Cultures by Altering the Myeloid Microenvironment

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3894-3894
Author(s):  
Angela Schulz ◽  
Claudia Dürr ◽  
Thorsten Zenz ◽  
Stephan Stilgenbauer ◽  
Peter Lichter ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Drug Effects ◽  
Surface Expression ◽  
Lymphocytic Leukemia ◽  
Cell Surface Expression ◽  
Clinical Activity ◽  
Altered Expression

Abstract Abstract 3894 Chronic lymphocytic leukemia (CLL) cells are highly dependent on their microenvironment. External stimuli provided by bone marrow stromal cells or non-malignant leukocytes are required for their survival and proliferation. Interestingly, peripheral blood-derived monocytes differentiate in the presence of CLL cells to so-called Nurse-like cells (NLCs), which are round or fibroblast-shaped adherent cells that were shown to promote survival of CLL cells in vitro and to exist in lymph nodes of CLL patients. In search of new therapeutic options for patients with CLL, the immunomodulatory drug lenalidomide turned out to have significant clinical activity in CLL. Lenalidomide does not induce apoptosis in CLL cells directly, but is rather believed to act via the microenvironment. Several studies described that it alters cytokine levels and the activation status of the cells. Further, a CLL-specific T-cell defect was shown to be repaired by lenalidomide, which might represent a major activity of this drug in CLL. However, its mechanism of action seems to be complex and is not well understood. As monocytes as well as NLCs are very effective in maintaining survival of CLL cells, we aimed to investigate whether lenalidomide interferes with these supportive cell-cell interactions. To do this, we established primary co-cultures of monocytes and CLL cells in the presence or absence of lenalidomide and observed a significantly decreased viability of CLL cells after 14 days of treatment, suggesting an impact of this drug on the survival support of NLCs. Therefore, we analyzed the immunophenotype of NLCs by flow cytometry, as well as the secretion of cytokines in the co-cultures by ELISA and antibody-coupled bead arrays. Among the effects induced by lenalidomide, we observed reduced cell surface expression of the MHC II protein HLA-DR on NLCs as well as lower levels of the chemokine CCL2, but higher levels of IL-10 in the culture supernatant, indicating an altered inflammatory milieu in the co-cultures. The enhanced IL-10 levels resulted in an increase in STAT1 phosphorylation in CLL cells as measured by Western blot analysis. As a consequence, enhanced expression of the adhesion molecule ICAM-1 (CD54) and an altered expression of cytoskeletal genes (e.g. RHOC and CORO1B) were observed in CLL cells after lenalidomide treatment. Chemotaxis assays using transwell culture dishes and SDF1-α as chemoattractant revealed an impaired migratory potential of lenalidomide-treated CLL cells, which was not due to reduced expression of the SDF1-α receptor CXCR4. In summary, our data show that lenalidomide reduces the survival support of NLCs for CLL cells in vitro, suggesting that this drug effects the myeloid microenvironment in CLL in vivo. Furthermore, lenalidomide impairs the migratory potential of CLL cells which may affect circulation and homing of CLL cells in vivo. Disclosures: No relevant conflicts of interest to declare.

Microrna-34a As a Marker for Fludarabine Resistance and Impairment of p53-Pathway in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3883-3883
Author(s):  
Marek Mraz ◽  
Katerina Cerna ◽  
Veronika Mayerova ◽  
Katerina Musilova ◽  
Karla Plevova ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Absolute Quantification ◽  
P53 Mutation ◽  
Fold Change ◽  
Lymphocytic Leukemia ◽  
Apoptotic Pathway ◽  
P53 Pathway

Abstract Abstract 3883 Background. MicroRNAs (miRNAs) are known to be involved in the pathogenesis of chronic lymphocytic leukemia (CLL) and affect its clinical course (Calin et al. NEJM, 2005; Mraz et al. Blood, 2012). Moreover, we and others have shown that several miRNAs are down-regulated in the aggressive CLL subtype harboring p53 aberration (Mraz et al. Leukemia, 2009). The role of miRNAs in primary or acquired resistance to therapy in CLL, however, is poorly understood. Aim. In this study, we screened for miRNAs that are induced by fludarabine-mediated apoptosis in vitro, and we suggested that differences in the expression of one of the identified miRNAs (miR-34a) can be used to distinguish patients with impairment of the p53-apoptotic pathway. Results. Ten primary CLL B-cell samples (purity>95% of CD5+19+ cells) were treated in vitro with fludarabine dose (IC50 dose of 3.5 ug/mL, 48hrs), and the expression of 750 miRNAs was subsequently profiled (TaqMan miRNA Cards, ABI). In comparison with untreated control samples, 15 miRNAs were induced by fludarabine (fold change>1.5, SAM FDR<0.05). The most prominently up-regulated miRNA was miR-34a (fold change 3.7, P=0.003), which is a known p53 down-stream target (He et al. Nature, 2007). We then compared miR-34a up-regulation post fludarabine treatment to the decrease in cell viability (wt-p53 samples, N=20). This revealed that miR-34a induction was significantly higher in CLL samples more sensitive to fludarabine and suggested its role in the apoptotic effects of fludarabine in B-cells. Moreover, the up-regulation of miR-34a was also observed in vivo in samples obtained from fifty FCR-treated CLL patients (fold change 2.2, P<0.0001, analyzed at day 0 and 3 of FCR). These data encouraged us to develop an assay for absolute quantification of miR-34a which would allow determining the copy numbers of miR-34a, defining precise cut-offs, and comparing miR-34a levels during the course of the disease in one patient. We designed synthetic RNA oligos that were used to construct standard curves for both miR-34a and a normalization gene (RNU48). Using this assay, we profiled the expression of miR-34a in a cohort of CLL patients (N=200) to define a cut-off value that would discriminate therapy resistant cases. The distribution of miR-34a expression in the cohort ranged from 1 to 81820 molecules (per 10e6 copies of RNU48). Significantly, miR-34a levels below 2500 copies (N=47) were correlated with shorter overall survival (9.6 years vs. not-reached, HR 2.2 [CI 1.1–4.5], P=0.03). Subsequently, the expression of miR-34a was compared in samples stratified by known prognostic markers: chromosomal aberrations (del17p13, del11q23, tris.12, and del13q14), IgHV status, expression of CD38, CD49d, age, gender, and Rai stage. The lower miR-34a levels were only associated with the deletion of 17p13 locus that includes the p53 gene (N=18, fold change −3.4, P=0.003). Remarkably, CLL samples with sole p53 mutation not accompanied by p53 deletion (N=13) also expressed low levels of miR-34a compared to wt-p53 (P=0.005, fold change −2.7). Most notably, 77% (10/13) of these samples had miR-34a levels below the cut-off of 2500 copies. We further validated our observations and assay by analyzing miR-34a expression in paired samples from 12 CLL cases that acquired p53 aberration during the course of the disease. This emphasized that miR-34a expression decreased in all cases after occurrence of p53 mutation (P=0.0008, fold change −6.1). Additionally, the effect of miR-34a up-regulation on therapy response is currently being investigated in a cohort of FCR-treated patients (N=50). Conclusions. Our data provide complex evidence for the use of miR-34a as a marker of fludarabine-resistant disease. MicroRNA-34a quantification can identify p53 mutated cases that would not be recognized by FISH (mutation not accompanied by del17p13), and miR-34a down-regulation can be used as a sensor for acquisition of p53 abnormality during the course of the disease. This can be accomplished without treatment of cells with gamma-irradiation, which was previously used to identify functional impairment of the p53-pathway (Pettitt et al. Blood, 2001; Mous et al. Leukemia, 2009; Lin et al. CCR, 2012). The described assay for absolute quantification of miR-34a encourages further inter-laboratory validation. IGA MZCR NT11218–6/2010, CZ.1.07/2.3.00/20.0045, CZ.1.05/1.1.00/02.0068 Disclosures: No relevant conflicts of interest to declare.

Divergence in CD19-Mediated Signaling Unfolds Intra-Clonal Diversity in Chronic Lymphocytic Leukemia Which Correlates with Disease Progression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4570-4570
Author(s):  
Yair Herishanu ◽  
Sigi Kay ◽  
Nili Dezorella ◽  
Chava Perry ◽  
Varda Deutsch ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Disease Progression ◽  
Conflicts Of Interest ◽  
Clonal Diversity ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Cell Fraction ◽  
Akt Phosphorylation

Abstract Abstract 4570 Emerging data on intra-clonal diversity imply that this phenomenon may play a role in the clinical outcome of patients with chronic lymphocytic leukemia (CLL), where subsets of the CLL clone responding more robustly to external stimuli may gain a growth and survival advantage. Here we report intra-clonal diversity resolved by responses to CD19 engagement in CLL cells, which can be classified into responding (CD19-R) and non-responding (CD19-N) sub-populations. Engagement of CD19 by anti-CD19 antibody rapidly induced cellular aggregation in the CD19-R CLL cells. The CD19-R CLL cells expressed higher surface levels of CD19 and c-myc mRNA and exhibited distinct morphological features. Both sub-populations reacted to IgM stimulation in a similar manner and exhibited similar levels of Akt phosphorylation, pointing to functional signaling divergence within the B-cell receptor. CD19 unresponsiveness was partially reversible, where CD19-N cells spontaneously recover their signaling capacity following incubation in vitro, pointing to possible in vivo CD19-signaling attenuating mechanisms. This concept was supported by the lower CD19-R occurrence in bone marrow-derived samples compared with cells derived from the peripheral blood of the same patients. CLL patients with more than 18.5% of CD19-R cell fraction had a shorter median time to treatment compared to patients with less than 18.5% of CD19-R cell fraction. Conclusions: Divergence in CD19-mediated signaling unfolds both inter-patient and intra-clonal diversity in CLL. This signaling diversity is associated with physiological implications including the location of the cells and disease progression. 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.

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