scholarly journals VLA-4 Expression and Activation in B Cell Malignancies: Functional and Clinical Aspects

2020 ◽  
Vol 21 (6) ◽  
pp. 2206 ◽  
Author(s):  
Andrea Härzschel ◽  
Antonella Zucchetto ◽  
Valter Gattei ◽  
Tanja Nicole Hartmann
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Types ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Clinical Aspects ◽  
Neoplastic Disease ◽  
B Cell Differentiation ◽  
B Cell Malignancies ◽  
Bcr Signaling

Lineage commitment and differentiation of hematopoietic cells takes place in well-defined microenvironmental surroundings. Communication with other cell types is a vital prerequisite for the normal functions of the immune system, while disturbances in this communication support the development and progression of neoplastic disease. Integrins such as the integrin very late antigen-4 (VLA-4; CD49d/CD29) control the localization of healthy as well as malignant B cells within the tissue, and thus determine the patterns of organ infiltration. Malignant B cells retain some key characteristics of their normal counterparts, with B cell receptor (BCR) signaling and integrin-mediated adhesion being essential mediators of tumor cell homing, survival and proliferation. It is thus not surprising that targeting the BCR pathway using small molecule inhibitors has proved highly effective in the treatment of B cell malignancies. Attenuation of BCR-dependent lymphoma–microenvironment interactions was, in this regard, described as a main mechanism critically contributing to the efficacy of these agents. Here, we review the contribution of VLA-4 to normal B cell differentiation on the one hand, and to the pathophysiology of B cell malignancies on the other hand. We describe its impact as a prognostic marker, its interplay with BCR signaling and its predictive role for novel BCR-targeting therapies, in chronic lymphocytic leukemia and beyond.

ZAP-70 Is Not Phosphorylated on the Activating Tyrosine Residue (Tyr319) in Chronic Lymphocytic Leukemia and B-Cell Lymphoma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 178-178
Author(s):  
Stefania Gobessi ◽  
Aleksandar Petlickovski ◽  
Luca Laurenti ◽  
Dimitar G. Efremov
Keyword(s):  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Progressive Disease ◽  
Cell Receptor ◽  
Kinase Domain ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling ◽  
Cell Receptor Signaling

Abstract The protein tyrosine kinase ZAP-70 is expressed at high levels in leukemic B-cells from chronic lymphocytic leukemia (CLL) patients with progressive disease and short survival. ZAP-70 is a key component of the proximal T-cell receptor signaling pathway and is highly homologous to Syk, an important B-cell receptor signaling (BCR) molecule. Recent studies indicate that ZAP-70 may participate in BCR signaling as well, but the mechanism of action is still not well understood. In T-cells, upon TCR stimulation ZAP-70 becomes phosphorylated on Tyr319 by the Src-like kinase Lck, which results in the release of the ZAP-70 kinase domain from an autoinhibited state to a fully active conformation. The Tyr319 site in ZAP-70 corresponds to the Tyr352 site in Syk, which is phosphorylated in B-cells following BCR stimulation. We therefore investigated the activation status of ZAP-70 and Syk in BCR stimulated CLL B-cells, using phosphorylation of Tyr319 and Tyr352 as markers of their activation. Analysis of 10 ZAP-70-positive CLL samples by immunoblotting with the phospho-ZAP70Tyr319/SykTyr352 antibody revealed that ZAP-70 is not phosphorylated at this site either before or after BCR stimulation, although in control experiments with Jurkat T-cells ZAP-70 became phosphorylated on Tyr319 upon TCR stimulation. Moreover, the Tyr352 site in Syk was phosphorylated following BCR stimulation in 6 of the 10 CLL B-cell samples. To further investigate the reasons for the unexpected lack of ZAP-70 activation in CLL B-cells, we produced stable transfectants of the BJAB lymphoma B-cell line that expressed ZAP-70 at levels similar to those found in CLL cases with progressive disease. In agreement with the CLL B-cell experiments, the Tyr319 site in ZAP-70 was not phosphorylated either before or after BCR stimulation. Since phosphorylation of Tyr319 is Lck-dependent in T-cells, and this kinase is expressed also in CLL B-cells, we ectopically expressed Lck in the ZAP-70-positive BJAB clones. Again, the Tyr319 site was not phosphorylated, indicating that ZAP-70 does not undergo activation of the kinase domain also in this cellular system. In contrast, BCR crosslinking in BJAB cells induced significant phosphorylation of Tyr352 in Syk, which was further enhanced in the clones that coexpressed ZAP-70. Furthermore, analysis of downstream signaling pathways following BCR stimulation showed stronger and prolonged activation of ERK and to a lesser extent Akt in the ZAP-70 positive clones, whereas no difference was observed in terms of activation of PLC-γ 2, JNK and degradation of the NF-kB inhibitor IkB. These data indicate that ZAP-70 does not undergo full activation in B-cells, but can still enhance activation of certain downstream BCR signaling pathways, possibly by affecting the activity of the related PTK Syk.

Involvement of PKC-δ in the Regulation of Notch2 in B-CLL.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4990-4990
Author(s):  
Rainer Hubmann ◽  
Markus Duechler ◽  
Martin Hilgarth ◽  
Susanne Schnabl ◽  
Josef D. Schwarzmeier ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Negative Selection ◽  
Cell Receptor ◽  
Immunoglobulin M ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Mobility Shift ◽  
Bcr Signaling ◽  
Electrophoretic Mobility Shift Assays

Abstract We have recently shown that NOTCH2 signaling is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia (B-CLL) cells (Hubmann et al., BLOOD 2002). There is an increasing evidence that NOTCH2 plays a determining role in the development/homeostasis of self-reactive CD5+ B-cells, suggesting a potential function of NOTCH2 in B-cell leukemogenesis. Here we study the regulation of NOTCH2 signaling in B-CLL cells and its possible function in B-lymphocytes using NOTCH2 transduced BL41 cells as a model system. Cultured B-CLL samples (n=30) lose their nuclear NOTCH2 (N2IC) activity within one day as demonstrated by electrophoretic mobility shift assays (EMSA). However, DNA-bound NOTCH2 complexes could be maintained in culture by exposure to the phorbolester TPA and is accompained by increased cell viability. The effect of TPA is prevented by the PKC-δ inhibitor Rottlerin indicating that PKC-δ is involved in the regulation of NOTCH2 signaling in B-CLL cells. The activity of N2IC in the leukemic cells appeared to be resistant to the γ-secretase inhibitors DAPT and compound E, two substances known to block ligand mediated release of the NOTCH2 intracellular domain (N2IC). Since B-CLL cells are locked in an anergic state, we next asked whether NOTCH2 modulates B-cell receptor (BCR) signaling and found that retrovirally transduced N2IC rescues the B-cell line BL41 from surface immunoglobulin M (sIgM) mediated apoptosis, a mechanism proposed to prevent the uncontrolled expansion of self-reactive CD5+ B-cells. In summary, our data suggest that B-CLL cells express an activated form of NOTCH2 which might be involved in the protection of the malignant clone from peripheral negative selection.

Expression of ZAP-70 in Chronic Lymphocytic Leukemia Cells Enhances CD79b Phosphorylation Following Surface IgM Ligation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2799-2799
Author(s):  
Liguang Chen ◽  
John Apgar ◽  
Li Tang ◽  
Thomas J. Kipps
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Intracellular Signaling ◽  
Critical Role ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Cell Surface Molecule ◽  
Bcr Signaling ◽  
Accessory Molecules

Abstract CD79b is B-cell surface molecule that non-covalently associates with CD79a and surface immunoglobulin (sIg), which together serve as the B-cell receptor complex (BCR). Both CD79a and CD79b have cytosolic immunoreceptor tyrosine-based activation motifs (ITAMs) that can become phosphorylated following sIg ligation, thereby allowing for recruitment to the BCR complex of cytosolic kinases, such as p72Syk , which then can initiate downstream intracellular signaling events. Compared to normal B cells, chronic lymphocytic leukemia (CLL) B cells typically expresses low levels of CD79b, which is speculated to contribute to the relatively poor capacity of CLL cells to initiate intracellular signaling following BCR ligation despite having apparently adequate levels of p72Syk. BCR signaling in CLL cells can be enhanced by expression of the zeta-associated protein of 70 kD (ZAP-70), a tyrosine kinase that initially was identified in T cells, where it plays a critical role in the phosphorylation of ITAMs of the accessory molecules of the T-cell receptor (TCR) complex for antigen following TCR ligation. We investigated for phosphorylation of CD79b following BCR ligation with F(ab)2 anti- μ antibody in CLL cell samples that did or did not express ZAP-70. All CLL cell samples expressed similar amounts of surface IgM and p72Syk, as assessed via flow cytometry and immunoblot analysis. Within 10 minutes after treatment with anti-μ the CLL cell samples that expressed ZAP-70 (n = 28) experienced a mean increase in phosphorylation of CD79b of 21.5% (± 14.0% S.D.), which was significantly greater than the 7.5% increase (± 7.9% S.D.) experienced by similarly treated CLL cell samples that did not express ZAP-70 (n = 19) (P< 0.01). Immune precipitation studies demonstrated association of CD79b with p72Syk in CLL B cells. CLL cell samples (n = 5) lacking expression of ZAP-70 were transfected with a control vector or an expression vector encoding ZAP-70, allowing us to examine the effect that engineered-expression of ZAP-70 has on CD79 phosphorylation following treatment with anti-μ. Anti-μ treatment induced significantly higher mean levels of CD79b phosphorylation in CLL samples made to express ZAP-70 (33% ± 16%) than in control mock-transfected CLL cells (4% ± 2%). This also was associated with enhanced anti-μ induced phosphorylation of p72Syk. We conclude that expression of ZAP-70 in CLL B cells enhances phosphorylation of the accessory molecules in the BCR complex following sIg ligation, potentially allowing for improved recruitment of cytosolic kinases and adapter proteins to these accessory molecules for enhanced BCR signaling.

Targeting Early Events of B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia: Suppressed Syk and PLCγ2 Activities Predict Apoptotic Response of Leukemic Cells to Dasatinib

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5023-5023
Author(s):  
Y. Lynn Wang ◽  
Zibo Song ◽  
Pin Lu ◽  
John P. Leonard ◽  
Morton Coleman ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Bcr Signaling

Abstract B cell receptor (BCR) signaling plays an essential role in the pathogenesis of chronic lymphocytic leukemia. In a subset of patients with a poor clinical outcome, BCR ligation leads to increased cell metabolism and cell survival (Cancer Research66, 7158–66, 2006). Based on these findings, we tested whether targeting BCR signaling with dasatinib, an inhibitor of Src kinase, would interfere with the signaling cascade and cause death of CLL B cells. CLL leukemic cells were isolated from 34 patients and were incubated with or without dasatinib at a low dose of 128 nM. Among 34 cases, viability of leukemic cells was reduced by 2% to 90%, with an average of ~50% reduction on day 4 of ex vivo culture. Further study showed that CLL B cells undergo death by apoptosis via the intrinsic pathway which involves the generation of reactive oxygen species. Analysis of the Src family kinases showed that phosphorylation of Src, Lyn and Hck was inhibited by dasatinib not only in those cases that responded to dasatinib with apoptosis, but also in those that did not respond well (&lt;20% apoptosis). Further analysis revealed that suppressed activity of two downstream molecules, Syk and PLC Statistical analysis showed a significant correlation between CLL dasatinib response and their IgVH mutation and ZAP70 status. Cases with worse prognoses by these criteria have a better response to the kinase inhibitor. Lastly, we have also found that ZAP70 positive cases showed a greater degree of PLC

scholarly journals Patterns of microRNA expression characterize stages of human B-cell differentiation

Blood ◽  
2009 ◽  
Vol 113 (19) ◽  
pp. 4586-4594 ◽  
Author(s):  
Jenny Zhang ◽  
Dereje D. Jima ◽  
Cassandra Jacobs ◽  
Randy Fischer ◽  
Eva Gottwein ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Microrna Expression ◽  
Lymphocytic Leukemia ◽  
Cell Phenotype ◽  
Tumor Type ◽  
B Cell Differentiation ◽  
B Cell Malignancies

Abstract Mature B-cell differentiation provides an important mechanism for the acquisition of adaptive immunity. Malignancies derived from mature B cells constitute the majority of leukemias and lymphomas. These malignancies often maintain the characteristics of the normal B cells that they are derived from, a feature that is frequently used in their diagnosis. The role of microRNAs in mature B cells is largely unknown. Through concomitant microRNA and mRNA profiling, we demonstrate a potential regulatory role for microRNAs at every stage of the mature B-cell differentiation process. In addition, we have experimentally identified a direct role for the microRNA regulation of key transcription factors in B-cell differentiation: LMO2 and PRDM1 (Blimp1). We also profiled the microRNA of B-cell tumors derived from diffuse large B-cell lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia. We found that, in contrast to many other malignancies, common B-cell malignancies do not down-regulate microRNA expression. Although these tumors could be distinguished from each other with use of microRNA expression, each tumor type maintained the expression of the lineage-specific microRNAs. Expression of these lineage-specific microRNAs could correctly predict the lineage of B-cell malignancies in more than 95% of the cases. Thus, our data demonstrate that microRNAs may be important in maintaining the mature B-cell phenotype in normal and malignant B cells.

scholarly journals Autoimmunity Checkpoints As Therapeutic Targets in B-Cell Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1587-1587
Author(s):  
Zhengshan Chen ◽  
Markus Muschen
Keyword(s):  
Drug Resistance ◽  
B Cells ◽  
B Cell ◽  
Negative Selection ◽  
Cell Types ◽  
Oncogenic Signaling ◽  
B Cell Malignancies ◽  
Minimum Threshold ◽  
Bcr Signaling ◽  
Novel Strategy

Abstract Concept. Targeted therapy of cancer typically focuses on inhibitors (e.g. tyrosine kinase inhibitors) that suppress oncogenic signaling below a minimum threshold required for survival and proliferation of cancer cells. Acute lymphoblastic leukemia (ALL) and B cell lymphomas originate from various stages of development of B cells, which unlike other cell types are under intense selective pressure. The vast majority of newly generated B cells are autoreactive and die by negative selection at autoimmunity checkpoints (AIC). Owing to ubiquitous encounter of self-antigen, autoreactive B cells are eliminated by overwhelming signaling strength of their autoreactive B cell antigen receptor (BCR). A series of recent findings suggests that, despite malignant transformation, AIC are fully functional in B cell malignancies. We propose that targeted engagement of AIC represents a previously unrecognized therapeutic opportunity to overcome conventional mechanisms of drug-resistance in pre-B ALL and other B cell malignancies. Results: Oncogenic drivers in B- cell malignancies function as mimics of B-cell receptor (BCR) signaling. Oncogenic activation of BCR-signaling represents the functional equivalent of positive selection during normal lymphocyte development. Addiction to survival and proliferation signals (or the equivalent of positive selection) is a common feature in many types of cancer. However, B-cell malignancies are unique in that they are also subject to an active negative selection process. B-cells expressing autoreactive BCRs or antibodies can cause systemic autoimmunity. As a safeguard against autoimmune diseases, lymphocyte development evolved autoimmunity checkpoints (AIC) to eliminate autoreactive clones. Owing to negative selection of autoreactive B-cells through AIC activation, lymphoid cells fundamentally differ in their signaling requirements from other cell types. Recent studies from our group showed that despite malignant transformation, B-cell leukemia and lymphoma cells are fully sensitive to negative selection and AIC-activation resulting (Chen et al., Nature 2015; Shojaee et al., Nature Med 2016; Chan et al., Nature 2017; Xiao et al., Cell 2018). AIC-activation in various lymphoid malignancies is achievable by pharmacological hyperactivation of BCR-signaling above a maximum threshold (see Schematic below). Unlike other types of cancer, B-cell malignancies are uniquely susceptible to clonal deletion induced by hyperactive signaling from an autoreactive BCR. Hence, targeted AIC-activation can be leveraged for eradication of drug-resistant leukemia and lymphoma clones. Here, we propose a novel strategy to overcome drug-resistance in B-lymphoid malignancies based on targeted activation of autoimmunity checkpoints (AIC) for removal of autoreactive B-lymphocytes. We have recently discovered that targeted hyperactivation of SYK, PI3K and ERK in B cell malignancies represents the functional equivalent of an autoimmunity checkpoint (AIC) for elimination of autoreactive clones among normal B cells. B cell tumors are uniquely vulnerable to AIC activation, suggesting that targeted activation of this checkpoint represents a novel strategy to induce cell death in otherwise drug-resistant B cell malignancies. Conclusion: Normal B-cells are positively selected for BCR signaling of intermediate strength (moderate activation of SYK, PI3K and ERK). In the absence of a functional BCR, SYK, PI3K and ERK activity fall below a minimum threshold, resulting in death by neglect. Hyperactivation above maximum thresholds (e.g. autoreactive BCR) triggers negative selection and cell death via AIC-activation. Targeted therapy of cancer typically focuses on agents that suppress oncogenic signaling below a minimum threshold. Our results support a novel strategy to overcome drug-resistance in B-cell malignancies based on targeted activation of autoimmunity checkpoints (AIC) for removal of autoreactive cells. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Sustained Signaling through the B-Cell Receptor Induces Mcl-1 and Promotes Survival of Chronic Lymphocytic Leukemia B-Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 178-178
Author(s):  
Dimitar G. Efremov ◽  
Aleksandar Petlickovski ◽  
Luca Laurenti ◽  
Xiaoping Li ◽  
Sara Marietti ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Spontaneous Apoptosis ◽  
B Cell Survival ◽  
Bcr Signaling ◽  
Induced Apoptosis

Abstract The clinical course of chronic lymphocytic leukemia (CLL) differs significantly between patients with mutated (M-CLL) and unmutated (U-CLL) immunoglobulin V genes, implying a role for B-cell receptor (BCR) signaling in the pathogenesis of this disease. BCR stimulation in normal B-cells triggers several crucial signaling pathways, including PI3K/Akt, IKK/NF- κB and the mitogen-activated protein kinases Erk, JNK and p38 MAPK, which can induce proliferation, survival, differentiation or apoptosis, depending on the nature and context of the antigenic stimulation. We have now investigated activation of these downstream signaling pathways, as well as induction of anti-apoptotic proteins and survival of CLL B-cells stimulated with soluble (sol-IgM) and immobilized (imm-IgM) anti-IgM antibodies, which were used to mimic stimulation with soluble and particulate/membrane-bound antigen, respectively. Stimulation with sol-IgM revealed similar activation patterns in the 10 U-CLL and 12 M-CLL cases that partially resembled the pattern described for tolerant B-cells. The response in the U-CLL cases was characterized by transient (<45 minutes) phosphorylation of Akt and Erk, no activation of JNK and p38 MAPK, and activation of IKKβ in 50% of the cases. Most M-CLL cases showed similar activation of Akt and Erk, but lacked activation of IKKβ, whereas three M-CLL cases were completely non-responsive. To investigate the effects on CLL B-cell survival, 14 U-CLL and 19 M-CLL cases were analyzed by Annexin V/PI staining after 48 hours stimulation with sol-IgM. A 10–40% increase in apoptotic cells was observed in the majority of cases from both CLL subsets (p<0.001 with respect to spontaneous apoptosis). Induction of apoptosis was confirmed by analyzing cleavage of the Caspase 3 substrate PARP, and was accompanied by an approximately 50% reduction in the levels of Mcl-1, an antiapoptotic protein implicated in CLL B-cell survival and resistance to chemotherapy. A markedly different response was induced by imm-IgM, which was characterized by activation of IKKβ in all cases and sustained Akt and Erk phosphorylation that persisted over 24 hours. This response resulted in a 2.5 fold mean increase in the levels of Mcl-1, whereas no changes were observed in the levels of Bcl-2 and Bcl-xL. Imm-IgM slightly reduced the percentage of cells undergoing spontaneous apoptosis after 48 hours, but significantly protected from fludarabine- and methylprednisolone-induced apoptosis. To investigate which of the three imm-IgM activated pathways is responsible for induction of Mcl-1 and protection from chemotherapy-induced apoptosis, we incubated CLL B-cells with LY294002, U0126 and BAY-11 (inhibitors of PI3K, ERK and NF- κB, respectively) prior to stimulation with imm-IgM and addition of fludarabine. Induction of Mcl-1 and inhibition of fludarabine-induced PARP cleavage were significantly abrogated only by LY294002, indicating that the PI3K/Akt pathway is the major link between the BCR and apoptosis resistance of CLL B-cells. In conclusion, this study shows that the response of CLL B-cells to BCR stimulation primarily depends on the nature of the antigenic stimulus. Moreover, it shows that only sustained BCR signaling can promote survival of CLL B-cells, and raises the possibility that the distinct clinical and biological behavior of U-CLL and M-CLL is determined by the availability of such stimulation.

Sustained NF-Kappa B Activity In Chronic Lymphocytic Leukemia Is Independent of Genetic and Epigenetic Modifications of the Tumor Suppressor Genes CYLD and A20

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3597-3597
Author(s):  
Lukas P. Frenzel ◽  
Janine Schwamb ◽  
Rainer Claus ◽  
Julia Claasen ◽  
Reinhild Brinker ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
B Cell Malignancies ◽  
Healthy Donors ◽  
B Cell Receptor Signaling ◽  
Cell Receptor Signaling ◽  
Stimulation Of

Abstract Abstract 3597 Introduction: The microenvironment and especially the antigenic stimulation of the B-cell receptor on the surface of the malignant cell play a crucial role in the pathogenesis of chronic lymphocytic leukemia (CLL). Aberrant Nuclear Factor kappa B (NFκB) activity is another major hallmark of B-cell malignancies as well as of CLL. NFκB-dependent genes are involved in anti-apoptotic regulation, cell proliferation and metastasis and are responsible for survival and proliferation of tumors. However, the mechanisms of NFκB over-expression in CLL still remain to be elucidated. Prior studies revealed that cylindromatosis (CYLD) function might be of special interest in CLL since it inhibits signaling via TRAF2 and c-IAP1/2, which are known to be over-expressed in CLL. CYLD inactivation might therefore result in sustained NFκB signaling. The enzyme CYLD, a tumor suppressor that functions as a deubiquitinase, plays a role in other physiological aspects such as cell cycle response, inflammatory and immune processes. Moreover, it could be shown that impaired CYLD activity leads to increased NFκB activity in multiple myeloma cells demonstrating the negative regulatory function of CYLD regarding NFκB. Aside from CYLD, which is constitutively active preventing uncontrolled transcription factor activation, the enzyme A20, a key player in negative feedback loop regulation of NFκB, operates via induction, supposing that both enzymes might proceed at different phases of NFκB signaling. A20, also known as tumor necrosis factor alpha-induced protein 3 (TNFAIP3), acts as an ubiquitin-editing enzyme. Its inactivation is involved in immunopathologies (e.g. Crohn's disease, rheumatoid arthritis, systemic lupus erythematodes, psoriasis and type 1 diabetes mellitus) and in tumorigenesis. Frequent mutations in the A20 locus – leading to sustained NFκB activity – could be shown to play a dominant role in development of different B-cell malignancies. Experimental design and results: Based on genome-wide gene expression profiling analysis of CLL samples (n=8) compared to healthy donor B-cells (n=5), CYLD is expressed and its expression was reduced following B-cell receptor cross-linking (24 hours) (p=0,0036) contrary to A20 that could be induced after receptor stimulation (p=0,044). These results underline the role of B-cell receptor signaling in survival regulation of CLL cells and also its in-direct influence on NFκB activity. Recently, our report revealed by methylation analysis and additional sequence analysis that the A20 region neither contains any methylation (64 CLL patients versus 10 healthy donors) nor mutation (55 CLL patients with sequence analysis of exons 2–9 of the tnfaip3 gene) contrary to reports from other B-cell malignancies. Moreover, A20 expression could be confirmed by immunoblotting showing comparable results to healthy B-cells. In order to check if such alterations in the enzyme CYLD might occur in CLL leading to sustained activity of NFκB similar to other B-cell entities, we performed analysis of the methylation status of the promoter region of CYLD in 64 CLL patients compared to 10 DNAs of CD19-selected B-cells from healthy donors. Epigenetic alterations of the CYLD promoter could not be identified. Conclusions: Here we present the first report of epigenetic and mRNA expression analysis concerning the deubiquitinase CYLD in CLL. We identified that CYLD as well as A20 are regulated by B-cell receptor signaling. The opposed expression of CYLD and A20 after stimulation of the receptor might contribute to an almost balanced and well-adjusted NFκB activity. Our results of lacking epigenetic alteration in both proteins (A20 and CYLD) and absence of mutations in A20 indicate that malignant development in CLL differs from most of other B-cell malignancies, which show frequent inactivation of either CYLD or A20. Disclosures: No relevant conflicts of interest to declare.

scholarly journals IGLV3-21R110 identifies an aggressive biological subtype of chronic lymphocytic leukemia with intermediate epigenetics

Blood ◽  
2020 ◽  
Author(s):  
Ferran Nadeu ◽  
Romina Royo ◽  
Guillem Clot ◽  
Martí Duran-Ferrer ◽  
Alba Navarro ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Immunoglobulin Gene ◽  
Biological Features ◽  
Mutational Status ◽  
Bcr Signaling

B-cell receptor (BCR) signaling is crucial for chronic lymphocytic leukemia (CLL) biology. IGLV3-21-expressing B-cells may acquire a single point mutation (R110) that triggers autonomous BCR signaling conferring aggressive behavior. Epigenetic studies have defined three CLL subtypes based on methylation signatures reminiscent of naïve-like (n-CLL), intermediate (i-CLL) and memory-like B-cells (m-CLL) with different biological features. i-CLL carry a borderline IGHV mutational load and a significant higher usage of IGHV3-21/IGLV3-21. To determine the clinical and biological features of IGLV3-21R110 CLL and its relationship to these epigenetic subtypes we have characterized the immunoglobulin gene of 584 CLL cases using whole-genome/exome and RNA sequencing. IGLV3-21R110 was detected in 6.5% of cases, being 30/79 (38%) i-CLL, 5/291 (1.7%) m-CLL and 1/189 (0.5%) n-CLL. All stereotype subset #2 cases carried IGLV3-21R110 while 62% of IGLV3-21R110 i-CLL had non-stereotyped B-cell receptor immunoglobulins. IGLV3-21R110 i-CLL had significantly higher number of SF3B1 and ATM mutations, and total number of driver alterations. Nonetheless, the R110 mutation was the sole alteration in one i-CLL and accompanied only by del(13q) in three. Although composite regarding IGHV mutational status, IGLV3-21R110 i-CLL transcriptomically resembled naïve-like/unmutated IGHV CLL with a specific signature including WNT5A/B overexpression. Contrarily, i-CLL lacking the IGLV3-21R110 mirrored memory-like/mutated IGHV cases. IGLV3-21R110 i-CLL had a short time to first treatment and overall survival similar to n-CLL/unmutated IGHV cases whereas non-IGLV3-21R110 i-CLL had a good prognosis similar to memory-like/mutated IGHV. Altogether, IGLV3-21R110 defines a CLL subgroup with specific biological features and an unfavorable prognosis independent of the IGHV mutational status and epigenetic subtypes.

scholarly journals Aberrations of the B-Cell Receptor B29 (CD79b) Gene in Chronic Lymphocytic Leukemia

Blood ◽  
1997 ◽  
Vol 90 (4) ◽  
pp. 1387-1394 ◽  
Author(s):  
Alexis A. Thompson ◽  
Jeaniene A. Talley ◽  
Ha Nancy Do ◽  
H. Lee Kagan ◽  
Lori Kunkel ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Point Mutations ◽  
Cell Receptor ◽  
Surface Expression ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Cdna Clones ◽  
Bcr Signaling

Abstract Leukemic B cells in chronic lymphocytic leukemia (B-CLL) typically exhibit low or undetectable surface Ig. Because the B29 (CD79b and Igβ) and mb-1 (CD79a and Igα) gene products are required for surface Ig display in the B-cell receptor complex (BCR), we analyzed the expression of these genes in B-CLL cells. The majority (83%) of the randomly selected B-CLL patient samples analyzed exhibited low or undetectable surface BCR measured by μ heavy chain and B29 expression. Levels of mb-1 mRNA in these B-CLL samples with low surface BCR were similar to those in normal B cells. Among those with decreased surface expression, B29 mRNA was not detected in half of these B-CLL samples. The remaining B-CLL samples with diminished surface BCR contained normal levels of B29 mRNA. Further analysis of cDNA clones from the majority of these latter samples contained point mutations, insertions, or deletions that were largely located in the B29 transmembrane and cytoplasmic domains. These results indicate the occurrence of somatic mutations predicted to affect B29 expression and/or function in the majority of B-CLL and suggest that these aberrations underlie the diminished surface BCR display and loss of BCR signaling characteristic of this leukemia.

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