SLP76 Is Ectopically Expressed in Chronic Lymphocytic Leukemia Cells and Involves in B-Cell Receptor Signaling

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1719-1719
Author(s):  
Yair Herishanu ◽  
Nili Dorozella ◽  
Mika Shapiro ◽  
Chava Perry ◽  
Ben-Zion Katz
Keyword(s):  
B Cell ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Tcr Signaling ◽  
Bcr Signaling

Abstract In the last decade, the B-cell receptor (BCR) has emerged as a pivotal stimulus in CLL pathogenesis. The BCR responsiveness in CLL cells is heterogeneous among patients and correlates with disease aggressiveness. Here we show for the first time, that SLP76 a key scaffold protein in T-cell receptor (TCR) signaling, is ectopically expressed in CD19+ purified CLL cells (purity >95%) in the majority of patients, and is co-expressed with other components of the TCR pathway, including LCK and ZAP70. SLP76 mRNA levels correlated with its protein expression and SLP76 protein levels were higher in unmutated IGHV and ZAP70+ CLL cells. SLP76 was found to be functionally active in CLL cells, as it becomes phosphorylated in response to BCR engagement in a time dependent manner. Activation with anti-IgM antibody results in phosphorylation of SLP76 on the positive regulatory tyrosine residue Y128 residue with increased physical association with Btk, peaking after 15 minutes. The negative regulatory residue S376 became phosphorylated only after 45', concomitantly with downregulation of the tyrosine phosphorylation. SLP76 phosphorylation in response to BCR engagement did not correlate with total ZAP70 expression. Pre-incubation of CLL cells with the LCK kinase inhibitor LCKi and with the SYK inhibitor R406, inhibited SLP76 phosphorylation in response to BCR activation, while the BTK inhibitor-ibrutinib had no effect. These suggest that LCK and SYK, but not ZAP70, play a central role in the upstream signaling involved in SLP76 activation in CLL cells. Knockdown of SLP76 in CLL cells resulted in decreased induction of BTK and PLCγ2 phosphorylation after BCR activation with anti-IgM. Consistent with our findings that SLP76 in involved in BCR signaling in CLL cells, we found that high total SLP76 expression was associated with a shorter time to first progression or need for treatment. In conclusion, SLP76 is ectopically expressed in CLL cells and plays a role in BCR signaling in those cells. Disclosures No relevant conflicts of interest to declare.

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 (<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 The multi-kinase inhibitor TG02 induces apoptosis and blocks B-cell receptor signaling in chronic lymphocytic leukemia through dual mechanisms of action

2021 ◽  
Vol 11 (3) ◽  
Author(s):  
Rong Chen ◽  
Jennifer Tsai ◽  
Philip A. Thompson ◽  
Yuling Chen ◽  
Ping Xiong ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Rna Polymerase Ii ◽  
Kinase Inhibitor ◽  
Leukemia Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Rapid Induction ◽  
Bcr Signaling

AbstractThe constitutive activation of B-cell receptor (BCR) signaling, together with the overexpression of the Bcl-2 family anti-apoptotic proteins, represents two hallmarks of chronic lymphocytic leukemia (CLL) that drive leukemia cell proliferation and sustain their survival. TG02 is a small molecule multi-kinase inhibitor that simultaneously targets both of these facets of CLL pathogenesis. First, its inhibition of cyclin-dependent kinase 9 blocked the activation of RNA polymerase II and transcription. This led to the depletion of Mcl-1 and rapid induction of apoptosis in the primary CLL cells. This mechanism of apoptosis was independent of CLL prognostic factors or prior treatment history, but dependent on the expression of BAX and BAK. Second, TG02, which inhibits the members of the BCR signaling pathway such as Lck and Fyn, blocked BCR-crosslinking-induced activation of NF-κB and Akt, indicating abrogation of BCR signaling. Finally, the combination of TG02 and ibrutinib demonstrated moderate synergy, suggesting a future combination of TG02 with ibrutinib, or use in patients that are refractory to the BCR antagonists. Thus, the dual inhibitory activity on both the CLL survival pathway and BCR signaling identifies TG02 as a unique compound for clinical development in CLL and possibly other B cell malignancies.

ZAP-70 enhances B-cell–receptor signaling despite absent or inefficient tyrosine kinase activation in chronic lymphocytic leukemia and lymphoma B cells

Blood ◽  
2006 ◽  
Vol 109 (5) ◽  
pp. 2032-2039 ◽  
Author(s):  
Stefania Gobessi ◽  
Luca Laurenti ◽  
Pablo G. Longo ◽  
Simona Sica ◽  
Giuseppe Leone ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Antigen Receptor ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Tcr Signaling ◽  
Bcr Signaling

Abstract Expression of ZAP-70 is an important negative prognostic factor in chronic lymphocytic leukemia (CLL). This protein tyrosine kinase is a key mediator of T-cell receptor (TCR) signaling and is structurally homologous to Syk, which plays an analogous role in B-cell receptor (BCR) signaling. Recent studies indicate that ZAP-70 may participate in BCR signaling as well, but the mechanism of action is not completely understood. We have now compared antigen receptor-induced activation of ZAP-70 in B cells and T cells by analyzing phosphorylation of critical regulatory tyrosine residues. We show that BCR-mediated activation of ZAP-70 is very inefficient in CLL and lymphoma B cells and is negligible when compared to activation of Syk. Despite the inefficient catalytic activation, the ability of ZAP-70 to recruit downstream signaling molecules in response to antigen receptor stimulation appeared relatively preserved. Moreover, ectopic expression of ZAP-70 enhanced and prolonged activation of several key mediators of BCR signaling, such as the Syk, ERK, and Akt kinases, and decreased the rate of ligand-mediated BCR internalization. We conclude that the role of ZAP-70 in BCR signaling is quite distinct from its role in TCR signaling and is likely mediated by inhibition of events that terminate the signaling response.

scholarly journals Targeted inhibition of eIF4A suppresses B-cell receptor-induced translation and expression of MYC and MCL1 in chronic lymphocytic leukemia cells

2021 ◽  
Author(s):  
Sarah Wilmore ◽  
Karly-Rai Rogers-Broadway ◽  
Joe Taylor ◽  
Elizabeth Lemm ◽  
Rachel Fell ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mrna Translation ◽  
Cell Receptor ◽  
Memory B Cells ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Mrna Stabilization

AbstractSignaling via the B-cell receptor (BCR) is a key driver and therapeutic target in chronic lymphocytic leukemia (CLL). BCR stimulation of CLL cells induces expression of eIF4A, an initiation factor important for translation of multiple oncoproteins, and reduces expression of PDCD4, a natural inhibitor of eIF4A, suggesting that eIF4A may be a critical nexus controlling protein expression downstream of the BCR in these cells. We, therefore, investigated the effect of eIF4A inhibitors (eIF4Ai) on BCR-induced responses. We demonstrated that eIF4Ai (silvestrol and rocaglamide A) reduced anti-IgM-induced global mRNA translation in CLL cells and also inhibited accumulation of MYC and MCL1, key drivers of proliferation and survival, respectively, without effects on upstream signaling responses (ERK1/2 and AKT phosphorylation). Analysis of normal naïve and non-switched memory B cells, likely counterparts of the two main subsets of CLL, demonstrated that basal RNA translation was higher in memory B cells, but was similarly increased and susceptible to eIF4Ai-mediated inhibition in both. We probed the fate of MYC mRNA in eIF4Ai-treated CLL cells and found that eIF4Ai caused a profound accumulation of MYC mRNA in anti-IgM treated cells. This was mediated by MYC mRNA stabilization and was not observed for MCL1 mRNA. Following drug wash-out, MYC mRNA levels declined but without substantial MYC protein accumulation, indicating that stabilized MYC mRNA remained blocked from translation. In conclusion, BCR-induced regulation of eIF4A may be a critical signal-dependent nexus for therapeutic attack in CLL and other B-cell malignancies, especially those dependent on MYC and/or MCL1.

scholarly journals IVIg modulates BCR signaling through CD22 and promotes apoptosis in mature human B lymphocytes

Blood ◽  
2010 ◽  
Vol 116 (10) ◽  
pp. 1698-1704 ◽  
Author(s):  
Jean-François Séïté ◽  
Divi Cornec ◽  
Yves Renaudineau ◽  
Pierre Youinou ◽  
Rizgar A. Mageed ◽  
...  
Keyword(s):  
B Cell ◽  
B Lymphocytes ◽  
B Lymphocyte ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Time Dependent ◽  
G1 Phase ◽  
Dependent Manner ◽  
Bcr Signaling ◽  
Sustained Activation

Abstract Among various mechanisms for interactions with B cells, intravenous immunoglobulin (IVIg) may operate through the insertion of its Fc part into the Fc-γ receptor, or the binding of its sialic acid (SA)–bearing glycans to the negatively regulating CD22 lectin. It appeared that IVIg reduces B lymphocyte viability in a dose- and time-dependent manner. Furthermore, we show by confocal microscopy that SA-positive IgG, but not SA-negative IgG bind to CD22. This interaction reduces the strength of B-cell receptor–mediated signaling trough down-regulating tyrosine phosphorylation of Lyn and the B-cell linker proteins, and up-regulating phospholipase Cγ2 activation. This cascade resulted in a sustained activation of Erk 1/2 and arrest of the cell cycle at the G1 phase. These changes may be accounted for the efficacy of IVIg in autoimmune diseases.

High ZAP-70 and Differential Expression of B-Cell Receptor Related Genes in Chronic Lymphocytic Leukemia with V3-21 Gene Usage.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 773-773
Author(s):  
Dirk Kienle ◽  
Alexander Kröber ◽  
Dirk Winkler ◽  
Daniel Mertens ◽  
Annett Habermann ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mutation Status ◽  
Bcr Signaling ◽  
Gene Usage ◽  
Lower Expression

Abstract V3-21 gene usage defines a distinct genetic subgroup of chronic lymphocytic leukemia (CLL) characterized by a poor clinical outcome regardless of the VH mutation status. V3-21 cases exhibit a highly characteristic B-cell receptor (BCR) structure as demonstrated by homologous CDR3 sequences and a restricted use of VL genes implicating a common antigen involved in tumor pathogenesis of this specific CLL subgroup. To investigate the role of antigenic stimulation in the pathogenesis of V3-21 using CLL, we analyzed the quantitative expression of genes involved in BCR signaling (ZAP-70, SYK, BLNK, LYN, PI3K, PLCG2, FOS), B-cell activation (TRAF3, STAT6, NFKB), and cell cycle or apoptosis control (ATM, BCL-2, BAX, CDK4, CCND1, CCND2, CCND3, p27, E2F1, MYC) in V3-21 cases in comparison to VH mutated (VH MUT) and VH unmutated (VH UM) cases not using the V3-21 gene. To obtain native expression signatures we studied a non-CD19-purified (nPU) cohort (V3-21: 18 cases, equally divided into VH mutated and VH unmutated cases; VH MUT: 17; VH UM: 19) and, for verification, a CD19-purified (PU) cohort (V3-21: 10 cases, equally divided into VH mutated and unmutated; VH MUT: 12; VH UM: 16) to exclude a contamination of the results by non-tumor cells. All cases were analyzed by FISH for +3q, 6q-, +8q, 11q-, +12q, 13q-, 17p-, and t(11;14) to avoid major imbalances of genomic alterations between the subgroups under study. As expected, ZAP-70 expression was higher in VH UM as compared to VH MUT cases in the nPU (p=0.007) as well as the PU cohort (p=0.009). V3-21 cases showed a higher ZAP-70 expression as compared to VH MUT (nPU: p=0.033; PU: p=0.038). This applied also when restricting this comparison to V3-21 mutated cases (nPU: p=0.018). Median ZAP-70 expression in the PU cohort was 1.15 in VH MUT vs. 7.69 in VH UM cases, as compared to 7.05 in V3-21 cases (V3-21 mutated cases: 10.69; V3-21 unmutated: 6.7). Other genes differentially expressed between the V3-21 and VH MUT subgroups in nPU cases were PI3K (p=0.048), PLCG2 (p=0.007), CCND2 (p=0.003), p27 (p=0.003), BCL-2 (p=0.025), and ATM (p=0.006). In addition, a set of genes was detected with a differential expression between V3-21 and VH UM (nPU) including PLCG2 (p=0.014), NFKB (p=0.023), CCND2 (p=0.001), p27 (0.002), and BAX (p=0.028). Notably, except for ZAP-70, all of the differentially expressed genes showed a lower expression in V3-21 as compared to the other subgroups. When comparing the V3-21 mutated and V3-21 unmutated subgroups (nPU), there were no significant gene expression differences except for CDK4, which showed a lower expression in V3-21 unmutated cases. Therefore, cases with V3-21 usage appear to show a rather homogeneous gene expression pattern independently of the VH mutation status, which can be distinguished from VH MUT and VH UM cases not using V3-21. The expression differences observed suggest a role of differential BCR signaling in the pathogenesis of this distinct CLL subgroup. Deregulation of cell cycle, apoptosis, and candidate genes such as ATM indicate the involvement of additional pathways in the pathogenesis of CLL cases using V3-21.

B-Cell Receptor Signaling Enhances Migration of B-Cell Chronic Lymphocytic Leukemia Cells in Response to the Chemokine Stromal Cell-Derived Factor-1 (SDF-1/CXCL12).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1187-1187
Author(s):  
Jan A. Burger ◽  
Myriam Krome ◽  
Andrea Bürkle ◽  
Tanja N. Hartmann
Keyword(s):  
Dendritic Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Stromal Cells ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Follicular Dendritic Cells ◽  
Accessory Cells ◽  
Bcr Signaling

Abstract There is growing evidence that the microenvironment confers survival signals to Chronic Lymphocytic Leukemia (CLL) B-cells that may result in disease progression and resistance to therapy. In the marrow or secondary lymphoid tissues, CLL cells are in close contact with non-tumoral accessory cells, such as mesenchymal stromal cells or nurselike cells. We previously characterized SDF-1 (CXCL12) as a central mediator for CLL cell migration and interaction with the protective microenvironment. Constitutive secretion of CXCL12 attracts CLL cells to stroma or NLC through its cognate receptor, CXCR4. These accessory cells protect CLL cells from spontaneous or drug-induced apoptosis, which is contact-dependent and partially mediated by CXCL12. B-cell receptor (BCR) signaling has been considered another important regulator of CLL cell survival. Typically, CLL cell that lack somatic mutations in the immunoglobulin (Ig) variable region (V) genes and display high levels of the tyrosine kinase ZAP-70 strongly responds to anti-IgM stimulation. Because both, CXCL12 stimulation and BCR signaling may represent important mechanism for maintenance of CLL cell within the microenvironment, we examined whether anti-IgM stimulation affects CXCL12 responses in correlation with the ZAP-70 status. BCR signaling was modulated either by crosslinking the BCR with IgM or by blocking the tyrosine kinase Syk. Effective BCR cross-linking with anti-IgM antibodies was demonstrated by phosphorylation of Syk and p44/42 MAP kinase. In ZAP-70 positive cells, BCR crosslinking resulted in a robust activation of Syk, p44/42 MAP kinases, and protein kinase B (Akt). ZAP-70 negative CLL cells displayed a weaker activation of p44/42 upon IgM crosslinking. Pretreatment of CLL cells with anti-IgM resulted in an enhanced calcium mobilization upon CXCL12 stimulation. This was not due to changes in surface expression of CXCR4. Accordingly, Syk inhibition by piceatannol resulted in a loss of calcium response upon CXCL12 stimulation. Furthermore, anti-IgM stimulation significantly increased CLL cell chemotaxis towards CXCL12 1.4 ± 1.2fold (n=9, p=0.027), and Syk inhibition by piceatannol decreased chemotaxis to 0.6 ± 0.2fold of controls (n=8). In these experiments, we could not detect differences between ZAP-70 positive or negative cells. However, there was a strong difference regarding the spontaneous, CXCL12-dependent migration of CLL cells beneath marrow stromal cells (pseudoemperipolesis). BCR crosslinking significantly increased pseudoemperipolesis of ZAP-70 expressing CLL cells 13.4 ± 21.0fold (n=7, p=0.043), whereas there was no significant increase in pseudoemperipolesis of ZAP-70 negative cells (1.4 ± 0.2fold increase, n=8). Syk inhibition by piceatannol significantly decreased the pseudoemperipolesis of ZAP-70 positive as well as ZAP-70 negative CLL cells to 0.4 ± 0.07 of controls (n=5, p=0.043). Interestingly, spontaneous migration of CLL cells beneath follicular dendritic cells (HK cells) was also significantly enhanced by anti-IgM stimulation, in particular in ZAP-70 positive cases. In summary, BCR signaling enhances calcium mobilization, CLL cell migration to CXCL12, and pseudoemperipolesis beneath marrow stroma or follicular dendritic cells. These data suggest that BCR stimulation co-operates with CXCL12 for localization and/or maintenance of CLL cells within distinct tissue microenvironments.

Influence of Mir-155 On B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2343-2343
Author(s):  
Liguang Chen ◽  
Bing Cui ◽  
George Chen ◽  
Michelle Salcedo ◽  
Carlo M. Croce ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Calcium Influx ◽  
Critical Role ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Calcium Flux ◽  
Expression Levels ◽  
Bcr Signaling

Abstract Abstract 2343 Poster Board II-320 B-cell receptor (BCR) signaling arguably plays an important role in the pathogenesis and/or progression of chronic lymphocytic leukemia. Ligation of the BCR by F(ab)2 anti-μ can induce phosphorylation of p72Syk, BLNK, phospholipase C-gamma (PLCγ) and other downstream adapter/signaling molecules, inducing intracellular calcium flux and cellular activation. Prior studies found that CLL cells that expressed unmutated Ig heavy-chain variable region genes (IGHV) and the zeta-associated protein of 70 kD (ZAP-70) generally experienced greater levels of activation following treatment with anti-μ than did CLL cells that lacked expression of ZAP-70. However, we found unusual cases that lacked expression of ZAP-70 that also responded vigorously to treatment with anti-μ, suggesting that other factors contribute to the noted differences in BCR-signaling. Analyses for expression of microRNAs by microarray revealed that CLL cells that used unmutated IGHV and that expressed ZAP-70 expressed higher levels of certain microRNAs than did cases that used mutated IGHV and that lacked expression of ZAP-70. One of such microRNA, miR-155, was found to target mRNA encoding SHIP-1, a phosphatase that plays a critical role in modulating the level of BCR signaling in normal B cells. Using quantitative assays for miR-155 we found high-level expression of this microRNA was associated with proficient BCR signaling in CLL. To examine whether miR-155 could modulate the levels of SHIP-1 and/or BCR signaling in CLL cells we transfected primary leukemia cells from each of multiple patients with control oligo-RNAs, miR-155, or a specific inhibitor of miR-155 (miR-155 inhibitor). Twenty-four hours later the cells were stimulated with anti-μ or control antibody and then examined 10 minutes later for expression of SHIP-1, induced calcium influx, or phosphorylation of kinases and adapter proteins that are involved in BCR signaling. CLL cells that had low expression levels of miR-155 and that were poorly responsive BCR had significantly higher levels of calcium influx and phosphorylated p72Syk, BLNK, and PLCγ in response to anti-μ following transfection with miR-155 than following mock transfection or transfection with control oligo-RNA. Conversely, CLL cells that had high expression levels of miR-155 and highly responsive BCR were made to have significantly higher amounts of SHIP-1 protein and to have significantly lower relative levels of phosphorylated protein and calcium influx in response to anti-μ following transfection with the miR-155 inhibitor than did mock transfected CLL cells. These results identify miR-155 as a factor that can modulate BCR signaling in CLL in part by regulating the relative expression level of SHIP-1. These results demonstrate that differential expression of microRNAs in CLL can influence physiologic features that potentially contribute to disease progression. Disclosures: No relevant conflicts of interest to declare.

Evidence for Autostimulatory Mechanisms in Chronic Lymphocytic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2880-2880
Author(s):  
Martin Trepel ◽  
Fabian Muller ◽  
Mareike Frick ◽  
Janina Rahlff ◽  
Claudia Wehr ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Phage Display ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Specific Binding ◽  
Variable Region ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Fab Fragment

Abstract Abstract 2880 Background: The development and / or course of chronic lymphocytic leukemia (CLL) may be driven by the recognition of antigens through the B cell receptor (BCR). While it has been recognized that the diversity of epitope recognition may be astonishingly confined in CLL, knowledge on antigens recognized by CLL BCRs is still limited. Here, we identified and characterized an epitope recognized by a defined CLL BCR which may broaden our view on potential mechanisms of antigenic drive in CLL. Methods: The B- cell receptor of a random CLL-patient was cloned and expressed as Fab fragment in E.coli. Random phage display reptile litanies we skeletal on the immobilized Fab and landed peptides were tested for specific binding. Specific clones we sequenced and sequences were analyzed for homology to known proteins. Recognition of candidate proteins was verified in brooding assays or recombinant proteins. Results: Screening random phage display peptide libraries, we identified a CLL BCR epitope mimic that displayed a high degree of homology to a conserved peptide string in the variable region of immunoglobulin heavy and light chains. CLL BCR binding to this epitope as well as binding to full length heavy and light immunoglobulin chains was verified by binding assays and a protein array screening. Interestingly, the CLL BCR also interacted with itself, as the identified epitope was also present in its own primary amino acid sequence. Conclusions: These findings suggest the possibility of self-recognition of BCRs within the CLL cell membrane or BCR interactions between neighboring CLL cells. This may potentially result in autostimulation of the leukemic cell independent of “exogenous” antigens and may account for self-sufficient signaling of some CLL-BCRs in driving disease progression. As the peptide mimicking this immunoglobulin epitope is known to be recognized by BCRs of other CLL cases in addition to the index case investigated here, such autostimulatory mechanisms may be relevant to a large number of CLL patients. Disclosures: No relevant conflicts of interest to declare.

B-Cell Receptor Configuration and Adverse Cytogenetics Are Associated with Autoimmune Hemolytic Anemia in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1780-1780
Author(s):  
Francesco Maura ◽  
Carlo Visco ◽  
Erika Falisi ◽  
Reda Gianluigi ◽  
Sonia Fabris ◽  
...  
Keyword(s):  
Multivariate Analysis ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Hemolytic Anemia ◽  
Autoimmune Hemolytic Anemia ◽  
Germ Line ◽  
Conflicts Of Interest ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia

Abstract Abstract 1780 Background: Biological features related to the development of autoimmune hemolytic anemia (AHIA) in patients with chronic lymphocytic leukemia (CLL) are crucial insights in the understanding of the pathogenesis of autoimmune phenomena in the course of the disease. Design and Methods: We retrospectively analyzed 585 CLL patients with available immunoglobulin heavy-chain variable (IGHV) gene status and B-cell receptor (BCR) configuration (HCDR3). Of them, 73 developed AIHA. The clinical characteristics at CLL diagnosis and follow-up were available in all patients, while cytogenetic analysis at the time of diagnosis was available in 409 patients. Results: Occurrence of AIHA was significantly associated with an IGHV unmutated (UM) status (p<0.0001) and unfavorable cytogenetic lesions [del(17)(p13) and del(11)(q23)] (p<0.0001). Stereotyped HCDR3 sequences were identified in 173 of 585 patients (29.6%) and were similarly represented among patients developing AIHA (28,7%) or not (29.6%). Of the stereotyped subsets, subset #3 was associated with a significantly higher risk of AIHA occurrence than the other HCDR3 configurations (p=0.004). Restricting the analysis to UM patients, a strong association was found between AIHA and “truly” UM patients, defined as patients carrying a 100% identity with the germ line configuration. Multivariate analysis showed that “truly” UM IGHV, del(17)(p13) and del(11)(q23) were the strongest independent variables associated with risk of developing AIHA (p=0.02, p=0.0002 and p=0.01, respectively). Based on the results of the multivariate analysis, we constructed a risk score of developing AIHA during time, according to the presence of none (low risk = favorable cytogenetics and mutated (M) IGHV), one (intermediated risk = unfavorable cytogenetic or UM), or two (high risk = unfavorable cytogenetic and UM) risk factors. This scoring system allowed a significant patient risk stratification (Figure 1). Conclusions: Taken together, our data indicate that an UM IGHV status and/or unfavorable cytogenetic lesions are associated with the risk of developing secondary AIHA in CLL patients and suggest a possible role of specific stereotyped BCR subsets in a proportion of cases. Disclosures: No relevant conflicts of interest to declare.

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