B cell receptor antigen receptor expression and phosphatidylinositol 3-kinase signaling regulate genesis and maintenance of mouse chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a frequent lymphoproliferative disorder of B cells. Although inhibitors targeting signal proteins involved in B cell antigen receptor (BCR) signaling constitute an important part of the current therapeutic protocols for CLL patients, the exact role of BCR signaling, as compared to genetic aberration, in the development and progression of CLL is controversial. To investigate whether BCR expression per se is pivotal for the development and maintenance of CLL B cells, we used the TCL1 mouse model. By ablating the BCR in CLL cells from TCL1 transgenic mice, we show that CLL cells cannot survive without BCR signaling and are lost within eight weeks in diseased mice. Furthermore, we tested whether mutations augmenting B cell signaling influence the course of CLL development and its severity. The Phosphatidylinositol-3-kinase (PI3K) signaling pathway is an integral part of the BCR signaling machinery and its activity is indispensable for B cell survival. It is negatively regulated by the lipid phosphatase PTEN, whose loss mimics PI3K pathway activation. Herein, we show that PTEN has a key regulatory function in the development of CLL, as deletion of the Pten gene resulted in greatly accelerated onset of the disease. By contrast, deletion of the gene TP53, which encodes the tumor suppressor p53 and is highly mutated in CLL, did not accelerate disease development, confirming that development of CLL was specifically triggered by augmented PI3K activity through loss of PTEN and suggesting that CLL driver consequences most likely affect BCR signaling. Moreover, we could show that in human CLL patient samples, 64% and 81% of CLL patients with a mutated and unmutated IgH VH, respectively, show downregulated PTEN protein expression in CLL B cells if compared to healthy donor B cells. Importantly, we found that B cells derived from CLL patients had higher expression levels of the miRNA-21 and miRNA-29, which suppresses PTEN translation, compared to healthy donors. The high levels of miRNA-29 might be induced by increased PAX5 expression of the B-CLL cells. We hypothesize that downregulation of PTEN by increased expression levels of miR-21, PAX5 and miR-29 could be a novel mechanism of CLL tumorigenesis that is not established yet. Together, our study demonstrates the pivotal role for BCR signaling in CLL development and deepens our understanding of the molecular mechanisms underlying the genesis of CLL and for the development of new treatment strategies.

Download Full-text

Mir-155 Contributed to Chronic Lymphocytic Leukemia Survival by Modulation of BCR-Signalling

Blood ◽

10.1182/blood.v118.21.620.620 ◽

2011 ◽

Vol 118 (21) ◽

pp. 620-620

Author(s):

Liguang Chen ◽

Bing Cui ◽

George Chen ◽

Michelle Salcedo ◽

Carlo M. Croce ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Survival ◽

Stromal Cells ◽

Initial Therapy ◽

Lymphocytic Leukemia ◽

Expression Levels ◽

Bcr Signaling ◽

Median Level

Abstract Abstract 620 The microRNA miR-155 is encoded within the B-cell integration cluster bic, which is a frequent integration site for avian leucosis virus. This microRNA is over-expressed in certain B cell lymphomas, including chronic lymphocytic leukemia (CLL), suggesting miR-155 contributes to lymphoma development. Consistent with this are observations that mice made transgenic for miR-155 under a B-cell-specific promoter develop pre-B cell lymphomas. One of the target genes regulated by miR-155 is SHIP-1, a phosphatase that plays a critical role in down-modulating B-cell receptor (BCR) signaling, which appears to provide a growth and/or survival stimulus for CLL B cells. To test for this, we examined for relationships between the CLL-cell expression of miR-155, SHIP-1, magnitude of BCR-signaling, and time from diagnosis to initial therapy (TFS) in 33 pts with CLL. The relative level of miR-155 was determined by real-time PCR. SHIP-1 protein was determined by flow cytometry and immunoblot analyses. We noted variations in expression levels of miR-155 among patient samples studied. CLL cells that expressed levels of miR-155 higher than the median level of miR-155 for all patients studied had significantly lower expression levels of SHIP-1 protein than CLL cells that expressed levels of miR-155 lower than the median level of miR-155 had (7.0 ± 0.2 vs. 8.3 ± 0.47, mean ± SEM, P<0.05). CLL B cells were stimulated with anti-μ or control Ig and then examined for relative protein phosphorylation and calcium influx. CLL cases were segregated into groups with high-BCR signaling versus low BCR-signaling based on their responses to anti-μ stimulation. We found that CLL cells with high-BCR signaling expressed significantly higher levels of miR-155 (1.54 ± 0.22) than did CLL cells with low-BCR signaling (0.90 ± 0.13, p<0.05). CLL cells with high BCR-signaling had significantly lower amounts of SHIP-1 protein (7.1 ± 0.39) than did CLL cells with low BCR-signaling (9.12 ± 0.7, p<0.05). Moreover, Kaplan-Meier survival analysis revealed an association between higher expression levels or miR-155 and shorter survival times from diagnosis to initial therapy (TFS) (P<0.05). CLL patients with greater miR-155 expression had a median TFS of 25.9 months that was significantly shorter than the median TFS of 112.8 months for patients with low miR-155 expression. To further investigate its function role, CLL cells were transfected with miR155 mimic or a miR-155 inhibitor and examined for SHIP-1 expression, BCR signaling, and cell survival, with or without stromal cells. Introduction of miR-155 to CLL cells that had low expression levels of miR-155 significantly reduced SHIP-1 protein expression, enhanced BCR signaling and improved cell survival relative to that of mock transfect CLL cells or CLL cells tranfected with micro RNA. Conversely, transfection of CLL cells that had high expression levels of miR-155 with miR-155 inhibitor resulted in significantly increased SHIP-1 expression, reduced BCR signaling, and poor survival than mock transfected CLL cells. Moreover, transfection of miR-155 inhibitor significantly reduced the capacity of stromal cells to enhance CLL cell survival. We found that the viability of cells transfected with miR-155 inhibitor was significantly lower than that noted for cells transfected with control miRs when cocultured with stromal cells. These results demonstrate that expression of miR-155 can modulate CLL expression of SHIP-1, CLL cell BCR signaling, and survival. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Association Between the Proficiency of B-Cell Receptor Signaling and the Relative Expression Levels of ZAP-70, SHIP-1, and Mir-155 in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v112.11.3155.3155 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3155-3155 ◽

Cited By ~ 3

Author(s):

Liguang Chen ◽

Bing Cui ◽

Suping Zhang ◽

George Chen ◽

Carlo M. Croce ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Calcium Flux ◽

Expression Levels ◽

Relative Expression ◽

Bcr Signaling

Abstract The immunoglobulin (Ig) repertoire expressed in chronic lymphocytic leukemia (CLL) appears highly selected, suggesting that stimulation of the B-cell receptor (BCR) by unknown self or environmental antigen(s) likely contributes to the pathogenesis and/or progression of this disease. Ligation of the BCR by F(ab)2 anti-μ can induce phosphorylation of p72Syk, BLNK, phospholipase Cgamma 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/or 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 use mutated IGHV and/or that lacked expression of ZAP-70. However, unusual cases that expressed mutated IGHV or that lack expression of ZAP-70 also were well stimulated by treatment with anti-μ, suggesting that other factors contribute to the noted differences in BCR-signaling observed between cases of CLL. We found that cases that used unmutated IGHV and that expressed ZAP-70 could be distinguished from cases that used mutated IGHV and that lacked expression of ZAP-70 by interrogating for differences in expression of selected microRNA, which are short non-coding RNA that each govern the post-transcriptional expression of a discrete set of genes. We focused attention on expression of miR-155, which generally is expressed at higher levels in CLL cells that express unmutated IGHV and ZAP-70 than CLL cells that use mutated IGHV and that lack ZAP-70. One of the putative target genes regulated by this microRNA is SHIP-1, a phosphatase that plays a critical role in modulating BCR signaling. We examined the MicroRNA-155 expression in CLL B cells and compared these values with the relative expression levels of SHIP-1 protein or ZAP-70 and use of unmutated IGHV. The relative levels of miR-155 were determined by real-time PCR. CLL B cells were stimulated with anti-μ or control Ig for 10 minutes and then examined for relative protein phosphorylation by flow cytometric and immunoblot analyses. CLL cases were segregated into groups with high-BCR signaling versus low BCR-signaling based on the relative levels of phosphorylation observed on signaling/adapter proteins following treatment with anti-μ. CLL cells with high-BCR signaling potential expressed significantly higher levels of miR-155 (1.62±0.33) than did CLL cells with low-BCR signaling potential (0.42±0.13, p<0.05). We also examined for SHIP-1 protein by flow cytometry and phosphorylated SHIP-1 by immunoblot analyses. These analyses revealed that the expression levels of SHIP-1 protein inversely correlated with the expression levels of miR-155 in CLL and the proficiency of BCR-signaling. Moreover, CLL cells with high BCR-signaling potential had significantly lower amounts of SHIP-1 protein, and significantly higher relative levels of phosphorylated SHIP-1 following treatment with anti-μ, than did CLL cells with low BCR-signaling potential. Although SHIP-1 protein was significantly more abundant in cases that lacked ZAP-70 than in cases that expressed ZAP-70, we identified cases that lacked ZAP-70 and had low levels of SHIP-1 that also experienced high-levels of BCRsignaling following treatment with anti-μ. These results indicate that the proficiency of BCR-signaling in CLL could be influenced by the relative levels of ZAP-70 and SHIP-1, at least the latter of which appears regulated by microRNA that are differentially expressed in aggressive versus indolent cases of CLL.

Download Full-text

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

Blood ◽

10.1182/blood-2006-03-011759 ◽

2006 ◽

Vol 109 (5) ◽

pp. 2032-2039 ◽

Cited By ~ 111

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.

Download Full-text

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

Blood ◽

10.1182/blood.v106.11.178.178 ◽

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.

Download Full-text

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

Blood ◽

10.1182/blood.v114.22.2343.2343 ◽

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.

Download Full-text

The Target Genes and Prognostic Significance of Mir-150 in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v120.21.3859.3859 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3859-3859

Author(s):

Marek Mraz ◽

Laura Z. Rassenti ◽

Emanuela M. Ghia ◽

Liguang Chen ◽

Jessie-Farah Fecteau ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Biology ◽

Fold Change ◽

Lymphocytic Leukemia ◽

Inverse Association ◽

Expression Of Genes ◽

Bcr Signaling ◽

Indolent Disease

Abstract Abstract 3859 Chronic lymphocytic leukemia (CLL) is the first disease in which miRNAs (hsa-miR-15a-16–1) were directly linked to cancer pathogenesis (Calin et al. PNAS, 2002). We and others have also shown that expression of certain miRNAs associates with disease activity in patients with CLL (Calin et al. NEJM, 2005; Mraz et al. Blood, 2012; Mraz et al. Leukemia, 2009). Moreover, patients with more aggressive disease have CLL cells that generally express unmutated IGHV and/or ZAP-70 and have a miRNA expression profile that differs from that of CLL cells from patients with indolent disease (Calin et al. NEJM, 2005). However, we still have very limited understanding of how miRNAs affect CLL cell-biology and expression of genes that play a critical role in either promoting or arresting the disease. We used pooled samples from 10 CLL patients to screen (TaqMan miRNA Cards-ABI, 750 miRNAs) for abundantly expressed miRNAs that could hypothetically influence CLL B cell biology. We identified miR-150 as the most abundant miRNA in CLL cells and also as being strongly expressed when compared to CD19+ blood lymphocytes of normal adults (N=5, P=0.008). This miRNA already has been reported to influence the differentiation and gene expression of normal B cells (Xiao et al. Cell, 2007) suggesting its possible relevance for CLL B cell biology. We examined additional CLL cell samples (N=168) and confirmed high miR-150 levels and also noted heterogeneity in its expression between CLL cells of patients with aggressive versus indolent disease. In our cohort, CLL cells of patients that expressed ZAP-70 (20% cut-off, N=74) or had unmutated IGHV (N=72) expressed significantly lower median-levels of miR-150 (fold change −1.7 and −2.0 respectively, p<0.005). Moreover, the lower levels of miR-150 also directly associated with higher response to stimulation of B-cell receptor (BCR) on CLL cells with anti-IgM (P<0.05, N=36, quantified by flow cytometric measurement of calcium mobilization). To understand the gene network regulated by miR-150 in CLL we performed array-based transcriptome analyses (HG-U133 Plus 2.0, Affymetrix) of 110 patient samples, which identified differential expression of 215 genes between CLL cells expressing low versus high levels of miR-150 (SAM analysis of upper and lower terciles). Thirty-eight of these 215 genes (17%) are predicted targets of miR-150 (determined by TargetScan, www.targetscan.org). Two well annotated genes (GAB1 and FOXP1) have evolutionary conserved binding sides for miR-150 in their 3‘UTRs, suggesting the possible importance of miR-150 in their regulation. GAB1 is an adaptor molecule and plays a key role in variety of cell signaling pathways (PLCγ, Ras/Erk, PI3K/Akt, CrkL). Interestingly, GAB1 modulates PI3K/Akt-pathway through binding domain identical to Bruton’s tyrosine kinase (Rameh et al. JBC, 1997) and is a key molecule involved in regulating BCR-signaling (Ingham et al. JBC, 1998, 2001), a process that factors prominently in the pathogenesis and progression of CLL. FOXP1 is an essential participant in the transcriptional regulatory network of B lymphopoiesis and has been identified as playing a role in disease progression of other B-cell lymphomas (Hu et al. Nat Immunol, 2006). The immunoblot analysis of GAB1 and FOXP1 in CLL cells confirmed their higher protein levels in cases with low miR-150 expression (P<0.005, fold change >10.0). Importantly, cells with higher expression of GAB1 or FOXP1 were more responsive to BCR stimulation in vitro (P<0.01, N=36) and higher expression of each associates with shorter overall survival (OS) (13.9 vs. 22.7 years, 13.9 vs. 21.1 years; N=168; P<0.05). Most notably, a reverse trend was observed for miR-150, where higher levels (>median) were associated with significantly longer OS (not-reached vs. 13.9 years, N=168, P=0.006). Additionally, the expression level of miR-150 was an independent predictor of OS and time to first treatment (TTFT) in multivariate analyses, which included IGHV status, ZAP-70, CD38, Rai stage, gender, and age (OS HR: 3.4 [CI 1.4–8.6], P=0.009; TTFT HR: 2.3 [CI 1.3–4.2], P=0.004). We conclude that there is an inverse association between high-risk disease and expression of miR-150, which may reflect its capacity to regulate the expression of genes encoding proteins that may contribute to BCR-signaling and/or survival of CLL B cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The role of phosphatidylinositol 3-kinase-δ in the immunomodulatory effects of lenalidomide in chronic lymphocytic leukemia

Blood ◽

10.1182/blood-2010-11-315705 ◽

2011 ◽

Vol 117 (16) ◽

pp. 4323-4327 ◽

Cited By ~ 67

Author(s):

Sarah E. M. Herman ◽

Rosa Lapalombella ◽

Amber L. Gordon ◽

Asha Ramanunni ◽

Kristie A. Blum ◽

...

Keyword(s):

B Cells ◽

Growth Factor ◽

Chronic Lymphocytic Leukemia ◽

Immune Modulation ◽

Cell Activation ◽

Costimulatory Molecule ◽

Immunoglobulin M ◽

Lymphocytic Leukemia ◽

Phosphatidylinositol 3 Kinase ◽

Phosphatidylinositol 3

Abstract In patients with chronic lymphocytic leukemia (CLL), lenalidomide can promote humoral immune responses but also induces a distinct disease-specific toxicity of tumor flare and cytokine release. These CLL-specific events result from increased expression of costimulatory molecules on B cells. Here we demonstrate that lenalidomide activation of CLL cells depends on the phosphatidylinositol 3-kinase p110δ (PI3K-δ) pathway. Inhibition of PI3K-δ signaling by the PI3K-δ-inhibiting drug, CAL-101, or by siRNA knockdown of p110δ, abrogates CLL cell activation, costimulatory molecule expression, and vascular endothelial growth factor and basic fibroblast growth factor gene expression that is induced by lenalidomide. In addition, CAL-101 attenuates lenalidomide-mediated increases in immunoglobulin M production by normal B cells. Collectively, these data demonstrate the importance of PI3K-δ signaling for lenalidomide immune modulation. These findings may guide development of strategies for the treatment of CLL that combine lenalidomide with CAL-101, with other inhibitors of the PI3K-δ pathway, or with other agents that target downstream kinases of this signaling pathway.

Download Full-text

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

Blood ◽

10.1182/blood.v108.11.2799.2799 ◽

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.

Download Full-text

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 ◽

10.1182/blood.v112.11.5023.5023 ◽

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

Download Full-text

Early generated B1 B cells with restricted BCRs become chronic lymphocytic leukemia with continued c-Myc and low Bmf expression

Journal of Experimental Medicine ◽

10.1084/jem.20160712 ◽

2016 ◽

Vol 213 (13) ◽

pp. 3007-3024 ◽

Cited By ~ 24

Author(s):

Kyoko Hayakawa ◽

Anthony M. Formica ◽

Joni Brill-Dashoff ◽

Susan A. Shinton ◽

Daiju Ichikawa ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Subset ◽

Lymphocytic Leukemia ◽

Transgenic Expression ◽

Bcr Signaling ◽

B Cell Subsets ◽

A Minor ◽

B1 B Cells

In mice, generation of autoreactive CD5+ B cells occurs as a consequence of BCR signaling induced by (self)-ligand exposure from fetal/neonatal B-1 B cell development. A fraction of these cells self-renew and persist as a minor B1 B cell subset throughout life. Here, we show that transfer of early generated B1 B cells from Eμ-TCL1 transgenic mice resulted in chronic lymphocytic leukemia (CLL) with a biased repertoire, including stereotyped BCRs. Thus, B1 B cells bearing restricted BCRs can become CLL during aging. Increased anti-thymocyte/Thy-1 autoreactive (ATA) BCR cells in the B1 B cell subset by transgenic expression yielded spontaneous ATA B-CLL/lymphoma incidence, enhanced by TCL1 transgenesis. In contrast, ATA B-CLL did not develop from other B cell subsets, even when the identical ATA BCR was expressed on a Thy-1 low/null background. Thus, both a specific BCR and B1 B cell context were important for CLL progression. Neonatal B1 B cells and their CLL progeny in aged mice continued to express moderately up-regulated c-Myc and down-regulated proapoptotic Bmf, unlike most mature B cells in the adult. Thus, there is a genetic predisposition inherent in B-1 development generating restricted BCRs and self-renewal capacity, with both features contributing to potential for progression to CLL.

Download Full-text