An Alternatively Spliced Form of CD79b Gene May Account for Altered B-Cell Receptor Expression in B-Chronic Lymphocytic Leukemia

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2327-2335 ◽  
Author(s):  
A. Alfarano ◽  
S. Indraccolo ◽  
P. Circosta ◽  
S. Minuzzo ◽  
A. Vallario ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Receptor Expression ◽  
Rt Pcr ◽  
Alternatively Spliced

Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Ig/Igβ (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19+ cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5+ and CD5− B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5+ and CD5− B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 ± 0.20 SD in normal donors and 0.44 ± 0.27 SD in B-CLL (P = .01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT → TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.

An Alternatively Spliced Form of CD79b Gene May Account for Altered B-Cell Receptor Expression in B-Chronic Lymphocytic Leukemia

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2327-2335 ◽  
Author(s):  
A. Alfarano ◽  
S. Indraccolo ◽  
P. Circosta ◽  
S. Minuzzo ◽  
A. Vallario ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Receptor Expression ◽  
Rt Pcr ◽  
Alternatively Spliced

Abstract Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Ig/Igβ (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19+ cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5+ and CD5− B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5+ and CD5− B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 ± 0.20 SD in normal donors and 0.44 ± 0.27 SD in B-CLL (P = .01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT → TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.

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.

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

Lower levels of surface B-cell-receptor expression in chronic lymphocytic leukemia are associated with glycosylation and folding defects of the μ and CD79a chains

Blood ◽  
2005 ◽  
Vol 105 (7) ◽  
pp. 2933-2940 ◽  
Author(s):  
Françoise Vuillier ◽  
Gérard Dumas ◽  
Christian Magnac ◽  
Marie-Christine Prevost ◽  
Ana Inés Lalanne ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cellular Localization ◽  
Cell Receptor ◽  
Surface Expression ◽  
B Cell Receptor ◽  
Immunoglobulin M ◽  
Lymphocytic Leukemia ◽  
Receptor Expression ◽  
Surface Immunoglobulin

AbstractLow levels of B-cell-receptor (BCR) expression are the hallmark of tumoral B lymphocytes in B-cell chronic lymphocytic leukemia (B-CLL). These cells also respond inadequately to stimulation through the BCR. This receptor consists of a surface immunoglobulin associated with a CD79a/CD79b heterodimer. We previously showed that the intracellular synthesis of BCR components, from transcription onward, is normal. Here, we investigated the glycosylation status and cellular localization of μ, CD79a, and CD79b chains in 10 CLL patients differing in surface immunoglobulin M (IgM) expression. We reported a severe impairment of the glycosylation and folding of μ and CD79a. These defects were associated with the retention of both chains in the endoplasmic reticulum and lower levels of surface IgM expression. In contrast, no clear impairment of glycosylation and folding was observed for CD79b. No sequence defects were identified for BCR components and for the chaperone proteins involved in BCR folding processes. These data show, for the first time, that lower levels of BCR surface expression observed in CLL are accounted for by an impaired glycosylation and folding of the μ and CD79a chains.

scholarly journals Engagement of the B-cell receptor of chronic lymphocytic leukemia cells drives global and MYC-specific mRNA translation

Blood ◽  
2016 ◽  
Vol 127 (4) ◽  
pp. 449-457 ◽  
Author(s):  
Alison Yeomans ◽  
Stephen M. Thirdborough ◽  
Beatriz Valle-Argos ◽  
Adam Linley ◽  
Sergey Krysov ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mrna Translation ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Key Points ◽  
Specific Mrna

Key Points BCR stimulation promotes mRNA translation in CLL cells, including of the oncoprotein, MYC, and is inhibited by ibrutinib or tamatinib. Differences in mechanisms of regulation of mRNA translation in CLL and normal blood B cells may highlight potential targets for therapy.

scholarly journals Reduced expression of the tumor suppressor PHLPP1 enhances the antiapoptotic B-cell receptor signal in chronic lymphocytic leukemia B-cells

Leukemia ◽  
2010 ◽  
Vol 24 (12) ◽  
pp. 2063-2071 ◽  
Author(s):  
M Suljagic ◽  
L Laurenti ◽  
M Tarnani ◽  
M Alam ◽  
S N Malek ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Tumor Suppressor ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Receptor Signal

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.

Chronic Lymphocytic Leukemia B-Cells Lack or Express Markedly Reduced Levels of the Phosphatase PHLPP, Which Regulates B-Cell Receptor Induced AKT Activation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2080-2080
Author(s):  
Mirza Suljagic ◽  
Luca Laurenti ◽  
Muhammad Alam ◽  
Pablo G Longo ◽  
Sami N Malek ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Akt Pathway ◽  
Immunoblotting Analysis ◽  
Akt Phosphorylation ◽  
Akt Activation

Abstract The PI3K/AKT pathway plays a central role in regulating cellular growth and survival. This pathway is activated by signals derived from various receptors and is tightly regulated through the action of several phosphatases, including SHIP and PTEN, which hydrolyze the PI3K product PIP3, and the recently identified PHLPP, which directly dephosphorylates AKT. Hyperactivation of the PI3K/AKT pathway has been implicated in the pathogenesis of many types of cancer, including chronic lymphocytic leukemia (CLL) and B-cell lymphoma. In addition, gene expression profiling and real-time RT/PCR analysis have recently shown differential expression of PHLPP mRNA in CLL subsets classified according to the presence of the 13q14 abnormality, with many CLL cases demonstrating absent PHLPP expression altogether. These findings prompted us to compare the levels of PHLPP expression in primary CLL B-cells (n=17) with normal tonsillar B-cells (n=4) and various lymphoma cell lines, including the diffuse large B-cell lymphomas (DLBCL) DHL-4, DHL-6, DHL-8, DHL-10, WSU, Toledo, Ly1, Ly3, Ly7 and Ly18, the Burkitt’s lymphoma BJAB and the prolymphocytic leukemia MEC1. Immunoblotting analysis revealed abundant and uniform expression of PHLPP in normal B-cells and in 7 out of 12 investigated lymphoma cell lines. Higher levels were observed in the BJAB, Ly1 and Ly18 cell lines, whereas PHLPP was undetectable in the DLBCL cell lines WSU and Toledo. Remarkably, PHLPP was either not expressed or was expressed at markedly reduced levels in all of the investigated CLL samples, with levels of expression ranging from 0 to 10% of the levels in normal B-cells. In contrast, the levels of expression of the phosphatase SHIP were relatively similar between CLL and normal B-cells. To determine what are the consequences of reduced PHLPP expression on signaling through AKT in malignant B-lymphocytes, we downregulated PHLPP in BJAB and DHL-4 cells by RNA interference. A significant reduction in the levels of PHLPP was achieved in both cell lines, which amounted to 20–40% of the levels in cells transfected with the control siRNA. Immunoblotting analysis of protein extracts from cells transfected with PHLPP and control siRNA did not show a difference in AKT phosphorylation on Ser473 and Thr308, indicating that a reduction in PHLPP expression is not sufficient to augment basal AKT activity. To determine the effects of PHLPP downregulation on agonist-induced AKT activation, we investigated phosphorylation on Ser473 and Thr308 in BJAB and DHL-4 cells stimulated through the B-cell receptor. In both cell lines downregulation of PHLPP resulted in more than a 50% increase in BCR-induced AKT phosphorylation. In contrast, phosphorylation of other signaling molecules that are also activated by BCR crosslinking, such as PLCγ2 and ERK, appeared unaffected by PHLPP downregulation. These data confirm the functional relevance of PHLPP in AKT regulation in B-lymphoid cells and implicate reduced or absent PHLPP expression in CLL B-cells as a potential determinant of BCR-induced AKT signaling in CLL.

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