In Vivo Inhibition of BCR Activation in High-Risk CLL Patients On Therapy with Bruton's Tyrosine Kinase Inhibitor Ibrutinib: Correlative Studies from an Ongoing Phase 2 Clinical Trial

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 186-186
Author(s):  
Julia Hoellenriegel ◽  
Susan O'Brien ◽  
Michael J. Keating ◽  
William G. Wierda ◽  
Joseph J. Buggy ◽  
...  
Keyword(s):  
High Risk ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Bcr Signaling ◽  
Correlative Studies ◽  
Pre Treatment

Abstract Abstract 186 B cell receptor (BCR) signaling is essential for normal B cell development and plays an important role in several B cell malignancies, including chronic lymphocytic leukemia (CLL). BrutonÕs tyrosine kinase (Btk) transmits B cell receptor (BCR) signaling and can be inhibited by ibrutinib, a selective, covalent Btk inhibitor. Because of highly encouraging results with ibrutinib in high-risk CLL patients in the Phase 1/2 trial, we explored the combination of ibrutinib and rituximab in forty high-risk CLL patients, characterized by the presence of 17p deletion or TP53 mutation (treated or untreated), previously treated patients with 11q deletion, or patients with a short remission duration (< 3 years) after first-line chemo-immunotherapy. Here, we present early correlative studies from this trial, focusing on BCR-related CLL responses (chemokine secretion, viability) and CLL cell migration, based on our recent experience with ibrutinib in preclinical CLL models (Ponader S et al., Blood 119:1182–9, 2012). The aim of this study was to determine if preclinical results of Btk inhibition with ibrutinib can be recapitulated in vivo with specimen from the high-risk population enrolled on this trial. Plasma levels of CLL3 and CCL4 (MIP-1α/β), two chemokines secreted by CLL cells in response to BCR activation, were assessed before, at 14, and at 28 days on treatment with ibrutinib. In 28 analyzed patients, we demonstrate robust, significant reductions in CCL3 and CCL4 plasma concentrations after 14 and 28 days of treatment. As shown in Figure 1, plasma CCL3 levels were reduced from 139.6 (± 40.4) pg/mL before treatment to 6.9 (± 0.9) pg/mL or 6.5 (± 0.7) pg/mL after 14 and 28 days of treatment, respectively. We also evaluated in vitro secretion of CCL3 and CCL4 into supernatants of CLL cells isolated from 12 patients before and during ibrutinib therapy in response to stimulation with anti-IgM. Compared to CLL cells from pre-treatment specimen, CLL cells from patients on Ibrutinib therapy showed reduced levels of CLL3 and CCL4 secretion, and additional treatment with Ibrutinib (0.5 μM – 1μM) led to reduced chemokine levels only in pre-treatment samples, indicating complete Btk target inhibition. We next evaluated the effect of ibrutinib on CLL cell viability after anti-IgM stimulation. In pre-treatment samples, ibrutinib abrogated BCR-triggered CLL cell survival. Surprisingly, CLL cells from ibrutinib-treated patients remained anti-IgM responsive in these viability assays. These Btk-independent pro-survival effects could not be inhibited by in vitro treatment with ibrutinib, indicating that some of the anti-IgM-triggered pro-survival signaling can bypass Btk. Next, we analyzed migration of CLL cells towards the chemokines CXCL12 and CXCL13 in transwell chemotaxis assays. Pre-treatment samples displayed significant higher chemotaxis towards CXCL12 and CXCL13 when compared to CLL samples from patients on therapy with ibrutinib. The mean relative migration of such samples toward CXCL12 or CXCL13 was reduced to 28% (± 5%) or 35% (±15%) of respective CLL cells isolated before ibrutinib therapy (100%), n=6. Collectively, our results demonstrate that Ibrutinib blocks BCR-dependent survival and migration responses in high-risk CLL patients in vivo. They also corroborate the validity and robustness of CCL3 and CCL4 as biomarkers for BCR inhibition in CLL patients. Figure 1 Figure 1. Disclosures: O'Brien: Pharmacyclics: Research support Other. Buggy:Pharmacyclics: Employment, Equity Ownership. Burger:Pharmacyclics: Consultancy, Research Funding.

scholarly journals A B Cell Receptor with Two Igα Cytoplasmic Domains Supports Development of Mature But Anergic B Cells

2004 ◽  
Vol 199 (6) ◽  
pp. 855-865 ◽  
Author(s):  
Amy Reichlin ◽  
Anna Gazumyan ◽  
Hitoshi Nagaoka ◽  
Kathrin H. Kirsch ◽  
Manfred Kraus ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Cytoplasmic Domain ◽  
B Cell Receptor ◽  
B1a Cells ◽  
Bcr Signaling ◽  
Membrane Bound

B cell receptor (BCR) signaling is mediated through immunoglobulin (Ig)α and Igβ a membrane-bound heterodimer. Igα and Igβ are redundant in their ability to support early B cell development, but their roles in mature B cells have not been defined. To examine the function of Igα–Igβ in mature B cells in vivo we exchanged the cytoplasmic domain of Igα for the cytoplasmic domain of Igβ by gene targeting (Igβc→αc mice). Igβc→αc B cells had lower levels of surface IgM and higher levels of BCR internalization than wild-type B cells. The mutant B cells were able to complete all stages of development and were long lived, but failed to differentiate into B1a cells. In addition, Igβc→αc B cells showed decreased proliferative and Ca2+ responses to BCR stimulation in vitro, and were anergic to T-independent and -dependent antigens in vivo.

Chronic lymphocytic leukemia: revelations from the B-cell receptor

Blood ◽  
2004 ◽  
Vol 103 (12) ◽  
pp. 4389-4395 ◽  
Author(s):  
Freda K. Stevenson ◽  
Federico Caligaris-Cappio
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Cell Of Origin ◽  
Regulatory B Cell ◽  
V Genes

Abstract The finding that chronic lymphocytic leukemia (CLL) consists of 2 clinical subsets, distinguished by the incidence of somatic mutations in the immunoglobulin (Ig) variable region (V) genes, has clearly linked prognosis to biology. Antigen encounter by the cell of origin is indicated in both subsets by selective but distinct expression of V genes, with evidence for continuing stimulation after transformation. The key to distinctive tumor behavior likely relates to the differential ability of the B-cell receptor (BCR) to respond. Both subsets may be undergoing low-level signaling in vivo, although analysis of blood cells limits knowledge of critical events in the tissue microenvironment. Analysis of signal competence in vitro reveals that unmutated CLL generally continues to respond, whereas mutated CLL is anergized. Differential responsiveness may reflect the increased ability of post-germinal center B cells to be triggered by antigen, leading to long-term anergy. This could minimize cell division in mutated CLL and account for prognostic differences. Unifying features of CLL include low responsiveness, expression of CD25, and production of immunosuppressive cytokines. These properties are reminiscent of regulatory T cells and suggest that the cell of origin of CLL might be a regulatory B cell. Continuing regulatory activity, mediated via autoantigen, could suppress Ig production and lead to disease-associated hypogammaglobulinemia. (Blood. 2004;103:4389-4395)

Nurture versus Nature: The Microenvironment in Chronic Lymphocytic Leukemia

Hematology ◽  
2011 ◽  
Vol 2011 (1) ◽  
pp. 96-103 ◽  
Author(s):  
Jan A. Burger
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Disease Progression ◽  
Drug Testing ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
In Vivo Models

Abstract Intrinsic factors such as genetic lesions, anti-apoptotic proteins, and aberrant signaling networks within leukemia cells have long been the main focus of chronic lymphocytic leukemia (CLL) research. However, over the past decade, it became increasingly clear that external signals from the leukemia microenvironment make pivotal contributions to disease progression in CLL and other B-cell malignancies. Consequently, increasing emphasis is now placed on exploring and targeting the CLL microenvironment. This review highlights critical cellular and molecular pathways of CLL-microenvironment cross-talk. In vitro and in vivo models for studying the CLL microenvironment are discussed, along with their use in searching for therapeutic targets and in drug testing. Clinically, CXCR4 antagonists and small-molecule antagonists of B cell receptor (BCR)-associated kinases (spleen tyrosine kinase [Syk], Bruton's tyrosine kinase [Btk], and PI3Kδ) are the most advanced drugs for targeting specific interactions between CLL cells and the miocroenvironment. Preclinical and first clinical evidence suggests that high-risk CLL patients can particularly benefit from these alternative agents. These findings indicate that interplay between leukemia-inherent and environmental factors, nature and nurture determines disease progression in CLL.

The Role of the Microenvironment for CLL Proliferation and Survival: Gene Expression Profiling of Leukemic Cells Derived from Blood, Bone Marrow and Lymph Nodes Reveals the B-Cell Receptor and NF κB as Dominant Signaling Pathways

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 356-356 ◽  
Author(s):  
Yair Herishanu ◽  
Berengere Vire ◽  
Delong Liu ◽  
Federica Gibellini ◽  
Gerald E Marti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Cells ◽  
Expression Profiling ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Leukemic Cells ◽  
Activation Markers ◽  
Bcr Signaling

Abstract The host microenvironment is important for proliferation and survival of leukemic cells in chronic lymphocytic leukemia (CLL). Numerous molecules, signaling pathways and cell types have been reported to enhance CLL cell survival. To date, most reports on such interactions are derived from in-vitro studies, where each study focused on a specific ligand/receptor interaction or candidate pathway. Here, we adopted a more global approach to evaluate in-vivo effects of the microenvironment on leukemic cell biology. CLL cells from 15 patients were obtained on the same day from 3 different compartments: peripheral blood (PB), bone marrow (BM) and lymph node (LN), from which a single cell suspension was prepared. Tumor cells from all three compartments were purified by CD19 selection to purity &gt;98%. Patients were assigned to prognostic subtypes based on immunoglobulin sequencing (Ig) and ZAP70 expression: 10 patients had the more progressive subtype (Ig-unmutated, ZAP70+) and 5 patients belonged to the more indolent subtype. Cells were analyzed for surface markers by flow cytometry and by gene expression profiling on Affymetrix HG U133 Plus 2.0 arrays. By flow cytometry, CLL cells in LN expressed higher levels of activation markers including CD69 and CD38 compared to CLL cells in PB (% CD19+/69+; 71 ±27 vs. 35 ±28, p&lt;0.001 and % CD19+/CD38+; 33 ±28 vs. 20±19, p&lt;0.001, respectively). The expression of activation markers in BM derived cells was less consistent and did not reach statistically significant differences. We therefore focused our analysis on a comparison between LN and PB derived cells. First, we confirmed that the expression of a diagnostic CLL gene expression signature established previously for PB derived cells (Klein et al, 2001) was equally present in leukemic cells derived from all three compartments. We then identified a set of about 275 genes that were differentially expressed between LN resident and circulating tumor cells, most of which were up-regulated (fold change &gt;2, FDR &lt;0.2). A large number of these genes encode proteins important for cell cycle control and proliferation: different cyclins, PCNA, Ki67, TOP2A and MYC. We also detected a significant increase in the expression of NF-κB target genes in LN resident tumor cells, including CD83, CD69, JunB, Cyclin D2, GADD45B, CCL3, CCL4 and others. Consistent with activation of the NF-κB pathway in LN, IκB-beta protein levels in tumor cells from LN were lower than levels in matching PB cells. Next we identified genes differentially expressed between CLL subtypes based on Ig-mutation status separately for each of the 3 compartments. Interestingly, these subtype identifying gene sets were only partially overlapping. In Ig-unmutated, ZAP70+ cells several genes were more strongly regulated by the microenvironment then in Ig-mutated, ZAP70 negative cells. Among these genes is LPL, which has been reported to distinguish the CLL subtypes, and other genes induced by B-cell receptor (BCR) signaling. Using in-vitro IgM activation, we show that these genes are indeed induced by BCR stimulation but not by CD40 ligation and that their induction is confined to ZAP70+ CLL cells. In conclusion: interactions between CLL cells and elements of the microenvironment in LN induce cell proliferation and NF-κB activation. The preferential upregulation of BCR regulated genes in ZAP70+ CLL demonstrates a more efficient in-vivo response of ZAP-70+ cells to BCR stimulation. Our results highlight the importance of NFκ κB and BCR signaling in CLL and provide a rationale to focus treatment approaches on these central pathways.

Single Cell Profiling of B Cell Receptor Signaling and Apoptosis Networks in Chronic Lymphocytic Leukemia: Association with in Vitro Sensitivity to Fludarabine Monophosphate (F-ara-A).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1263-1263
Author(s):  
Erik Evensen ◽  
Adam Palazzo ◽  
Ying-Wen Huang ◽  
Alessandra Cesano ◽  
Laura Z. Rassenti ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
B Cell ◽  
Single Cell ◽  
Phosphatase Activity ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Employment Equity ◽  
Bcr Signaling ◽  
Equity Ownership

Abstract Abstract 1263 Poster Board I-285 Background In conjunction with antigen-driven responses, ligand-independent signaling (termed tonic signaling) through both the pre-B cell receptor and B-cell receptor has an important role in B cell development, maturation and survival. In addition to the recognized role of CD79 alpha and CD79 beta BCR signaling, tyrosine phosphatases can impact tonic BCR signaling (Wienands et al. PNAS, 93 p.7865 (1996), Monroe Nat. Rev. Immunol. 6 p.283 (2006)). We previously subjected chronic lymphocytic leukemia (CLL) cells with modulators of BCR signaling and monitored their responses using flow cytometry-based Single Cell Network Profiling (SCNP). Of the many signaling modulators studied, hydrogen peroxide treatment (a general inhibitor of tyrosine phosphatase activity) augmented BCR signaling in a subset of CLL patient samples evaluated. In the remaining samples there was an apparent lack of response to hydrogen peroxide. These data suggested that differential phosphatase activity proximal to BCR signaling was driving the biology of these two patient groups. Objectives Studies were designed to evaluate whether there were any associations between tonic and/or ligand-dependent BCR signaling and in vitro sensitivity to fludarabine, as well as whether such response profiles showed a relationship to the hydrogen peroxide-dependent signaling we observed previously. Methods 23 CLL samples and 7 healthy PBMCs were treated with anti-m alone, hydrogen peroxide alone or the combination for 10 minutes. Separate aliquots of the same sample were exposed to F-ara-A for 48 hours. SCNP was carried out on gated B cells with quantitation of single cell measures of intracellular phosphorylated kinases and adaptor proteins downstream of the BCR. Additionally, the relative activation status of several protein markers of the apoptotic cascade (cytoplasmic cytochrome C, cleaved caspase 3, and cleaved PARP) was measured. Results As previously observed, CLL samples could be segregated into one of two groups exhibiting either responsive or refractory signaling after exposure to hydrogen peroxide alone. Moreover, responsive signaling in CLL cells was correlated in that all the measured components of the canonical B cell receptor network (p-Lyn, p-Syk, p-BLNK, p-PLC-gamma-2, p-Erk and p-Akt) showed the same phosphorylation response: either augmented in unison, or not activated at all. In vitro F-ara-A treatment (48 hours in the presence of 1mM F-ara-A) of parallel samples from these same CLL patients identified distinct populations of apoptosis responsive and refractory cells. Surprisingly, the capacity of patient samples to show augmented BCR signaling in response to hydrogen peroxide was associated prominently with the ability of cells in these patients to exhibit apoptotic proficiency to F-ara-A in vitro. This implies a link between mechanisms governing apoptosis in these CLL cells, survival pathways, and cell states that govern the role of phosphatase activity and BCR signaling potential. Conclusions This study reveals a link between tonic BCR signaling and regulation of apoptosis pathways. This suggests that the subgroup of CLL patients with active phosphatase activity (which suppresses BCR responses) have cell populations that are responsive to F-ara-A, a standard drug in CLL therapy. Conversely, the presence of CLL cells in a patient sample that remain unresponsive to hydrogen peroxide repression of phosphatase activity appear to identify patient samples which cannot undergo apoptosis in response to in vitro F-Ara-A exposure. The clinical implications of this work will be the focus of future translational studies. Disclosures Evensen: Nodality Inc.: Employment, Equity Ownership. Palazzo:Nodality Inc.: Employment, Equity Ownership. Huang:Nodality Inc.: Employment, Equity Ownership. Cesano:Nodality Inc.: Employment, Equity Ownership. Fantl:Nodality, Inc.: Employment, Equity Ownership.

Rational Combinations Including a Novel Syk Inhibitor, Fostamatinib Disodium (FosD) in Diffuse Large B Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 283-283
Author(s):  
Randall M Rossi ◽  
Valerie Grose ◽  
Polly Pine ◽  
Richard I Fisher ◽  
Craig T. Jordan ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Cell Viability ◽  
B Cell ◽  
Cell Line ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
In Vivo Studies

Abstract Abstract 283 Certain malignant B-cells rely upon B-cell receptor-mediated survival signals. Spleen tyrosine kinase (Syk) initiates and amplifies the B-cell receptor-mediated signal. We and others have demonstrated that fostamatinib disodium (FosD: a prodrug of R406, a potent and specific inhibitor of Syk) induces apoptosis in lymphoma cell lines and primary tumors. A recent clinical trial has demonstrated significant clinical activity of FosD in relapsed/refractory B-cell non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia, and minimal overlap in toxicities with conventional agents. Given this background, future development in B-cell NHL will include rational combinations of FosD and currently available therapies. Therefore, we conducted in vitro and in vivo studies of rational combinations including FosD, in anticipation of clinical trial development. First, using a human DLBCL cell line of GCB genotype, (OCI-Ly19), we analyzed in vitro the combination of R406 with the following agents: fludarabine, rapamycin, rituximab, bendamustine and bortezomib. Increased cytotoxicity was observed using in vitro culture assays with the addition of fludarabine, rapamycin, or rituximab to R406. Cell viability at 72 hours was 25% with R406 alone, 27% for fludarabine alone, and only 9% for the fludarabine/R406. At 48 hours, cell viability was 49% using R406 alone, 31% using rituximab alone, and 21% for rituximab/R406. At 120 hours using primary lymphoma cells (DLCL27), there were no viable cells treated with the rapamycin/FosD combination, compared with rapamycin alone (7%) or FosD alone (25%) The addition of bortezomib or bendamustine to FosD resulted in only a minimal additive increase in cytotoxicity. Results with all combinations were similar with the OCI-Ly10 human DLBCL line of ABC genotype. We then performed in vivo studies by subcutaneous transplantation of the DLBCL cell line OCI-Ly19, (engineered to express luciferase allowing for real time in vivo imaging) into immune deficient NOD/SCID mice which reproducibly formed tumors. Recipient animals were separated into uniform cohorts when the tumors were less than or equal to 500 mm3 in size. The animals were then simultaneously treated with FosD (n=7; 3 gm/kg ad. lib.; translates into 2-5 micromolar R406 systemically throughout the 24h period) and either bortezomib, (n=6; 0.4 mg/kg weekly IP), or rituximab, (n=13; 3 mg/kg, 2x weekly IP). Analysis of the OCI-Ly19 tumor volumes at day 46 showed a median of 2364 mm3 with bortezomib alone compared with 1823 mm3 with bortezomib and FosD. When FosD was combined with rituximab the most significant cytotoxicity was observed: (p=0.01; median tumor volume of 497 mm3 following the combination) in comparison to either FosD alone (3150 mm3) or rituximab alone (1764 mm3). We conclude that the addition of FosD appears to increase activity against NHL of several drugs, including fludarabine and rapamycin. These agents have significant activity in indolent and mantle cell NHL as well as CLL. Moreover, there is no evidence that FosD impedes rituximab responses in vitro or in vivo; in fact we have suggested possible synergy with the combination of rituximab and FosD. Based upon the documented single agent activity of FosD in humans, and this data, clinical trials are now indicated using these promising combinations in NHL and CLL. Disclosures: Pine: Rigel: Employment. Friedberg:Rigel: Research Funding.

Inhibitors Of B-Cell Receptor Molecules Affect Surface CD20 and Impair Antitumor Activity Of Anti-CD20 Monoclonal Antibodies

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4217-4217
Author(s):  
Kamil Bojarczuk ◽  
Magdalena Winiarska ◽  
Malgorzata Bobrowicz ◽  
Michal Dwojak ◽  
Nina Miazek ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Antitumor Activity ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Bcr Signaling ◽  
Anti Cd20 ◽  
Constitutively Active

Abstract Background Anti-CD20 monoclonal antibodies (mAbs) are widely used in the treatment of non-Hodgkin's lymphomas (NHL) and chronic lymphocytic leukemia (CLL). Combining new agents with already used anti-CD20 mAbs seems to be a reasonable approach to further improve current therapeutic options. It seems that signaling via the aberrantly activated B-cell receptor (BCR) plays a key role in the pathogenesis of certain types of B-cell tumors. Blocking BCR pro-survival pathway holds a great therapeutic potential in both NHL and CLL. Several trials are currently being conducted to investigate the effects of combination of BCR-targeting agents with anti-CD20 mAbs–based therapies. To improve these therapeutic approaches in a planned manner it will be utterly important to decipher actual mechanisms of interactions between BCR-targeted therapies and anti-CD20 mAbs in established in vitro models. Aims The aim of this study is to elucidate the role of BCR signaling pathways in the regulation of CD20 levels in B-cell-derived tumor cells and antitumor activity of anti-CD20 mAbs. Methods The project is undertaken fully in in vitro settings in the models of human lymphoma cells as well as primary cells from patients with B-cell tumors. Cells are pre-incubated for 48h with inhibitors of BCR signaling (SYK, BTK, PI3K, AKT, PLC-γ, PKC, mTOR, ERK 1/2) and subsequently tested using flow cytometry for their susceptibility to antitumor activity of anti-CD20 mAbs. Membrane level of CD20 antigen is assessed with FITC-conjugated anti-CD20 antibody staining, total level of CD20 protein is assessed in Western blotting. Transcription processes are analyzed with qPCR, ChIP and EMSA. Moreover, stably transduced lymphoma cells with silenced or constitutively active proteins of interest are employed. Results The results of our preliminary experiments show that blocking BCR network at many stages of the signaling cascade with specific chemical inhibitors or selective shRNA-mediated silencing of SYK or BTK results in considerable down-regulation of CD20 level as determined with flow cytometry. Moreover, a 48-hour incubation with BCR inhibitors leads to a substantial impairment of antitumor activity of anti-CD20 mAbs. Selected inhibitors of BCR signaling considerably decrease CD20 protein level in total cellular lysates as analyzed using Western blotting. In Raji cells incubated with selected BCR inhibitors quantitative real-time PCR shows a significant decrease in CD20 mRNA level. Noteworthy, washout experiments showed that surface CD20 reaches level of control after 96 h from the time that inhibitors were eliminated from the culture media. Studies performed on cell line expressing constitutively active AKT showed up-regulation of CD20 levels at both levels of protein and mRNA. Moreover, constitutively active AKT protects cells from BCR inhibitors-induced decrease of surface CD20. Summary/conclusions Blocking BCR complex network on nearly every step of signal initiation and propagation considerably down-regulates CD20 levels what might have extremely important consequences for the anti-cancer therapy that is based on the use of anti-CD20 mAbs. These studies should provide us with extensive knowledge on the biology of CD20 protein and pathways involved in CD20 regulation. In light of our recent experiments therapeutic combinations of BCR inhibitors and anti-CD20 mAbs-based modalities should be rationally and consciously introduced into clinic in optimized therapeutic schemes. We hypothesize that results of our experiments may lead to identification of the most beneficial therapeutic modalities and schedules that would improve the quality of life of patients suffering from B-cells originating tumors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Syk activation by the Src-family tyrosine kinase in the B cell receptor signaling.

1994 ◽  
Vol 179 (5) ◽  
pp. 1725-1729 ◽  
Author(s):  
T Kurosaki ◽  
M Takata ◽  
Y Yamanashi ◽  
T Inazu ◽  
T Taniguchi ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
B Cell ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Cell Receptor ◽  
B Cell Antigen Receptor ◽  
Syk Kinase ◽  
Bcr Signaling ◽  
B Cell Receptor Signaling

Signaling through the B cell antigen receptor (BCR) results in rapid increases in tyrosine phosphorylation on a number of proteins. The BCR associates with two classes of tyrosine kinase: Src-family kinase (Src-protein-tyrosine kinase [PTK]; Lyn, Fyn, Blk, or Lck) and Syk kinase. We have investigated the interaction between the Src-PTK and the Syk kinase in the BCR signaling. In contrast to wild-type B cells, BCR-mediated tyrosine phosphorylation of Syk and activation of its in vitro kinase activity were profoundly reduced in lyn-negative cells. The requirement of the Src-PTK to induce tyrosine phosphorylation and activation of Syk was also demonstrated by cotransfection of syk and src-PTK cDNAs into COS cells. These results suggest that the Src-PTK associated with BCR phosphorylates the tyrosine residue(s) of Syk upon receptor stimulation, enhancing the activity of Syk.

scholarly journals B-Cell Receptor-Associated Protein 31 Regulates the Expression of Valosin-Containing Protein Through Elf2

2018 ◽  
Vol 51 (4) ◽  
pp. 1799-1814 ◽  
Author(s):  
Cong-cong Jia ◽  
Juan Du ◽  
Xia Liu ◽  
Rui Jiang ◽  
Yongye Huang ◽  
...  
Keyword(s):  
Western Blot ◽  
B Cell ◽  
Real Time Pcr ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Cns Diseases ◽  
Receptor Associated Protein ◽  
Protein Levels

Background/Aims: B-cell receptor-associated protein 31 (Bap31) is an evolutionarily conserved, ubiquitously expressed, polytopic integral membrane protein in the endoplasmic reticulum (ER) that is involved in the regulation of apoptosis, protein transport and degradation. Patients with Bap31 mutations exhibit symptoms similar to those exhibited by patients with central nervous system (CNS) diseases, such as deafness, dystonia, and intellectual disability. The present study aimed to investigate the function of Bap31 in CNS diseases by identifying a CNS disease-related gene regulated by Bap31 and exploring the underlying molecular mechanism. Methods: ShRNA-Bap31 and siRNA-Bap31 were used to knockdown Bap31 in N2a cells, and real-time PCR was performed to detect the mRNA levels of genes involved in CNS diseases. Western blot analyses were used to examine the protein levels of the candidate gene (valosin-containing protein, VCP) both in vivo and in vitro. The functions of Bap31 and VCP in mediating the degradation of the hyper-unstable mutant of cystic fibrosis trans-membrane conductance regulator (CFTRΔF508) were studied. Moreover, real-time PCR, Western blot and dual luciferase reporter analyses were conducted to investigate the molecular mechanism by which Bap31 regulates the expression levels of VCP. Results: VCP was identified as a candidate gene based on its differential expression in N2a cells following both shRNA- and siRNA-mediated knockdown of Bap31. Both the mRNA and protein levels of VCP were regulated by Bap31 in vivo and in vitro. In the ER-associated degradation (ERAD) pathway, Bap31 also regulated VCP expression and caused differences in the binding quantities of CFTRΔF508 and VCP. Furthermore, a transcription factor of VCP (E74-like factor 2, Elf2) was regulated by Bap31, and Elf2 mediated the changes in VCP transcription and expression in cells with altered Bap31 expression. Conclusion: These results indicate that Bap31 regulates the expression of VCP possibly via Elf2 and support the potential molecular function of Bap31 in CNS diseases.

The Syk inhibitor fostamatinib disodium (R788) inhibits tumor growth in the Eμ- TCL1 transgenic mouse model of CLL by blocking antigen-dependent B-cell receptor signaling

Blood ◽  
2010 ◽  
Vol 116 (23) ◽  
pp. 4894-4905 ◽  
Author(s):  
Mirza Suljagic ◽  
Pablo G. Longo ◽  
Sara Bennardo ◽  
Emerald Perlas ◽  
Giuseppe Leone ◽  
...  
Keyword(s):  
Mouse Model ◽  
B Cell ◽  
Transgenic Mouse ◽  
Cell Receptor ◽  
Transgenic Mouse Model ◽  
B Cell Receptor ◽  
Promising Therapeutic Approach ◽  
Bcr Signaling ◽  
Syk Inhibitor

Abstract Inhibition of antigen-dependent B-cell receptor (BCR) signaling is considered a promising therapeutic approach in chronic lymphocytic leukemia (CLL), but experimental in vivo evidence to support this view is still lacking. We have now investigated whether inhibition of BCR signaling with the selective Syk inhibitor fostamatinib disodium (R788) will affect the growth of the leukemias that develop in the Eμ-TCL1 transgenic mouse model of CLL. Similarly to human CLL, these leukemias express stereotyped BCRs that react with autoantigens exposed on the surface of senescent or apoptotic cells, suggesting that they are antigen driven. We show that R788 effectively inhibits BCR signaling in vivo, resulting in reduced proliferation and survival of the malignant B cells and significantly prolonged survival of the treated animals. The growth-inhibitory effect of R788 occurs despite the relatively modest cytotoxic effect in vitro and is independent of basal Syk activity, suggesting that R788 functions primarily by inhibiting antigen-dependent BCR signals. Importantly, the effect of R788 was found to be selective for the malignant clones, as no disturbance in the production of normal B lymphocytes was observed. Collectively, these data provide further rationale for clinical trials with R788 in CLL and establish the BCR-signaling pathway as an important therapeutic target in this disease.

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