PAX5/TEL Has An Opposite Dominant Effect on Endogenous PAX5, Affecting Its Transcriptional Regulation, B Cell Adhesion and BCR Signaling In Murine Pre-BI Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2482-2482
Author(s):  
Grazia Fazio ◽  
Valeria Cazzaniga ◽  
Chiara Palmi ◽  
Marta Galbiati ◽  
Marco Giordan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
B Cell ◽  
Cell Receptor ◽  
Cellular Processes ◽  
Transcription Process ◽  
Bcr Signaling ◽  
B Cell Receptor Signaling ◽  
In Vitro Adhesion

Abstract Abstract 2482 Introduction. PAX5 is a transcription factor with both activation and repression functions, essential for B-cell development. Recently, it has been found as frequent target of abnormalities in B-cell precursor ALL, showing point mutations, deletions or involvement in chromosomal translocations. The functional role of these lesions is still poorly understood. In previous experiments in mouse pre-BI cells, we showed that the PAX5/TEL protein acts as an aberrant transcription factor with repressor function, causing a block on B-cell differentiation, short-term IL-7 independence and resistance to the anti-proliferative and pro-apoptotic effects of TGFbeta1. Moreover, PAX5/TEL enhances cell migration towards CXCL12, with the over expression of CXCR4. Aim. The aim of the present study was to comprehensively understand how PAX5/TEL affects the transcription process and eventually interferes with PAX5 and TEL pathways and how these modulate cellular processes. Methods. We analyzed gene expression profiles in pre-BI cells transduced either by MIGR-PAX5/TEL-IRES-GFP or by MIGR-GFP (Affymetrix Gene Chip technology, Mouse array 430A 2.0). Validation of Differentially Expressed Genes has been performed by quantitative RQ-PCR and FACS analyses. In vitro adhesion assays have been performed on VCAM1-coated slides. Results. PAX5/TEL significantly modulated the transcription process: among 340 differentially expressed genes, 61% were down- and 39% up-regulated. Both up and down-regulated genes encompass numerous PAX5-target genes; in particular, PAX5/TEL represses genes which are normally activated by PAX5, and, vice versa, it activates genes physiologically repressed by PAX5. Moreover, gene function classification analyses suggested that PAX5/TEL modulates molecules which are related to fundamental cellular processes, such as phosphorylation, transcription, B cell receptor signaling, as well as adhesion. In particular, we demonstrated the modulation of surface antigens responsible of extra cellular binding as well as the modulation of intracellular molecules involved in the signaling of adhesion regulation, such as CD44, SDC4, EDG1, NEDD9, BCAR3, PLEKHA2, SPHK1. Moreover, in vitro adhesion assays on VCAM1-coated slides showed a significant reduction of adhesion capacities in PAX5/TEL positive cells. In agreement with our previous results, which showed down-regulation of CD19, BLNK/SLP-65 and MB-1/CD79a, both genes involved in BCR signaling, we demonstrated the additional repression of numerous key molecules fundamental for this pathway, such as SIGLECG/CD22, IRF4, LCP2/SLP-75, SLAMF6/LY108, PLEKHA2, PRKD2, IKZF2, IKZF3. Furthermore, we functionally validated the impairment of BCR-signaling and demonstrated that PAX/TEL transduced pre-BI cells are completely blocked in IgM protein expression, loosing the ability to complete the VDJ rearrangement and consequently express the m-chain on the cells surface, compared to the control cells. Conclusions. These analyses further sustain the role of PAX5/TEL as an aberrant transcription factor. Its effect on endogenous PAX5 does not represent a classical dominant negative role; indeed, we defined this effect as an ‘opposite dominance’, since PAX5/TEL caused the up-regulation of PAX5-repressed genes and, vice versa, the down-regulation of PAX5-activated targets. The biological consequences of this aberrant transcriptional activity are the impairment of B cell receptor signaling and the reduced adhesion capacity, both fundamental processes in B-cells, potentially involved in tumor transformation and in leukemia. 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 Induction of In Vivo Antipolysaccharide Immunoglobulin Responses to Intact Streptococcus pneumoniae Is More Heavily Dependent on Btk-Mediated B-Cell Receptor Signaling than Antiprotein Responses

2006 ◽  
Vol 74 (2) ◽  
pp. 1419-1424 ◽  
Author(s):  
Abdul Q. Khan ◽  
Goutam Sen ◽  
Shuling Guo ◽  
Owen N. Witte ◽  
Clifford M. Snapper
Keyword(s):  
Streptococcus Pneumoniae ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Relative Role ◽  
Bcr Signaling ◽  
B Cell Receptor Signaling ◽  
Extracellular Bacterium

ABSTRACT The relative role of Btk-dependent B-cell receptor (BCR) signaling in the induction of antipolysaccharide (anti-PS) and antiprotein immunoglobulin (Ig) responses to an intact extracellular bacterium in vivo is unknown. Btklow mice exhibit reduced BCR signaling but largely restore B-cell development. Btklow mice immunized with intact Streptococcus pneumoniae elicit reduced anti-PS but normal antiprotein Ig responses. Immunization of Btklow mice with PS-protein conjugate in saline results in an even more profound defect in the anti-PS but not antiprotein response, which is largely restored by use of a CpG-containing oligodeoxynucleotide as an adjuvant. These data demonstrate a greater dependence on Btk-mediated BCR signaling for physiologic anti-PS relative to antiprotein responses, as well as the existence of a compensatory Toll-like-receptor-mediated signaling pathway naturally triggered in response to intact bacterial pathogens.

scholarly journals Lipid rafts interaction of the ARID3A transcription factor with EZRIN and G-Actin regulates B cell receptor signaling

2020 ◽  
Author(s):  
Christian Schmidt ◽  
Laura Christian ◽  
Josephine Tidwell ◽  
Dongkyoon Kim ◽  
Haley O Tucker
Keyword(s):  
Transcription Factor ◽  
B Cell ◽  
Lipid Rafts ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Actin Binding ◽  
Cortical Actin ◽  
Downstream Signaling ◽  
Bcr Signaling ◽  
B Cell Receptor Signaling

ABSTRACTPreviously we demonstrated that the ARID3A transcription factor shuttles between the nucleus and the plasma membrane, where it localizes within lipid rafts. There it interacts with components of the B Cell Receptor (BCR) to reduce its ability to transmit downstream signaling. We demonstrate in this report that a direct component of ARID3A-regulated BCR signal strength is cortical Actin. ARID3A interacts with Actin exclusively within lipid rafts via the Actin binding protein EZRIN, which confines unstimulated BCRs within lipid rafts. BCR ligation discharges the ARID3A-EZRIN complex from lipid rafts allowing the BCR to initiate downstream signaling events. The ARID3A-EZRIN interaction occurs almost exclusively within unpolymerized G-Actin where EZRIN interacts with the multifunctional ARID3A REKLES domain. These observations provide a novel mechanism by which a transcription factor directly regulates BCR signaling.

scholarly journals The signaling adaptor BCAP inhibits NLRP3 and NLRC4 inflammasome activation in macrophages through interactions with Flightless-1

2019 ◽  
Vol 12 (581) ◽  
pp. eaau0615 ◽  
Author(s):  
Samuel J. Carpentier ◽  
Minjian Ni ◽  
Jeffrey M. Duggan ◽  
Richard G. James ◽  
Brad T. Cookson ◽  
...  
Keyword(s):  
B Cell ◽  
Yersinia Pseudotuberculosis ◽  
Cell Receptor ◽  
Pi3k Pathway ◽  
Inflammasome Activation ◽  
Interacting Protein ◽  
Caspase 1 ◽  
B Cell Receptor Signaling ◽  
Phosphoinositide 3 Kinase

B cell adaptor for phosphoinositide 3-kinase (PI3K) (BCAP) is a signaling adaptor that activates the PI3K pathway downstream of B cell receptor signaling in B cells and Toll-like receptor (TLR) signaling in macrophages. BCAP binds to the regulatory p85 subunit of class I PI3K and is a large, multidomain protein. We used proteomic analysis to identify other BCAP-interacting proteins in macrophages and found that BCAP specifically associated with the caspase-1 pseudosubstrate inhibitor Flightless-1 and its binding partner leucine-rich repeat flightless-interacting protein 2. Because these proteins inhibit the NLRP3 inflammasome, we investigated the role of BCAP in inflammasome function. Independent of its effects on TLR priming, BCAP inhibited NLRP3- and NLRC4-induced caspase-1 activation, cell death, and IL-1β release from macrophages. Accordingly, caspase-1–dependent clearance of a Yersinia pseudotuberculosis mutant was enhanced in BCAP-deficient mice. Mechanistically, BCAP delayed the recruitment and activation of pro–caspase-1 within the NLRP3/ASC preinflammasome through its association with Flightless-1. Thus, BCAP is a multifunctional signaling adaptor that inhibits key pathogen-sensing pathways in macrophages.

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.

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.

Anergic Response to B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia: The Differing Roles of IgD and IgM in the Microenvironment and Peripheral Blood.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2889-2889
Author(s):  
Tom Butler ◽  
Alexander Montoya ◽  
Andrew James Clear ◽  
Rita Coutinho ◽  
David C Taussig ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling ◽  
B Cell Receptor Signaling ◽  
Cell Receptor Signaling

Abstract Abstract 2889 Chronic Lymphocytic Leukemia (CLL) cells depend on B cell receptor signaling as well as other microenvironmental survival signals (1). Drugs targeting the BCR signaling pathways are showing exciting results in CLL clinical trials. A peculiarity of CLL is that IgD signaling is generally preserved, whilst IgM signaling is decreased and it has been suggested that this pattern mimics anergic B-cells, and might be consistent with chronic autoantigen exposure. We examined the differing roles of IgM and IgD signaling in CLL using a theoretical framework of anergy. Peripheral blood (PB) CLL cells exhibited higher IgD expression, as compared to IgM (n=204, p<0.0001), but this did not have prognostic impact. When we examined IgM and IgD expression in LN biopsies compared to paired PB (n=10) expression, IgM expression was lower in LN (p=0.002) whilst IgD expression was unchanged. Although the number of these paired samples is small, cases with lower LN IgM levels had poorer prognosis, and we are investigating this further with a larger cohort. We hypothesize that reduced LN IgM expression reflects antigen engagement and an anergic response in the microenvironment. We sought to replicate Mockridge et al' s model of reversible anergy (2) by monitoring the dynamic changes in IgM/D expression after in vitro incubation. Most (18/20) PB CLL samples underwent calcium (Ca) flux after IgD crosslinking, whereas only 13/20 cases underwent IgM Ca flux, and the level of Ca flux was less than with IgD, a well recognized anergic pattern. Incubation for 24h in vitro led to partial restoration of IgM Ca flux and some improvement in IgD Ca flux. This was impaired by treatment with anti-IgD or IgM F(ab)2 fragments, mimicking antigen exposure, and in keeping with a model of CLL cells engaging autoantigen in vivo. Further support for the pro-survival role of the BCR in CLL was demonstrated by the finding that both IgD and IgM ligation was associated with reduced apoptosis in vitro, with a significant decrease in apoptosis with IgD ligation as compared to IgM. To examine the mechanistic differences of signaling via IgM and IgD further, we used high-throughput mass-spectrometry based phosphoproteomics. This allows analysis of multiple active signaling pathways without a priori knowledge of which pathways to investigate. 6 CLL samples were compared to 5 tonsil controls. 4,575 unique phosphopeptides were identified using MASCOT proteomics software and quantified using a label-free technique based on extracted ion currents. 174 phosphoproteins (p<0.001, fold change up to >4000-fold) were over-expressed in CLL relative to healthy B-cells. These included components of RNA processing complexes, cytoskeletal regulators and MAPK signaling pathway components. Kinase prediction based on phosphoprotein substrates confirmed activation of kinases known to be active in CLL (such as AKT1, ERK1/2, CK2), but several novel kinases (such as CaMK1, CRIK, ROCK1 and BCKDK) were also active in CLL relative to healthy controls. Evaluation of differentially expressed phosphoproteins after BCR ligation included components of the spliceosome, regulators of the cytoskeleton, as well as known BCR signaling components. BCR-induced kinase activities included mTOR, CDK family members, MAPKs, BCKDK and others. There was much overlap between kinases active after IgM and IgD ligation, but also marked differences in CLL and tonsil BCR signaling. CONCLUSIONS Anergic IgM signaling is contrasted with IgD as a dynamic and plastic process that appears different in the LN and PB compartments in CLL. Mass-spectrometry based phosphoproteomics offers a powerful tool for interrogating intracellular signaling, with networks of phosphorylation characterizing the topology of pathways. BCR signaling in healthy B-cells has not previously been studied using this approach and comparisons with CLL highlight known pathways as well as suggesting novel treatment targets. The ultimate goal is to identify kinases active in CLL that will provide rational and effective drug combinations. Disclosures: Gribben: Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

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.

Self-Enforcing Feedback Activation Between BCL6 and Tonic Pre-B Cell Receptor Signaling in Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 284-284
Author(s):  
Huimin Geng ◽  
Christian Hurtz ◽  
Dirk Baumjohann ◽  
Zhengshan Chen ◽  
Wei-Yi Chen ◽  
...  
Keyword(s):  
B Cell ◽  
Tyrosine Kinases ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Bcr Signaling ◽  
Btk Inhibitor ◽  
Bcl6 Expression ◽  
Targeting Strategy

Abstract Background and hypothesis: Like mature B cell lymphoma, pre-B ALL originates from B cell precursors that critically depend on survival signals emanating from a functional (pre-) B cell receptor (BCR). While recent work successfully introduced BCR signaling inhibitors into patient care for various subtypes of mature B cell lymphoma, it is not known whether pre-BCR signaling represents a therapeutic target in pre-B ALL and in which cytogenetic subsets targeting of pre-BCR signaling will be effective. In this study we demonstrated that ALL can be subdivided into two groups that fundamentally differ with respect to pre-BCR signalling. We identified a novel mechanism of self-enforcing feedback activation between the transcription factor BCL6 and tonic pre-BCR signaling in pre-BCR+ ALL and proposed a dual targeting strategy of both BCL6 and pre-BCR related tyrosine kinases for the treatment of patients with pre-BCR+ ALL. Results: Flow cytometry analysis of surface pre-BCR expression (λ5, VpreB), cytoplasmic μ heavy chain (μHC) expression and intracellular Ca2+ signal in 29 patient-derived pre-B ALL xenograft samples and cell lines showed pre-BCR expression and activity in a subset of pre-B ALL, including all TCF3-PBX1 cases studied (n=4) and two cases with deletions at 6q21. Studying 830 pre-B ALL cases from four clinical trials (MDACC, St. Jude, COG P9906 and ECOG E2993), tonic pre-BCR signaling and constitutive PI3K-AKT activation was found in 112 cases (13.5%), including 93% TCF3-PBX1 (53 of 57), del (6)(q21) (7 of 7), PBX1 (1q23) duplication (4 of 4), MLL-rearrangement (3 of 86), hyperdiploid (2 of 43) and other (43 of 406) pre-B ALL cases. In other major ALL subtypes, we found no evidence for pre-BCR expression and activity, including BCR-ABL1 (0 of 196) and ETV6-RUNX1 (0 of 31). We found frequent 1q23 (PBX1) duplication, TCF3-PBX1 or other PBX1-rearrangement, 6q21 (PRDM1) deletion in ALL cells with tonic pre-BCR signaling. Development of a genetic mouse model for inducible ablation of Bcl6. Pre-BCR-induced activation of BCL6 relieves PRDM1-mediated repression of pre-BCR signaling components and positively regulates pre-BCR signaling output at the transcriptional level. The clinical data (COG P9906, ECOG E2993) revealed that high mRNA levels of BCL6 at the time of diagnosis is predictive of poor clinical outcome specifically in patients with pre-BCR+ ALL but not ALL cells lacking pre-BCR expression. These findings suggest an important role of BCL6 as a cofactor of pre-BCR signaling in a large subset of ALL. To directly test the role of Bcl6- and pre-BCR interactions, we generated a novel mouse model for inducible Cre-mediated deletion of Bcl6 exons 5-10, flanked by loxP sites. For lineage-specific deletion in vivo, we crossed these mice with an Mb1-Cre deleter strain, in which Bcl6 was deleted in pro-B cells, resulting in a differentiation block at the pre-B cell stage. Deletion of Bcl6 in mouse pre-BCR+ ALL and expression of a dominate-negative form of BCL6 in human primary pre-BCR+ALL cells, both rapidly induced cell death, indicating BCL6 cooperates with the pre-BCR in leukemic transformation. Cooperation between pre-BCR and BCL6 signaling. Inhibition of BCL6 via the specific BCL6 inhibitor RI-BPI showed compromised colony formation and induced cell cycle arrest. Interestingly, constitutive BCL6 expression was sensitive to inhibition of SYK and SRC tyrosine kinases downstream of the pre-BCR. Treating 6 pre-BCR+ and 8 pre-BCR- patient-derived ALL samples with the SYK inhibitor (PRT06207), BTK inhibitor (Ibrutinib) or a broader SRC and BTK inhibitor Dasatinib, we observed remarkably decreased BCL6 expression and increased apoptosis in pre-BCR+ but not pre-BCR- ALL cells. In vivo treatments with Dasatinib prevented leukemia initiation and significantly prolonged survival of the recipient mice that were injected with primary pre-BCR+ ALL cells, compared to non-treatment or Nilotinib-treatment. These data demonstrate that both inhibition of BCL6 and pre-BCR signaling selectively killed patient-derived pre-BCR+ ALL cells. Conclusions: Our study identified two distinct subtypes of pre-B ALL that fundamentally differ with respect to pre-BCR signaling. Tonic pre-BCR signaling engages a BCL6-dependent, self-enforcing amplification loop. Based on these findings, we propose a dual targeting strategy of BCL6 and pre-BCR tyrosine kinases for the treatment of patients with pre-BCR+ALL. Disclosures No relevant conflicts of interest to declare.

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