BLNK Research Articles

Syk mediates BCR- and CD40-signaling integration during B cell activation

CD40 is essential for optimal B cell activation. It has been shown that CD40 stimulation can augment BCR-induced B cell responses, but the molecular mechanism(s) by which CD40 regulates BCR signaling is poorly understood. In this report, we attempted to characterize the signaling synergy between BCR- and CD40-mediated pathways during B cell activation. We found that spleen tyrosine kinase (Syk) is involved in CD40 signaling, and is synergistically activated in B cells in response to BCR/CD40 costimulation. CD40 stimulation alone also activates B cell linker (BLNK), Bruton tyrosine kinase (Btk), and Vav-2 downstream of Syk, and significantly enhances BCR-induced formation of complex consisting of, Vav-2, Btk, BLNK, and phospholipase C-gamma2 (PLC-γ2) leading to activation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase, Akt, and NF-κB required for optimal B cell activation. Therefore, our data suggest that CD40 can strengthen BCR-signaling pathway and quantitatively modify BCR signaling during B cell activation.

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

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

Cloning and functional characterization of chicken stem cell antigen 2

Stem cell antigen 2 (SCA2) is a Ly6 family member whose function is largely unknown. To characterize biological properties and tissue distribution of chicken SCA2, SCA2 was expressed in E. coli, purified, and a polyclonal antibody developed. Utilizing the polyclonal antibody, SCA2 is a 13 kDa cell surface protein anchored by a glycosyl-phosphatidylinositol (GPI) moiety. SCA2 is expressed in connective tissues of thymus and bursa based on immunohistochemistry, immunoprecipitation, and western blots. In bursal follicles, SCA2 is specifically expressed on the cortical-medullary epithelial cells (CMEC) surrounded by MHC class II presenting cells. Expression profiles of bursal cells induced by contact with SCA2-expressing cells shows down-regulation of numerous genes including CD79B, B cell linker ( BLNK), spleen tyrosine kinase ( SYK), and gamma 2-phospholipase C ( PLCG2) that are involved in the B cell receptor (BCR) and immune response signaling pathways. These results suggest chicken SCA2 plays a role in regulating B lymphocytes.

The Pax5 Fusion Product Pax5-C20orf112 Causes Downregulation of Pre-B Cell Receptor Genes and Induces Differential Proliferation Patterns in B-Lymphoblastic Cell Lines.

Abstract 1284Poster Board I-306Recent SNP array analyses of B-acute lymphoblastic leukemia (B-ALL) have identified disruptions of the gene encoding the B-cell specific transcription factor Pax5 as one of the most common genomic lesions in this disease (> 30%); it being hemizygously deleted, mutated or involved in translocations. Pax5 translocates and forms fusion products with at least 12 different partners including C20orf112, leading to a chimeric Pax5/C20orf112 (Pax5/C20s) protein. Pax5 fusion products act as dominant negatives, competing for promoter binding sites with wild type (wt) Pax5 and thereby deregulating expression of target genes. In order to elucidate the molecular effects of fusion products involving the Pax5 gene, we performed a global gene expression analysis in the Nalm-6 B-ALL cell line. The cells were transfected with MSCV expression plasmids containing either empty vector, wild type Pax5 or a short fusion product of Pax5 and C20orf112 (Pax5/C20s), each containing IRES sequences for co-expression of GFP. Overexpression of Pax5 and Pax5/C20s was confirmed by western blot and quantitative RT PCR. RNA was extracted from cells sorted by FACS for GFP and processed for hybridization on Affymetrix HG-U133 plus 2 gene expression microarrays. Candidate genes were validated with RT real time PCR. Among the most differentially downregulated genes by the Pax5/C20s fusion product were candidate genes such as pleckstrin homology domain containing, family A member 2 (PLEKHA2) (12.64-fold), B-cell associated transcription factors POU class 2 associating factor 1 (POU2AF1) (4.4-fold) and transcription factor 3 (TCF3, E2A) (3.9-fold). Another intriguing observation was the downregulation of a group of genes associated with signaling through the pre-B cell receptor such as phosphoinositide-3-kinase adaptor protein 1 (BCAP) (3.35 fold), immunoglobulin heavy locus (IGH) (2.8 fold), pre-B lymphocyte genes -3 and -1 (VPREB3, VPREB1) (2.6-fold and 1.75-fold, respectively), spleen tyrosine kinase (SYK) (1.6 fold) and B-cell linker (SLP65, BLNK) (1.5-fold) by the Pax5/C20s fusion product. For stable expression and growth curves, Nalm6, 697, Kasumi2, RCH-ACV, SEM, HPB-Null, BV173 and BEL1 B-lymphoblastic cell lines were infected with retroviruses expressing the above mentioned retroviral expression constructs. We noted that forced expression of the PAX5/C20s fusion product inhibited growth in cell lines, which had functional pre-B cell receptor signaling. In contrast, the fusion gene either did not affect or enhanced growth of B-ALL cell lines, in which expression of a functional pre-B cell receptor was missing. Of note, Pax5 wt caused growth inhibition in B-ALL cell lines lacking functional pre-B cell receptor signaling. In cells with functional pre-B cell signaling, the response to engagement of the receptor as measured by calcium flux assay was diminished by overexpression of the Pax5/C20s fusion product as compared to empty vector control or PAX5 wt. These results suggest that the mechanisms of leukemogenesis of Pax5/C20s in ALL cells may be dependent on the functionality of the pre-B cell receptor pathway. This could be of great therapeutic value as it would potentially allow ALL cells to be divided into two different subtypes depending on pre-B cell receptor functionality and possibly identify the pre-B cell receptor pathway as a new therapeutic target. DisclosuresNo relevant conflicts of interest to declare.

SLP-76 Couples Syk to the Osteoclast Cytoskeleton

The capacity of the osteoclast (OC) to resorb bone is dictated by cytoskeletal organization, which in turn emanates from signals derived from the alpha(v)beta(3) integrin and c-Fms. Syk is key to these signals and, in other cells, this tyrosine kinase exerts its effects via intermediaries including the SLP adaptors, SLP-76 and BLNK (B cell linker). Thus, we asked whether these two SLP proteins regulate OC function. We find BLNK-deficient OCs are normal, whereas cytoskeletal organization of those lacking SLP-76 is delayed, thus modestly reducing bone resorption in vitro. Cytoskeletal organization and bone resorption are more profoundly arrested in cultured OCs deficient in BLNK and SLP-76 double knockout (DKO) phenotypes. In contrast, stimulated bone resorption in vivo is inhibited approximately 40% in either SLP-76(-/-) or DKO mice. This observation, taken with the fact that DKO OCs are rescued by retroviral transduction of only SLP-76, indicates that SLP-76 is the dominant SLP family member in the resorptive process. We also find SLP-76 is phosphorylated in a Syk-dependent manner. Furthermore, in the absence of the adaptor protein, integrin-mediated phosphorylation of Vav3, the OC cytoskeleton-organizing guanine nucleotide exchange factor, is abrogated. In keeping with a central role of SLP-76/Vav3 association in osteoclastic resorption, retroviral transduction of SLP-76, in which the Vav binding site is disrupted (3YF), fails to normalize the cytoskeleton of DKO OCs and the resorptive capacity of the cells. Finally, c-Fms-activated Syk also exerts its OC cytoskeleton-organizing effect in a SLP-76/Vav3-dependent manner.

Signaling in Human B-Lymphoma Rafts

In normal and tumoral B-lymphocytes, proximal signaling requires assemblies of proteins to generate pro- survival and anti-apoptotic information. B-cell receptor (BCR) engagement by antigen results in the formation of raft- based microclusters that incorporate the Lyn kinase. Further involvement of the Syk kinase and the B-cell linker (BLNK) adaptor depend on the kinase activity of Lyn to phosphorylate specific motifs in the cytoplasmic portion of the BCR complex, and to maintain this complex in its open conformation. Signaling proceeds by way of phospholipase-C acti- vation, protein kinase C activation and phosphorylation of the caspase-recruitement domain (CARD)-membrane- associated guanylate kinase (MAGUK) protein-1 (CARMA1) protein. Oligomerization of phosphorylated CARMA1 proteins generates a CARMA1 signalosome, which is anchored in rafts and permits phosphorylation of the inhibitor of nuclear factor kappa-B (NF- B), its degradation in the proteasome and activation of NF- B. Constitutively active signalo- somes have been identified in B-lymphoma cell membranes that either lock the Lyn kinase in a permanently active confi- guration by association with the Csk-binding protein (Cbp) Cbp/phosphoprotein associated with glycosphingolipid-rich domains (PAG) adaptor, or maintain the CD40 receptor permanently active by engagement with its ligand CD154. The constitutively active Lyn is directly connected with pro-survival and anti-apoptotic pathways, as Lyn inhibition results in lymphoma cell death and the CD40-CD40 ligand signalosome sends co-stimulatory signals via the NF- B pathway. The constitutively active signalosomes exploit raft microdomains to assemble signaling proteins that generate inappropriately durable signals. Such signalosomes reflect the dependence of lymphoma cells on a narrow spectrum of signaling proteins for the generation of survival signals, and could be exploited therapeutically.

B‐cell linker protein prevents aneuploidy by inhibiting cytokinesis

Aneuploidy is a hallmark of human cancers. Although the maintenance of genomic integrity by p53 is important in preventing aneuploidy, its mechanism remains to be elucidated. Here we report evidence that B-cell linker protein (BLNK) mediates the inhibition of cytokinesis, which generates tetraploidy but prevents aneuploidy. We identified BLNK as a transcriptional target of p53. Surprisingly, ectopic expression of exogenous BLNK inhibited cytokinesis, resulting in the formation of tetraploid cells. Indeed, BLNK was involved in the generation of spontaneously arising binucleate tetraploid cells. Interestingly, cytokinesis after DNA damage was inhibited in p21(-/-) and p53+/+ cells, but not in p53(-/-) cells. BLNK knockdown in p53+/+ and p21(-/-) cells enhanced cytokinesis after DNA damage, leading to the generation of aneuploid cells. In addition, a BLNK-downregulated human pre-B leukemia cell line showed increased cytokinesis and aneuploidy after DNA damage compared with two other pre-B leukemia cell lines expressing higher levels of BLNK. These results suggest that BLNK acts as a mediator of p53 in the inhibition of cytokinesis, which prevents aneuploidy. We propose that the inhibition of cytokinesis is crucial for the maintenance of genomic integrity.

Identification of differential gene expression during porcine conceptus rapid trophoblastic elongation and attachment to uterine luminal epithelium

Early embryonic development in the pig is characterized by a rapid elongation of the conceptus trophectoderm on days 11-12 of gestation. Initially, the conceptus trophoblast is morphologically rearranged from a 10-mm sphere into a tubular shape, transitioning into a thin filamentous form >150 mm in length in 2-3 h, followed by continued expansion within the uterine lumen for several days. Conceptus elongation is critical for establishing adequate placental surface area needed for embryo and fetal survival throughout gestation. The objective of this study was to characterize conceptus gene expression during trophoblastic elongation and the early attachment to the uterine endometrium on days 11-14 of gestation with the GeneChip Porcine Genome Array. In all, 3,759 different probe sets were statistically different in at least one comparison [spherical vs. tubular, spherical vs. day 12 filamentous (D12F), spherical vs. day 14 filamentous (D14F), tubular vs. D12F, tubular vs. D14F, and D12F vs. D14F]. When restricted to the spherical vs. D12F and D12F vs. D14F comparisons, 482 and 232 genes, respectively, were statistically different with greater than twofold change in expression. Utilization of k-means clustering, in addition to the Database for Annotation, Visualization, and Integrated Discovery (DAVID), identified genes of interest. Quantitative RT-PCR expression profiles for interferon-gamma (IFNG), heat shock protein 27 kDa (HSPB1), angiomotin, B-cell linker (BLNK), chemokine ligand 14 (CXCL14), parathyroid hormone-like hormone (PTHLH), and maspin were supportive of the GeneChip Porcine Genome Array data.

PKCη directs induction of IRF-4 expression and Ig κ gene rearrangement in pre-BCR signaling pathway

Pre-B cell receptor (pre-BCR) signals promote pre-B cell differentiation, in which the adaptor protein B-cell linker (BLNK) plays a crucial role. However, the molecular pathways downstream of BLNK are currently unclear. Utilizing pre-B leukemia cell lines (BKO84 and others) derived from BLNK-deficient mice as in vitro models of the pre-B cell differentiation, we have demonstrated that reconstitution of BLNK as well as an active form of protein kinase C (PKC)eta induces the differentiation events, such as pre-BCR down-regulation and kappa gene rearrangement. Here we show that the same events are induced by cross-linking of pre-BCR with anti-mu antibody in these pre-B cell lines, as well as in ex vivo pre-B cells from BLNK-deficient mice, suggesting a function of BLNK as an internal cross-linker of pre-BCR. Anti-mu treatment of BKO84 cells up-regulated membrane recruitment of PKC eta and the expression of IRF-4, a transcription factor known to promote light chain gene rearrangements. Anti-mu induction of surface kappa chain on BKO84 cells was blocked by reagents that inhibit phospholipase C or PKC. Enforced expression of the active PKC eta in BKO84 cells resulted in up-regulation of IRF-4 expression. Conversely, siRNA-mediated silencing of PKC eta expression strikingly attenuated the anti-mu-induced IRF-4 expression and kappa gene rearrangement, which were restored by PKC eta reconstitution. Finally, enforced expression of IRF-4, but not of BLNK, in the PKC eta-silenced BKO84 cells resulted in kappa gene rearrangement. These results indicate that PKC eta directs the induction of IRF-4 expression downstream of BLNK in the pre-BCR signaling pathway promoting kappa gene rearrangement.

SYK-dependent tonic B-cell receptor signaling is a rational treatment target in diffuse large B-cell lymphoma

The role of B-cell receptor (BCR)-mediated survival signals in diffuse large B-cell lymphoma (DLBCL) remains undefined. Ligand-induced BCR signaling induces receptor oligomerization, Igalpha/beta immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation, and activation of the spleen tyrosine kinase (SYK), which initiates downstream events and amplifies the initial BCR signal. BCRs also transmit low-level tonic survival signals in the absence of receptor engagement. Herein, we assess the role of SYK-dependent tonic BCR survival signals in DLBCL cell lines and primary tumors and evaluate the efficacy of an ATP-competitive inhibitor of SYK, R406, in vitro. R406 induced apoptosis of the majority of examined DLBCL cell lines. In R406-sensitive DLBCL cell lines, R406 specifically inhibited both tonic- and ligand-induced BCR signaling (autophosphorylation of SYK525/526 and SYK-dependent phosphorylation of the B-cell linker protein [BLNK]). The majority of examined primary DLBCLs also exhibited tonic- and ligand-induced BCR signaling; in these primary tumors, BCR signaling was also inhibited by R406. Of note, BCR-dependent and R406-sensitive DLBCL cell lines were independently identified as "BCR-type" tumors by transcriptional profiling. Therefore, SYK-dependent tonic BCR signaling is an important and potentially targetable survival pathway in some, but not all, DLBCLs. In addition, R406-sensitive DLBCLs can be identified by their transcriptional profiles.

Repression of B-Cell Linker (BLNK) and B-Cell Adaptor for Phosphoinositide 3-Kinase (BCAP) Is Important for Lymphocyte Transformation by Rel Proteins

Persistent Rel/nuclear factor-kappaB (NF-kappaB) activity is a hallmark of many human cancers, and the Rel proteins are implicated in leukemia/lymphomagenesis but the mechanism is not fully understood. Microarray analysis to identify transformation-impacting genes regulated by NF-kappaB's oncogenic v-Rel and c-Rel proteins uncovered that Rel protein expression leads to transcriptional repression of key B-cell receptor (BCR) components and signaling molecules like B-cell linker (BLNK), the B-cell adaptor for phosphoinositide 3-kinase (BCAP) and immunoglobulin lambda light chain (Ig lambda), and is accompanied by a block in BCR-mediated activation of extracellular signal-regulated kinase, Akt, and c-Jun-NH(2)-kinase in response to anti-IgM. The BLNK and BCAP proteins were also down-regulated in lymphoid cells expressing a transformation-competent chimeric RelA/v-Rel protein, suggesting a correlation with the capacity of Rel proteins to transform lymphocytes. DNA-binding studies identified functional NF-kappaB-binding sites, and chromatin immunoprecipitation (ChIP) data showed binding of Rel to the endogenous blnk and bcap promoters in vivo. Importantly, restoration of either BLNK or BCAP expression strongly inhibited transformation of primary chicken lymphocytes by the potent NF-kappaB oncoprotein v-Rel. These findings are interesting because blnk and other BCR components and signaling molecules are down-regulated in primary mediastinal large B-cell lymphomas and Hodgkin's lymphomas, which depend on c-Rel for survival, and are consistent with the tumor suppressor function of BLNK. Overall, our results indicate that down-regulation of BLNK and BCAP is an important contributing factor to the malignant transformation of lymphocytes by Rel and suggest that gene repression may be as important as transcriptional activation for Rel's transforming activity.

ZAP-70 enhances IgM signaling independent of its kinase activity in chronic lymphocytic leukemia

We transduced chronic lymphocytic leukemia (CLL) cells lacking ZAP-70 with vectors encoding ZAP-70 or various mutant forms of ZAP-70 and monitored the response of transduced CLL cells to treatment with F(ab)(2) anti-IgM (anti-mu). CLL cells made to express ZAP-70, a kinase-defective ZAP-70 (ZAP-70-KA(369)), or a ZAP-70 unable to bind c-Cbl (ZAP-YF(292)) experienced greater intracellular calcium flux and had greater increases in the levels of phosphorylated p72(Syk), B-cell linker protein (BLNK), and phospholipase C-gamma, and greater activation of the Ig accessory molecule CD79b in response to treatment with anti-mu than did mock-transfected CLL cells lacking ZAP-70. Transfection of CLL cells with vectors encoding truncated forms of ZAP-70 revealed that the SH2 domain, but not the SH1 domain, was necessary to enhance intracellular calcium flux in response to treatment with anti-mu. We conclude that ZAP-70 most likely acts as an adapter protein that facilitates B-cell receptor (BCR) signaling in CLL cells independent of its tyrosine kinase activity or its ability to interact with c-Cbl.

High Level Phosphatase Activity Revealed in Chronic Lymphocytic Leukemia Cells That Use Mutated Immunoglobulin Heavy Chain Variable Region Genes and Lack High-Level Expression of the Zeta-Associated Protein 70 (ZAP-70).

Signals propagated through the B cell receptor (BCR) guide the maturation and survival of B cells and might factor in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). BCR signaling in CLL cells was investigated at the single-cell level using multiparametric flow cytometry. Concurrent analysis was performed using fluorochrome-conjugated antibodies specific for B-cell surface antigens and a panel of antibodies recognizing specific phospho-peptide epitopes within a selected group of intracellular signaling proteins. CLL samples from patients (N=6) showed weak or minimal signaling activity at p72SYK/p70ZAP, Erk1/2, B-Cell linker protein (BLNK) and phospholipase-Cγ-2, (PLCγ2) when stimulated only at the BCR with anti-μ crosslinking, whereas a robust signal was observed in a control Ramos B cell line. The low-level signaling in CLL cells could be accounted for by either a defect in activation of a key protein required for signaling, or by enhanced inhibition mediated by phosphatases such as SHIP-1, SHIP-2, SHP-1, or SHP-2. To determine whether phosphatases were preventing or dampening BCR activation in CLL samples, CLL cells were treated with hydrogen peroxide (H2O2), a physiologic phosphatase inhibitor generated during BCR signaling that has been used previously to reveal dysregulated BCR signaling in follicular lymphoma (Sing et al., Cell 2005, Reth., Nat. Immunol. 2002, Irish et al., Blood 2006). H2O2 treatment of CLL cells that had molecular features associated with indolent disease (e.g. use of mutated immunoglobulin heavy chain variable region genes (IgVH) and a low level expression of ZAP70) induced high-levels of phosphorylated p72SYK/p70ZAP, ERK1/2, BLNK, and PLCγ2, independent of surface F(ab)2anti-μ ligation. In contrast, CLL-B cells that had molecular features associated with more aggressive disease (expression of unmutated IgVH and high-level expression of ZAP-70) were significantly less responsive to this treatment, even in the presence of F(ab)2anti-μ. Exposure of blood B cells from healthy donors to H2O2 failed to elicit a substantial increase in phosphorylation of these same intracellular signaling proteins. These studies reveal a previously unrecognized, constitutive high-level phosphatase activity in CLL cells that express mutated IgVH and low levels of ZAP-70, possibly contributing to the attenuated signaling observed in these cells following surface IgM ligation. Because this high level of constitutive phosphatase activity was not observed in CLL cells that express unmutated IgVH with high-level expression of ZAP70, flow cytometric measurement of the increased phosphorylation triggered by H2O2 could provide for a useful means with which to distinguish leukemia cells from patients with indolent versus aggressive CLL. Evaluation of additional samples is ongoing to test this hypothesis.

ZAP-70 Enhances IgM Signaling in Chronic Lymphocytic Leukemia Cells Independent of Its Tyrosine Kinase Activity.

We found that expression of the zeta-associated protein of 70 kD (ZAP-70) by chronic lymphocytic leukemia (CLL) B cells enhanced IgM-receptor signaling, even though such CLL cells also expressed a highly-related and more active tyrosine kinase, p72Syk. To investigate whether the kinase activity of ZAP-70 is necessary for this effect, we transfected ZAP-70-negative primary leukemia cells with expression vectors encoding either wild-type ZAP-70 or ZAP-70-KI, a mutant ZAP-70 that has an inactivating point mutation in the ATP-binding site (Lys369-Ala) and that lacks tyrosine kinase activity. We achieved high-level expression of ZAP-70 or ZAP-70-KI in transfected CLL cell samples and compared these cells with each other and with the same CLL cell samples that had been mock transfected with a control vector and that remained negative for expression of ZAP-70 (n = 7). To examine the B-cell-receptor (BCR) signaling potential of these cells we assessed the extent of tyrosine phosphorylation of p72Syk, B-cell linker protein (BLNK), and phospholipase C gamma (PLC-γ), and measured intracellular calcium flux ([Ca2+]I) before and 5–10 minutes after surface IgM ligation with F(ab)2 anti- μantibody. Treatment of control mock-transfected ZAP-70-negative CLL cells with anti-μ resulted in negligible-to-minimal increases in phosphorylation of these cytosolic proteins, with mean increases in phosphorylation of p72Syk, BLNK, and PLC-γ of only 46% ± 47% (S.D), 173% ± 112%, and 73% ± 76%, respectively. CLL cells engineered to express non-mutated ZAP-70 experienced significantly higher levels of protein tyrosine phosphorylation following treatment with anti-μ, with mean increases in phosphorylation of p72Syk, BLNK, and PLC-γ of 186% ± 102%, 490% ± 323%, and 722% ± 836%, respectively, as had been noted in earlier studies. Surprisingly, CLL cells made to express the tyrosine-kinase-defective mutant ZAP-70KI also experienced significantly higher levels of protein tyrosine phosphorylation following treatment with anti-μ than did the mock-transfected CLL cells, with mean increases in phosphorylation of p72Syk, BLNK, and PLC-γ of 172% ± 94%, 431% ± 261%, and 759% ± 637%, respectively. These values were similar to those noted for anti-μ treated CLL cells that expressed the wild-type ZAP-70 protein. These results were reflected also in the ([Ca2+]I) induced by anti-μ in each of the three groups of CLL cells. Whereas anti-μ treatment of mock-transfected CLL cells resulted in negligible-to-minimal increases in ([Ca2+]I) of 0.25 units ± 0.19, anti-μ treatment of ZAP-70-transfected or ZAP-70KI-transfected CLL cells resulted in increases in ([Ca2+]I) of 1.05 units ± 0.53 and 0.95 units ± 0.46, respectively. These values each were significantly higher than that noted for mock-transfected CLL cells. We conclude that the tyrosine-kinase activity of ZAP-70 is not required for ZAP-70 to enhance BCR signaling in CLL cells.

Identification of Genes Abnormally Expressed in Human MLL-AF4 Leukemia.

The t(4;11)(q21;q23) translocation is a hallmark of infant acute lymphoblastic leukemia (ALL), which results in the fusion of the MLL gene on chromosome 11 and the AF4 gene on chromosome 4. MLL-AF4 fusion is the most common consequence of chromosomal translocations in infant leukemia and is associated with a poor prognosis. To identify leukemia-related genes, we used the SAGE technique to compare gene expression profiles between two MLL-AF4 patient samples and one normal sample (CD19+ progenitor B cells; 216,464 tags in total). We identified 61 candidate genes that appear to be abnormally expressed in the leukemia samples (29 up- and 32 down-regulated). Remarkably, we found that many candidate genes appear to play important role in the development of B cells. In addition, many candidate genes can bind with and/or regulate other candidates in the candidate gene list. For example, SYK, BTK and BLNK can bind directly and regulate each other. SYK can also bind directly with TNFRSF1B. In addition, EBF may positively regulate BLK, while BLK can bind directly with BTK. All six of these genes are significantly down-regulated in MLL-AF4 leukemia samples. BTK, SYK and BLK are tyrosine kinases. BTK (B-cell progenitor tyrosine kinase) is a key regulator in B-lymphocyte differentiation and activation. BLK (B-lymphocyte-specific tyrosine kinase) is expressed only in B lymphocytes, and controls pre-B cell development. SYK (spleen tyrosine kinase) is widely expressed in hematopoietic cells, which can phosphorylate BLNK (B-cell linker protein). BLNK represents a central linker protein that bridges the B-cell receptor-associated kinases and may regulate B-cell function and development. EBF (early B-cell factor) is a tissue-specific and differentiation stage-specific DNA-binding protein, and mice lacking Ebf are unable develop B lymphoid cells. TNFRSF1B is strongly expressed on stimulated T and B lymphocytes. Moreover, previous studies indicate that BTK, BLK, SYK, BLNK and TNFRSF1B can positively regulate apoptosis, while BTK can also positively regulate differentiation. Thus, their down-regulation may inhibit apoptosis and differentiation, and thereby contribute to leukemogenesis. In contrast, GNA12, a transforming oncogene which can enhance proliferation and transformation and can bind directly with BTK, is significantly up-regulated in MLL-AF4 leukemia cells. Its up-regulation may also be important to leukemogenesis. Taken together, the deregulation of the important candidate genes may contribute to leukemogenesis through inhibiting apoptosis and differentiation while promoting proliferation of hematopoietic cells. We have validated the expression patterns of the candidate genes with real-time quantitative RT-PCR and are studying the functions and pathways of the validated candidate genes using RNAi and retrovirus transduction over-expression methods. In addition, we will also establish knock-in or knock-out mouse models for the most promising functional candidate genes to see the effect on the development of leukemia. Our studies will provide important insights into the complex functional pathways related to MLL rearrangements in the development of acute lymphoblastic leukemia, which may lead to more effective therapy for these leukemias.

Aberrant Pax5 Production with Deletion of Exon 8 in Childhood B-Lineage Acute Lymphoblastic Leukemia.

Background. B-lineage acute lymphoblastic leukemia (ALL) is a common subtype of childhood leukemia. Among numerous transcription factors that are required for B-cell development, Pax5 regulates the expression of B-cell-specific genes including BLNK, CD19, LEF-1, blk and mb-1 of the Ig genes, and plays a central role in B-cell development and differentiation. Recently, it has been reported that aberrant expression of Pax5 is implicated in the development of a certain type of B-cell lymphomas. These observations suggest that the alteration of Pax5 function might contribute to leukemogenesis by aberrantly regulating the differentiation of early B-cell progenitors.Procedure. Leukemic cells in children with B-lineage ALL, B-lineage ALL cell lines, and normal B lymphocytes from different age groups were used for the study. RNA was purified and cDNA was synthesized for amplification of Pax5, BLNK mRNA. Pax-5 variants were cloned and nucleotide sequences were determined. The production of Pax5 protein was confirmed with anti-human Pax5 antibody specific for the N terminal region of Pax5 to detect eight variant proteins.Results. Twelve of 21 ALL patients (57%) displayed multiple Pax5 mRNA fragments. These fragments corresponded to the full-length human Pax5 mRNA (Pax5 FL) and spliced variants with deletions of exon 7 (ΔE7, exon 8 (ΔE8, exons 7 and 8 (ΔE7/8), exons 8 and 9 (ΔE8/9), exons 6, 7 and 8 (ΔE6/7/8), exons 7, 8 and 9 (ΔE7/8/9), and exons 6, 7, 8 and 9 (ΔE6/7/8/9). Normal B lymphocytes expressed only Pax5 FL and lower level of Pax5 ΔE8. By Western blot analysis, normal B lymphocytes displayed a single band at a molecular weight of 50 kDa corresponding to Pax5 FL, whereas all B-lineage ALL patients except two revealed one protein band at a lower molecular weight of 46 kDa corresponding to Pax5 ΔE8. The production Pax5 and Pax5 ΔE8 proteins was also analyzed in normal B lymphocytes from different age groups. The dominant production of Pax5 ΔE8 was detected in neonates and infants, whereas only Pax5 FL was observed in children and in adults. Since Pax5 regulates the BLNK gene, which endows B lymphocytes with the ability to respond to B-cell-specific signals, BLNK expression and protein production was also analyzed in B-lineage ALL cells. Among 33 ALL samples, 16 (48%) either lacked or weakly expressed BLNK. All ALL samples with multiple Pax5 variants showed low or negative expression of BLNK, regardless of the expression of Pax5 ΔE8.Conclusions. Most of B-lineage ALL displayed multiple Pax5 mRNA transcripts, which were generated through alternative splicing in the 3′ region of the Pax5. On the other hand, all B-lineage ALL patients except two showed only Pax5 ΔE8 protein. The dominant production of Pax5 ΔE8 was also detected in normal B lymphocytes from neonates and infants. These results indicate that Pax5 ΔE8 participates in the early stage of B-cell differentiation predominantly detected in very young children. The altered Pax5 expression might disrupt B-cell hematopoiesis due to the absence or reduction of Pax5-regulated genes and cause the arrest at the stage of early B-cell differentiation and an abnormal expansion of immature B cells in B-lineage ALL in children.

R406, an Orally Available Spleen Tyrosine Kinase Inhibitor Blocks Fc Receptor Signaling and Reduces Immune Complex-Mediated Inflammation

Recent compelling evidence has lead to renewed interest in the role of antibodies and immune complexes in the pathogenesis of several autoimmune disorders, such as rheumatoid arthritis. These immune complexes, consisting of autoantibodies to self-antigens, can mediate inflammatory responses largely through binding and activating the immunoglobulin Fc receptors (FcRs). Using cell-based structure activity relationships with cultured human mast cells, we have identified the small molecule R406 [N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine] as a potent inhibitor of immunoglobulin E (IgE)- and IgG-mediated activation of Fc receptor signaling (EC(50) for degranulation = 56-64 nM). Here we show that the primary target for R406 is the spleen tyrosine kinase (Syk), which plays a key role in the signaling of activating Fc receptors and the B-cell receptor (BCR). R406 inhibited phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 bound to the ATP binding pocket of Syk and inhibited its kinase activity as an ATP-competitive inhibitor (K(i) = 30 nM). Furthermore, R406 blocked Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and BCR-mediated activation of B lymphocytes. R406 was selective as assessed using a large panel of Syk-independent cell-based assays representing both specific and general signaling pathways. Consistent with Syk inhibition, oral administration of R406 to mice reduced immune complex-mediated inflammation in a reverse-passive Arthus reaction and two antibody-induced arthritis models. Finally, we report a first-inhuman study showing that R406 is orally bioavailable, achieving exposures capable of inhibiting Syk-dependent IgE-mediated basophil activation. Collectively, the results show R406 potential for modulating Syk activity in human disease.

Primary immunodeficiency diseases: An update from the International Union of Immunological Societies Primary Immunodeficiency Diseases Classification Committee Meeting in Budapest, 2005

Although relatively rare, primary immunodeficiency diseases (PIDs) provide an excellent window into the functioning of the immune system. In the late 1960s, observations on these diseases, with their associated infections and genetics, bisected the immune system into humoral immunity and cell-mediated immunity. These diseases also represent a challenge in their diagnosis and treatment. Beginning in 1970, a unified nomenclature for the then-known PIDs was created by a committee convoked by the World Health Organization. Since then, and later under the aegis of the International Union of Immunological Societies, an international committee of experts has met every 2 to 3 years to update the classification of PIDs. During the past 15 years, the molecular basis of more than 120 PIDs has been elucidated. This update results from the latest meeting of this committee in Budapest, Hungary, in June 2005, which followed 2 1/2 days of scientific discussions. As a result of this work, new entities have been included, and the nomenclature of some PIDs (specifically of the various forms of class-switch recombination defects, previously known as hyper-IgM syndromes) has been changed.

Deficiency of BLNK hampers PLC-gamma2 phosphorylation and Ca2+ influx induced by the pre-B-cell receptor in human pre-B cells.

B-cell linker protein (BLNK) is a component of the B-cell receptor (BCR) as well as of the pre-BCR signalling pathway, and BLNK(-/-) mice have a block in B lymphopoiesis at the pro-B/pre-B cell stage. A recent report described the complete loss or drastic reduction of BLNK expression in approximately 50% of human childhood pre-B acute lymphoblastic leukaemias (ALL), therefore we investigated BLNK expression in human pre-B ALL cell lines. One of the four cell lines tested, HPB-NULL cells, was found to lack BLNK expression, and we used these human pre-B ALL cell lines that express and do not express BLNK to investigate the intracellular signalling events following pre-BCR cross-linking. When pre-BCR was cross-linked with anti-micro heavy-chain antibodies, significant phosphorylation of intracellular molecules, including Syk, Shc, ERK MAP kinase, and AKT, and an activation of Ras were observed without regard to deficiency of BLNK expression, suggesting that BLNK is not required for pre-BCR-mediated activation of MAP kinase and phosphatidyl-inositol 3 (PI3) kinase signalling. By contrast, phospholipase C-gamma2 (PLC-gamma2) phosphorylation and an increase in intracellular Ca(2+) level mediated by pre-BCR cross-linking were observed only in the BLNK-expressing cells, indicating that BLNK is essential for PLC-gamma2-induced Ca(2+) influx. Human pre-B cell lines expressing and not expressing BLNK should provide an in vitro model for investigation of the role of BLNK in the pre-BCR-mediated signalling mechanism.

Requirements for distinct steps of phospholipase Cgamma2 regulation, membrane-raft-dependent targeting and subsequent enzyme activation in B-cell signalling.

Studies of PLCgamma (phospholipase Cgamma) have identified a number of regulatory components required for signalling; however, molecular mechanisms and the relationship between events leading to translocation and an increase of substrate hydrolysis have not been well defined. The addition of a membrane-targeting tag to many signal transducers results in constitutive activation, suggesting that these processes could be closely linked and difficult to dissect. The present study of PLCgamma2 regulation by cross-linking of the BCR (B-cell antigen receptor) or H2O2 stress in DT40 B-cells, demonstrated that the membrane targeting is a separate step from further changes that result in enzyme activation and substrate hydrolysis. Furthermore, we have defined the roles of different domains of PLCgamma2 and, using a panel of cell lines deficient in components linked to PLCgamma2 regulation, the involvement of signalling molecules with respect to each of the steps. We have found that only the lipid-raft-targeted Lyn-PLCgamma2 construct, unlike non-specific membrane targeting, overcame the requirement for the adapter protein BLNK (B-cell linker). The stable expression of Lyn-PLCgamma2 was not accompanied by an increase in substrate hydrolysis in resting cells, which followed stimulation and specifically required the presence and/or activation of Syk, Btk, phosphoinositide 3-kinase but not BLNK, as established using deficient cell lines or specific inhibitors. Based on mutational analysis of the specific tyrosine residues [Tyr753-->Phe (Y753F)/Y759F] and SH2 (Src homology 2) domains (R564A/R672A) in the context of Lyn-PLCgamma2, we found that Tyr753/Tyr759 were essential, whereas the PLCgamma2 SH2 domains did not have an important role in the transient activation of Lyn-PLCgamma2 but may serve to stabilize an activated form in sustained activation.