Differentiation Of pre-B Cells Research Articles

High Salt-Induced Hyperosmolality Reduces in Vitro Survival and Proliferation of Pre-B Cells

Aim: B cells of the adaptive immunity are critical for protection against the vast majority of pathogens through the production of specific antibodies. A number of signaling pathways and transcription factors control B cell development. Environmental factors, including diet, are also important in determining how B cell develop and function. Here, the effects of hyperosmolality induced by elevated salt on the survival, IL-7-induced proliferation and differentiation of pre-B cells were tested in vitro. 
 Material and Methods: The wk3 pre-B cell line generated from SLP65–/– mice was used. Hyperosmolality in the cell culture medium was created by increasing the salt concentration with the addition of 40 mM NaCl. Wk3 pre-B cells were cultured in standard (normal NaCl) and high salt (+40 mM NaCl) medium, followed by flow cytometric analysis.
 Results: It was found that hyperosmolality caused by high salt reduced survival and induced apoptosis in wk3 pre-B cells. In addition, hyperosmolality inhibited IL-7-induced proliferation of pre-B cells. Conversely, pre-B cells treated with high salt were able to differentiate normally into IgM+ immature B cells when IL-7 was removed. 
 Conclusion: These findings suggest that the hyperosmolar microenvironment induced by high salt may play a key role in B cell development in the bone marrow.

Identification of key transcription factors and their functional role involved in Salmonella typhimurium infection in chicken using integrated transcriptome analysis and bioinformatics approach

Salmonella enterica serovar typhimurium is the cause of significant morbidity and mortality worldwide that causes economic losses to poultry and is able to cause infection in humans. Indigenous chicken breeds are a potential source of animal protein and have the added advantage of being disease resistant. An indigenous chicken, Kashmir favorella and commercial broiler were selected for understanding the mechanism of disease resistance. Following infection in Kashmir favorella, three differentially expressed genes Nuclear Factor Kappa B (NF-κB1), Forkhead Box Protein O3 (FOXO3) and Paired box 5 (Pax5) were identified. FOXO3, a transcriptional activator, is the potential marker of host resistance in Salmonella infection. NF-κB1 is an inducible transcription factor which lays the foundation for studying gene network of the innate immune response of Salmonella infection in chicken. Pax5 is essential for differentiation of pre-B cells into mature B cell. The real time PCR analysis showed that in response to Salmonella Typhimurium infection a remarkable increase of NF-κB1 (P˂0.01), FOXO3 (P˂0.01) gene expression in liver and Pax5 (P˂0.01) gene expression in spleen of Kashmir favorella was observed. The protein–protein interaction (PPI) and protein-TF interaction network by STRINGDB analysis suggests that FOXO3 is a hub gene in the network and is closely related to Salmonella infection along with NF-κB1. All the three differentially expressed genes (NF-κB1, FOXO3 and PaX5) showed their influence on 12 interacting proteins and 16 TFs, where cyclic adenosine monophosphate Response Element Binding protein (CREBBP), erythroblast transformation-specific (ETSI), Tumour-protein 53(TP53I), IKKBK, lymphoid enhancer-binding factor-1 (LEF1), and interferon regulatory factor-4 (IRF4) play role in immune responses. This study shall pave the way for newer strategies for treatment and prevention of Salmonella infection and may help in increasing the innate disease resistance.

ATP11C promotes the differentiation of pre-B cells into immature B cells but does not affect their IL-7-dependent proliferation.

The P4-type ATPases are believed to function as flippases that contribute to the organization of the asymmetric aminophospholipid distribution on the plasma membranes of eukaryotes by their ability to internalize specific phospholipids from the outer leaflet to the inner leaflet. Despite the existence of 14 members of the P4-type ATPases in humans and 15 in mice, their roles in the immune system have not been fully understood. So far, ATP11C was shown to be important for B cells, and mice deficient for ATP11C had a developmental arrest at the pro-B to pre-B cell transition stageof B cell development. Using an ATP11C-deficient pre-B cell line generated through CRISPR/Cas9 engineering, we here tested the role of ATP11C in pre-B cells in vitro and showed that ablation of ATP11C in pre-B cells causes a defect in the flippase activity. We further demonstrated that loss of ATP11C does not impede the proliferation of pre-B cells in response to IL-7. However, pre-B cells lacking ATP11C failed to differentiate into immature B cells upon removal of IL-7. These results suggest that disruption of lipid asymmetry by loss of ATP11C in pre-B cells may control the switch from proliferation to differentiation in pre-B cells.

COX-2/PGE2 upregulation contributes to the chromosome 17p-deleted lymphoma

Deletions of chromosome 17p, where TP53 gene locates, are the most frequent chromosome alterations in human cancers and associated with poor outcomes in patients. Our previous work suggested that there were p53–independent mechanisms involved in chromosome 17p deletions-driven cancers. Here, we report that altered arachidonate metabolism, due to the deficiency of mouse Alox8 on chromosome 11B3 (homologous to human ALOX15B on chromosome 17p), contributes to the B cell malignancy. While the metabolites produced from lipoxygenase pathway reduced, chromosome 11B3 deletions or Alox8 loss, lead to upregulating its paralleling cyclooxygenase pathway, indicated by the increased levels of oncometabolite prostaglandin E2. Ectopic PGE2 prevented the apoptosis and differentiation of pre-B cells. Further studies revealed that Alox8 deficiency dramatically and specifically induced Cox-2(Ptgs2) gene expression. Repressing Cox-2 by its shRNAs impaired the tumorigenesis driven by Alox8 loss. And, in turn, tumor cells with Alox8 or 11B3 loss were sensitive to the COX-2 inhibitor celecoxib. This correlation between COX-2 upregulation and chromosome 17p deletions was consistent in human B-cell lymphomas. Hence, our studies reveal that the arachidonate metabolism abnormality with unbalanced ALOX and COX pathways underlies human cancers with 17p deletions and suggest new susceptibility for this disease.

Coexpression of CCR7 and CXCR4 During B Cell Development Controls CXCR4 Responsiveness and Bone Marrow Homing.

The CXCL12–CXCR4 axis plays a key role in the retention of stem cells and progenitors in dedicated bone marrow niches. It is well-known that CXCR4 responsiveness in B lymphocytes decreases dramatically during the final stages of their development in the bone marrow. However, the molecular mechanism underlying this regulation and whether it plays a role in B-cell homeostasis remain unknown. In the present study, we show that the differentiation of pre-B cells into immature and mature B cells is accompanied by modifications to the relative expression of chemokine receptors, with a two-fold downregulation of CXCR4 and upregulation of CCR7. We demonstrate that expression of CCR7 in B cells is involved in the selective inactivation of CXCR4, and that mature B cells from CCR7−/− mice display higher responsiveness to CXCL12 and improved retention in the bone marrow. We also provide molecular evidence supporting a model in which upregulation of CCR7 favors the formation of CXCR4–CCR7 heteromers, wherein CXCR4 is selectively impaired in its ability to activate certain G-protein complexes. Collectively, our results demonstrate that CCR7 behaves as a novel selective endogenous allosteric modulator of CXCR4.

The endoplasmic reticulum–resident E3 ubiquitin ligase Hrd1 controls a critical checkpoint in B cell development in mice

Humoral immunity involves multiple checkpoints that occur in B cell development, maturation, and activation. The pre-B-cell receptor (pre-BCR) is expressed following the productive recombination of the immunoglobulin heavy-chain gene, and sSignalsing through the pre-BCR are required for the differentiation of pre-B cells into immature B cells. However, the molecular mechanisms controlling the pre-BCR expression and signaling strength remain undefined. Herein, we probed the role of the endoplasmic reticulum-associated, stress-activated E3 ubiquitin ligase HMG-CoA reductase degradation 1 (Hrd1) in B cell differentiation. Using mice with a specific Hrd1 deletion in pro-B cells and subsequent B cell developmental stages, we showed that the E3 ubiquitin ligase Hrd1 governs a critical checkpoint during B cell development. We observed that Hrd1 is required for degradation of the pre-BCR complex during the early stage of B cell development. As a consequence, loss of Hrd1 in the B cell lineage resulted in increased pre-BCR expression levels and a developmental defect in the transition from large to small pre-B cells. This defect, in turn, resulted in reduced fewer mature B cells in bone marrow and peripheral lymphoid organs. Our results revealed a novel critical role of Hrd1 in controlling a critical checkpoint in B cell-mediated immunity and suggest that Hrd1 may functioning as an E3 ubiquitin ligase of the pre-BCR complex.

The survival and differentiation of pro-B and pre-B cells in the bone marrow is dependent on IL-7Rα Tyr449.

IL-7 is critical for murine T and B cell development and survival and plays a significant role in lymphoblastic leukemia in both humans and mice. We evaluated the role of the IL-7Rα Tyr(449) cytoplasmic SH2-binding motif in IL-7-mediated B cell development using a knock-in mouse with a Tyr to Phe mutation (IL-7Rα(449F/449F) mouse). IL-7Rα(449F/449F) and IL-7Rα(-/-) mice showed no defect in the number of pre-pro-B cells, although IL-7Rα(449F/449F) mice had decreased Ebf1 in pre-pro-B cells and impairment in B cell-committed CLPs. We identified that IL-7Rα Tyr(449) was critical for both pro-B and pre-B stages of development in the bone marrow. IL-7Rα(449F/449F) and IL-7Rα(-/-) mice had comparable precursor B cell defects, indicating that signaling from the IL-7Rα required this motif. Although the defect in IL-7Rα(449F/449F) pro-B cells was associated with loss of STAT5 activation and diminished expression of Mcl1, this was not rescued by overexpression of Bcl-2. IL-7Rα(449F/449F) and IL-7Rα(-/-) pre-B cells also showed defective cyto-Igμ and CD25 expression, associated with reduced levels of Rag1, Rag2, and Irf4. Pre-B cells from IL-7Rα(449F/449F) mice also failed to proliferate, perhaps as a result of the failure to rearrange Igμ. Our data suggest that IL-7Rα Tyr(449) was essential for IL-7Rα signaling in bone marrow B cell development and survival.

Loss of Ikaros DNA-binding function confers integrin-dependent survival on pre-B cells and progression to acute lymphoblastic leukemia

Deletion of the Ikaros (Ikzf1) DNA-binding domain generates dominant-negative isoforms that interfere with Ikaros family activity and correlate with poor prognosis in human precursor B cell acute lymphoblastic leukemias (B-ALL). Here, we show that conditional inactivation of the Ikaros DNA binding domain in early pre-B cells arrests their differentiation at a stage where integrin-dependent niche adhesion augments mitogen-activated protein kinase signaling, proliferation, and self-renewal, and attenuates pre-B cell receptor signaling and differentiation. Transplantation of polyclonal Ikzf1 mutant pre-B cells results in long-latency oligoclonal pre-B-ALL, demonstrating that loss of Ikaros contributes to multistep B-leukemogenesis. These results explain how normal pre-B cells transit from a highly proliferative and stromal-dependent to a stromal-independent phase where differentiation is enabled, providing potential therapeutic strategies for IKZF1 mutant B-ALL.

Small-molecule inhibition of CBP/catenin interactions eliminates drug-resistant clones in acute lymphoblastic leukemia

Drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem warranting new treatment strategies. Wnt/catenin signaling is critical for the self-renewal of normal hematopoietic progenitor cells. Deregulated Wnt signaling is evident in chronic and acute myeloid leukemia, however little is known about ALL. Differential interaction of catenin with either the Kat3 coactivator CREBBP (CBP) or the highly homologous EP300 (p300) is critical to determine divergent cellular responses and provides a rationale for the regulation of both proliferation and differentiation by the Wnt signaling pathway. Usage of the coactivator CBP by catenin leads to transcriptional activation of cassettes of genes that are involved in maintenance of progenitor cell self-renewal. However, the use of the coactivator p300, leads to activation of genes involved in the initiation of differentiation. ICG-001 is a novel small molecule modulator of Wnt/catenin signaling, which specifically binds to the N-terminus of CBP and not p300, within amino acids 1–110, thereby disrupting the interaction between CBP and catenin. Here, we report that selective disruption of the CBP/β- and γ-catenin interactions using ICG-001 leads to differentiation of pre-B ALL cells and loss of self-renewal capacity. Survivin, an inhibitor-of-apoptosis protein, was also downregulated in primary ALL after treatment with ICG-001. Using ChIP assay, we demonstrate occupancy by CBP of the survivin promoter, which is decreased by ICG-001 in primary ALL. CBP-mutations have been recently identified in a significant percentage of ALL patients, however, almost all of the identified mutations reported occur C-terminal to the binding site for ICG-001. Importantly, ICG-001, regardless of CBP mutational status and chromosomal aberration, leads to eradication of drug-resistant primary leukemia in combination with conventional therapy in vitro and significantly prolongs the survival of NOD/SCID mice engrafted with primary ALL. Therefore, specifically inhibiting CBP/catenin transcription represents a novel approach to overcome relapse in ALL.

YB-1 Is Activated by IL-7 and Is Overexpressed in Pediatric Pre-B Acute Lymphoblastic Leukemia

Abstract 1468 Background:Increased YB-1 expression correlates with poor prognosis, drug resistance and metastasis in several different cancers including B cell lymphoma. Phosphorylation and nuclear localization of YB-1 in response to growth factors leads to increased survival through expression of proteins such as survivin and multidrug resistance protein 1. Until now, its role in leukemia has not been investigated. We hypothesized that YB-1 expression is aberrantly regulated in pediatric pre-B acute lymphoblastic leukemia (pre-B ALL), and that YB-1 may be activated downstream of IL-7. This cytokine facilitates the differentiation and survival of pre-B cells and has been implicated in the drug resistance of pre-B ALL. Methods:YB-1 and IL-7Ra protein expression was investigated by flow cytometry in normal pre-B cells (CD19+CD10+CD20−), and mature B cells (CD19+CD10−CD20+) as well as diagnostic and relapsed pre-B ALL (CD19+CD10+/−). Cell surface and cytoplasmic expression was quantified by mean fluorescent intensity (MFI). Bone marrow from healthy donors was used as a source of normal pre-B cells, while mature B cells were derived from PBMCs; leukemic cells at presentation and relapse were obtained following local IRB approval and informed consent. Activation of YB-1 downstream of IL-7 stimulation (25 ng/ml) was examined in pre-B ALL cell lines or NSG (NOD scid gamma) mice-expanded pre-B ALL by Western blotting using anti-phosphoYB-1(S012). Pre-B ALL cell lines used in these experiments were 697, 380, RCH and RS-4;11. Signaling pathways were investigated by pre-treatment of cells with pharmacological inhibitors followed by Western analyses. For the transient overexpression of YB-1, pEGFP or a pEGFP/YB-1 fusion protein was electroporated into freshly isolated mature B cells (which have a low basal expression of YB-1) and YB-1 and IL-7Ra expression was assessed by flow cytometry after 24 h. Results:While intracellular YB-1 expression was significantly higher in leukemia samples at presentation compared to normal pre-B cells, the highest YB-1 levels were found in relapsed pre-B ALL (see figure, part A). All examined pre-B ALL cell lines had levels comparable to those of the relapse samples. Similarly, surface IL-7Ra (CD127) levels (MFI medians; upper-lower range) were increased in onset (221; 150–286), and relapsed (1840; 651–2030) ALL compared to normal pre-B cells (528; 333–2673). (normal pre-B vs. leukemia at presentation, p<0.001, Mann-Whitney). Overexpression of YB-1-GFP in normal mature B cells resulted in increased expression of IL-7Ra (see figure, part B), suggesting an link between the YB-1 and IL-7 signaling pathways.Activated YB-1 is phosphorylated on S102 and relocated to the nucleus. Addition of IL-7 to pre-B ALL cell lines led to phosphorylation of YB-1 within 30 min. Similar results were shown for patient-derived, NSG mice-expanded pre-B ALL samples. Intracellular immunostaining using Imagestream technology (Amnis) showed that IL-7 treatment of pre-B ALL cell lines increased nuclear YB-1 levels 4-fold.As PI3K and MEK1 are involved in signaling downstream of IL-7, we investigated their role in YB-1 signaling in both pre-B ALL cell lines and NSG-mouse expanded pre-B ALL using pharmacological inhibitors. Western analyses showed that inhibition of PI3K using LY294002 did not prevent IL-7-mediated phosphorylation of YB-1 but the MEK1 inhibitor U0126 did, indicating the involvement of MAPK (see figure, part C). Conclusion:We show that YB-1, which is highly expressed in pediatric pre-B ALL compared to normal pre-B cells, is expressed at even higher levels after relapse. We demonstrate a link between the YB-1 and IL-7 signaling pathways which could offer a novel target for the treatment of refractory leukemia. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Reduction of Prep1 Levels Affects Differentiation of Normal and Malignant B Cells and Accelerates Myc Driven Lymphomagenesis

The Prep1 homeodomain transcription factor has recently been recognized as a tumor suppressor. Among other features, haploinsufficiency of Prep1 is able to strongly accelerate the B-lymphomagenesis in EμMyc mice. Now we report that this occurs concomitantly with a change in the type of B-cell lymphomas generated by the Myc oncogene. Indeed, the tumors generated in the EμMyc-Prep1+/− mice are much more immature, being mostly made up of Pro-B or Pre-B cells, while those in the EμMyc-Prep1+/+ mice are more differentiated being invariably IgM+. Moreover, we show that Prep1 is in fact required for the differentiation of Pro-B and Pre-B cells into IgM+ lymphocytes and/or their proliferation, thus showing also how a normal function of Prep1 affects EμMyc lymphomagenesis. Finally, we show that the haploinsufficiency of Prep1 is accompanied with a major decrease of Myc-induced apoptosis and that the haploinsufficieny is sufficient for all these effects because the second allele of Prep1 is not lost even at late stages. Therefore, the tumor-suppressive activity of Prep1 is intertwined with both the interference with Myc-induced apoptosis as well as with natural developmental functions of the protein.

Species-specific 5′-genomic structure and multiple transcription start sites in the chicken Pax5 gene

Master differentiation transcription factors (MDFs) play decisive roles in cell lineage commitment. Paired box 5 (Pax5) is one of MDFs essential for differentiation of pre-B cells into mature B cells. Here, we analyzed the 5′-genomic structure and transcription of the chicken Pax5 (c Pax5) gene in the chicken mature B cell line, DT40. We showed that the c Pax5 gene has two first exons: exon 1A contains long AG repeats, while exon 1B has high GC contents. The exons 1A and 1B had one and three major transcription start sites, respectively. Semi-quantitative RT-PCR revealed that comparable amounts of mRNA are transcribed from the exons 1A and 1B. Interestingly, the transcription start site of the c Pax5 exon 1A was chicken-specific. In addition, the c Pax5 promoter upstream of the exon 1A had no homology with the human and mouse Pax5 promoters. Thus, the mechanisms regulating transcription of the Pax5 exon 1A might not be conserved among species. Furthermore, we determined the physical structure of the exons 1A, 1B, and 2 in the genome of DT40 cells. Our results will be useful for elucidating mechanisms that control c Pax5 transcription and B cell lineage commitment, which is conserved or not conserved among different species.

Mechanisms of Pre-B Cell Receptor-Inactivation In Acute Lymphoblastic Leukemia

AbstractAbstract 147 Background:We recently established that the pre-B cell receptor functions as a tumor suppressor in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). The pre-B cell receptor promotes differentiation of normal pre-B cells and couples the immunoglobulin μ -chain to activating tyrosine kinases (e.g. SYK) via linker molecules (e.g. BLNK). In virtually all cases of Ph+ ALL, pre-B cell receptor function is compromised and its reconstitution induces rapid cell cycle arrest. However, genomic deletions in pre-B cell receptor pathway are rare and the mechanisms of inactivation are not known. Here we report that pre-B cell receptor inactivation occurs at multiple levels and involves at least four different mechanisms, namely (1) deleterious immunoglobulin gene rearrangement, (2) defective splicing of pre-B cell receptor signaling molecules, (3) expression of dominant-negative PAX5 fusion genes and (4) overexpression of inhibitory signaling molecules. Result:(1) Studying progressive transformation of pre-B cells in BCR-ABL1-transgenic mice, we observed that surface expression of the immunoglobulin μ -chain was downregulated after 60 days of age, which was a prerequisite for the onset of full-blown leukemia. While the repertoire of immunoglobulin gene rearrangements was polyclonal in wildtype pre-B cells, BCR-ABL1-transgenic pre-B cells show clonal expansions, which are derived from one ancestral productive immunoglobulin gene rearrangement in the transformed pre-B cell. However, the ancestral immunoglobulin gene rearrangements were rendered non-functional through deleterious secondary rearrangements. Likewise, in 47 of 57 cases of primary human Ph+ ALL, we detected traces of pre-B cell receptor-inactivation through secondary deleterious recombination events at the immunoglobulin μ -chain locus.(2) We studied pre-B cell receptor signaling molecules in primary human pre-B cells and 10 patient-derived Ph+ ALL samples by Western blotting and RT-PCR. As opposed to normal bone marrow pre-B cells, in all 10 cases of Ph+ ALL defective splice variants of the SYK tyrosine kinase and its linker molecule BLNK were found. Sequence analysis revealed a frequent 4 bp slippage during SYK pre-mRNA splicing which resulted in a truncated protein lacking the kinase domain, as confirmed by Western blot. To study the functional significance of defective Syk expression in Ph+ ALL cells, we transformed pre-B cells from Syk-fl/fl mice with BCR-ABL1 and deleted the Syk kinase using tamoxifen-inducible Cre. As opposed to Syk-fl/fl leukemia cells, inducible ablation of Syk rendered the leukemia cells insensitive to forced expression of the pre-B cell receptor. Multiple defective transcript variants of BLNK were found that all lacked exon 16 encoding the central part of the BLNK SH2 domain. In the absence of exon 16, BLNK splice variants were detached from the pre-B cell receptor and function in a dominant-negative way as they reduce Ca2+-mobilization in response to pre-B cell receptor stimulation. In a titration experiment, BLNK−/− leukemia cells were reconstituted with full-length and exon 16-deficient BLNK. Dominant-negative BLNK interfered with pre-B cell receptor-mediated tumor suppression at a ratio of 0.1 relative to full-length BLNK. Of note, we found somatic mutations within the splice site of exon 16 in 2 of 6 primary Ph+ ALL cases.(3) Ph+ ALL cells often carry chromosomal translocations leading to the expression of dominant-negative PAX5-fusion molecules. In a systematic gene expression analysis, we observed that ectopic expression of the dominant-negative PAX5-C20orf112 fusion led to downregulation of immunoglobulin μ -chain and the signaling molecules including SYK and BLNK. As a consequence, Ca2+-mobilization in response to pre-B cell receptor stimulation was significantly diminished.(4) Correction of defective immunoglobulin-μ chain and BLNK expression results in compensatory overexpression of a broad array of inhibitory signaling molecules. These molecules share an ITIM signaling motif, which attenuates pre-B cell receptor signal transduction through recruitment of inhibitory phosphatases. Conclusion:Even though loss of pre-B cell receptor function represents the uniform outcome of a diverse spectrum of lesions, individual Ph+ ALL subclones exhibit a complex pattern of shared and distinct defects involving one or more of these 4 mechanisms. Disclosures:No relevant conflicts of interest to declare.

IKAROS and BCL6 Limit Pre-B Cell Expansion and Prevent Leukemogenesis Downstream of the Pre-B Cell Receptor

AbstractAbstract 146 Background:The pre-B cell receptor promotes differentiation of normal pre-B cells and induces cell cycle arrest at the transition from large cycling pre-B cells (Fraction C') to small resting pre-B cells (Fraction D). While pre-B cell receptor-induced cell cycle arrest represents a critical safeguard against pre-B cell leukemogenesis, the mechanism of pre-B cell receptor-dependent tumor suppression is only poorly understood. We recently established that pre-B cell receptor signaling leads to upregulation of Ikaros (Trageser et al., J Exp Med, 2009). Ikaros functions as a tumor suppressor in BCR-ABL1 pre-B ALL and is deleted in >80% of the cases. In addition, we recently reported that BCL6 is upregulated during pre-B cell receptor-induced cell cycle arrest (Duy et al., J Exp Med 2010). Result:To elucidate the mechanism of pre-B cell receptor-dependent tumor suppression in BCR-ABL1-driven B cell lineage leukemia, we studied regulation of Stat5-phosphorylation as a central mediator of survival and proliferation downstream of the BCR-ABL1 kinase. Forced expression of the pre-B cell receptor resulted in rapid dephosphorylation of Stat5 Y694 and concomitant upregulation of BCL6. Pre-B cell receptor-mediated upregulation of BCL6 was sensitive to expression of a constitutively active mutant of Stat5. Therefore, upregulation of BCL6 likely occurs indirectly through dephosphorylation of Stat5 downstream of the pre-B cell receptor. Upregulation of BCL6 is indeed causally linked to pre-B cell receptor-induced cell cycle arrest: By genome-wide ChIP-on-chip analysis and single-locus qChIP verification, we observed direct recruitment of the BCL6 transcriptional repressor to the promoter regions of CCND2 and MYC, which represent central mediators of cell cycle progression in BCR-ABL1 ALL. The negative effect of BCL6 on cell cycle progression was confirmed by retroviral overexpression, which induced cell cycle arrest in the vast majority of BCR-ABL1 ALL cells. In addition, overexpression of Myc could rescue BCL6-dependent cell cycle arrest downstream of the pre-B cell receptor. To verify the role of BCL6 in negative cell cycle regulation in a genetic experiment, we tested the function of the pre-B cell receptor in BCL6+/+ and BCL6-Null BCR-ABL1-transformed pre-B ALL cells. Forced expression of the pre-B cell receptor rapidly induced cell cycle arrest in BCL6+/+ but not BCL6-Null pre-B ALL cells. We conclude that upregulation of BCL6 leads to transcriptional repression of Myc/CCND2 and is required for pre-B cell receptor-mediated cell cycle arrest. Since our experiments established that BCL6 upregulation required Stat5-dephosphorylation, we next studied how pre-B cell receptor signaling leads to dephosphorylation of Stat5 and, hence, transcriptional activation of BCL6 as key effector to induce cell cycle arrest. Surprisingly, this analysis identified Ikaros as the key-mediator of Stat5-dephosphorylation is direct cooperation with the pre-B cell receptor signaling cascade. Reconstitution of Ikaros expression resulted in dramatic Stat5-dephosphorylation, which was comparable to the effect of Imatinib. Ikaros-dependent Stat5-dephosphorylation directly intersects with the pre-B cell receptor signaling pathway, because the pre-B cell receptor-associated linker molecule BLNK (SLP65) is required for Ikaros-mediated dephosphorylation. In BLNK-Null BCR-ABL1 pre-B ALL cells, Ikaros expression did neither affect Stat5-phosphorylation nor proliferation and survival of leukemia cells. As an indirect consequence of Stat5-dephosphorylation, Ikaros/BLNK signaling resulted in upregulation of BCL6 and subsequent cell cycle arrest. Conclusion:The Ikaros (IKZF1) tumor suppressor is deleted in >80% of the cases of BCR-ABL1-driven pre-B ALL, however, the mechanisms of Ikaros-dependent tumor suppression remained elusive. Here we describe for the first time that Ikaros functions as tumor suppressor via dephosphorylation of Stat5. Thereby, the Ikaros tumor suppressor requires direct interaction with the pre-B cell receptor signaling pathway including BLNK. Ikaros/BLNK inactivate Stat5 and, hence, a critical survival and proliferation signal. In addition, Ikaros/BLNK signaling leads to activation of BCL6, which functions as negative regulator of Myc/CCND2-dependent proliferation. Disclosures:No relevant conflicts of interest to declare.

SYK Is a Tumor Suppressor In Pre-B Cell Acute Lymphoblastic Leukemia and Not a Therapeutic Target

AbstractAbstract 4199 Background:The pre-B cell receptor promotes differentiation of normal pre-B cells and couples the immunoglobulin μ-chain to activating tyrosine kinases (e.g. SYK) via linker molecules (e.g. BLNK). We recently established that the pre-B cell receptor functions as a tumor suppressor in pre-B acute lymphoblastic leukemia (ALL) including ALL cells carrying the BCR-ABL1 oncogene (Trageser et al., J Exp Med, 2009). In virtually all cases of BCR-ABL1 ALL, pre-B cell receptor function is compromised and its reconstitution induces rapid cell cycle arrest. Given that the SYK tyrosine kinase represents a critical signaling molecule in the pre-B cell receptor pathway, one would expect that SYK tyrosine kinase activity has a tumor suppressive effect. It therefore seems counterintuitive that pharmacologic targeting of SYK was recently proposed as a new treatment approach for pre-B ALL (Uckun et al., Br J Haematol. 2010). While there is solid evidence for a role of Syk as a target in B cell lymphoma (Friedberg et al., Blood 2010) and B cell lineage CLL (Buchner et al., Blood 2010), where tonic B cell receptor signaling delivers critical survival signals, the role of Syk downstream of the pre-B cell receptor in ALL is unclear. Results:To clarify the role of SYK downstream of the pre-B cell receptor in pre-B ALL, we performed a genetic experiment to inducibly delete the Syk gene in pre-B ALL cells. To this end, pre-B cells from Syk-fl/fl mice were propagated in the presence of IL7 and then transformed with retroviral BCR-ABL1 or MLL-ENL oncogenes. After transformation, pre-B leukemia cells were transduced with 4-hydroxy-tamoxyfen (4-OHT)-inducible retroviral Cre-ERT2 or an ERT2 empty vector control. After puromycin-selection of Cre-ERT2 and ERT2 transduced leukemia cells, Cre-ERT2 or the ERT2 control were induced by addition of 4-OHT and deletion of Syk was studied at different time points. As assessed by Western blot and PCR, deletion of Syk was near complete after two days and undetectable after six days. We then studied changes in cell viability upon inducible deletion of Syk: Acute deletion of the Syk tyrosine kinase had no significant impact on the viability of pre-B ALL cells, even after prolonged cell culture over several weeks. We then reasoned that the effect of Syk-deletion may be subtle yet important, so we studied in BCR-ABL1-transformed Syk-fl/fl pre-B leukemia cells whether Syk-deletion sensitizes to Imatinib-treatment. Deletion of Syk was again confirmed by Western blot, yet the dose-response curves to Imatinib-treatment were superimposable for Syk-fl/fl and Syk-del/del pre-B leukemia cells. We conclude that SYK does not contribute important survival signals in our mouse model for pre-B ALL, nor does deletion of Syk sensitize BCR-ABL1-driven pre-B leukemia cells to Imatinib-treatment.We next investigated the counter-hypothesis that Syk functions as a tumor suppressor downstream of the pre-B cell receptor. To test this possibility, we tested the effect of forced pre-B cell receptor expression in the presence or absence of Syk. Syk-fl/fl and Syk-del/del pre-B leukemia cells were transduced with CD8/μ-chain or a CD8 empty vector control. The μ-chain represents the central component of the pre-B cell receptor. Forced expression of the CD8 empty vector control had no effect regardless of whether Syk was deleted or not. When pre-B cell receptor signaling was reconstituted in Syk-fl/fl cells by expression of CD/μ-chain, viability of the leukemia decreased by >80%. By contrast, deletion of Syk greatly attenuated the tumor suppressive effect of CD8/μ-chain expression and less than 25% of the leukemia cells underwent cell cycle arrest and cell death. Background:Genetic deletion of Syk unequivocally demonstrates that Syk does not deliver critical survival signals downstream of the pre-B cell receptor in ALL. This is unlike B cell lymphoma, where tonic signaling from the B cell receptor promotes cell survival via Syk (Friedberg et al., 2010; Buchner et al., 2010). On the contrary, in pre-B ALL, the Syk kinase mediates pre-B cell receptor-induced cell cycle arrest. These findings are in direct conflict with a recent report on the therapeutic usefulness of pharmacological inhibition of Syk in pre-B ALL cell lines (Uckun et al., Br J Haematol.; 149: 508-17; 2010). The compound (C-61) used in this study may have unrecognized off-target effects, which might account for the discrepancies. Disclosures:No relevant conflicts of interest to declare.

All oligosaccharide moieties of the μ chains in the pre-BCR are of the high-mannose type

Although it is well established that pre-BCR signaling governs proliferation and differentiation during B cell development, the components of the pre-BCR that are important for signaling are a matter of controversy. It has been suggested that signaling by the μ heavy chains of the pre-BCR induces survival and differentiation of pre-B cells, while the λ5 part of the pre-BCR is essential for proliferation and clonal expansion. However, the mechanism by which pre-BCR μ chains initiate differentiation signals is not clear. Using two variants of a murine B-lymphocyte cell line that differ only in surface expression of either BCR or pre-BCR, we demonstrated that surface μ chains in the pre-BCR are of the high-mannose type only, while those in the BCR are of the complex type. It is hypothesized that mannose-specific lectin-like molecules on accessory cells or in solution may function as the non-antigen ligand that triggers the pre-BCR.

Inactivation of Pre-B Cell Receptor-Mediated Tumor Suppression by Aberrant Splicing in Ph+ Acute Lymphoblastic Leukemia.

Abstract 579The pre-B cell receptor instructs differentiation of normal pre-B cells and functions as a tumor suppressor in pre-B acute lymphoblastic leukemia (ALL). Its function is impaired in virtually all cases of pre-B ALL carrying the Philadelphia chromosome (Ph), which encodes the BCR-ABL1 kinase. However, genomic deletions in pre-B cell receptor pathway are rare and the mechanism of its inactivation in ALL is not known. Here we report that aberrant splicing of SLP65 (BLNK, BASH) and the pre-B cell receptor-related linker molecules LCP2, SYK and LAT represents a major mechanism of pre-B cell receptor-inactivation, which is consistent with deregulated expression of splice factors in Ph+ ALL. In contrast to normal pre-B cells, BCR-ABL1-transformed pre-B ALL expressed multiple splice variants of the pre-B cell receptor-related linker molecules SLP65, LCP2, SYK and LAT. A detailed sequence analysis of these splice variants revealed that aberrant splicing resulted in functional inactivation of important protein domains in SLP65, LCP2, SYK and LAT transcripts. Of note, the vast majority of SLP65 splice variants lacked a functional SH2 domain, which is required to anchor SLP65 to the Igαa signaling chain of the pre-B cell receptor. We then focused our analysis on the SH2 domain of SLP65 for the following reasons: (1) SLP65 functions as a powerful tumor suppressor downstream of the pre-B cell receptor. (2) Exon 16 which encodes the central part of the SH2 domain was affected in the vast majority of SLP65 splice variants. (3) The SH2 domain of SLP65 is required to link downstream effector molecules (BTK, SYK, PLCγ2) to the Igαa signaling chain of the pre-B cell receptor. We found that deletion of the SH2 domain represents a frequent feature in Ph+ ALL.In reconstitution experiments with pre-B cells from SLP65-/- mice, SLP65 forms lacking the SH2 domain failed to support pre-B cell receptor signaling. By coexpression with full-length SLP65, we demonstrated that SH2-deficient variants have a dominant-negative effect by reducing the Ca2+-release and the leukemia-suppressive function of SLP65. These effects are dose-dependent as demonstrated in a titration experiment, in which SLP65 and SH2-deficient SLP65 were expressed at different ratios. SH2-deficient SLP65 also compromised the leukemia-suppressive function of the pre-B cell receptor in vivo transplantation model: While reconstitution of full-length SLP65 in SLP65-/- BCR-ABL1 ALL cells suppressed leukemia growth and bone marrow infiltration, loss of the SLP65 SH2 domain abolished the tumor suppressor function of the pre-B cell receptor.To address potential mechanisms of aberrant pre-mRNA splicing, we studied expression levels of splicing factors in Ph+ ALL and searched for splice site mutations: Consistent with aberrant splicing of pre-B cell receptor-related linker molecules including SLP65, SYK, LAT and LCP2, we found evidence of splice factor deregulation in Ph+ ALL: Among the 94 pre-mRNA splice factors studied, 24 were differentially expressed between Ph+ ALL (n=15) and normal bone marrow B cell precursors (n=11). Of note, the SRPK1, hnRPC, hnRNP-E2 (PCBP2) and hnRNP-A1 pre-mRNA splice factors that were previously found as BCR-ABL1-regulated, were also identified in our analysis. Sequence analysis of the 3'prime splice site of exon 16 in three Ph+ ALL cell lines and six primary cases of Ph+ ALL revealed one clonal splice site mutation in one primary Ph+ ALL case. In one cell line and one primary Ph+ ALL case, each one additional mutation was found within 2 basepairs distance from the splice site. It is not clear, however, whether and to which extent these mutations affect splice site selection. Interestingly, we found that clones amplified from one primary Ph+ ALL case and one Ph+ ALL cell line harbor mutations encoding premature translation stops close to the end of exon 16. While these mutations do not lead to aberrant splicing of exon 16, they reinforce the notion that Ph+ ALL cells are selected for inactivation of the SLP65 SH2 domain.We conclude that aberrant splicing frequently results in the expression of dominant-negative SH2-deficient SLP65, which is sufficient to compromise the tumor suppressor function of the pre-B cell receptor in Ph+ ALL. Whereas mutations and deletions of the SLP65 gene are rare, we conclude that aberrant splicing of the SLP65 SH2 domain represents a common mechanism in ALL cells to compromise the leukemia-suppressive function of the pre-B cell receptor. Disclosures:Martinelli:Novartis: Research Funding.

Regulation of B-cell proliferation and differentiation by pre-B-cell receptor signalling

The pre-B-cell receptor (pre-BCR) is expressed following the productive recombination of the immunoglobulin heavy chain gene. Signals through the pre-BCR are required for initiating diverse processes in pre-B cells, including proliferation and recombination of the light chain gene, which eventually lead to the differentiation of pre-B cells to immature B cells. However, the molecular mechanisms by which the pre-BCR promotes these processes remain largely unresolved. Recent findings suggest that forkhead box O (FOXO) transcription factors connect pre-BCR signalling to the activation of the recombination machinery. In this Review, we discuss how FOXO transcription factors are regulated by the pre-BCR to allow the progression of the cell cycle and the recombination of the light chain gene.

A Unique Role for the λ5 Nonimmunoglobulin Tail in Early B Lymphocyte Development

Precursor BCR (pre-BCR) signaling governs proliferation and differentiation of pre-B cells during B lymphocyte development. However, it is controversial as to which parts of the pre-BCR, which is composed of Igmu H chain, surrogate L chain (SLC), and Igalpha-Igbeta, are important for signal initiation. Here, we show in transgenic mice that the N-terminal non-Ig-like (unique) tail of the surrogate L chain component lambda5 is critical for enhancing pre-BCR-induced proliferation signals. Pre-BCRs with a mutated lambda5 unique tail are still transported to the cell surface, but they deliver only basal signals that trigger survival and differentiation of pre-B cells. Further, we demonstrate that the positively charged residues of the lambda5 unique tail, which are required for pre-BCR self-oligomerization, can also mediate binding to stroma cell-associated self-Ags, such as heparan sulfate. These findings establish the lambda5 unique tail as a pre-BCR-specific autoreactive signaling motif that could increase the size of the primary Ab repertoire by selectively expanding pre-B cells with functional Igmu H chains.

Visfatin (pre-B cell colony-enhancing factor) gene expression in patients with rheumatoid arthritis

Visfatin, also known as pre-B cell colony enhancing factor, is a cytokine-like protein originally cloned from peripheral blood mononuclear cells (PBMC) as a factor that enhances differentiation of pre-B cells...