Spatial Stochastic Model of the Pre-B Cell Receptor.

Survival and proliferation of immature B lymphocytes requires expression and tonic signaling of the pre-B cell receptor (pre-BCR). This low level, ligand-independent signaling is likely achieved through frequent, but short-lived, homo interactions. Tonic signaling is also central in the pathology of precursor B acute lymphoblastic leukemia (B-ALL). In order to understand how repeated, transient events can lead to sustained signaling and to assess the impact of receptor accumulation induced by the membrane landscape, we developed a spatial stochastic model of receptor aggregation and downstream signaling events. Our rule- and agent-based model builds on previous mature BCR signaling models and incorporates novel parameters derived from single particle tracking of pre-BCR on surfaces of two different B-ALL cell lines, 697 and Nalm6. Live cell tracking of receptors on the two cell lines revealed characteristic differences in their dimer dissociation rates and diffusion coefficients. We report here that these differences affect pre-BCR aggregation and consequent signal initiation events. Receptors on Nalm6 cells, which have a lower off-rate and lower diffusion coefficient, more frequently form higher order oligomers than pre-BCR on 697 cells, resulting in higher levels of downstream phosphorylation in the Nalm6 cell line.

Loss of early B cell protein λ5 decreases bone mass and accelerates skeletal aging.

The early B cell protein λ5 is an essential component of the surrogate light chain and the preB cell receptor (preBCR), which is critical for optimal B cell development. To investigate the effect of λ5 and/or B cells on bone acquisition over time, we developed a panel of JH -/- , λ5-/-, JH -/- λ5-/-, and wild-type (WT) BALB/c mice and then studied postnatal bone development and aging in these mice at one, six, twelve, and twenty-two months of age. The trabecular bone volume over total volume (BV/TV) in JH -/- mice was similar to WT mice at all ages. In contrast, at six months of age and thereafter, λ5-/- and JH -/- λ5-/- mice demonstrated a severe decrease in trabecular bone mass. Surprisingly, bone mass in six-month-old λ5-/- and JH -/- λ5-/- mice was similar to or even lower than in aged (twenty-two-months) WT mice, suggesting accelerated skeletal aging. The postnatal development and the acquisition of cortical bone mass in JH -/- λ5-/- mice were generally comparable to WT. However, JH -/- λ5-/- mice showed a significant decrease in cortical BV/TV at six- and twelve months of age. To examine the contribution of λ5 and B cells to postnatal bone synthesis, we separately transplanted whole bone marrow cells from JH -/- λ5-/- and WT mice into irradiated JH -/- λ5-/- and WT recipients. WT recipients of JH -/- λ5-/- marrow cells failed to show acquisition of trabecular bone mass, whereas transplanting WT marrow cells into JH -/- λ5-/- recipients led to the recovery of trabecular bone mass. Transfer of WT marrow cells into JH -/- λ5-/- mice promoted synthesis of new cortical and trabecular bone. Our findings indicate that λ5 plays a major role in preserving bone mass during postnatal development and skeletal aging which is distinct from its role in B cell development. The absence of both λ5 and B cells in JH -/- λ5-/- mice leads to delayed acquisition of cortical bone during postnatal development. Dissecting the mechanism(s) by which λ5 regulates bone homeostasis may provide new avenues for the treatment of age-related loss of bone mass and osteoporosis.

B-1a cells acquire their unique characteristics by bypassing the pre-BCR selection stage.

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.

Disruption of the preB Cell Receptor Complex Leads to Decreased Bone Mass.

In the bone marrow, preB cells are found adjacent to the bone endosteum where bone synthesizing osteoblast and bone resorbing osteoclasts reside. Although there is evidence of interactions between preB and bone cells, the factors that contribute to such interactions are poorly understood. A critical checkpoint for preB cell development assesses the integrity of the nascent immunoglobulin µ heavy chain (HC) by testing whether it can participate in the formation of a preB cell receptor (preBCR), composed of the µ HC and surrogate light chain (LC). In this work, we tested whether loss of preBCR components can affect bone synthesis. A panel of gene targeted mice with sequential blocks in preBCR formation or function [surrogate light chain component lambda 5 deleted (λ5-/-), transmembrane domain of µHC deleted (IgM-mem-/-), and CD19 preBCR co-receptor deleted (CD19-/-)] were evaluated for effects on postnatal bone synthesis. Postnatal bone mass was analyzed in 6 month old mice using µ-CT, histomorphometry and double calcein labeling. Both cortical and trabecular bone mass were significantly decreased in the femurs of the λ5 and IgM-mem deficient mice. Histomorphometric analysis showed a decrease in the numbers of osteoblasts and osteoclasts in all three mutant strains. Double calcein labeling revealed a significant decrease in dynamic synthesis and mineralization of bone in λ5-/- mice. Our data strongly suggest that interference with preBCR formation or function affects bone homeostasis independent of the presence or absence of mature B cells, and that components of the preBCR play important, and potentially distinct, roles in regulating adult bone mass.

The Role of the Pre-B Cell Receptor in B Cell Development, Repertoire Selection, and Tolerance.

Around four decades ago, it had been observed that there were cell lines as well as cells in the fetal liver that expressed antibody µ heavy (µH) chains in the apparent absence of bona fide light chains. It was thus possible that these cells expressed another molecule(s), that assembled with µH chains. The ensuing studies led to the discovery of the pre-B cell receptor (pre-BCR), which is assembled from Ig µH and surrogate light (SL) chains, together with the signaling molecules Igα and ß. It is expressed on a fraction of pro-B (pre-BI) cells and most large pre-B(II) cells, and has been implicated in IgH chain allelic exclusion and down-regulation of the recombination machinery, assessment of the expressed µH chains and shaping the IgH repertoire, transition from the pro-B to pre-B stage, pre-B cell expansion, and cessation.

Science Signaling Podcast for 29 November 2016: Pre-B cell receptor signaling in leukemia.

This Podcast features an interview with Bridget Wilson, author of a Research Article that appears in the 29 November 2016 issue of Science Signaling, about pre-B cell receptor (pre-BCR) signaling in B cell precursor acute lymphoblastic leukemia (BCP-ALL). Signaling through the pre-BCR, an immature form of the BCR, promotes the survival of B cell progenitors and has been implicated in the pathology of BCP-ALL. Erasmus et al found that pre-BCRs formed transient homomeric complexes that correlated with pro-survival signaling. Preventing homotypic interactions between pre-BCRs sensitized B cells to chemotherapeutic agents, suggesting that interfering with such interactions may improve the efficacy of existing chemotherapies for BCP-ALL.Listen to Podcast.

The Expression Pattern of the Pre-B Cell Receptor Components Correlates with Cellular Stage and Clinical Outcome in Acute Lymphoblastic Leukemia.

Precursor-B cell receptor (pre-BCR) signaling represents a crucial checkpoint at the pre-B cell stage. Aberrant pre-BCR signaling is considered as a key factor for B-cell precursor acute lymphoblastic leukemia (BCP-ALL) development. BCP-ALL are believed to be arrested at the pre-BCR checkpoint independent of pre-BCR expression. However, the cellular stage at which BCP-ALL are arrested and whether this relates to expression of the pre-BCR components (IGHM, IGLL1 and VPREB1) is still unclear. Here, we show differential protein expression and copy number variation (CNV) patterns of the pre-BCR components in pediatric BCP-ALL. Moreover, analyzing six BCP-ALL data sets (n = 733), we demonstrate that TCF3-PBX1 ALL express high levels of IGHM, IGLL1 and VPREB1, and are arrested at the pre-B stage. By contrast, ETV6-RUNX1 ALL express low levels of IGHM or VPREB1, and are arrested at the pro-B stage. Irrespective of subtype, ALL with high levels of IGHM, IGLL1 and VPREB1 are arrested at the pre-B stage and correlate with good prognosis in high-risk pediatric BCP-ALL (n = 207). Our findings suggest that BCP-ALL are arrested at different cellular stages, which relates to the expression pattern of the pre-BCR components that could serve as prognostic markers for high-risk pediatric BCP-ALL patients.

Charged Amino Acid-rich Leucine Zipper-1 (Crlz-1) as a Target of Wnt Signaling Pathway Controls Pre-B Cell Proliferation by Affecting Runx/CBFß-targeted VpreB and λ5 Genes.

The proliferation of pre-B cells is known to further increase the clonal diversity of B cells at the stage of pre-B cells by allowing the same rearranged heavy chains to combine with differently rearranged light chains in a subsequent developmental stage. Crlz-1 (charged amino acid-rich leucine zipper-1) was found to control this proliferation of pre-B cells by working as a Wnt (wingless-related mouse mammary tumor virus integration site) target gene in these cells. Mechanistically, Crlz-1 protein functioned by mobilizing cytoplasmic CBFß (core binding factor ß) into the nucleus to allow Runx (runt-related transcription factor)/CBFß heterodimerization. Runx/CBFß then turned on its target genes such as EBF (early B cell factor), VpreB, and λ5 and thereby pre-B cell receptor signaling, leading to the expression of cyclins D2 and D3 Actually, the proliferative function of Crlz-1 was demonstrated by not only Crlz-1 or ß-catenin knockdown but also Crlz-1 overexpression. Furthermore, the mechanistic view that the proliferative function of Crlz-1 is caused by relaying Wnt/ß-catenin to pre-B cell receptor signaling pathways through the regulation of Runx/CBFß heterodimerization was also verified by employing niclosamide, XAV939, and LiCl as Wnt inhibitors and activator, respectively.

RAG-mediated DNA double-strand breaks activate a cell type-specific checkpoint to inhibit pre-B cell receptor signals.

DNA double-strand breaks (DSBs) activate a canonical DNA damage response, including highly conserved cell cycle checkpoint pathways that prevent cells with DSBs from progressing through the cell cycle. In developing B cells, pre-B cell receptor (pre-BCR) signals initiate immunoglobulin light (Igl) chain gene assembly, leading to RAG-mediated DNA DSBs. The pre-BCR also promotes cell cycle entry, which could cause aberrant DSB repair and genome instability in pre-B cells. Here, we show that RAG DSBs inhibit pre-BCR signals through the ATM- and NF-κB2-dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor, resulting in suppression of pre-BCR signaling. This regulatory circuit prevents the pre-BCR from inducing additional Igl chain gene rearrangements and driving pre-B cells with RAG DSBs into cycle. We propose that pre-B cells toggle between pre-BCR signals and a RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes.

ZFP521 contributes to pre-B-cell lymphomagenesis through modulation of the pre-B-cell receptor signaling pathway.

ZFP521 was previously identified as a putative gene involved in induction of B-cell lymphomagenesis. However, the contribution of ZFP521 to lymphomagenesis has not been confirmed. In this study, we sought to elucidate the role of ZFP521 in B-cell lymphomagenesis. To this end, we used a retroviral insertion method to show that ZFP521 was a target of mutagenesis in pre-B-lymphoblastic lymphoma cells. The pre-B-cell receptor (pre-BCR) signaling molecules BLNK, BTK and BANK1 were positively regulated by the ZFP521 gene, leading to enhancement of the pre-BCR signaling pathway. In addition, c-myc and c-jun were upregulated following activation of ZFP521. Stimulation of pre-BCR signaling using anti-Vpreb antibodies caused aberrant upregulation of c-myc and c-jun and of Ccnd3, which encodes cyclin D3, thereby inducing the growth of pre-B cells. Stimulation with Vpreb affected the growth of pre-B cells, and addition of interleukin (IL)-7 receptor exerted competitive effects on pre-B-cell growth. Knockdown of BTK and BANK1, targets of ZFP521, suppressed the effects of Vpreb stimulation on cell growth. Furthermore, in human lymphoblastic lymphoma, analogous to pre-B-cell lymphoma in mice, the expression of ZNF521, the homolog of ZFP521 in humans, was upregulated. In conclusion, our data showed that the ZFP521 gene comprehensively induced pre-B-cell lymphomagenesis by modulating the pre-B-cell receptor signaling pathway.

The pre-B-cell receptor checkpoint in acute lymphoblastic leukaemia.

The B-cell receptor (BCR) and its immature form, the precursor-BCR (pre-BCR), have a central role in the control of B-cell development, which is dependent on a sequence of cell-fate decisions at specific antigen-independent checkpoints. Pre-BCR expression provides the first checkpoint, which controls differentiation of pre-B to immature B-cells in normal haemopoiesis. Pre-BCR signalling regulates and co-ordinates diverse processes within the pre-B cell, including clonal selection, proliferation and subsequent maturation. In B-cell precursor acute lymphoblastic leukaemia (BCP-ALL), B-cell development is arrested at this checkpoint. Moreover, malignant blasts avoid clonal extinction by hijacking pre-BCR signalling in favour of the development of BCP-ALL. Here, we discuss three mechanisms that occur in different subtypes of BCP-ALL: (i) blocking pre-BCR expression; (ii) activating pre-BCR-mediated pro-survival and pro-proliferative signalling, while inhibiting cell cycle arrest and maturation; and (iii) bypassing the pre-BCR checkpoint and activating pro-survival signalling through pre-BCR independent alternative mechanisms. A complete understanding of the BCP-ALL-specific signalling networks will highlight their application in BCP-ALL therapy.

Rationale for targeting the pre-B-cell receptor signaling pathway in acute lymphoblastic leukemia.

Inhibitors of B-cell receptor (BCR) and pre-BCR signaling were successfully introduced into patient care for various subtypes of mature B-cell lymphoma (e.g., ibrutinib, idelalisib). Acute lymphoblastic leukemia (ALL) typically originates from pre-B cells that critically depend on survival signals emanating from a functional pre-BCR. However, whether patients with ALL benefit from treatment with (pre-) BCR inhibitors has not been explored. Recent data suggest that the pre-BCR functions as tumor suppressor in the majority of cases of human ALL. However, a distinct subset of human ALL is selectively sensitive to pre-BCR antagonists.

Pre-B Lymphocyte Protein 3 (VPREB3) Expression in the Adrenal Cortex: Precedent for non-Immunological Roles in Normal and Neoplastic Human Tissues.

The pre-B lymphocyte protein 3 (VPREB3) is expressed during B cell differentiation and in subsets of mature B lymphocytes and is mainly found in bone marrow and lymphoid tissue germinative centers. So far, its function in B cells remains to be clarified. The messenger RNA (mRNA) of VPREB3 was previously detected in aldosterone-producing adenomas (APA); however, further information about this protein in human adrenocortical cells and tissues is currently unavailable. Therefore, in the present study, we, for the first time, investigate the protein expression of VPREB3 in human adrenocortical tissues. In addition, we approach the previously suggested similarities in expression patterns of aldosterone-producing cells and Purkinje neurons. Immunohistochemical analysis of VPREB3 was performed in 13 nonpathological adrenals (NA), 6 adrenal glands with idiopathic hyperaldosteronism (IHA), 18 APA, 5 cortisol-producing adenomas (CPA), and 5 nonpathological human cerebellum specimens. The mRNA levels of VPREB3, steroidogenic enzymes, and other aldosterone biosynthesis markers were detected in 53 APA samples using real-time RT-PCR (qPCR) and compared to the clinical data of APA patients. In our results, the VPREB3 protein was diffusely detected in APA, partially or weakly detected in CPA, and immunolocalized in the zona glomerulosa of NA and IHA, as well as in the cytoplasm of cerebellar Purkinje cells. In APA, VPREB3 mRNA levels were significantly correlated to plasma aldosterone (P = 0.026; R = 0.30), KCNJ5 mutations (P = 0.0061; mutated 34:19 wild type), CYP11B2 (P < 0.0001; R = 0.65), Purkinje cell protein 4 (PCP4; P < 0.0001; R = 0.53), and voltage-dependent calcium channels CaV1.3 (P = 0.023; R = 0.31) and CaV3.2 (P = 0.0019; R = 0.42). Based on our data, we hypothesize a possible role for VPREB3 in aldosterone biosynthesis, and present ideas for future functional studies.

Pre-B cell receptor binding to galectin-1 modifies galectin-1/carbohydrate affinity to modulate specific galectin-1/glycan lattice interactions.

Galectins are glycan-binding proteins involved in various biological processes including cell/cell interactions. During B-cell development, bone marrow stromal cells secreting galectin-1 (GAL1) constitute a specific niche for pre-BII cells. Besides binding glycans, GAL1 is also a pre-B cell receptor (pre-BCR) ligand that induces receptor clustering, the first checkpoint of B-cell differentiation. The GAL1/pre-BCR interaction is the first example of a GAL1/unglycosylated protein interaction in the extracellular compartment. Here we show that GAL1/pre-BCR interaction modifies GAL1/glycan affinity and particularly inhibits binding to LacNAc containing epitopes. GAL1/pre-BCR interaction induces local conformational changes in the GAL1 carbohydrate-binding site generating a reduction in GAL1/glycan affinity. This fine tuning of GAL1/glycan interactions may be a strategic mechanism for allowing pre-BCR clustering and pre-BII cells departure from their niche. Altogether, our data suggest a novel mechanism for a cell to modify the equilibrium of the GAL1/glycan lattice involving GAL1/unglycosylated protein interactions.

Examination of galectin-induced lattice formation on early B-cell development.

Galectin-1 (GAL1) is a pre-B cell receptor (pre-BCR) ligand that induces pre-BCR clustering and leads to efficient pre-B cell proliferation and differentiation in the bone marrow. To study pre-BCR-GAL1 interactions and its functional consequence on the early steps of the B cell development, we combine structural nuclear magnetic resonance (NMR) approaches and B cell biology techniques. NMR is applied to identify the residues involved in pre-BCR-GAL1 interactions by monitoring chemical shift perturbations when the complex is formed. This structural information is then used at the cellular level to target specifically the complex formation during GAL1-induced pre-BCR clustering and lattice formation, using immunofluorescence techniques. Moreover, an in vivo assay was set up to study the consequence of synapse formation on the early steps of B cell development.

Mechanisms of pre-B-cell receptor checkpoint control and its oncogenic subversion in acute lymphoblastic leukemia.

Pre-B cells within the bone marrow represent the normal counterpart for most acute lymphoblastic leukemia (ALL). During normal early B-cell development, survival and proliferation signals are dominated by cytokines, particularly interleukin-7 (IL-7) for murine developing B cells. With expression of a functional pre-B-cell receptor (BCR), cytokine signaling is attenuated and the tonic/autonomous pre-BCR signaling pathway provides proliferation as well as differentiation signals. In this review, we first describe checkpoint mechanisms during normal B-cell development and then discuss how genetic lesions in these pathways function as oncogenic mimicries and allow transformed pre-B cells to bypass checkpoint control. We focus on cytokine receptor signaling that is mimicked by activating lesions in receptor subunits or downstream mediators as well as aberrant activation of non-B lymphoid cytokine receptors. Furthermore, we describe the molecular switch from cytokine receptor to pre-BCR signaling, how this pathway is of particular importance for certain ALL subtypes, and how pre-BCR signaling is engaged by genetic lesions, such as BCR-ABL1. We discuss the transcriptional control mechanisms downstream of both cytokine- and pre-BCR signaling and how normal checkpoint control mechanisms are circumvented in pre-B ALL. Finally, we highlight new therapeutic concepts for targeted inhibition of oncogenic cytokine or pre-BCR signaling pathways.

Transcriptional and metabolic pre-B cell receptor-mediated checkpoints: implications for autoimmune diseases.

At the pre-B cell stage of lymphocyte development, immunoglobulin light-chains are not yet produced, and heavy-chains are covalently linked to surrogate light-chains composed of VpreB and λ5 to form the pre-B cell receptor (pre-BCR) in a non-covalent association with signal-transducing modules. Even tough the pre-BCR does not have the potential to bind conventional antigens, accumulating evidence indicates that pre-BCR-mediated checkpoints are important both for negative and positive selection of self-reactivity, and that defects in these regulatory nodes may be associated with autoimmune disease. Thus, the transcription factor BACH2, which represents a susceptibility locus for rheumatoid arthritis, has recently emerged as a crucial mediator of negative selection at a pre-BCR checkpoint. The lysosome-associated protein LAPTM5, which is highly expressed in an animal model of Sjögren's syndrome, plays a role in down-modulation of the pre-BCR. Studies of copy number variation in rheumatoid arthritis suggest that a reduced dosage of the VPREB1 gene is involved in disease pathogenesis. Notably, animal models of autoimmune disease exhibit defects in pre-B to naïve B cell checkpoints. Administration of a pre-BCR ligand, which also plays a role in anergy both in human and murine B lymphocytes, ameliorates disease in experimental models of autoimmunity. Further investigation is required to gain a better insight into the molecular mechanisms of pre-BCR-mediated checkpoints and to determine their relevance to autoimmune diseases.

The combined expression of VPREB3 and ID3 represents a new helpful tool for the routine diagnosis of mature aggressive B-cell lymphomas.

Genomic studies, such as gene expression profiling and next-generation sequencing studies, have provided new insights into the phenotypic characteristics and pathogenesis of mature aggressive B-cell lymphomas. In particular, mutations in the transcription factors ID3 and TCF3, leading to overexpression of B-cell receptor components such as VPREB3, have been shown to be specific for Burkitt lymphoma (BL) and play an important tumourigenic role by mediating the activation of the pro-survival phosphatidylinositol-3-OH kinase pathway. We performed immunohistochemical analysis by applying commercially available anti-VPREB3 antibody to a large cohort of 185 genetically and immunophenotypically characterized mature aggressive B-cell lymphomas and analyzed these results together with recent data on ID3 expression. The combined expression of both VPREB3 and ID3 was associated with a diagnosis of BL with high sensitivity (0.77), high specificity (0.75) and high negative predictive values (0.96), however, with lower positive predictive value (0.30). Double negative cases were absent in the group of BLs but could be found in approximately one third of the remaining cases of mature aggressive B-cell lymphomas. Further, we could not identify a correlation with MYC, BCL2 or BCL6 aberrations with neither VPREB3 nor ID3 expression in each of the diagnostic groups analyzed. Our results, which are in line with recently discovered mutations in next-generation sequencing studies, suggest that the combined immunohistochemical detection of VPREB3 and ID3 is applicable to the routine diagnostic in case of mature aggressive B-cell lymphomas. In particular, it represents a useful and routinely applicable diagnostic tool to exclude BL diagnosis in case of single positive or double negative cases.

BACH2-BCL6 balance regulates selection at the pre-B cell receptor checkpoint.

At the pre-B cell receptor (BCR) checkpoint, developing pre-B cells are selected for successful rearrangement of V(H)-DJ(H) gene segments and expression of a pre-BCR. Reduced stringency at this checkpoint may obstruct the B cell repertoire with nonfunctional B cell clones. Earlier studies have described that activation of B cell lymphoma/leukemia (BCL)6 by a functional pre-BCR mediates positive selection of pre-B cells that have passed the checkpoint. This concept is now further elaborated by the recent finding that the BTB and CNC homology 1 basic leucine zipper transcription factor 2 (BACH2) induces negative selection and opposes BCL6 function prior to the pre-BCR checkpoint. Here, we discuss the antagonism between BCL6 and BACH2 during early B cell development, as well as its implications in both repertoire selection and counter-selection of premalignant clones for leukemia suppression.

A Bach2 link between pre-B cell receptor checkpoint and pre-B cell ALL.

Bach2 is a transcription factor required for affinity maturation of B cells. A recent study reveals, quite unexpectedly, that Bach2 also plays a key role in the pre-B cell receptor checkpoint and functions as a tumor suppressor in pre-B cell acute lymphocytic leukemia.