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

Deregulated Syk inhibits differentiation and induces growth factor–independent proliferation of pre–B cells

The nonreceptor protein spleen tyrosine kinase (Syk) is a key mediator of signal transduction in a variety of cell types, including B lymphocytes. We show that deregulated Syk activity allows growth factor–independent proliferation and transforms bone marrow–derived pre–B cells that are then able to induce leukemia in mice. Syk-transformed pre–B cells show a characteristic pattern of tyrosine phosphorylation, increased c-Myc expression, and defective differentiation. Treatment of Syk-transformed pre–B cells with a novel Syk-specific inhibitor (R406) reduces tyrosine phosphorylation and c-Myc expression. In addition, R406 treatment removes the developmental block and allows the differentiation of the Syk-transformed pre–B cells into immature B cells. Because R406 treatment also prevents the proliferation of c-Myc–transformed pre–B cells, our data indicate that endogenous Syk kinase activity may be required for the survival of pre–B cells transformed by other oncogenes. Collectively, our data suggest that Syk is a protooncogene involved in the transformation of lymphocytes, thus making Syk a potential target for the treatment of leukemia

BCL6 is critical for the development of a diverse primary B cell repertoire

BCL6 protects germinal center (GC) B cells against DNA damage–induced apoptosis during somatic hypermutation and class-switch recombination. Although expression of BCL6 was not found in early IL-7–dependent B cell precursors, we report that IL-7Rα–Stat5 signaling negatively regulates BCL6. Upon productive VH-DJH gene rearrangement and expression of a μ heavy chain, however, activation of pre–B cell receptor signaling strongly induces BCL6 expression, whereas IL-7Rα–Stat5 signaling is attenuated. At the transition from IL-7–dependent to –independent stages of B cell development, BCL6 is activated, reaches expression levels resembling those in GC B cells, and protects pre–B cells from DNA damage–induced apoptosis during immunoglobulin (Ig) light chain gene recombination. In the absence of BCL6, DNA breaks during Ig light chain gene rearrangement lead to excessive up-regulation of Arf and p53. As a consequence, the pool of new bone marrow immature B cells is markedly reduced in size and clonal diversity. We conclude that negative regulation of Arf by BCL6 is required for pre–B cell self-renewal and the formation of a diverse polyclonal B cell repertoire

B cell antigen receptor assembly and Syk activation in the S2 cell reconstitution system

Signal transduction from the B cell antigen receptor (BCR) involves a multitude of signaling molecules often organized in dynamic protein complexes. The molecular mechanisms operating during signaling are difficult to study solely by loss-of-function analysis. For a better understanding of the transient interaction of signaling molecules and their regulation by feedback loops, as well as their dynamic behavior in living cells, new techniques are required. We have developed a method allowing the reconstitution of the BCR complex and several of its key signaling elements in the evolutionary distant environment of the Drosophila S2 Schneider cell line. With this gain-of-function approach, we study here the assembly of the BCR complex and the control of its transport to the cell surface of S2 cells. We find that without binding to a light chain, the membrane-bound μm heavy chain (μmHC) homodimer, together with the Ig-α/Ig-β heterodimer, can come to the cell surface where it is signaling competent. This finding could have implications for potential signaling functions of such a receptor molecule during pro-/pre-B cell development. We also studied the activation of the BCR-proximal kinase Syk. We found that a truncated Syk mutant lacking the first (N-terminal) SH2 domain and the linker regions, is still regulated by autoinhibition and can only become activated in the presence of the BCR. This indicates that the C-terminal SH2 domain of Syk is the dominant regulatory subunit of this kinase

Amplification of B Cell Antigen Receptor Signaling by a Syk/ITAM Positive Feedback Loop

We have established a protocol allowing transient and inducible coexpression of many foreign genes in Drosophila S2 Schneider cells. With this powerful approach of reverse genetics, we studied the interaction of the protein tyrosine kinases Syk and Lyn with the B cell antigen receptor (BCR). We find that Lyn phosphorylates only the first tyrosine whereas Syk phosphorylates both tyrosines of the BCR immunoreceptor tyrosine-based activation motif (ITAM). Furthermore, we show that Syk is a positive llosteric enzyme, which is strongly activated by the binding to the phosphorylated ITAM tyrosines, thus initiating a positive feedback loop at the receptor. The BCR-dependent Syk activation and signal amplification is efficiently counterbalanced by protein tyrosine phosphatases, the activity of which is regulated by H₂O₂ and the redox equilibrium inside the cell

The Role of the BCR Class Expressed by Eμ-TCL1tg Mice and Human CLL

Abstract Abstract 182 The B-cell antigen receptor (BCR) plays a critical role in the development and progression of B-cell lymphomas. In chronic lymphocytic leukemia (CLL), the existence of stereotyped heavy-chain complementarity determining regions (HCDR3) suggested that binding of external antigens might play a role in CLL pathogenesis. In contrast, we recently reported that BCRs derived from both human CLL patients and from Eμ-TCL1tg mice have the unique function to induce antigen-independent signaling. This capacity is mediated by the HCDR3 through binding to a BCR internal motif in adjacent BCRs on the same cell (Dühren-von Minden et al., Nature 2012). Mature B cells as well as CLL B cells co-express IgM and IgD with the same HCDR3. In this study, we address the respective roles of these expressed BCR classes in lymphoma pathogenesis in Eμ-TCL1tg mice and in human CLL. By mating Eμ-TCL1tg-mice with IgM−/− mice, which lacks the μ constant heavy domain (μCH) and instead expresses IgD in all developmental stages, we demonstrate a significantly lower frequency of CD19+CD5+IgM+IgDlow lymphoma cells in heterozygous IgM+/−TCL1 mice compared to conventional IgM+/+TCL1 mice (p=0.007). Furthermore, IgM+/−TCL1 mice show a delayed or slowed disease progression compared to TCL1tg mice carrying both IgM alleles. In both TCL1tg mice strains, lymphoma development was exclusively linked to expression of surface IgM, since no IgD single positive lymphoma was detected in IgM+/−TCL1 mice (n=12). TCL1tg mice that lack both μCH alleles showed an accumulation of CD19+CD5+ cells in the spleen at the age of 6 months. According to the genotype of these mice, this population was indeed IgD single positive. However, no further progression could be observed during follow-up to an age of 8 months, indicating a benign form of lymphoproliferation. In contrast to BCRs derived from Eμ-TCL1 mice, analysis of the signaling properties of BCRs derived from IgM−/−TCL1 mice failed to show any autonomous signaling capacity, even when they were expressed as IgM. To address whether IgD in general is able to mediate autonomous signaling reported for TCL1tg- and CLL-derived BCRs, we tested these receptors for autonomous signaling capacity when expressed as IgD. However, expression as IgD led to a complete loss of autonomous signaling capacity in all cases (n=10). In conclusion, whereas autonomous signaling is a characteristic feature of TCL1tg- and CLL-derived BCRs, the pathogenesis of CLL is dependent on the expression of their BCR as the IgM isotype. Expression of IgD-BCRs leads to loss of autonomous signaling capacity, and mice that lack μCH fail to develop malignant lymphoproliferation. To address the question if differential expression of IgD and IgM also had an impact on the clinical behavior of human CLL, we measured the relative expression levels of surface IgD and IgM on circulating lymphoma cells from 67 CLL patients by flow cytometry with simultaneous staining. According to previous reports (Mockridge et al., Blood 2007), unmutated (UM-CLL) cases (n=22) had a higher level of total surface Ig compared to mutated CLL (M-CLL) cases (n=45). Based on our results that IgM is more potent to drive lymphoproliferation, we calculated the ratio of mean fluorescence intensities for IgD over IgM, further called DvM-Score, for every case. A significant difference in the expression pattern as represented by the DvM-Score was observed for UM-CLL and M-CLL (p=0.003) as well as for ZAP70+ and ZAP70- cases (p=0.0002). Both UM-CLL cases as well as ZAP70+ cases show a higher amount of IgM compared to IgD represented by a DvM-Score of &lt;1, whereas the majority of M-CLL and ZAP70− cases express less IgM than IgD and show a DvM-Score of &gt;1. Based on receiver operating characteristics, an DvM cut-off value of 1.15 was identified to optimally discriminate mutational status (AUC: 0.72) and ZAP70 expression (AUC: 0.78). Preliminary data show that CLL samples with a DvM-Score &lt;1.15 had a more aggressive disease course as indicated by a median time to first treatment (TTFT) of 39 months, whereas cases with a DvM-Score of &gt;1.15 show a median TTFT of 154 months (log rank test, p=0.0014). In summary, our results demonstrate an important role of the BCR class, especially with respect to the pathogenetic role of autonomously active IgM BCRs expressed by CLL B cells, for the outcome of the disease. In addition, the DvM score may represent a convenient and novel prognostic marker for CLL. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Conventional Light Chains Inhibit the Autonomous Signaling Capacity of the B Cell Receptor

SummarySignals from the B cell antigen receptor (BCR), consisting of μ heavy chain (μHC) and conventional light chain (LC), and its precursor the pre-BCR, consisting of μHC and surrogate light chain (SLC), via the adaptor protein SLP-65 regulate the development and function of B cells. Here, we compare the effect of SLC and conventional LC expression on receptor-induced Ca2+ flux in B cells expressing an inducible form of SLP-65. We found that SLC expression strongly enhanced an autonomous ability of μHC to induce Ca2+ flux irrespective of additional receptor crosslinking. In contrast, LC expression reduced this autonomous μHC ability and resulted in antigen-dependent Ca2+ flux. These data indicate that autonomous ligand-independent signaling can be induced by receptor forms other than the pre-BCR. In addition, our data suggest that conventional LCs play an important role in the inhibition of autonomous receptor signaling, thereby allowing further B cell differentiation