PU.1 and Spi-B Oppose Transformation Of Pre-B Cells Through Activation Of Key Genes Involved In B Cell Receptor Signalling

Abstract B cell development is controlled by stage-specific expression of transcription factors. Aberrant expression of such factors can lead to B cell acute lymphoblastic leukemia (B-ALL). Deletion of genes encoding the E26 transformation-specific (ETS) transcription factors, PU.1 and Spi-B, in B cells (CD19+/CreSfpi1lox/loxSpib-/- mice, abbreviated to CD19-CreΔPB) leads to B-ALL at 100% incidence and with a median survival of 21 weeks. However, little is known about the target genes of PU.1 and Spi-B that explain leukemic transformation in these mice. In the current study, we investigated the developmental origins and mechanisms of leukemogenesis in CD19-CreΔPB mice. We found that B-ALL cells in CD19-CreΔPB mice had frequently rearranged both their heavy and light chain genes, but retained cell surface expression of interleukin-7 receptor (IL-7R), suggesting aberrant pre-B cell differentiation. Preleukemic CD19-CreΔPB mice had increased frequencies of pre-B cells compared to wild type mice. Pre-B cells, but not mature B cells, purified from the bone marrow of preleukemic CD19-CreΔPB mice could rapidly transfer disease to transplanted recipient mice. B-ALL cells from established tumors had uniform expression of markers indicating a pre-B cell phenotype and contained a high-frequency of leukemia-initiating cells as measured by transplantation assays. Genome-wide analysis of gene expression showed that B cell receptor signalling was the top impaired pathway in B-ALL cells from CD19-CreΔPB mice. Bone marrow cells from CD19-CreΔPB mice had increased responsiveness to IL-7R signalling and could be cultured as IL-7-dependent cell lines. Preleukemic or leukemic cells from CD19-CreΔPB mice expressed reduced levels of the gene encoding Bruton’s tyrosine kinase (Btk), which we show is a target gene of PU.1 and/or Spi-B that in combination with reduced BLNK is sufficient to explain increased IL-7R responsiveness. We conclude that mutation of PU.1 and Spi-B predispose developing B cells to leukemogenesis by impairing expression of key genes, such as Btk, that are required for BCR signalling and are involved in attenuation of IL-7 receptor signaling. Disclosures: No relevant conflicts of interest to declare.

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Pre-B Cell Receptor Signaling Prevents Leukemic Transformation by BCR-ABL1.

Blood ◽

10.1182/blood.v110.11.2795.2795 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2795-2795

Author(s):

Daniel Trageser ◽

Lars Klemm ◽

Sebastian Herzog ◽

Yong-mi Kim ◽

Cihangir Duy ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Transgenic Mice ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Receptor Signaling ◽

Leukemic Transformation ◽

B Cell Receptor Signaling ◽

Cell Receptor Signaling

Abstract Pre-B cells within the bone marrow are destined to die unless they are rescued through survival signals from the pre-B cell receptor. Studying the configuration of the immunoglobulin heavy chain locus (IGHV) in sorted human bone marrow pre-B cells by single-cell PCR, we detected a functional IGHV allele consistent with the expression of a functional pre-B cell receptor in the vast majority of normal human pre-B cells. However, only in 10 of 44 cases of BCR-ABL1-transformed pre-B cell-derived acute lymphoblastic leukemia (ALL), we detected a functional IGHV allele. For this reason, we studied the function of the pre-B cell receptor during early B cell development and progressive transformation in a BCR-ABL1-transgenic mouse model: Interestingly, BCR-ABL1-transgenic mice that have not yet undergone leukemic transformation show almost normal pre-B cell receptor selection. In these “pre-leukemic” pre-B cells, however, expression of the BCR-ABL1-transgene is extremely low as compared to full-blown ALL, suggesting that high levels of BCR-ABL1 expression are not compatible with normal expression of the pre-B cell receptor. Consistent with our observations in human ALL, full-blown ALL clones in BCR-ABL1-transgenic mice indeed show defective pre-B cell receptor selection and the pre-B cell receptors expressed on few leukemic cells are not functional. Treatment of leukemic mice with the BCR-ABL1 kinase inhibitor AMN107, however, reinstated normal pre-B cell receptor selection and pre-B cell receptor function within seven days. These data suggest that the transforming signal through BCR-ABL1 and normal survival signals through the pre-B cell receptor are mutually exclusive. In support of this hypothesis, we found that the full-blown leukemia only comprises one to four independent clones of “crippled” pre-B cells - even though all B cell precursors in these mice carry the BCR-ABL1-transgene. To test whether functional pre-B cell receptor signaling vetoes transformation by BCR-ABL1, we transformed murine pre-B cells carrying a deletion of the SLP65 gene, which is required for functional pre-B cell receptor signaling. Unlike SLP65-wildtype pre-B cells, SLP65−/− pre-B cells can be transformed by BCR-ABL1 at a high efficiency. Reconstitution of SLP65 using a retroviral vector, however, induced rapid cell death of BCR-ABL1-transformed pre-B cells. Next, we identified human BCR-ABL1-negative ALL cases with a functional or defective pre-B cell receptor signaling cascade. Transduction of pre-B cell receptor-deficient ALL cells resulted in rapid outgrowth while ALL cells with a functional pre-B cell receptor were not permissive to transduction with BCR-ABL1. We conclude that the pre-B cell receptor represents a potent tumor suppressor and a safeguard against BCR-ABL1-mediated transformation. Only “crippled” pre-B cells with a non-functional pre-B cell receptor are susceptible to BCR-ABL1-mediated transformation.

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The Human Pre-B Cell Receptor Signaling Cascade Is Regulated Via PI-3Kinase and MAPK Pathway

Blood ◽

10.1182/blood.v118.21.1314.1314 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1314-1314

Author(s):

Kolandaswamy Anbazhagan ◽

Vincent Fuentes ◽

Eliane Bissac ◽

Remy Nyga ◽

Naomi Taylor ◽

...

Keyword(s):

Transcription Factors ◽

B Cells ◽

B Cell ◽

Nuclear Translocation ◽

Accessory Pathway ◽

Cell Receptor ◽

B Cell Receptor ◽

Signaling Cascade ◽

Down Regulation ◽

Bcr Signaling

Abstract Abstract 1314 Background: Pre-B cell receptor (pre-BCR) constitutes a major check point in the early steps of mouse and human B cell development. Several functions have been attributed to this receptor which include a delivery of proliferation and survival signals, increased sensitivity to interleukin-7 (IL-7) and down modulation of recombinase activating genes (RAG) and surrogate light chain (SLC) encoding genes. Pre-BCR is also involved in shaping the VH repertoire and preventing autoimmunity. Finally, there is increasing evidence that pre-BCR might be implicated in leukemogenesis. Most of the functions of pre-BCR have been predicted based on studies in knockout mice and leukemic cell lines. In a previous study we have shown that pre-BCR aggregation resulted in the activation of src and Syk kinases which in turn activated the PI-3K/Akt, Btk, PLCγ-2 and Ras/MAPK. In this study, we examined the pre-BCR signalling cascade using human normal primary pre-B cells with a particular focus on transcription factors activation and Rag modulation and their regulatory aspects. Methods: Pre-B cells were sorted from adult human bone marrow samples, treated or not with inhibitors of Syk (BAY61–3606), Akt (LY294002) and MEKK1 (UO126) prior to crosslink the pre-BCR by means of F(ab')2 anti-μHC. The effect of Pre-BCR signaling was examined by quantifying the transcript levels of Rag1, Rag2, E2A, EBF1, Pax5, FoxO1 and FoxO3, IRF4/8. Activation of transcription factors such as NF-κB p50, c-Fos, IRF4 and FoxO3A, was assessed by analyzing their nuclear translocation by immunofluorescence microscopy. Results: We show that NF-κB p50 is translocated into nucleus within 3h after pre-BCR stimulation. Crosslinking of pre-BCR also resulted in an enhancement of nuclear c-Fos translocation. BAY61-3606 (Syk inhibitor) treatment resulted in complete apoptosis (100 % cell death within 48h). Although treatment of normal pre-B cells with LY294002 or U0126 did not alter cell survival, nuclear translocation of pre-BCR-induced p50 NF-κB was prevented by former and enhanced by later. Conversely, c-Fos nuclear expression was inhibited by U0126 and slightly but consistently enhanced by LY294002 in association with a decrease in its cytoplasmic location. Pre-BCR stimulation also induced IRF4 translocation to the nucleus. Pre-BCR stimulation also resulted in the down regulation of Rag1 (− 48 %, P<0.01), Pax5 (− 40%, P<0.01) and E2A (− 35 %, P< 0.01) transcripts, whereas EBF1 and FoxO1 and 3 expression remained unchanged. In LY294002-treated cells, Rag1/Rag2 expression was up regulated (+130%, P< 0.01 and +251%, P< 0.01, respectively) following pre-BCR crosslinking, whereas in the presence of U0126 the pre-BCR induced Rag1/Rag2 down modulation remained unchanged. Conclusion: Our results indicate that the pre-BCR has the potential to promote pre-B cell proliferation, survival and differentiation by activating NF-kB, c-Fos and IRF4. It also has the ability to protect pre-B cells from genome instability by down-regulating Rag1/2, probably through down modulation of Pax5 and E2A. We bring evidence that PI-3 K/Akt pathway plays a crucial role in the regulation of the pre-BCR signaling cascade and that Akt-mediated NF-kB and c-Fos activation is antagonized by MAPK. Up-regulation of Rag transcripts upon Akt inhibition suggests either a feed-back negative loop or a dual effect of pre-BCR on Rag expression with an Akt-dependent Rag down regulation and an accessory pathway that enhances Rag expression. Disclosures: No relevant conflicts of interest to declare.

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Epstein–Barr virus latent membrane protein 2A mimics B-cell receptor-dependent virus reactivation

Journal of General Virology ◽

10.1099/vir.0.80440-0 ◽

2005 ◽

Vol 86 (3) ◽

pp. 551-559 ◽

Cited By ~ 20

Author(s):

Eveline Schaadt ◽

Barbara Baier ◽

Josef Mautner ◽

Georg W. Bornkamm ◽

Barbara Adler

Keyword(s):

B Cells ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Lytic Cycle ◽

Virus Reactivation ◽

Latent Membrane Protein 2A ◽

Barr Virus ◽

Receptor Signalling ◽

Epstein Barr

Latent membrane protein 2A (LMP2A) of Epstein–Barr virus (EBV) shares protein motifs with the B-cell receptor that play a role in B-cell receptor signalling and has been shown to mimic an activated B-cell receptor by providing a survival signal for mature B cells in transgenic mice. Conversely, LMP2A has been reported not to support but to inhibit B-cell receptor signalling with respect to virus reactivation and to block lytic virus induction after anti-Ig treatment of EBV-infected B cells. To solve this apparent paradox, the role of LMP2A in lytic-cycle induction was re-examined in B cells conditionally immortalized by EBV. It was shown that, in the absence of other stimuli, LMP2A expression alone could lead to induction of the virus lytic cycle. Similarly to B-cell receptor stimulation by anti-Ig treatment, this LMP2A-mediated reactivation was dependent on the mitogen-activated protein kinase pathway and could be inhibited by the viral LMP1. Our data reinforce the notion that LMP2A is a functional homologue of the B-cell receptor, not only with respect to B-cell survival but also with respect to regulation of the lytic cycle.

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Pre-B Cell Receptor Signaling Prevents Leukemic Transformation by BCR-ABL1

Blood ◽

10.1182/blood.v112.11.15.15 ◽

2008 ◽

Vol 112 (11) ◽

pp. 15-15

Author(s):

Daniel Trageser ◽

Cihangir Duy ◽

Lars Klemm ◽

Tanja Gruber ◽

Rahul Nahar ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Receptor Signaling ◽

Leukemic Transformation ◽

B Cell Receptor Signaling ◽

Cell Receptor Signaling

Abstract Pre-B cells within the bone marrow are destined to die unless they are rescued through survival signals from the pre-B cell receptor. Studying the configuration of the immunoglobulin heavy chain locus (IGHM) in sorted human bone marrow pre-B cells by single-cell PCR, we detected a functional IGHM allele consistent with the expression of a functional pre-B cell receptor in the vast majority of normal human pre-B cells. However, only in 10 of 57 cases of BCR-ABL1-transformed pre-B cell-derived acute lymphoblastic leukemia (ALL), we detected a functional IGHM allele. While normal pre-B cells respond vigorously to pre-B cell receptor engagement by Ca2+ release, the pre-B cell receptor was unresponsive even in the few cases of BCR-ABL1-driven ALL, in which we amplified a productively rearranged IGHM allele. For this reason, we studied the function of the pre-B cell receptor during early B cell development and progressive transformation in a BCR-ABL1-transgenic mouse model: Interestingly, BCR-ABL1-transgenic mice that have not yet undergone leukemic transformation show almost normal pre-B cell receptor selection. In these pre-leukemic pre-B cells, however, expression of the BCR-ABL1-transgene is very low as compared to full-blown ALL, suggesting that high levels of BCR-ABL1 expression are not compatible with normal expression of the pre-B cell receptor. Consistent with our observations in human ALL, full-blown ALL clones in BCR-ABL1-transgenic mice show defective pre-B cell receptor selection and the pre-B cell receptors expressed on few leukemic cells are not functional. Treatment of leukemic mice with the BCR-ABL1 kinase inhibitor AMN107, however, reinstated normal pre-B cell receptor selection and pre-B cell receptor function within seven days. These data suggest that the transforming signal through BCR-ABL1 and normal survival signals through the pre-B cell receptor are mutually exclusive. To test whether functional pre-B cell receptor signaling prevents transformation by BCR-ABL1, we transformed murine pre-B cells carrying a deletion of the SLP65 gene, which is required for functional pre-B cell receptor signaling. Unlike SLP65-wildtype pre-B cells, SLP65−/− pre-B cells can be transformed by BCR-ABL1 at a high efficiency. Reconstitution of SLP65 using a retroviral vector, however, induced rapid cell death of BCR-ABL1-transformed pre-B cells. We next investigated the potential impact of Slp65-reconstitution on leukemic growth of BCR-ABL1-transformed pre-B cells from SLP65−/− mice in vivo. To this end, SLP65−/− BCR-ABL1-transformed pre-B cells were labeled with firefly-luciferase and then transduced with retroviral vectors encoding SLP65/GFP or GFP alone. NOD/SCID mice were sublethally irradiated and injected with either SLP65/GFP+ or GFP+ ALL cells. Engraftment as monitored by bioluminescence imaging was delayed by more than three weeks in mice injected with SLP65/GFP+ ALL cells as compared to mice injected with GFP+ ALL cells. 36 days after injection, the first mice that were inoculated with GFP-transduced leukemia cells, became terminally ill and also the other mice in this group showed weight loss at that time. In contrast, the mice injected with SLP65-GFP-transduced ALL cells showed no signs of disease and no significant weight loss. At this time, all mice were sacrificed: Whereas mice injected with GFP-transduced ALL cells showed splenomegalia and leukemic infiltration into multiple organs, there was only mild splenic enlargement, when SLP65-reconstituted ALL cells were injected. Reconstitution of SLP65 also reduced the frequency of BCR-ABL1-transformed leukemia cells about 15-fold in the bone marrow, 5-fold in the spleen and >100-fold in the peripheral blood. We conclude that deficiency of the pre-B cell receptor-related signaling molecule SLP65 not only represents a frequent feature in human ALL cells but also represents a critical requirement for BCR-ABL1-driven leukemic growth in vivo. We conclude that pre-B cell receptor signaling renders B cell progenitor cells non-permissive to BCR-ABL1-mediated transformation. Only crippled pre-B cells with a non-functional pre-B cell receptor are susceptible to BCR-ABL1-mediated transformation.

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Tim-1 is induced on germinal centre B cells through B-cell receptor signalling but is not essential for the germinal centre response

Immunology ◽

10.1111/j.1365-2567.2010.03276.x ◽

2010 ◽

pp. no-no ◽

Cited By ~ 3

Author(s):

See Heng Wong ◽

Jillian L. Barlow ◽

Stephen Nabarro ◽

Padraic G. Fallon ◽

Andrew N. J. McKenzie

Keyword(s):

B Cells ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Germinal Centre ◽

Receptor Signalling

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Bone Marrow Dendritic Cell-Mediated Regulation of TLR and B Cell Receptor Signaling in B Cells

The Journal of Immunology ◽

10.4049/jimmunol.1101352 ◽

2012 ◽

Vol 189 (7) ◽

pp. 3355-3367 ◽

Cited By ~ 11

Author(s):

Vishal J. Sindhava ◽

Halide Tuna ◽

Beth W. Gachuki ◽

David J. DiLillo ◽

Margarita G. Avdiushko ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Dendritic Cell ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Receptor Signaling ◽

B Cell Receptor Signaling ◽

Cell Receptor Signaling

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EBV LMP2A provides a surrogate pre-B cell receptor signal through constitutive activation of the ERK/MAPK pathway

Journal of General Virology ◽

10.1099/vir.0.2008/001461-0 ◽

2008 ◽

Vol 89 (7) ◽

pp. 1563-1568 ◽

Cited By ~ 28

Author(s):

Leah J. Anderson ◽

Richard Longnecker

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Current Knowledge ◽

Mapk Pathway ◽

Cell Receptor ◽

B Cell Receptor ◽

Barr Virus ◽

Erk Mapk ◽

Interleukin 7

Latent membrane protein 2A (LMP2A) of Epstein–Barr virus (EBV) provides developmental and survival signals that mimic those of a B-cell receptor (BCR). Expression of LMP2A during B-cell development results in the ability of B cells to exit the bone marrow in the absence of a BCR and persist in the periphery, where they would normally undergo apoptosis. This study extends the current knowledge of LMP2A function by examining the growth properties of bone marrow B cells from TgE LMP2A mice. Despite the lack of pre-BCR expression, bone marrow B cells from TgE LMP2A mice proliferate and survive in low concentrations of interleukin 7, similar to wild-type cells. Constitutive phosphorylation of ERK/MAPK and PI3K/Akt in TgE LMP2A bone marrow B cells is also reminiscent of signalling through the pre-BCR, altogether demonstrating that LMP2A provides a pre-BCR-like signal to developing B cells.

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