The Experts below are selected from a list of 1061943 Experts worldwide ranked by ideXlab platform
Markus Müschen - One of the best experts on this subject based on the ideXlab platform.
-
Targeting the B-Cell Receptor signaling pathway in B lymphoid malignancies.
Current Opinion in Hematology, 2014Co-Authors: Maike Buchner, Markus MüschenAbstract:PURPOSE OF REVIEW Normal B cells that failed to productively rearrange immunoglobulin V region genes, encoding a functional B cell Receptor (BCR) are destined to die. Likewise, the majority of B cell malignancies remain dependent on functional BCR signaling, while in some subtypes BCR expression is missing and, apparently, counterselected. Here we summarize recent the experimental evidence for the importance of BCR signaling and clinical concepts to target the BCR pathway in B cell leukemia and lymphoma.
-
Pre-B Cell Receptor-Mediated Activation of BCL6 Induces Pre-B Cell Quiescence Through Transcriptional Repression of MYC
Blood, 2011Co-Authors: Rahul Nahar, Maximilian Mossner, Cihangir Duy, Hassan Jumaa, Parham Ramezani-rad, Ari Melnick, Leandro Cerchietti, Huimin Geng, Markus MüschenAbstract:Abstract Abstract 1406 Background: Pre-B cell Receptor signaling is critical to induce initial expansion of the pool of differentiating B cell precursors but also mediates subsequent cell cycle exit and quiescence. Initial cell surface expression of the pre-B cell Receptor induces a series of tyrosine phosphorylation events resulting in MYC and CCND2-mediated proliferation. After 2–5 divisions, however, large pre-BII (Fraction C') cells exit cell cycle to become resting, small pre-BII cells (Fraction D). While the signaling pathway resulting in pre-B cell Receptor-induced proliferation is well understood, the mechanism by which pre-BII cells exit cell cycle, however, is currently unclear. Results: This checkpoint and cell cycle exit at the Fraction C'-D transition is critical for immunoglobulin light chain gene recombination and to prevent malignant transformation into acute lymphoblastic leukemia. Here we demonstrate that inducible activation of pre-B cell Receptor signaling recapitulates the initial proliferative burst followed by cell cycle exit, which is characterized by strong upregulation of BCL6 and subsequent loss of MYC and CCND2 expression. ChIP-on-chip analysis revealed that the transcriptional repressor BCL6 is directly recruited to the MYC promoter. Inducible activation of pre-B cell Receptor signaling in pre-B cells from BCL6-deficient mice failed to induce cell cycle exit and quiescence. We conclude that activation of BCL6 downstream of the pre-B cell Receptor is crucial for the induction of quiescence at the Fraction C'-D checkpoint. As expected, inducible activation of BCL6 downstream of the pre-B cell Receptor results in transcriptional repression of MYC and CCND2. Overexpression of MYC prevented pre-B cell Receptor/BCL6-induced cell cycle exit. Conclusion: Hence, pre-B cell Receptor signaling induces cellular quiescence of Fraction C' pre-B cells through activation of BCL6 and BCL6-mediated transcriptional repression of MYC and CCND2. Disclosures: No relevant conflicts of interest to declare.
-
Pre-B Cell Receptor Signaling Distinguishes E2A-PBX1 From Other Subtypes of Acute Lymphoblastic Leukemia
Blood, 2010Co-Authors: Rahul Nahar, Daniel Trageser, Lars Klemm, Cihangir Duy, Yong-mi Kim, Nora Heisterkamp, Wolf-karsten Hofmann, Eugene Park, Hassan Jumaa, Markus MüschenAbstract:Abstract 274 Background: The E2A-PBX1 [t(1;19)(q23;p13)] fusion is found in ≂f4% of cases of childhood ALL and involves a rearrangement of the TCF3 gene (encoding the E2A transcription factor). TCF3 (E2A) is not only a critical regulator of B cell lineage commitment and early B cell development (Muschen et al., 2002; Sigvardsson et al., 2002), it also cooperates with LEF1 to activate canonical WNT/β-catenin signaling (Hovanes et al., 2001; Merrill et al., 2001). Pre-B cells in human bone marrow are destined to die unless they are rescued through survival signals from a successfully assembled pre-B cell Receptor. Congenital defects in pre-B cell Receptor-related signaling molecules cause a severe differentiation block at an early pre-B cell stage. Likewise, B cell lineage acute lymphoblastic leukemia (ALL) cells are arrested at an early pre-B cell stage in the vast majority of cases. Given that the pre-B cell Receptor drives both proliferation and differentiation of normal B cell precursors, we test here the hypothesis that pre-B cell Receptor signaling represents a critical target for malignant transformation towards ALL. Results: Studying 148 cases of pre-B cell-derived human ALL, we found that pre-B cell Receptor expression and function is linked to specific cytogenetic subgroups: ALL cells carrying an E2A-PBX1- gene rearrangement are, like normal pre-B cells, highly selected for the expression of a functional pre-B cell Receptor. In all 8 ALL cases with E2A-PBX1 fusion, engagement of the pre-B cell Receptor resulted in a strong Ca 2+ signal, which strongly suggests that E2A-PBX1 leukemia clones are selected for active pre-B cell Receptor signaling. In striking contrast, ALL cells carrying BCR-ABL1- or MLL-AF4 fusion genes and ALL cells with hyperdiploid karyotype lack expression of a functional pre-B cell Receptor in virtually all cases. Only 10 of 57 cases with BCR-ABL1 , 0 of 7 cases with MLL-AF4 and 1 of 30 cases with hyperdiploid karyotype a productively rearranged μ -heavy chain locus encoding the central component of the pre-B cell Receptor, was found. Even in the few BCR-ABL1 ALL cases, in which a productively rearranged μ -chain was amplified, no pre-B cell Receptor was expressed. Based on these findings, we hypothesize that ALL can be subdivided into two groups based on whether pre-B cell Receptor signaling enables ( E2A-PBX1 ) or suppresses ( BCR-ABL1 , MLL-AF4 , Hyperdiploid, likely other subtypes) leukemic growth. In a proof-of-concept experiment, we super-transformed E2A-PBX1 -induced ALL cells (active pre-B cell Receptor signaling) and MLL-AF4 -ALL cells lacking pre-B cell Receptor function with the BCR-ABL1 oncogene. The BCR-ABL1 oncogene was chosen, because it is only found in leukemia cells that lack pre-B cell Receptor function. Whereas growth of pre-B cell Receptor-negative MLL-AF4 ALL cells was strongly accelerated by BCR-ABL1-transformation, pre-B cell Receptor-positive E2A-PBX1 ALL cells were rapidly eliminated within 9 days after BCR-ABL1 -transduction. Interestingly, incubation of E2A-PBX1 ALL cells survived BCR-ABL1-transduction only in the presence of 10 μ mol/l of the BCR-ABL1 kinase inhibitor Imatinib. To identify factors that distinguish E2A-PBX1 and other ALL subtypes ( BCR-ABL1 , MLL-AF4 , Hyperdiploid) that may explain the divergent role of pre-B cell Receptor signaling in these groups, we performed a comparative gene expression including a meta-analysis of published microarray data and quantitative RT-PCR. In this analysis, E2A-PBX1 ALL cells were distinguished by high expression levels of pre-B cell Receptor-related signaling molecules (e.g. BLNK, SYK, BTK). The most prominent gene expression differences involve canonical WNT/β-catenin signaling. As opposed to other ALL subtypes, E2A-PBX1 ALL cells express the β-catenin cofactors TCF3 and LEF1 at >5-fold higher levels and WNT16 at >12-fold higher levels compared to BCR-ABL1 , MLL-AF4 , Hyperdiploid and TEL-AML1 -driven ALL subtypes. Conclusions: Constitutive activation of the canonical WNT/β-catenin signaling pathway in E2A-PBX1 ALL cells may explain the distinct role of pre-B cell Receptor signaling in this ALL subset: Since pre-B cell Receptor signaling via BTK negatively regulates WNT/β-catenin-dependent survival and self-renewal signaling (James et al., 2009), the level of constitutive WNT/β-catenin-signaling may determine permissiveness of ALL cells to pre-B cell Receptor function. 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
Blood, 2010Co-Authors: Rahul Nahar, Cihangir Duy, Parham Ramezani-rad, Sinisa Dovat, Ari Melnick, Markus MüschenAbstract:Abstract 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 C9) 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.
-
Pre-B cell Receptor signaling in acute lymphoblastic leukemia
Cell Cycle, 2009Co-Authors: Rahul Nahar, Markus MüschenAbstract:B cell lineage ALL represents by far the most frequent malignancy in children and is also common in adults. Despite significant advances over the past four decades, cytotoxic treatment strategies have recently reached a plateau with cure rates at 80 percent for children and 55 percent for adults. Relapse after cytotoxic drug treatment, initial drug-resistance and dose-limiting toxicity are among the most frequent complications of current therapy approaches. For this reason, pathway-specific treatment strategies in addition to cytotoxic drug treatment seem promising to further improve therapy options for ALL patients. In a recent study on 111 cases of pre-B cell-derived human ALL, we found that ALL cells carrying a BCR-ABL1-gene rearrangement lack expression of a functional pre-B cell Receptor in virtually all cases. In a proof-of-principle experiment, we studied pre-B cell Receptor function during progressive leukemic transformation of pre-B cells in BCR-ABL1-transgenic mice: Interestingly, signaling from...
James R. Drake - One of the best experts on this subject based on the ideXlab platform.
-
the syk binding ubiquitin ligase c cbl mediates signaling dependent b cell Receptor ubiquitination and b cell Receptor mediated antigen processing and presentation
Journal of Biological Chemistry, 2012Co-Authors: Bhuvana Katkere, Sarah J Rosa, James R. DrakeAbstract:B cell Receptor (BCR)-mediated antigen (Ag) processing and presentation lead to B cell-T cell interactions, which support affinity maturation and immunoglobulin class switching. These interactions are supported by generation of peptide-MHC class II complexes in multivesicular body-like MIIC compartments of B cells. Previous studies have shown that trafficking of Ag·BCR complexes to MVB-like MIIC occurs via an ubiquitin-dependent pathway and that ubiquitination of Ag·BCR complexes occurs by an Src family kinase signaling-dependent mechanism that is restricted to lipid raft-resident Ag·BCR complexes. This study establishes that downstream Syk-dependent BCR signaling is also required for BCR ubiquitination and BCR-mediated antigen processing and presentation. Knockdown studies reveal that of the two known Syk-binding E3 ubiquitin ligases c-Cbl and Cbl-b, only c-Cbl appears to have a central role in BCR ubiquitination, trafficking to MIIC, and ubiquitin-dependent BCR-mediated antigen processing and presentation. These results establish the novel role for Syk signaling and the Syk-binding ubiquitin ligase c-Cbl in the BCR-mediated processing and presentation of cognate antigen and define one mechanism by which antigen-induced BCR ubiquitination is modulated to impact the initiation and maturation of the humoral immune response.
-
Low-level signaling generated by FcγRIIB-B cell Receptor co-ligation establishes a state of global B cell Receptor nonresponsiveness
Immunological investigations, 2005Co-Authors: Mark T. Preissler, Laura Kaiser, James R. Drake, Edmund J. GosselinAbstract:In addition to the stimulatory, antigen-specific B cell Receptor (BCR), B lymphocytes also express multiple inhibitory Receptors, including Fc gamma Receptor type IIB (FcγRIIB). Moreover, many laboratories have demonstrated that co-ligation of BCR molecules to inhibitory FcγRIIB molecules with high concentrations (10–15 µg/ml) of ligand results in altered BCR signaling. However, there are no reports on the effect of low concentrations of ligand on BCR-FcγRIIB co-ligation and subsequent signaling. This knowledge will be critical for optimizing the in vivo use of such reagents. Accordingly, the effect of low ligand concentration on the level of BCR-FcγRIIB co-ligation and subsequent BCR signaling was analyzed. The results demonstrate that co-ligation of BCR and FcγRIIB molecules at low concentrations (0.5–1.5 µg/ml) of cross-linking reagent, establishes a condition that prevents the B cell from responding to subsequent stimulation, even when the initial exposure to cross-linking reagent fails to generate a ...
Adrian Wiestner - One of the best experts on this subject based on the ideXlab platform.
-
Targeting B cell Receptor signalling in cancer: preclinical and clinical advances
Nature Reviews Cancer, 2018Co-Authors: Jan A Burger, Adrian WiestnerAbstract:B cell Receptor (BCR) signalling is indispensable for normal B cell development and adaptive immunity. In some B cell leukaemias and lymphomas, malignant B cells utilize BCR signalling for growth and survival. The mechanism of activation of BCR signalling includes continuous BCR stimulation by microbial antigens and/or autoantigens that are present in the tissue microenvironment, oncogenic mutations within the BCR complex or downstream signalling components and ligand-independent tonic BCR signalling. Bruton tyrosine kinase (BTK) inhibitors and/or PI3Kδ selective inhibitors are effective against chronic lymphocytic leukaemia (CLL), mantle cell lymphoma, follicular lymphoma, Waldenstrom macroglobulinaemia (WM) and other selective B cell malignancies. In CLL and WM, BTK inhibitors are increasingly replacing chemotherapy. BTK and PI3Kδ inhibitors cause redistribution of malignant B cells from tissue sites into the peripheral blood, especially in patients with CLL. How much this redistribution, resulting in a form of programmed cell death (anoikis) as a consequence of detachment of the malignant cells from their supportive tissue microenvironment, contributes to the efficacy of these agents remains unclear. The involvement of BTK, PI3Kδ and other BCR-related kinases such as spleen tyrosine kinase (SYK) in the signalling and function of homing Receptors (chemokine Receptors and integrins) appears to be the molecular basis for this B cell redistribution. BTK and PI3Kδ are also expressed in non-malignant cells in the microenvironment, such as T cells and monocytes and macrophages. The effects of BTK and PI3Kδ inhibitors extend to these cell lineages, which may contribute to antitumour effects but can give rise to side effects. In addition, ibrutinib, the most widely used BTK inhibitor, also targets inducible T cell kinase (ITK), a related kinase that promotes T helper 2 (T_H 2) cell differentiation. B cells and macrophages are part of a tumour-supportive microenvironment in solid tumours, including pancreatic cancer. Targeting B cell and/or macrophage function yields antitumour effects in preclinical models, and this strategy is being investigated in ongoing clinical trials. In this Review, Burger and Wiestner describe the latest insights into B cell Receptor (BCR) signalling with respect to its contribution to B cell malignancies and discuss how inhibitors that target kinases downstream of the BCR are changing treatment outcomes for patients with B cell neoplasms. B cell Receptor (BCR) signalling is crucial for normal B cell development and adaptive immunity. BCR signalling also supports the survival and growth of malignant B cells in patients with B cell leukaemias or lymphomas. The mechanism of BCR pathway activation in these diseases includes continuous BCR stimulation by microbial antigens or autoantigens present in the tissue microenvironment, activating mutations within the BCR complex or downstream signalling components and ligand-independent tonic BCR signalling. The most established agents targeting BCR signalling are Bruton tyrosine kinase (BTK) inhibitors and PI3K isoform-specific inhibitors, and their introduction into the clinic is rapidly changing how B cell malignancies are treated. B cells and BCR-related kinases, such as BTK, also play a role in the microenvironment of solid tumours, such as squamous cell carcinoma and pancreatic cancer, and therefore targeting B cells or BCR-related kinases may have anticancer activity beyond B cell malignancies.
-
the role of b cell Receptor inhibitors in the treatment of patients with chronic lymphocytic leukemia
Haematologica, 2015Co-Authors: Adrian WiestnerAbstract:Chronic lymphocytic leukemia is a malignancy of mature auto-reactive B cells. Genetic and functional studies implicate B-Cell Receptor signaling as a pivotal pathway in its pathogenesis. Full B-Cell Receptor activation requires tumor-microenvironment interactions in lymphoid tissues. Spleen tyrosine kinase, Bruton’s tyrosine kinase, and the phosphatidylinositol 3-kinase (PI3K) δ isoform are essential for B-Cell Receptor signal transduction but also mediate the effect of other pathways engaged in chronic lymphocytic leukemia cells in the tissue-microenvironment. Orally bioavailable inhibitors of spleen tyrosine kinase, Bruton’s tyrosine kinase, or PI3Kδ, induce high rates of durable responses. Ibrutinib, a covalent inhibitor of Bruton’s tyrosine kinase, and idelalisib, a selective inhibitor of PI3Kδ, have obtained regulatory approval in chronic lymphocytic leukemia. Ibrutinib and idelalisib are active in patients with high-risk features, achieving superior disease control in difficult-to-treat patients than prior best therapy, making them the preferred agents for chronic lymphocytic leukemia with TP53 aberrations and for patients resistant to chemoimmunotherapy. In randomized trials, both ibrutinib, versus ofatumumab, and idelalisib in combination with rituximab, versus placebo with rituximab improved survival in relapsed/refractory chronic lymphocytic leukemia. Responses to B-Cell Receptor inhibitors are mostly partial, and within clinical trials treatment is continued until progression or occurrence of intolerable side effects. Ibrutinib and idelalisib are, overall, well tolerated; notable adverse events include increased bruising and incidence of atrial fibrillation on ibrutinib and colitis, pneumonitis and transaminase elevations on idelalisib. Randomized trials investigate the role of B-Cell Receptor inhibitors in first-line therapy and the benefit of combinations. This review discusses the biological basis for targeted therapy of chronic lymphocytic leukemia with B-Cell Receptor inhibitors, and summarizes the clinical experience with these agents.
Simone Rizzetto - One of the best experts on this subject based on the ideXlab platform.
-
b cell Receptor reconstruction from single cell rna seq with vdjpuzzle
Bioinformatics, 2018Co-Authors: Simone Rizzetto, David Koppstein, Jerome Samir, Joanne H Reed, Andrew R Lloyd, Mandeep Singh, Auda A EltahlaAbstract:Motivation: The B-Cell Receptor (BCR) performs essential functions for the adaptive immune system including recognition of pathogen-derived antigens. The vast repertoire and adaptive variation of BCR sequences due to V(D)J recombination and somatic hypermutation necessitates single-cell characterization of BCR sequences. Single-cell RNA sequencing presents the opportunity for simultaneous capture of paired BCR heavy and light chains and the transcriptomic signature. Results: We developed VDJPuzzle, a novel bioinformatic tool that reconstructs productive, full-length B-Cell Receptor sequences of both heavy and light chains and extract somatic mutations on the VDJ region. VDJPuzzle successfully reconstructed BCRs from 100% (n=117) human and 96.5% (n=200) murine B cells. The reconstructed BCRs were successfully validated with single-cell Sanger sequencing. Availability and implementation: VDJPuzzle is available at https://bitbucket.org/kirbyvisp/vdjpuzzle2. Supplementary information: Supplementary data are available at Bioinformatics online.
-
b cell Receptor reconstruction from single cell rna seq with vdjpuzzle
bioRxiv, 2017Co-Authors: Simone Rizzetto, David Koppstein, Jerome Samir, Joanne H Reed, Andrew R Lloyd, Auda A Eltahla, Mandeep Singh, Christopher C Goodnow, Fabio LucianiAbstract:The B-Cell Receptor (BCR) performs essential functions for the adaptive immune system including recognition of pathogen-derived antigens. Cell-to-cell variability of BCR sequences due to V(D)J recombination and somatic hypermutation (SHM) necessitates single-cell characterization of BCR sequences. Single-cell RNA sequencing (scRNA-seq) presents the opportunity for simultaneous capture of the BCR sequence and transcriptomic signature for a detailed understanding of the dynamics of an immune response. We developed VDJPuzzle 2.0, a bioinformatic tool that reconstructs productive, full-length B-Cell Receptor sequences of both heavy and light chains. VDJPuzzle successfully reconstructs BCRs from 98.3% (n=117) of human and 96.5% (n=200) from murine B cells. 92.0% of clonotypes and 90.3% of mutations were concordant with single-cell Sanger sequencing of the immunoglobulin chains. VDJPuzzle is available at https://bitbucket.org/kirbyvisp/vdjpuzzle2
Rahul Nahar - One of the best experts on this subject based on the ideXlab platform.
-
Pre-B Cell Receptor-Mediated Activation of BCL6 Induces Pre-B Cell Quiescence Through Transcriptional Repression of MYC
Blood, 2011Co-Authors: Rahul Nahar, Maximilian Mossner, Cihangir Duy, Hassan Jumaa, Parham Ramezani-rad, Ari Melnick, Leandro Cerchietti, Huimin Geng, Markus MüschenAbstract:Abstract Abstract 1406 Background: Pre-B cell Receptor signaling is critical to induce initial expansion of the pool of differentiating B cell precursors but also mediates subsequent cell cycle exit and quiescence. Initial cell surface expression of the pre-B cell Receptor induces a series of tyrosine phosphorylation events resulting in MYC and CCND2-mediated proliferation. After 2–5 divisions, however, large pre-BII (Fraction C') cells exit cell cycle to become resting, small pre-BII cells (Fraction D). While the signaling pathway resulting in pre-B cell Receptor-induced proliferation is well understood, the mechanism by which pre-BII cells exit cell cycle, however, is currently unclear. Results: This checkpoint and cell cycle exit at the Fraction C'-D transition is critical for immunoglobulin light chain gene recombination and to prevent malignant transformation into acute lymphoblastic leukemia. Here we demonstrate that inducible activation of pre-B cell Receptor signaling recapitulates the initial proliferative burst followed by cell cycle exit, which is characterized by strong upregulation of BCL6 and subsequent loss of MYC and CCND2 expression. ChIP-on-chip analysis revealed that the transcriptional repressor BCL6 is directly recruited to the MYC promoter. Inducible activation of pre-B cell Receptor signaling in pre-B cells from BCL6-deficient mice failed to induce cell cycle exit and quiescence. We conclude that activation of BCL6 downstream of the pre-B cell Receptor is crucial for the induction of quiescence at the Fraction C'-D checkpoint. As expected, inducible activation of BCL6 downstream of the pre-B cell Receptor results in transcriptional repression of MYC and CCND2. Overexpression of MYC prevented pre-B cell Receptor/BCL6-induced cell cycle exit. Conclusion: Hence, pre-B cell Receptor signaling induces cellular quiescence of Fraction C' pre-B cells through activation of BCL6 and BCL6-mediated transcriptional repression of MYC and CCND2. Disclosures: No relevant conflicts of interest to declare.
-
Pre-B cell Receptor-mediated activation of BCL6 induces pre-B cell quiescence through transcriptional repression of MYC
Blood, 2011Co-Authors: Rahul Nahar, Maximilian Mossner, Cihangir Duy, Hassan Jumaa, Parham Ramezani-rad, Sinisa Dovat, Leandro Cerchietti, Huimin Geng, Ari MelnickAbstract:Initial cell surface expression of the pre-B cell Receptor induces proliferation. After 2 to 5 divisions, however, large pre-BII (Fraction C') cells exit cell cycle to become resting, small pre-BII cells (Fraction D). The mechanism by which pre-BII cells exit cell cycle, however, is currently unclear. The checkpoint at the Fraction C'-D transition is critical for immunoglobulin light chain gene recombination and to prevent malignant transformation into acute lymphoblastic leukemia. Here we demonstrate that inducible activation of pre-B cell Receptor signaling induces cell-cycle exit through up-regulation of the transcriptional repressor BCL6. Inducible activation of BCL6 downstream of the pre-B cell Receptor results in transcriptional repression of MYC and CCND2 . Hence, pre-B cell Receptor-mediated activation of BCL6 limits pre-B cell proliferation and induces cellular quiescence at the small pre-BII (Fraction D) stage.
-
Mechanisms of Pre-B Cell Receptor-Inactivation In Acute Lymphoblastic Leukemia
Blood, 2010Co-Authors: Cihangir Duy, Daniel Nowak, Rahul Nahar, Lars Klemm, Nora Heisterkamp, Wolf-karsten Hofmann, Emily Elliott, Clifford A. Lowell, Phillip H. KoefflerAbstract:Abstract 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 Ca 2+ -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, Ca 2+ -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.
-
Pre-B Cell Receptor Signaling Distinguishes E2A-PBX1 From Other Subtypes of Acute Lymphoblastic Leukemia
Blood, 2010Co-Authors: Rahul Nahar, Daniel Trageser, Lars Klemm, Cihangir Duy, Yong-mi Kim, Nora Heisterkamp, Wolf-karsten Hofmann, Eugene Park, Hassan Jumaa, Markus MüschenAbstract:Abstract 274 Background: The E2A-PBX1 [t(1;19)(q23;p13)] fusion is found in ≂f4% of cases of childhood ALL and involves a rearrangement of the TCF3 gene (encoding the E2A transcription factor). TCF3 (E2A) is not only a critical regulator of B cell lineage commitment and early B cell development (Muschen et al., 2002; Sigvardsson et al., 2002), it also cooperates with LEF1 to activate canonical WNT/β-catenin signaling (Hovanes et al., 2001; Merrill et al., 2001). Pre-B cells in human bone marrow are destined to die unless they are rescued through survival signals from a successfully assembled pre-B cell Receptor. Congenital defects in pre-B cell Receptor-related signaling molecules cause a severe differentiation block at an early pre-B cell stage. Likewise, B cell lineage acute lymphoblastic leukemia (ALL) cells are arrested at an early pre-B cell stage in the vast majority of cases. Given that the pre-B cell Receptor drives both proliferation and differentiation of normal B cell precursors, we test here the hypothesis that pre-B cell Receptor signaling represents a critical target for malignant transformation towards ALL. Results: Studying 148 cases of pre-B cell-derived human ALL, we found that pre-B cell Receptor expression and function is linked to specific cytogenetic subgroups: ALL cells carrying an E2A-PBX1- gene rearrangement are, like normal pre-B cells, highly selected for the expression of a functional pre-B cell Receptor. In all 8 ALL cases with E2A-PBX1 fusion, engagement of the pre-B cell Receptor resulted in a strong Ca 2+ signal, which strongly suggests that E2A-PBX1 leukemia clones are selected for active pre-B cell Receptor signaling. In striking contrast, ALL cells carrying BCR-ABL1- or MLL-AF4 fusion genes and ALL cells with hyperdiploid karyotype lack expression of a functional pre-B cell Receptor in virtually all cases. Only 10 of 57 cases with BCR-ABL1 , 0 of 7 cases with MLL-AF4 and 1 of 30 cases with hyperdiploid karyotype a productively rearranged μ -heavy chain locus encoding the central component of the pre-B cell Receptor, was found. Even in the few BCR-ABL1 ALL cases, in which a productively rearranged μ -chain was amplified, no pre-B cell Receptor was expressed. Based on these findings, we hypothesize that ALL can be subdivided into two groups based on whether pre-B cell Receptor signaling enables ( E2A-PBX1 ) or suppresses ( BCR-ABL1 , MLL-AF4 , Hyperdiploid, likely other subtypes) leukemic growth. In a proof-of-concept experiment, we super-transformed E2A-PBX1 -induced ALL cells (active pre-B cell Receptor signaling) and MLL-AF4 -ALL cells lacking pre-B cell Receptor function with the BCR-ABL1 oncogene. The BCR-ABL1 oncogene was chosen, because it is only found in leukemia cells that lack pre-B cell Receptor function. Whereas growth of pre-B cell Receptor-negative MLL-AF4 ALL cells was strongly accelerated by BCR-ABL1-transformation, pre-B cell Receptor-positive E2A-PBX1 ALL cells were rapidly eliminated within 9 days after BCR-ABL1 -transduction. Interestingly, incubation of E2A-PBX1 ALL cells survived BCR-ABL1-transduction only in the presence of 10 μ mol/l of the BCR-ABL1 kinase inhibitor Imatinib. To identify factors that distinguish E2A-PBX1 and other ALL subtypes ( BCR-ABL1 , MLL-AF4 , Hyperdiploid) that may explain the divergent role of pre-B cell Receptor signaling in these groups, we performed a comparative gene expression including a meta-analysis of published microarray data and quantitative RT-PCR. In this analysis, E2A-PBX1 ALL cells were distinguished by high expression levels of pre-B cell Receptor-related signaling molecules (e.g. BLNK, SYK, BTK). The most prominent gene expression differences involve canonical WNT/β-catenin signaling. As opposed to other ALL subtypes, E2A-PBX1 ALL cells express the β-catenin cofactors TCF3 and LEF1 at >5-fold higher levels and WNT16 at >12-fold higher levels compared to BCR-ABL1 , MLL-AF4 , Hyperdiploid and TEL-AML1 -driven ALL subtypes. Conclusions: Constitutive activation of the canonical WNT/β-catenin signaling pathway in E2A-PBX1 ALL cells may explain the distinct role of pre-B cell Receptor signaling in this ALL subset: Since pre-B cell Receptor signaling via BTK negatively regulates WNT/β-catenin-dependent survival and self-renewal signaling (James et al., 2009), the level of constitutive WNT/β-catenin-signaling may determine permissiveness of ALL cells to pre-B cell Receptor function. 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
Blood, 2010Co-Authors: Rahul Nahar, Cihangir Duy, Parham Ramezani-rad, Sinisa Dovat, Ari Melnick, Markus MüschenAbstract:Abstract 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 C9) 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.
