The Experts below are selected from a list of 288 Experts worldwide ranked by ideXlab platform
Markus Müschen - One of the best experts on this subject based on the ideXlab platform.
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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.
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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.
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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.
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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...
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Pre-B Cell Receptor signaling in acute lymphoblastic leukemia
Cell cycle (Georgetown Tex.), 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 the pre-B Cell Receptor and the oncogenic BCR-ABL1 kinase are mutually exclusive and only "crippled" pre-B Cells that fail to express a functional pre-B Cell Receptor are permissive to transformation by BCR-ABL1.
Mark M Davis - One of the best experts on this subject based on the ideXlab platform.
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An Analysis of T Cell Receptor–Ligand Interaction Using a Transgenic Antigen Model for T Cell Tolerance and T Cell Receptor Mutagenesis
Molecular Mechanisms of Immunological Self-Recognition, 1993Co-Authors: Barbara Fazekas De St. Groth, Phillip A. Patten, Edwin P. Rock, Mark M DavisAbstract:Publisher Summary This chapter presents an analysis of T Cell Receptor-ligand interaction using a transgenic antigen model for T-Cell tolerance and T-Cell Receptor mutagenesis. It presents an analysis of the molecular basis of T-Cell Receptor (TCR) recognition of foreign peptide-major histocompatibility complexes (MHC). The chapter presents a few experiments that succeeded in transferring Staphylococcus aureus enterotoxin B (SEB) reactivity. T-Cell Receptor sequences, particularly V regions, are very similar to immunoglobulins, and the consensus of a number of experts is that they probably fold and pair in much the same way. However, the preponderance of their sequence diversity lies in the V(D)J junctional or CDR3-equivalent region, exceeding the diversity of immunoglobulin CDR3s by many orders of magnitude in the absence of somatic mutation.
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Topology and affinity of T-Cell Receptor mediated recognition of peptide-MHC complexes.
Current opinion in immunology, 1993Co-Authors: Mark M Davis, Yueh-hsiu ChienAbstract:Significant progress has been made on several long-standing issues regarding T-Cell Receptor mediated recognition of antigen-MHC complexes. For one, early data suggest that the affinity of the T-Cell Receptor for the peptide-MHC complex is extremely low, with a KID of approximately 10−4−10−5SM, much weaker than most antibody-antigen interactions. The fact that this affinity is lower than that of some T-Cell adhesion molecules for their ligands could have important implications for immune surveillance. A second area of interest is the topology of T-Cell Receptor recognition; evidence of direct contact between the third complementarity determining region of the T-Cell Receptor and peptide determinants has been obtained. In addition, the orientation of the T-Cell Receptor with respect to several antigen-MHC complexes has been predicted. They suggest that whereas most or all peptides seem to bind in the same orientation in both class I and class II MHC molecules, the orientation of the T-Cell Receptor over the peptide-MHC complex may not be fixed.
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mapping t Cell Receptor peptide contacts by variant peptide immunization of single chain transgenics
Nature, 1992Co-Authors: Jeffrey L Jorgensen, Barbara Fazekas De St. Groth, Ursula Esser, Philip A Reay, Mark M DavisAbstract:To test models of T-Cell recognition, mice transgenic for T-Cell Receptor α or β chain have been immunized with variant peptides that force changes in the resulting T-Cell response. In particular, charge substitutions on the peptide often elicit reciprocal charges in the junctional (CDR3) sequences of T-Cell Receptor Vαor Vβ chains, indicating direct T-Cell Receptor–peptide contact, and allowing derivation of a topology for the T-Cell Receptor–MHC interaction. At one position on the peptide, variants transformed a homogeneous Vβ response into a very heterogeneous one.
Marie-paule Lefranc - One of the best experts on this subject based on the ideXlab platform.
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TRG@ (T Cell Receptor gamma)
Atlas of Genetics and Cytogenetics in Oncology and Haematology, 2011Co-Authors: Marie-paule LefrancAbstract:Review on TRG (T Cell Receptor gamma), with data on DNA, on the protein encoded, and where the gene is implicated.
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T Cell Receptor structural and biological properties
The T Cell Receptor FactsBook, 2001Co-Authors: Marie-paule Lefranc, Gérard LefrancAbstract:The T-Cell Receptors, such as the immunoglobulins (Ig), are clonotypic antigen-specific Receptors that are essential to the immune response. However, they are different in several ways from the Ig. This chapter discusses the structural and biological properties of various T-Cell Receptors. The T-Cell Receptor chains are thoroughly described. The chapter mentions the structure and the glycosylation of T-Cell Receptors. The chapter also mentions the T-Cell Receptor-CD3 complex, which comprises additional proteins. This is followed by the biosynthesis and assembly of the T-Cell Receptor-CD3 complex. Finally, the activation of the T-Cell Receptor-CD3 complex is mentioned. This activation leads to membrane reorganization and the formation of a supramolecular activation cluster or mature immunological synapse with the antigen-presenting Cells (APC).
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Current Protocols in Immunology - Nomenclature of the Human T Cell Receptor Genes
Current Protocols in Immunology, 2000Co-Authors: Marie-paule LefrancAbstract:The human T Cell Receptors (TcR) alpha-beta and gamma-delta are the products of four sets of genes on two chromosomes: T Cell Receptors alpha (TRA) and delta (TRD) on chromosome 14 at 14q11.2, T Cell Receptor beta (TRB) on chromosome 7 at 7q35, and T Cell Receptor gamma (TRG) on chromosome 7 at 7p15-p14. This appendix presents tabulated lists of the human TcR alpha, beta, gamma, and delta genes named in accordance with the International ImMunoGeneTics database and approved by the Human Genome Organization Nomenclature Committee in 1999. Two additional tables list corresponding nomenclatures for these genes.
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Genetic Organization of the Human T Cell Receptor Gamma Locus
Current Topics in Microbiology and Immunology, 1991Co-Authors: Marie-paule Lefranc, Terence H. RabbittsAbstract:The T-Cell gamma/delta Receptor is expressed on about 3–5 % of the circulating T lymphocytes in human. In this report, we will review the genetic organization of the human T Cell Receptor gamma locus.
Daniel Trageser - One of the best experts on this subject based on the ideXlab platform.
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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.
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Pre-B Cell Receptor-mediated Cell cycle arrest in Philadelphia chromosome-positive acute lymphoblastic leukemia requires IKAROS function
Journal of Experimental Medicine, 2009Co-Authors: Daniel Trageser, Rahul Nahar, Lars Klemm, Cihangir Duy, Tanja A. Gruber, Eugene Park, Ilaria Iacobucci, Gregor Von Levetzow, Wolfgang Schuh, Sebastian HerzogAbstract:B Cell lineage acute lymphoblastic leukemia (ALL) arises in virtually all cases from B Cell precursors that are arrested at pre-B Cell Receptor-dependent stages. The Philadelphia chromosome-positive (Ph(+)) subtype of ALL accounts for 25-30% of cases of adult ALL, has the most unfavorable clinical outcome among all ALL subtypes and is defined by the oncogenic BCR-ABL1 kinase and deletions of the IKAROS gene in >80% of cases. Here, we demonstrate that the pre-B Cell Receptor functions as a tumor suppressor upstream of IKAROS through induction of Cell cycle arrest in Ph(+) ALL Cells. Pre-B Cell Receptor-mediated Cell cycle arrest in Ph(+) ALL Cells critically depends on IKAROS function, and is reversed by coexpression of the dominant-negative IKAROS splice variant IK6. IKAROS also promotes tumor suppression through cooperation with downstream molecules of the pre-B Cell Receptor signaling pathway, even if expression of the pre-B Cell Receptor itself is compromised. In this case, IKAROS redirects oncogenic BCR-ABL1 tyrosine kinase signaling from SRC kinase-activation to SLP65, which functions as a critical tumor suppressor downstream of the pre-B Cell Receptor. These findings provide a rationale for the surprisingly high frequency of IKAROS deletions in Ph(+) ALL and identify IKAROS-mediated Cell cycle exit as the endpoint of an emerging pathway of pre-B Cell Receptor-mediated tumor suppression.
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The Pre-B Cell Receptor Suppresses Leukemogenesis by Censoring MYC Expression.
Blood, 2008Co-Authors: Rahul Nahar, Daniel Trageser, Lars Klemm, Cihangir Duy, Peter Van Essen, Yong-mi Kim, Nora Heisterkamp, Giovanni Martinelli, Wolf-karsten Hofmann, Hans-martin JackAbstract: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. 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. 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; Type I) or suppresses ( BCR-ABL1 , MLL-AF4 , Hyperdiploid; Type II) leukemic growth. In a proof-of-concept experiment, we super-transformed E2A-PBX1 -induced Type I ALL Cells (active pre-B Cell Receptor signaling) and MLL-AF4 -induced Type II ALL Cells (lack of pre-B Cell Receptor expression) with the BCR-ABL1 oncogene. Whereas growth of pre-B Cell Receptor-negative Type II ALL Cells was accelerated by BCR-ABL1-transformation, pre-B Cell Receptor-positive Type I ALL Cells rapidly eliminated by apoptosis within 9 days after BCR-ABL1 -transduction. To identify factors that distinguish Type I ( E2A-PBX1 ) and Type II ( BCR-ABL1 , MLLAF4 , Hyperdiploid) ALL and that may explain the divergent role of pre-B Cell Receptor signaling in these groups, we performed a comparative gene expression including a metaanalysis of published microarray data and quantitative RT-PCR. Compared to E2A-PBX1 Type I ALL, MYC mRNA levels are on average 4-fold, 6-fold and 2.5-fold higher in BCR-ABL1 , MLL-AF4 and Hyperdiploid ALL Cells, respectively. To test whether high expression levels of MYC render leukemia Cells non-permissive to pre-B Cell Receptor expression, we studied bone marrow B Cell precursors from Rag2 −/− mice that carry a tetracycline-inducible μ-chain transgene (Hess et al. , 2001) as Cell culture model for inducible pre-B Cell Receptor expression. When expression of the pre-B Cell Receptor was induced in normal IL7-dependent B Cell precursors, the Cells were induced to first proliferate and subsequently differentiate, hence mirroring normal stages of early B Cell development. We then transformed Rag2 −/− Tet-μ-chain B Cell precursors by retroviral transduction with MYC. MYC-transformed Cells rapidly outcompeted untransduced normal IL7-dependent B Cell precursors in Cell culture. Induction of pre-B Cell Receptor expression, however, completely reversed growth kinetics and within a few days, normal untransduced pre-B Cell Receptor-positive Cells had a substantial growth advantage over MYC-transduced pre-B Cell Receptor-positive Cells that were progressively lost in Cell culture. Interestingly, this growth pattern was reversible by subsequent ablation of pre-B Cell Receptor expression: After Tet-mediated ablation of pre-B Cell Receptor expression, the initial growth kinetics were restored and MYC-transduced pre-B Cell Receptor-negative Cells regained a substantial growth advantage. These findings demonstrate that different levels of MYC expression determine permissiveness of ALL Cells for pre-B Cell Receptor signaling. Hence, the pre-B Cell Receptor suppresses outgrowth of Type II leukemia by censoring high levels of MYC expression.
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Pre-B Cell Receptor signaling prevents malignant transformation by BCR-ABL1
2008Co-Authors: Daniel TrageserAbstract:The pre-B Cell Receptor plays a critical role in the early B Cell development. As a failure of expression results in the lack of survival signal it represents an important checkpoint for further proliferation and differentiation. It has been shown that Cells in acute lymphoblastic leukemia frequently carry defects in important pre-B Cell Receptor related signaling molecules like BTK or SLP65, indicating a tumor suppressive function. In contrast pre-B Cell Receptor expression is required in malignant lymphoproliferation and at least in mouse pre-B Cells for transformation by the Myc oncogene. Therefore we investigated the role of the pre-B Cell Receptor as tumor suppressor vs. a requirement for leukemic transformation. We found that only E2A-PBX1-mediated ALL carry a functional pre-B Cell Receptor and therefore seems to be essential for this type of leukemic transformation. The remaining investigated ALL subtypes are Receptor independent or even show strong negative selection against a functional pre-B Cell Receptor. To clarify the role of the pre-B Cell Receptor during leukemogenesis, we studied the progressive transformation of pre-B Cells in a transgenic mouse model for BCR-ABL1-induced ALL. Interestingly, before the onset of leukemia BCR-ABL1-transgenic pre-B Cells respond normally to pre-B Cell Receptor engagement and express BCR-ABL1 at low levels. In full-blown leukemia, ALL Cells do not respond to pre-B Cell Receptor engagement and express BCR-ABL1 at high levels. Signaling from the pre-B Cell Receptor and BCR-ABL1 are mutually exclusive: i) Treatment of leukemic mice with the BCR-ABL1 kinase inhibitor, AMN107, reinstates normal pre-B Cell Receptor signaling within seven days. ii) Reconstitution of pre-B Cell Receptor signaling in BCR-ABL1-transformed pre-B Cells induces rapid Cell death. iii) Transduction of human ALL Cells with the BCR-ABL1 oncogene induces apoptosis in the presence of pre-B Cell Receptor signaling and accelerates proliferation in its absence. We conclude that pre-B Cell Receptor signaling -while compatible with expression of E2A-PBX1- renders B Cell precursors non-permissive to transformation by BCR-ABL1 and likely other leukemogenic fusion genes.
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Pre-B Cell Receptor Signaling Prevents Leukemic Transformation by BCR-ABL1 .
Blood, 2007Co-Authors: Daniel Trageser, Rahul Nahar, Lars Klemm, Cihangir Duy, Yong-mi Kim, Nora Heisterkamp, Tanja A. Gruber, Eugene Park, John Groffen, Wolf-karsten HofmannAbstract: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.
Rahul Nahar - One of the best experts on this subject based on the ideXlab platform.
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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.
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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.
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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.
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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.
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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...