open access

Vol 2, No 2 (2009)
ROZPRAWA HABILITACYJNA
Published online: 2009-05-18
Get Citation

Pathogenetic mechanisms and therapeutic targets in diffuse large B-cell lymphomas characterized by constitutive tonic B-cell receptor activity and BCL6 transcriptional program: from DNA microarrays to rational therapy

Przemysław Juszczyński
Journal of Transfusion Medicine 2009;2(2):41-72.

open access

Vol 2, No 2 (2009)
ROZPRAWA HABILITACYJNA
Published online: 2009-05-18

Abstract

Diffuse large B-cell lymphomas (DLBCL) are the most common lymphoid malignancy in adults and an extremely heterogeneous group of disorders. To delineate functionally relevant DLBCL subsets, consensus clustering methods were previously applied to the transcriptional profiles of two large independent series of primary DLBCL to identify the dominant substructure a priori. The obtained consensus clusters were highly reproducible and included a group of DLBCL, termed B-cell receptor (“BCR”) that was characterized by increased expression of components of the BCR signaling cascade including SYK (Spleen Tyrosine Kinase) and certain B-cell specific transcription factors such as BCL6; these DLBCL also exhibit more frequent translocations of the BCL6 locus. These observations suggested that the “BCR” molecular category of DLBCL might be reliant on either, or both, BCR signaling and BCL6 transcriptional program. To test this hypothesis, the studies were undertaken to specifically investigate the role of tonic SYK-dependent signaling in “BCR”-type DLBCL and the biological consequences of the pathway inhibition in vitro, mechanisms controlling SYK activity, role of transcriptional program controlled by BCL6 and the biological consequences of its inhibition in vitro and relationship between BCL6 mediated repression and SYK-dependent signaling in “BCR”-type DLBCL.
These studies demonstrated that inhibition of the BCR signaling pathway with an ATPcompetitive inhibitor of SYK, R406, induced apoptosis of the majority of examined DLBCL cell lines and primary DLBCL in vitro. R406-sensitive DLBCL cell lines and primary tumors exhibited tonic activity of SYK and its direct substrate, B-cell linker protein (BLNK). In these R406-sensitive lines and primary tumors, R406 specifically inhibited both tonic and ligandinduced BCR signaling (SYK-dependent phosphorylation of BLNK). Therefore, SYK-dependent tonic BCR signaling is an important and potentially targetable survival pathway in some, but not all, DLBCL. In addition, R406-sensitive DLBCL can be identified by their transcriptional profiles.
Consistent with critical role of SYK in modulating BCR signaling, its activity remains under tight control. SYK is a major substrate of a tissue-specific and developmentally regulated PTP, PTP receptor–type O truncated (PTPROt). The overexpression of PTPROt inhibited BCR-triggered SYK tyrosyl phosphorylation and downstream signaling events, including extracellular signal–regulated kinase (ERK1/2) activation. PTPROt overexpression also inhibited lymphoma cell proliferation and induced apoptosis in the absence of BCR cross-linking, suggesting that the phosphatase modulates tonic BCR signaling.
“BCR” tumors also exhibit more abundant BCL6 expression and more frequent BCL6 translocations, suggesting that these tumors likely rely on BCL6 transcriptional program. It could be predicted therefore that DLBCL dependent upon BCL6-regulated pathways would exhibit coordinate repression of BCL6 target genes. For this reason, genomic array ChIP-on-chip was utilized to identify the cohort of direct BCL6 target genes. In primary DLBCL classified on the basis of gene expression profiles, these BCL6 target genes were differentially regulated in “BCR” tumors. In a panel of DLBCL cell lines analyzed by expression arrays and classified according to their gene expression profiles, only “BCR” tumors were highly sensitive to the BCL6 peptide inhibitor (BPI). These studies identify a discrete subset of DLBCL that are reliant upon BCL6 signaling and uniquely sensitive to BCL6 inhibitors.
Since the same transcriptionally defined subset of DLBCL relies upon SYK-dependent BCR signaling and exhibits coordinate BCL6-mediated transcriptional repression, the relationship between these two processes was subsequently explored. In transcriptionally profiled normal B-cell subsets (naïve, germinal center [GC], and memory B cells) and in primary DLBCL, there were reciprocal patterns of expression of BCL6 and the SYK tyrosine phosphatase, PTPROt. BCL6 repressed PTPROt transcription via a direct interaction with functional BCL6 binding sites in PTPROt promoter. Enforced expression of BCL6 in normal naïve B cells and RNAi-mediated depletion of BCL6 in GC B-cells directly modulated PTPROt expression. In “BCR”-type DLBCL, BCL6 depletion increased PTPROt expression and decreased phosphorylation of SYK and the downstream adaptor protein, BLNK, demonstrating that BCL6 augments BCR signaling. Since BCL6 and SYK are both promising therapeutic targets in many DLBCL, combined inhibition of these functionally related pathways warrants further study.

Abstract

Diffuse large B-cell lymphomas (DLBCL) are the most common lymphoid malignancy in adults and an extremely heterogeneous group of disorders. To delineate functionally relevant DLBCL subsets, consensus clustering methods were previously applied to the transcriptional profiles of two large independent series of primary DLBCL to identify the dominant substructure a priori. The obtained consensus clusters were highly reproducible and included a group of DLBCL, termed B-cell receptor (“BCR”) that was characterized by increased expression of components of the BCR signaling cascade including SYK (Spleen Tyrosine Kinase) and certain B-cell specific transcription factors such as BCL6; these DLBCL also exhibit more frequent translocations of the BCL6 locus. These observations suggested that the “BCR” molecular category of DLBCL might be reliant on either, or both, BCR signaling and BCL6 transcriptional program. To test this hypothesis, the studies were undertaken to specifically investigate the role of tonic SYK-dependent signaling in “BCR”-type DLBCL and the biological consequences of the pathway inhibition in vitro, mechanisms controlling SYK activity, role of transcriptional program controlled by BCL6 and the biological consequences of its inhibition in vitro and relationship between BCL6 mediated repression and SYK-dependent signaling in “BCR”-type DLBCL.
These studies demonstrated that inhibition of the BCR signaling pathway with an ATPcompetitive inhibitor of SYK, R406, induced apoptosis of the majority of examined DLBCL cell lines and primary DLBCL in vitro. R406-sensitive DLBCL cell lines and primary tumors exhibited tonic activity of SYK and its direct substrate, B-cell linker protein (BLNK). In these R406-sensitive lines and primary tumors, R406 specifically inhibited both tonic and ligandinduced BCR signaling (SYK-dependent phosphorylation of BLNK). Therefore, SYK-dependent tonic BCR signaling is an important and potentially targetable survival pathway in some, but not all, DLBCL. In addition, R406-sensitive DLBCL can be identified by their transcriptional profiles.
Consistent with critical role of SYK in modulating BCR signaling, its activity remains under tight control. SYK is a major substrate of a tissue-specific and developmentally regulated PTP, PTP receptor–type O truncated (PTPROt). The overexpression of PTPROt inhibited BCR-triggered SYK tyrosyl phosphorylation and downstream signaling events, including extracellular signal–regulated kinase (ERK1/2) activation. PTPROt overexpression also inhibited lymphoma cell proliferation and induced apoptosis in the absence of BCR cross-linking, suggesting that the phosphatase modulates tonic BCR signaling.
“BCR” tumors also exhibit more abundant BCL6 expression and more frequent BCL6 translocations, suggesting that these tumors likely rely on BCL6 transcriptional program. It could be predicted therefore that DLBCL dependent upon BCL6-regulated pathways would exhibit coordinate repression of BCL6 target genes. For this reason, genomic array ChIP-on-chip was utilized to identify the cohort of direct BCL6 target genes. In primary DLBCL classified on the basis of gene expression profiles, these BCL6 target genes were differentially regulated in “BCR” tumors. In a panel of DLBCL cell lines analyzed by expression arrays and classified according to their gene expression profiles, only “BCR” tumors were highly sensitive to the BCL6 peptide inhibitor (BPI). These studies identify a discrete subset of DLBCL that are reliant upon BCL6 signaling and uniquely sensitive to BCL6 inhibitors.
Since the same transcriptionally defined subset of DLBCL relies upon SYK-dependent BCR signaling and exhibits coordinate BCL6-mediated transcriptional repression, the relationship between these two processes was subsequently explored. In transcriptionally profiled normal B-cell subsets (naïve, germinal center [GC], and memory B cells) and in primary DLBCL, there were reciprocal patterns of expression of BCL6 and the SYK tyrosine phosphatase, PTPROt. BCL6 repressed PTPROt transcription via a direct interaction with functional BCL6 binding sites in PTPROt promoter. Enforced expression of BCL6 in normal naïve B cells and RNAi-mediated depletion of BCL6 in GC B-cells directly modulated PTPROt expression. In “BCR”-type DLBCL, BCL6 depletion increased PTPROt expression and decreased phosphorylation of SYK and the downstream adaptor protein, BLNK, demonstrating that BCL6 augments BCR signaling. Since BCL6 and SYK are both promising therapeutic targets in many DLBCL, combined inhibition of these functionally related pathways warrants further study.
Get Citation

Keywords

Diffuse large B-cell lymphomas; Microarray gene expression profiling; B-cell receptor; SYK; PTPROt; BCL6 transcriptional program; Targeted therapy

About this article
Title

Pathogenetic mechanisms and therapeutic targets in diffuse large B-cell lymphomas characterized by constitutive tonic B-cell receptor activity and BCL6 transcriptional program: from DNA microarrays to rational therapy

Journal

Journal of Transfusion Medicine

Issue

Vol 2, No 2 (2009)

Pages

41-72

Published online

2009-05-18

Bibliographic record

Journal of Transfusion Medicine 2009;2(2):41-72.

Keywords

Diffuse large B-cell lymphomas
Microarray gene expression profiling
B-cell receptor
SYK
PTPROt
BCL6 transcriptional program
Targeted therapy

Authors

Przemysław Juszczyński

References (65)
  1. Abramson JS, Shipp MA. Advances in the biology and therapy of diffuse large B-cell lymphoma: moving toward a molecularly targeted approach. Blood. 2005; 106(4): 1164–1174.
  2. Küppers R, Klein U, Hansmann ML, et al. Cellular origin of human B-cell lymphomas. N Engl J Med. 1999; 341(20): 1520–1529.
  3. Klein U, Goossens T, Fischer M, et al. Somatic hypermutation in normal and transformed human B cells. Immunol Rev. 1998; 162: 261–280.
  4. Klein U, Dalla-Favera R. Germinal centres: role in B-cell physiology and malignancy. Nat Rev Immunol. 2008; 8(1): 22–33.
  5. Kramer MH, Hermans J, Wijburg E, et al. Clinical relevance of BCL2, BCL6, and MYC rearrangements in diffuse large B-cell lymphoma. Blood. 1998; 92(9): 3152–3162.
  6. Pasqualucci L, Bhagat G, Jankovic M, et al. AID is required for germinal center-derived lymphomagenesis. Nat Genet. 2008; 40(1): 108–112.
  7. Pasqualucci L, Neumeister P, Goossens T, et al. Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas. Nature. 2001; 412(6844): 341–346.
  8. International Non-Hodgkin's Lymphoma Prognostic Factors Project. A predictive model for aggressive non-Hodgkin's lymphoma. N Engl J Med. 1993; 329(14): 987–994.
  9. Monti S, Savage KJ, Kutok JL, et al. Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response. Blood. 2005; 105(5): 1851–1861.
  10. Rosenwald A, Wright G, Chan WC, et al. Lymphoma/Leukemia Molecular Profiling Project. The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med. 2002; 346(25): 1937–1947.
  11. Wright G, Tan B, Rosenwald A, et al. A gene expression-based method to diagnose clinically distinct subgroups of diffuse large B cell lymphoma. Proc Natl Acad Sci U S A. 2003; 100(17): 9991–9996.
  12. Takahashi H, Feuerhake F, Kutok JL, et al. FAS death domain deletions and cellular FADD-like interleukin 1beta converting enzyme inhibitory protein (long) overexpression: alternative mechanisms for deregulating the extrinsic apoptotic pathway in diffuse large B-cell lymphoma subtypes. Clin Cancer Res. 2006; 12(11 Pt 1): 3265–3271.
  13. Gauld SB, Dal Porto JM, Cambier JC. B cell antigen receptor signaling: roles in cell development and disease. Science. 2002; 296(5573): 1641–1642.
  14. Monroe JG. ITAM-mediated tonic signalling through pre-BCR and BCR complexes. Nat Rev Immunol. 2006; 6(4): 283–294.
  15. Rolli V, Gallwitz M, Wossning T, et al. Amplification of B cell antigen receptor signaling by a Syk/ITAM positive feedback loop. Mol Cell. 2002; 10(5): 1057–1069.
  16. Kulathu Y, Hobeika E, Turchinovich G, et al. The kinase Syk as an adaptor controlling sustained calcium signalling and B-cell development. EMBO J. 2008; 27(9): 1333–1344.
  17. Kraus M, Alimzhanov MB, Rajewsky N, et al. Survival of resting mature B lymphocytes depends on BCR signaling via the Igalpha/beta heterodimer. Cell. 2004; 117(6): 787–800.
  18. Lam KP, Kühn R, Rajewsky K. In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death. Cell. 1997; 90(6): 1073–1083.
  19. Smith SH, Reth M. Perspectives on the nature of BCR-mediated survival signals. Mol Cell. 2004; 14(6): 696–697.
  20. Wienands J, Larbolette O, Reth M. Evidence for a preformed transducer complex organized by the B cell antigen receptor. Proc Natl Acad Sci U S A. 1996; 93(15): 7865–7870.
  21. Küppers R. Mechanisms of B-cell lymphoma pathogenesis. Nat Rev Cancer. 2005; 5(4): 251–262.
  22. Cattoretti G, Chang CC, Cechova K, et al. BCL-6 protein is expressed in germinal-center B cells. Blood. 1995; 86(1): 45–53.
  23. Ye BH, Cattoretti G, Shen Q, et al. The BCL-6 proto-oncogene controls germinal-centre formation and Th2-type inflammation. Nat Genet. 1997; 16(2): 161–170.
  24. Fearon DT, Manders PM, Wagner SD. Bcl-6 uncouples B lymphocyte proliferation from differentiation. Adv Exp Med Biol. 2002; 512: 21–28.
  25. Fujita N, Jaye DL, Kajita M, et al. MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth pathway in breast cancer. Cell. 2003; 113(2): 207–219.
  26. Tunyaplin C, Shaffer AL, Angelin-Duclos CD, et al. Direct repression of prdm1 by Bcl-6 inhibits plasmacytic differentiation. J Immunol. 2004; 173(2): 1158–1165.
  27. Saito M, Gao J, Basso K, et al. A signaling pathway mediating downregulation of BCL6 in germinal center B cells is blocked by BCL6 gene alterations in B cell lymphoma. Cancer Cell. 2007; 12(3): 280–292.
  28. Ye BH, Lista F, Lo Coco F, et al. Alterations of a zinc finger-encoding gene, BCL-6, in diffuse large-cell lymphoma. Science. 1993; 262(5134): 747–750.
  29. Baron BW, Anastasi J, Montag A, et al. The human BCL6 transgene promotes the development of lymphomas in the mouse. Proc Natl Acad Sci U S A. 2004; 101(39): 14198–14203.
  30. Cattoretti G, Pasqualucci L, Ballon G, et al. Deregulated BCL6 expression recapitulates the pathogenesis of human diffuse large B cell lymphomas in mice. Cancer Cell. 2005; 7(5): 445–455.
  31. Polo JM, Dell'Oso T, Ranuncolo SM, et al. Specific peptide interference reveals BCL6 transcriptional and oncogenic mechanisms in B-cell lymphoma cells. Nat Med. 2004; 10(12): 1329–1335.
  32. Cerchietti LC, Yang SN, Shaknovich R, et al. A peptomimetic inhibitor of BCL6 with potent antilymphoma effects in vitro and in vivo. Blood. 2009; 113(15): 3397–3405.
  33. Chen L, Monti S, Juszczynski P, et al. SYK-dependent tonic B-cell receptor signaling is a rational treatment target in diffuse large B-cell lymphoma. Blood. 2008; 111(4): 2230–2237.
  34. Polo JM, Juszczynski P, Monti S, et al. Transcriptional signature with differential expression of BCL6 target genes accurately identifies BCL6-dependent diffuse large B cell lymphomas. Proc Natl Acad Sci U S A. 2007; 104(9): 3207–3212.
  35. Braselmann S, Taylor V, Zhao H, et al. R406, an orally available spleen tyrosine kinase inhibitor blocks fc receptor signaling and reduces immune complex-mediated inflammation. J Pharmacol Exp Ther. 2006; 319(3): 998–1008.
  36. Irish JM, Czerwinski DK, Nolan GP, et al. Altered B-cell receptor signaling kinetics distinguish human follicular lymphoma B cells from tumor-infiltrating nonmalignant B cells. Blood. 2006; 108(9): 3135–3142.
  37. Phan RT, Dalla-Favera R. The BCL6 proto-oncogene suppresses p53 expression in germinal-centre B cells. Nature. 2004; 432(7017): 635–639.
  38. Fujita N, Jaye DL, Geigerman C, et al. MTA3 and the Mi-2/NuRD complex regulate cell fate during B lymphocyte differentiation. Cell. 2004; 119(1): 75–86.
  39. Niu H, Ye BH, Dalla-Favera R. Antigen receptor signaling induces MAP kinase-mediated phosphorylation and degradation of the BCL-6 transcription factor. Genes Dev. 1998; 12(13): 1953–1961.
  40. Cornall RJ, Cheng AM, Pawson T, et al. Role of Syk in B-cell development and antigen-receptor signaling. Proc Natl Acad Sci U S A. 2000; 97(4): 1713–1718.
  41. Shaffer AL, Yu X, He Y, et al. BCL-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control. Immunity. 2000; 13(2): 199–212.
  42. Dent AL, Shaffer AL, Yu X, et al. Control of inflammation, cytokine expression, and germinal center formation by BCL-6. Science. 1997; 276(5312): 589–592.
  43. Oberley MJ, Tsao J, Yau P, et al. High-throughput screening of chromatin immunoprecipitates using CpG-island microarrays. Methods Enzymol. 2004; 376: 315–334.
  44. Trinklein ND, Aldred SF, Hartman SJ, et al. An abundance of bidirectional promoters in the human genome. Genome Res. 2004; 14(1): 62–66.
  45. Juszczynski P, Kutok JL, Li C, et al. BAL1 and BBAP are regulated by a gamma interferon-responsive bidirectional promoter and are overexpressed in diffuse large B-cell lymphomas with a prominent inflammatory infiltrate. Mol Cell Biol. 2006; 26(14): 5348–5359.
  46. Castillo-Davis CI, Hartl DL. GeneMerge--post-genomic analysis, data mining, and hypothesis testing. Bioinformatics. 2003; 19(7): 891–892.
  47. Benjamini Y, Drai D, Elmer G, et al. Controlling the false discovery rate in behavior genetics research. Behav Brain Res. 2001; 125(1-2): 279–284.
  48. Reiner A, Yekutieli D, Benjamini Y. Identifying differentially expressed genes using false discovery rate controlling procedures. Bioinformatics. 2003; 19(3): 368–375.
  49. Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005; 102(43): 15545–15550.
  50. Aguiar RC, Yakushijin Y, Kharbanda S, et al. PTPROt: an alternatively spliced and developmentally regulated B-lymphoid phosphatase that promotes G0/G1 arrest. Blood. 1999; 94(7): 2403–2413.
  51. Klein U, Tu Y, Stolovitzky GA, et al. Transcriptional analysis of the B cell germinal center reaction. Proc Natl Acad Sci U S A. 2003; 100(5): 2639–2644.
  52. Ranuncolo SM, Polo JM, Dierov J, et al. Bcl-6 mediates the germinal center B cell phenotype and lymphomagenesis through transcriptional repression of the DNA-damage sensor ATR. Nat Immunol. 2007; 8(7): 705–714.
  53. Irish JM, Czerwinski DK, Nolan GP, et al. Kinetics of B cell receptor signaling in human B cell subsets mapped by phosphospecific flow cytometry. J Immunol. 2006; 177(3): 1581–1589.
  54. Chen L, Juszczynski P, Takeyama K, et al. Protein tyrosine phosphatase receptor-type O truncated (PTPROt) regulates SYK phosphorylation, proximal B-cell-receptor signaling, and cellular proliferation. Blood. 2006; 108(10): 3428–3433.
  55. Campbell K. Signal transduction from the B cell antigen-receptor. Current Opinion in Immunology. 1999; 11(3): 256–264.
  56. Dal Porto JM, Gauld SB, Merrell KT, et al. B cell antigen receptor signaling 101. Mol Immunol. 2004; 41(6-7): 599–613.
  57. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science. 2002; 298(5600): 1911–1912.
  58. Amoui M, Baylink DJ, Tillman JB, et al. Expression of a structurally unique osteoclastic protein-tyrosine phosphatase is driven by an alternative intronic, cell type-specific promoter. J Biol Chem. 2003; 278(45): 44273–44280.
  59. Friedberg JW, Sharman J, Schaefer-Cutillo J, et al. Fostamatinib Disodium (FosD), An Oral Inhibitor of Syk, Is Well-Tolerated and Has Significant Clinical Activity in Diffuse Large B Cell Lymphoma (DLBCL) and Chronic Lymphocytic Leukemia (SLL/CLL). Blood (ASH Annual Meeting Abstracts. 2008; 112: Abstract.
  60. Seimiya H, Sawabe T, Inazawa J, et al. Cloning, expression and chromosomal localization of a novel gene for protein tyrosine phosphatase (PTP-U2) induced by various differentiation-inducing agents. Oncogene. 1995; 10(9): 1731–1738.
  61. Wiggins RC, Wiggins JE, Goyal M, et al. Molecular cloning of cDNAs encoding human GLEPP1, a membrane protein tyrosine phosphatase: characterization of the GLEPP1 protein distribution in human kidney and assignment of the GLEPP1 gene to human chromosome 12p12-p13. Genomics. 1995; 27(1): 174–181.
  62. Cheng AM, Rowley B, Pao W, et al. Syk tyrosine kinase required for mouse viability and B-cell development. Nature. 1995; 378(6554): 303–306.
  63. Turner M, Gulbranson-Judge A, Quinn ME, et al. Syk tyrosine kinase is required for the positive selection of immature B cells into the recirculating B cell pool. J Exp Med. 1997; 186(12): 2013–2021.
  64. Colucci F, Guy-Grand D, Wilson A, et al. A new look at Syk in alpha beta and gamma delta T cell development using chimeric mice with a low competitive hematopoietic environment. J Immunol. 2000; 164(10): 5140–5145.
  65. Ci W, Polo JM, Cerchietti L, et al. The BCL6 transcriptional program features repression of multiple oncogenes in primary B cells and is deregulated in DLBCL. Blood. 2009; 113(22): 5536–5548.

Important: This website uses cookies. More >>

The cookies allow us to identify your computer and find out details about your last visit. They remembering whether you've visited the site before, so that you remain logged in - or to help us work out how many new website visitors we get each month. Most internet browsers accept cookies automatically, but you can change the settings of your browser to erase cookies or prevent automatic acceptance if you prefer.

Czasopismo Journal of Transfusion Medicine dostęne jest również w Ikamed - księgarnia medyczna

Wydawcą serwisu jest Via Medica sp. z o.o. sp. komandytowa, ul. Świętokrzyska 73, 80–180 Gdańsk

tel.:+48 58 320 94 94, faks:+48 58 320 94 60, e-mail: viamedica@viamedica.pl