Role of RS/κDE in B Cell Receptor Editing

Abstract V3-21 gene usage defines a distinct genetic subgroup of chronic lymphocytic leukemia (CLL) characterized by a poor clinical outcome regardless of the VH mutation status. V3-21 cases exhibit a highly characteristic B-cell receptor (BCR) structure as demonstrated by homologous CDR3 sequences and a restricted use of VL genes implicating a common antigen involved in tumor pathogenesis of this specific CLL subgroup. To investigate the role of antigenic stimulation in the pathogenesis of V3-21 using CLL, we analyzed the quantitative expression of genes involved in BCR signaling (ZAP-70, SYK, BLNK, LYN, PI3K, PLCG2, FOS), B-cell activation (TRAF3, STAT6, NFKB), and cell cycle or apoptosis control (ATM, BCL-2, BAX, CDK4, CCND1, CCND2, CCND3, p27, E2F1, MYC) in V3-21 cases in comparison to VH mutated (VH MUT) and VH unmutated (VH UM) cases not using the V3-21 gene. To obtain native expression signatures we studied a non-CD19-purified (nPU) cohort (V3-21: 18 cases, equally divided into VH mutated and VH unmutated cases; VH MUT: 17; VH UM: 19) and, for verification, a CD19-purified (PU) cohort (V3-21: 10 cases, equally divided into VH mutated and unmutated; VH MUT: 12; VH UM: 16) to exclude a contamination of the results by non-tumor cells. All cases were analyzed by FISH for +3q, 6q-, +8q, 11q-, +12q, 13q-, 17p-, and t(11;14) to avoid major imbalances of genomic alterations between the subgroups under study. As expected, ZAP-70 expression was higher in VH UM as compared to VH MUT cases in the nPU (p=0.007) as well as the PU cohort (p=0.009). V3-21 cases showed a higher ZAP-70 expression as compared to VH MUT (nPU: p=0.033; PU: p=0.038). This applied also when restricting this comparison to V3-21 mutated cases (nPU: p=0.018). Median ZAP-70 expression in the PU cohort was 1.15 in VH MUT vs. 7.69 in VH UM cases, as compared to 7.05 in V3-21 cases (V3-21 mutated cases: 10.69; V3-21 unmutated: 6.7). Other genes differentially expressed between the V3-21 and VH MUT subgroups in nPU cases were PI3K (p=0.048), PLCG2 (p=0.007), CCND2 (p=0.003), p27 (p=0.003), BCL-2 (p=0.025), and ATM (p=0.006). In addition, a set of genes was detected with a differential expression between V3-21 and VH UM (nPU) including PLCG2 (p=0.014), NFKB (p=0.023), CCND2 (p=0.001), p27 (0.002), and BAX (p=0.028). Notably, except for ZAP-70, all of the differentially expressed genes showed a lower expression in V3-21 as compared to the other subgroups. When comparing the V3-21 mutated and V3-21 unmutated subgroups (nPU), there were no significant gene expression differences except for CDK4, which showed a lower expression in V3-21 unmutated cases. Therefore, cases with V3-21 usage appear to show a rather homogeneous gene expression pattern independently of the VH mutation status, which can be distinguished from VH MUT and VH UM cases not using V3-21. The expression differences observed suggest a role of differential BCR signaling in the pathogenesis of this distinct CLL subgroup. Deregulation of cell cycle, apoptosis, and candidate genes such as ATM indicate the involvement of additional pathways in the pathogenesis of CLL cases using V3-21.

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SYK Is a Tumor Suppressor In Pre-B Cell Acute Lymphoblastic Leukemia and Not a Therapeutic Target

Blood ◽

10.1182/blood.v116.21.4199.4199 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4199-4199

Author(s):

Carina Ng ◽

Rahul Nahar ◽

Emily Elliott ◽

Clifford A. Lowell ◽

Markus Muschen

Keyword(s):

Cell Cycle ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Leukemia Cells ◽

Imatinib Treatment ◽

Empty Vector Control ◽

Empty Vector ◽

Cycle Arrest

Abstract Abstract 4199 Background: 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). We recently established that the pre-B cell receptor functions as a tumor suppressor in pre-B acute lymphoblastic leukemia (ALL) including ALL cells carrying the BCR-ABL1 oncogene (Trageser et al., J Exp Med, 2009). In virtually all cases of BCR-ABL1 ALL, pre-B cell receptor function is compromised and its reconstitution induces rapid cell cycle arrest. Given that the SYK tyrosine kinase represents a critical signaling molecule in the pre-B cell receptor pathway, one would expect that SYK tyrosine kinase activity has a tumor suppressive effect. It therefore seems counterintuitive that pharmacologic targeting of SYK was recently proposed as a new treatment approach for pre-B ALL (Uckun et al., Br J Haematol. 2010). While there is solid evidence for a role of Syk as a target in B cell lymphoma (Friedberg et al., Blood 2010) and B cell lineage CLL (Buchner et al., Blood 2010), where tonic B cell receptor signaling delivers critical survival signals, the role of Syk downstream of the pre-B cell receptor in ALL is unclear. Results: To clarify the role of SYK downstream of the pre-B cell receptor in pre-B ALL, we performed a genetic experiment to inducibly delete the Syk gene in pre-B ALL cells. To this end, pre-B cells from Syk-fl/fl mice were propagated in the presence of IL7 and then transformed with retroviral BCR-ABL1 or MLL-ENL oncogenes. After transformation, pre-B leukemia cells were transduced with 4-hydroxy-tamoxyfen (4-OHT)-inducible retroviral Cre-ERT2 or an ERT2 empty vector control. After puromycin-selection of Cre-ERT2 and ERT2 transduced leukemia cells, Cre-ERT2 or the ERT2 control were induced by addition of 4-OHT and deletion of Syk was studied at different time points. As assessed by Western blot and PCR, deletion of Syk was near complete after two days and undetectable after six days. We then studied changes in cell viability upon inducible deletion of Syk: Acute deletion of the Syk tyrosine kinase had no significant impact on the viability of pre-B ALL cells, even after prolonged cell culture over several weeks. We then reasoned that the effect of Syk-deletion may be subtle yet important, so we studied in BCR-ABL1-transformed Syk-fl/fl pre-B leukemia cells whether Syk-deletion sensitizes to Imatinib-treatment. Deletion of Syk was again confirmed by Western blot, yet the dose-response curves to Imatinib-treatment were superimposable for Syk-fl/fl and Syk-del/del pre-B leukemia cells. We conclude that SYK does not contribute important survival signals in our mouse model for pre-B ALL, nor does deletion of Syk sensitize BCR-ABL1-driven pre-B leukemia cells to Imatinib-treatment. We next investigated the counter-hypothesis that Syk functions as a tumor suppressor downstream of the pre-B cell receptor. To test this possibility, we tested the effect of forced pre-B cell receptor expression in the presence or absence of Syk. Syk-fl/fl and Syk-del/del pre-B leukemia cells were transduced with CD8/μ-chain or a CD8 empty vector control. The μ-chain represents the central component of the pre-B cell receptor. Forced expression of the CD8 empty vector control had no effect regardless of whether Syk was deleted or not. When pre-B cell receptor signaling was reconstituted in Syk-fl/fl cells by expression of CD/μ-chain, viability of the leukemia decreased by >80%. By contrast, deletion of Syk greatly attenuated the tumor suppressive effect of CD8/μ-chain expression and less than 25% of the leukemia cells underwent cell cycle arrest and cell death. Background: Genetic deletion of Syk unequivocally demonstrates that Syk does not deliver critical survival signals downstream of the pre-B cell receptor in ALL. This is unlike B cell lymphoma, where tonic signaling from the B cell receptor promotes cell survival via Syk (Friedberg et al., 2010; Buchner et al., 2010). On the contrary, in pre-B ALL, the Syk kinase mediates pre-B cell receptor-induced cell cycle arrest. These findings are in direct conflict with a recent report on the therapeutic usefulness of pharmacological inhibition of Syk in pre-B ALL cell lines (Uckun et al., Br J Haematol.; 149: 508-17; 2010). The compound (C-61) used in this study may have unrecognized off-target effects, which might account for the discrepancies. Disclosures: No relevant conflicts of interest to declare.

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Characterisation of B Cell Receptor-Induced NF-KB Activation In Chronic Lymphocytic Leukaemia Cells

Blood ◽

10.1182/blood.v116.21.375.375 ◽

2010 ◽

Vol 116 (21) ◽

pp. 375-375 ◽

Cited By ~ 1

Author(s):

Fatima Talab ◽

Victoria Thompson ◽

John C Allen ◽

Ke Lin ◽

Joseph R Slupsky

Keyword(s):

B Cells ◽

Tyrosine Kinase ◽

Chronic Lymphocytic Leukaemia ◽

B Cell ◽

Cell Cultures ◽

Lymphocytic Leukaemia ◽

Cell Receptor ◽

B Cell Receptor ◽

Nfkb Pathway

Abstract Abstract 375 B cell receptor (BCR) signaling promotes survival of the malignant clone in chronic lymphocytic leukaemia (CLL) through its ability to stimulate NFkB pathway signaling. In lymphoid cells, antigen receptor stimulation of this pathway is achieved by engaging the Carma-1 – Bcl10 – MALT1 (CBM) complex for eventual activation of I-kB kinases (IKKs). In B cells, protein kinase C beta (PKCbeta) is an important mediator of CBM complex activation. However, in CLL cells we found that PKCs do not appear to have a role in BCR-mediated NFkB pathway signaling, despite high expression levels of PKCbeta, because the presence of specific inhibitors of this kinase (LY379196 and bisindolylmaleimide-I) has no effect on the induction of IKK phosphorylation during BCR crosslinking. Examination of CBM complex expression suggests an explanation for this phenomenon; the expression levels of Carma-1 and MALT-1 are largely similar in CLL and normal B cells, but the expression of Bcl10 is much reduced in CLL cells. These findings, taken together with the established role of Bcl10 in the pathway of BCR-induced NFkB activation, suggest that CLL cells may employ a different mechanism to activate this pathway during BCR stimulation. Tyrosine kinases are known to play a role in BCR-induced IKK activation in CLL cells because compounds like dasatinib and PP2 inhibit NFkB pathway activation by BCR. One possible tyrosine kinase is c-Abl because we have shown this protein to be overexpressed in CLL cells, where it plays a role in activation of the NFkB pathway. To investigate the role of c-Abl in BCR-induced IKK activation, we used the inhibitor imatinib and found that the presence of this compound partially inhibited IKK phosphorylation in BCR-stimulated CLL cells. However, imatinib can also inhibit Lck, a T cell-specific src-family tyrosine kinase that is expressed by CLL cells. To differentiate between Lck- and c-Abl-mediated BCR signals we used the specific inhibitor 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[3,2d] pyrimidin-7-yl-cyclopentane (Lck-i). We found that the presence of this compound in CLL cell cultures undergoing BCR stimulation almost completely inhibited the induction of IKK activation. Investigation of Lck-i specificity revealed this compound did not inhibit either c-Abl or Lyn at the concentration used to inhibit Lck in CLL cell cultures. Further investigation of the effects of Lck-i showed that this compound was also effective in inhibiting BCR-induced activation of the Akt and ERK signaling pathways. Taken together, these data suggest a major role for Lck in BCR-mediated signaling in CLL cells, and question the existing paradigm on the importance of Lyn. Disclosures: No relevant conflicts of interest to declare.

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The Role of Canonical Transient Receptor Potential 7 in B-cell Receptor-activated Channels

Journal of Biological Chemistry ◽

10.1074/jbc.m507606200 ◽

2005 ◽

Vol 280 (42) ◽

pp. 35346-35351 ◽

Cited By ~ 40

Author(s):

Jean-Philippe Lievremont ◽

Takuro Numaga ◽

Guillermo Vazquez ◽

Loïc Lemonnier ◽

Yuji Hara ◽

...

Keyword(s):

B Cell ◽

Transient Receptor Potential ◽

Receptor Potential ◽

Cell Receptor ◽

B Cell Receptor ◽

Canonical Transient Receptor Potential ◽

Transient Receptor

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Essential Role of Phospholipase Cγ2 in Early B-Cell Development and Myc-Mediated Lymphomagenesis

Molecular and Cellular Biology ◽

10.1128/mcb.00839-06 ◽

2006 ◽

Vol 26 (24) ◽

pp. 9364-9376 ◽

Cited By ~ 22

Author(s):

Renren Wen ◽

Yuhong Chen ◽

Li Bai ◽

Guoping Fu ◽

James Schuman ◽

...

Keyword(s):

B Cells ◽

Transgenic Mice ◽

B Cell ◽

Cell Receptor ◽

Cell Development ◽

B Cell Development ◽

B Cell Receptor ◽

Wild Type ◽

Interleukin 7

ABSTRACT Phospholipase Cγ2 (PLCγ2) is a critical signaling effector of the B-cell receptor (BCR). Here we show that PLCγ2 deficiency impedes early B-cell development, resulting in an increase of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B cells. PLCγ2 deficiency impairs pre-BCR-mediated functions, leading to enhanced interleukin-7 (IL-7) signaling and elevated levels of RAGs in the selected large pre-B cells. Consequently, PLCγ2 deficiency renders large pre-B cells susceptible to transformation, resulting in dramatic acceleration of Myc-induced lymphomagenesis. PLCγ2 −/− Eμ-Myc transgenic mice mainly develop lymphomas of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B-cell origin, which are uncommon in wild-type Eμ-Myc transgenics. Furthermore, lymphomas from PLCγ2 −/− Eμ-Myc transgenic mice exhibited a loss of p27Kip1 and often displayed alterations in Arf or p53. Thus, PLCγ2 plays an important role in pre-BCR-mediated early B-cell development, and its deficiency leads to markedly increased pools of the most at-risk large pre-B cells, which display hyperresponsiveness to IL-7 and express high levels of RAGs, making them prone to secondary mutations and Myc-induced malignancy.

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