Retention and defective assembly of the B-cell receptor in the endoplasmic reticulum of chronic lymphocytic leukaemia B cells cannot be reverted upon CD40 ligand stimulation

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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Does B Cell Receptor Signaling in Chronic Lymphocytic Leukaemia Cells Differ from That in Other B Cell Types?

Scientifica ◽

10.1155/2014/208928 ◽

2014 ◽

Vol 2014 ◽

pp. 1-14 ◽

Cited By ~ 3

Author(s):

Joseph R. Slupsky

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukaemia ◽

B Cell ◽

Lymphocytic Leukaemia ◽

Signal Transduction Pathway ◽

Cell Types ◽

Cell Receptor ◽

B Cell Receptor ◽

Incurable Disease ◽

B Cell Receptor Signaling

Chronic lymphocytic leukaemia (CLL) is an incurable malignancy of mature B cells. CLL is important clinically in Western countries because of its commonality and because of the significant morbidity and mortality associated with the progressive form of this incurable disease. The B cell receptor (BCR) expressed on the malignant cells in CLL contributes to disease pathogenesis by providing signals for survival and proliferation, and the signal transduction pathway initiated by engagement of this receptor is now the target of several therapeutic strategies. The purpose of this review is to outline current understanding of the BCR signal cascade in normal B cells and then question whether this understanding applies to CLL cells. In particular, this review studies the phenomenon of anergy in CLL cells, and whether certain adaptations allow the cells to overcome anergy and allow full BCR signaling to take place. Finally, this review analyzes how BCR signals can be therapeutically targeted for the treatment of CLL.

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Subversion of Pkca and Pkcbii Upregulation Play an Essential Role in Chronic Lymphocytic Leukaemia Development, Inducing Constitutive B Cell Receptor-Mediated Signals

Blood ◽

10.1182/blood.v126.23.488.488 ◽

2015 ◽

Vol 126 (23) ◽

pp. 488-488

Author(s):

Anuradha Tarafdar ◽

Ashfia Fatima Khan ◽

Emilio Cosimo ◽

Hothri A. Moka ◽

Karen Dunn ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukaemia ◽

B Cell ◽

Progenitor Cells ◽

Essential Role ◽

Lymphocytic Leukaemia ◽

Cell Receptor ◽

B Cell Receptor

Abstract B cell receptor (BCR)-mediated signals orchestrate key events during the life cycle of B lymphocytes enabling normal B cell development and maturation. Chronic lymphocytic leukaemia (CLL), an incurable malignancy of mature B cells, displays deregulated BCR-mediated signalling, the intensity of which correlates disease pathogenesis. As such, signals generated upon BCR engagement represent promising targets for novel therapies. Of note, the expression profile of selected protein kinase C (PKC) isoforms, which link proximal BCR mediated signals with downstream pathways, exhibit altered expression patterns in CLL cells: upregulation of PKCβII, PKCε, PKCζ and downregulation of PKCα and PKCβI compared with normal B cells. We previously demonstrated that introduction of a kinase inactive PKCα (PKCα-KR) construct in mouse lymphoid progenitor cells resulted in development of a CLL-like disease both in vitro and in vivo. This model resembles the more aggressive subset of CLL, exhibiting an upregulation of ZAP-70 and elevated ERK-MAPK-mTOR signalling resulting in enhanced proliferation and increased tumor load in the lymphoid organs. Interestingly, reduced PKCα function resulted in PKCβII upregulation, a key pathogenic feature of CLL. Inhibition of PKCβII in these cells with enzastaurin resulted in cell cycle arrest in vitro, reduced tumour load and elevated apoptosis in vivo indicating that PKCβII plays a vital role in maintaining cell survival in our model. To further elucidate the role of PKCβ in leukaemogenesis, we have performed sequential prkcb knockdown (KD) and PKCα-KR expression in the lymphoid progenitor cells. prkcb KD resulted in reduced proliferation and survival concurrent with reduced expression of leukaemic markers (CD23 and CD5) compared to control cells indicating that prkcb plays an essential role in initiation of leukaemogenesis in our model. To identify targets responsible for the regulation for prkcb transcription we performed global gene analysis (Affymetrix GeneChip mouse gene 1.0 ST) comparing MIEV (empty vector control) and PKCα-KR transduced cells at successive time-points mapping critical stages of B-cell transformation, pre- and post PKCβII upregulation. MetaCoreTM analysis revealed that upon upregulation of PKCβII, the BCR-mediated signalling pathway was significantly upregulated in PKCα-KR expressing cells. At the protein level, key hubs proximal to the BCR - Lyn, Btk and Akt - were upregulated, indicating constitutive activation of BCR signalling in the CLL-like PKCα-KR expressing cells. Additionally, proliferative signals downstream of the BCR (mTOR, NF-kB and c-myc) were also upregulated. Treatment of PKCα-KR expressing cells with the Btk inhibitor Ibrutinib (PCI-32765), which has recently been approved for the treatment of CLL, reduced cellular proliferation and inhibited phosphorylation of BtkY223, AktS473 and S6S235/236. Overall, we demonstrate that PKCβII expression is essential for leukaemogenesis and identify key signalling pathways that drive the initiation/development of CLL in the PKCα-KR model. Disclosures No relevant conflicts of interest to declare.

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