High Efficiency Of the PDPK1-Inhibitor, BX912, In MCL

Abstract Background Mantle cell lymphoma (MCL) is an aggressive lymphoid malignancy with a median survival of 3-5 years. New strategies including proteasome inhibitors, immune modulatory drugs (IMiDs) and mTOR inhibitors achieve high response rate. Emerging data support the clinical efficiency of inhibitors of the B-cell receptor pathway. PDPK1 (3-phosphoinositide dependent protein kinase-1) is an important downstream target of this crucial pathway. Methods MCL cell lines (Granta 519, Jeko-1, Rec-1) were exposed to various PDK1-Inhibitors (OSU-03012, BX912; BX517; GSK2334470; 0,0625-1µM) and cell proliferation was analysed by WST assay. The effect of BX912 was tested on 6 MCL. Cell proliferation (trypanblue staining), induction of apoptosis (Annexin V PE/7-AAD staining) and cell cycle (FACS) were investigated. In MCL cell lines protein expression of the PI3K/Akt/mTOR pathway candidates (Akt, mTOR, eIF4E, PDK) was analysed after 24h BX912 exposure. Combined approaches were evaluated by cell proliferation analysis (WST-assay, trypan blue staining). In an alternative approach PDPK1 expression was downregulated by siRNA and consequently investigated in detail. Results BX912 appeared to be the most potent PDPK1-Inhibitor in the MCL cell lines tested. Sensitivity to BX912 was detected in 4 out of 6 MCL- (IC50: 0,25 -0,75µM). In addition sensitive MCL cells showed strong G2 arrest. In contrast healthy donor lymphocytes did not respond to PDPK1-inhibition. In MCL cell lines response to BX912 correlated with the PDPK1 protein expression status. Treatment with BX912 led to downregulation of PDPK1 protein expression and dephosphorylation of Rictor, Raptor, RSK and eIF4E proteins in the most sensitive to the inhibitor MCL cell line, Z138, suggesting a mode of action of BX912 mainly through the mTOR pathway. Combination of the PDPK1-inhibitors (BX912; OSU-03012, GSK2334470) with each other revealed synergism especially in combinations with GSK2334470 (BX912 and GSK2334470: CI 0,89; OSU-03012 and GSK2334470: CI 0,484; BX912 and OSU-03012: CI: 1,3), substantiating the therapeutic benefit of comprehensive PDPK1 – inhibition in MCL. Combination experiments of BX912 with inhibitors of the B-cell receptor pathway (PI3K, mTOR, PKCß) and the JAK/STAT-pathway (PIM1, JAK1/2) exhibited BX912 and the PI3K-inhibitor, CAL101, as the most potent combination (CI 0,7 -0,91) in MCL cell lines. To uncover the molecular mode of action of this combination, results of protein expression analysis will be shown. Conclusions The PDPK1 inhibitor BX912 shows high efficiency in MCL cells. Our data let suggest PDPK1 inhibition as a new targeted approach in MCL. However further exploration of the underlying molecular mechanisms is warranted to guide the future development of combined treatment approaches. Disclosures: Dreyling: Janssen: Support of IITs, Scientiffic advisory board, Speakers honoraria Other; Hoffmann-La Roche: Support of IITs, Speakers honoraria, Support of IITs, Speakers honoraria Other.

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B-Cell Receptor Signaling Suppresses TAp63-Induced Apoptosis Via PI3-K/mTOR Pathway and Upregulation of MiR-21 in Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood.v120.21.562.562 ◽

2012 ◽

Vol 120 (21) ◽

pp. 562-562

Author(s):

Leigh Ann Humphries ◽

Darcy Bates ◽

Claire Godbersen ◽

Prabhjot Kaur ◽

Alexey V. Danilov

Keyword(s):

B Cells ◽

B Cell ◽

Cell Lines ◽

B Cell Lymphoma ◽

Cell Receptor ◽

Mtor Pathway ◽

B Cell Receptor ◽

Lymphoma Cell ◽

Raji Cells ◽

Transcript Levels

Abstract Abstract 562 p63, an ancestral homolog of p53, encodes two major variants that have variable expression and context-specific functions in malignant tissues. We and others have shown that N-terminally truncated ΔNp63 promotes tumor growth in carcinomas. Meanwhile, the full-length TAp63 variant, which predominates in lymphoid malignancies, is anti-oncogenic in solid tumor models, where it mediates Ras-induced cellular senescence, suppresses anchorage-independent growth, and induces apoptosis. In hepatoma cells, TAp63 activates both extrinsic and intrinsic apoptosis pathways and enhances chemosensitivity. CLL clonal B cells have a low proliferative potential and disrupted apoptotic mechanism as a result of intrinsic defects and interaction with the microenvironment. At the crossroads of those pathways, the B-cell receptor (BCR) serves as a key survival molecule in CLL. Little is known about whether p63 regulates B-cell survival in CLL. Here we sought to investigate the role of TAp63 in regulation of apoptosis in CLL B cells and lymphoma cell lines and determined whether B-cell receptor signaling modulates p63. Forty-eight patients with B-CLL were enrolled in this study. CLL B cells were isolated from peripheral blood using standard Ficoll-Hypaque technique and co-cultured with M210B4 murine stroma cell line layers in RPMI supplemented with 15% fetal bovine serum (FBS). B-cell lymphoma cell lines Daudi, DOHH, Raji, OCI-LY3, OCI-LY19, SU DHL-4 and SUDHL-10 were maintained in RPMI with 10% FBS. CLL B cells and Raji cells were transfected with TAp63α expression vector or with siRNAs targeting p63 or miR-21 using Lonza Nucleofector with B-cell nucleofection solution (CLL B cells) and Solution V (Raji cells). Apoptosis was quantified by means of Annexin V/7-AAD staining and flow cytometry. B-cell receptor was engaged with 5 mg/mL (Raji cells) or 10 mg/mL IgM (CLL B cells). Expression of p63 and miR-21 was evaluated by immunoblotting and real-time RT-PCR. Median age of patients was 63 years. Median follow up was 3 years. Most patients presented in Rai stage 0–1 (80%). TAp63α was the predominantly expressed p63 variant in CLL cells and 7 lymphoma cell lines. Compared with normal B cells, TAp63 mRNA transcript levels were low in 28 CLL patient samples (using an arbitrary cutoff of <10% normal; 58.3%) and normal/elevated in 20 samples (41.7%). Patients with high expression of p63 were more likely to exhibit unmutated IGHV (U-CLL; p=0.016). siRNA-mediated knockdown of p63 in CLL cells resulted in protection from spontaneous apoptosis of CLL cells cultured on M210B4 murine bone marrow stroma (p<0.01) and was accompanied by a reduction in Noxa, Puma and Bax transcript levels. By contrast, enforced expression of TAp63α enhanced apoptosis. p63 knockdown in the Raji lymphoma cells resulted in increased proliferation and metabolic activity (p<0.05). B-cell receptor engagement suppressed p63 expression in CLL cells and Raji lymphoma cells. Pre-incubation of Raji cells with Syk inhibitor R406 and inhibitors of the PI-3K/mTOR pathway (LY294002, rapamycin, and CAL-101) resulted in the reversal of this phenomenon. Meanwhile, chemical inhibition of MEK, Erk, JNK, and p38 and siRNA-mediated knockdown of the transcription factor FOXO (a downstream targets of PI-3K) had no effect on p63 expression. Since TAp63α is a known target of miR-21, a microRNA that has been implicated in the pathogenesis of CLL, we examined their relationship in CLL and lymphoma. We found that TAp63 transcript levels inversely correlated with the expression of miR-21 in CLL B cells, but not in lymphoma cell lines. BCR stimulation led to increased miR-21 levels in CLL B cells, whereas knockdown of miR-21 resulted in upregulation of TAp63 in 3 out of 5 tested samples. TAp63α is the predominantly expressed p63 variant in the peripheral blood CLL cells and B-cell lymphoma cell lines, where it modulates the apoptosis program. BCR signaling repressed TAp63α via PI-3K/mTOR pathway and via upregulation of miR-21. This may be particularly relevant in U-CLL, where baseline p63 levels were higher. These data provide additional insights and rationale for targeting the BCR pathway and miR-21 in CLL and lymphoma. Disclosures: No relevant conflicts of interest to declare.

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Synergistic Blockade Of Activated B Cell-Like DLBCL Proliferation With a Selective Inhibitor Of IRAK4 In Combination With Inhibition Of The B-Cell Receptor Signaling Network

Blood ◽

10.1182/blood.v122.21.3833.3833 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3833-3833 ◽

Cited By ~ 7

Author(s):

Divya Chaudhary ◽

Nancy Wood ◽

Donna L. Romero ◽

Shaughnessy D. Robinson ◽

Jeremy R Greenwood ◽

...

Keyword(s):

Cell Proliferation ◽

B Cell ◽

Cell Survival ◽

Cell Lines ◽

Immune Cells ◽

Cell Receptor ◽

B Cell Receptor ◽

Inflammatory Signaling ◽

Bcr Signaling ◽

Equity Ownership

Abstract Toll-Like Receptor (TLR) and IL-1 signaling is mediated by the adaptor protein MyD88 through IRAK4 activation. TLR and IL-1 family ligands activate NFkB through this pathway and stimulate proliferation and cell survival, as well as induce cytokine and chemokine production that can amplify tumor cell survival. The gain-of-function L265P mutation in MyD88 occurs in ∼30% of patients with activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) and ∼90% of Waldenström’s macroglobulinemia. Therefore, inhibition of IRAK4 may be therapeutically relevant in hematologic malignancies containing MyD88 mutations. Recent clinical results with kinase inhibitors strongly support a role for signaling through the B-cell receptor (BCR) pathway in the progression of hematological malignancies including ABC-DLBCL. We were interested to understand the potential utility of selective IRAK4 inhibitors in combination with inhibition of the BCR signaling networks. We have reported previously the identification and characterization of potent and selective IRAK4 inhibitors that are effective in blocking inflammatory signaling in immune cells and demonstrate efficacy in vivo in models of autoimmune disease. ND-2158, a potent (Ki of 1.2 nM) and highly selective IRAK4 inhibitor has been shown to be effective in reducing the proliferation of ABC-DLBCL cell lines. ND-2158 does not decrease cell viability for other cell lines that lack the MyD88 mutation including a germinal center-like DLBCL cell line, BJAB, suggesting that the anti-proliferative effects in ABC-DLBCL cells relate in part to the activating MyD88 mutation. Complete cross-over dose-response proliferation studies of the ABC-DLBCL cell line, OCI-LY10, were conducted using ND-2158 in combination with blockade of key BCR signaling network nodes, using inhibitors of either Btk (ibrutinib), PI3Kdelta (GS-1101), or Syk (P505-15). Isobologram analysis using the Chou-Talalay method revealed that ND-2158 was able to synergistically block cell proliferation in combination with ibrutinib, P505-15, or GS-1101. Interestingly, we find that blockade of SYK, PI3Kdelta, or BTK signaling enhances the potency of ND-2158 in ABC-DLBCL cells. The IC50 values observed in this context are comparable to the potency of ND-2158 when used as a single agent to inhibit inflammatory signaling in immune cells that are not dependent on BCR signaling. The cell proliferation blockade IC50for ND-2158 shifted from an average value of ∼7 μM to 0.19, 0.05, or 0.15 μM, when combined with the IC50 concentrations of the inhibitors of BTK, PI3Kdelta or SYK kinases, respectively. These results suggest that inhibition of both BCR signaling pathways that are amplified in ABC-DLBCL, and IRAK4 signaling activated through MyD88 mutations, are required for a more complete blockade of ABC-DLBCL proliferation. Moreover, we explored ND-2158 combination with lenalidomide, known to be synergistic with BCR and NFkB pathway inhibitors. In contrast to combinations with BCR signaling inhibition, studies with lenalidomide failed to demonstrate an additive or synergistic activity when combined with IRAK4 inhibition in ABC-DLBCL cell lines. Therefore, we conclude that IRAK4 activation, as well as aberrant BCR signaling, are likely to contribute to the proliferative capacity of ABC-DLBCL. We propose that combinatorial therapeutic approaches, including inhibition of IRAK4, may provide benefit for patients with ABC-DLBCL. Disclosures: Chaudhary: Nimbus Discovery Inc.: Employment. Off Label Use: Exploratory inhibitor of IRAK4 for research purposes. Wood:Nimbus Discovery Inc.: Employment. Romero:Nimbus Discovery Inc.: Consultancy, Equity Ownership. Robinson:Schrodinger Inc. Consultant to Nimbus Discovery Inc.: Consultancy. Greenwood:Schrodinger Inc. Consultant to Nimbus Discovery Inc.: Consultancy. Shelley:Schrodinger Inc. Consultant to Nimbus Discovery Inc.: Consultancy. Morin:Nimbus Discovery Inc.: Consultancy. Kapeller:Nimbus Discovery Inc.: Employment. Westlin:Nimbus Discovery Inc.: Employment, Equity Ownership.

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The PI3K Delta Inhibitor Idelalisib Interferes With Pre-B Cell Receptor Signaling In Acute Lymphoblastic Leukemia (ALL): A New Therapeutic Concept

Blood ◽

10.1182/blood.v122.21.2632.2632 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2632-2632

Author(s):

Nathalie Y. Rosin ◽

Ekaterina Kim ◽

Stefan Koehrer ◽

Zhiqiang Wang ◽

Susan O'Brien ◽

...

Keyword(s):

Gene Expression ◽

Protein Expression ◽

B Cell ◽

Cell Lines ◽

Cell Receptor ◽

B Cell Receptor ◽

Differentially Expressed ◽

B Cell Malignancies ◽

Bcr Signaling ◽

Dose Dependent

Abstract Phosphoinositide-3-kinases (PI3K) play an important role in transmitting signals from surface receptors such as the B-cell receptor (BCR), cytokine receptors and receptor tyrosine kinases, that result in survival and growth of normal and malignant B cells. In mature B-cell malignancies such as CLL and indolent B-NHL, the PI3K pathway is constitutively upregulated and is dependent on PI3Kδ. Idelalisib is a p110δ-isoform-specific PI3K inhibitor that is highly active in patients with CLL and indolent NHL. In contrast to mature B cell malignancies, expression and function of PI3Kδ in B-ALL has not been well characterized. RNA expression of the PI3K isoforms α, β, γ, and δ was detected in all B-ALL cell lines. Protein expression was analyzed by immunoblotting. We noted that in vitro responsiveness to idelalisib treatment was associated with protein expression of the δ isoform and presence of the Pre-BCR. We found that treatment of B-ALL cell lines with idelalisib at concentrations between 10 nM and 5 µM inhibited metabolism and growth of B-ALL cells expressing a pre-BCR, whereas only minor effects were observed in Pro-B. To monitor the expression and phosphorylation of proteins (CD72, Akt, Plcγ2, S6, Vav) involved in BCR and PI3K signaling, we prepared protein lysates of B-ALL cell lines. Cells were treated with idelalisib with/without stimulation of the µ-heavy chain of the (Pre-) BCR. We were able to show an increase of pAktSer473 and pS6Ser235/236 after stimulation, as well as a decrease in a dose dependent manner with idelalisib. The decreased amount of Akt phosphorylation was linear with the sensitivity to idelalisib treatment of the cell lines. To investigate intracellular calcium mobilization which occurs in B cells through pre-BCR stimulation, we treated cells with idelalisib and stimulated them with anti-Igµ. A dose dependent decrease in calcium flux was observed in 5 of 6 pre-B cell lines. To examine the effects of idelalisib treatment on the gene expression of pre-B ALL cells, gene expression profiling (GEP) was performed. This revealed down regulation of several genes involved in MAP-Kinase signaling, (Pre-) BCR signaling and Natural Killer cell (NK-cell) mediated cytotoxicity. For the Pre-BCR signaling pathway several genes were differentially expressed, including genes encoding the following proteins, which were found to be down regulated after 3 days of 1 µM idelalisib treatment: BCL-6, CD72, CD79a, Vav, and Plcγ2. To verify the data of the GEP, qPCR analysis was performed. To further investigate the effects of idelalisib on proteins involved in BCR signaling, six Pre-B-ALL cell lines, as well as one mature and one Pro-B cell line were treated with 1 µM idelalisib and the protein expression was quantified after immunoblotting. Most of the proteins that were differentially expressed on genomic levels have also been differentially expressed on proteomic levels and therefore confirmed the effect on Pre-BCR signaling. In summary, these experiments demonstrate inhibition of Pre-BCR signaling on both gene and protein expression levels via idelalisib treatment of Pre-B-ALL cell lines and support the importance of clinical development of the δ isoform specific PI3K inhibitor idelalisib. Disclosures: Burger: Gilead Sciences Inc: Research Funding.

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Positive Signaling Through CD72 Induces Mitogen-Activated Protein Kinase Activation and Synergizes with B Cell Receptor Signals to Induce X-Linked Immunodeficiency B Cell Proliferation

The Journal of Immunology ◽

10.4049/jimmunol.167.3.1263 ◽

2001 ◽

Vol 167 (3) ◽

pp. 1263-1273 ◽

Cited By ~ 27

Author(s):

Hsin-Jung Wu ◽

Chandrasekar Venkataraman ◽

Steven Estus ◽

Chen Dong ◽

Roger J. Davis ◽

...

Keyword(s):

Cell Proliferation ◽

Protein Kinase ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Mitogen Activated Protein Kinase ◽

Kinase Activation ◽

Protein Kinase Activation ◽

Mitogen Activated Protein

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Co-engaging CD47 and CD19 with a bispecific antibody abrogates B-cell receptor/CD19 association leading to impaired B-cell proliferation

mAbs ◽

10.1080/19420862.2018.1558698 ◽

2019 ◽

Vol 11 (2) ◽

pp. 322-334 ◽

Cited By ~ 11

Author(s):

Eric Hatterer ◽

Leticia Barba ◽

Nelly Noraz ◽

Bruno Daubeuf ◽

Jean-Pierre Aubry-Lachainaye ◽

...

Keyword(s):

Cell Proliferation ◽

B Cell ◽

Bispecific Antibody ◽

Cell Receptor ◽

B Cell Receptor

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Antigens Varying in Affinity for the B Cell Receptor Induce Differential B Lymphocyte Responses

Journal of Experimental Medicine ◽

10.1084/jem.188.8.1453 ◽

1998 ◽

Vol 188 (8) ◽

pp. 1453-1464 ◽

Cited By ~ 109

Author(s):

Valerie Kouskoff ◽

Sara Famiglietti ◽

Georges Lacaud ◽

Paul Lang ◽

James E. Rider ◽

...

Keyword(s):

Cell Proliferation ◽

B Cell ◽

Cell Activation ◽

B Lymphocyte ◽

Receptor Occupancy ◽

Cell Receptor ◽

Interferon Γ ◽

B Cell Receptor ◽

Multiple Parameters ◽

Biological Responses

The B cell receptor (BCR) triggers a variety of biological responses that differ depending upon the properties of the antigen. A panel of M13 phage-displayed peptide ligands with varying affinity for the 3-83 antibody was generated to explore the role of antigen-BCR affinity in cell activation studies using primary 3-83 transgenic mouse B cells. Multiple parameters of activation were measured. T cell–independent B cell proliferation, antibody secretion, induction of germline immunoglobulin γ1 transcripts, and B cell production of interleukin (IL) 2 and interferon γ responses were better correlated with antigen-BCR affinity than with receptor occupancy. In contrast, other responses, such as upregulation of major histocompatibility complex class II and B7.2 (CD86), secretion of IL-6, and B cell proliferation in the context of CD40 signaling were only weakly dependent on antigen affinity. Biochemical analysis revealed that at saturating ligand concentrations the ability of phage to stimulate some early signaling responses, such as Ca++ mobilization and tyrosine phosphorylation of syk or Igα, was highly affinity dependent, whereas the ability to stimulate Lyn phosphorylation was less so. These data suggest that the BCR is capable of differential signaling. The possibility that differential BCR signaling by antigen determines whether an antibody response will be T independent or dependent is discussed.

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Rituximab But Not GA101 Is Partially Antagonistic With Inhibitors Of The B-Cell Receptor Pathway In Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood.v122.21.4420.4420 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4420-4420

Author(s):

Anna-Katharina Zoellner ◽

Nico Peter ◽

Grit Hutter ◽

Yvonne Zimmermann ◽

Wolfgang Hiddemann ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cell Receptor ◽

Advisory Board ◽

B Cell Receptor ◽

Additive Effects ◽

Anti Cd20 ◽

The Impact

Introduction Anthracyclin-containing immuno-chemotherapy represents the current standard approach in diffuse large cell B cell lymphoma (DLBCL). However, especially in ABC lymphoma new therapeutic approaches are warranted. Small molecule inhibitors of the B- cell receptor pathway have recently achieved high response rates in several lymphoma subtypes. Since rituximab has been previously described to influence the PI3K- AKT pathway, we investigated the impact of rituximab as well as the novel glycoengineered type II anti-CD20 antibody GA101 (obinutuzumab) in combination with the PI3K- delta inhibitor idelalisib and BTK inhibitor ibrutinib. Methods Established DLBCL ABC (U2932, OCI-Ly10, OCI-Ly3, HBL-1) and GCB (HT, WILL-2, SU-DHL-5, SU-DHL-4, ULA) cell lines were cultivated under standard conditions and exposed to previously determined doses of compounds (rituximab [R]: 1 µg/ml, GA101 [G]: 1 µg/ml, idelalisib [ID]: 5 µM, ibrutinib [I]: 5 nM). Viable cells were determined after 24, 48 and 72 hours based on trypane blue exclusion test. Western blot analysis was performed after 1, 6, 12 and 24 h. All experiments were performed at least in triplicates. Results Rituximab in combination with idelalisib showed differential effects in ABC and GCB cell lines. In ABC cell lines the combination was not superior to single substances after 48 h (OCI-LY10: R: 70%, ID: 83%, R+ID: 76%; U2932: R: 63%, ID: 88%, R+ID: 58%) whereas after 72 h additive effects were observed (OCI-LY10: R: 78%, ID: 77%, R+ID: 57%; U2932: R: 58%, ID: 87%, R+ID: 47%). In GCB cell lines, rituximab and idelalisib again were partially antagonistic and did not increase the effect of single drugs (48 h: ULA: R: 79%, ID: 76%, R+ID: 76%; 72 h: SU-DHL-5: R: 87%, ID: 69%, R+ID: 64%). Combination treatment with GA101 and idelalisib was more effective in both subtypes. In ABC cell lines cell counts were additively reduced after 72 h (U2932: G: 40%, ID: 47%, G+ID: 33%; HBL-1: G: 83%, ID: 86%, G+ID: 63%). Similar additive effects were detected in GCB cell lines (SU-DHL-5: G: 91%, ID: 69%, G+ID: 52%; ULA: G: 59%, ID: 68%, G+ID: 50%). As expected, ibrutinib was not effective in GCB cell lines. In ABC cell lines effects of the combination with rituximab or GA101 were comparable to ibrutinib only (OCI-LY10: R: 84%, I: 51%, R+I: 49%; HBL-1: G: 76%, I: 76%, G+I: 77%). In contrast to published data downregulation of p-AKT was detected after antibody treatment in neither ABC nor GCB cell lines. Idelalisib significantly reduced expression of p-AKT already after 1 h in GCB cell lines (ULA). The combination of idelalisib and GA101 also downregulated potently p-AKT whereas the rituximab combination did not reduce p-AKT expression as pronounced. Similar differences were observed In the ABC cell line U2932. Conclusion The combination of rituximab and idelalisib induced a partially antagonistic effect in GCB cell lines. In contrast, in ABC an additive effect of the combination was observed at all time points. Combination of GA101 and idelalisib was more effective in ABC and GCB lymphoma cell lines potentially due to the more pronounced down regulation of p-AKT. These in vitro data suggest that GA101 may overcome the previously reported antagonism of anti CD20 antibodies and inhibitors of the B-cell receptor pathway. However, the relevance of these data has to be validated in clinical trials. Disclosures: Hiddemann: Hoffmann-La Roche: Support of IITs, Scientiffic advisory board, Speakers honoraria Other. Dreyling:Hoffmann-La Roche: Support of IITs, Speakers honoraria, Support of IITs, Speakers honoraria Other; Janssen: Support of IITs, Scientiffic advisory board, Speakers honoraria Other.

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An Alternatively Spliced Form of CD79b Gene May Account for Altered B-Cell Receptor Expression in B-Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v93.7.2327.407a08_2327_2335 ◽

1999 ◽

Vol 93 (7) ◽

pp. 2327-2335 ◽

Cited By ~ 1

Author(s):

A. Alfarano ◽

S. Indraccolo ◽

P. Circosta ◽

S. Minuzzo ◽

A. Vallario ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Lines ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Receptor Expression ◽

Rt Pcr ◽

Alternatively Spliced

Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Ig/Igβ (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19+ cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5+ and CD5− B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5+ and CD5− B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 ± 0.20 SD in normal donors and 0.44 ± 0.27 SD in B-CLL (P = .01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT → TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.

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