scholarly journals Kinobead Profiling Reveals Reprogramming of B-cell Receptor Signaling in Response to Therapy Within Primary Chronic Lymphocytic Leukemia Cells

2019 ◽  
Author(s):  
AJ Linley ◽  
LI Karydis ◽  
A Mondru ◽  
A D’Avola ◽  
S Cicconi ◽  
...  
Keyword(s):  
B Cell ◽  
Disease Progression ◽  
Cell Receptor ◽  
Malignant Cell ◽  
B Cell Receptor ◽  
Response To Therapy ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling ◽  
B Cell Receptor Signaling ◽  
Primary Chronic Lymphocytic Leukemia

ABSTRACTSignaling via the B-cell receptor (BCR) is critical for driving CLL pathobiology, promoting both malignant cell survival and disease progression. However, understanding of this pathway is limited, particularly in relation to potential changes in response to therapy. Here, we describe a kinobead-based protocol, used in conjunction with mass-spectrometry to study surface-IgM signaling in primary CLL cells. We identified a ‘fingerprint’ of over 30 kinases which displayed unique, patientspecific response following sIgM stimulation. Matched analysis of CLL cells in samples taken from clinical trials showed that BCR-induced kinome responses altered between baseline and disease progression in patients who relapsed from chemoimmunotherapy. Moreover, adaptive changes to BCR signaling were also observed in CLL cells from clinical trial patients receiving ibrutinib; longitudinal profiling revealed increased signaling despite BTK inhibition. Collectively, these data comprise the first comprehensive investigation into BCR signaling response within CLL where kinobead profiling reveals unique evidence of adaptive reprogramming in response to therapy.

High ZAP-70 and Differential Expression of B-Cell Receptor Related Genes in Chronic Lymphocytic Leukemia with V3-21 Gene Usage.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 773-773
Author(s):  
Dirk Kienle ◽  
Alexander Kröber ◽  
Dirk Winkler ◽  
Daniel Mertens ◽  
Annett Habermann ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mutation Status ◽  
Bcr Signaling ◽  
Gene Usage ◽  
Lower Expression

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.

B-Cell Receptor Signaling Enhances Migration of B-Cell Chronic Lymphocytic Leukemia Cells in Response to the Chemokine Stromal Cell-Derived Factor-1 (SDF-1/CXCL12).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1187-1187
Author(s):  
Jan A. Burger ◽  
Myriam Krome ◽  
Andrea Bürkle ◽  
Tanja N. Hartmann
Keyword(s):  
Dendritic Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Stromal Cells ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Follicular Dendritic Cells ◽  
Accessory Cells ◽  
Bcr Signaling

Abstract There is growing evidence that the microenvironment confers survival signals to Chronic Lymphocytic Leukemia (CLL) B-cells that may result in disease progression and resistance to therapy. In the marrow or secondary lymphoid tissues, CLL cells are in close contact with non-tumoral accessory cells, such as mesenchymal stromal cells or nurselike cells. We previously characterized SDF-1 (CXCL12) as a central mediator for CLL cell migration and interaction with the protective microenvironment. Constitutive secretion of CXCL12 attracts CLL cells to stroma or NLC through its cognate receptor, CXCR4. These accessory cells protect CLL cells from spontaneous or drug-induced apoptosis, which is contact-dependent and partially mediated by CXCL12. B-cell receptor (BCR) signaling has been considered another important regulator of CLL cell survival. Typically, CLL cell that lack somatic mutations in the immunoglobulin (Ig) variable region (V) genes and display high levels of the tyrosine kinase ZAP-70 strongly responds to anti-IgM stimulation. Because both, CXCL12 stimulation and BCR signaling may represent important mechanism for maintenance of CLL cell within the microenvironment, we examined whether anti-IgM stimulation affects CXCL12 responses in correlation with the ZAP-70 status. BCR signaling was modulated either by crosslinking the BCR with IgM or by blocking the tyrosine kinase Syk. Effective BCR cross-linking with anti-IgM antibodies was demonstrated by phosphorylation of Syk and p44/42 MAP kinase. In ZAP-70 positive cells, BCR crosslinking resulted in a robust activation of Syk, p44/42 MAP kinases, and protein kinase B (Akt). ZAP-70 negative CLL cells displayed a weaker activation of p44/42 upon IgM crosslinking. Pretreatment of CLL cells with anti-IgM resulted in an enhanced calcium mobilization upon CXCL12 stimulation. This was not due to changes in surface expression of CXCR4. Accordingly, Syk inhibition by piceatannol resulted in a loss of calcium response upon CXCL12 stimulation. Furthermore, anti-IgM stimulation significantly increased CLL cell chemotaxis towards CXCL12 1.4 ± 1.2fold (n=9, p=0.027), and Syk inhibition by piceatannol decreased chemotaxis to 0.6 ± 0.2fold of controls (n=8). In these experiments, we could not detect differences between ZAP-70 positive or negative cells. However, there was a strong difference regarding the spontaneous, CXCL12-dependent migration of CLL cells beneath marrow stromal cells (pseudoemperipolesis). BCR crosslinking significantly increased pseudoemperipolesis of ZAP-70 expressing CLL cells 13.4 ± 21.0fold (n=7, p=0.043), whereas there was no significant increase in pseudoemperipolesis of ZAP-70 negative cells (1.4 ± 0.2fold increase, n=8). Syk inhibition by piceatannol significantly decreased the pseudoemperipolesis of ZAP-70 positive as well as ZAP-70 negative CLL cells to 0.4 ± 0.07 of controls (n=5, p=0.043). Interestingly, spontaneous migration of CLL cells beneath follicular dendritic cells (HK cells) was also significantly enhanced by anti-IgM stimulation, in particular in ZAP-70 positive cases. In summary, BCR signaling enhances calcium mobilization, CLL cell migration to CXCL12, and pseudoemperipolesis beneath marrow stroma or follicular dendritic cells. These data suggest that BCR stimulation co-operates with CXCL12 for localization and/or maintenance of CLL cells within distinct tissue microenvironments.

Influence of Mir-155 On B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2343-2343
Author(s):  
Liguang Chen ◽  
Bing Cui ◽  
George Chen ◽  
Michelle Salcedo ◽  
Carlo M. Croce ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Calcium Influx ◽  
Critical Role ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Calcium Flux ◽  
Expression Levels ◽  
Bcr Signaling

Abstract Abstract 2343 Poster Board II-320 B-cell receptor (BCR) signaling arguably plays an important role in the pathogenesis and/or progression of chronic lymphocytic leukemia. Ligation of the BCR by F(ab)2 anti-μ can induce phosphorylation of p72Syk, BLNK, phospholipase C-gamma (PLCγ) and other downstream adapter/signaling molecules, inducing intracellular calcium flux and cellular activation. Prior studies found that CLL cells that expressed unmutated Ig heavy-chain variable region genes (IGHV) and the zeta-associated protein of 70 kD (ZAP-70) generally experienced greater levels of activation following treatment with anti-μ than did CLL cells that lacked expression of ZAP-70. However, we found unusual cases that lacked expression of ZAP-70 that also responded vigorously to treatment with anti-μ, suggesting that other factors contribute to the noted differences in BCR-signaling. Analyses for expression of microRNAs by microarray revealed that CLL cells that used unmutated IGHV and that expressed ZAP-70 expressed higher levels of certain microRNAs than did cases that used mutated IGHV and that lacked expression of ZAP-70. One of such microRNA, miR-155, was found to target mRNA encoding SHIP-1, a phosphatase that plays a critical role in modulating the level of BCR signaling in normal B cells. Using quantitative assays for miR-155 we found high-level expression of this microRNA was associated with proficient BCR signaling in CLL. To examine whether miR-155 could modulate the levels of SHIP-1 and/or BCR signaling in CLL cells we transfected primary leukemia cells from each of multiple patients with control oligo-RNAs, miR-155, or a specific inhibitor of miR-155 (miR-155 inhibitor). Twenty-four hours later the cells were stimulated with anti-μ or control antibody and then examined 10 minutes later for expression of SHIP-1, induced calcium influx, or phosphorylation of kinases and adapter proteins that are involved in BCR signaling. CLL cells that had low expression levels of miR-155 and that were poorly responsive BCR had significantly higher levels of calcium influx and phosphorylated p72Syk, BLNK, and PLCγ in response to anti-μ following transfection with miR-155 than following mock transfection or transfection with control oligo-RNA. Conversely, CLL cells that had high expression levels of miR-155 and highly responsive BCR were made to have significantly higher amounts of SHIP-1 protein and to have significantly lower relative levels of phosphorylated protein and calcium influx in response to anti-μ following transfection with the miR-155 inhibitor than did mock transfected CLL cells. These results identify miR-155 as a factor that can modulate BCR signaling in CLL in part by regulating the relative expression level of SHIP-1. These results demonstrate that differential expression of microRNAs in CLL can influence physiologic features that potentially contribute to disease progression. Disclosures: No relevant conflicts of interest to declare.

Anergic Response to B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia: The Differing Roles of IgD and IgM in the Microenvironment and Peripheral Blood.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2889-2889
Author(s):  
Tom Butler ◽  
Alexander Montoya ◽  
Andrew James Clear ◽  
Rita Coutinho ◽  
David C Taussig ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling ◽  
B Cell Receptor Signaling ◽  
Cell Receptor Signaling

Abstract Abstract 2889 Chronic Lymphocytic Leukemia (CLL) cells depend on B cell receptor signaling as well as other microenvironmental survival signals (1). Drugs targeting the BCR signaling pathways are showing exciting results in CLL clinical trials. A peculiarity of CLL is that IgD signaling is generally preserved, whilst IgM signaling is decreased and it has been suggested that this pattern mimics anergic B-cells, and might be consistent with chronic autoantigen exposure. We examined the differing roles of IgM and IgD signaling in CLL using a theoretical framework of anergy. Peripheral blood (PB) CLL cells exhibited higher IgD expression, as compared to IgM (n=204, p<0.0001), but this did not have prognostic impact. When we examined IgM and IgD expression in LN biopsies compared to paired PB (n=10) expression, IgM expression was lower in LN (p=0.002) whilst IgD expression was unchanged. Although the number of these paired samples is small, cases with lower LN IgM levels had poorer prognosis, and we are investigating this further with a larger cohort. We hypothesize that reduced LN IgM expression reflects antigen engagement and an anergic response in the microenvironment. We sought to replicate Mockridge et al' s model of reversible anergy (2) by monitoring the dynamic changes in IgM/D expression after in vitro incubation. Most (18/20) PB CLL samples underwent calcium (Ca) flux after IgD crosslinking, whereas only 13/20 cases underwent IgM Ca flux, and the level of Ca flux was less than with IgD, a well recognized anergic pattern. Incubation for 24h in vitro led to partial restoration of IgM Ca flux and some improvement in IgD Ca flux. This was impaired by treatment with anti-IgD or IgM F(ab)2 fragments, mimicking antigen exposure, and in keeping with a model of CLL cells engaging autoantigen in vivo. Further support for the pro-survival role of the BCR in CLL was demonstrated by the finding that both IgD and IgM ligation was associated with reduced apoptosis in vitro, with a significant decrease in apoptosis with IgD ligation as compared to IgM. To examine the mechanistic differences of signaling via IgM and IgD further, we used high-throughput mass-spectrometry based phosphoproteomics. This allows analysis of multiple active signaling pathways without a priori knowledge of which pathways to investigate. 6 CLL samples were compared to 5 tonsil controls. 4,575 unique phosphopeptides were identified using MASCOT proteomics software and quantified using a label-free technique based on extracted ion currents. 174 phosphoproteins (p<0.001, fold change up to >4000-fold) were over-expressed in CLL relative to healthy B-cells. These included components of RNA processing complexes, cytoskeletal regulators and MAPK signaling pathway components. Kinase prediction based on phosphoprotein substrates confirmed activation of kinases known to be active in CLL (such as AKT1, ERK1/2, CK2), but several novel kinases (such as CaMK1, CRIK, ROCK1 and BCKDK) were also active in CLL relative to healthy controls. Evaluation of differentially expressed phosphoproteins after BCR ligation included components of the spliceosome, regulators of the cytoskeleton, as well as known BCR signaling components. BCR-induced kinase activities included mTOR, CDK family members, MAPKs, BCKDK and others. There was much overlap between kinases active after IgM and IgD ligation, but also marked differences in CLL and tonsil BCR signaling. CONCLUSIONS Anergic IgM signaling is contrasted with IgD as a dynamic and plastic process that appears different in the LN and PB compartments in CLL. Mass-spectrometry based phosphoproteomics offers a powerful tool for interrogating intracellular signaling, with networks of phosphorylation characterizing the topology of pathways. BCR signaling in healthy B-cells has not previously been studied using this approach and comparisons with CLL highlight known pathways as well as suggesting novel treatment targets. The ultimate goal is to identify kinases active in CLL that will provide rational and effective drug combinations. Disclosures: Gribben: Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

An HSP90 Inhibitor, PU-H71, Antagonizes Stroma-Induced Pro-Survival Effects in CLL through Its Inhibition of Multi-Component B-Cell Receptor Signaling Pathway

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5289-5289
Author(s):  
Ailin Guo ◽  
Pin Lu ◽  
Chaojie Zhen ◽  
Gabriela Chiosis ◽  
Yue Lynn Wang
Keyword(s):  
B Cell ◽  
Signaling Pathway ◽  
Cell Receptor ◽  
Hsp90 Inhibitor ◽  
B Cell Receptor ◽  
Receptor Signaling ◽  
Bcr Signaling ◽  
Receptor Signaling Pathway ◽  
B Cell Receptor Signaling ◽  
Cell Receptor Signaling

Abstract Background: Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B-cells in the hematopoietic system. The B-cell receptor (BCR) plays an essential role in the pathogenesis of CLL and many components of the BCR signaling pathway are known clients of HSP90. HSP90 is a highly conserved molecular chaperone that ensures the proper folding and stabilization of its client proteins. In this study, we investigated whether PU-H71 a novel purine-scaffold HSP90 Inhibitor, has anti-tumor activity in CLL by destabilizing BCR signaling pathway constituents. Design: Fresh CLL cells were isolated and cultured ex vivo with or without stromal co-culture. Molecular and cellular events were studied in PU-H71-treated and control CLL cells. Results: Immunoblotting revealed that a significantly higher amount of HSP90 is present in CLL cells than in peripheral blood mononuclear cells (PBMC), suggesting the chaperone is pathogenically relevant. We found that PU-H71 caused the death of CLL cells in a dose and time dependent manner while the viability of either PBMC or normal B lymphocytes were not affected. PU-H71 induced apoptosis resulting in CLL cell death as it caused mitochondrial cytochrome C release and a decrease in the abundance of several anti-apoptotic proteins. Interestingly, PU-H71 has the ability to counteract the pro-survival effects of the stroma and caused apoptosis in CLL cells co-cultured with stroma. To gain mechanistic insights into how PU-H71 acts, we examined the BCR signaling pathway. We found that the amounts of several key components of the pathway were reduced by PU-H71 treatment. This occurred even in the presence of stromal co-culture. The results suggest that PU-H71 antagonizes the function of HSP90 leading to the destabilization of the BCR signaling transducers. A chemical pull-down experiment revealed the co-existence of the BCR components and HSP90 in the same complex, suggesting these BCR constituents are indeed clients of HSP90 in CLL cells. Further, specific genetic knock-down of the signal transducers by siRNA confirmed their key roles in mediating the survival of CLL cells. Conclusions: PU-H71 antagonizes stroma-induced pro-survival effects in CLL through its inhibition of the B-cell receptor signaling pathway. Our results suggest that PU-H71 may serve as a useful therapy against CLL and is worth further clinical development. Disclosures No relevant conflicts of interest to declare.

scholarly journals The outcome of B-cell receptor signaling in chronic lymphocytic leukemia: proliferation or anergy

Haematologica ◽  
2014 ◽  
Vol 99 (7) ◽  
pp. 1138-1148 ◽  
Author(s):  
G. Packham ◽  
S. Krysov ◽  
A. Allen ◽  
N. Savelyeva ◽  
A. J. Steele ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Receptor Signaling ◽  
B Cell Receptor Signaling ◽  
Cell Receptor Signaling
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