Dataset Information

Multidimensional single-cell analysis of BCR signaling reveals proximal activation defect as a hallmark of chronic lymphocytic leukemia B cells.

ABSTRACT:

Purpose

Chronic Lymphocytic Leukemia (CLL) is defined by a perturbed B-cell receptor-mediated signaling machinery. We aimed to model differential signaling behavior between B cells from CLL and healthy individuals to pinpoint modes of dysregulation.

Experimental design

We developed an experimental methodology combining immunophenotyping, multiplexed phosphospecific flow cytometry, and multifactorial statistical modeling. Utilizing patterns of signaling network covariance, we modeled BCR signaling in 67 CLL patients using Partial Least Squares Regression (PLSR). Results from multidimensional modeling were validated using an independent test cohort of 38 patients.

Results

We identified a dynamic and variable imbalance between proximal (pSYK, pBTK) and distal (pPLC?2, pBLNK, ppERK) phosphoresponses. PLSR identified the relationship between upstream tyrosine kinase SYK and its target, PLC?2, as maximally predictive and sufficient to distinguish CLL from healthy samples, pointing to this juncture in the signaling pathway as a hallmark of CLL B cells. Specific BCR pathway signaling signatures that correlate with the disease and its degree of aggressiveness were identified. Heterogeneity in the PLSR response variable within the B cell population is both a characteristic mark of healthy samples and predictive of disease aggressiveness.

Conclusion

Single-cell multidimensional analysis of BCR signaling permitted focused analysis of the variability and heterogeneity of signaling behavior from patient-to-patient, and from cell-to-cell. Disruption of the pSYK/pPLC?2 relationship is uncovered as a robust hallmark of CLL B cell signaling behavior. Together, these observations implicate novel elements of the BCR signal transduction as potential therapeutic targets.

SUBMITTER: Palomba ML

PROVIDER: S-EPMC3906024 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Publications

Multidimensional single-cell analysis of BCR signaling reveals proximal activation defect as a hallmark of chronic lymphocytic leukemia B cells.

Palomba M Lia ML Piersanti Kelly K Ziegler Carly G K CG Decker Hugo H Cotari Jesse W JW Bantilan Kurt K Rijo Ivelise I Gardner Jeff R JR Heaney Mark M Bemis Debra D Balderas Robert R Malek Sami N SN Seymour Erlene E Zelenetz Andrew D AD van den Brink Marcel R M MR Altan-Bonnet Grégoire G

PloS one 20140129 1

<h4>Purpose</h4>Chronic Lymphocytic Leukemia (CLL) is defined by a perturbed B-cell receptor-mediated signaling machinery. We aimed to model differential signaling behavior between B cells from CLL and healthy individuals to pinpoint modes of dysregulation.<h4>Experimental design</h4>We developed an experimental methodology combining immunophenotyping, multiplexed phosphospecific flow cytometry, and multifactorial statistical modeling. Utilizing patterns of signaling network covariance, we model ...[more]

PMID: 24489640

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Project description:BACKGROUND:TOSO, also named Fas inhibitory molecule 3 (FAIM3), has recently been identified as an immunoglobulin M (IgM) Fc receptor (Fc?R). Previous studies have shown that TOSO is specifically over-expressed in chronic lymphocytic leukemia (CLL). However, the functions of TOSO in CLL remain unknown. The B-cell receptor (BCR) signaling pathway has been reported to be constitutively activated in CLL. Here, we aimed to investigate the functions of TOSO in the BCR signaling pathway and the pathogenesis of CLL. METHODS:We over-expressed TOSO in B-cell lymphoma cell lines (Granta-519 and Z138) by lentiviral transduction and knocked down TOSO by siRNA in primary CLL cells. The over-expression and knockdown of TOSO were confirmed at the RNA level by polymerase chain reaction and protein level by Western blotting. Co-immunoprecipitation with TOSO antibody followed by liquid chromatography coupled with tandem mass spectrometry (IP/LCMS) was used to identify TOSO interacting proteins. Western blotting was performed to detect the activation status of BCR signaling pathways as well as B-cell lymphoma 2 (BCL-2). Flow cytometry was used to examine the apoptosis of TOSO-over-expressing B lymphoma cell lines and TOSO-down-regulated CLL cells via the staining of Annexin V and 7-AAD. One-way analyses of variance were used for intergroup comparisons, while independent samples t tests were used for two-sample comparisons. RESULTS:From IP/LCMS, we identified spleen tyrosine kinase (SYK) as a crucial candidate of TOSO-interacting protein and confirmed it by co-immunoprecipitation. After stimulation with anti-IgM, TOSO over-expression increased the phosphorylation of SYK, and subsequently activated the BCR signaling pathway, which could be reversed by a SYK inhibitor. TOSO knockdown in primary CLL cells resulted in reduced SYK phosphorylation as well as attenuated BCR signaling pathway. The apoptosis rates of the Granta-519 and Z138 cells expressing TOSO were (8.46?±?2.90)% and (4.20?±?1.21)%, respectively, significantly lower than the rates of the control groups, which were (25.20?±?4.60)% and (19.72?±?1.10)%, respectively (P?<?0.05 for both). The apoptosis rate was reduced after knocking down TOSO in the primary CLL cells. In addition, we also found that TOSO down-regulation in primary cells from CLL patients led to decreased expression of BCL-2 as well as lower apoptosis, and vice versa in the cell line. CONCLUSIONS:TOSO might be involved in the pathogenesis of CLL by interacting with SYK, enhancing the BCR signaling pathway, and inducing apoptosis resistance.

Dataset Information

Multidimensional single-cell analysis of BCR signaling reveals proximal activation defect as a hallmark of chronic lymphocytic leukemia B cells.

Purpose

Experimental design

Results

Conclusion

Publications

Multidimensional single-cell analysis of BCR signaling reveals proximal activation defect as a hallmark of chronic lymphocytic leukemia B cells.

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