Multidimensional Single-Cell Analysis of BCR Signaling Reveals Proximal Activation Defect As a Hallmark of Chronic Lymphocytic Leukemia B Cells

M Lia Palomba,Ivelise Rijo,Kelly Piersanti,Grégoire Altan-Bonnet,Marcel R M Van Den Brink,Sami N Malek,Debra Bemis,Carly G K Ziegler,Jeff R Gardner,Kurt Bantilan,Erlene Seymour,Mark Heaney,Robert Balderas,Hugo Decker,Jesse W Cotari,Andrew D Zelenetz,Yolande Richard

doi:10.1371/journal.pone.0079987

Abstract

PurposeChronic 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 DesignWe 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.ResultsWe identified a dynamic and variable imbalance between proximal (pSYK, pBTK) and distal (pPLCγ2, pBLNK, ppERK) phosphoresponses. PLSR identified the relationship between upstream tyrosine kinase SYK and its target, PLCγ2, 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.ConclusionSingle-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/pPLCγ2 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.

Highlights

Chronic lymphocytic leukemia (CLL) results from the accumulation of mature monoclonal CD5+ B cells in the bone marrow, lymphoid organs and peripheral blood
Partial Least Squares Regression (PLSR) identified the relationship between upstream tyrosine kinase SYK and its target, PLCc2, 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
Disruption of the pSYK/pPLCc2 relationship is uncovered as a robust hallmark of CLL B cell signaling behavior

Summary

Introduction

Chronic lymphocytic leukemia (CLL) results from the accumulation of mature monoclonal CD5+ B cells in the bone marrow, lymphoid organs and peripheral blood. Clonal expansion of B cells depends on the efficient propagation of signal from the cell membrane to target genes following antigenic stimulation of the BCR [9,10]. It has been proposed that unmutated surface immunoglobulins in CLL are more responsive to antigenic stimulation, resulting in strong BCR-mediated signal transduction and induction of anti-apoptotic proteins such as XIAP and MCL-1 [11,12,13], while CLL cells with mutated IGHV more closely resembles anergic B cells [14], with incomplete responsiveness through the BCR pathway and induction of tolerogenic signals. BCR signaling aberrancy has been shown to correlate with prognostic clinical parameters or disease stage at the time of diagnosis. The target specificity, off-target activity and exact mechanism of action of these novel drugs, are not completely understood at this time [20,21,22,23,24,25]

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