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

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 gamma 2, pBLNK, ppERK) phosphoresponses. PLSR identified the relationship between upstream tyrosine kinase SYK and its target, PLC gamma 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 gamma 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.