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


METHODS
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 (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.


CONCLUSIONS
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/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.
Division of Hematology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America ; Center Cancer Systems Biology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
M Lia
ML
Palomba
Division of Hematology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America ; Center Cancer Systems Biology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
Kelly
K
Piersanti
Center Cancer Systems Biology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America ; Program in Computational Biology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
Carly G K
CG
Ziegler
Center Cancer Systems Biology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America ; Program in Computational Biology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
Hugo
H
Decker
Center Cancer Systems Biology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America ; Program in Computational Biology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
Jesse W
JW
Cotari
Division of Hematology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
Kurt
K
Bantilan
Division of Hematology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
Ivelise
I
Rijo
Division of Hematology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
Jeff R
JR
Gardner
Division of Hematology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
Mark
M
Heaney
Center Cancer Systems Biology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America ; Program in Computational Biology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
Debra
D
Bemis
BD Biosciences, San Diego, California, United States of America.
Robert
R
Balderas
Division of Hematology/Oncology, University of Michigan Health System, Ann Harbor, Michigan, United States of America.
Sami N
SN
Malek
Division of Hematology/Oncology, University of Michigan Health System, Ann Harbor, Michigan, United States of America.
Erlene
E
Seymour
Division of Hematology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
Andrew D
AD
Zelenetz
Division of Hematology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
Marcel R M
MR
van den Brink
Center Cancer Systems Biology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America ; Program in Computational Biology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
Grégoire
G
Altan-Bonnet
D000888:Antibodies, Anti-Idiotypic / Q000494:pharmacology; D001402:B-Lymphocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D015973:Gene Expression Regulation, Leukemic; D006801:Humans; D016130:Immunophenotyping; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D016018:Least-Squares Analysis; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008213:Lymphocyte Activation / Q000187:drug effects; D015233:Models, Statistical; D051966:Phospholipase C gamma / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D011947:Receptors, Antigen, B-Cell / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D059010:Single-Cell Analysis; D000072377:Syk Kinase
D000888:Antibodies, Anti-Idiotypic; D047908:Intracellular Signaling Peptides and Proteins; D011947:Receptors, Antigen, B-Cell; C044203:anti-IgM; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D051966:Phospholipase C gamma