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Novel Model of B-Cell Receptor Signaling Cascade Topology in Nonmalignant and CLL Cells

By: Emily Rhode
Posted: Tuesday, February 14, 2023

Treatment of chronic lymphocytic leukemia (CLL) typically involves the inhibition of B-cell receptor–related kinases; however, adverse events and relapse can occur when resistance mechanisms to treatment arise. A new way to model leukemic B-cell receptor signaling cascade topology may allow for a more targeted approach to treating CLL, according to research published in the International Journal of Cancer. Daniel Mertens, PhD, of the German Cancer Research Center, Heidelberg, and colleagues used a quantitative characterization model to compare healthy and malignant cells as well as CLL subgroups and observe the impact of the inhibition of certain protein kinases on signaling components.

Cells were isolated from 11 patients with previously untreated CLL who had normal karyotypes or chromosome 13q deletion, and CD19-positive B cells were isolated from healthy donor buffy coats. Inhibitor treatment was initiated by suspending cells in phosphate-buffered saline containing ibrutinib or idelalisib. CLL or normal CD19-positive B cells with biotin-coupled CD19 and/or immunoglobulin M antibodies were incubated, and receptors were crosslinked and then stimulated by adding streptavidin followed by a solution of streptavidin and H2O2. A mix of all phosphospecific antibodies was added to the cells before further incubation. Simultaneous phosphorylation of five phosphospecific antibodies (ZAP70/SYK, BTK, PLCγ2, AKT, ERK1/2) were then measured with single-cell phosphospecific flow cytometry.

No change was detected in the intracellular phosphorylation level of proteins involved in the B-cell receptor signaling cascades. The network circuit topology remained stable between malignant and nonmalignant cells. Strong interrelation among SYK, PLCy2, BTK, and ERK were shown, whereas AKT exhibited independent signaling properties. A stronger B-cell receptor response was shown in mutated CLL cells compared with unmutated CLL cells. The authors noted that B cells from healthy donors seemed to respond less to stimulation and H2O2 compared with both mutated and unmutated CLL cells.

“This [B-cell receptor] signaling quantification would also be applicable for studying long-term effects as well as for evaluating future therapeutic targets in signaling cascades,” concluded the authors.

Disclosure: For full disclosures of the study authors, visit

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