Responsiveness of chronic lymphocytic leukemia cells to B-cell receptor stimulation is associated with low expression of regulatory molecules of the nuclear factor-kappa B pathway

Chronic lymphocytic leukemia (CLL) is a disease with heterogeneous clinical and biological characteristics. Differences in Ca2+ levels among cases, both basal and upon B-cell receptor (BCR) stimulation, may reflect heterogeneity in the pathogenesis due to cell-intrinsic factors. Our aim was to elucidate cell-intrinsic differences between BCR-responsive and -unresponsive cases. We therefore determined BCR responsiveness ex vivo based on Ca2+ influx upon alpha-IgM stimulation of purified CLL cell fractions from 52 patients. Phosphorylation levels of various BCR signaling molecules, and expression of activation markers were assessed by flow cytometry. Transcription profiling of responsive (n=6) and unresponsive cases (n=6) was performed by RNA sequencing. Real-time quantitative polymerase chain reaction analysis was used to validate transcript level differences in a larger cohort. In 24 cases an alpha-IgM response was visible by Ca2+ influx which was accompanied by higher phosphorylation of PLC gamma 2 and Akt after alpha-IgM stimulation in combination with higher surface expression of IgM, IgD, CD19, CD38 and CD43 compared to the unresponsive cases (n=28). Based on RNA sequencing analysis several components of the canonical nuclear factor (NF)-kappa B pathway, especially those related to NF-kappa B inhibition, were expressed more highly in unresponsive cases. Moreover, upon alpha-IgM stimulation, the expression of these NF-kappa B pathway genes (especially genes coding for NF-kappa B pathway inhibitors but also NF-kappa B subunit REL) was upregulated in BCR-responsive cases while the level did not change, compared to basal level, in the unresponsive cases. These findings suggest that cells from CLL cases with enhanced NF-kappa B signaling have a lesser capacity to respond to BCR stimulation.