Csnk2β, the Regulatory Subunit of Protein Kinase CK2, modulates Peripheral B Cell Development Repressing Notch2 Signaling and Promoting a Proper B-Cell Receptor Signal Transmission

Background. Serine-threonine protein kinase CK2 has been recently involved in the pathogenesis of B-cell tumors, such as B acute lymphoblastic leukemia, B chronic lymphocytic leukemia, mantle cell lymphoma and multiple myeloma. CK2 acts through a “non-oncogene” addiction mechanism to propel tumor growth, protecting from apoptosis by a phosphorylation-dependent “shielding” mechanism of pro-survival molecules and stimulating oncogenic kinases by helping folding and enzymatic activity. In addition, CK2 has been shown to enhance the transactivation potential of several transcription factors, such as STAT3, NF-κB and c-Myc. The existing data on CK2 function in B cell tumors suggest that this kinase might act as a “hub” downstream signals from surface membrane molecules, like the B-cell (BCR), growth factor and cytokine receptors, as well as from cell-intrinsic pathways – like proteotoxic and DNA-damage-related stress cascades.

Aims and methods. To gain insights into the role of CK2 in B-lymphopoiesis and, consequently, in B-cell tumors, we generated CK2β conditional knockout (KO) mice in B-cells by crossing Csnk2β-Flox/Flox mice with CD19-CRE transgenic mice.

Results. CK2 kinase activity was decreased in Csnk2β KO B cells. In the bone marrow (BM), Csnk2β KO mice displayed a reduction of B-cells, especially of the B220^high IgM^int-high recirculating population of transitional and follicular (FO) B cells. Pro-B and pre-B-cell progenitors were slightly reduced in number. In peripheral blood, lymph-nodes, spleen and peritoneal cavity the number of B-cells was markedly reduced. Csnk2β KO mice had lower levels of all the immunoglobulin classes in the serum. The splenic IgD^low IgM^high B-cell subset was increased whereas the IgD^high IgM^int-low population was decreased. An imbalance between the amount of FO and marginal zone (MZ) B-cells was found with an absolute reduction of FO B cells by approximately 2-folds and an increase of MZ B-cells and MZB cell precursors by up to three folds, on average. Histological and immunofluorescence (IF) analysis revealed a change of size/shape of spleen follicles and a significant expansion of the inter-follicular, marginal zone areas, which appeared to invade the follicle with larger cells. In vitro class-switch recombination assays demonstrated impairment in IgG₁ and IgG₃ class-switch and a marked reduction of the generation of antibody-producing cells. Anti-IgM stimulation was uncoupled to Ca++ mobilization, indicating a disrupted transmission of the signal from the BCR to the release of Ca++ stores in the endoplasmic reticulum. In vivo sheep red blood cells (SRBC) treatment (T-cell dependent response) showed a conserved up-regulation of GC markers, such as CD38, GL7 and PNA. Nonetheless, the architecture of the reactive follicles was found markedly changed. The analysis of FO, GC and MZ-associated genes showed normal levels of Bcl6, elevated levels of Lrf mRNA and, more significantly, a marked up-regulation of Notch2 target genes, such as Hes1 and Deltex1, in Csnk2β KO B cells. In vivo Notch2 blockage with neutralizing antibodies markedly reduced the MZB cell number in Csnk2β KO mice, indicating a Notch2-dependent MZB expansion associated with Csnk2β loss. High throughput RNAseq analysis was also performed and revealed significant alteration in FOB and MZB-regulating pathways.

Conclusions. Here, we found that the β subunit of protein kinase CK2 is a novel regulator of peripheral B cell differentiation. CK2β sustains a proper BCR signal, controls the GC reaction and negatively regulates Notch2 signaling, acting as a master regulator of follicular/marginal zone architecture and terminal homeostasis of FOB and MZB cells. On one side our data enrich the knowledge on the mechanisms regulating B cell development, on the other side they inform about the potential mechanisms altered by CK2 during B-cell tumorigenesis.