Analysis of Protein Kinase CK2 in B-lymphopoiesis and lymphomagenesis in a mouse conditional Knockout model

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 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. 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 CK2β-Flox/Flox mice with CD19-CRE transgenic mice. CK2 kinase activity was decreased in CK2β KO B-cells. In the bone marrow (BM), CK2β KO mice displayed a reduction of B-cells, especially of the 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. CK2β KO mice displayed lower levels of all the immunoglobulin classes in the serum. In the spleen of CK2β KO we observed 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. 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. In vivo sheep red blood cells (SRBC) treatment (T-cell dependent response) showed a conserved up-regulation of germinal center (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 CK2β KO B-cells. In vivo Notch2 blockage with neutralizing antibodies markedly reduced the MZB cell number in CK2β KO mice, indicating a Notch2-dependent MZB expansion associated with CK2β loss. High throughput RNAseq analysis was also performed and revealed significant alterations in FOB and MZB-regulating pathways. 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