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1.
Front Immunol ; 13: 959138, 2022.
Article in English | MEDLINE | ID: mdl-36713383

ABSTRACT

Serine-Threonine kinase CK2 supports malignant B-lymphocyte growth but its role in B-cell development and activation is largely unknown. Here, we describe the first B-cell specific knockout (KO) mouse model of the ß regulatory subunit of CK2. CK2ßKO mice present an increase in marginal zone (MZ) and a reduction in follicular B cells, suggesting a role for CK2 in the regulation of the B cell receptor (BCR) and NOTCH2 signaling pathways. Biochemical analyses demonstrate an increased activation of the NOTCH2 pathway in CK2ßKO animals, which sustains MZ B-cell development. Transcriptomic analyses indicate alterations in biological processes involved in immune response and B-cell activation. Upon sheep red blood cells (SRBC) immunization CK2ßKO mice exhibit enlarged germinal centers (GCs) but display a limited capacity to generate class-switched GC B cells and immunoglobulins. In vitro assays highlight that B cells lacking CK2ß have an impaired signaling downstream of BCR, Toll-like receptor, CD40, and IL-4R all crucial for B-cell activation and antigen presenting efficiency. Somatic hypermutations analysis upon 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Chicken Gamma Globulin (NP-CGG) evidences a reduced NP-specific W33L mutation frequency in CK2ßKO mice suggesting the importance of the ß subunit in sustaining antibody affinity maturation. Lastly, since diffuse large B cell lymphoma (DLBCL) cells derive from GC or post-GC B cells and rely on CK2 for their survival, we sought to investigate the consequences of CK2 inhibition on B cell signaling in DLBCL cells. In line with the observations in our murine model, CK2 inactivation leads to signaling defects in pathways that are essential for malignant B-lymphocyte activation.


Subject(s)
Casein Kinase II , Lymphocyte Activation , Animals , Mice , Sheep , Casein Kinase II/genetics , Signal Transduction , Protein Serine-Threonine Kinases/metabolism , Mice, Knockout , Receptors, Antigen, B-Cell/genetics , Cell Differentiation
2.
Curr Cancer Drug Targets ; 18(6): 608-616, 2018.
Article in English | MEDLINE | ID: mdl-28460620

ABSTRACT

BACKGROUND: Approximately one third of Diffuse Large B cell Lymphomas (DLBCL) are refractory or relapse. Novel therapeutic approaches under scrutiny include inhibitors of B-cell receptor (BCR) signaling. Protein kinase CK2 propels survival, proliferation and stress response in solid and hematologic malignancies and promotes a "non-oncogene addiction" phenotype. Whether this kinase regulates BCR signaling, being a suitable pharmacological target in DLBCL, is unknown. OBJECTIVE: The objective was to establish if CK2 controls DLBCL cell survival and the BCR signaling, to check if the combination of CK2 inhibitor CX-4945 and BCR blockers Ibrutinib and Fostamatinib is more effectively cytotoxic for DLBCL cells than the single agents and to survey the changes in signaling molecules downstream BCR upon CK2 inhibition. METHOD: A panel of GC and ABC DLBCL cells was treated with CX-4945 and Fostamatinib or Ibrutinib. BCR signaling was assayed by intracellular Ca++ measurement and looking at the phosphorylation of signaling molecules. The effects on cell survival were assessed by flow cytometry, western blot and MTT assays. RESULTS: CK2 inhibition with CX-4945 causes DLBCL cell death. CX-4945 impaired AKT phosphorylation and intracellular Ca++ mobilization upon BCR engagement. The CK2 inhibitor acted synergistically with either the SYK inhibitor Fostamatinib or the BTK inhibitor Ibrutinib in inducing DLBCL cell death. CX-4945 was equally effective in GC and ABC DLBCL subtypes as well as in "double hit" DLBCL cell lines. CONCLUSION: These findings suggest a role for CK2 downstream of the BCR in controlling survival pathways crucial for cell growth of different DLBCL subtypes. Also, the use of CX-4945 in combination with BCR signaling blockers could represent a novel rational therapeutic approach in the DLBCL.


Subject(s)
Apoptosis/drug effects , Drug Synergism , Lymphoma, Large B-Cell, Diffuse/drug therapy , Naphthyridines/pharmacology , Oxazines/pharmacology , Pyrazoles/pharmacology , Pyridines/pharmacology , Pyrimidines/pharmacology , Receptors, Antigen, B-Cell/antagonists & inhibitors , Adenine/analogs & derivatives , Aminopyridines , Casein Kinase II/antagonists & inhibitors , Cell Proliferation , Humans , Lymphoma, Large B-Cell, Diffuse/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , Morpholines , Phenazines , Piperidines , Tumor Cells, Cultured
3.
Oncotarget ; 6(9): 6544-52, 2015 Mar 30.
Article in English | MEDLINE | ID: mdl-25788269

ABSTRACT

Serine-threonine kinase CK2 is highly expressed and pivotal for survival and proliferation in multiple myeloma, chronic lymphocytic leukemia and mantle cell lymphoma. Here, we investigated the expression of α catalytic and ß regulatory CK2 subunits by immunohistochemistry in 57 follicular (FL), 18 Burkitt (BL), 52 diffuse large B-cell (DLBCL) non-Hodgkin lymphomas (NHL) and in normal reactive follicles. In silico evaluation of available Gene Expression Profile (GEP) data sets from patients and Western blot (WB) analysis in NHL cell-lines were also performed. Moreover, the novel, clinical-grade, ATP-competitive CK2-inhibitor CX-4945 (Silmitasertib) was assayed on lymphoma cells. CK2 was detected in 98.4% of cases with a trend towards a stronger CK2α immunostain in BL compared to FL and DLBCL. No significant differences were observed between Germinal Center B (GCB) and non-GCB DLBCL types. GEP data and WB confirmed elevated CK2 mRNA and protein levels as well as active phosphorylation of specific targets in NHL cells. CX-4945 caused a dose-dependent growth-arresting effect on GCB, non-GCB DLBCL and BL cell-lines and it efficiently shut off phosphorylation of NF-κB RelA and CDC37 on CK2 target sites. Thus, CK2 is highly expressed and could represent a suitable therapeutic target in BL, FL and DLBCL NHL.


Subject(s)
B-Lymphocytes/enzymology , Burkitt Lymphoma/enzymology , Cell Proliferation , Lymphoma, Follicular/enzymology , Lymphoma, Large B-Cell, Diffuse/enzymology , Antineoplastic Agents/pharmacology , B-Lymphocytes/drug effects , B-Lymphocytes/pathology , Blotting, Western , Burkitt Lymphoma/genetics , Burkitt Lymphoma/pathology , Casein Kinase II/antagonists & inhibitors , Casein Kinase II/genetics , Casein Kinase II/metabolism , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Chaperonins/metabolism , Dose-Response Relationship, Drug , Gene Expression Profiling , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Lymphoma, Follicular/genetics , Lymphoma, Follicular/pathology , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/pathology , Naphthyridines/pharmacology , Phenazines , Phosphorylation , Protein Kinase Inhibitors/pharmacology , RNA, Messenger/metabolism , Retrospective Studies , Signal Transduction , Transcription Factor RelA/metabolism
4.
Cell Microbiol ; 16(6): 925-37, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24397470

ABSTRACT

Neisseria meningitidis is a human pathogen that can cause fatal sepsis and meningitis once it reaches the blood stream and the nervous system. Here we demonstrate that a fragment, released upon proteolysis of the surface-exposed protein Neisserial Heparin Binding Antigen (NHBA), by the bacterial protease NalP, alters the endothelial permeability by inducing the internalization of the adherens junction protein VE-cadherin. We found that C2 rapidly accumulates in mitochondria where it induces the production of reactive oxygen species: the latter are required for the phosphorylation of the junctional protein and for its internalization that, in turn, is responsible for the endothelial leakage. Our data support the notion that the NHBA-derived fragment C2 might contribute to the extensive vascular leakage typically associated with meningococcal sepsis.


Subject(s)
Adherens Junctions/drug effects , Antigens, Bacterial/metabolism , Endothelial Cells/drug effects , Neisseria meningitidis/physiology , Permeability/drug effects , Antigens, Bacterial/genetics , Cell Line , Humans , Virulence Factors/genetics , Virulence Factors/metabolism
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