Expression and function of protein kinase CK2 in Hodgkin lymphoma

Hodgkin lymphoma (HL) is a lymphoid tissue neoplasia accounting for almost 0.6% of all cancers. The neoplastic cells of Hodgkin and Reed-Sternberg cells (HRS) represent less than 1% of all the tumor bulk which is mainly composed by an heterogenous group of reactive cells of the immune system. The abnormalities of transcriptional factors and genes involved in hematopoietic cells differentiation justify the typical immunophenotype of HRS, being positive for CD30, CD15 and the immuno-checkpoint PD-L1 but usually negative for CD20. The survival of neoplastic cells is favored by the activation of NF-κB, JAK/STAT e PI3K pathways, which play a key role in the pathogenesis of this disease. Protein casein kinase 2 (CK2) is a serine/threonine kinase ubiquitously expressed in eukaryotic cells, constitutively active, consisting of two catalytic (α) and two non-catalytic (β) subunits assembled to form a tetramer. It is involved in a broad variety of cellular processes, among which survival, proliferation, differentiation, DNA damage and other stress responses. This kinase has been found overexpressed in several solid tumors and hematologic malignancies. It has been fully demonstrated that CK2 acts as a potent antiapoptotic factor that promotes a “non-oncogene addiction” phenotype in cancer cells. In particular, it was shown that many B-cell derived tumors, like multiple myeloma, mantle cell lymphoma and chronic lymphocytic leukemia, rely on high CK2 activity and that its genetic and chemical inhibition induces malignant cell death without significantly affecting normal B lymphocytes. However, the involvement of CK2 in the pathogenesis of HL is still unclear. By means of western blotting, real time PCR, immunofluorescence, flow cytometry and subcellular fractionation we analyzed the expression of CK2 subunit and the effect of Ck2 inhibition in four HL cell lines (L-428, L-540, KM-H2, HDLM-2), Kasumi-1 (a cell lines derived from a patient with acute myeloid leukemia) and age-matched B lymphocytes from a healthy donor, as controls. Immunohistochemistry on tissue microarray was also used. Apoptosis was assessed by flow cytometry using Annexin V/Propidium Iodine test and PARP cleavage by western blotting. We observed that CK2α was overexpressed while CK β was expressed at lower levels in all HL cell lines as compared to normal B cells. The α catalytic subunit CK2 was localized both in the nucleus and the cytosol, while the β subunit was mainly cytosolic in HL cell lines. These data were confirmed on primary nodes of patients with HL using tissue microarray. This aberrant expression of protein CK2 was not associated with different mRNA levels of CSNK2A1 and CSNK2B, genes coding for the α and β subunit respectively. We observed that CK2 substrates namely NF-κB, STAT3, AKT were constitutively phosphorylated on activatory serine amino acids (NF-κB Ser529, STAT3 Ser727, AKT Ser473, AKT Ser129) in HL cell lines. The pharmacological inhibition of CK2 with the clinical inhibitor CX-4945/silmitasertib mediates time- and dose-dependent apoptosis and dephosphorylation of CK2 targets at activatory residues. Moreover, we demonstrated that silmitasertib was able to decrease the expression of the immuno-checkpoint CD297/PD-L1 but not of CD30, and to enhance the cytotoxicity caused by monomethyl auristatin E (MMAE), the microtubule inhibitor conjugated to an anti-CD30 monoclonal antibody in the brentuximab vedotin drug. Comprehensively our data point out a pivotal role of CK2a in the survival, as well as the activation of some key signaling pathways, in HL and identify this kinase as a targetable kinase for the development of new effective therapy for this neoplastic disease. Remarkably a skewed expression between CK2 subunits has never been reported in other hematological cancers.