A. Visentin1,2, F. Frezzato1,2, F. Severin1,2, M. Pizzi3, V. Martini1,2, F. Raggi1,2, S. Imbergamo1, E. Scomazzon1, S. Pravato1, S. Manni1,2, M. Facco1,2, C. Carlo-Stella4, G. Semenzato1,2, F. Piazza1,2, L. Trentin1,2
1Hematology and Clinical Immunology Unit, Department of Medicine, University of Padua, Padua, Italy,2Venetian Institute of Molecular Medicine, Padua, Italy,3General Pathology & Cytopathology Unit, Department of Medicine, University of Padua, Padua, Italy,4Department of Biochemical Sciences, Humanitas University, Milan, Italy
Introduction: Most patients with Hodgkin lymphoma (HL) are cured with first line therapy however, treatment of relapsed patients still represents as unmet medical need requiring the development of new effective drugs. CK2 is a pleiotropic kinase consisting of 2 catalytic (a) and 2 regulatory (b) subunits that sustains cancer signaling cascades through the activation of NF- B, PI3K and STAT pathways. Despite CK2 have been extensively studied in non-Hodgkin lymphomas (NHL), this protein has not yet been investigated in HL. Considering that NF- B, STAT and PI3K pathways are key players in HL, it is likely that CK2 might have a role in the pathogenesis of this disease.
Methods: Experiments were performed and replicated at least for five times employing 4 HL cell lines (L-428, L-540, KM-H2 and HDML-1) cultured in RPMI for 24 h, 48 h and 72 h with or without the CK2 inhibitor silmitasertib. CK2a, CK2b, RelA-Serine (S)529, RelA, PARP, AKT-S473, AKT, STAT3-S727, STAT3 and tubulin expression levels were evaluated by western blot analysis (WB). Apoptosis was assessed by Annexin V/Propidium iodide (AV/PI) assay and PARP cleavage by WB. Immunohistochemistry (IHC) for CK2 and CK2 was performed on formalin fixed paraffin embedded sections of lymph-nodes from patients with HL, as well as indolent and aggressive NHL. Data were considered statistically significant when p values were < 0.05.
Results: By WB and IF we found that all the 4 HL cell lines expressed higher levels of CK2a, but not of CK2b, as compared with normal B lymphocytes. These data were confirmed by IHC on primary lymph-nodes derived from patients with HL (n = 15), showing that CK2a but not CK2b was highly expressed in Reed-Sternberg cells. No clinical data correlated with CK2a expression. This unbalance between a and b subunits was not observed in follicular or high-grade NHL (p < 0.01). In order to evaluate the activity of CK2, we performed WB analysis of the phosphorylation status of CK2 substrates. AKT, RelA and STAT3 were constitutively phosphorylated in HL at their activatory S-residue (S473, S529, and S727 respectively). Treatment of HL cell lines with silmitasertib caused down-regulation of AKT-S473, time and dose-dependent apoptosis (p < 0.01).
Conclusions: We demonstrated that CK2 is overexpressed, active and induces key pro-survival signals in HL. Chemical inhibition with silmitasterib was able to induce apoptosis of HL cell lines. These preliminary data suggest that CK2 could play a pivotal role in HL.