IDENTIFICAZIONE DI NUOVI BERSAGLI TERAPEUTICI NEL LINFOMA DI HODGKIN CLASSICO

Classical Hodgkin lymphoma (cHL) is a B cell derived cancer, characterized by the presence of a small number of malignant “Hodgkin and Reed-Sternberg” (HRS) cells, surrounded by a protective tumor microenvironment (TME). Although this tumor has a good prognosis, 20-30% of patients relapse or are refractory to therapy. To increase the treatment success rate, new therapeutic agents are needed. During this three-year PhD project, we studied the role of casein kinase 2 (CK2) in cHL. CK2 is a constitutively active and highly conserved serine/threonine protein kinase, resulting deregulated in different hematological malignancies, but never investigated in cHL. CK2 regulates crucial cellular processes, including gene expression, protein synthesis, cell proliferation, and apoptosis. Generally, it is assembled as tetrameric enzyme, consisted of two catalytic (α or α’) and two regulatory (β) subunits, which are also present in a non-associated form. CK2 is known to play as non-oncogene addiction factor. NF-B, PI3K/Akt, and JAK/STAT3, the main signaling pathways over-expressed in HRS cells, are known targets of CK2. Analyses were performed using four cHL cell lines (L-428, L-540, KM-H2, and HDLM-2) as in vitro model. By western blotting (WB) analysis, we found that CK2α catalytic subunit was over-expressed in the HL cell lines compared to healthy B lymphocytes (p<0.05, Student’s t test), whereas the regulatory CK2β subunit was expressed at significant lower levels (p<0.001, Student’s t test). Since subunit gene expressions did not differ, we showed that  subunit lower levels were, at least in part, due to CK2β ubiquitination and consequent proteasome degradation, as highlighted in the four HL cell lines. By CK2 inhibitor CX-4945, we analyzed the effects of CK2 inhibition on HL cell line viability, finding a significant increase of cell apoptosis (Annexin V/Propidium Iodide flow cytometry test, p<0.01, Kruskal-Wallis’s test). In addition, in the four HL cell lines, we studied the phosphorylation levels of CK2 serine targets on NF-κB (Ser529), STAT3 (Ser727), AKT (Ser473 and Ser129). At basal conditions, the above targets resulted phosphorylated. On the contrary, after CK2 inhibition, we demonstrated a significant reduction of the phosphorylation levels on CK2 targets, thus suggesting a basic and essential CK2 engagement in the pathways related to the survival of HRS cells. Noteworthy, CK2 inhibition induced the decrease of the expression on HL cell lines of the immune checkpoint PD-L1 ( p<0.01, Student’s t test), known to be regulated by STAT3 and NF-kB signals. Finally, we investigated the potential involvement of CK2 in TME formation. By migration assays performed on normal T and B lymphocytes, in presence of supernatants (conditioned medium - C.M.) derived from HL cell lines, cultured with or without CX-4945, we showed that CK2 inhibition significantly counteracted lymphocyte migration (p<0.05, One-way ANOVA). Furthermore, preliminary results suggested a modulation of the expression of the chemokine receptor CXCR3 on T cells, induced by HL cell cytokine release, an issue that needs to be further investigated. In conclusion, our data demonstrate CK2 strong involvement in HL development and support the studies on this “pleiotropic” molecule as new promising target for the therapy of HL.