Abstract P032

CK2 FAVORS T-CELL CHEMOTAXIS THROUGHT THE RELEASE OF SOLUBLE FACTORS BY HODGKIN AND REED–STERNBERG CELLS

In classical Hodgkin lymphoma (HL), Hodgkin and Reed–Sternberg (HRS) cells are surrounded by T cells. We recently identified CK2 as a key protein for the survival of HRS cells and how its inhibition triggers apoptosis. In this study, we assess the role of protein CK2 in sustaining T-cell recruitment in the tumor niche.

HL cell lines (KM-H2 and HDLM-2) were treated with 0, 5, and 10μM of CX-4945 (CX), a CK2 inhibitor, for 24/48h. Apoptosis was quantified by flow cytometry with the Annexin V/ Propidium iodide assay. Migration assays were performed using fibronectin-coated transwells. Conditioned media (CM) from the cell lines, collected after 24/48h treatment, was added to the bottom chamber. T-cells were purified from age-matched healthy donors. A multiplexed array was used to determine the concentration of 27 cytokines from the supernatants. CXCR3, CCR7 on T-cells, and AKT, STAT3, NF-kB on HL cells lines were assessed by western blot (WB). In vitro CK2 inhibition by CX was not toxic for donor-derived healthy T cells after 24 or 48 hours of culture as opposite to HL cells lines (p<0.01). CX-treated HL cell lines generate a CM with decreased chemoattractant effects on T lymphocytes. The percentage of migrated T lymphocytes toward the CM obtained from HDLM-2 and KM-H2 cells treated with CX 5μM and 10μM for 24h and 48h decreased by 12.1% and 18%, 25.3% and 34%, respectively, compared to untreated conditions (p<0.05, Figure 1).

In vitro treatment of HL cell lines with CX caused the dephosphorylation of AKT, STAT3 and NF-kB as assessed by WB, likely interfering with production of several cytokines and chemokines. We performed an array analysis to identify CK2-related molecules. Among the tested cytokines, IL-6, M-CSF, RANTES, TARC, TGF-β1, TNF-α, and VEGF, demonstrated a significant CK2 dependence. When HL cell lines were treated with 10μM CX, there was a significant reduction of IL-6, TARC, TGF-β1, TNF-α, and VEGF release (p<0.0001) and for some molecules also at 5μM.

We also found that CM from HL cell lines was able to modulate the expression of the T-cell surface receptor CXCR3 but not CCR7, assessed by WB (p<0.05), compared the untreated condition, which was not observed with the CM derived from CX-treated HL cells. In conclusion, CK2 emerged as a novel player in the formation of HL microenvironment by modulating the release of cytokines from HRS cells molecules that are able to chemoattract and shape chemokines receptor on the surface of T cells.

Authors

Andrea Visentin, Federica Frezzato, Guido Capasso, Nayla Mouawad, Maria Castronuovo, Alessandro Cellini, Francesco Angotzi, Andrea Serafin, Chiara Adele Cavarretta, Valeria Ruocco, Arianna Bevilacqua, Sabrina Manni, Monica Facco, Federico Scarmozzino, Marco Pizzi, Fabrizio Vianello, Francesco Piazza, Livio Trentin