The kinase inhibitors R406 and GS-9973 impair T cell functions in chronic lymphocytic leukemia (CLL) patients

ANA COLADO; DENISE RISNIK; RAIMUNDO BEZARES; MIRTA GIORDANO; MARÍA BELÉN ALMEJÚN; CARMEN STANGANELLI; HORACIO FERNÁNDEZ GRECO; ROMINA GAMBERALE; ENRIQUE PODAZA; IRMA SLAVUTSKY; MARÍA CABREJO; MERCEDES BORGE

Colonia

Workshop; Young Investigators´ Meeting 2016, Xth International Workshop of the German CLL Study Group; 2016

German CLL Study Group

Small molecules targeting kinases involved in B-cell receptor (BCR) signaling are showing encouraging clinical activity in CLL patients. Fostamatinib (R406) and Entospletinib (GS-9973) are ATP-competitive inhibitors designed to target Syk, that have shown both safety and efficacy in trials with CLL patients. Although most of pre-clinical studies with kinase inhibitors have focused on their effect on the leukemic clone, it has been recently shown that some of them can also affect cells from the tumor microenvironment such as T cells and macrophages. T cells are key players in CLL's tumor microenvironment by promoting leukemic cell survival and proliferation through the expression of molecules as CD40L, IL-4 and IFNɣ. In this work we asked if R406 and GS-9973 affect the activation, proliferation and migration of T cells from CLL patients.PBMC or purified T cells from CLL patients were cultured on immobilized anti-CD3 mAb in the presence of each inhibitor or vehicle (DMSO). Then, the expression of the activation markers CD25, CD69 and CD40L was evaluated on T cell populations by flow cytometry at 24h. We found that R406 and GS-9973 impaired the expression of the activation markers on CD4+ and CD8+ at 1 µM, which is a clinically relevant concentration for both drugs (n=12, p˂0.05, Friedman test followed by Dunn?s multiple comparison post-test). The secretion of IFNɣ, IL-10 and IL4 was also impaired by 1 µM of the inhibitors (n=8, p˂0.05). Proliferation of T cells in response to TCR/CD3 stimulation was also evaluated by CFSE-dilution assay and found to be inhibited by both inhibitors in CD4+ and CD8+ T cell (n=12, p˂0.05). We also found that R406 and GS-9973 inhibited T cell proliferation induced by IL15 (n=12, p˂0.05), a cytokine involved in the homeostatic proliferation of memory T cells. Importantly neither of these drugs induced the apoptosis of T cells at the concentrations used in our experiments. Chemotaxis of T cells towards CXCL12, CCL19 and CCL21 was impaired at high concentrations of R406 and GS-9973 (5 µM) (n=10, p˂0.05) without affecting the expression of their chemokines receptors (CXCR4 and CCR7). By western blot analysis we found that Syk is not expressed on freshly isolated or anti-CD3 activated T cells from CLL patients indicating that the effect of both R406 and GS-9973 inhibitors on T cell is due to the inhibition of kinase/s other than Syk.In conclusion we found that clinically relevant doses of R406 and GS-9973 significantly impair the activation and proliferation of T cells from CLL patients in response to TCR stimulation or IL-15. Given the relevance of activated T cell to support CLL-B progression, our results suggest that R406 and GS-9973 may be successful in the treatment of CLL patients because of their effect not only on the malignant B cell clone but also on the T cells from the supportive microenvironment. Nevertheless, the strong impairment in T cell functions induced by R406 and GS-9973 might increase the known susceptibility to infections in CLL patients and thus should be considered in long-term therapy with kinase inhibitors.