ASSESSMENT OF THE EFFECT OF SPHINGOSINE KINASE INHIBITORS ON APOPTOSIS, UNFOLDED PROTEIN RESPONSE AND AUTOPHAGY OF T-ACUTE LYMPHOBLASTIC LEUKEMIA CELLS: INDICATIONS FOR NOVEL THERAPEUTICS

Introduction. Sphingosine 1-phosphate (S1P) is involved in many processes such as cell survival, growth, migration, and cancer. S1P is formed by the phosphorylation of sphingosine by sphingosine kinase 1 (SK1) or sphingosine kinase 2 (SK2). While the role of SK1 in cancer is well established, the role of SK2 in regulating apoptosis is still a matter of debate, even though emerging evidence has highlighted the importance of SK2 in cancer. Therefore, SKs represent a promising target for cancer therapy. Recently, the importance of S1P in hematological malignancies has been described. Here, we analyze the therapeutic effects of 2-(p-Hydroxyanilino)-4-(p-chlorophenyl)thiazole (SKi), an SK1/2 inhibitor, and (R)-FTY720 methyl ether (ROMe), a SK2-selective inhibitor, in T-ALL cell lines. Methods. T- ALL cell lines (Molt-4, Jurkat, CEM-R) were treated with SKi and ROMe for 40 h, then MTT assays were performed to analyze cell viability. Apoptosis induction was evaluated by Annexin V/PI staining. Protein expression was studied by western blot (WB). Results. SKi and ROMe induced a decrease in cell viability, as demonstrated by MTT assays. Annexin-V/PI staining and flow cytometric analysis of cells treated with a SKi concentration equivalent to the IC50 documented the occurrence of apoptotic cell death. This was confirmed by WB analysis for caspase and PARP cleavage in all the cell lines. However, we also observed a SKi-induced autophagy by means of WB and transmission electron microscopy analysis in Jurkat and CEM-R cells. We then analyzed the expression of ER stress/UPR hallmarks. SKi activated the ER stress/UPR pathway and this occurred following apoptosis and correlated with autophagy. The combination of SKi and chloroquine (an autophagy inhibitor) induced a decrease in cell viability, indicating that the UPR/autophagic response is likely to be a protective mechanism. In Molt-4 cells, we detected an increase in SK1 expression after 40 h of treatment with SKi, that could represent an attempt of cells to escape SKi-induced apoptosis, as high SK1 expression is known to enhance cell growth and survival. Notably, SKi synergized with vincristine at concentrations of SKi that were much below its respective IC50, suggesting that vincristine sensitized T-ALL cells to SKi and that combining chemotherapeutic agents with SK inhibitors could be feasible in the treatment of T-ALL. Finally, ROMe treatment induced an autophagic type of cell death. Conclusions. We report herein that SKi and ROMe affect T-ALL cell viability, but they exert their effects through different mechanisms. While ROMe induces an autophagic cell death, SKi induces apoptosis. Moreover, for the first time, we demonstrated that SKi activates an ER stress/UPR pathway in T-ALL cells and this is linked with a protective autophagic response. Thus, our findings indicate that SK1 or SK2 may represent potential targets for treating T-ALL, thereby enabling better management of this cancer