Fingolimod (FTY720) is an immunosuppressive drug that was recently approved for the treatment of multiple sclerosis and is currently under pre-clinical investigation as a therapy for a number of haematological malignancies. Previous studies have indicated a role for FTY720 in inducing autophagy and caspase-independent cell death in cancer cells through incompletely characterized molecular mechanisms. Our study thus aims at a beeer understanding of the way of action of FTY720. In chronic lymphocytic leukaemia (CLL) cells, FTY720 induced cell death with typical features of apoptosis, including phosphatidylserine exposure and caspase-3 activation, and features of autophagy, including LC3 conversion, autophagolysosome formation and lysosomal cathepsins activation. However, neither caspase nor autophagy blockade prevented the cytotoxic effect of FTY720, suggesting another mechanism of cell death. Using electron and fluorescence microscopy, flow cytometry and biochemical analyses, we found that FTY720 treatment increased a fraction of annexin V-/7-AAD+ cells both in primary and transformed leukemic cells and induced morphological changes representative of necrosis, including oncosis, mitochondrial and plasma membrane alteration. FTY720 treatment resulted in increased plasma membrane permeability as shown by the extracellular translocation of the nuclear high mobility group box 1 (HMGB1) protein and by the release into the culture medium of the cytosolic enzyme lactate dehydrogenase (LDH). Interestingly, cell death induced by FTY720 was not prevented by pharmacological inhibition of RIPK1 and PP2A. In contrast, FTY720--‐induced necrosis was accompanied by an early relocation to the mitochondria of Dynamin Related Protein 1, DRP1. Importantly, FTY720 stimulation led to ma tior changes in the phosphorylation of serine residues associated with the mitochondrial fission activity of DRP1. Finally, siRNA--‐mediated knockdown of DRP1 significantly reduced necrotic cell death induced by FTY720. In this study, we thus demonstrate that in leukemic cells the cytotoxic effect of the immunosuppressive drug Fingolimod involves a DRP1--‐dependent regulated necrosis. These observations are important in line of the future development of Fingolimod as a new therapeutic agent in haematological malignancies.
