FTY720 (Fingolimod) Targets the Sphingolipid Pathway and Induces Autophagy-Related Apoptosis in Human Natural Killer Large Granular Lymphocyte Leukemia

Introduction: Natural killer large granular lymphocytic leukemia (NK-LGL) and chronic lymphoproliferative disorder of NK cells (CLPD-NK) are rare disorders of cytotoxic CD3-/CD56+ natural killer cells. Aggressive NK-LGL leukemia patients present with a malignant clinical course and a fatal outcome with median survival time of two months from diagnosis. Aggressive NK-LGL is refractory to conventional chemotherapy and pathogenetic mechanisms remain undefined.

The 'sphingolipid rheostat' has been identified as a key player in determining cell fate. The balance between pro-apoptotic ceramide and pro-survival sphingosine-1-phosphate (S1P) in this rheostat is determined by the expression of acid ceramidase (AC) which converts ceramide to sphingosine, and sphingosine kinase-1 and -2 (SPHK1 and SPHK2), which convert sphingosine to S1P. The S1P pathway is involved in cancer pathogenesis, cell survival, and resistance to chemotherapy and radiation, therefore it is considered a potential target for anti-cancer therapy. S1P signals both extracellularly through a family of five related GPCRs; the S1P receptors (S1PR_1-5) and intracellularly through various incompletely defined mechanisms. FTY720 is a sphingolipid modulator that targets the S1P system and has potent immunosuppressive and anti-cancer properties. We have previously shown that FTY720 shows therapeutic efficacy in a rat model of NK-LGL leukemia, but its mechanism-of-action in inducing cell death is incompletely defined. The aim of this study is to explore these molecular mechanisms and further characterize the actions of FTY720 in NK-LGL leukemia.

Results: We identified that the key enzymes in S1P biosynthesis, SPHK1 and AC, and the S1P receptor S1PR5 were all expressed in LGL cell lines and primary PBMCs from NK-LGL patients. Increased mRNA levels of AC, SPHK1 and S1PR5 were detected in NK-LGL samples (n=8) compared to PBMCs from healthy donors (n=8) (p < 0.05). These results were verified by immunoblot in NK-LGL samples (n=6) versus PBMCs from healthy donors (n=3) (p < 0.05). In vitro treatment of human and rat LGL cell lines (NKL, RNK16 and TL-1) resulted in time- and dose-dependent decreases in cell proliferation and increased apoptosis (IC50 5.0 - 12.5 μM). Interestingly, treatment of the LGL cell lines with the phosphorylated form of FTY720, FTY720-P, which acts antagonistically at the S1PRs, resulted in minimal effects on proliferation and apoptosis. This suggests that FTY720-induced cell death is independent of its effects on S1PRs. However, treatment of LGL cell lines with FTY720 did target multiple sphingolipid pathway members. We observed dose-dependent decreases in AC and S1PR5 protein expression. Interestingly, the combination of FTY720 with LCL204, an AC inhibitor, resulted in additive effects on cell proliferation and apoptosis in the NKL cells.

Further characterization of cell death mechanisms demonstrated involvement of autophagy-related apoptosis. We observed increased PARP and caspase-3 cleavage after FTY720 treatment, which occurred after increased expression of LC3A/B and the conversion of LC3-I to LC3-II, suggesting autophagy is being induced prior to apoptosis induction. We identified the mTOR pathway as a target of FTY720. The mTOR pathway negatively regulates autophagy and its inhibition can induce autophagy. FTY720 treatment suppressed mTOR signaling in LGL cell lines as demonstrated by decreased phosphorylation of mTOR, p70S6K and S6 Kinase. These effects preceded the autophagy-related changes in LC3A/B. Furthermore, PARP and caspase-3 cleavage could be blocked by pre-treatment with 3-methyladenine and BafilomycinA1, both inhibitors of autophagy. Interestingly, the effects of FTY720 in LGL cell lines could also be blocked by pre-treatment with autophagy inhibitors, further suggesting that autophagy is required for the FTY720-induced effects. Importantly, these key findings were also reproduced in PBMCs from NK-LGL patients (n=3).

Conclusions: Taken together, our findings demonstrate that FTY720 targets multiple sphingolipid pathway members in NK-LGL leukemia and that FTY720-induced cell death is autophagy-dependent. The down-stream targeting of mTOR by FTY720, combined with mTOR's prominent role in inhibiting autophagy, suggests that co-treatment with specific mTOR inhibitors in combination with FTY720 may be a promising and novel therapeutic strategy in LGL leukemia.