The immune modulator FTY720 targets sphingosine 1-phosphate receptors

Immunosuppressant drugs such as cyclosporin have allowed widespread organ transplantation, but their utility remains limited by toxicities, and they are ineffective in chronic management of autoimmune diseases such as multiple sclerosis. In contrast, the immune modulating drug FTY720 is efficacious in a variety of transplant and autoimmune models without inducing a generalized immunosuppressed state and is effective in human kidney transplantation. FTY720 elicits a lymphopenia resulting from a reversible redistribution of lymphocytes from circulation to secondary lymphoid tissues by unknown mechanisms. Using FTY720 and several analogs, we show now that FTY720 is phosphorylated by sphingosine kinase; the phosphorylated compound is a potent agonist at four sphingosine 1-phosphate receptors and represents the therapeutic principle in a rodent model of multiple sclerosis. Our results suggest that FTY720, after phosphorylation, acts through sphingosine 1-phosphate signaling pathways to modulate chemotactic responses and lymphocyte trafficking.     

Sphingosine kinase 2 is required for modulation of lymphocyte traffic by FTY720

Immunotherapeutic drugs that mimic sphingosine 1-phosphate (S1P) disrupt lymphocyte trafficking and cause T helper and T effector cells to be retained in secondary lymphoid tissue and away from sites of inflammation. The prototypical therapeutic agent, 2-alkyl-2-amino-1,3-propanediol (FTY720), stimulates S1P signaling pathways only after it is phosphorylated by one or more unknown kinases. We generated sphingosine kinase 2 (SPHK2) null mice to demonstrate that this kinase is responsible for FTY720 phosphorylation and thereby its subsequent actions on the immune system. Both systemic and lymphocyte-localized sources of SPHK2 contributed to FTY720 induced lymphopenia. Although FTY720 was selectively activated in vivo by SPHK2, other S1P pro-drugs can be phosphorylated to cause lymphopenia through the action of additional sphingosine kinases. Our results emphasize the importance of SPHK2 expression in both lymphocytes and other tissues for immune modulation and drug metabolism.     

An update on sphingosine-1-phosphate receptor 1 modulators

Sphingolipids represent an essential class of lipids found in all eukaryotes, and strongly influence cellular signal transduction. Autoimmune diseases like asthma and multiple sclerosis (MS) are mediated by the sphingosine-1-phosphate receptor 1 (S1P₁) to express a variety of symptoms and disease patterns. Inspired by its natural substrate, an array of artificial sphingolipid derivatives has been developed to target this specific G protein-coupled receptor (GPCR) in an attempt to suppress autoimmune disorders. FTY720, also known as fingolimod, is the first oral disease-modifying therapy for MS on the market. In pursuit of improved stability, bioavailability, and efficiency, structural analogues of this initial prodrug have emerged over time. This review covers a brief introduction to the sphingolipid metabolism, the mechanism of action on S1P₁, and an updated overview of synthetic sphingosine S1P₁ agonists.     

Involvement of vacuolar H+‐ATPase in killing of human melanoma cells by the sphingosine kinase analogue FTY720

Targeting the sphingosine 1‐phosphate (S1P)/S1P receptor (S1PR) signalling axis is emerging as a promising strategy in the treatment of cancer. However, the effect of such an approach on survival of human melanoma cells remains less understood. Here, we show that the sphingosine analogue FTY720 that functionally antagonises S1PRs kills human melanoma cells through a mechanism involving the vacuolar H⁺‐ATPase activity. Moreover, we demonstrate that FTY720‐triggered cell death is characterized by features of necrosis and is not dependent on receptor‐interacting protein kinase 1 or lysosome cathepsins, nor was it associated with the activation of protein phosphatase 2A. Instead, it is mediated by increased production of reactive oxygen species and is antagonized by activation of autophagy. Collectively, these results suggest that FTY720 and its analogues are promising candidates for further development as new therapeutic agents in the treatment of melanoma.     

Modulators of the Sphingosine 1-phosphate receptor 1

The Sphingosine 1-phosphate receptor (S1P-R) signaling system has proven to be of biological and medical importance in autoimmune settings. S1P₁-R is a validated drug target for multiple sclerosis (MS) for which FTY720 (Fingolimod), a S1P_1,3–5-R pan-agonist, was recently approved as the first orally active drug for the treatment of relapsing-remitting MS. Transient bradycardia and long half-life are the FTY720 critical pitfalls. This review provides the latest advances on next-generation S1P₁-R modulators from 2012 up to date, with an overview of the chemical structures, structure–activity relationships, and relevant biological and clinical properties.     

The immune modulator FTY720 inhibits sphingosine-1-phosphate lyase activity

FTY720 is a novel immunomodulatory agent that inhibits lymphocyte trafficking and prevents allograft rejection. FTY720 is phosphorylated in vivo, and the phosphorylated drug acts as agonist for a family of G protein-coupled receptors that recognize sphingosine 1-phosphate. Evidence suggests that FTY720-phosphate-induced activation of S1P1 is responsible for its mechanism of action. FTY720 was rationally designed by modification of myriocin, a naturally occurring sphingoid base analog that causes immunosuppression by interrupting sphingolipid metabolism. In this study, we examined interactions between FTY720, FTY720-phosphate, and sphingosine-1-phosphate lyase, the enzyme responsible for irreversible sphingosine 1-phosphate degradation. FTY720-phosphate was stable in the presence of active sphingosine-1-phosphate lyase, demonstrating that the lyase does not contribute to FTY720 catabolism. Conversely, FTY720 inhibited sphingosine-1-phosphate lyase activity in vitro. Treatment of mice with FTY720 inhibited tissue sphingosine-1-phosphate lyase activity within 12 h, whereas lyase gene and protein expression were not significantly affected. Tissue sphingosine 1-phosphate levels remained stable or increased throughout treatment. These studies raise the possibility that disruption of sphingosine 1-phosphate metabolism may account for some effects of FTY720 on immune function and that sphingosine-1-phosphate lyase may be a potential target for immunomodulatory therapy.     

Gilenya (FTY720) inhibits acid sphingomyelinase by a mechanism similar to tricyclic antidepressants

The immunomodulator drug Gilenya (FTY720), marketed as the first oral sphingosine-1-phosphate receptor (S1P-R) modulator for treatment of Multiple Sclerosis (MS) also inhibits lysosomal acid sphingomyelinase (ASMase). Treatment of cultured cells for 24 h with FTY720 (up to 10 μM) inhibited ASMase by >80% and this could be reversed by pre-treatment with the cathepsin protease inhibitor leupeptin (5 μM). In contrast, neutral sphingomyelinase activity was unaffected and sphingosine-1-phosphate treatment had no effect on ASMase. RT-PCR revealed no inhibition of ASMase mRNA and there was no direct (in vitro) inhibition of ASMase by either FTY720 or FTY720-phosphate. This suggests that its mechanism of inhibition is similar to that of tricyclic anti-depressants such as desipramine, which are also amphiphilic cationic drugs. Both Desipramine and FTY720 treatment reduced ASMase without significant inhibition of other lysosomal hydrolases but most hydrolases showed increased secretion (up to a 50% increase) providing more evidence of lysosomal disruption by these drugs.     

The Sphingosine 1-Phosphate Transporter,SPNS2, Functions as a Transporter of the Phosphorylated Form of the Immunomodulating Agent FTY720

FTY720 is a novel immunomodulating drug that can be phosphorylated inside cells; its phosphorylated form, FTY720-P, binds to a sphingosine 1-phosphate (S1P) receptor, S1P₁, and inhibits lymphocyte egress into the circulating blood. Although the importance of its pharmacological action has been well recognized, little is known about how FTY720-P is released from cells after its phosphorylation inside cells. Previously, we showed that zebrafish Spns2 can act as an S1P exporter from cells and is essential for zebrafish heart formation. Here, we demonstrate that human SPNS2 can transport several S1P analogues, including FTY720-P. Moreover, FTY720-P is transported by SPNS2 through the same pathway as S1P. This is the first identification of an FTY720-P transporter in cells; this finding is important for understanding FTY720 metabolism.     

Phosphorylation of the immunomodulatory drug FTY720 by sphingosine kinases

The immunomodulatory drug FTY720 is phosphorylated in vivo, and the resulting FTY720 phosphate as a ligand for sphingosine-1-phosphate receptors is responsible for the unique biological effects of the compound. So far, phosphorylation of FTY720 by murine sphingosine kinase (SPHK) 1a had been documented. We found that, while FTY720 is also phosphorylated by human SPHK1, the human type 2 isoform phosphorylates the drug 30-fold more efficiently, because of a lower Km of FTY720 for SPHK2. Similarly, murine SPHK2 was more efficient than SPHK1a. Among splice variants of the human SPHKs, an N-terminally extended SPHK2 isoform was even more active than SPHK2 itself. Further SPHK superfamily members, namely ceramide kinase and a "SPHK-like" protein, failed to phosphorylate sphingosine and FTY720. Thus, only SPHK1 and 2 appear to be capable of phosphorylating FTY720. Using selective assay conditions, SPHK1 and 2 activities in murine tissues were measured. While activity of SPHK2 toward sphingosine was generally lower than of SPHK1, FTY720 phosphorylation was higher under conditions favoring SPHK2. In human endothelial cells, while activity of SPHK1 toward sphingosine was 2-fold higher than of SPHK2, FTY720 phosphorylation was 7-fold faster under SPHK2 assay conditions. Finally, FTY720 was poorly phosphorylated in human blood as compared with rodent blood, in line with the low activity of SPHK1 and in particular of SPHK2 in human blood. To conclude, both SPHK1 and 2 are capable of phosphorylating FTY720, but SPHK2 is quantitatively more important than SPHK1.     

The effect of the sphingosine‐1‐phosphate analogue FTY720 on atrioventricular nodal tissue

The sphingosine‐1‐phosphate (S1P) receptor modulator, fingolimod (FTY720), has been used for the treatment of patients with relapsing forms of multiple sclerosis, but atrioventricular (AV) conduction block have been reported in some patients after the first dose. The underlying mechanism of this AV node conduction blockade is still not well‐understood. In this study, we hypothesize that expression of this particular arrhythmia might be related to a direct effect of FTY720 on AV node rather than a parasympathetic mimetic action. We, therefore, investigated the effect of FTY720 on AV nodal, using in vitro rat model preparation, under both basal as well as ischaemia/reperfusion conditions. We first look at the expression pattern of S1P receptors on the AV node using real‐time PCR. Although all three S1P receptor isoforms were expressed in AVN tissues, S1P1 receptor isoform expression level was higher than S1P2 and S1P3. The effect of 25 nM FTY720 on cycle length (CL) was subsequently studied via extracellular potentials recordings. FTY720 caused a mild to moderate prolongation in CL by an average 9% in AVN (n = 10, P < 0.05) preparations. We also show that FTY720 attenuated both ischaemia and reperfusion induced AVN rhythmic disturbance. To our knowledge, these remarkable findings have not been previously reported in the literature, and stress the importance for extensive monitoring period in certain cases, especially in patients taking concurrently AV node blocker agents.     

AKP-11 - A Novel S1P1 Agonist with Favorable Safety Profile Attenuates Experimental Autoimmune Encephalomyelitis in Rat Model of Multiple Sclerosis

Sphingosine-1-phosphate receptor 1 (S1P1) mediated regulation of lymphocyte egress from lymphoid organs is recognized as the mechanism of FTY720 (Fingolimod, Gilenya) efficacy in relapsing-remitting forms of multiple sclerosis (RRMS). In this study we describe a novel S1P1 agonist AKP-11, next generation of S1P1 agonist, with immunomodulatory activities in cell culture model and for therapeutic efficacy against an animal model of MS, i.e. experimental autoimmune encephalomyelitis (EAE) but without the adverse effects observed with FTY720. Like FTY720, AKP-11 bound to S1P1 is internalized and activates intracellular AKT and ERKs cellular signaling pathways. In contrast to FTY720, AKP-11 mediated S1P1 downregulation is independent of sphingosine kinase activity indicating it to be a direct agonist of S1P1. The S1P1 loss and inhibition of lymphocyte egress by FTY720 leads to lymphopenia. In comparison with FTY720, oral administration of AKP-11 caused milder and reversible lymphopenia while providing a similar degree of therapeutic efficacy in the EAE animal model. Consistent with the observed reversible lymphopenia with AKP-11, the S1P1 recycled back to cell membrane in AKP-11 treated cells following its withdrawal, but not with withdrawal of FTY720. Accordingly, a smaller degree of ubiquitination and proteolysis of S1P1 was observed in AKP-11 treated cells as compared to FTY720. Consistent with previous observations, FTY720 treatment is associated with adverse effects of bradycardia and lung vascular leaks in rodents, whereas AKP-11 treatment had undetectable effects on bradycardia and reduced lung vascular leaks as compared to FTY720. Taken together, the data documents that AKP-11 treatment cause milder and reversible lymphopenia with milder adverse effects while maintaining therapeutic efficacy similar to that observed with FTY720, thus indicating therapeutic potential of AKP-11 for treatment of MS and related autoimmune disorders.     

FTY720 induces necrotic cell death and autophagy in ovarian cancer cells: a protective role of autophagy

FTY720, a sphingosine analog, is a novel immunosuppressant currently undergoing multiple clinical trials for the prevention of organ transplant rejection and treatment of various autoimmune diseases. Recent studies indicate an additional cytotoxic effect of FTY720 and its preclinical efficacy in a variety of cancer models, yet the underlying mechanisms remain unclear. We demonstrate here for the first time that FTY720 exhibits a potent, dose- and time-dependent cytotoxic effect in human ovarian cancer cells, even in the cells that are resistant to cisplatin, a commonly prescribed chemotherapeutic drug for treatment of ovarian cancer. In contrast to the previously reported cytotoxicity of FTY720 in many other cancer cell types, FTY720 kills ovarian cancer cells independent of caspase 3 activity and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Furthermore, the presence of autophagic hallmarks, including an increased number of autophagosomes and the formation and accumulation of LC3-II, are observed in FTY720-treated cells before cell death. FTY720 treatment enhances autophagic flux as reflected in the increased LC3 turnover and p62 degradation. Notably, blockade of autophagy by either specific chemical inhibitors or siRNAs targeting Beclin 1 or LC3 resulted in aggravated necrotic cell death in response to FTY720, suggesting that FTY720-induced autophagy plays a self-protective role against its own cytotoxic effect. Thus, our findings not only demonstrate a new death pathway underlying the cytotoxic effect of FTY720, but also suggest that targeting autophagy could augment the anticancer potency, providing the framework for further development of FTY720 as a new chemotherapeutic agent for ovarian cancer treatment.     

FTY720 induces necrotic cell death and autophagy in ovarian cancer cells: A protective role of autophagy

FTY720, a sphingosine analog, is a novel immunosuppressant currently undergoing multiple clinical trials for the prevention of organ transplant rejection and treatment of various autoimmune diseases. Recent studies indicate an additional cytotoxic effect of FTY720 and its preclinical efficacy in a variety of cancer models, yet the underlying mechanisms remain unclear. We demonstrate here for the first time that FTY720 exhibits a potent, dose- and time-dependent cytotoxic effect in human ovarian cancer cells, even in the cells that are resistant to cisplatin, a commonly prescribed chemotherapeutic drug for treatment of ovarian cancer. In contrast to the previously reported cytotoxicity of FTY720 in many other cancer cell types, FTY720 kills ovarian cancer cells independent of caspase 3 activity and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Furthermore, the presence of autophagic hallmarks, including an increased number of autophagosomes and the formation and accumulation of LC3-II, are observed in FTY720-treated cells before cell death. FTY720 treatment enhances autophagic flux as reflected in the increased LC3 turnover and p62 degradation. Notably, blockade of autophagy by either specific chemical inhibitors or siRNAs targeting Beclin 1 or LC3 resulted in aggravated necrotic cell death in response to FTY720, suggesting that FTY720-induced autophagy plays a self-protective role against its own cytotoxic effect. Thus, our findings not only demonstrate a new death pathway underlying the cytotoxic effect of FTY720, but also suggest that targeting autophagy could augment the anticancer potency, providing the framework for further development of FTY720 as a new chemotherapeutic agent for ovarian cancer treatment.     

PTEN‐ and p53‐mediated apoptosis and cell cycle arrest by FTY720 in gastric cancer cells and nude mice

FTY720, a new immunosuppressant, derived from ISP‐1, has been studied for its putative anti‐cancer properties in the recent years. In this study, we have reported that FTY720 greatly inhibited gastric cancer cell proliferation for the first time, and found this effect was associated with G1 phase cell cycle arrest and apoptosis. Results from our Western blotting and Real‐time PCR showed that FTY720 induced obvious PTEN expression in a p53‐independent way, consistent with a substantial decrease in p‐Akt and MDM2. FTY720 dramatically increased the expression of Cip1/p21, p27, and BH3‐only proteins through the accumulation of p53 by PTEN‐mediated inhibition of the PI3K/Akt/MDM2 signaling. Suppression of PTEN expression with siRNA significantly reduced the p53 and p21 levels and activated Akt, resulting in decreased apoptosis and increased cell survival. Furthermore, we have observed an additive effect of FTY720 in killing gastric cancer cells when in combination with Cisplatin, partly through PTEN‐mediated Akt/MDM2 inhibition. In vivo study has also shown that tumor growth was significantly suppressed after FTY720 treatment. In conclusion, our results suggest that FTY720 induces a significant increase of PTEN, which inhibits p‐Akt and MDM2, and then increases the level of p53, thereby inducing G1 phase arrest and apoptosis. We have characterized a novel immunosuppressant, for the first time, which shows potential anti‐tumor effects on gastric cancer by PTEN activation through p53‐independent mechanism, especially in combination with Cisplatin. This PTEN target‐based therapy is worth further investigation and warrants clinical evaluation. J. Cell. Biochem. 111: 218–228, 2010. © 2010 Wiley‐Liss, Inc.     

Phosphorylation and action of the immunomodulator FTY720 inhibits vascular endothelial cell growth factor-induced vascular permeability

FTY720, a potent immunosuppressive agent, is phosphorylated in vivo into FTY720-P, a high affinity agonist for sphingosine 1-phosphate (S1P) receptors. The effects of FTY720 on vascular cells, a major target of S1P action, have not been addressed. We now report the metabolic activation of FTY720 by sphingosine kinase-2 and potent activation of vascular endothelial cell functions in vitro and in vivo by phosphorylated FTY720 (FTY720-P). Incubation of endothelial cells with FTY720 resulted in phosphorylation by sphingosine kinase activity and formation of FTY720-P. Sphingosine kinase-2 effectively phosphorylated FTY720 in the human embryonic kidney 293T heterologous expression system. FTY720-P treatment of endothelial cells stimulated extracellular signal-activated kinase and Akt phosphorylation and adherens junction assembly and promoted cell survival. The effects of FTY720-P were inhibited by pertussis toxin, suggesting the requirement for Gi-coupled S1P receptors. Indeed, transmonolayer permeability induced by vascular endothelial cell growth factor was potently reversed by FTY720-P. Furthermore, oral FTY720 administration in mice potently blocked VEGF-induced vascular permeability in vivo. These findings suggest that FTY720 or its analogs may find utility in the therapeutic regulation of vascular permeability, an important process in angiogenesis, inflammation, and pathological conditions such as sepsis, hypoxia, and solid tumor growth.     

FTY720-induced blockage of autophagy enhances anticancer efficacy of milatuzumab in mantle cell lymphoma

Inhibition of the autophagic pathway has recently revealed promising results in increasing pro-death activity of multiple cancer therapeutics. Here, we discuss our findings regarding the autophagy-blocking and anti-neoplastic effects of a synthetic sphingosine analog, FTY720, in mantle cell lymphoma (MCL). We also emphasize how FTY720 enhances the pro-death activity of the fully humanized monoclonal antibody milatuzumab by inhibiting the autophagy-lysosome dependent degradation of its therapeutic target, CD74. Our results provide justification for further evaluation of FTY720 and milatuzumab as a combination therapy for this aggressive B-cell malignancy.     

The immunosuppressant FTY720 is phosphorylated by sphingosine kinase type 2

The potent immunosuppressive drug FTY720, a sphingosine analog, induces redistribution of lymphocytes from circulation to secondary lymphoid tissues. FTY720 is phosphorylated in vivo and functions as an agonist for four G-protein-coupled sphingosine-1-phosphate receptors. The identity of the kinase that phosphorylates FTY720 is still not known. Here we report that although both sphingosine kinase type 1 (SphK1) and type 2 (SphK2) can phosphorylate FTY720 with low efficiency, SphK2 is much more effective than SphK1. FTY720 inhibited phosphorylation of sphingosine catalyzed by SphK2 to a greater extent than it inhibits SphK1. Thus, SphK2 may be the relevant enzyme that is responsible for in vivo phosphorylation of FTY720.     

FTY720-induced blockage of autophagy enhances anticancer efficacy of milatuzumab in mantle cell lymphoma: Is FTY720 the next autophagy-blocking agent in lymphoma treatment?

Inhibition of the autophagic pathway has recently revealed promising results in increasing pro-death activity of multiple cancer therapeutics. Here, we discuss our findings regarding the autophagy-blocking and anti-neoplastic effects of a synthetic sphingosine analog, FTY720, in mantle cell lymphoma (MCL). We also emphasize how FTY720 enhances the pro-death activity of the fully humanized monoclonal antibody milatuzumab by inhibiting the autophagy-lysosome dependent degradation of its therapeutic target, CD74. Our results provide justification for further evaluation of FTY720 and milatuzumab as a combination therapy for this aggressive B-cell malignancy.