Sphingosine-1-phosphate Receptor 1 Receptor Research Articles

SPHK1-S1P-S1PR1 signaling pathway is emerging as a potential therapeutic target in improving endothelial barrier function and prevent coronary artery disease.

Abstract Sphingosine kinases (SPHKs) catalyze the formation of sphingosine 1 phosphate (S1P) from sphingosine. S1P, a lipid mediator, was shown to be one such factor promoting endothelial barrier function. S1P binds to S1P receptor-1 (S1PR1) in endothelial cells, leading to activation of heterotrimeric G proteins (Gi), and signals enhancement of endothelial barrier function by the activation of small GTPase Rac1. We hypothesized that SPHK1 via generation of S1P induces reannealing of adherens junctions and thereby prevents thrombin-induced endothelial permeability. To understand the role of SPHK1 pathway, we depleted SPHK1 protein in endothelial cells using SPHK1 specific small interference (si) RNA. Depletion of SPHK1 significantly disrupted the adherens junction, VE-cadherin and increased the gap formation of endothelial cells. Upon depletion of SPHK1 we found decrease cell proliferation and migration, but no change in the expression of Sphingosine 1 Phosphate Receptor 1 (S1PR1) expression in endothelial cells. To determine if in the absence of SPHK1, does S1P has any pharmacological role, we treated the cells with Sphingosine 1 Phosphate (S1P) at the dose of 1μM. We found that S1P re-anneal the adherens junctions and improve cell proliferation and migration in SPHK1 depleted endothelial cells. This indicates that SPHK1 via generation of S1P prevents endothelial permeability through S1PR1 receptor activation. Upon treatment with S1P in SPHK1 depleted endothelia showed completely recovery in barrier function. In conclusion, our study for the first time shows that SPHK1 maintains endothelial barrier function and reanneals adherens junctions, thus hastens the recovery process. CTRE grant to Professor Nadeem Fazal, MD, PhD

Myeloid cells and sphingosine-1-phosphate are required for TCRαβ +intraepithelial lymphocyte recruitment to the colon epithelium

Abstract Intraepithelial lymphocytes (IELs) of the colon are essential for barrier maintenance and repair after injury. As bacterial depletion or global MyD88 deficiency results in a significant reduction of colon IELs and impaired barrier integrity, we aimed to connect the signals of how bacteria recruit colon IELs to the epithelium using conditional knockouts of MyD88 to target T cells (Lck-Cre), IECs (Villin-Cre), and myeloid cells (LysM-Cre). Only in the colons of LysM-Cre +mice were TCRαβ +IELs reduced, at 3.3-fold (p<0.005 compared to Cre −littermate controls) without effect on lamina propria T cells or myeloid cells. TCRγδ +IELs were unaffected in all knockouts, suggesting they are recruited via different mechanisms. We then examined upregulated cell surface gene expression of IELs during recruitment, identifying sphingosine-1-phosphate receptor 1 (S1PR1) as a candidate for further study. Indeed, S1PR1 expression is significantly increased at 3.7-fold in IELs (p<0.05 compared to lamina propria T cells). In accordance with these findings, we demonstrated that TCRαβ+ IEL recruitment is blocked by administration of the S1PR1 receptor agonist FTY720 (p<0.05 compared to vehicle treatment). Furthermore, myeloid cell production of sphingosine-1-phosphate (S1P) was required for colon TCRαβ +IEL recruitment in a MyD88-dependent manner. In the TNF ΔARE/+model of spontaneous colitis, loss of myeloid MyD88 resulted in protection from disease (p<0.05 compared to Cre-littermate controls). Overall, these data demonstrate the requirement for myeloid-produced S1P for TCRαβ+ IEL recruitment to the colon and suggest that TCRαβ +IELs can contribute to intestinal inflammation under a specific genetic predisposition.

Endothelial S1P1 Signaling Counteracts Infarct Expansion in Ischemic Stroke.

Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke. To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.

A label-free impedance assay in endothelial cells differentiates the activation and desensitization properties of clinical S1P1 agonists.

Sphingosine-1 phosphate receptor-1 (S1P1 ) activation maintains endothelial barrier integrity, whereas S1P1 desensitization induces peripheral blood lymphopenia. The latter is exploited in the approval and/or late-stage development of receptor-desensitizing agents targeting the S1P1 receptor in multiple sclerosis, such as siponimod, ozanimod, and ponesimod. SAR247799 is a recently described G protein-biased S1P1 agonist that activates S1P1 without desensitization and thus has endothelial-protective properties in patients without reducing lymphocytes. As SAR247799 demonstrated endothelial-protective effects at sub-lymphocyte-reducing doses, the possibility exists that other S1P1 modulators could also exhibit endothelial-protective properties at lower doses. To explore this possibility, we sought to quantitatively compare the biased properties of SAR247799 with the most advanced clinical molecules targeting S1P1 . In this study, we define the β-arrestin pathway component of the impedance profile following S1P1 activation in a human umbilical vein endothelial cell line (HUVEC) and report quantitative indices of the S1P1 activation-to-desensitization ratio of various clinical molecules. In a label-free impedance assay assessing endothelial barrier integrity and disruption, the mean estimates (95% confidence interval) of the activation-to-desensitization ratios of SAR247799, ponesimod, ozanimod, and siponimod were 114 (91.1-143), 7.66 (3.41-17.2), 6.35 (3.21-12.5), and 0.170 (0.0523-0.555), respectively. Thus, we show that SAR247799 is the most G protein-biased S1P1 agonist currently characterized. This rank order of bias among the most clinically advanced S1P1 modulators provides a new perspective on the relative potential of these clinical molecules for improving endothelial function in patients in relation to their lymphocyte-reducing (desensitization) properties.

Sphingosine‐1‐Phosphate Receptor 1 Mediated Vasodilation in Human Arterioles

Precise regulation of vascular resistance is critical to maintain proper tissue perfusion. Recent data in animal models have shown that sphingolipids, a ubiquitous class of bioactive lipid messengers, can influence vasomotor tone via dilation or constriction. It has been suggested that sphingosine‐1‐phosphate (S1P) elicits vasodilation through generation of endothelial cell nitric oxide (NO) via activation of sphingosine‐1‐phosphate receptor 1 (S1PR1). We therefore examined the hypothesis that S1P is a regulator of human microvascular tone and elicits dilation via S1PR1‐induced increase in NO. Arterioles from healthy human adipose tissue were dissected and prepared for videomicroscopy, equilibrated and pre‐constricted with endothelin 1. Changes in arteriolar luminal diameter were recorded in response to increasing concentrations of S1P (10−12 to 10−6M) in the presence or absence of the S1PR1 receptor antagonist W146 (10−5M), nitric oxide synthase (NOS) inhibitor Nω‐nitro‐ l‐arginine (L‐NAME, 10−4M) or hydrogen peroxide (H2O2) scavenger polyethylene glycol‐catalase (peg‐Cat, 500U/ml). Administration of exogenous S1P induced vasodilation in healthy vessels to a maximal dilation of 63.7%±5.0, n=8 (mean±SEM). As with previous animal studies, vascular constriction was observed with higher, but still physiological levels of S1P (10−6 M). Vasodilation due to S1P was reduced following inhibition of S1PR1 compared to S1P alone (1.8%±4, n=5). In the presence of L‐NAME, S1P‐induced dilation decreased (9%±7.9, n=6). Interestingly, addition of peg‐Cat, an H2O2 scavenger, also decreased S1P‐induced dilation (11.1%±6.6, n=3). Together these data suggest that S1PR1 is a critical mechanistic component of S1P‐induced vasodilation in the human vasculature. Further, the S1P vasodilatory pathway may require formation of H2O2 in addition to NO.Support or Funding InformationThis research was supported by National Institute of Health (NHLBI) K08 HL141562‐02 (JKF)

Abrogation of Pancreatic Cancer Using a Brain Malady Drug

Pancreatic cancer is the fourth preeminent cause of cancer-related deaths with a poor prognosis. Desmoplasia and drug resistance are few of the pivotal hurdles in pancreatic cancer management. Unlike breast cancer, awareness about pancreatic cancer still lacks in all the aspects. Our extensive bioinformatic and clinical data mining led us to notice an abnormal upregulation of a membrane protein Sphingosine-1-Phosphate Receptor-1 (S1PR1) in pancreatic cancer. S1PR1 is an important lipid signalling receptor whose ligand is a bioactive lipid, Sphingosine-1-phosphate. We were curious to know what happens to the cancer cell if we block this active lipid signaling. So, we targeted this protein using its antagonist, FTY720, which is already in the market for a known brain disorder called multiple sclerosis. FDA accreditation and known ADMET patters prompted us to choose this pre-existing drug. Accordingly, time-consuming clinical trials could be bypassed leading to rapid repurposing for pancreatic cancer. Our in vitro studies on numerous pancreatic cancer cell lines displayed a dose-dependent increase in cell death with low toxicity on normal cells. Although, gemcitabine is the first line chemotherapeutic drug for pancreatic cancer, the emergence of resistance among the patients was observed which leads to metastasis. In particular, combination therapy is an imminent approach to tackle the trouble of drug resistance. Indeed, an amalgamation of FTY720 and gemcitabine showed the enhanced anticancer effects on ex vivo experiments. Furthermore, two animal models showed the conspicuous abatement in tumor chunk as a result of blended drug treatment for 30 days. Besides, reduction of significant cell proliferation markers in the tumor microenvironment was observed. We found that FTY720 alone and also in commixture with gemcitabine effectively ameliorated the activity of PP2A which is the known target of FTY720. STAT3 is a transcription factor, which plays a remarkable role in carcinogenesis. We also observed a reduced S1PR1-STAT3 loop in vivo along with its downstream survival proteins due to strong affinity of FTY720 on the S1PR1 receptor. Since the combination of FTY720+Gem showed an astonishing role in abatement of tumor size than solitary treatment, the exact role of this combination strategy was explained by exploring the role of FTY720, which may act as a trooper for gemcitabine to cross the desmoplasia, an enlarged collagen area. As expected, we found a reduction in desmoplastic markers in the FTY720 treatment group. It infers that FTY720 loosens up the tumor tissue so that gemcitabine can readily reach the cancer cells. In conclusion, we propose that FTY720 can be a propitious molecule in pancreatic cancer management in combination with other chemotherapeutic drugs. Support or Funding Information University Grants Commision, Government of India and Department of Biotechnology, Government of India. The Mechanism of action of the combination therapy in pancreatic cancer. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

S1P1 Modulator-Induced Gαi Signaling and β-Arrestin Recruitment Are Both Necessary to Induce Rapid and Efficient Reduction of Blood Lymphocyte Count In Vivo

S1P1 (sphingosine-1-phosphate receptor 1) agonists prevent lymphocyte egress from secondary lymphoid organs and cause a reduction in the number of circulating blood lymphocytes. We hypothesized that S1P1 receptor modulators with pathway-selective signaling properties could help to further elucidate the molecular mechanisms involved in lymphocyte trapping. A proprietary S1P1 receptor modulator library was screened for compounds with clear potency differences in β-arrestin recruitment and G protein alpha i subunit (G αi) protein-mediated signaling. We describe here the structure-activity relationships of highly potent S1P1 modulators with apparent pathway selectivity for β-arrestin recruitment. The most differentiated compound, D3-2, displayed a 180-fold higher potency in the β-arrestin recruitment assay (EC50 0.9 nM) compared with the G αi-activation assay (167 nM), whereas ponesimod, a S1P1 modulator that is currently in advanced clinical development in multiple sclerosis, was equipotent in both assays (EC50 1.5 and 1.1 nM, respectively). Using these novel compounds as pharmacological tools, we showed that although a high potency in β-arrestin recruitment is required to fully internalize S1P1 receptors, the potency in inducing G αi signaling determines the rate of receptor internalization in vitro. In contrast to ponesimod, the compound D3-2 did not reduce the number or circulating lymphocytes in rats despite high plasma exposures. Thus, for rapid and maximal S1P1 receptor internalization a high potency in both G αi signaling and β-arrestin recruitment is mandatory and this translates into efficient reduction of the number of circulating lymphocytes in vivo.

Cenerimod, a novel selective S1P1 receptor modulator with unique signaling properties.

Sphingosine‐1‐phosphate receptor 1 (S1P1) modulators sequester circulating lymphocytes within lymph nodes, thereby preventing potentially pathogenic autoimmune cells from exiting into the blood stream and reaching inflamed tissues. S1P1 receptor modulation may thus offer potential to treat various autoimmune diseases. The first nonselective S1P1‐5 receptor modulator FTY720/fingolimod/Gilenya® has successfully demonstrated clinical efficacy in relapsing forms of multiple sclerosis. However, cardiovascular, hepatic, and respiratory side‐effects were reported and there is a need for novel S1P1 receptor modulators with better safety profiles. Here, we describe the discovery of cenerimod, a novel, potent and selective S1P1 receptor modulator with unique S1P1 receptor signaling properties and absence of broncho‐ and vasoconstrictor effects ex vivo and in vivo. Cenerimod dose‐dependently lowered circulating lymphocyte counts in rats and mice after oral administration and effectively attenuated disease parameters in a mouse experimental autoimmune encephalitis (EAE) model. Cenerimod has potential as novel therapy with improved safety profile for autoimmune diseases with high unmet medical need.

Antinociceptive effects of FTY720 during trauma-induced neuropathic pain are mediated by spinal S1P receptors

FTY720 (fingolimod) is, after its phosphorylation by sphingosine kinase (SPHK) 2, a potent, non-selective sphingosine-1-phosphate (S1P) receptor agonist. FTY720 has been shown to reduce the nociceptive behavior in the paclitaxel model for chemotherapy-induced neuropathic pain through downregulation of S1P receptor 1 (S1P1) in microglia of the spinal cord. Here, we investigated the mechanisms underlying the antinociceptive effects of FTY720 in a model for trauma-induced neuropathic pain. We found that intrathecal administration of phosphorylated FTY720 (FTY720-P) decreased trauma-induced pain behavior in mice, while intraplantar administered FTY720-P had no effect. FTY720-P, but not FTY720, reduced the nociceptive behavior in SPHK2-deficient mice, suggesting the involvement of S1P receptors. Fittingly, intrathecal administration of antagonists for S1P1 or S1P3, W146 and Cay10444 respectively, abolished the antinociceptive effects of systemically administered FTY720, demonstrating that activation of both receptors in the spinal cord is necessary to induce antinociceptive effects by FTY720. Accordingly, intrathecal administration of S1P1 receptor agonists was not sufficient to evoke an antinociceptive effect. Taken together, the data show that, in contrast to its effects on chemotherapy-induced neuropathy, FTY720 reduces trauma-induced neuropathic pain by simultaneous activation of spinal S1P1 and S1P3 receptor subtypes.

Bioactive lipids and cationic antimicrobial peptides as new potential regulators for trafficking of bone marrow-derived stem cells in patients with acute myocardial infarction.

Acute myocardial infarction (AMI) triggers mobilization of stem cells from bone marrow (BM) into peripheral blood (PB). Based on our observation that the bioactive sphingophospholipids, sphingosine-1 phosphate (S1P), and ceramide-1 phosphate (C1P) regulate trafficking of hematopoietic stem cells (HSCs), we explored whether they also direct trafficking of non-hematopoietic stem cells (non-HSCs). We detected a 3-6-fold increase in circulating CD34+, CD133+, and CXCR4+ lineage-negative (Lin-)/CD45- cells that are enriched in non-HSCs [including endothelial progenitors (EPCs) and very small embryonic-like stem cells (VSELs)] in PB from AMI patients (P<0.05 vs. controls). Concurrently, we measured a ∼3-fold increase in S1P and C1P levels in plasma from AMI patients. At the same time, plasma obtained at hospital admission and 6 h after AMI strongly chemoattracted human BM-derived CD34+/Lin- and CXCR4+/Lin- cells in Transwell chemotaxis assays. This effect of plasma was blunted after depletion of S1P level by charcoal stripping and was further inhibited by the specific S1P1 receptor antagonist such as W146 and VPC23019. We also noted that the expression of S1P receptor 1 (S1P1), which is dominant in naïve BM, is reduced after the exposure to S1P at concentrations similar to the plasma S1P levels in patients with AMI, thus influencing the role of S1P in homing to the injured myocardium. Therefore, we examined mechanisms, other than bioactive lipids, that may contribute to the homing of BM non-HSCs to the infarcted myocardium. Hypoxic cardiac tissue increases the expression of cathelicidin and β-2 defensin, which could explain why PB cells isolated from patients with AMI migrated more efficiently to a low, yet physiological, gradient of stromal-derived factor-1 in Transwell migration assays. Together, these observations suggest that while elevated S1P and C1P levels early in the course of AMI may trigger mobilization of non-HSCs into PB, cathelicidin and β-2 defensin could play an important role in their homing to damaged myocardium.

S1P1 localizes to the colonic vasculature in ulcerative colitis and maintains blood vessel integrity

Signaling through sphingosine-1-phosphate receptor₁ (S1P₁) promotes blood vessel barrier function. Degradation of S1P₁ results in increased vascular permeability in the lung and may explain side effects associated with administration of FTY720, a functional antagonist of the S1P₁ receptor that is currently used to treat multiple sclerosis. Ulcerative colitis (UC) is characterized by an increased density of abnormal vessels. The expression or role of S1P₁ in blood vessels in the colon has not been investigated. In the present study, we show that S1P₁ is overexpressed in the colonic mucosa of UC patients. This increase in S1P₁ levels reflects increased vascular density in the inflamed mucosa. Genetic deletion of S1pr1 in mice increases colonic vascular permeability under basal conditions and increases bleeding in experimental colitis. In contrast, neither FTY720 nor AUY954, two S1P receptor-targeting agents, increases bleeding in experimental colitis. Taken together, our findings demonstrate that S1P₁ is critical to maintaining colonic vascular integrity and may play a role in UC pathogenesis.

A Potent and Selective S1P1 Antagonist with Efficacy in Experimental Autoimmune Encephalomyelitis

Lymphocyte trafficking is critically regulated by the Sphingosine 1-phosphate receptor-1 (S1P(1)), a G protein-coupled receptor that has been highlighted as a promising therapeutic target in autoimmunity. Fingolimod (FTY720, Gilenya) is a S1P(1) receptor agonist that has recently been approved for the treatment of multiple sclerosis (MS). Here, we report the discovery of NIBR-0213, a potent and selective S1P(1) antagonist that induces long-lasting reduction of peripheral blood lymphocyte counts after oral dosing. NIBR-0213 showed comparable therapeutic efficacy to fingolimod in experimental autoimmune encephalomyelitis (EAE), a model of human MS. These data provide convincing evidence that S1P(1) antagonists are effective in EAE. In addition, the profile of NIBR-0213 makes it an attractive candidate to further study the consequences of S1P(1) receptor antagonism and to differentiate the effects from those of S1P(1) agonists.

The role of sphingosine-1-phosphate receptor-1 in antigen-specific CD8 T cell migration (173.14)

Abstract Our previous studies have helped illuminate the landscape in which the endogenous CD8 T cell immune response occurs. However, the factors that regulate the anatomical program that antigen-specific CD8 T follow after infection are not well understood. Although, it is known that sphingosine-1-phosphate receptor 1 (S1P1) controls naïve T cell trafficking, our understanding of how S1P1 regulates the migration and differentiation of CD8 T cells after infection remains poor. Here we investigated the role of S1P1 in the migration and localization of antigen-specific CD8 T cells in the lymph node (LN) after viral infection. We utilized pharmacological modulation of S1P1 and cell-specific S1P1 knock out mouse models to reveal a central role of S1P1 in controlling effector CD8 T cell migration from the draining LN. In the absence of S1P1 receptor signaling, antigen-specific CD8 T cells failed to utilize the lymphatic sinuses to egress from the draining LN. Furthermore, we used a novel mouse model that contains a mutated native S1P1 protein that cannot be efficiently internalized. Using this mouse model we show that receptor internalization can regulate effector CD8 T cell response early after infection. Therefore, our studies demonstrate that S1P1 receptor signaling is the dominant factor that regulates endogenous effector CD8 T cell migration in secondary lymphoid organs.

Fingolimod Treatment Is Associated with a Down-Modulation of S1P1 Receptor Protein in the CNS (P02.107)

Objective: To determine the effects of fingolimod treatment on the expression of S1P1 receptor protein in the CNS. Background Fingolimod (FTY720, Gilenya) has recently been approved as the first oral therapy for relapsing Multiple Sclerosis. Fingolimod causes internalization and proteasomal degradation of sphingosine 1-phosphate receptor-1 (S1P1) on T cells, thereby favoring their retention in lymph nodes. It remains to be determined whether fingolimod also down-modulates S1P1 receptors in other tissues, including the CNS. Design/Methods: Normal rats and S1P1 transgenic mice were treated with vehicle or fingolimod (0.3mg/kg, the minimal effective dose in Experimental Autoimmune Encephalomyelitis, EAE). Tissues were isolated at various time points after drug treatment and S1P1 protein expression determined in homogenized tissues using western blots, or in cell membrane preparations using competitive 33 P-S1P-S1P receptor binding assays. S1P1-specific binding of 33 P-S1P was detected by competing off binding with (cold) S1P1-selective AUY954. Results: In normal rats, S1P1 protein was detectable in brain and lung (by 33 P-S1P-S1P binding assays), and in S1P1 transgenic mice also in the cardiovascular system (by western blot). In fingolimod-treated rats and mice, the levels of S1P1 were down-modulated by approximately 80% in all tissues, including brain. S1P1 was more rapidly down-modulated in peripheral organs compared to brain tissue, but reductions were comparable after 7 days. Conclusions: It has been reported earlier that MS is associated with increased expression of S1P1 in astrocytes and of S1P in the CNS. Here we show that EAE-therapeutic dosing of fingolimod down-modulates S1P1 receptor protein levels in the brain, presumably in neural cells/glia which make up >90% of the brain cells, thereby antagonizing the pro-inflammatory S1P-S1P1 axis that is intimately involved in astrogliosis and may perturb neural cell function. Disclosure: Dr. Brinkmann has received personal compensation for activities with Novartis as an employee. Dr. Brinkmann has received research support from Novartis. Dr. Streiff has received personal compensation for activities with Novartis as an employee. Dr. Streiff has received research support from Novartis. Dr. Bigaud has received personal compensation from Novartis for activities as an employee. Dr. Bigaud has received research support from Novartis. Dr. Kinzel has received personal compensation for activities with Novartis as an employee. Dr. Kinzel has received research support from Novartis.

PP2A Activating Drugs (PAD): Anti-Leukemic and Non-Toxic Activity of Two Novel and Non-Immunosuppressive FTY720 Derivatives

AbstractAbstract 2901FTY720 is a sphingosine analog proposed by the FDA for treating Multiple Sclerosis patients because of its immunosuppressive activity, which depends on its ability to prevent lymphocyte egress into the peripheral blood. To act as an immunosuppressive drug, FTY720 undergoes sphingosine kinase 2 (SPHK2) phosphorylation and internalization upon interaction with the sphingosine-1-phosphate receptor 1 (S1PR1). FTY720 also acts as a potent activator of protein phosphatase 2A (PP2A), a tumor suppressor found inactivated in chronic and blast crisis CML with wild type or imatinib/dasatinib-resistant BCR-ABL1, Ph+ B-ALL, KitD816V AML, Jak2V617F+ MPDs and other leukemias/lymphomas. FTY720 treatment of cell lines and primary progenitors isolated from bone marrow of patients with these malignancies, markedly suppressed leukemic cell proliferation/survival and induced apoptosis in a PP2A-dependent manner. Notably, long-term treatment with FTY720 of mice carrying these hematopoietic malignancies significantly prolonged survival and restored normal myelopoiesis without exerting any toxic effect in hematopoietic and non-hematopoietic organs. However, in vivo administration of FTY720 strongly, albeit reversibly, decreases the number of circulating B and T lymphocytes.Here we report that a synthetically phosphorylated FTY720 (FTY720-P) is unable to induce neither PP2A activation nor apoptosis of BCR-ABL1-, Jak2V617F-, or KitD816V-expressing myeloid precursors, indicating that FTY720 phosphorylation is dispensable for its anti-leukemic activity. Thus, we functionally characterize two FTY720 derivatives, QC-FTYSM and OSU-2S, which were synthesized as molecules unable to undergo SPHK2 phosphorylation. Treatment (2.5 uM; 24h) of FTY720-sensitive 32D-BCR/ABL cells with QC-FTYSM and OSU2S results in ∼80% and 40%, respectively, more efficient suppression of BCR-ABL1 expression and kinase activity than that observed with FTY720. Moreover, QC-FTYSM, OSU-2S and FTY720 (2.5uM; 0–60h) induce a progressive block of proliferation and marked induction of apoptosis of 32D-BCR/ABL cells. In fact, a 96%, 98%, and 79% decrease in viability is observed after treatment with QC-FTYSM, OSU-2S and FTY720, respectively. Notably, viability of non-transformed myeloid 32Dcl3 cells is not significantly affected by treatment with FTY720 or its derivatives. Consistent with the ability of FTY720 to induce apoptosis through rescue of PP2A activity, phosphatase assays show identical ability of FTY720, QC-FTYSM and OSU-2S to restore PP2A functionality. In fact, comparable and marked decrease in the amount of inactive Y307-phosphorylated PP2Ac was detected in 32D-BCR/ABL cells treated with FTY720 or its derivatives. To formally demonstrate that QC-FTYSM and OSU-2S lack immunosuppressive activity, we first assessed their ability to be internalized upon interaction/association with the S1PR1 receptor. Thus, cells were transduced with a GFP-tagged S1PR1 and treated with either QC-FTYSM, OSU-2S, or, as positive control, FTY720-P. Confocal microscopy revealed that treatment FTY720-P resulted in a strong S1PR1 internalization. Conversely, exposure of the cells to QC-FTYSM and OSU-2S did not alter the S1PR1 membrane localization, indicating that these molecules did not undergo SHPK2 phosphorylation. Further demonstration of the inability of these compounds to act as immunosuppressive molecules was gained upon in vivo administration of a single dose of FTY720, QC-FTYSM or OSU-2C (10 mg/kg) to wild type FVB/N mice. As expected, percentage of B220+/CD19+ circulating B-cells decreased of ∼90% in FTY720-treated animals. Conversely, the percentage of B-cells after exposure to QC-FTYSM and OSU-2S remained unchanged (≤ 1% decrease). Likewise, the number or CD4+ and CD8+ cells also was not affect by treatment with the QC-FTYSM compound. Note that effect of OSU-2S on T-cells and the toxicity profile and anti-leukemic activity of these drugs in healthy animals and mouse models of deadly leukemias (e.g. T315I+ and blast crisis CML and Ph+ ALL) as well as Ph− MPDs are currently being assessed.Altogether our data indicate that QC-FTYSM and OSU-2S represent two potentially powerful and safe drugs which could be introduced in the current therapeutic protocols for different types of hematopoietic and non-hematopoietic malignancies characterized by functional inactivation of the PP2A tumor suppressor. Disclosures:No relevant conflicts of interest to declare.

Recent progress in the development of selective S1P1 receptor agonists for the treatment of inflammatory and autoimmune disorders

Background: The sphingosine-1-phosphate receptor 1 (S1P1) belongs to the endothelial differentiation gene family of G-protein-coupled receptors involved in embryonic development, cellular differentiation and survival, adherence and tight junction assembly, and leukocyte trafficking. In vivo S1P1 is required for egress of thymocytes into the blood and of naive lymphocytes from secondary lymphoid organs back into the lymph. Downregulation of S1P1 disrupts lymphocyte migration, tissue homing and recirculation. To explore the immunomodulatory potential of S1P1, pharma is developing S1P1-selective agonists for treating autoimmune and inflammatory disorders. FTY720 (fingolimod, Novartis AG), a nonselective S1P1 agonist that induces sustained lymphopenia, is a promising pseudo-lipid aminodiol prodrug that has been shown in preclinical and early clinical trials to be effective in suppressing various autoimmune conditions and to aid in acute organ transplant, and is currently in pivotal Phase III clinical trials for relapsing-remitting multiple sclerosis. Objective: In this review article, we outline recent advances made in the discovery and patenting of new small-molecule S1P1 selective agonists and summarize a rapidly growing body of intellectual property. Conclusion: Fingolimod-like S1P1 agonists with a novel mechanism of action have emerged as promising immunosuppressants. S1P1 agonists induce T-cell sequestration in lymph nodes and Peyer's patches, diminishing their ability to reach distant sites and cause pathology. Preclinical studies have already provided preliminary evidence that low-dose treatment with fingolimod in conjunction with subtherapeutic doses of ciclosporin A and tacrolimus enable potent immunosuppression in the absence of immediately serious side effects. Future efforts will determine whether S1P1 agonists do indeed synergize with existing DMAs to create new mutidrug paradigms with superior efficacy and safety for the treatment of autoimmune diseases and the prevention of organ transplant rejection.

To be or not to be protease activated receptor-1 in activated protein C-initiated endothelial barrier protection?

The review by Looney and Matthay summarized recent progress in basic and preclinical research of activated protein C (APC), a novel therapeutic agent for the treatment of severe sepsis [1]. APC is traditionally an anticoagulant and profibrinolytic agent, and has been appreciated to possess anti-inflammatory and anti-apoptotic properties, yet the exact mechanisms by which APC executes its clinical effects in sepsis are not defined [2]. Emerging evidence has suggested that APC might exert its clinical benefit, at least in part, by maintaining endothelial barrier function [3,4]. The authors reviewed conflicting results of endothelial barrier protection of APC in the literature and pointed out that caution must be taken to interpret cell culture experiments and their relationship to in vivo conditions. Indeed, one should exercise caution while reviewing controversial data. Looney and Matthay stated that 'in two different in vitro investigations [3,4], APC promoted endothelial barrier protection in a PAR-1- and S1P1-dependent mechanism', which represents a misinterpretation. These two papers did conclude that APC enhanced endothelial barrier function in different cell lines by a sphingosine 1-phosphate receptor-1 (S1P1)-dependent signaling pathway, but it is still arguable whether this is protease activated receptor-1 (PAR-1) dependent. Finigan and colleagues showed that anti-PAR-1 blocking antibody did not interfere with either APC-mediated myosin light-chain phosphorylation or S1P1 receptor phosphorylation/activation, and concluded that it was endothelial protein C receptor (EPCR) ligation with APC and the resulting S1P1 transactivation that led toendothelial cell cytoskeletal rearrangement and barrier protection [4]. There is therefore no evidence so far to support the notion that the 'APC ... effect on endothelial cytoskeletal rearrangement ... appears to operate in a PAR-1- ... dependent manner' [1].

Cell migration activated by platelet‐derived growth factor receptor is blocked by an inverse agonist of the sphingosine 1‐phosphate receptor‐1

We have previously identified a novel complex between the platelet-derived growth factor (PDGF)beta receptor and the sphingosine 1-phosphate receptor-1 (S1P1). The complex permits the utilization of active G-protein subunits (made available by constitutively active S1P1 receptor) by the PDGFbeta receptor kinase to transmit signals to p42/p44 MAPK in response to PDGF. Therefore, an inverse agonist of the S1P1 receptor is predicted to reduce signal transduction from PDGFbeta receptor tyrosine kinase by blocking the constitutive activity of the G-protein coupled receptor. SB649146 is a novel inverse agonist of the S1P1 receptor. First, SB649146 displaced the S1P1 receptor agonist dihydrosphingosine 1-phosphate from membranes expressing the recombinant S1P1 receptor. Second, SB649146 reduced basal recombinant S1P1 receptor-induced GTPgammaS binding and S1P-induced GTPgammaS binding in membranes. Third, SB649146 blocked the S1P-induced activation of p42/p44 MAPK in airway smooth muscle cells, a response that is mediated by the S1P1 receptor. We now report that inverse agonism of the S1P1 receptor with SB649146 reduced the endocytosis of the PDGFbeta receptor-S1P1 receptor complex and the stimulation of p42/p44 MAPK and cell migration in response to PDGF. These findings are the first to report that a GPCR inverse-agonist reduces growth factor-induced receptor tyrosine kinase signaling, fundamentally broadening their mechanism of action. The data obtained with SB649146 also suggest that the constitutively active endogenous S1P1 receptor enhances PDGF-induced cell migration.