Abstract
Sphingosine 1-phosphate (S1P) is a signaling lipid that has broad roles, working either intracellularly through various protein targets, or extracellularly via a family of five G-protein coupled receptors. Agents that selectively and specifically target each of the S1P receptors have been sought as both biological tools and potential therapeutics. JTE-013, a small molecule antagonist of S1P receptors 2 and 4 (S1P2 and S1P4) has been widely used in defining the roles of these receptors in various biological processes. Indeed, our previous studies showed that JTE-013 had anti-acute myeloid leukaemia (AML) activity, supporting a role for S1P2 in the biology and therapeutic targeting of AML. Here we examined this further and describe lipidomic analysis of AML cells that revealed JTE-013 caused alterations in sphingolipid metabolism, increasing cellular ceramides, dihydroceramides, sphingosine and dihydrosphingosine. Further examination of the mechanisms behind these observations showed that JTE-013, at concentrations frequently used in the literature to target S1P2/4, inhibits several sphingolipid metabolic enzymes, including dihydroceramide desaturase 1 and both sphingosine kinases. Collectively, these findings demonstrate that JTE-013 can have broad off-target effects on sphingolipid metabolism and highlight that caution must be employed in interpreting the use of this reagent in defining the roles of S1P2/4.
Highlights
The sphingolipid pathway is comprised of a range of bioactive lipids that contribute to cellular membranes and can act as signaling molecules
While these studies called into question the selectivity, and the usefulness of JTE-013 as a tool to probe the functions of S1P215,17, debate remains if these observations arose due to “off-targets” of JTE-013, or from the complex pharmacology of G protein-coupled r eceptors[18]
We have previously demonstrated that SK1 inhibition induced Mcl-1 degradation and cell death in acute myeloid leukaemia (AML) cells, and that this could be recapitulated by the S 1P2 antagonist JTE-013, implicating the SK1-Sphingosine 1-phosphate (S1P)-S1P2 axis in controlling Mcl-1 protein levels[13]
Summary
The sphingolipid pathway is comprised of a range of bioactive lipids that contribute to cellular membranes and can act as signaling molecules. Our further analysis demonstrated that these effects were due, at least in part to JTE-013 directly inhibiting dihydroceramide desaturase 1 (Des1), SK1 and SK2 Inhibition of these enzymes by JTE-013 occurs at concentrations previously used in many published studies for selective targeting of S1P2 (10 μM). These findings suggest considerable caution is required in the interpretation of findings gained from the use of JTE-013, at higher concentrations, and that some previous findings may warrant further investigation to distinguish the effects of S1P2 antagonism versus other enzymes in the sphingolipid pathway such as Des[1] and the SKs
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