FTY720 requires vitamin B12-TCN2-CD320 signaling in astrocytes to reduce disease in an animal model of multiple sclerosis

FTY720 (fingolimod) is a sphingosine 1-phosphate (S1P) receptor modulator and sphingosine analogue approved for multiple sclerosis (MS) therapy, which can functionally antagonize the S1P receptor, S1P1. Vitamin B12 (B12) deficiency produces neurological manifestations resembling MS. Here, we report a new mechanism where FTY720 suppresses neuroinflammation by regulating B12 metabolic pathways. Nuclear RNA-seq of c-Fos-activated astrocytes (called ieAstrocytes) from experimental autoimmune encephalomyelitis (EAE) spinal cords identified up-regulation of CD320, a transcobalamin 2 (TCN2)-B12 receptor, by S1P1 inhibition. CD320 was reduced in MS plaques. Deficiency of CD320 or dietary B12 worsened EAE and eliminated FTY720 efficacy, while concomitantly down-regulating type I interferon signaling. TCN2 functioned as a chaperone for FTY720 and sphingosine, which induced astrocytic CD320 internalization. An accompanying paper identified a requirement for astrocyte sphingosine kinases in FTY720 efficacy and its altered expression in MS brains, molecularly linking MS and B12 deficiency that can be accessed by sphingolipid/fingolimod metabolic pathways.

Single-nucleus RNA-seq of normal-appearing brain regions in relapsing-remitting vs. secondary progressive multiple sclerosis

Multiple sclerosis (MS) is an immune-mediated demyelinating disease that alters central nervous system (CNS) functions. Relapsing-remitting MS (RRMS) is the most common form, which can transform into secondary-progressive MS (SPMS) that is associated with progressive neurodegeneration. Single-nucleus RNA sequencing (snRNA-seq) of MS lesions identified disease-related transcriptomic alterations; however, their relationship to non-lesioned MS brain regions has not been reported and which could identify prodromal or other disease susceptibility signatures. Here, snRNA-seq was used to generate high-quality RRMS vs. SPMS datasets of 33,197 nuclei from 8 normal-appearing MS brains, which revealed divergent cell type-specific changes. Notably, SPMS brains downregulated astrocytic sphingosine kinases (SPHK1/2), the enzymes required to phosphorylate and activate the MS drug, fingolimod. This reduction was modeled with astrocyte-specific Sphk1/2 null mice in which fingolimod lost activity, supporting functionality of observed transcriptomic changes. These data provide an initial resource for studies of single cells from non-lesioned RRMS and SPMS brains.

The sphingosine 1-phosphate receptor 2/4 antagonist JTE-013 elicits off-target effects on sphingolipid metabolism

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.

Downregulation of Lipid Phosphate Phosphatase 3 correlates with Tumor-Infiltrating Immune Cells in Oral Cancer

Purpose Sphingosine-1-phosphate (S1P), a potent oncogenic lipid. Intracellular levels of S1P are tightly regulated by eight S1P metabolizing enzymes. S1P is synthesized by phosphorylation of sphingosine which is catalyzed by two sphingosine kinases (SphK1 and SphK2). Five lipid phosphatases (two S1P phosphatases and three lipid phosphate phosphatases) reversibly convert S1P back to sphingosine. S1P is ultimately irreversibly degraded by S1P lyase. The role of sphingosine-1-phosphate (S1P) metabolizing enzymes in oral squamous cell carcinoma (OSCC) has not been fully studied. Methods In the current study, we have determined the protein expression of four S1P metabolizing enzymes, namely sphingosine Kinase (SphK) -1, SphK2, S1P phosphatase 1 (SGPP1), and lipid phosphate phosphatase 3 (LPP3) by immunohistochemistry (IHC) and western botting in tumor tissues of 46 OSCC patients and normal oral mucosa (N = 6). Further, we determined the associations of expression of S1P metabolizing enzymes with clinicopathological features of OSCC patients. Results SphK2 and LPP3 exhibit low IRS in OSCC tumors. Importantly, expression of SphK2 and LPP3 was downregulated in malignant cells compared to non-malignant mucosa. Further, LPP3 expression negatively correlated with TNM staging of patients (ρ = -0.307, p = 0.043). Importantly, TCGA analysis revealed that LPP3 expression was positively correlated with infiltration of B cells, neutrophils, macrophages, and dendritic cells in the HNSCC tumors. Conclusion In conclusion, our data show that expression of SphK2 and LPP3 is decreased in OSCC tumors compared to normal mucosa. Thus, LPP3 could represent a potential prognostic marker and therapeutic target for OSCC.

Sphingosine Kinase 1 Signaling in Breast Cancer: A Potential Target to Tackle Breast Cancer Stem Cells

Sphingosine kinases (SPHKs) are conserved lipid enzymes that catalyze the formation of sphingosine-1-phosphate (S1P) through ATP-dependent phosphorylation of sphingosine. Two distinct SPHK isoforms, namely SPHK1 and SPHK2, have been identified to date, and the former has been implicated for its oncogenic roles in cancer development and progression. While SPHK1 signaling axis has been extensively studied in non-stem breast cancer cells, recent evidence has emerged to suggest a role of SPHK1 in regulating cancer stem cells (CSCs). With the clinical implications of CSCs in disease relapse and metastasis, it is believed that therapeutic approaches that can eradicate both non-stem cancer cells and CSCs could be a key to cancer cure. In this review, we first explore the oncogenic functions of sphingosine kinase 1 in human cancers and summarize current research findings of SPHK1 signaling with a focus on breast cancer. We also discuss the therapeutic potentials and perspectives of targeting SPHK1 signaling in breast cancer and cancer stem cells. We aim to offer new insights and inspire future studies looking further into the regulatory functions of SPHK1 in CSC-driven tumorigenesis, uncovering novel therapeutic avenues of using SPHK1-targeted therapy in the treatment of CSC-enriched refractory cancers.

DDRE-06. TARGETING THE SPHINGOLIPID BALANCE VIA ACID CERAMIDASE INHIBITION TO DECREASE GROWTH OF TMZ-RESISTANT GLIOBLASTOMA AND BLOCK MIGRATION

Dysregulated sphingolipid metabolism is associated with many cancers; allowing cells to evade apoptosis through increases in sphingosine-1-phosphate (S1P) and decreases in ceramides. Ceramides can be hydrolyzed by ceramidases to sphingosine, which can then be phosphorylated by sphingosine kinases to S1P. S1P allows cells to evade apoptosis and increase migration, while shifts toward ceramides favor cell death. Glioblastoma (GBM) exhibits shifts in the sphingolipid balance towards S1P, contributing to chemoresistance and migration. Understanding of sphingolipid metabolism in GBM is still limited, and currently, there are no approved treatments to target the dysregulation. Acid ceramidase (ASAH1), a key enzyme in the production of S1P, is highly expressed in GBM and is associated with worse survival of GBM patients, as per The Cancer Genome Atlas data. To address the altered sphingolipid metabolism and therapeutic resistance in GBM, we explored the efficacy of pharmacologic and genetic inhibition of ASAH1 in both parental and temozolomide (TMZ)-resistant patient-derived xenografts. Cells were infected with ASAH1 shRNA or treated with ASAH1 inhibitors and assessed for cell growth and migration. Our work suggests that pharmacologic inhibition of ASAH1 induces cell death and that this effect is maintained in TMZ-resistant cells. Furthermore, we find a novel role for carmofur, an ASAH1 inhibitor, in the inhibition of GBM migration. Together, these data suggest the potential utility of normalizing the sphingolipid balance in the context of GBM TMZ resistance.

Interaction of microRNAs with sphingosine kinases, sphingosine-1 phosphate, and sphingosine-1 phosphate receptors in cancer

Sphingosine-1-phosphate (S1P), a pleiotropic lipid mediator, participates in various cellular processes during tumorigenesis, including cell proliferation, survival, drug resistance, metastasis, and angiogenesis. S1P is formed by two sphingosine kinases (SphKs), SphK1 and SphK2. The intracellularly produced S1P is delivered to the extracellular space by ATP-binding cassette (ABC) transporters and spinster homolog 2 (SPNS2), where it binds to five transmembrane G protein-coupled receptors to mediate its oncogenic functions (S1PR1-S1PR5). MicroRNAs (miRNAs) are small non-coding RNAs, 21–25 nucleotides in length, that play numerous crucial roles in cancer, such as tumor initiation, progression, apoptosis, metastasis, and angiogenesis via binding to the 3′‐untranslated region (3′‐UTR) of the target mRNA. There is growing evidence that various miRNAs modulate tumorigenesis by regulating the expression of SphKs, and S1P receptors. We have reviewed various roles of miRNAs, SphKs, S1P, and S1P receptors (S1PRs) in malignancies and how notable miRNAs like miR-101, miR-125b, miR-128, and miR-506, miR-1246, miR-21, miR-126, miR499a, miR20a-5p, miR-140-5p, miR-224, miR-137, miR-183-5p, miR-194, miR181b, miR136, and miR-675-3p, modulate S1P signaling. These tumorigenesis modulating miRNAs are involved in different cancers including breast, gastric, hepatocellular carcinoma, prostate, colorectal, cervical, ovarian, and lung cancer via cell proliferation, invasion, angiogenesis, apoptosis, metastasis, immune evasion, chemoresistance, and chemosensitivity. Therefore, understanding the interaction of SphKs, S1P, and S1P receptors with miRNAs in human malignancies will lead to better insights for miRNA-based cancer therapy.

Novelty of Sphingolipids in the Central Nervous System Physiology and Disease: Focusing on the Sphingolipid Hypothesis of Neuroinflammation and Neurodegeneration

For decades, lipids were confined to the field of structural biology and energetics as they were considered only structural constituents of cellular membranes and efficient sources of energy production. However, with advances in our understanding in lipidomics and improvements in the technological approaches, astounding discoveries have been made in exploring the role of lipids as signaling molecules, termed bioactive lipids. Among these bioactive lipids, sphingolipids have emerged as distinctive mediators of various cellular processes, ranging from cell growth and proliferation to cellular apoptosis, executing immune responses to regulating inflammation. Recent studies have made it clear that sphingolipids, their metabolic intermediates (ceramide, sphingosine-1-phosphate, and N-acetyl sphingosine), and enzyme systems (cyclooxygenases, sphingosine kinases, and sphingomyelinase) harbor diverse yet interconnected signaling pathways in the central nervous system (CNS), orchestrate CNS physiological processes, and participate in a plethora of neuroinflammatory and neurodegenerative disorders. Considering the unequivocal importance of sphingolipids in CNS, we review the recent discoveries detailing the major enzymes involved in sphingolipid metabolism (particularly sphingosine kinase 1), novel metabolic intermediates (N-acetyl sphingosine), and their complex interactions in CNS physiology, disruption of their functionality in neurodegenerative disorders, and therapeutic strategies targeting sphingolipids for improved drug approaches.