AF1q is a universal marker of neuroblastoma that sustains N-Myc expression and drives tumorigenesis.

Neuroblastoma is the most common extracranial malignant tumor of childhood, accounting for 15% of all pediatric cancer deaths. Despite significant advances in our understanding of neuroblastoma biology, five-year survival rates for high-risk disease remain less than 50%, highlighting the importance of identifying novel therapeutic targets to combat the disease. MYCN amplification is the most frequent and predictive molecular aberration correlating with poor outcome in neuroblastoma. N-Myc is a short-lived protein primarily due to its rapid proteasomal degradation, a potentially exploitable vulnerability in neuroblastoma. AF1q is an oncoprotein with established roles in leukemia and solid tumor progression. It is normally expressed in brain and sympathetic neurons and has been postulated to play a part in neural differentiation. However, no role for AF1q in tumors of neural origin has been reported. In this study, we found AF1q to be a universal marker of neuroblastoma tumors. Silencing AF1q in neuroblastoma cells caused proteasomal degradation of N-Myc through Ras/ERK and AKT/GSK3ß pathways, activated p53 and blocked cell cycle progression, culminating in cell death via the intrinsic apoptotic pathway. Moreover, silencing AF1q attenuated neuroblastoma tumorigenicity in vivo signifying AF1q's importance in neuroblastoma oncogenesis. Our findings reveal AF1q to be a novel regulator of N-Myc and potential therapeutic target in neuroblastoma.

Gene therapy with AAV9-SGPL1 in an animal model of lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung that leads rapidly to respiratory failure. Novel approaches to treatment are urgently needed. The bioactive lipid sphingosine-1-phosphate (S1P) is increased in IPF lungs and promotes proinflammatory and profibrotic TGF-ß signaling. Hence, decreasing lung S1P represents a potential therapeutic strategy for IPF. S1P is degraded by the intracellular enzyme S1P lyase (SPL). Here we find that a knock-in mouse with a missense SPL mutation mimicking human disease resulted in reduced SPL activity, increased S1P, increased TGF-ß signaling, increased lung fibrosis, and higher mortality after injury compared to wild type (WT). We then tested adeno-associated virus 9 (AAV9)-mediated overexpression of human SGPL1 (AAV-SPL) in mice as a therapeutic modality. Intravenous treatment with AAV-SPL augmented lung SPL activity, attenuated S1P levels within the lungs, and decreased injury-induced fibrosis compared to controls treated with saline or only AAV. We confirmed that AAV-SPL treatment led to higher expression of SPL in the epithelial and fibroblast compartments during bleomycin-induced lung injury. Additionally, AAV-SPL decreased expression of the profibrotic cytokines TNFα and IL1ß as well as markers of fibroblast activation, such as fibronectin (Fn1), Tgfb1, Acta2, and collagen genes in the lung. Taken together, our results provide proof of concept for the use of AAV-SPL as a therapeutic strategy for the treatment of IPF. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

AAV-SPL 2.0, a Modified Adeno-Associated Virus Gene Therapy Agent for the Treatment of Sphingosine Phosphate Lyase Insufficiency Syndrome.

Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is an inborn error of metabolism caused by inactivating mutations in SGPL1, the gene encoding sphingosine-1-phosphate lyase (SPL), an essential enzyme needed to degrade sphingolipids. SPLIS features include glomerulosclerosis, adrenal insufficiency, neurological defects, ichthyosis, and immune deficiency. Currently, there is no cure for SPLIS, and severely affected patients often die in the first years of life. We reported that adeno-associated virus (AAV) 9-mediated SGPL1 gene therapy (AAV-SPL) given to newborn Sgpl1 knockout mice that model SPLIS and die in the first few weeks of life prolonged their survival to 4.5 months and prevented or delayed the onset of SPLIS phenotypes. In this study, we tested the efficacy of a modified AAV-SPL, which we call AAV-SPL 2.0, in which the original cytomegalovirus (CMV) promoter driving the transgene is replaced with the synthetic "CAG" promoter used in several clinically approved gene therapy agents. AAV-SPL 2.0 infection of human embryonic kidney (HEK) cells led to 30% higher SPL expression and enzyme activity compared to AAV-SPL. Newborn Sgpl1 knockout mice receiving AAV-SPL 2.0 survived ≥ 5 months and showed normal neurodevelopment, 85% of normal weight gain over the first four months, and delayed onset of proteinuria. Over time, treated mice developed nephrosis and glomerulosclerosis, which likely resulted in their demise. Our overall findings show that AAV-SPL 2.0 performs equal to or better than AAV-SPL. However, improved kidney targeting may be necessary to achieve maximally optimized gene therapy as a potentially lifesaving SPLIS treatment.

Efficacy of AAV9-mediated SGPL1 gene transfer in a mouse model of S1P lyase insufficiency syndrome.

Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is a rare metabolic disorder caused by inactivating mutations in sphingosine-1-phosphate lyase 1 (SGPL1), which is required for the final step of sphingolipid metabolism. SPLIS features include steroid-resistant nephrotic syndrome and impairment of neurological, endocrine, and hematopoietic systems. Many affected individuals die within the first 2 years. No targeted therapy for SPLIS is available. We hypothesized that SGPL1 gene replacement would address the root cause of SPLIS, thereby serving as a universal treatment for the condition. As proof of concept, we evaluated the efficacy of adeno-associated virus 9-mediated transfer of human SGPL1 (AAV-SPL) given to newborn Sgpl1-KO mice that model SPLIS and die in the first weeks of life. Treatment dramatically prolonged survival and prevented nephrosis, neurodevelopmental delay, anemia, and hypercholesterolemia. STAT3 pathway activation and elevated proinflammatory and profibrogenic cytokines observed in KO kidneys were attenuated by treatment. Plasma and tissue sphingolipids were reduced in treated compared with untreated KO pups. SGPL1 expression and activity were measurable for at least 40 weeks. In summary, early AAV-SPL treatment prevents nephrosis, lipidosis, and neurological impairment in a mouse model of SPLIS. Our results suggest that SGPL1 gene replacement holds promise as a durable and universal targeted treatment for SPLIS.

Mutations in sphingosine-1-phosphate lyase cause nephrosis with ichthyosis and adrenal insufficiency.

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1Δ yeast strains, whereas expression of disease-associated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS.

Sphingosine phosphate lyase regulates myogenic differentiation via S1P receptor-mediated effects on myogenic microRNA expression.

S1P lyase (SPL) catalyzes the irreversible degradation of sphingosine-1-phosphate (S1P), a bioactive lipid whose signaling activities regulate muscle differentiation, homeostasis, and satellite cell (SC) activation. By regulating S1P levels, SPL also controls SC recruitment and muscle regeneration, representing a potential therapeutic target for muscular dystrophy. We found that SPL is induced during myoblast differentiation. To investigate SPL's role in myogenesis at the cellular level, we generated and characterized a murine myoblast SPL-knockdown (SPL-KD) cell line lacking SPL. SPL-KD cells accumulated intracellular and extracellular S1P and failed to form myotubes under conditions that normally stimulate myogenic differentiation. Under differentiation conditions, SPL-KD cells also demonstrated delayed induction of 3 myogenic microRNAs (miRNAs), miR-1, miR-206, and miR-486. SPL-KD cells successfully differentiated when treated with an S1P1 agonist, S1P2 antagonist, and combination treatments, which also increased myogenic miRNA levels. SPL-KD cells transfected with mimics for miR-1 or miR-206 also overcame the differentiation block. Thus, we show for the first time that the S1P/SPL/S1P-receptor axis regulates the expression of a number of miRNAs, thereby contributing to myogenic differentiation.

Sphingosine-1-phosphate enhances satellite cell activation in dystrophic muscles through a S1PR2/STAT3 signaling pathway.

Sphingosine-1-phosphate (S1P) activates a widely expressed family of G protein-coupled receptors, serves as a muscle trophic factor and activates muscle stem cells called satellite cells (SCs) through unknown mechanisms. Here we show that muscle injury induces dynamic changes in S1P signaling and metabolism in vivo. These changes include early and profound induction of the gene encoding the S1P biosynthetic enzyme SphK1, followed by induction of the catabolic enzyme sphingosine phosphate lyase (SPL) 3 days later. These changes correlate with a transient increase in circulating S1P levels after muscle injury. We show a specific requirement for SphK1 to support efficient muscle regeneration and SC proliferation and differentiation. Mdx mice, which serve as a model for muscular dystrophy (MD), were found to be S1P-deficient and exhibited muscle SPL upregulation, suggesting that S1P catabolism is enhanced in dystrophic muscle. Pharmacological SPL inhibition increased muscle S1P levels, improved mdx muscle regeneration and enhanced SC proliferation via S1P receptor 2 (S1PR2)-dependent inhibition of Rac1, thereby activating Signal Transducer and Activator of Transcription 3 (STAT3), a central player in inflammatory signaling. STAT3 activation resulted in p21 and p27 downregulation in a S1PR2-dependent fashion in myoblasts. Our findings suggest that S1P promotes SC progression through the cell cycle by repression of cell cycle inhibitors via S1PR2/STAT3-dependent signaling and that SPL inhibition may provide a therapeutic strategy for MD.

Protection of LPS-induced murine acute lung injury by sphingosine-1-phosphate lyase suppression.

A defining feature of acute lung injury (ALI) is the increased lung vascular permeability and alveolar flooding, which leads to associated morbidity and mortality. Specific therapies to alleviate the unremitting vascular leak in ALI are not currently clinically available; however, our prior studies indicate a protective role for sphingosine-1-phosphate (S1P) in animal models of ALI with reductions in lung edema. As S1P levels are tightly regulated by synthesis and degradation, we tested the hypothesis that inhibition of S1P lyase (S1PL), the enzyme that irreversibly degrades S1P via cleavage, could ameliorate ALI. Intratracheal instillation of LPS to mice enhanced S1PL expression, decreased S1P levels in lung tissue, and induced lung inflammation and injury. LPS challenge of wild-type mice receiving 2-acetyl-4(5)-[1(R),2(S),3(R),4-tetrahydroxybutyl]-imidazole to inhibit S1PL or S1PL(+/-) mice resulted in increased S1P levels in lung tissue and bronchoalveolar lavage fluids and reduced lung injury and inflammation. Moreover, down-regulation of S1PL expression by short interfering RNA (siRNA) in primary human lung microvascular endothelial cells increased S1P levels, and attenuated LPS-mediated phosphorylation of p38 mitogen-activated protein kinase and I-κB, IL-6 secretion, and endothelial barrier disruption via Rac1 activation. These results identify a novel role for intracellularly generated S1P in protection against ALI and suggest S1PL as a potential therapeutic target.

Cancer treatment strategies targeting sphingolipid metabolism.

Ceramide and sphingosine-1-phosphate are related sphingolipid metabolites that can be generated through a de novo biosynthetic route or derived from the recycling of membrane sphingomyelin. Both these lipids regulate cellular responses to stress, with generally opposing effects. Sphingosine-1-phosphate functions as a growth and survival factor, acting as a ligand for a family of G protein-coupled receptors, whereas ceramide activates intrinsic and extrinsic apoptotic pathways through receptor-independent mechanisms. A growing body of evidence has implicated ceramide, sphingosine-1-phosphate and the genes involved in their synthesis, catabolism and signaling in various aspects of oncogenesis, cancer progression and drug- and radiation resistance. This may be explained in part by the finding that both lipids impinge upon the PI3K/ AKT pathway, which represses apoptosis and autophagy. In addition, sphingolipids influence cell cycle progression, telomerase function, cell migration and stem cell biology. Considering the central role of ceramide in mediating physiological as well as pharmacologically stimulated apoptosis, ceramide can be considered a tumor-suppressor lipid. In contrast, sphingosine-1-phosphate can be considered a tumor-promoting lipid, and the enzyme responsible for its synthesis functions as an oncogene. Not surprisingly, genetic mutations that result in reduced ceramide generation, increased sphingosine-1-phosphate synthesis or which reduce steady state ceramide levels and increase sphingosine-1-phosphate levels have been identified as mechanisms of tumor progression and drug resistance in cancer cells. Pharmacological tools for modulating sphingolipid pathways are being developed and represent novel therapeutic strategies for the treatment of cancer.

Natural sphingadienes inhibit Akt-dependent signaling and prevent intestinal tumorigenesis.

Sphingolipid metabolites regulate cell proliferation, migration, and stress responses. Alterations in sphingolipid metabolism have been proposed to contribute to carcinogenesis, cancer progression, and drug resistance. We identified a family of natural sphingolipids called sphingadienes and investigated their effects in colon cancer. We find that sphingadienes induce colon cancer cell death in vitro and prevent intestinal tumorigenesis in vivo. Sphingadienes exert their influence by blocking Akt translocation from the cytosol to the membrane, thereby inhibiting protein translation and promoting apoptosis and autophagy. Sphingadienes are orally available, are slowly metabolized through the sphingolipid degradative pathway, and show limited short-term toxicity. Thus, sphingadienes represent a new class of therapeutic and/or chemopreventive agents that blocks Akt signaling in neoplastic and preneoplastic cells.

Sphingosine-1-phosphate metabolism and intestinal tumorigenesis: lipid signaling strikes again.

Sphingolipids are an evolutionary conserved class of membrane lipids synthesized by all eukaryotic cells. The biological functions of sphingolipids are diverse, encompassing structural roles through their participation in membrane lipid rafts, and informational roles via the involvement of their metabolites in signal transduction pathways. An important sphingolipid metabolite is sphingosine-1-phosphate (S1P), which acts through G protein-coupled receptors present on mammalian cells, thereby stimulating cell proliferation, angiogenesis and inhibiting apoptosis. The main enzyme responsible for S1P synthesis, sphingosine kinase 1 (Sphk1), behaves as an oncogene in experimental systems and is required for polyp enlargement in the Min mouse model of intestinal tumorigenesis. S1P is irreversibly degraded by S1P lyase (SPL), an enzyme that is highly expressed in enterocytes, where it is involved in metabolism of dietary sphingolipids. Forced expression of SPL sensitizes human cells to various stressful stimuli and enhances apoptotic cell death. SPL expression is induced in response to DNA damaging agents in a time- and concentration-dependent manner. On the other hand, SPL is downregulated in human colon cancers and in Min mouse adenomas compared to adjacent uninvolved tissues. These observations suggest that SPL, like Sphk1, may play a role in tumorigenesis. Added support for this notion comes from the fact that S1P-specific antibodies slow tumor progression and angiogenesis in murine xenograft and allograft models. Together, these recent studies have established a link between S1P signaling, metabolism and carcinogenesis that may have implications regarding colon cancer screening, dietary chemoprevention and therapeutics.

Sphingosine-1-phosphate lyase potentiates apoptosis via p53- and p38-dependent pathways and is down-regulated in colon cancer.

Sphingolipid metabolites such as sphingosine-1-phosphate (S1P) and ceramide modulate apoptosis during development and in response to stress. In general, ceramide promotes apoptosis, whereas S1P stimulates cell proliferation and protects against apoptosis. S1P is irreversibly degraded by the enzyme S1P lyase (SPL). In this study, we show a crucial role for SPL in mediating cellular responses to stress. SPL expression in HEK293 cells potentiated apoptosis in response to stressful stimuli including DNA damage. This effect seemed to be independent of ceramide generation but required SPL enzymatic activity and the actions of p38 MAP kinase, p53, p53-inducible death domain protein (PIDD), and caspase-2 as shown by molecular and chemical inhibition of each of these targets. Further, SPL expression led to constitutive activation of p38. Endogenous SPL expression was induced by DNA damage in WT cells, whereas SPL knockdown diminished apoptotic responses. Importantly, SPL expression was significantly down-regulated in human colon cancer tissues in comparison with normal adjacent tissues, as determined by quantitative real-time PCR (Q-PCR) and immunohistochemical analysis. Down-regulation of S1P phosphatases was also observed, suggesting that colon cancer cells manifest a block in S1P catabolism. In addition, SPL expression and activity were down-regulated in adenomatous lesions of the Min mouse model of intestinal tumorigenesis. Taken together, these results indicate that endogenous SPL may play a physiological role in stress-induced apoptosis and provide an example of altered SPL expression in a human tumor. Our findings suggest that genetic or epigenetic changes affecting intestinal S1P metabolism may correlate with and potentially contribute to carcinogenesis.

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.

Regulation of sphingosine-1-phosphate lyase gene expression by members of the GATA family of transcription factors.

Sphingosine-1-phosphate is a bioactive sphingolipid that regulates proliferation, differentiation, migration, and apoptosis. Sphingosine-1-phosphate is irreversibly degraded by the highly conserved enzyme sphingosine-1-phosphate lyase. Recent studies have suggested that sphingosine-1-phosphate lyase expression affects animal development and cell fate decisions. Despite its crucial role, mechanisms affecting expression of sphingosine-1-phosphate lyase remain poorly understood. In this study, regulation of sphingosine-1-phosphate lyase gene expression was investigated in Caenorhabditis elegans, where lyase expression is spatially restricted to cells of the developing and adult gut and is essential for normal development. Deletion analysis and generation of transgenic worms combined with fluorescence microscopy identified a 350-nucleotide sequence upstream of the ATG start site necessary for maximal lyase expression in adult worms. Site-specific mutagenesis of a GATA transcription factor-binding motif in the promoter led to loss of reporter expression. Knockdown of the gut-specific GATA transcription factor ELT-2 by RNA interference similarly led to loss of reporter expression. ELT-2 interacted with the GATA factor-binding motif in vitro and was also capable of driving expression of a Caenorhabditis elegans lyase promoter-beta-galactosidase reporter in a heterologous yeast system. These studies demonstrate that ELT-2 regulates sphingosine-1-phosphate lyase expression in vivo. Additionally, we demonstrate that the human sphingosine-1-phosphate lyase gene is regulated by a GATA transcription factor. Overexpression of GATA-4 led to both an increase in activity of a reporter gene as well as an increase in endogenous sphingosine-1-phosphate lyase protein.

Death and taxis: what non-mammalian models tell us about sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) is a signaling molecule that regulates critical events including mammalian cell proliferation, survival, migration and cell-cell interactions. Most of these signals are triggered by engagement of sphingosine-1-phosphate receptors of the Edg family. However, accumulating evidence derived from investigation of non-mammalian models that lack Edg receptors suggests that sphingosine-1-phosphate-like molecules can act through alternative mechanisms and thereby contribute to morphogenesis, development, reproduction and survival. This review provides an overview of sphingosine-1-phosphate metabolism, the isolation of genes in this pathway employing yeast genetics, the evidence for its influence on non-mammalian development, and the pertinence of these findings to human disease.

Sphingosine-phosphate lyase enhances stress-induced ceramide generation and apoptosis.

Sphingosine-1-phosphate lyase is a widely expressed enzyme that catalyzes the essentially irreversible cleavage of the signaling molecule sphingosine 1-phosphate. To investigate whether sphingosine-1-phosphate lyase influences mammalian cell fate decisions, a recombinant human sphingosine-1-phosphate lyase fused to green fluorescent protein was expressed in HEK293 cells. The recombinant enzyme was active, localized to the endoplasmic reticulum, and reduced baseline sphingosine and sphingosine 1-phosphate levels. Stable overexpression led to diminished viability under stress, which was attributed to an increase in apoptosis and was reversible in a dose-dependent manner by exogenous sphingosine 1-phosphate. In contrast to sphingosine 1-phosphate, the products of the lyase reaction had no effect on apoptosis. Lyase enzymatic activity was required to potentiate apoptosis, because cells expressing a catalytically inactive enzyme behaved like controls. Stress increased the amounts of long- and very long-chain ceramides in HEK293 cells, and this was enhanced in cells overexpressing wild type but not catalytically inactive lyase. The ceramide increases appeared to be required for apoptosis, because inhibition of ceramide synthase with fumonisin B1 decreased apoptosis in lyase-overexpressing cells. Thus, sphingosine-1-phosphate lyase overexpression in HEK293 cells decreases sphingosine and sphingosine 1-phosphate amounts but elevates stress-induced ceramide generation and apoptosis. This identifies sphingosine-1-phosphate lyase as a dual modulator of sphingosine 1-phosphate and ceramide metabolism as well as a regulator of cell fate decisions and, hence, a potential target for diseases with an imbalance in these biomodulators, such as cancer.