Sphingolipid Dysregulation Research Articles

Abstract 4740: Acid ceramidase inhibition enhances venetoclax sensitivity in preclinical models of acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is an aggressive hematologic malignancy in urgent need of improved therapeutic strategies. AML blasts frequently rely on the anti-apoptotic protein, Bcl-2, for survival. The specific Bcl-2 inhibitor, venetoclax (VEN), is FDA-approved in combination with low-dose cytarabine (LDAC) or azacitidine for patients unfit for intensive induction chemotherapy. Unfortunately, responses are transient due to upregulation of compensatory survival proteins (e.g., Bcl-xL or Mcl-1) or resistance characterized by reprogrammed mitochondrial bioenergetics. Our research aims to improve current AML treatments and understanding of AML pathogenesis by targeting dysregulated sphingolipid metabolism. Ceramides are tumor-suppressor sphingolipids that mediate therapy-induced cell death. Sphingolipid dysregulation that results in decreased ceramide content and/or enhanced ceramide catabolism is an emerging AML hallmark. We’ve previously shown that acid ceramidase (AC), a ceramide catabolizing lysosomal enzyme, is overexpressed in AML and contributes to drug resistance. Here, we demonstrated that AC inhibition enhanced VEN cytotoxicity. Combining the AC inhibitor/ceramide analog, SACLAC, with VEN resulted in synergistic lethality in multiple AML cell lines in cell viability and apoptosis assays. Genetic inhibition of AC also improved VEN cytotoxicity. The SACLAC+VEN combination achieved Bliss scores (SynergyFinder 2.0) between 22 and 32 in cell lines, which indicate a highly synergistic combination. SACLAC+VEN efficacy was comparable to the FDA-approved combination of VEN+LDAC when tested in 67 primary AML patient samples. Pharmacological inhibition of AC also increased the efficacy of the VEN+LDAC combination. Mechanistically, the observed synergistic lethality was independent of changes to Bcl-2, Mcl-1, and Bcl-xL protein levels. Combined AC and Bcl-2 inhibition resulted in synergistic ceramide accumulation, integrated stress response (ISR) activation, and caspase-dependent apoptosis characterized by impaired mitochondrial function. Pretreatment with the pan-caspase inhibitor, zVAD-FMK, or ISR inhibitor, ISRIB, significantly rescued cell death. Ongoing studies aim to further characterize mitochondrial impairment and ISR activation induced by inhibiting AC and Bcl-2. Taken together, these results detail the first report of ISR activation and mitochondrial defects elicited by combined AC and Bcl-2 inhibition as contributory to cytotoxic impact (mechanism) and warrant additional studies of AC inhibitors with frontline AML therapeutics. Citation Format: Johnson Ung, Su-Fern Tan, Jeremy J. Shaw, Todd E. Fox, Maansi Taori, David F. Claxton, Kelsey H. Fisher-Wellman, Myles C. Cabot, David J. Feith, Thomas P. Loughran. Acid ceramidase inhibition enhances venetoclax sensitivity in preclinical models of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4740.

A protective role of ABCA5 in response to elevated sphingomyelin levels in Parkinson’s disease

Parkinson’s disease (PD) is a chronic neurodegenerative disorder that affects the motor system. Increasing evidence indicates that lysosomal dysfunction is pivotal in the pathogenesis of PD, typically characterized by dysregulation of sphingolipids in lysosomes. ATP-binding cassette subfamily A member 5 (ABCA5) is a lysosomal transporter that mediates the removal of excess sphingomyelin from lysosomes. We therefore investigated whether the expression levels of ABCA5 are associated with sphingomyelin levels and α-synuclein pathology in PD. Firstly, we undertook a comprehensive assessment of the six sphingolipid classes that are part of the lysosomal salvage pathway in the disease-affected amygdala and disease-unaffected visual cortex using liquid chromatography-mass spectrometry. We found that sphingomyelin levels were significantly increased in PD compared to controls and correlated with disease duration only in the amygdala, whereas, the five other sphingolipid classes were slightly altered or unaltered. Concomitantly, the expression of ABCA5 was upregulated in the PD amygdala compared to controls and correlated strongly with sphingomyelin levels. Using neuronal cells, we further verified that the expression of ABCA5 was dependent on cellular levels of sphingomyelin. Interestingly, sphingomyelin levels were strongly associated with α-synuclein in the amygdala and were related to α-synuclein expression. Finally, we revealed that sphingomyelin levels were also increased in PD plasma compared to controls, and that five identical sphingomyelin species were increased in both the brain and the plasma. When put together, these results suggest that in regions accumulating α-synuclein in PD, ABCA5 is upregulated to reduce lysosomal sphingomyelin levels potentially as a protective measure. This process may provide new targets for therapeutic intervention and biomarker development for PD.

Untargeted metabolomics identifies metabolic dysregulation of sphingolipids associated with aggressive chronic lymphocytic leukaemia and poor survival.

Metabolic dependencies of chronic lymphocytic leukaemia (CLL) cells may represent new personalized treatment approaches in patients harbouring unfavourable features. Here, we used untargeted metabolomics and lipidomics analyses to isolate metabolomic features associated with aggressive CLL and poor survival outcomes. We initially focused on profiles associated with overexpression of the adverse metabolic marker glycosyltransferase (UGT2B17) associated with poor survival and drug resistance. Leukaemic B-cell metabolomes indicated a significant perturbation in lipids, predominantly bio-active sphingolipids. Expression of numerous enzyme-encoding genes of sphingolipid biosynthesis pathways was significantly associated with shorter patient survival. Targeted metabolomics further exposed higher circulating levels of glucosylceramides (C16:0 GluCer) in CLL patients relative to healthy donors and an aggressive cancer biology. In multivariate analyses, C16:0 GluCer and sphinganine were independent prognostic markers and were inversely linked to treatment-free survival. These two sphingolipid species function as antagonistic mediators, with sphinganine being pro-apoptotic and GluCer being pro-proliferative, tested in leukemic B-CLL cell models. Blocking GluCer synthesis using ceramide glucosyltransferase inhibitors induced cell death and reduced the proliferative phenotype, which further sensitized a leukaemic B-cell model to the anti-leukaemics fludarabine and ibrutinib in vitro. Specific sphingolipids may serve as prognostic markers in CLL, and inhibiting enzymatic pathways involved in their biosynthesis has potential as a therapaeutic approach.

S1PR2 inhibition mitigates cognitive deficit in diabetic mice by modulating microglial activation via Akt-p53-TIGAR pathway

Cognitive deficit is one of the challenging complications of type 2 diabetes. Sphingosine 1- phosphate receptors (S1PRs) have been implicated in various neurodegenerative and metabolic disorders. The association of S1PRs and cognition in type 2 diabetes remains elusive. Microglia-mediated neuronal damage could be the thread propagating cognitive deficit. The effects of S1PR2 inhibition on cognition in high-fat diet and streptozotocin-induced diabetic mice were examined in this work. We further assessed microglial activation and putative microglial polarisation routes. Cognitive function loss was observed after four months of diabetes induction in Type 2 diabetes animal model. JTE013, an S1PR2 inhibitor, was used to assess neuroprotection against cognitive decline and neuroinflammation in vitro and in vivo diabetes model. JTE013 (10 mg/kg) improved synaptic plasticity by upregulating psd95 and synaptophysin while reducing cognitive decline and neuroinflammation. It further enhanced anti-inflammatory microglia in the hippocampus and prefrontal cortex (PFC), as evidenced by increased Arg-1, CD206, and YM-1 levels and decreased iNOS, CD16, and MHCII levels. TIGAR, TP53-induced glycolysis and apoptosis regulator, might facilitate the anti-inflammatory microglial phenotype by promoting oxidative phosphorylation and decreasing apoptosis. However, since p53 is a TIGAR suppressor, inhibiting p53 could be beneficial. S1PR2 inhibition increased p-Akt and TIGAR levels and reduced the levels of p53 in the PFC and hippocampus of type 2 diabetic mice, thereby decreasing apoptosis. In vitro, palmitate was used to imitate sphingolipid dysregulation in BV2 cells, followed by conditioned media exposure to Neuro2A cells. JTE013 rescued the palmitate-induced neuronal apoptosis by promoting the anti-inflammatory microglia. In the present study, we demonstrate that the inhibition of S1PR2 improves cognitive function and skews microglia toward anti-inflammatory phenotype in type 2 diabetic mice, thereby promising to be a potential therapy for neuroinflammation.

Nasal Transcriptome and Epigenome Analysis Identifies the Pathogenic Features of Aspirin-Exacerbated Respiratory Disease.

Dysregulation of the arachidonic acid metabolic pathway is the most widely known pathomechanism of aspirin-exacerbated respiratory disease (AERD). This study aimed to perform integrative analysis of transcriptomic and epigenomic profiling with network analysis to determine the novel pathogenic features of AERD. Ten patients with asthma including 5 patients with AERD and another 5 patients with aspirin tolerant asthma (ATA) were enrolled. Nasal scraping was performed and nasal mucosa was used in omics profiling. Peripheral eosinophil counts, sputum eosinophil counts, fractional exhaled nitric oxide levels, and pulmonary function test results were evaluated. Differentially expressed genes (DEGs), differentially methylated probes (DMPs) and differentially correlated genes (DCGs) between patients with AERD and those with ATA were analyzed. Network analysis using ingenuity pathway analysis (IPA) was performed to determine the gene connection network and signaling pathways. In total, 1,736 DEGs, 1,401 DMPs, and 19 pairs for DCGs were identified. Among DCGs, genes related to vesicle transport (e.g., RAB3B and STX2) and sphingolipid dysregulation (e.g., SMPD3) were found to be hypo-methylated and up-regulated in AERD. Using the canonical pathway analysis of IPA with 78 asthma-related DEGs, signaling pathways of T helper cell differentiation/activation and Fcε receptor I were generated. Up-regulation of RORγt and FcER1A were noted in AERD. Gene expression levels of RAB3B, SYNE1, STX2, SMPD3 and RORγt were significantly associated with sputum eosinophil counts. Quantitative real-time polymerase chain reaction was performed and mRNA expression levels of STX2, SMPD3, RORγt, and FcER1A were significantly higher in AERD compared to ATA. Distinct pathogenic features were identified by using integrative multi-omics data analysis in patients with AERD.

Fingolimod Alleviates Cognitive Deficit in Type 2 Diabetes by Promoting Microglial M2 Polarization via the pSTAT3-jmjd3 Axis.

Sphingosine receptors (S1PRs) are implicated in the progression of neurodegenerative diseases and metabolic disorders like obesity and type 2 diabetes (T2D). The link between S1PRs and cognition in type 2 diabetes, as well as the mechanisms that underpin it, are yet unknown. Neuroinflammation is the common pathology shared among T2D and cognitive impairment. However, the interplay between the M1 and M2 polarization state of microglia, a primary driver of neuroinflammation, could be the driving factor for impaired learning and memory in diabetes. In the present study, we investigated the effects of fingolimod (S1PR1 modulator) on cognition in high-fat diet and streptozotocin-induced diabetic mice. We further assessed the potential pathways linking microglial polarization and cognition in T2D. Fingolimod (0.5mg/kg and 1mg/kg) improved M2 polarization and synaptic plasticity while ameliorating cognitive decline and neuroinflammation. Sphingolipid dysregulation was mimicked in vitro using palmitate in BV2 cells, followed by conditioned media exposure to Neuro2A cells. Mechanistically, type 2 diabetes induced microglial activation, priming microglia towards the M1 phenotype. In the hippocampus and cortex of type 2 diabetic mice, there was a substantial drop in pSTAT3, which was reversed by fingolimod. This protective effect of fingolimod on microglial M2 polarization was primarily suppressed by selective jmjd3 blockade in vitro using GSK-J4, revealing that jmjd3 was involved downstream of STAT3 in the fingolimod-enabled shift of microglia from M1 to M2 polarization state. This study suggested that fingolimod might effectively improve cognition in type 2 diabetes by promoting M2 polarization.

Abstract P038: Tissue-specific Regulation Of Ceramide De Novo Biosynthesis In Type 1 Diabetes. Role Of Metformin

Endothelial cell (EC) dysfunction precedes the onset of cardiovascular diseases (CVD), main complications of diabetes. Sphingolipids (SL) are essential components of cell membranes and bioactive lipids regulating different cellular functions in health and diseases. Dysregulation of SL, such as ceramides (Cer) accrual, has been implicated in diabetes and CVD, although specific mechanisms remain poorly understood. Whereas Cer accrual impairs endothelial nitric oxide synthase (eNOS) activity, low ceramides disrupt endothelial signal transduction and vascular tone regulation. To date, how Cer changes in diabetic EC in vivo is unknown. Metformin (Met), first-line therapy for diabetes, was shown to lower Cer in liver and muscle in mice. This study investigates the role of sphingolipid metabolism in diabetes and whether Met directly regulates this pathway.We discovered that Met can directly downregulate serine palmitoyltransferase activity, catalyzing the first step of the de novo biosynthesis of SL, thus refraining Cer from accrual. To assess this effect in vivo , we used non-obese diabetic mice, a model of type 1 diabetes, and nondiabetic mice as control. Diabetic mice were treated with Met (300mg/Kg/d) or vehicle for 5 weeks. Diabetic mesenteric arteries showed an impaired vasodilation to acetylcholine and exaggerated response to vasoconstrictors. Met treatment significantly improved endothelial function, by restoring acetylcholine-vasodilation and increasing phosphorylation of vasodilator stimulated phosphoprotein, index of eNOS-cGMP signaling. Interestingly, whereas Cer and sphingomyelins (SM) increased in the heart and liver, they were significantly decreased in myocardial EC, suggesting that in diabetes SL are differentially regulated. Met refrained Cer/SM from accrual in the heart and liver, while further decreased Cer/SM in EC. In conclusions, Met prevents Cer accrual in the heart and liver, thus limiting its deleterious effects. Contrary to the current believe, Cer are suppressed in diabetic EC in vivo , and might contribute to the progression of dysfunction. Most likely, Met downregulation of sphingolipid de novo biosynthesis, already suppressed in diabetic EC, does not underlie beneficial effects of Met on the endothelium.

CFTR modulator therapy alters plasma sphingolipid profiles in people with cystic fibrosis

BackgroundSphingolipids, in particular ceramides, play an important role in the pathogenesis of cystic fibrosis (CF) lung disease. Ceramides seem to be dysregulated in people with CF (PWCF): An elevated ratio of ceramides C16Cer/ C24Cer has been linked to inflammation and disease severity. CFTR modulators might influence sphingolipid dysregulation in PWCF. MethodsSphingolipid profiles were retrospectively analyzed in serum from 112 PWCF and 96 healthy controls as well as in plasma from 25 PWCF before and after treatment with the CFTR modulator elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Lipid data were correlated with clinical parameters. ResultsThere were significantly higher levels of long-chain ceramides C18Cer and C20Cer and of the very long-chain ceramide C24:1Cer in PWCF versus healthy controls. Sphingosine levels were significantly reduced and accurately distinguished PWCF from healthy controls. Treatment with ELX/TEZ/IVA was associated with a decrease in levels of long-chain ceramides C16Cer, C18Cer and C20Cer and very long-chain ceramide C24:1Cer. Plasma levels of the most abundant very long-chain ceramide C24Cer as well as sphingosine-1-phosphate increased. Consequently, the ratio of ceramides C16Cer/ C24Cer decreased. Sphingolipid levels showed weak correlations with clinical parameters. ConclusionsThese findings highlight the existence of a distinctive sphingolipid profile in blood from PWCF, which appears to be altered by ELX/TEZ/IVA therapy. Thus, strategies for sphingolipid remodeling need to be reassessed and adjusted in the light of highly effective CFTR modulator therapies.

The Role of Sphingolipids Metabolism in Cancer Drug Resistance.

Drug resistance continues to be one of the major challenges to cure cancer. As research in this field evolves, it has been proposed that numerous bioactive molecules might be involved in the resistance of cancer cells to certain chemotherapeutics. One well-known group of lipids that play a major role in drug resistance are the sphingolipids. Sphingolipids are essential components of the lipid raft domains of the plasma membrane and this structural function is important for apoptosis and/or cell proliferation. Dysregulation of sphingolipids, including ceramide, sphingomyelin or sphingosine 1-phosphate, has been linked to drug resistance in different types of cancer, including breast, melanoma or colon cancer. Sphingolipid metabolism is complex, involving several lipid catabolism with the participation of key enzymes such as glucosylceramide synthase (GCS) and sphingosine kinase 1 (SPHK1). With an overview of the latest available data on this topic and its implications in cancer therapy, this review focuses on the main enzymes implicated in sphingolipids metabolism and their intermediate metabolites involved in cancer drug resistance.

Regulation of hepatic insulin signaling and glucose homeostasis by sphingosine kinase 2

Sphingolipid dysregulation is often associated with insulin resistance, while the enzymes controlling sphingolipid metabolism are emerging as therapeutic targets for improving insulin sensitivity. We report herein that sphingosine kinase 2 (SphK2), a key enzyme in sphingolipid catabolism, plays a critical role in the regulation of hepatic insulin signaling and glucose homeostasis both in vitro and in vivo. Hepatocyte-specific Sphk2 knockout mice exhibit pronounced insulin resistance and glucose intolerance. Likewise, SphK2-deficient hepatocytes are resistant to insulin-induced activation of the phosphoinositide 3-kinase (PI3K)-Akt-FoxO1 pathway and elevated hepatic glucose production. Mechanistically, SphK2 deficiency leads to the accumulation of sphingosine that, in turn, suppresses hepatic insulin signaling by inhibiting PI3K activation in hepatocytes. Either reexpressing functional SphK2 or pharmacologically inhibiting sphingosine production restores insulin sensitivity in SphK2-deficient hepatocytes. In conclusion, the current study provides both experimental findings and mechanistic data showing that SphK2 and sphingosine in the liver are critical regulators of insulin sensitivity and glucose homeostasis.

Fumonisin B1-Induced Toxicity Was Not Exacerbated in Glutathione Peroxidase-1/Catalase Double Knock Out Mice.

Fumonisin B1 (FB1) structurally resembles sphingolipids and interferes with their metabolism leading to sphingolipid dysregulation. We questioned if FB1 could exacerbate liver or kidney toxicities in glutathione peroxidase 1 (Gpx1) and catalase (Cat) knockout mice. While higher serum levels of thiobarbituric acid reactive substances (TBARS) and sphinganine (Sa) were measured in Gpx1/Cat knockout mice (Gpx1/Cat KO) than wild type mice after 5 days of FB1 treatment, serum levels of alanine aminotransferase (ALT), sphingosine-1 phosphate (So-1-P), and sphinganine-1 phosphate (Sa-1-P) were found to be relatively low. Although Sa was highly elevated in Gpx1/Cat KO mice and wild mice, lower levels of So and Sa were found in both the kidney and liver tissues of Gpx/Cat KO mice than wild type mice after FB1 treatment. Paradoxically, FB1-induced cellular apoptosis and necrosis were hastened under oxidative stress in Gpx1/Cat KO mice.

Effects of constant light exposure on sphingolipidomics and progression of NASH in high‐fat‐fed rats

Non-alcoholic fatty liver disease (NAFLD) is a growing public health concern worldwide. With the progression of urbanization, light pollution is becoming an inevitable risk factor for NAFLD. However, the role of light pollution on NAFLD is insufficiently understood, and the underlying mechanism remains unclear. The present study explored effects of constant light exposure on NAFLD and elucidated its related mechanisms. Thirty-two male Sprague Dawley rats were divided into four groups (n=8 each): (i) rats on a normal diet exposed to standard light-dark cycle (ND-LD); (ii) rats on a normal diet exposed to constant light (ND-LL); (iii) rats on a high-fat diet exposed to standard light-dark cycle (HFD-LD); and (iv) and rats on a high-fat diet exposed to constant light (HFD-LL). After 12weeks of treatment, rats were sacrificed and pathophysiological assessments were performed. Targeted lipidomics was used to measure sphingolipids, including ceramides, glucosylceramides, and lactosylceramides, sphingomyelins, and sphingosine-1-phosphates in plasma and liver tissues. In normal chow rats, constant light exposure led to glucose abnormalities and dyslipidemia. In high-fat-fed rats, constant light exposure exacerbated glucose abnormalities, dyslipidemia, insulin resistance, and inflammation and aggravated steatohepatitis. Compared with HFD-LD rats, HFD-LL had decreased plasma sphingosine-1-phosphate and elevated liver concentrations of total ceramide and specific ceramide species (ceramide d18:0/24:0, ceramide d18:1/22:0, ceramide d18:1/24:0, and ceramide d18:1/24:1), which were associated with increased hepatocyte apoptosis. Constant light exposure causes dysregulation of sphingolipids and promotes steatohepatitis in high-fat-fed rats.

Quantitative Systems Pharmacology Model for Alzheimer Disease Indicates Targeting Sphingolipid Dysregulation as Potential Treatment Option.

Alzheimer disease (AD) is a devastating neurodegenerative disorder with high unmet medical need. Drug development is hampered by limited understanding of the disease and its driving factors. Quantitative Systems Pharmacology (QSP) modeling provides a comprehensive quantitative framework to evaluate the relevance of biological mechanisms in the context of disease and to predict the efficacy of novel treatments. Here, we report a QSP model for AD with a particular focus on investigating the relevance of dysregulation of cholesterol and sphingolipids. We show that our model captures the modulation of several biomarkers in subjects with AD, as well as the response to pharmacological interventions. We evaluate the impact of targeting the sphingosine‐1‐phosphate 5 receptor (S1PR5) as a potential novel treatment option for AD, and model predictions increase our confidence in this novel disease pathway. Future applications for the QSP model are in validation of further targets and identification of potential treatment response biomarkers.

Sphingolipid accumulation causes mitochondrial dysregulation and cell death.

Sphingolipids are structural components of cell membranes that have signaling roles to regulate many activities, including mitochondrial function and cell death. Sphingolipid metabolism is integrated with numerous metabolic networks, and dysregulated sphingolipid metabolism is associated with disease. Here, we describe a monogenic yeast model for sphingolipid accumulation. A csg2Δ mutant cannot readily metabolize and accumulates the complex sphingolipid inositol phosphorylceramide (IPC). In these cells, aberrant activation of Ras GTPase is IPC-dependent, and accompanied by increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial mass. Survival or death of csg2Δ cells depends on nutritional status. Abnormal Ras activation in csg2Δ cells is associated with impaired Snf1/AMPK protein kinase, a key regulator of energy homeostasis. csg2Δ cells are rescued from ROS production and death by overexpression of mitochondrial catalase Cta1, abrogation of Ras hyperactivity or genetic activation of Snf1/AMPK. These results suggest that sphingolipid dysregulation compromises metabolic integrity via Ras and Snf1/AMPK pathways.

Acid ceramidase is upregulated in AML and represents a novel therapeutic target.

There is an urgent unmet need for new therapeutics in acute myeloid leukemia (AML) as standard therapy has not changed in the past three decades and outcome remains poor for most patients. Sphingolipid dysregulation through decreased ceramide levels and elevated sphingosine 1-phosphate (S1P) promotes cancer cell growth and survival. Acid ceramidase (AC) catalyzes ceramide breakdown to sphingosine, the precursor for S1P. We report for the first time that AC is required for AML blast survival. Transcriptome analysis and enzymatic assay show that primary AML cells have high levels of AC expression and activity. Treatment of patient samples and cell lines with AC inhibitor LCL204 reduced viability and induced apoptosis. AC overexpression increased the expression of anti-apoptotic Mcl-1, significantly increased S1P and decreased ceramide. Conversely, LCL204 induced ceramide accumulation and decreased Mcl-1 through post-translational mechanisms. LCL204 treatment significantly increased overall survival of C57BL/6 mice engrafted with leukemic C1498 cells and significantly decreased leukemic burden in NSG mice engrafted with primary human AML cells. Collectively, these studies demonstrate that AC plays a critical role in AML survival through regulation of both sphingolipid levels and Mcl-1. We propose that AC warrants further exploration as a novel therapeutic target in AML.

Sphingolipid profile alters in retinal dystrophic P23H-1 rats and systemic FTY720 can delay retinal degeneration

Retinal degeneration (RD) affects millions of people and is a major cause of ocular impairment and blindness. With a wide range of mutations and conditions leading to degeneration, targeting downstream processes is necessary for developing effective treatments. Ceramide and sphingosine-1-phosphate, a pair of bioactive sphingolipids, are involved in apoptosis and its prevention, respectively. Apoptotic cell death is a potential driver of RD, and in order to understand the mechanism of degeneration and potential treatments, we studied rhodopsin mutant RD model, P23H-1 rats. Investigating this genetic model of human RD allows us to investigate the association of sphingolipid metabolites with the degeneration of the retina in P23H-1 rats and the effects of a specific modulator of sphingolipid metabolism, FTY720. We found that P23H-1 rat retinas had altered sphingolipid profiles that, when treated with FTY720, were rebalanced closer to normal levels. FTY720-treated rats also showed protection from RD compared with their vehicle-treated littermates. Based on these data, we conclude that sphingolipid dysregulation plays a secondary role in retinal cell death, which may be common to many forms of RDs, and that the U.S. Food and Drug Administration-approved drug FTY720 or related compounds that modulate sphingolipid metabolism could potentially delay the cell death.

Targeting glucosylceramide synthase upregulation reverts sorafenib resistance in experimental hepatocellular carcinoma.

Evasive mechanisms triggered by the tyrosine kinase inhibitor sorafenib reduce its efficacy in hepatocellular carcinoma (HCC) treatment. Drug-resistant cancer cells frequently exhibit sphingolipid dysregulation, reducing chemotherapeutic cytotoxicity via the induction of ceramide-degrading enzymes. However, the role of ceramide in sorafenib therapy and resistance in HCC has not been clearly established. Our data reveals that ceramide-modifying enzymes, particularly glucosylceramide synthase (GCS), are upregulated during sorafenib treatment in hepatoma cells (HepG2 and Hep3B), and more importantly, in sorafenib-resistant cell lines. GCS silencing or pharmacological GCS inhibition sensitized hepatoma cells to sorafenib exposure. GCS inhibition, combined with sorafenib, triggered cytochrome c release and ATP depletion in sorafenib-treated hepatoma cells, leading to mitochondrial cell death after energetic collapse. Conversely, genetic GCS overexpression increased sorafenib resistance. Of interest, GCS inhibition improved sorafenib effectiveness in a xenograft mouse model, recovering drug sensitivity of sorafenib-resistant tumors in mice. In conclusion, our results reveal GCS induction as a mechanism of sorafenib resistance, suggesting that GCS targeting may be a novel strategy to increase sorafenib efficacy in HCC management, and point to target the mitochondria as the subcellular location where sorafenib therapy could be potentiated.

Acid Ceramidase Inhibition Impairs Tumor Progression in a Rat Model of LGL Leukemia

Natural killer cell large granular lymphocytic (NK-LGL) leukemia is a disorder characterized by the expansion of clonal CD3-, CD56+ cells in the peripheral blood and marrow. NK-LGL leukemia could manifest in both aggressive and chronic forms. Currently, no curative treatment is available for patients with NK-LGL leukemia. The pathogenetic mechanisms of NK-LGL leukemia are also poorly understood, which highlights the need to increase our understanding of the disorder in order to develop novel therapeutic targets. Sphingolipid dysregulation has been shown to promote tumor growth and survival in several types of cancers, including LGL leukemia. In cancer cells, the accumulation of pro-survival sphingosine-1-phosphate (S1P) throws the S1P/ceramide rheostat off balance, which leads to tumor growth and activated signaling pathways. Our laboratory previously discovered that the reversal of sphingolipid dysregulation in NK-LGL leukemia, either through targeting sphingolipid enzymes or by the addition of antiproliferative ceramide, leads to programmed cell death in leukemic cells.Acid ceramidase, a cysteine hydrolase, catalyzes the breakdown of ceramide into sphingosine and fatty acid. We found that cells from chronic NK-LGL patients have lower pro-death ceramide levels and higher S1P levels when compared to normal NK donors. Furthermore, the mRNA expression and activity of acid ceramidase (AC) were elevated in leukemic NK cells compared to normal NK cells. We further demonstrate the importance of AC in NK-LGL leukemic cells through AC knockdown in human (NKL) and rat (RNK-16) NK-LGL leukemic cell lines. AC knockdown decreased cell viability and increased ceramide levels, significantly ceramide species C16 and C24 (p<0.05), showing that AC is essential for the growth of leukemic NK cells. We further confirm that the accumulation of both C16 and C24 ceramide mediated the decreased viability of NKL and RNK-16 cells by treating the cells with C16 and C24 ceramide (6.25µM, 12.5µM and 25µM) for 24 hours.As our data showed that AC mediates the survival of NK-LGL, we did further analysis on the effects of AC knockdown on survival signals. Protein expression of AC knockdown- NKL and RNK-16 cell lines showed that AC knockdown decreased survival signaling mediators, including Bcl-2, Mcl-1 and survivin. Conversely, pro-apoptotic puma increased with AC knockdown in both cell lines. We further extended our in vitro findings in an established in vivo NK-LGL Fischer rat model. RNK-16 cells stably expressing AC shRNA (RNK-16/shRNA) or stably expressing scrambled shRNA (RNK-16/scr) were transplanted into 6-week old male rats (7 per group). The rats engrafted with RNK-16/shRNA survived significantly longer than those engrafted with RNK-16/scr (Mantel-Cox test, p<0.05). Interestingly, RNK-16/shRNA rats did not exhibit organomegaly compared to their leukemic counterparts. Splenocytes isolated from RNK-16/shRNA engrafted animals showed 50 percent reduction in AC expression compared to splenocytes from RNK-16/scr. Lipid analysis on splenocytes isolated from both RNK-16/scr and RNK-16/shRNA showed that RNK-16/shRNA cells had significant increases in total ceramide, C16 and C24 ceramide species, and a significant decrease in S1P. Collectively, our data show that targeting AC slows the progression of NK-LGL leukemia by reversing sphingolipid dysregulation and warrants further investigation as a therapeutic target in this incurable disease. DisclosuresNo relevant conflicts of interest to declare.

P6 - Impaired metabolism of senescent muscle satellite cells is associated with oxidative modifications of glycolytic enzymes

Accumulation of damaged macromolecules, including irreversibly oxidized proteins, is a hallmark of cellular and organismal ageing. Failure of protein homesotasis is a major contributor to the age-related accumulation of damaged proteins. In skeletal muscle, tissue maintenance and regeneration is assured by resident adult stem cells known as satellite cells. During senescence their replication and differentiation is compromised contributing to sarcopenia. In this study we have addressed the impact of oxidatively modified proteins in the impaired metabolism of senescent human satellite cells. By using a targeted proteomics analysis we have found that proteins involved in protein quality control and glycolytic enzymes are the main targets of oxidation (carbonylation) and modification with advanced glycation/lipid peroxidation end products during replicative senescence of satellite cells. Inactivation of the proteasome in aged cells appeared as a key contributor to the accumulation of such damaged proteins. Untargeted metabolomic profiling and functional analyses indicated glucose metabolism impairment in senescent cells, although mitochondrial respiration remained unaffected. A metabolic shift leading to increased mobilization of non-carbohydrate substrates as branched chain amino acids or long chain fatty acids was observed in senescent cells. In addition, phospho-and glycerolipids metabolism was altered. Increased levels of acyl-carnitines indicated augmented turnover of storage and membrane lipids for energy production. Such changes reflect alterations in membrane composition and dysregulation of sphingolipids signaling during senescence. This study establishes a new concept connecting oxidative protein modifications with the altered cellular metabolism associated with the senescent phenotype. In addition, these findings highlight the molecular mechanisms implicated in satellite cells dysfunction during ageing, paving the road for future therapeutic interventions aimed at preventing oxidative modifications of proteins and/or stimulating their elimination.