Altered Sphingolipid Metabolism Research Articles

The shifting lipidomic landscape of blood monocytes and neutrophils during pneumonia.

The lipidome of immune cells during infection has remained unexplored, although evidence of the importance of lipids in the context of immunity is mounting. In this study, we performed untargeted lipidomic analysis of blood monocytes and neutrophils from patients hospitalized for pneumonia and age- and sex-matched noninfectious control volunteers. We annotated 521 and 706 lipids in monocytes and neutrophils, respectively, which were normalized to an extensive set of internal standards per lipid class. The cellular lipidomes were profoundly altered in patients, with both common and distinct changes between the cell types. Changes involved every level of the cellular lipidome: differential lipid species, class-wide shifts, and altered saturation patterns. Overall, differential lipids were mainly less abundant in monocytes and more abundant in neutrophils from patients. One month after hospital admission, lipidomic changes were fully resolved in monocytes and partially in neutrophils. Integration of lipidomic and concurrently collected transcriptomic data highlighted altered sphingolipid metabolism in both cell types. Inhibition of ceramide and sphingosine-1-phosphate synthesis in healthy monocytes and neutrophils resulted in blunted cytokine responses upon stimulation with lipopolysaccharide. These data reveal major lipidomic remodeling in immune cells during infection, and link the cellular lipidome to immune functionality.

Therapeutic Potential for Sphingolipids in Inflammatory Bowel Disease and Colorectal Cancer.

Inflammatory bowel disease (IBD), characterized by chronic inflammation in the intestinal tract, increases the risk for the development of colorectal cancer (CRC). Sphingolipids, which have been implicated in IBD and CRC, are a class of bioactive lipids that regulate cell signaling, differentiation, apoptosis, inflammation, and survival. The balance between ceramide (Cer), the central sphingolipid involved in apoptosis and differentiation, and sphingosine-1-phosphate (S1P), a potent signaling molecule involved in proliferation and inflammation, is vital for the maintenance of normal cellular function. Altered sphingolipid metabolism has been implicated in IBD and CRC, with many studies highlighting the importance of S1P in inflammatory signaling and pro-survival pathways. A myriad of sphingolipid analogues, inhibitors, and modulators have been developed to target the sphingolipid metabolic pathway. In this review, the efficacy and therapeutic potential for modulation of sphingolipid metabolism in IBD and CRC will be discussed.

Evaluation of plasma sphingolipids as mediators of the relationship between kidney disease and cardiovascular events

Sphingolipids are a family of circulating lipids with regulatory and signaling roles that are strongly associated with both eGFR and cardiovascular disease. Patients with chronic kidney disease (CKD) are at high risk for cardiovascular events, and have different plasma concentrations of certain plasma sphingolipids compared to patients with normal kidney function. We hypothesize that circulating sphingolipids partially mediate the associations between eGFR and cardiovascular events. We measured the circulating concentrations of 8 sphingolipids, including 4 ceramides and 4 sphingomyelins with the fatty acids 16:0, 20:0, 22:0, and 24:0, in plasma from 3,463 participants in a population-based cohort (Cardiovascular Health Study) without prevalent cardiovascular disease. We tested the adjusted mediation effects by these sphingolipids of the associations between eGFR and incident cardiovascular disease via quasi-Bayesian Monte Carlo method with 2,000 simulations, using a Bonferroni correction for significance. The mean (±SD) eGFR was 70 (±16) mL/min/1.73m2; 62% of participants were women. Lower eGFR was associated with higher plasma ceramide-16:0 and sphingomyelin-16:0, and lower ceramides and sphingomyelins-20:0 and -22:0. Lower eGFR was associated with risk of incident heart failure and ischemic stroke, but not myocardial infarction. Five of eight sphingolipids partially mediated the association between eGFR and heart failure. The sphingolipids associated with the greatest proportion mediated were ceramide-16:0 (proportion mediated 13%, 95% CI 8-22%) and sphingomyelin-16:0 (proportion mediated 10%, 95% CI 5-17%). No sphingolipids mediated the association between eGFR and ischemic stroke. Plasma sphingolipids partially mediated the association between lower eGFR and incident heart failure. Altered sphingolipids metabolism may be a novel mechanism for heart failure in patients with CKD. This study was supported by T32 DK007467 and a KidneyCure Ben J. Lipps Research Fellowship (Dr.Lidgard). Sphingolipid measurements were supported by R01 HL128575 (Dr. Lemaitre) and R01 HL111375 (Dr.Hoofnagle) from the National Heart, Lung, and Blood Institute (NHLBI).

Metabolic indicators of an altered sphingolipid metabolism are biomarkers for non-alcoholic fatty liver disease

Metabolic indicators of an altered sphingolipid metabolism are biomarkers for non-alcoholic fatty liver disease

Acetylcysteine modulates sphingolipid levels by inhibiting neutral sphingomyelinase during aging in rat tissues

Old-age-associated pathologies usually coincide with altered sphingolipid metabolism. Changes in the level of sphingomyelin (SM) and ceramide are largely determined by the activation of neutral sphingomyelinase (nSMase) during aging. nSMase is a key enzyme in sphingolipid turnover; it generates ceramide through the hydrolysis of SM. While N—acetylcysteine (NAC) is a powerful antioxidant which has anti-inflammatory properties and it is the precursor of glutathione (GSH). The aim of this study is to determine the impact of NAC on nSMase activity in the age-dependent changes of sphingolipid (SM and ceramide) levels and GSH content in blood serum, gastrocnemius muscle and kidney tissue of 24-month-old rats. It was found that the effect of NAC on the tissue of 24-month-old rats increased SM content; GSH content and decreased the ceramide content. The gastrocnemius muscle and kidney were the most sensitive to the effects of NAC. The results show that GSH inhibit the activity of nSMase. These results emphasize that nSMase. plays a key role in age-associated ceramide accumulation in rat tissues during aging. Furthermore, it was indicated that NAC is an effective modulator of nSMase and has pleiotropic effects that may alter sphingolipid metabolism. The nSMase inhibition enhances the benefits of NAC treatment.

Abstract 1585: Targeting acid ceramidase-1 to inhibit sphingolipid metabolism and tumor growth in pancreatic ductal carcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related deaths in the United States. PDAC is an aggressive disease with a poor survival rate of <10%. Hence, there is an urgent need to find novel drug targets for the clinical management of PDAC. Altered sphingolipid metabolism (SM) is a hallmark of cancer. The major sphingolipid metabolites are ceramide, sphingosine and sphingosine-1-phosphate (S1P). We found that S1P is the predominant SM metabolite in PDAC cells and tissues. The equilibrium between ceramide and S1P levels within the body is an important factor to determine cell fate. Ceramide induces apoptosis, while S1P promotes cancer growth. The IHC and ELISA assays showed that S1P levels are higher in PDAC cells and tissues compared to normal pancreatic tissue. We found that S1P induces spheroid formation in PDAC cells at nanomolar concentrations when compared to C1P and sphingosine. S1P binds to S1P receptors (SIPR1-5) to activate downstream signaling. Mechanistically, S1P treatment activates beta-catenin and notch signaling pathway proteins in PDAC cells. Our data suggest that S1P promotes tumor growth and stemness in PDAC and inhibition of S1P production can be a novel strategy for PDAC treatment. The cancer genome atlas (TCGA) data analysis indicated that genes involved in S1P production, especially acid ceramidase (ASAH1) and sphingosine kinase 1 (SPHK1) are upregulated in PDAC. ASAH1 converts ceramide to sphingosine which further gets converted to S1P by SPHK1. SPHK1 has been targeted in cancers, but kinase inhibitors are traditionally known for their non-selectivity and toxicity. Moreover, selective targeting of S1PR1-5 has been unsuccessful. Hence, there is a need to identify other targets to inhibit the production of S1P. Therefore, we decided to target Acid ceramidase 1 (ASAH1). Inhibiting ASAH1 will result in the accumulation of proapoptotic ceramide and inhibition of S1P production. Our data showed that ASAH1 is overexpressed in PDAC tissue and cells, while the ASAH1 knockdown in PDAC cells resulted in reduced proliferation, colony formation, migration and spheroid formation. Hence, targeting ASAH1 represents a novel strategy to inhibit PDAC progression by inhibiting sphingolipid metabolism and S1P production. Next, we developed a series of ASAH1 inhibitors based on Cerenib-2 (known ASAH1 inhibitor). One of the analogs, Cer-IVA inhibited the proliferation, colony and spheroid formation in PDAC cells at nanomolar concentration. Moreover, it also induced apoptosis and G2/M-cell cycle arrest in a dose and time-dependent manner. Further, it inhibited S1P production and increased ceramide production in PDAC cells. We are currently investigating the antitumor effects of Cer-IVA in an orthotopic model in C57BL/6 mice. In conclusion, targeting ASAH1 by Cer-IVA is an attractive strategy to inhibit S1P production, growth and stemness in PDAC cells. Citation Format: Krishan G. Jain, Hindole Ghosh, Sangita Bhattacharyya, Anup Kasi, Ameer Hamza, Scott J. Weir, Michael VanSaun, Stefan Bossmann, Shrikant Anant, Bernhard Biersack, Prasad R. Dandawate. Targeting acid ceramidase-1 to inhibit sphingolipid metabolism and tumor growth in pancreatic ductal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1585.

Abstract 3042: BXQ-350 may protect peripheral nerves from the direct cytotoxicity of chemotherapeutic agents leading to chemotherapy induced peripheral neuropathy

Abstract Background: Chemotherapy Induced Peripheral Neuropathy (CIPN) is a debilitating side effect associated with many antineoplastic chemotherapeutic agents including cytotoxic and targeted agents. It significantly impacts cancer patients’ quality of life during treatment, and may cause lasting neuropathy and shorten treatment regimen, potentially impacting clinical benefit. The pathology of CIPN is complex and still not completely understood. Damages to nerve cells are believed to be directly resulting from the antineoplastic agents’ cytotoxicity, and inflammation and the immune system are believed to be involved as well. Evidence suggests that intervention of certain GPCRs (e.g., cannabinoid receptors and others) may be useful as potential treatments. Altered neuronal sphingolipid metabolism has been linked to neuropathic pain and elevated plasma levels of sphingosine-1-phosphate (S1P) have also been associated with patients receiving chemotherapy and developing CIPN; therefore, modulating S1P levels may also be a potential treatment. Method: BXQ-350 is a nanovesicle of Saposin C, an allosteric activator of sphingolipid metabolism, that lowers systemic S1P; Saposin C has been reported to activate certain GPCRs, including GPR37. BXQ-350 was investigated in an adult Phase 1 dose-escalation safety study in heavily pretreated all-comer cancer patients with advanced solid malignancies (NCT02859857). The cytoprotective properties of BXQ-350 against multiple agents known to induce CIPN were investigated in vitro in neuronal PC12 cells and BXQ-350’s CIPN mitigation properties were investigated in vivo in a CIPN preclinical model. Results: A pancreatic cancer patient with chronic CIPN at time of enrollment in the Phase 1 study spontaneously reported a significant improvement of her neuropathic symptoms shortly after receiving BXQ-350; several patients had similar improvements. Results from preclinical experiments in PC12 cells revealed that BXQ-350 at concentration as low as 50 nM protected cells from oxaliplatin, paclitaxel, bortezomib or MMAE and promoted neurite growth. BXQ-350 was subsequently investigated in a murine oxaliplatin-induced CIPN preclinical model, showing a dose-dependent reduction of thermal and mechanical allodynia correlating with decreasing systemic S1P levels. Conclusions: Preclinical results demonstrated that BXQ-350 was highly effective at protecting neuronal cells from antineoplastic agents known to induce CIPN and prevent CIPN in a preclinical model. These results appeared to support the clinical observation that BXQ-350 alleviated CIPN symptoms in several patients soon after receiving BXQ-350. Additional studies are on-going. Citation Format: Nikhil Wilkins, Tim Stephens, Robin Furnish, Charlie Cruze, Darren Wolfe, Gilles H. Tapolsky, Ray Takigiku. BXQ-350 may protect peripheral nerves from the direct cytotoxicity of chemotherapeutic agents leading to chemotherapy induced peripheral neuropathy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3042.

Blocking SphK/S1P/S1PR1 axis signaling pathway alleviates remifentanil-induced hyperalgesia in rats

Recent research shows a correlation between altered sphingolipid metabolism and nociceptive processing. Activation of the sphingosine-1-phosphate receptor 1 subtype (S1PR1) by its ligand, sphingosine-1-phosphate (S1P), causes neuropathic pain. However, its role in remifentanil-induced hyperalgesia (RIH) has not been investigated. The purpose of this research was to establish if the SphK/S1P/S1PR1 axis mediated remifentanil-induced hyperalgesia and identify its potential targets. This study examined the protein expression of ceramide, sphingosine kinases (SphK), S1P, and S1PR1 in the spinal cord of rats treated with remifentanil (1.0 μg/kg/min for 60 min). Prior to receiving remifentanil, rats were injected with SK-1 (a SphK inhibitor); LT1002 (a S1P monoclonal antibody); CYM‐5442, FTY720, and TASP0277308（the S1PR1 antagonists); CYM-5478 (a S1PR2 agonist); CAY10444 (a S1PR3 antagonist); Ac-YVAD-CMK (a caspase-1 antagonist); MCC950 (the NOD-like receptor protein 3 (NLRP3) inflammasome antagonist); and N-tert-Butyl-α-phenylnitrone (PBN, a reactive oxygen species (ROS) scavenger). Mechanical and thermal hyperalgesia were evaluated at baseline (24 h prior to remifentanil infusion) and 2, 6, 12, and 24 h following remifentanil administration. The expression of the NLRP3-related protein (NLRP3, caspase-1), pro-inflammatory cytokines (interleukin-1β(IL-1β), IL-18), and ROS was found in the spinal dorsal horns. In the meantime, immunofluorescence was used to ascertain if S1PR1 co-localizes with astrocytes. Remifentanil infusion induced considerable hyperalgesia in addition to increased ceramide, SphK, S1P, and S1PR1, NLRP3-related protein (NLRP3, Caspase-1, IL-1β, IL-18) and ROS expression, and S1PR1 localized astrocytes. By blocking the SphK/S1P/S1PR1 axis, remifentanil-induced hyperalgesia was reduced, as was the expression of NLRP3, caspase-1, pro-inflammatory cytokines (IL-1β, IL-18) and ROS in the spinal cord. In addition, we observed that suppressing NLRP3 or ROS signal attenuated the mechanical and thermal hyperalgesia induced by remifentanil. Our findings indicate that the SphK/SIP/S1PR1 axis regulates the expression of NLRP3, Caspase-1, IL-1β, IL-18 and ROS in the spinal dorsal horn to mediate remifentanil-induced hyperalgesia. These findings may contribute to pain and SphK/S1P/S1PR1 axis research positively, and inform the future study of this commonly used analgesic.

BXQ-350 may alleviate symptoms of chemotherapy- induced peripheral neuropathy via modulation of S1P.

93 Background: Chemotherapy Induced Peripheral Neuropathy (CIPN) is a debilitating side effect associated with many chemotherapeutic agents. It significantly impacts quality of life during treatment, causes lasting neuropathy, and may also shorten the treatment regimen, potentially impacting clinical benefit. The pathology of CIPN is still not completely understood, however, increasing evidence suggests sphingosine-1-phosphate (S1P) may be an important signaling molecule. Altered neuronal sphingolipid metabolism has been linked to neuropathic pain, evidenced by elevated plasma levels of S1P in patients receiving chemotherapy. Methods: BXQ-350 is a nanovesicle of Saposin C, an allosteric activator of sphingolipid metabolism, that lowers systemic S1P. BXQ-350 was investigated in an adult Phase 1 dose-escalation safety study in heavily pretreated all-comer cancer patients with advanced solid malignancies ( NCT02859857 ). The primary objective was to determine the safety profile and potential clinical activity of BXQ-350 as monotherapy. Samples were collected to explore potential biomarkers. Results: BXQ-350 was safe and well tolerated. Clinical signs of activity were observed in 13 patients (~17.8% of evaluable patients) experiencing a clinical benefit (PR, SD) up to cycle 6 and beyond including: 4 CRC, 1 pancreatic, and 1 GIST patient. Two patients are still on study six years after enrollment, including 1 CRC. Interestingly, a pancreatic cancer patient with chronic CIPN at time of enrollment spontaneously reported a significant improvement of her neuropathic symptoms shortly after receiving BXQ-350. Investigation of potential improvements in patients with chronic CIPN at time of enrollment revealed that 4 out of 10 patients experienced an improvement of their symptoms that seemed to be associated with a decrease in S1P systemic levels following BXQ-350 administration. BXQ-350 was subsequently investigated in a murine oxaliplatin-CIPN preclinical model with results showing a dose-dependent prevention/resolution of CIPN correlating with decreasing systemic S1P levels. Conclusions: Results of this Phase 1 study in heavily pretreated patients shows that BXQ-350 was well tolerated and seems to generate a clinical benefit via modulation of S1P. There were preliminary signs that BXQ-350 may alleviate symptoms of CIPN in relation to decreasing S1P concentration. Additional studies are underway to better understand this novel mechanism of action. Clinical trial information: 02859857 .

Identification and validation of a prognostic risk-scoring model based on sphingolipid metabolism-associated cluster in colon adenocarcinoma.

Colon adenocarcinoma (COAD) is the primary factor responsible for cancer-related mortalities in western countries, and its development and progression are affected by altered sphingolipid metabolism. The current study aimed at investigating the effects of sphingolipid metabolism-related (SLP) genes on multiple human cancers, especially on COAD. We obtained 1287 SLP genes from the GeneCard and MsigDb databases along with the public transcriptome data and the related clinical information. The univariate Cox regression analysis suggested that 26 SLP genes were substantially related to the prognosis of COAD, and a majority of SLP genes served as the risk genes for the tumor, insinuating a potential pathogenic effect of SLP in COAD development. Pan-cancer characterization of SLP genes summarized their expression traits, mutation traits, and methylation levels. Subsequently, we focused on the thorough research of COAD. With the help of unsupervised clustering, 1008 COAD patients were successfully divided into two distinct subtypes (C1 and C2). C1 subtype is characterized by a poor prognosis, activation of SLP pathways, high expression of SLP genes, disordered carcinogenic pathways, and immune microenvironment. Based on the clusters of SLP, we developed and validated a novel prognostic model, consisting of ANO1, C2CD4A, EEF1A2, GRP, HEYL, IGF1, LAMA2, LSAMP, RBP1, and TCEAL2, to quantitatively evaluate the clinical outcomes of COAD. The Kaplain-Meier survival curves and ROC curves highlighted the accuracy of our SLP model in both internal and external cohorts. Compared to normal colon tissues, expression of C2CD4A was detected to be significantly higher in COAD; whereas, expression levels of EEF1A2, IGF1, and TCEAL2 were detected to be significantly lower in COAD. Overall, our research emphasized the pathogenic role of SLP in COAD and found that targeting SLP might help improve the clinical outcomes of COAD. The risk model based on SLP metabolism provided a new horizon for prognosis assessment and customized patient intervention.

Alteration of glycosphingolipid metabolism by ozone is associated with exacerbation of allergic asthma characteristics in mice.

Asthma is a common chronic respiratory disease exacerbated by multiple environmental factors. Acute ozone exposure has previously been implicated in airway inflammation, airway hyperreactivity, and other characteristics of asthma, which may be attributable to altered sphingolipid metabolism. This study tested the hypothesis that acute ozone exposure alters sphingolipid metabolism within the lung, which contributes to exacerbations in characteristics of asthma in allergen-sensitized mice. Adult male and female BALB/c mice were sensitized intranasally to house dust mite (HDM) allergen on days 1, 3, and 5 and challenged on days 12-14. Mice were exposed to ozone following each HDM challenge for 6 h/day. Bronchoalveolar lavage, lung lobes, and microdissected lung airways were collected for metabolomics analysis (N = 8/sex/group). Another subset of mice underwent methacholine challenge using a forced oscillation technique to measure airway resistance (N = 6/sex/group). Combined HDM and ozone exposure in male mice synergistically increased airway hyperreactivity that was not observed in females and was accompanied by increased airway inflammation and eosinophilia relative to control mice. Importantly, glycosphingolipids were significantly increased following combined HDM and ozone exposure relative to controls in both male and female airways, which was also associated with both airway resistance and eosinophilia. However, 15 glycosphingolipid species were increased in females compared with only 6 in males, which was concomitant with significant associations between glycosphingolipids and airway resistance that ranged from R2 = 0.33-0.51 for females and R2 = 0.20-0.34 in male mice. These observed sex differences demonstrate that glycosphingolipids potentially serve to mitigate exacerbations in characteristics of allergic asthma.

Clinical relevance of CERK and SPHK1 in breast cancer and their association with metastasis and drug resistance

Despite numerous reports on the altered sphingolipids metabolism in human cancers, their clinical significance in breast cancer remains obscure. Previously, we identified the high levels of sphingolipids, ceramide phosphates and sphingosine phosphates, and the genes involved in their synthesis, CERK and SPHK1, in breast cancer patients. The present study aimed to determine the correlations of CERK and SPHK1 with clinical outcomes as well as metastasis and drug resistance markers. Both local and TCGA cohorts were analysed. High-confidence regulatory interaction network was constructed to find association of target genes with metastasis and drug resistance. Furthermore, correlations of CERK and SPHK1 with selected metastasis and drug resistance markers were validated in both cohorts. Overexpression of CERK and SPHK1 was associated with nodal metastasis, late tumor stage and high proliferation potency. In addition, increased CERK expression was also indicative of poor patient survival. Computational network analysis revealed the association of CERK and SPHK1 with known metastasis markers MMP-2 and MMP-9 and drug resistance markers ABCC1 and ABCG2. Correlation analysis confirmed the associations of target genes with these markers in both local as well as TCGA cohort. The above findings suggest clinical utility of CERK and SPHK1 as potential biomarkers in breast cancer patients and thus could provide novel leads in the development of therapeutics.

Metabolic indicators of an altered sphingolipid metabolism are biomarkers for non-alcoholic fatty liver disease

Background and Aims : Non-alcoholic fatty liver disease (NAFLD) is the most frequent cause of chronic liver disease in the western world. Steatosis can be accompanied by inflammation and cell damage (non-alcoholic steatohepatitis, NASH) as well as fibrosis. Sphingolipids and in particular an atypical class of 1-deoxysphingolipids have been associated with NAFLD. Increased 1-deoxySL formation is typically related to an altered amino acid homeostasis. We therefore analyzed the metabolic background that is associated with the increased formation of these lipids in NAFLD.

Ceramide and Sphingosine-1-Phosphate in Neurodegenerative Disorders and Their Potential Involvement in Therapy.

Neurodegenerative disorders (ND) are progressive diseases of the nervous system, often without resolutive therapy. They are characterized by a progressive impairment and loss of specific brain regions and neuronal populations. Cellular and animal model studies have identified several molecular mechanisms that play an important role in the pathogenesis of ND. Among them are alterations of lipids, in particular sphingolipids, that play a crucial role in neurodegeneration. Overall, during ND, ceramide-dependent pro-apoptotic signalling is promoted, whereas levels of the neuroprotective spingosine-1-phosphate are reduced. Moreover, ND are characterized by alterations of the metabolism of complex sphingolipids. The finding that altered sphingolipid metabolism has a role in ND suggests that its modulation might provide a useful strategy to identify targets for possible therapies. In this review, based on the current literature, we will discuss how bioactive sphingolipids (spingosine-1-phosphate and ceramide) are involved in some ND (Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis) and their possible involvement in therapies.

The Role of Obesity, Inflammation and Sphingolipids in the Development of an Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is a local dilatation of the vessel equal to or exceeding 3 cm. It is a disease with a long preclinical period commonly without any symptoms in its initial stage. Undiagnosed for years, aneurysm often leads to death due to vessel rupture. The basis of AAA pathogenesis is inflammation, which is often associated with the excess of adipose tissue, especially perivascular adipose tissue, which synthesizes adipocytokines that exert a significant influence on the formation of aneurysms. Pro-inflammatory cytokines such as resistin, leptin, and TNFα have been shown to induce changes leading to the formation of aneurysms, while adiponectin is the only known compound that is secreted by adipose tissue and limits the development of aneurysms. However, in obesity, adiponectin levels decline. Moreover, inflammation is associated with an increase in the amount of macrophages infiltrating adipose tissue, which are the source of matrix metalloproteinases (MMP) involved in the degradation of the extracellular matrix, which are an important factor in the formation of aneurysms. In addition, an excess of body fat is associated with altered sphingolipid metabolism. It has been shown that among sphingolipids, there are compounds that play an opposite role in the cell: ceramide is a pro-apoptotic compound that mediates the development of inflammation, while sphingosine-1-phosphate exerts pro-proliferative and anti-inflammatory effects. It has been shown that the increase in the level of ceramide is associated with a decrease in the concentration of adiponectin, an increase in the concentration of TNFα, MMP-9 and reactive oxygen species (which contribute to the apoptosis of vascular smooth muscle cell). The available data indicate a potential relationship between obesity, inflammation and disturbed sphingolipid metabolism with the formation of aneurysms; therefore, the aim of this study was to systematize the current knowledge on the role of these factors in the pathogenesis of abdominal aortic aneurysm.

Virtual Screening Approach to Repurpose FDA‐approved Drugs for Targeting Acid Ceramidase‐1 in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer‐related deaths in the US. Despite extensive research and advances in molecular pathogenesis, PDAC remains a major problem in the US. It is also projected to be the second leading cause of cancer‐related deaths in the US by 2030. PDAC is an aggressive disease and characterized by poor prognosis, rapid progression, drug resistance and recurrence. The current chemotherapy treatment only improved survival to <10%, hence there is a need to identify a novel target for drug discovery.Recently, altered sphingolipid metabolism was identified in PDAC patients. Sphingolipids are critical constituents of the cell membrane that regulate several signaling pathways related to cell survival. The major sphingolipid metabolites are ceramide and sphingosine which are interconvertible, while ceramide is pro‐apoptotic and sphingosine is anti‐apoptotic. Ceramidases are a heterogeneous enzyme family that convert ceramides into sphingosine and fatty acids. The five human ceramidases are identified are the acid ceramidase (ASAH1); neutral ceramidase (ASAH2); alkaline ceramidase 1 (ACER1); alkaline ceramidase 2 (ACER2); and alkaline ceramidase 3 (ACER3). To understand the role of ceramides in PDAC, we first interrogated the cancer genome atlas database and found that all ceramides are expressed in lower levels in PDAC except ASAH1 which is uniquely overexpressed in PDAC patients compared to normal individuals. Further, immunocytochemistry of PDAC patient tissue microarray showed higher expression of ASAH1 in PDAC tumors compared to normal pancreas. Western blot analysis also showed overexpression of ASAH1 protein in PDAC cell lines. Studies indicate that ASAH1 plays an important role in PDAC progression. Our data indicate that ASAH1 is a potential target for the treatment of PDAC. For faster translation of this target to the clinic, we used virtual screening and molecular docking approach to identify FDA‐approved drugs for targeting ASAH1. We have downloaded the 3D‐protein structure of ASAH1 from the protein data bank and currently screening a library of small molecular drugs against ASAH1 protein using multiple software programs such as Autodock vina and PyRx. We will select the top 10 drug candidates from virtual screening based on their binding energies and the ability to form hydrogen bonds with key amino acids. We will confirm these findings using a cellular thermal shift assay to analyze the binding of these drugs to ASAH1 in PDAC cell lines and test their anticancer activities in cell culture system and orthotopic PDAC mouse model.

Cytotoxic mechanisms of doxorubicin at clinically relevant concentrations in breast cancer cells.

Doxorubicin (DOX) is a chemotherapeutic agent frequently used for the treatment of a variety of tumor types, such as breast cancer. Despite the long history of DOX, the mechanistic details of its cytotoxic action remain controversial. Rather than one key mechanism of cytotoxic action, DOX is characterized by multiple mechanisms, such as (1) DNA intercalation and adduct formation, (2) topoisomerase II (TopII) poisoning, (3) the generation of free radicals and oxidative stress, and (4) membrane damage through altered sphingolipid metabolism. Many past reviews of DOX cytotoxicity are based on supraclinical concentrations, and several have addressed the concentration dependence of these mechanisms. In addition, most reviews lack a focus on the time dependence of these processes. We aim to update the concentration and time-dependent trends of DOX mechanisms at representative clinical concentrations. Furthermore, attention is placed on DOX behavior in breast cancer cells due to the frequent use of DOX to treat this disease. This review provides insight into the mechanistic pathway(s) of DOX at levels found within patients and establishes the magnitude of effect for each mechanism.

AKT1/FOXP3 axis-mediated expression of CerS6 promotes p53 mutant pancreatic tumorigenesis.

Ceramide synthases (CerSs) catalyze the formation of ceramides from sphingoid bases and acyl-CoA substrates. Increasing evidence suggests that cancer cells generally exhibit altered sphingolipid metabolism in the tumorigenesis of multiple cancers. However, there is no evidence that CerSs are associated with pancreatic ductal carcinoma (PDAC). In the present study, we examined CerS expression in clinical tissue and conducted data mining to investigate the clinical significance of CerSs in the TCGA-PAAD database. We found that high CerS6 expression positively correlated with progression and predicted worse prognosis in PDAC patients, establishing CerS6 as a potential biomarker for PDAC. Furthermore, CerS6 promoted cell proliferation, colony formation and invasion by producing C16-ceramide and was required for tumor formation. Mechanistically, AKT1 interacted with and phosphorylated FOXP3 at S418, which decreased the binding of FOXP3 to the CERS6 promoter and in turn induced CerS6 expression by reconstituting an activated state on the CERS6 promoter. The AKT1/FOXP3 axis mediated the CerS6 expression and promoted p53 mutant pancreatic tumorigenesis by producing excessive C16-ceramide, which induced the accumulation of mutant p53. Thus, our study explores the relationship between PI3K/AKT signaling and sphingolipid metabolism, revealing an oncogenic role for CerS6, which may represent a potential target for PDAC treatment.

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

Abstract 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.

Drug triggered pruritus, rash, papules, and blisters \u2013 is AGEP a clash of an altered sphingolipid-metabolism and lysosomotropism of drugs accumulating in the skin?

Rash, photosensitivity, erythema multiforme, and the acute generalized exanthematous pustulosis (AGEP) are relatively uncommon adverse reactions of drugs. To date, the etiology is not well understood and individual susceptibility still remains unknown. Amiodarone, chlorpromazine, amitriptyline, and trimipramine are classified lysosomotropic as well as photosensitizing, however, they fail to trigger rash and pruritic papules in all individuals. Lysosomotropism is a common charcteristic of various drugs, but independent of individuals. There is evidence that the individual ability to respond to external oxidative stress is crosslinked with the elongation of long-chain fatty acids to very long-chain fatty acids by ELOVLs. ELOVL6 and ELOVL7 are sensitive to ROS induced depletion of cellular NADPH and insufficient regeneration via the pentose phosphate pathway and mitochondrial fatty acid oxidation. Deficiency of NADPH in presence of lysosomotropic drugs promotes the synthesis of C16-ceramide in lysosomes and may contribute to emerging pruritic papules of AGEP. However, independently from a lysosomomotropic drug, severe depletion of ATP and NAD(P)H, e.g., by UV radiation or a potent photosensitizer can trigger likewise the collapse of the lysosomal transmembrane proton gradient resulting in lysosomal C16-ceramide synthesis and pruritic papules. This kind of papules are equally present in polymorphous light eruption (PMLE/PLE) and acne aestivalis (Mallorca acne). The suggested model of a compartmentalized ceramide metabolism provides a more sophisticated explanation of cutaneous drug adverse effects and the individual sensitivity to UV radiation. Parameters such as pKa and ClogP of the triggering drug, cutaneous fatty acid profile, and ceramide profile enables new concepts in risk assessment and scoring of AGEP as well as prophylaxis outcome.