Dividing cells regulate their lipid composition and localization.

Although massive membrane rearrangements occur during cell division, little is known about specific roles that lipids might play in this process. We report that the lipidome changes with the cell cycle. LC-MS-based lipid profiling shows that 11 lipids with specific chemical structures accumulate in dividing cells. Using AFM, we demonstrate differences in the mechanical properties of live dividing cells and their isolated lipids relative to nondividing cells. In parallel, systematic RNAi knockdown of lipid biosynthetic enzymes identified enzymes required for division, which highly correlated with lipids accumulated in dividing cells. We show that cells specifically regulate the localization of lipids to midbodies, membrane-based structures where cleavage occurs. We conclude that cells actively regulate and modulate their lipid composition and localization during division, with both signaling and structural roles likely. This work has broader implications for the active and sustained participation of lipids in basic biology.

Development of a Liquid Chromatography-Mass Spectrometry-Based In Vitro Assay to Assess Changes in Steroid Hormones Due to Exposure to Per- and Polyfluoroalkyl Substances.

Per- and poly-fluorinated substances (PFASs) are organic pollutants that have been linked to numerous health effects, including diabetes, cancers, and dysregulation of the endocrine system. This study aims to develop a liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay to measure changes in 17 hormones in H295R cell line (a steroid producing adrenocortical cells) upon exposure to PFASs. Due to the challenges in the analysis of steroid hormones using electrospray ionization MS, a chemical derivatization method was employed to achieve 0.07-2 µg/L detection limits in LC-MS/MS. Furthermore, a 10-fold concentration factor through solid-phase extraction (SPE) allows for consistent sub-parts per billion detections. Optimization of the derivatization conditions showed doubly-derivatized products in some hormone analytes, including progesterone, corticosterone, and cortisol, and gave improved ionization efficiency up to 20-fold higher signal than the singly-derivatized product. The use of SPE for sample cleanup to analyze hormones from cellular media using weak anion exchange sorbent yielded 80-100% recovery for the 17 targeted hormones. The method was validated by exposing H295R cells to two known endocrine disruptors, forskolin and prochloraz, which showed expected changes in hormones. An initial exposure of H295R cells with various PFAS standards and their mixtures at 1 µM showed significant increases in progestogens with some PFAS treatments, which include PFBS, PFHxA, PFOS, PFDA, and PFDS. In addition, modest changes in hormone levels were observed in cells treated with other sulfonated or carboxylated headgroup PFASs. This sensitive LC-MS/MS method for hormone analysis in H295R cells will allow for the investigations of the alterations in the hormone production caused by exposure to various environmental insults in cell-based assays and other in vitro models.

Cellular Interactions and Fatty Acid Transporter CD36-Mediated Uptake of Per- and Polyfluorinated Alkyl Substances (PFAS).

Per- and polyfluorinated alkyl substances (PFAS) are a class of widely used compounds in an array of commercial and industrial applications. Due to their extensive use and chemical stability, PFAS persist in the environment and bioaccumulate in humans and wildlife. PFAS exposure have been linked to several negative health effects, including the formation of various cancers, disruption of the endocrine system, and obesity. However, there is a major gap in understanding how structural differences in PFAS impact their interactions within a biological system. In this study, we examined the toxicity of PFAS with differences in chain length, head group, and degree of fluorination in human retinal epithelial cells. We focused on fluorotelomeric and fully fluorinated sulfonates and carboxylates and measured their uptake. Our results showed that sulfonates are taken up at higher levels as compared to their fluorotelomer and carboxylate counterparts. Furthermore, PFAS with 8 and 10 carbons (C8 and C10) are taken up at a higher level compared to those with six carbons (C6). We also investigated the role of the fatty acid transporter CD36 in PFAS uptake and found that increased CD36 levels result in higher levels of PFAS in cells. Overall, our results suggest that the head group structure of PFAS impacts toxicity, with sulfonates inducing a higher decrease in cell viability (∼50%) than carboxylates. Our results also link the activity of CD36 to PFAS uptake into cells.

Emerging Roles of Ceramides in Breast Cancer Biology and Therapy.

One of the classic hallmarks of cancer is the imbalance between elevated cell proliferation and reduced cell death. Ceramide, a bioactive sphingolipid that can regulate this balance, has long been implicated in cancer. While the effects of ceramide on cell death and therapeutic efficacy are well established, emerging evidence indicates that ceramide turnover to downstream sphingolipids, such as sphingomyelin, hexosylceramides, sphingosine-1-phosphate, and ceramide-1-phosphate, is equally important in driving pro-tumorigenic phenotypes, such as proliferation, survival, migration, stemness, and therapy resistance. The complex and dynamic sphingolipid network has been extensively studied in several cancers, including breast cancer, to find key sphingolipidomic alterations that can be exploited to develop new therapeutic strategies to improve patient outcomes. Here, we review how the current literature shapes our understanding of how ceramide synthesis and turnover are altered in breast cancer and how these changes offer potential strategies to improve breast cancer therapy.

A Single-Organelle Optical Omics Platform for Cell Science and Biomarker Discovery.

Research in fundamental cell biology and pathology could be revolutionized by developing the capacity for quantitative molecular analysis of subcellular structures. To that end, we introduce the Ramanomics platform, based on confocal Raman microspectrometry coupled to a biomolecular component analysis algorithm, which together enable us to molecularly profile single organelles in a live-cell environment. This emerging omics approach categorizes the entire molecular makeup of a sample into about a dozen of general classes and subclasses of biomolecules and quantifies their amounts in submicrometer volumes. A major contribution of our study is an attempt to bridge Raman spectrometry with big-data analysis in order to identify complex patterns of biomolecules in a single cellular organelle and leverage discovery of disease biomarkers. Our data reveal significant variations in organellar composition between different cell lines. We also demonstrate the merits of Ramanomics for identifying diseased cells by using prostate cancer as an example. We report large-scale molecular transformations in the mitochondria, Golgi apparatus, and endoplasmic reticulum that accompany the development of prostate cancer. Based on these findings, we propose that Ramanomics datasets in distinct organelles constitute signatures of cellular metabolism in healthy and diseased states.

Removal of Serum Lipids and Lipid-Derived Metabolites to Investigate Breast Cancer Cell Biology.

The use of cultured cells has been instrumental in studying biochemical, molecular, and cellular processes. The composition of serum that cells are maintained in can have a profound impact on important cellular checkpoints. Cell growth and apoptosis are analyzed in an estrogen receptor positive breast cancer cell line in the presence of serum that have been treated to remove steroids or lipids, as well-described in the literature. It is shown that maintaining cells in the presence of charcoal-dextran-treated serum causes reduced growth rate, which can be reversed by the addition of estradiol. Silica-treated-serum also slows down cell growth and induces apoptosis. In order to investigate the role of lipids in these phenotypes, the levels of a wide range of lipids in different sera are investigated. It is shown that silica-treatment significantly depletes phosphatidylcholines and cholesterol. It is also shown that lipogenesis is stimulated when cells are cultured with silica-treated-serum and this is reversed by the addition of exogenous lipids, which also restores growth rate and apoptosis. The results show that cultured cells are sensitive to different serum, most likely due to the differences in levels of structural and signaling metabolites present in their growth environment.

A Protective Role for Triacylglycerols during Apoptosis.

Triacylglycerols (TAGs) are one of the major constituents of the glycerolipid family. Their main role in cells is to store excess fatty acids, and they are mostly found within lipid droplets. TAGs contain acyl chains that vary in length and degree of unsaturation, resulting in hundreds of chemically distinct species. We have previously reported that TAGs containing polyunsaturated fatty acyl chains (PUFA-TAGs) accumulate via activation of diacylglycerol acyltransferases during apoptosis. In this work, we show that accumulation of PUFA-TAGs is a general phenomenon during this process. We further show that the accumulated PUFA-TAGs are stored in lipid droplets. Because membrane-residing PUFA phospholipids can undergo oxidation and form reactive species under increased levels of oxidative stress, we hypothesized that incorporation of PUFAs into PUFA-TAGs and their localization within lipid droplets during apoptosis limit the toxicity during this process. Indeed, exogenous delivery of a polyunsaturated fatty acid resulted in a profound accumulation of PUFA phospholipids and rendered cells more sensitive to oxidative stress, causing reduced viability. Overall, our results support the concept that activation of TAG biosynthesis protects cells from lipid peroxide-induced membrane damage under increased levels of oxidative stress during apoptosis. As such, targeting triacylglycerol biosynthesis in cancer cells might represent a new approach to promoting cell death during apoptosis.

Noncanonical Roles of Lipids in Different Cellular Fates.

Lipids are a diverse class of biomolecules. The biosynthesis and transport of these molecules are controlled by a considerable number of proteins, which facilitate spatiotemporal regulation of lipids during different fundamental cellular processes. Although lipids are traditionally considered as molecules for energy storage and as structural components of membranes, they are being increasingly recognized for their signaling roles. There is a growing appreciation of lipids' chemical diversity, which approaches that of proteins. In this Perspective, we discuss recent studies that suggest novel functions for distinct lipid species during different cellular processes. In particular, we discuss findings from our laboratory that illuminate the involvement of ceramides, polyunsaturated triacylglycerols, and very long chain fatty acids in different cellular fates. We also highlight recent innovative methods that have enabled the recognition of previously unknown lipid classes and/or roles of these molecules in different biological processes. We envision that advances in lipid identification, visualization, and perturbation will pave the way for broader investigations into this fascinating and influential class of biomolecules.

CBR1 rs9024 genotype status impacts the bioactivation of loxoprofen in human liver.

Loxoprofen is an anti-inflammatory drug that requires bioactivation into the trans-OH metabolite to exert pharmacological activity. Evidence suggests that carbonyl reductase 1 (CBR1) is important during the bioactivation of loxoprofen. This study examined the impact of the functional single nucleotide polymorphism CBR1 rs9024 on the bioactivation of loxoprofen in a collection of human liver samples. The synthesis ratios of trans-OH loxoprofen/cis-OH loxoprofen were 33% higher in liver cytosols from donors homozygous for the CBR1 rs9024 G allele in comparison with the ratios in samples from donors with heterozygous GA genotypes. Complementary studies examined the impact of CBR1 rs9024 on the bioactivation of loxoprofen in lymphoblastoid cell lines. CBR1 rs9024 genotype status impacts the synthesis of the bioactive trans-OH metabolite of loxoprofen in human liver.

Glycosphingolipids on Human Myeloid Cells Stabilize E-Selectin-Dependent Rolling in the Multistep Leukocyte Adhesion Cascade.

OBJECTIVE: Recent studies suggest that the E-selectin ligands expressed on human leukocytes may differ from those in other species, particularly mice. To elaborate on this, we evaluated the impact of glycosphingolipids expressed on human myeloid cells in regulating E-selectin-mediated cell adhesion. APPROACH AND RESULTS: A series of modified human cell lines and primary neutrophils were created by targeting UDP-Glucose Ceramide Glucosyltransferase using either lentivirus-delivered shRNA or CRISPR-Cas9-based genome editing. Enzymology and mass spectrometry confirm that the modified cells had reduced or abolished glucosylceramide biosynthesis. Glycomics profiling showed that UDP-Glucose Ceramide Glucosyltransferase disruption also increased prevalence of bisecting N-glycans and reduced overall sialoglycan expression on leukocyte N- and O-glycans. Microfluidics-based flow chamber studies demonstrated that both the UDP-Glucose Ceramide Glucosyltransferase knockouts and knockdowns display ≈60% reduction in leukocyte rolling and firm adhesion on E-selectin bearing stimulated endothelial cells, without altering cell adhesion to P-selectin. Consistent with the concept that the glycosphingolipids support slow rolling and the transition to firm arrest, inhibiting UDP-Glucose Ceramide Glucosyltransferase activity resulted in frequent leukocyte detachment events, skipping motion, and reduced diapedesis across the endothelium. Cells bearing truncated O- and N-glycans also sustained cell rolling on E-selectin, although their ability to be recruited from free fluid flow was diminished. CONCLUSIONS: Glycosphingolipids likely contribute to human myeloid cell adhesion to E-selectin under fluid shear, particularly the transition of rolling cells to firm arrest.

Rapid Light-Triggered Drug Release in Liposomes Containing Small Amounts of Unsaturated and Porphyrin-Phospholipids.

Prompt membrane permeabilization is a requisite for liposomes designed for local stimuli-induced intravascular release of therapeutic payloads. Incorporation of a small amount (i.e., 5 molar percent) of an unsaturated phospholipid, such as dioleoylphosphatidylcholine (DOPC), accelerates near infrared (NIR) light-triggered doxorubicin release in porphyrin-phospholipid (PoP) liposomes by an order of magnitude. In physiological conditions in vitro, the loaded drug can be released in a minute under NIR irradiation, while liposomes maintain serum stability otherwise. This enables rapid laser-induced drug release using remarkably low amounts of PoP (i.e., 0.3 molar percent). Light-triggered drug release occurs concomitantly with DOPC and cholesterol oxidation, as detected by mass spectrometry. In the presence of an oxygen scavenger or an antioxidant, light-triggered drug release is inhibited, suggesting that the mechanism is related to singlet oxygen mediated oxidization of unsaturated lipids. Despite the irreversible modification of lipid composition, DOPC-containing PoP liposome permeabilization is transient. Human pancreatic xenograft growth in mice is significantly delayed with a single chemophototherapy treatment following intravenous administration of 6 mg kg(-1) doxorubicin, loaded in liposomes containing small amounts of DOPC and PoP.

Application of metabolite profiling tools and time-of-flight mass spectrometry in the identification of transformation products of iopromide and iopamidol during advanced oxidation.

The efficiency of wastewater treatment systems in removing pharmaceuticals is often assessed on the basis of the decrease in the concentration of the parent compound. However, what is perceived as "removal" during treatment may not necessarily mean mineralization of the pharmaceutical compound but simply conversion into different transformation products (TPs). Using liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer (LC-QToF-MS), we demonstrated conversion of iopromide in wastewater to at least 14 TPs after an advanced oxidation process (AOP) using UV (fluence = 1500 mJ/cm(2)) and H2O2 (10 mg/L). Due to the complexity of the wastewater matrix, the initial experiments were performed using a high concentration (10 mg/L) of iopromide in order to facilitate the identification of TPs. Despite the high concentration of iopromide used, cursory inspection of UV and mass spectra only revealed four TPs in the chromatograms of the post-AOP samples. However, the use of METLIN database and statistics-based profiling tools commonly used in metabolomics proved effective in discriminating between background signals and TPs derived from iopromide. High-resolution mass data allowed one to predict molecular formulas of putative TPs with errors below 5 ppm relative to the observed m/z. Tandem mass spectrometry (MS/MS) data and isotope pattern comparisons provided necessary information that allowed one to elucidate the structure of iopromide TPs. The presence of the proposed iopromide TPs was determined in unspiked wastewater from a municipal wastewater treatment plant, but no iopromide and TPs were detected. Using analogous structural modifications and oxidation that results from the AOP treatment of iopromide, the potential TPs of iopamidol (a structurally similar compound to iopromide) were predicted. The same mass fragmentation pattern observed in iopromide TPs was applied to the predicted iopamidol TPs. LC-QToF-MS revealed the presence of two iopamidol TPs in unspiked AOP-treated wastewater.

Acylation of MLKL Impacts Its Function in Necroptosis.

Mixed lineage kinase domain-like (MLKL) is a key signaling protein of necroptosis. Upon activation by phosphorylation, MLKL translocates to the plasma membrane and induces membrane permeabilization, which contributes to the necroptosis-associated inflammation. Membrane binding of MLKL is initially initiated by electrostatic interactions between the protein and membrane phospholipids. We previously showed that MLKL and its phosphorylated form (pMLKL) are S-acylated during necroptosis. Here, we characterize the acylation sites of MLKL and identify multiple cysteines that can undergo acylation with an interesting promiscuity at play. Our results show that MLKL and pMLKL undergo acylation at a single cysteine, with C184, C269, and C286 as possible acylation sites. Using all-atom molecular dynamic simulations, we identify differences that the acylation of MLKL causes at the protein and membrane levels. Through investigations of the S-palmitoyltransferases that might acylate pMLKL in necroptosis, we showed that zDHHC21 activity has the strongest effect on pMLKL acylation, inactivation of which profoundly reduced the pMLKL levels in cells and improved membrane integrity. These results suggest that blocking the acylation of pMLKL destabilizes the protein at the membrane interface and causes its degradation, ameliorating the necroptotic activity. At a broader level, our findings shed light on the effect of S-acylation on MLKL functioning in necroptosis and MLKL-membrane interactions mediated by its acylation.

Building Chemical Intuition about Physicochemical Properties of C8-Per-/Polyfluoroalkyl Carboxylic Acids through Computational Means.

We have predicted acid dissociation constants (pK a), octanol-water partition coefficients (K OW), and DMPC lipid membrane-water partition coefficients (K lipid-w) of 150 different eight-carbon-containing poly-/perfluoroalkyl carboxylic acids (C8-PFCAs) utilizing the COnductor-like Screening MOdel for Realistic Solvents (COSMO-RS) theory. Different trends associated with functionalization, degree of fluorination, degree of saturation, degree of chlorination, and branching are discussed on the basis of the predicted values for the partition coefficients. In general, functionalization closest to the carboxylic headgroup had the greatest impact on the value of the predicted physicochemical properties.

Perfluorooctane Sulfonate (PFOS) Negatively Impacts Prey Capture Capabilities in Larval Zebrafish.

Per- and polyfluoroalkyl substances (PFAS) are widely used in many industrial and domestic applications, which has resulted in unintentional human exposures and bioaccumulation in blood and other organs. Perfluorooctane sulfonate (PFOS) is among the most prevalent PFAS in the environment and has been postulated to affect brain functions in exposed organisms. However, the impacts of PFOS in early neural development have not been well described. We used zebrafish larvae to assess the effects of PFOS on two fundamental complex behaviors, prey capture and learning. Zebrafish exposed to PFOS concentrations ranging from 2 to 20 µM for differing 48-h periods were viable through early larval stages. In addition, PFOS uptake was unaffected by the presence of a chorion. We employed two different experimental paradigms; first we assessed the impacts of increasing organismal PFOS bioaccumulation on prey capture and learning, and second, we probed stage-specific sensitivity to PFOS by exposing zebrafish at different developmental stages (0-2 vs. 3-5 days post fertilization). Following both assays we measured the amount of PFOS present in each larva and found that PFOS levels varied in larvae from different groups within each experimental paradigm. Significant negative correlations were observed between larval PFOS accumulation and percentage of captured prey, whereas nonsignificant negative correlations were observed between PFOS accumulation and experienced-induced prey capture learning. These findings suggest that PFOS accumulation negatively affects larval zebrafish's ability to perform complicated multisensory behaviors and highlights the potential risks of PFOS exposure to animals in the wild, with implications for human health. Environ Toxicol Chem 2024;43:847-855. © 2023 SETAC.

Mitochondrial Calcium Signaling Regulates Branched-Chain Amino Acid Catabolism in Fibrolamellar Carcinoma.

Metabolic adaptations in response to changes in energy supply and demand are essential for survival. The mitochondrial calcium uniporter coordinates metabolic homeostasis by regulating TCA cycle activation, mitochondrial fatty acid oxidation and cellular calcium signaling. However, a comprehensive analysis of uniporter-regulated mitochondrial metabolic pathways has remained unexplored. Here, we investigate the metabolic consequences of uniporter loss- and gain-of-function, and identify a key transcriptional regulator that mediates these effects. Using gene expression profiling and proteomic, we find that loss of uniporter function increases the expression of proteins in the branched-chain amino acid (BCAA) catabolism pathway. Activity is further augmented through phosphorylation of the enzyme that catalyzes this pathway's committed step. Conversely, in the liver cancer fibrolamellar carcinoma (FLC)-which we demonstrate to have high mitochondrial calcium levels- expression of BCAA catabolism enzymes is suppressed. We also observe uniporter-dependent suppression of the transcription factor KLF15, a master regulator of liver metabolic gene expression, including those involved in BCAA catabolism. Notably, loss of uniporter activity upregulates KLF15, along with its transcriptional target ornithine transcarbamylase (OTC), a component of the urea cycle, suggesting that uniporter hyperactivation may contribute to the hyperammonemia observed in FLC patients. Collectively, we establish that FLC has increased mitochondrial calcium levels, and identify an important role for mitochondrial calcium signaling in metabolic adaptation through the transcriptional regulation of metabolism.

Cellular Lipidome Changes during Retinoic Acid (RA)-Induced Differentiation in SH-SY5Y Cells: A Comprehensive In Vitro Model for Assessing Neurotoxicity of Contaminants.

The SH-SY5Y, neuroblastoma cell line, is a common in vitro model used to study physiological neuronal function and the neuronal response to different stimuli, including exposure to toxic chemicals. These cells can be differentiated to neuron-like cells by administration of various reagents, including retinoic acid or phorbol-12-myristate-13-acetate. Despite their common use, there is an incomplete understanding of the molecular changes that occur during differentiation. Therefore, there is a critical need to fully understand the molecular changes that occur during differentiation to properly study neurotoxicity in response to various environmental exposures. Previous studies have investigated the proteome and transcriptome during differentiation; however, the regulation of the cellular lipidome in this process is unexplored. In this work, we conducted liquid chromatography-mass spectrometry (LC-MS)-based untargeted lipidomics in undifferentiated and differentiated SH-SY5Y cells, induced by retinoic acid. We show that there are global differences between the cellular lipidomes of undifferentiated and differentiated cells. Out of thousands of features detected in positive and negative electrospray ionization modes, 44 species were identified that showed significant differences (p-value ≤0.05, fold change ≥2) in differentiated cells. Identification of these features combined with targeted lipidomics highlighted the accumulation of phospholipids, sterols, and sphingolipids during differentiation while triacylglycerols were depleted. These results provide important insights into lipid-related changes that occur during cellular differentiation of SH-5YSY cells and emphasize the need for the detailed characterization of biochemical differences that occur during differentiation while using this in vitro model for assessing ecological impacts of environmental pollutants.

Omics approaches to better understand the molecular mechanism of necroptosis and their translational implications.

Necroptosis is a type of programed cell death characterized by an inflammatory phenotype due to extensive membrane permeabilization and rupture. Initiation of necroptosis involves activation of tumor necrosis factor receptors by tumor necrosis factor alpha (TNFα) followed by coordinated activities of receptor-interacting protein kinases and mixed lineage kinase-like protein (MLKL). Subsequently, MLKL undergoes phosphorylation and translocates to the plasma membrane, leading to permeabilization. Such permeabilization results in the release of various cytokines and causes extensive inflammatory activity at the organismal level. This inflammatory activity is one of the major differences between apoptosis and necroptosis and links necroptosis to several human pathologies that exhibit inflammation, in addition to the ultimate cell death phenotype. Given the crosstalk between the activation of cell death pathway and inflammatory activity, approaches that provide insights on the regulation of transcripts, proteins and their processing at the global level have substantially improved our understanding of necroptosis and its involvement in different disease states. In this review, we highlight recent omic studies probing the transcriptome, proteome and lipidome which elucidate potential new mechanisms and signaling pathways during necroptosis and the necroptosis-associated inflammatory activity observed in various diseases. We specifically focus on studies investigating the transcriptome and intracellular and released proteome that contribute to inflammatory nature of necroptotic cells. We also highlight different lipids that have been implicated in necroptosis and lipidomic studies identifying lipid players in necroptosis. Finally, we review studies which suggest certain necroptosis-related genes as potential prognosis markers for different cancers and discuss their translational implications.

SREBP activation contributes to fatty acid accumulations in necroptosis.

Necroptosis is a type of programmed cell death. It is characterized by membrane permeabilization and is associated with the release of intracellular components due to compromised membrane integrity which induces a strong inflammatory response. We recently showed that the accumulation of very long chain fatty acids (VLCFAs) contributes to membrane permeabilization during necroptosis. However, the mechanisms that result in the accumulation of these cytotoxic lipids remain unknown. Using comparative transcriptomics and digital PCR validations, we found that several target genes of sterol regulatory element-binding proteins (SREBPs) were upregulated during necroptosis, suggesting that they might be responsible for the accumulation of VLCFA in this process. We demonstrated that activation of SREBPs during necroptosis exacerbates the permeability of the plasma membrane and cell death. Consistent with these observations, targeting sterol regulatory element-binding protein cleavage-activating protein (SCAP), a protein involved in SREBP activation, reversed the accumulation of VLCFAs, and restored cell death and membrane permeabilization during necroptosis. Collectively, our results highlight a role for SREBP in regulating lipid changes during necroptosis and suggest SREBP-mediated lipid remodeling as a potential target for therapeutics to reduce membrane permeabilization during necroptosis.

Ceramide-1-Phosphate Is Involved in Therapy-Induced Senescence.

Sphingolipids are key signaling lipids and their dysregulation has been associated with various cellular processes. We have previously shown significant changes in sphingolipids in therapy-induced senescence, a state of cell cycle arrest as a response to chemotherapy, including the accumulation of ceramides, and provided evidence suggesting that ceramide processing is important for this process. Herein, we conducted a focused small molecule inhibitor screen targeting the sphingolipid pathway, which highlighted a new lipid regulator of therapy-induced senescence. Among the inhibitors tested, the inhibition of ceramide kinase by NVP-231 reduced the levels of senescent cells. Ceramide kinase knockdown exhibited similar effects, strongly supporting the involvement of ceramide kinase during this process. We showed that ceramide-1-phosphate was upregulated in therapy-induced senescence and that NVP-231 reduced ceramide-1-phosphate levels in different cell line models of therapy-induced senescence. Finally, ceramide-1-phosphate addition to NVP-231-treated cells reversed the effects of NVP-231 during senescence. Overall, our results identify a previously unknown lipid player in therapy-induced senescence and highlight a potential targetable enzyme to reduce the levels of therapy-induced senescent cells.