Glioblastoma (GBM) is marked by cellular heterogeneity through microenvironments of a tumor, including metabolic heterogeneity. While altered cellular metabolism in cancer is well-known, how lipid metabolism is altered in different GBM microenvironmental conditions and cancer stem cell (CSC) states within a tumor remains an open question. We developed 3-dimensional GBM organoid models that mimic the transition zone between nutrient-rich cellular tumor and nutrient-poor psuedopalisading/perinecrotic tumor regions and performed spatially defined RNA-sequencing to investigate lipid metabolism. Spatial analysis revealed striking differences in metabolism between diverse cell populations from the same patient, with lipid enrichment in the hypoxic organoid cores and the pseudopalisading regions of patient tumors. This was accompanied by regionally restricted upregulation of lipid droplets and Hypoxia Inducible Lipid Droplet Associated gene expression in organoid cores and in the pseudopalisading regions of clinical GBM tumors. Using targeted lipidomic analysis, we assessed differences in acutely enriched CSC and non-CSCs from patient-derived models to explore the link between stem cell state and lipid metabolism. CSCs have low lipid droplet accumulation compared to non-CSCs in organoids and xenograft tumors, and prospectively sorted lipid-low GBM cells are functionally enriched for stem cell activity. This suggests lipid metabolism may not be simply a product of the microenvironment but also may be a reflection of cellular state. CSCs had decreased levels of major classes of neutral lipids compared to non-CSCs, but had significantly increased polyunsaturated fatty acid production due to increased expression of fatty acid desaturases FADS1 and FADS2. FADS1 and FADS2 expression are both essential to maintain CSC viability and self-renewal. Our data demonstrate that spatially and hierarchically distinct lipid metabolism phenotypes occur clinically in the majority of patients, can be recapitulated in laboratory models, and these altered lipid metabolic pathways may represent therapeutic targets for GBM.

Glioblastoma (GBM) displays marked cellular and metabolic heterogeneity that varies among cellular microenvironments within a tumor. Metabolic targeting has long been advocated as a therapy against many tumors including GBM, but how lipid metabolism is altered to suit different microenvironmental conditions and whether cancer stem cells (CSCs) have altered lipid metabolism are outstanding questions in the field. We interrogated gene expression in separate microenvironments of GBM organoid models that mimic the transition between nutrient-rich and nutrient-poor pseudopalisading/perinecrotic tumor zones using spatial-capture RNA-sequencing. We revealed a striking difference in lipid processing gene expression and total lipid content between diverse cell populations from the same patient, with lipid enrichment in hypoxic organoid cores and also in perinecrotic and pseudopalisading regions of primary patient tumors. This was accompanied by regionally restricted upregulation of hypoxia-inducible lipid droplet-associated (HILPDA) gene expression in organoid cores and pseudopalisading regions of clinical GBM specimens, but not lower-grade brain tumors. CSCs have low lipid droplet accumulation compared to non-CSCs in organoid models and xenograft tumors, and prospectively sorted lipid-low GBM cells are functionally enriched for stem cell activity. Targeted lipidomic analysis of multiple patient-derived models revealed a significant shift in lipid metabolism between GBM CSCs and non-CSCs, suggesting that lipid levels may not be simply a product of the microenvironment but also may be a reflection of cellular state. CSCs had decreased levels of major classes of neutral lipids compared to non-CSCs, but had significantly increased polyunsaturated fatty acid production due to high fatty acid desaturase (FADS1/2) expression which was essential to maintain CSC viability and self-renewal. Our data demonstrate spatially and hierarchically distinct lipid metabolism phenotypes occur clinically in the majority of patients, can be recapitulated in laboratory models, and may represent therapeutic targets for GBM.

This letter describes the on-going SAR efforts to develop PLD1, PLD2 and dual PLD1/2 inhibitors with improved physiochemical and disposition properties as well as securing intellectual property position. Previous PLD inhibitors, based on a triazaspiro[4.5]decanone core proved to be highly selective PLD2 inhibitors, but with low plasma free fraction (rat, human fu < 0.03), high predicted hepatic clearance (rat CLhep > 65 mL/min/kg) and very short half-lives in vivo (t1/2 < 0.15 h). Removal of a nitrogen atom from this core generated a 2,8-diazaspiro[4.5]decanone core, harboring a new chiral center, as well as increased sp3 character. This new core demonstrated enantioselective inhibition of the individual PLD isoforms, enhanced free fraction (rat, human fu < 0.13), engendered moderate predicted hepatic clearance (rat CLhep ∼ 43 mL/min/kg), improved half-lives in vivo (t1/2 > 3 h), and led to the first issued US patent claiming composition of matter for small molecule PLD inhibitors.

Lipid second messengers have essential roles in cellular function and contribute to the molecular mechanisms that underlie inflammation, malignant transformation, invasiveness, neurodegenerative disorders, and infectious and other pathophysiological processes. The phospholipase D (PLD) isoenzymes PLD1 and PLD2 are one of the major sources of signal-activated phosphatidic acid (PtdOH) generation downstream of a variety of cell-surface receptors, including G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and integrins. Recent advances in the development of isoenzyme-selective PLD inhibitors and in molecular genetics have suggested that PLD isoenzymes in mammalian cells and pathogenic organisms may be valuable targets for the treatment of several human diseases. Isoenzyme-selective inhibitors have revealed complex inter-relationships between PtdOH biosynthetic pathways and the role of PtdOH in pathophysiology. PLD enzymes were once thought to be undruggable owing to the ubiquitous nature of PtdOH in cell signalling and concerns that inhibitors would be too toxic for use in humans. However, recent promising discoveries suggest that small-molecule isoenzyme-selective inhibitors may provide novel compounds for a unique approach to the treatment of cancers, neurodegenerative disorders and other afflictions of the central nervous system, and potentially serve as broad-spectrum antiviral and antimicrobial therapeutics.

Determining the impact of lipid electrophile-mediated protein damage that occurs during oxidative stress requires a comprehensive analysis of electrophile targets adducted under pathophysiological conditions. Incorporation of ω-alkynyl linoleic acid into the phospholipids of macrophages prior to activation by Kdo2-lipid A, followed by protein extraction, click chemistry, and streptavidin affinity capture, enabled a systems-level survey of proteins adducted by lipid electrophiles generated endogenously during the inflammatory response. Results revealed a dramatic enrichment for membrane and mitochondrial proteins as targets for adduction. A marked decrease in adduction in the presence of MitoTEMPO demonstrated a primary role for mitochondrial superoxide in electrophile generation and indicated an important role for mitochondria as both a source and target of lipid electrophiles, a finding that has not been revealed by prior studies using exogenously provided electrophiles.

A reliable method for purifying envelope-stripped nuclei from immortalized murine embryonic fibroblasts (iMEFs) was established. Quantitative profiling of the glycerophospholipids (GPLs) in envelope-free iMEF nuclei yields several conclusions. First, we find the endonuclear glycerophospholipidome differs from that of bulk membranes, and phosphatidylcholine (PtdCho) and phosphatidylethanolamine species are the most abundant endonuclear GPLs by mass. By contrast, phosphatidylinositol (PtdIns) represents a minor species. We also find only a slight enrichment of saturated versus unsaturated GPL species in iMEF endonuclear fractions. Moreover, much lower values for GPL mass were measured in the iMEF nuclear matrix than those reported for envelope-stripped IMF-32 nuclei. The collective results indicate that the nuclear matrix in these cells is a GPL-poor environment where GPL occupies only approximately 0.1% of the total nuclear matrix volume. This value suggests GPL accommodation in this compartment can be satisfied by binding to resident proteins. Finally, we find no significant role for the PtdIns/PtdCho-transfer protein, PITPα, in shuttling PtdIns into the iMEF nuclear matrix.

Lenz-Majewski syndrome (LMS) is a rare disease characterized by complex craniofacial, dental, cutaneous, and limb abnormalities combined with intellectual disability. Mutations in thePTDSS1gene coding one of the phosphatidylserine (PS) synthase enzymes, PSS1, were described as causative in LMS patients. Such mutations render PSS1 insensitive to feedback inhibition by PS levels. Here we show that expression of mutant PSS1 enzymes decreased phosphatidylinositol 4-phosphate (PI4P) levels both in the Golgi and the plasma membrane (PM) by activating the Sac1 phosphatase and altered PI4P cycling at the PM. Conversely, inhibitors of PI4KA, the enzyme that makes PI4P in the PM, blocked PS synthesis and reduced PS levels by 50% in normal cells. However, mutant PSS1 enzymes alleviated the PI4P dependence of PS synthesis. Oxysterol-binding protein-related protein 8, which was recently identified as a PI4P-PS exchanger between the ER and PM, showed PI4P-dependent membrane association that was significantly decreased by expression of PSS1 mutant enzymes. Our studies reveal that PS synthesis is tightly coupled to PI4P-dependent PS transport from the ER. Consequently, PSS1 mutations not only affect cellular PS levels and distribution but also lead to a more complex imbalance in lipid homeostasis by disturbing PI4P metabolism.

Historically, lipids have been defined in many ways, but at the most elementary level, they are biologically derived molecules soluble in organic solvents. Lipid species include chemical entities that are among the fundamental building blocks of life. The chemical and structural heterogeneity of lipids contributes to innumerable biochemical and biophysical processes and thereby presents challenges in analysis. Advances in chromatography, mass spectrometry and computational biology have enabled the progression of lipidomics within the larger field of analytical chemistry. Advancements in mass spectrometry-based lipid analysis have contributed to remarkable discoveries in both basic and translational research and any well-conducted study of biological processes involving lipids is now expected to include lipidomic analysis. In this chapter we introduce readers to the basic concepts of identification and measurements of lipid species that constitute the modern field of lipidomics.

Cancer cells reorganize their metabolic pathways to fuel demanding rates of proliferation. Oftentimes, these metabolic phenotypes lie downstream of prominent oncogenes. The lipid signaling molecule phosphatidic acid (PtdOH), which is produced by the hydrolytic enzyme phospholipase D (PLD), has been identified as a critical regulatory molecule for oncogenic signaling in many cancers. In an effort to identify novel regulatory mechanisms for PtdOH, we screened various cancer cell lines, assessing whether treatment of cancer models with PLD inhibitors altered production of intracellular metabolites. Preliminary findings lead us to focus on how deoxyribonucleoside triphosphates (dNTPs) are altered upon PLD inhibitor treatment in gliomas. Using a combination of proteomics and small molecule intracellular metabolomics, we show herein that PtdOH acutely regulates the production of these pyrimidine metabolites through activation of CAD via mTOR signaling pathways independently of Akt. These changes are responsible for decreases in dNTP production after PLD inhibitor treatment. Our data identify a novel regulatory role for PLD activity in specific cancer types.

Phospholipase D is a ubiquitous protein in eukaryotes that hydrolyzes phospholipids to generate the signaling lipid phosphatidic acid (PtdOH). PldA, a Pseudomonas aeruginosa PLD, is a secreted protein that targets bacterial and eukaryotic cells. Here we have characterized the in vitro factors that modulate enzymatic activity of PldA, including divalent cations and phosphoinositides. We have identified several similarities between the eukaryotic-like PldA and the human PLD isoforms, as well as several properties in which the enzymes diverge. Notable differences include the substrate preference and transphosphatidylation efficiency for PldA. These findings offer new insights into potential regulatory mechanisms of PldA and its role in pathogenesis.

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and its ester analog, 2-arachidonoylglycerol (2-AG), to prostaglandins (PGs) and prostaglandin glyceryl esters (PG-Gs), respectively. Although the efficiency of oxygenation of these substrates by COX-2 in vitro is similar, cellular biosynthesis of PGs far exceeds that of PG-Gs. Evidence that the COX enzymes are functional heterodimers suggests that competitive interaction of AA and 2-AG at the allosteric site of COX-2 might result in differential regulation of the oxygenation of the two substrates when both are present. Modulation of AA levels in RAW264.7 macrophages uncovered an inverse correlation between cellular AA levels and PG-G biosynthesis. In vitro kinetic analysis using purified protein demonstrated that the inhibition of 2-AG oxygenation by high concentrations of AA far exceeded the inhibition of AA oxygenation by high concentrations of 2-AG. An unbiased systems-based mechanistic model of the kinetic data revealed that binding of AA or 2-AG at the allosteric site of COX-2 results in a decreased catalytic efficiency of the enzyme toward 2-AG, whereas 2-AG binding at the allosteric site increases COX-2's efficiency toward AA. The results suggest that substrates interact with COX-2 via multiple potential complexes involving binding to both the catalytic and allosteric sites. Competition between AA and 2-AG for these sites, combined with differential allosteric modulation, gives rise to a complex interplay between the substrates, leading to preferential oxygenation of AA.

Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early steps of virus replication. Low levels of both deoxyribonucleotide triphosphates (dNTPs) and the biosynthetic enzymes required for their de novo synthesis provide one barrier to infection. CD4+ T cell activation induces metabolic reprogramming that reverses this block and facilitates HIV-1 replication. Here, we show that phospholipase D1 (PLD1) links T cell activation signals to increased HIV-1 permissivity by triggering a c-Myc-dependent transcriptional program that coordinates glucose uptake and nucleotide biosynthesis. Decreasing PLD1 activity pharmacologically or by RNA interference diminished c-Myc-dependent expression during T cell activation at the RNA and protein levels. PLD1 inhibition of HIV-1 infection was partially rescued by adding exogenous deoxyribonucleosides that bypass the need for de novo dNTP synthesis. Moreover, the data indicate that low dNTP levels that impact HIV-1 restriction involve decreased synthesis, and not only increased catabolism of these nucleotides. These findings uncover a unique mechanism of action for PLD1 inhibitors and support their further development as part of a therapeutic combination for HIV-1 and other viral infections dependent on host nucleotide biosynthesis.

If critical steps in the influenza infection process are identified, druggable targets could be discovered. We have uncovered the role of lipid signaling through phospholipase D (PLD) during influenza infection. Our findings suggest that influenza virus takes advantage of PLD activity to quickly and quietly enter the host cell. By inhibiting PLD with small molecules, we found that endocytosis kinetics were subdued. Slowing influenza entry allowed innate immune proteins to sense the pathogen and mount an effective defense. The molecules required for defense included Rig-I, MxA, and IRF3. When these proteins were knocked down in cells treated with PLD2 inhibitor, protection was lost. Inhibition of PLD2 protected cells from several influenza subtypes, including H5N1 and the newly emerged H7N9. Additionally, viral growth in mice treated with the PLD2 inhibitor was reduced, and mice were more likely to survive when treated with PLD2 inhibitor. Our data suggest that signaling through PLD during influenza infection plays a critical role in innate immune detection and defense mechanisms, and that drugging PLD2, allows a better chance to defend against pathogens using innate molecules. Currently, second generation small molecule inhibitors of PLD2 are proving to be more efficacious and potent. Additionally, further insights are being gained regarding the temporal-spatial regulation of innate immune molecules during the first hours of infection.

Circulating levels of the gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) have recently been linked to cardiovascular disease (CVD) risk. Here, we performed transcriptional profiling in mouse models of altered reverse cholesterol transport (RCT) and serendipitously identified the TMAO-generating enzyme flavin monooxygenase 3 (FMO3) as a powerful modifier of cholesterol metabolism and RCT. Knockdown of FMO3 in cholesterol-fed mice alters biliary lipid secretion, blunts intestinal cholesterol absorption, and limits the production of hepatic oxysterols and cholesteryl esters. Furthermore, FMO3 knockdown stimulates basal and liver X receptor (LXR)-stimulated macrophage RCT, thereby improving cholesterol balance. Conversely, FMO3 knockdown exacerbates hepatic endoplasmic reticulum (ER) stress and inflammation in part by decreasing hepatic oxysterol levels and subsequent LXR activation. FMO3 is thus identified as a central integrator of hepatic cholesterol and triacylglycerol metabolism, inflammation, and ER stress. These studies suggest that the gut microbiota-driven TMA/FMO3/TMAO pathway is a key regulator of lipid metabolism and inflammation.