Lipid Deprivation Research Articles

Impaired T-Cell Proliferation in Chronic Lymphocytic Leukemia Patients Is Linked to Disturbed Lipid Metabolism and Homeostasis

Background Successful T-cell based anti-cancer therapies require proliferation and longevity of effector T cells, features that highly depend on metabolic processes. T cells from chronic lymphocytic leukemia (CLL) patients are characterized by acquired T-cell dysfunction, resulting in impaired activation, proliferation and cytotoxicity upon in-vitro activation (van Bruggen et al, Blood 2019). Whilst the role of glucose metabolism in activated T-cells is extensively studied, the contribution of other energy sources is less clear. Lipid metabolism has only recently come into focus as an important source of energy for immune cells. Aims Molecular and functional analysis of the lipid metabolic pathway in T cells from CLL patients. Methods Peripheral blood mononuclear cells (PBMCs) of untreated CLL patients and, as controls, age-matched healthy donors (HD) were studied. T cells at baseline and after TCR stimulation were analyzed molecularly using transcriptomics and lipidomics, and extensively characterized at protein level by flow cytometry and confocal imaging. Lipid metabolism was manipulated through culturing cells in lipoprotein deficient serum (LPDS), or in the presence of agonists and inhibitors of specific regulatory pathways. Results We found the uptake of lipids, specifically cholesterol, during T-cell activation essential for proliferation, which was evidenced by a nearly absent proliferative capacity of HD T cells in the lack of exogenous lipids which was fully rescued by re-addition of cholesterol-rich low-density lipoproteins (LDL). As CLL T cells portray an altered proliferation pattern even when cultured in normal medium, transcriptomic analysis was performed to investigate the expression of key lipid metabolism genes. CLL T-cells had reduced expression of the master transcription factors SREBP1/2, governing lipid and cholesterol metabolism, when stimulated in the presence of their autologous CLL cells (Figure 1A). Accordingly, downstream protein involved in the synthesis and desaturation of lipids and LDL uptake (specifically FASN, LDL-R and SCD) were significantly reduced in CLL compared to HD T cells. Remarkably, despite reduced capacity for LDL uptake, an accumulation of neutral lipids was found in CLL T cells especially upon stimulation (Figure 1B), a feature earlier described in dysfunctional macrophages. The lipid accumulation observed in CLL T cells was supported by the lack of expression of ATGL, a crucial enzyme for the utilization of neutral lipids for energy production and building of new membranes. In line with this, mitochondrial lipid oxidation was decreased in stimulated CLL T cells compared to HD. To investigate the composition of the accumulated lipids, lipidomics analysis was performed, revealing differential abundance of specific lipid classes in stimulated CLL versus HD T cells. Triglycerides were increased while cholesterol esters and phospholipids were reduced in CLL T cells. Triglycerides likely constitute the neutral lipids accumulation found in these cells. As cholesterol esters and phospholipids are essential for proper membrane formation, reduced levels of these species can contribute to the reduced proliferative capacity of CLL T cells. Summary/conclusion Altogether, the results presented here demonstrate a disturbed lipid composition and utilization in CLL T-cells, which contributes to T-cell dysfunction on at least two levels: decreased bioenergetics and decreased proliferation. Likewise, lipid deprivation due to both decreased uptake and a misbalanced lipidome leads to altered T-cell function. Therefore, strategies to increase lipid uptake, synthesis and utilization in order to rebalance homeostasis could ameliorate CLL T-cell dysfunction and specifically proliferation-defects in CLL.

Drosophila embryos allocate lipid droplets to specific lineages to ensure punctual development and redox homeostasis.

Lipid droplets (LDs) are ubiquitous organelles that facilitate neutral lipid storage in cells, including energy-dense triglycerides. They are found in all investigated metazoan embryos where they are thought to provide energy for development. Intriguingly, early embryos of diverse metazoan species asymmetrically allocate LDs amongst cellular lineages, a process which can involve massive intracellular redistribution of LDs. However, the biological reason for asymmetric lineage allocation is unknown. To address this issue, we utilize the Drosophila embryo where the cytoskeletal mechanisms that drive allocation are well characterized. We disrupt allocation by two different means: Loss of the LD protein Jabba results in LDs adhering inappropriately to glycogen granules; loss of Klar alters the activities of the microtubule motors that move LDs. Both mutants cause the same dramatic change in LD tissue inheritance, shifting allocation of the majority of LDs to the yolk cell instead of the incipient epithelium. Embryos with such mislocalized LDs do not fully consume their LDs and are delayed in hatching. Through use of a dPLIN2 mutant, which appropriately localizes a smaller pool of LDs, we find that failed LD transport and a smaller LD pool affect embryogenesis in a similar manner. Embryos of all three mutants display overlapping changes in their transcriptome and proteome, suggesting that lipid deprivation results in a shared embryonic response and a widespread change in metabolism. Excitingly, we find abundant changes related to redox homeostasis, with many proteins related to glutathione metabolism upregulated. LD deprived embryos have an increase in peroxidized lipids and rely on increased utilization of glutathione-related proteins for survival. Thus, embryos are apparently able to mount a beneficial response upon lipid stress, rewiring their metabolism to survive. In summary, we demonstrate that early embryos allocate LDs into specific lineages for subsequent optimal utilization, thus protecting against oxidative stress and ensuring punctual development.

Abstract 2308: Inhibition of fatty acid synthesis and concurrent lipid deprivation decreases neuroendocrine cancer cell proliferation and colony formation

Abstract Introduction. Gastroenteropancreatic neuroendocrine tumors (GEP-NETs), the second most common digestive cancer in terms of prevalence, can be particularly challenging to treat due to resistance to current clinical regimens. Lipid metabolism provides an important energy source for cancer cells; cells increase lipid supply by endogenous synthesis or exogenous uptake. Both processes can become dysregulated in cancer, but little is known about the role of either pathway in neuroendocrine tumors (NETs). The goal of this project was to determine whether inhibition of lipid synthesis with concurrent lipid deprivation affects NET cell proliferation and survival. Methods. (i) Expression and localization of fatty acid synthase (FASN) in NET patient samples (n=39) and human cell lines (BON, QGP-1, and NT-3) were examined with immunohistochemistry and immunoblotting, respectively. (ii) NET neutral lipid stores were determined with immunofluorescent analysis of Bodipy 493/503 staining. (iii) To study the role of lipid synthesis and lipid uptake on colony formation, NET cells were treated with TVB-3664 with or without FA depletion for 10d. Colonies were visualized with sulforhodamine B (SRB) dye staining and quantified with photospectrometry. (iv) To study the effect of lipid synthesis and uptake on proliferation, NET cells were treated with TVB-3664 with or without FA depletion for 72h, 96h, and 120h. Cellular growth was determined with SRB assays. (v) The effect of TVB-3664 on cyclin D1 and β-catenin expression in NET cells was determined with immunoblotting. Results. (i) FASN was detected in the cytoplasm of all 39 NET primary and metastatic tissue samples; scores of 5 or 6 indicated robust expression in 38/39 samples. Among cell lines, FASN expression was highest in BON cells followed by similar levels in QGP-1 and NT-3 cells. (ii) TVB-3664 treatment or lipid deprivation for 5d reduced neutral lipid storage in BON cells; combined TVB-3664 treatment with FA depletion for 5d fully depleted BON lipid stores. (iii) TVB-3664 treatment, but not FA deprivation, reduced NET colony formation by ~25% in BON cells and ~50% in QGP-1 cells. TVB-3664 treatment with FA deprivation reduced colony formation by ~65% in BON cells and ~75% in QGP-1 cells. (iv) TVB-3664 treatment or FA deprivation reduced NET cell proliferation at 72h, 96h, and 120h. (v) TVB-3664 treatment reduced cyclin D1 expression in BON and QGP-1 cells and β-catenin levels in BON cells. Conclusions. Our findings indicate that FASN is robustly expressed in NET patient samples and cell lines. Inhibition of endogenous lipid synthesis with concurrent lipid deprivation reduces colony formation and proliferation of NET cells. Importantly, our findings show that lipid metabolism promotes NET cell growth and that targeting de novo lipogenesis may be a potential treatment strategy for advanced GEP-NETs. Citation Format: Jeremy Johnson, Yekaterina Y. Zaytseva, Jennifer Castle, B. Mark Evers, Piotr Rychahou. Inhibition of fatty acid synthesis and concurrent lipid deprivation decreases neuroendocrine cancer cell proliferation and colony formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2308.

Restriction of extracellular lipids renders pancreatic cancer dependent on autophagy

BackgroundKRAS is the predominant oncogene mutated in pancreatic ductal adenocarcinoma (PDAC), the fourth cause of cancer-related deaths worldwide. Mutant KRAS-driven tumors are metabolically programmed to support their growth and survival, which can be used to identify metabolic vulnerabilities. In the present study, we aimed to understand the role of extracellularly derived fatty acids in KRAS-driven pancreatic cancer.MethodsTo assess the dependence of PDAC cells on extracellular fatty acids we employed delipidated serum or RNAi-mediated suppression of ACSL3 (to inhibit the activation and cellular retention of extracellular fatty acids) followed by cell proliferation assays, qPCR, apoptosis assays, immunoblots and fluorescence microscopy experiments. To assess autophagy in vivo, we employed the KrasG12D/+;p53flox/flox;Pdx1-CreERT2 (KPC) mice crossed with Acsl3 knockout mice, and to assess the efficacy of the combination therapy of ACSL3 and autophagy inhibition we used xenografted human cancer cell-derived tumors in immunocompromised mice.ResultsHere we show that depletion of extracellularly derived lipids either by serum lipid restriction or suppression of ACSL3, triggers autophagy, a process that protects PDAC cells from the reduction of bioenergetic intermediates. Combined extracellular lipid deprivation and autophagy inhibition exhibits anti-proliferative and pro-apoptotic effects against PDAC cell lines in vitro and promotes suppression of xenografted human pancreatic cancer cell-derived tumors in mice. Therefore, we propose lipid deprivation and autophagy blockade as a potential co-targeting strategy for PDAC treatment.ConclusionsOur work unravels a central role of extracellular lipid supply in ensuring fatty acid provision in cancer cells, unmasking a previously unappreciated metabolic vulnerability of PDAC cells.

Expression of lipid transport-associated genes in lipid-deprived cancer cells.

Cancer cells are known to significantly alter their lipid profiles in response to changes in extracellular lipid availability. Recent studies have shown that in response to lipid deprivation, cancer cells display significant changes in their cellular lipid homeostasis. These changes have been linked to the modulation of de novo lipid synthesis pathways that are markedly altered under lipid-deprived growth conditions. However, the effects of such environment on intracellular lipid trafficking-that could also affect cellular lipid homeostasis-have not been widely investigated. The presented work studies the effect of lipid deprivation on expression of genes for lipid transport proteins (LTPs) in cancer cell lines.

A Dual Omics Approach to Evaluate Transcriptional and Metabolic Responses During Lipid Deprivation in an Oyster Parasite, Perkinsus marinus.

Perkinsus marinus, a protozoan and the causative agent of perkinsosis (dermo disease) is a prevalent parasite found within the eastern oyster (Crassostrea virginica). In this study, we explore metabolic processes of P. marinus cells under lipid-depleted medium conditions to elucidate the interchanging flux of lipid and carbohydrate metabolism. Although P. marinus can synthesize their own lipids from available nutrients, they display a slower growth in medium not supplemented with lipids as opposed to medium with lipids. Under these conditions, using transcriptomics, we surprisingly observed evidence of stimulated lipid degradation through increased transcription of two core β-oxidation pathway enzymes. Simultaneously, phospholipid biosynthetic pathways were downregulated. Metabolomic analysis supported the transcriptomic results. Most fatty acids were decreased in lipid-deplete medium as opposed to lipid-replete medium, and available glucose was fermented to lactate. A significant increase in the cholesterol derivative zymosterol further supported a downregulation of membrane synthesis under the experimental conditions. A robust tricarboxylic acid (TCA) cycle was apparent by enhanced citrate synthase transcription, and a simultaneous reduction in branched chain amino acids. It is concluded that although P. marinus has the capacity for synthesizing its own lipids, it can respond to lipid deprivation in medium by oxidizing readily available stores, and likely transitioning into a resting stage.

Influence of Oxidative Stress on Time-Resolved Oxygen Detection by [Ru(Phen)3]2+ In Vivo and In Vitro.

Detection of tissue and cell oxygenation is of high importance in fundamental biological and in many medical applications, particularly for monitoring dysfunction in the early stages of cancer. Measurements of the luminescence lifetimes of molecular probes offer a very promising and non-invasive approach to estimate tissue and cell oxygenation in vivo and in vitro. We optimized the evaluation of oxygen detection in vivo by [Ru(Phen)3]2+ in the chicken embryo chorioallantoic membrane model. Its luminescence lifetimes measured in the CAM were analyzed through hierarchical clustering. The detection of the tissue oxygenation at the oxidative stress conditions is still challenging. We applied simultaneous time-resolved recording of the mitochondrial probe MitoTrackerTM OrangeCMTMRos fluorescence and [Ru(Phen)3]2+ phosphorescence imaging in the intact cell without affecting the sensitivities of these molecular probes. [Ru(Phen)3]2+ was demonstrated to be suitable for in vitro detection of oxygen under various stress factors that mimic oxidative stress: other molecular sensors, H2O2, and curcumin-mediated photodynamic therapy in glioma cancer cells. Low phototoxicities of the molecular probes were finally observed. Our study offers a high potential for the application and generalization of tissue oxygenation as an innovative approach based on the similarities between interdependent biological influences. It is particularly suitable for therapeutic approaches targeting metabolic alterations as well as oxygen, glucose, or lipid deprivation.

Non-coding RNAs, metabolic stress and adaptive mechanisms in cancer.

Metabolic reprogramming in cancer describes the multifaceted alterations in metabolism that contribute to tumorigenesis. Major determinants of metabolic phenotypes are the changes in signalling pathways associated with oncogenic activation together with cues from the tumor microenvironment. Therein, depleted oxygen and nutrient levels elicit metabolic stress, requiring cancer cells to engage adaptive mechanisms. Non-coding RNAs (ncRNAs) act as regulatory elements within metabolic pathways and their widespread dysregulation in cancer contributes to altered metabolic phenotypes. Indeed, ncRNAs are the regulatory accomplices of many prominent effectors of metabolic reprogramming including c-MYC and HIFs that are activated by metabolic stress. By example, this review illustrates the range of ncRNAs mechanisms impacting these effectors throughout their DNA-RNA-protein lifecycle along with presenting the mechanistic roles of ncRNAs in adaptive responses to glucose, glutamine and lipid deprivation. We also discuss the facultative activation of metabolic enzymes by ncRNAs, a phenomenon which may reflect a broad but currently invisible level of metabolic regulation. Finally, the translational challenges associated with ncRNA discoveries are discussed, emphasizing the gaps in knowledge together with importance of understanding the molecular basis of ncRNA regulatory mechanisms.

Putting Stem Cells on a Low-Fat Diet Switches Their Pluripotent State.

Previous work demonstrating the importance of stem cell metabolism in regulating development ignited a debate on how intrinsic versus extrinsic conditions influence metabolic programming of epigenetics. In this issue of Cell Stem Cell, Cornacchia etal. show that exogenous lipids regulate an acetyl-CoA-dependent switch to influence the intrinsic pluripotent state of hPSCs.

Lipid Deprivation Induces a Stable, Naive-to-Primed Intermediate State of Pluripotency in Human PSCs.

Current challenges in capturing naive human pluripotent stem cells (hPSCs) suggest that the factors regulating human naive versus primed pluripotency remain incompletely defined. Here we demonstrate that the widely used Essential 8minimal medium (E8) captures hPSCs at a naive-to-primed intermediate state of pluripotency expressing several naive-like developmental, bioenergetic, and epigenomic features despite providing primed-state-sustaining growth factor conditions. Transcriptionally, E8 hPSCs are marked by activated lipid biosynthesis and suppressed MAPK/TGF-β gene expression, resulting in endogenous ERK inhibition.These features are dependent on lipid-freeculture conditions and are lost upon lipid exposure, whereas short-term pharmacological ERK inhibition restores naive-to-primed intermediate traits even in the presence of lipids. Finally, we identify de novo lipogenesis as a common transcriptional signature of E8 hPSCs and the pre-implantation human epiblast invivo. These findings implicate exogenous lipid availability in regulating human pluripotency and define E8 hPSCs as a stable, naive-to-primed intermediate (NPI) pluripotent state.

Membrane cholesterol is essential for triterpenoid saponin augmentation of a saporin-based immunotoxin directed against CD19 on human lymphoma cells

Triterpenoid saponins from Saponinum Album (SA) exert potent lytic effects on eukaryotic cell plasma membranes and, when used at sub-lytic concentrations, significantly augment the cytotoxicity of saporin-based immunotoxins (IT). To help elucidate the mechanism(s) behind these two phenomena we investigated the role of cholesterol to both.Human Daudi lymphoma cells were lipid deprived using a combination of three different approaches. Following treatment, the total cellular lipid content was analyzed by electrospray ionization mass spectrometry (ESI-MS) and plasma membrane (PM) cholesterol content measured using the lipophilic fluorescent probe NR12S. Maximal lipid deprivation of cells resulted in a complete loss of sensitivity to lysis by SA. Similarly augmentation of the anti-CD19 immunotoxin (IT) BU12-SAPORIN by SA was lost but without a concomitant loss of intrinsic IT cytotoxicity. The lytic activity of SA was restored following incubation of lipid deprived Daudi cells with Synthecol or LDL. The augmentative effect of SA on IT cytotoxicity for Daudi cells was restored following repletion of PM cholesterol levels with LDL. NR12S fluorescence and ESI-MS analysis of cellular lipids demonstrated that restoration of SA lytic activity by Synthecol was entirely due to increased PM cholesterol levels. Restoration of cellular and PM cholesterol levels by LDL also restored the augmentative effect of SA for IT, an effect associated with repletion of PM cholesterol with minor changes in some phospholipid species. These results indicate that the lytic and IT augmentative properties of SA are cholesterol-dependent in contrast to intrinsic IT cytotoxicity that is at least partially cholesterol independent.

Abstract A61: CAF-derived ANXA6+-exosomes support pancreatic cancer aggressiveness and serve as a circulating biomarker

Abstract Pancreatic ductal adenocarcinoma (PDA) is one of deadliest human malignancies with a median survival barely reaching 6 months post-diagnosis. Such epidemiologic data highlight the urgent need to refine current therapies by integrating recent available knowledge on PDA biology. The malignant progression of PDA is characterized by the presence of an exuberant intra-tumoral microenvironment mainly composed of cancer associated fibroblasts (CAFs) and immune cells. This compartment interacts with pancreatic tumor cells surrounded by dense matrix and hypoxic areas, and affects their behavior through a poorly understood network. Indeed, cellular crosstalk between stromal and tumor cells is of major importance in pancreatic ductal adenocarcinoma (PDA). Here, by analyzing the proteomic stromal signature of human PDA, we highlight a possible contribution of the ANXA6/LRP1/TSP1 complex in such crosstalk. Following in vivo validations and stromal localization of each three candidates, we demonstrated that the formation of this complex is restricted to cancer associated fibroblasts (CAFs) cultured under physiopathological conditions (co-culture with macrophages, under hypoxia and lipid deprivation). Increased PDA aggressiveness is dependent on the uptake by tumor cells of CAFs-derived ANXA6+-exosomes. Targeting of ANXA6, and the relevant complex, in CAFs impairs PDA and metastasis occurrence while injection of ANXA6+-exosomes enhances tumorigenesis. Detection of ANXA6+-exosomes in serum is restricted to PDA patients and can distinguish PDA grade. Our data reveal a new CAF-tumor cell crosstalk supported by ANXA6+-exosomes and highlight a therapeutic target and an interesting biomarker for PDA. Citation Format: Julie Leca, Sebastien Martinez, Sophie Lac, Jeremy Nigri, Veronique Secq, Marion Rubis, Nelson Dusetti, Celine Loncle, Julie Roques, Daniel Pietrasz, Stéphane Garcia, Samuel Granjeaud, Mehdi Ouaissi, Jean-Baptiste Bachet, Christine Brun, Juan L. Iovanna, Pascale Zimmermann, Sophie Vasseur, Richard Tomasini.{Authors}. CAF-derived ANXA6+-exosomes support pancreatic cancer aggressiveness and serve as a circulating biomarker. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A61.

FASN regulates cellular response to genotoxic treatments by increasing PARP-1 expression and DNA repair activity via NF-κB and SP1

Fatty acid synthase (FASN), the sole cytosolic mammalian enzyme for de novo lipid synthesis, is crucial for cancer cell survival and associates with poor prognosis. FASN overexpression has been found to cause resistance to genotoxic insults. Here we tested the hypothesis that FASN regulates DNA repair to facilitate survival against genotoxic insults and found that FASN suppresses NF-κB but increases specificity protein 1 (SP1) expression. NF-κB and SP1 bind to a composite element in the poly(ADP-ribose) polymerase 1 (PARP-1) promoter in a mutually exclusive manner and regulate PARP-1 expression. Up-regulation of PARP-1 by FASN in turn increases Ku protein recruitment and DNA repair. Furthermore, lipid deprivation suppresses SP1 expression, which is able to be rescued by palmitate supplementation. However, lipid deprivation or palmitate supplementation has no effect on NF-κB expression. Thus, FASN may regulate NF-κB and SP1 expression using different mechanisms. Altogether, we conclude that FASN regulates cellular response against genotoxic insults by up-regulating PARP-1 and DNA repair via NF-κB and SP1.

Abstract 2716: Lipid mediated “nutrients sensing” checkpoint in G1 phase of the cell cycle

Abstract One of the hallmarks of cancers is dysregulation of G1 phase cell cycle progression- where cells decide their fate whether they should divide, arrest or undergo apoptosis. There is a growth factor-dependent Restriction point (R), where cells commit to replicate its genome or to enter a state of quiescence. Unicellular organisms only respond to the nutrients, a point known as START. In mammalian system, we have shown equivalent to START, “Cell Growth” checkpoints, where it senses Essential Amino Acids (EAAs), a “Conditionally” EAA glutamine (Q), and at last a checkpoint regulated by mTOR, which we think it senses nutrients and is the final arbitrator of the G1-S phase progression. mTOR- mammalian/mechanistic target of rapamycin integrates the growth factor signaling with nutrient sensing signaling and known to be involved in protein translation and proliferation. Another key nutrient needed for cell growth is lipids which is required as building blocks for plasma membrane synthesis and signaling molecules. Phosphatidic acid has been shown to be required for the activity of mTORC1 and mTORC2. In this study, we investigated the involvement of lipids in the cell cycle progression and how the signals are integrated. Upon lipid deprivation, normal fibroblasts cells arrest in G1-phase of the cell cycle. There is a distinct and distinguishable lipid sensing checkpoint from EAAs and Q. Temporally it is different from other nutrient sensing checkpoints in the late G1-phase of the cell cycle. In summary, there is a series of “nutrient sensing” checkpoints late in the G1-phase followed by mTOR “Growth” checkpoint. Dysregulation of these checkpoints in cancer cells may present better opportunity to target them with different therapeutic approach. Citation Format: Deven Patel, David Foster. Lipid mediated “nutrients sensing” checkpoint in G1 phase of the cell cycle. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2716.

Lipid Addiction of Diffuse Large B-Cell Lymphoma (DLBCL): Fatty Acid Synthase (FASN) and PI3K Dependent Cell Death Identifies a Novel Therapeutic Paradigm

Introduction: Fatty acid (FA) metabolism is altered in many solid tumor cancers in part through increased de novo synthesis of lipids via up-regulation of FASN as well as via increased utilization of lipids via β-oxidation. The dependence of DLBCL on FA metabolism, and potential associated oncogenic signaling pathways, are largely unknown.Methods: Cerulenin (FASN inhibitor), orlistat (combined FASN and lipoprotein lipase inhibitor), and BKM120 (PI3K inhibitor) small molecules were studied in OCI-LY3, OCI-LY19, SUDHL4, SUDHL6, and SUDHL10 DLBCL cell lines as well as in primary DLBCL cells for the impact on FA inhibition signaling and the induction of cell death (by MTT, cleaved caspases, and AnnexinV/PI by flow cytometric analysis). Synergy was assessed via Calcusyn software. All effects were compared by DLBCL cell of origin (COO). In addition, global transcriptome analyses of FASN inhibition were examined on Affymetrix Human 2.0 ST Genechip.Results: FASN protein was constitutively upregulated across all DLBCL cell lines, independent of COO. Treatment with pharmacological inhibitors of FASN (i.e., cerulenin or orlistat) resulted in dose- and time-dependent reduction in cell viability in all DLBCL cell lines. To further examine the dependence of DLBCL on fatty acids, OCI-LY3, SUDHL4, and SUDHL6 grown in presence of lipoprotein-depleted serum showed exquisite sensitivity to lipid deprivation resulting in near complete cytotoxicity (i.e., >98%) as determined by MTT, cleaved caspase 3 and PARP, and AnnexinV/PI. Moreover, these effects were completely rescued by Very Low Density Lipoprotein (VLDL) supplementation to growth medium. These effects were markedly more prominent in primary DLBCL cells compared with control non-malignant lymphocytes. Analysis of differentially expressed gene sets comparing germinal center (GC) and non-germinal center (non-GC) B-cell tumors with normal cells showed significant over expression of genes related to PI3K and lipid metabolism in GC B-cell tumors. Furthermore, global transcriptome analysis following cerulenin exposure revealed activation of carbohydrate and oxidative stress metabolism in GC-derived SUDHL6 and SUDHL10 cells. In the non-GC cell line, OCI-LY3, there was favorable biology induced with inhibition of progression when treated with cerulenin, with downregulated significant key genes representing growth promoting cytokines (Figure 1). Analysis of key significant genes in cerulenin-treated SUDHL10 cells resulted in induction of antioxidant genes (GSR, TXNRD1, NQO1, AIFM1) and oxidative stress responsive PHB, DHX9, HSP90 and PMPCA genes, which are upstream components of PI3K signaling. Western blot analysis with prohibitin (PHB) inhibitor rocoglamide in the presence of cerulenin inhibited phosphorylation of PI3K-p85 and also prominently downregulated FASN expression of GC-derived DLBCL, suggesting the role of PHB in PI3K response to cerulenin. In addition, inhibition of PI3K with BKM120 was shown to strongly down-regulate FASN expression in OCI-LY3, SUDHL6, and SUDHL10 DLBCL cells. Furthermore, treatment with BKM120 in combination with cerulenin was strongly synergistic (CI<1), by MTT and potentiated apoptosis (by Annexin/V flow cytometry) in GC-derived SUDHL10 cells.Conclusions: Multiple FA pathways are constitutively activated in DLBCL and cell survival appears strongly dependent on lipid metabolism, which appeared to occur independent of COO. Additionally, inhibition of FASN was regulated in part through PHB/PI3K-dependent pathways. Collectively, this work indicates that targeting FA metabolism may be considered as a valid therapeutic strategy for DLBCL. Further investigation is required to delineate the precise mechanisms through which PHB/PI3K regulates FASN and also to identify potential rational therapeutic combinations leveraging the addiction of DLBCL to FA signaling. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Fatty acid synthase is a metabolic oncogene targetable in malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas with minimal therapeutic opportunities. We observed that lipid droplets (LDs) accumulate in human MPNST cell lines and in primary human tumor samples. The goal of this study was to investigate the relevance of lipid metabolism to MPNST survival and as a possible therapeutic target. Based on preliminary findings that MPNSTs accumulate LDs, we hypothesized that a deregulated lipid metabolism supports MPNST cell survival/proliferation rate. To test this, we examined respiration, role of fatty acid oxidation (FAO), and the enzyme fatty acid synthase involved in de novo fatty acid synthesis in MPNSTs using both genetic and pharmacological tools. We demonstrate that LDs accumulate in MPNST cell lines, primary human and mouse MPNST tumors, and neural crest cells. LDs from MPNST cells disappear on lipid deprivation, indicating that LDs can be oxidized as a source of energy. Inhibition of FAO decreased oxygen consumption and reduced MPNST survival, indicating that MPNST cells likely metabolize LDs through active FAO. FAO inhibition reduced oxygen consumption and survival even in the absence of exogenous lipids, indicating that lipids synthesized de novo can also be oxidized. Consequently, inhibition of de novo fatty acid synthesis, which is overexpressed in human MPNST cell lines, effectively reduced MPNST survival and delayed induction of tumor growth in vivo. Our results show that MPNSTs depend on lipid metabolic pathways and suggest that disrupting lipid metabolism could be a potential new strategy for the development of MPNST therapeutics.

The Lipid Addiction of Diffuse Large B-Cell Lymphoma (DLBCL) and Potential Treatment Strategies with Novel Fatty Acid Synthase (FASN) Small Molecule Inhibitors

Background: Fatty acid (FA) metabolism is altered in several cancers through increased de novo synthesis of lipids via up-regulation fatty acid synthase (FASN) and increased utilization of lipids via β-oxidation. We investigated the dependence of DLBCL survival on FA metabolism. In addition, we examined novel FASN inhibitors TVB3567 and TVB3166 in comparison with cerulenin for the effects on cell survival and PI3K and MAPK-related biological pathways associated with tumor-related FA metabolism in DLBCL.Methods: FASN inhibitors, TVB3567 and TVB3166 (3V Biosciences, CA), cerulenin (FASN inhibitor), orlistat (anti-lipoprotein lipase (LPL) and FASN), PI3K/mTOR, and MEK small molecule inhibitors were studied in OCI-LY3, OCI-LY19, SUDHL4, SUDHL6, and SUDHL10 DLBCL cell lines for the effects of FA inhibition on lipid metabolism, cell signaling, and cell death. The effects of FASN inhibition on global gene expression profile (GEP) were also determined with Affymetrix Human 2.0 ST Genechip with Gene set enrichment analysis (GSEA). We also utilized short hairpin RNA interference (shRNA) to study interactions between FASN and PI3K/MAPK signaling. Finally, AutoDock Vina software (autodock.scripps.edu) was utilized to analyze drug target (FASN enzyme) binding affinity and assist in the design of FASN inhibitors with higher target binding affinity.Results: DLBCL cell lines OCI-LY3, SUDHL4, and SUDHL6 grown in the presence of lipoprotein-depleted serum showed exquisite sensitivity to lipid deprivation resulting in near complete cytotoxicity by MTT. Lipid deprivation-induced apoptotic cell death, detected as cleaved caspase 3 and PARP and Annexin-V/PI positivity, in these cells. Further, these effects were completely rescued by Very Low Density Lipoprotein (VLDL) supplementation to growth medium in SUDHL4 confirming the high lipid-dependency on cell survival in DLBCL. Treatment with pharmacological inhibitors of FASN (ie, TVB3567, TVB3166, cerulenin, or orlistat) resulted in a dose- and time-dependent reduction in cell viability in all DLBCL cell lines. Ingenuity Pathway Analysis (IPA) from GEP with cerulenin-treated OCI-LY3 showed prominent suppression of CD40, TNF, and NFκB dependent inflammatory responses as well as activation of apoptosis as predominant biological activities including significant down-regulation of genes involved in Krebs cycle and p38 MAPK pathways. Interestingly, upstream regulation by IPA predicted activation of MEK/ERK and MYC-dependent functions; and in OCI-LY3 with shRNA knock down of FASN, we observed constitutive activation of ERK as detected with increased phosphorylation by western blot. Activation of MEK/ERK and MYC is expected in part owing to metabolic stress induced by FASN inhibition. Considering the impact of MEK/ERK pathways on lipid metabolism, we next investigated the impact of MEK/ERK on FA metabolism. FASN was significantly decreased following MEK or ERK shRNA in OCILY-3 and SUDHL10 cells. Similarly, pharmacological inhibition of MEK or PI3K/mTOR (using novel small molecule agents AZD6244 (selumetinib) or BEZ235, respectively) resulted in marked down-regulation of FASN expression. Based on these results, FASN inhibition appears to a promising therapeutic target for the treatment of DLBCL, however attaining clinical efficacy with existing compounds require the effective drug concentration to be within the nanomolar range. Thus, we utilized AutoDock to determine drug docking enzyme inhibition constant (ki). We identified high ki values of 33μM and 180μM for Cerulenin and Orilstat, respectively. Therefore, we have developed/constructed modified novel and potent anti-FA compounds with ki <1μM that are currently being investigated.Conclusions: Collectively, we demonstrated that DLBCL cell survival is highly dependent on FA metabolism and that targeting lipid metabolism may be harnessed as a potential therapeutic strategy. We also showed that MEK/ERK-dependent mechanisms are intimately involved in promoting lipid addiction in DLBCL cells. Further investigation is warranted to delineate the mechanisms through which MEK/ERK regulate FASN expression and to determine in vivo implications of FASN inhibition on DLBCL tumor growth. In addition, continued development, design, and enhancement are needed to construct the most optimal anti-FA therapeutic agents. DisclosuresLansigan:Teva Pharmaceuticals: Research Funding; Spectrum Pharmaceuticals: Research Funding.

Female rats are relatively more sensitive to reduced lipid versus reduced carbohydrate availability

Objectives:Because females have blunted counterregulatory responses to hypoglycemia relative to males, we hypothesized that females would have greater sensitivity to changes in lipid availability.Design and subjects:To assess this, we examined the feeding response to glucoprivation (2-deoxyglucose; 2DG) and lipoprivation (mercaptoacetate; MA) in age-matched male and female Long-Evans rats.Results:Males versus females had significantly greater food intake after 250 mg kg−1 of 2DG, but there were no sex differences with the 750 mg kg−1 dose of 2DG. Glucose responses to 250 mg kg−1 of 2DG were also significantly greater in males versus females. In contrast, females had a significant increase in food intake with all doses of MA versus saline, and had significantly greater food intake compared with males at the lowest and highest doses of MA with a trend towards significance with the intermediate dose. To determine whether estradiol (E2) is the mechanism underlying this sexual dimorphism, ovariectomized females were injected with vehicle or 2 μg of E2 every fourth day to mimic the variations in across the estrous cycle. Ovariectomized females significantly increased feeding and glucose after 250 mg kg−1 of 2DG over intact females and E2 had no effect on these responses. Although the feeding response to 2DG was not different, the glucose response to 2DG was still significantly greater in males versus ovariectomies females. However, ovariectomized females also did not increase food intake after MA, regardless of E2 treatment.Conclusions:These data collectively suggest that males are relatively more sensitive to glucose deprivation and females are relatively more sensitive to lipid deprivation. Further, these data rule out a role for cyclic changes in E2 in these sex differences.

Lipid Synthetic Transcription Factor SREBP-1a Activates p21WAF1/CIP1, a Universal Cyclin-Dependent Kinase Inhibitor

Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that regulate lipid synthetic genes. In contrast to SREBP-2, which regulates cellular cholesterol level in normal cells, SREBP-1a is highly expressed in actively growing cells and activates entire programs of genes involved in lipid synthesis such as cholesterol, fatty acids, triglycerides, and phospholipids. Previously, the physiological relevance of this potent activity of SREBP-1a has been thought to regulate the supply of membrane lipids in response to cell growth. Here we show that nuclear SREBP-1a and SREBP-2 bind directly to a novel SREBP binding site in the promoter of the p21(WAF1/CIP1) gene, the major cyclin-dependent kinase inhibitor, and strongly activate its promoter activity. Only the SREBP-1a isoform consistently causes induction of p21 at both the mRNA and protein levels. Colony formation assays and polyploidy of livers from transgenic mice suggest that activation of p21 by SREBP-1a could inhibit cell growth. Activation of endogenous SREBPs in lipid deprivation conditions was associated with induction of p21 mRNA and protein. Expression of p21 was reduced in SREBP-1 null mice. These data suggest a physiological role of SREBP-1a in p21 regulation. Identification of p21 as a new SREBP target might implicate a new paradigm in the link between lipid synthesis and cell growth.

Transcriptional regulation of phagocytosis-induced membrane biogenesis by sterol regulatory element binding proteins

In the process of membrane biogenesis several dozen proteins must operate in precise concert to generate approximately 100 lipids at appropriate concentrations. To study the regulation of bilayer assembly in a cell cycle-independent manner, we have exploited the fact that phagocytes replenish membranes expended during particle engulfment in a rapid phase of lipid synthesis. In response to phagocytosis of latex beads, human embryonic kidney 293 cells synthesized cholesterol and phospholipids at amounts equivalent to the surface area of the internalized particles. Lipid synthesis was accompanied by increased transcription of several lipogenic proteins, including the low-density lipoprotein receptor, enzymes required for cholesterol synthesis (3-hydroxy-3-methylglutaryl CoA synthase, 3-hydroxy-3-methylglutaryl CoA reductase), and fatty acid synthase. Phagocytosis triggered the proteolytic activation of two lipogenic transcription factors, sterol regulatory element binding protein-1a (SREBP-1a) and SREBP-2. Proteolysis of SREBPs coincided with the appearance of their transcriptionally active N termini in the nucleus and 3-fold activation of an SREBP-specific reporter gene. In previous studies with cultured cells, proteolytic activation of SREBP-1a and SREBP-2 has been observed in response to selective starvation of cells for cholesterol and unsaturated fatty acids. However, under the current conditions, SREBP-1a and SREBP-2 are induced without lipid deprivation. SREBP activation is inhibited by high levels of the SREBP-interacting proteins Insig1 or the cytosolic domain of SREBP cleavage-activating protein. Upon overexpression of these proteins, phagocytosis-induced transcription and lipid synthesis were blocked. These results identify SREBPs as essential regulators of membrane biogenesis and provide a useful system for further studies on membrane homeostasis.