Inhibition Of Fatty Acid Oxidation Research Articles

Twist1 downregulation of PGC-1α decreases fatty acid oxidation in tubular epithelial cells, leading to kidney fibrosis.

Rationale: A deficiency of fatty acid oxidation (FAO) is the metabolic hallmark in proximal tubular cells (PTCs) in renal fibrosis owing to utilization of fatty acids by PTCs as the main energy source. Lipid accumulation may promote lipotoxicity-induced pathological injury in renal tissue. However, the molecular mechanism underlying lipotoxicity and renal tubulointerstitial fibrosis (TIF) remains unclear. Twist1 has been identified to play an essential role in fatty acid metabolism. We hypothesized that Twist1 may regulate FAO in PTCs and consequently facilitate lipotoxicity-induced TIF.Methods: We used hypoxia-induced Twist1 overexpression to incite defective mitochondrial FAO in PTCs, and used renal ischemia-reperfusion or unilateral ureteral obstruction to induce renal injury in mice. We used knockout cells, mice of Twist1, and Harmine to determine the role of Twist1 in FAO and TIF.Results: Overexpression of Twist1 downregulates the transcription of PGC-1α and further inhibits the expression of FAO-associated genes, such as PPARα, CPT1 and ACOX1. Consequently, reduced FAO and increased intracellular lipid droplet accumulation in a human PTC line (HK-2), leads to mitochondrial dysfunction, and production of increased profibrogenic factors. Twist1 knockout mice with renal injury had increased expression of PGC-1α, which restored FAO and obstructed progression of TIF. Strikingly, pharmacological inhibition of Twist1 by using Harmine reduced lipid accumulation and restored FAO in vitro and in vivo.Conclusion: Our findings suggest that Twist1-mediated inhibition of FAO in PTCs results in TIF and suggest that Twist1-targeted inhibition could provide a potential strategy for the treatment of renal fibrosis.

Abstract 12092: IMB-1018792, an Investigational Agent Designed to Augment Cardiac Glucose Utlization and Energetics Reduces Cardiac Remodelling and Preserves Cardiac Function in a Model of Pressure Overload-Induced Heart Failure

Introduction: Energetic impairments play a key role in heart failure (HF). Contributors include reduced pyruvate dehydrogenase activity partially uncoupling glycolysis from glucose oxidation, increased dependence on fatty acid (FA) metabolism yielding less ATP than glucose per O 2 molecule consumed, a lower phosphate-to-O 2 ratio, mitochondrial uncoupling and futile cycling of FA intermediates. IMB-1018972 (IMB-101) is a novel investigational agent designed to increase cardiac glucose uptake to promote glucose oxidation (Randle effect) through partial inhibition of FA oxidation enhancing cardiac energetics and mechanical efficiency. Hypotheses: Administration of IMB-101 will: 1) increase cardiac glucose uptake and 2) attenuate LV hypertrophic remodelling and progression of HF resulting from chronic pressure overload. Methods: 1) Cardiac glucose metabolism was evaluated using dynamic 18 F-FDG PET/CT imaging in vivo in fasted normal rats following a subcutaneous dose of IMB-101 or saline. Each animal had 2 randomized sequenced scan sessions (n=16). 2) Male C57BL/6 mice aged 12 weeks underwent transverse aortic constriction (TAC) or sham surgery (n=15) and were treated with IMB-101 (10 mg/kg/day, n=15) or saline (n=15) from day 1 (D1) for 6 weeks by osmotic minipump. Cardiac function was assessed by conscious echocardiography (D1, week 3 and 6). Cardiac fibrosis was measured by Masson’s Trichrome staining. Results: IMB-101 robustly increased in vivo cardiac 18 F-FDG uptake vs saline in rats. In mice subjected to TAC, compared to saline controls, IMB-101 treatment: preserved LV systolic function (fractional shortening 46%±3% vs 34%±3%, at week 6, p<0.05); improved LV relaxation with less prolonged isovolumic relaxation time (32±1 ms vs 36±1 ms, p<0.05); reduced cardiac hypertrophy (LV mass 156±10 mg vs 195±12 mg, p<0.05; and heart:body weight ratio 7.4±0.3 mg/g vs. 9.1±0.5 mg/g, p<0.05); and attenuated cardiac fibrosis (6.6±0.6% vs 10.7±1%, p<0.01). Conclusions: IMB-101 increases cardiac glucose uptake and protects against LV dysfunction, hypertrophic remodelling and interstitial fibrosis after chronic pressure overload.

A Role for Lipid Metabolism in Tyrosine Kinase Inhibitor (TKI) Resistance of Chronic Myeloid Leukemia (CML)

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 are remarkably effective therapies in chronic myeloid leukemia (CML). Despite success, TKIs do not target the CML leukemic stem cell (LSC), meaning patients must be treated for life at a high economic burden and often with significant side effects. Our previous work demonstrated a tumor suppressor role for G0/G1 switch gene 2 (G0S2) in CML, which is profoundly downregulated in TKI resistance (>3-fold, p<0.02) (Gonzalez MA, et al. Cancer Res 2020 #648). Low G0S2 expression not only correlated with TKI resistance, but also with a shorter overall survival in chronic phase (p=0.036), and transformation from the chronic to the blast phase of disease (p<0.05). G0S2 is known to regulate apoptosis, quiescence, lipid metabolism, oxidative phosphorylation, and also acts as an inhibitor of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in intracellular lipolysis. We hypothesized that G0S2-mediated ATGL inhibition underlies its tumor suppressor activity in CML and TKI resistance.To address this hypothesis, we first assessed ATGL protein expression in CML cell lines (K562 and KU812) in the presence and absence of the BCR-ABL TKI, imatinib. Importantly, ATGL protein expression remained unchanged in graded doses of imatinib, suggesting that ATGL expression occurs in a BCR-ABL1-independent manner. To assess the functional role of ATGL in CML and TKI resistance, we performed shRNA-mediated ATGL knockdown (shATGL) in both cell lines. Although ectopic G0S2 impaired survival of CML cell lines and patient samples, we observed a different phenotype upon ATGL knockdown. Rather, shATGL alone increased colony formation by ~30% in both cell lines in the absence but not presence of imatinib (K562, p=0.04; KU812, p=0.0024). Knockdown of ATGL had no effect on colony formation of normal cord blood CD34 + progenitors (p=0.742). Interestingly, when we ectopically expressed G0S2 into K562 cells with simultaneous ATGL knockdown, the phenotype in colony formation assays mimicked ectopic G0S2 expression, reducing survival by ~50% (p=0.05). These data suggest that the tumor suppressor role of G0S2 in CML is independent of ATGL. However, while ectopic G0S2 expression alone had no effect on apoptosis of CML cell lines, when combined with ATGL knockdown, imatinib-mediated apoptosis was markedly increased. This was similar to data observed in blast phase CML patient samples upon ectopic G0S2 expression. Consistently, RNA sequencing (RNAseq) data for CML patients revealed that ATGL mRNA is highly downregulated in blast phase (n=14) compared with patients in the chronic (n=52) or accelerated (n=11) phases of the disease (p<0.0001). These data suggest that ATGL abrogates G0S2-mediated apoptosis in the presence of imatinib. We next performed colony formation assays of K562 cells with G0S2 knockdown (shG0S2) versus a non-targeting control (shNT) in the absence and presence of an ATGL inhibitor (Atglistatin, 50 µM) and a fatty acid oxidation (FAO) inhibitor (TMZ, 10 µM). Atglistatin had no effect on colony formation in the shNT-expressing cells, but reduced survival by ~40% upon G0S2 knockdown. Similarly, TMZ reduced survival of shNT cells by ~30%, while it reduced survival by ~90% upon G0S2 knockdown. These data suggest that the phenotypic effects of G0S2 knockdown in TKI resistance in part requires FAO.The effect of G0S2 on lipid metabolism was further confirmed with RNAseq and metabolomics/lipidomics analyses. RNAseq revealed no overlapping pathways between K562 cells expressing ectopic G0S2 versus shATGL. Lipidomics analyses revealed that G0S2 knockdown reduced expression of tri- and di-glycerides, whereas ectopic G0S2 promoted triglyceride accumulation in K562 cells. G0S2 knockdown also resulted in substantial changes of phosphatidylethanolamine and phosphatidylcholine expression, implicating G0S2 in the production of lipid bilayer components. Finally, metabolomics data implicated a role for G0S2 as a negative regulator of the mitochondrial electron transport chain. Altogether, these findings suggest that G0S2 and lipid metabolism play a role in regulating leukemic stem and progenitor cell survival as well as TKI response in vitro. Therefore, restoring G0S2 expression and inhibiting FAO, combined with BCR-ABL1 inhibition, may be a novel clinical strategy to induce treatment-free remission in CML patients and eradicate the CML LSC. DisclosuresNo relevant conflicts of interest to declare.

Mannose Metabolism Is a Metabolic Vulnerability Unveiled By Standard and Novel Therapies in Acute Myeloid Leukemia

IntroductionThe development of resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukemia (AML). Metabolic rewiring has emerged as a therapeutically actionable vulnerability in AML, where specific metabolic adaptations arising as a result of driver mutations or in response to therapy have been reported. Mannose phosphoisomerase (MPI) is the first enzyme in the mannose metabolism (MM) pathway, that leads to the production of GDP-Mannose, a key sugar donor for N-glycosylation reactions. MPI was amongst the top drop-out genes in our published CRISPR-Cas9 screen aiming to identify sensitizers to FLT3 tyrosine kinase inhibitors (TKI) in AML carrying activating FLT3 internal tandem duplication (ITD) mutations (Gallipoli et al., Blood 2018). Moreover, analysis of published genomic datasets indicates that MPI expression levels are higher in AML compared to normal samples, correlate with patient outcome (A) and further increase in paired relapsed to diagnosis samples. We therefore hypothesized that MM and MPI inhibition sensitize AML cells to both FLT3-TKI and standard chemotherapy and tested this in preclinical models.MethodsExperiments were performed in human AML cell lines, primary AML mononuclear cells and normal CD34 + stem/progenitor cells. Liquid chromatography coupled to mass spectrometry and oxygen consumption/extracellular acidification rate as measured by a Seahorse analyser were employed to assess metabolic changes. Gene-expression was measured by RNA-sequencing and confirmed by RT-qPCR. Viability, surface protein expression and lipid peroxidation were assessed by flow-cytometry. Protein expression and localisation was measured by western blot and immunofluorescence. Gene silencing was performed using CRISPR-Cas9 gene editing and inducible short hairpin RNA interference.ResultsWe show that genetic and chemical MPI inhibition sensitizes FLT3 WT and FLT3 ITD AML cell lines in vitro to standard cytarabine chemotherapy and FLT3-TKI respectively and these effects are rescued by the addition of MPI downstream product mannose. We validate these findings in vivo using MPI knock-out (KO) cell line xenografts and in vitro using primary AML samples following MPI knock-down (B). We also demonstrate a lack of toxicity to normal CD34 + cells.Surprisingly, global metabolomic analysis show that MPI KO cells accumulate fatty acids and particularly polyunsaturated fatty acids (PUFA) (C), due to both increased uptake and reduced fatty acid oxidation (FAO). MPI KO cells downregulate several FAO metabolism genes and this is corroborated by the strong positive correlation of MPI expression levels with multiple genes involved in FAO across multiple primary AML datasets. Metabolic profiling demonstrates that MPI KO cells have reduced oxidative phosphorylation metabolism and in particular are unable to oxidise palmitate, an effect rescued by mannose or the FAO activator fenofibrate (D).We link this metabolic defect to the specific activation of the ATF6 arm of the unfolded protein response (UPR) due to a reduction in protein glycosylation in MPI KO cells. We validate this by demonstrating that activating or inhibiting ATF6 respectively phenocopies or rescues the effects of MPI KO (E) and show this to be secondary to ATF6 driving FAO inhibition, via transcriptional downregulation of PPARα, a master regulator of lipid catabolism.Finally, we show that accumulation of PUFA in MPI KO cells is accompanied by reduced cysteine levels and increased markers of oxidative stress and lipid peroxidation, such as 4-hydroxynonenal (4-HNE), all features associated with ferroptosis (F). We validate this by showing that combining FLT3-TKI with MPI KO leads to ferroptotic cell death in AML cells, which can be rescued by the radical-trapping antioxidant ferrostatin.ConclusionsWe provide further evidence to support the role of metabolic rewiring in driving therapy resistance in AML and show for the first time that targeting MPI and MM sensitizes AML cells to cytarabine and FLT3-TKI. Mechanistically we unveil a novel connection between MM and fatty acid metabolism, via activation of the ATF6 arm of the UPR, leading to cellular PUFA accumulation, lipid peroxidation and ferroptotic cell death. Finally, our findings also suggest that triggering ferroptosis can be leveraged as a therapeutically actionable mechanism driving cell death in therapy-resistant AML cells. [Display omitted] DisclosuresVassiliou: Kymab Ltd: Divested equity in a private or publicly-traded company in the past 24 months; STRM.BIO: Consultancy; Astrazeneca: Consultancy.

Aqueous Metabolite Trends for the Progression of Nonalcoholic Fatty Liver Disease in Female Bariatric Surgery Patients by Targeted 1H-NMR Metabolomics.

Determining biomarkers and better characterizing the biochemical progression of nonalcoholic fatty liver disease (NAFLD) remains a clinical challenge. A targeted 1H-NMR study of serum, combined with clinical variables, detected and localized biomarkers to stages of NAFLD in morbidly obese females. Pre-surgery serum samples from 100 middle-aged, morbidly obese female subjects, grouped on gold-standard liver wedge biopsies (non-NAFLD; steatosis; and fibrosis) were collected, extracted, and analyzed in aqueous (D2O) buffer (1H, 600 MHz). Profiled concentrations were subjected to exploratory statistical analysis. Metabolites varying significantly between the non-NAFLD and steatosis groups included the ketone bodies 3-hydroxybutyrate (↓; p = 0.035) and acetone (↓; p = 0.012), and also alanine (↑; p = 0.004) and a putative pyruvate signal (↑; p = 0.003). In contrast, the steatosis and fibrosis groups were characterized by 2-hydroxyisovalerate (↑; p = 0.023), betaine (↓; p = 0.008), hypoxanthine (↓; p = 0.003), taurine (↓; p = 0.001), 2-hydroxybutyrate (↑; p = 0.045), 3-hydroxyisobutyrate (↑; p = 0.046), and increasing medium chain fatty acids. Exploratory classification models with and without clinical variables exhibited overall success rates ca. 75–85%. In the study conditions, inhibition of fatty acid oxidation and disruption of the hepatic urea cycle are supported as early features of NAFLD that continue in fibrosis. In fibrosis, markers support inflammation, hepatocyte damage, and decreased liver function. Complementarity of NMR concentrations and clinical information in classification models is shown. A broader hypothesis that standard-of-care sera can yield metabolomic information is supported.

Heat Shock Protein 27, a Novel Downstream Target of Collagen Type XI alpha 1, Synergizes with Fatty Acid Oxidation to Confer Cisplatin Resistance in Ovarian Cancer Cells.

Simple SummaryCollagen type XI alpha 1 (COL11A1) is a novel biomarker associated with poor survival in ovarian cancer and a promoter of ovarian cancer cell resistance to cisplatin. However, it is poorly understood how COL11A1 promotes ovarian cancer cisplatin resistance. We performed assays to discover the biological molecules that are activated by COL11A1 in ovarian cancer cells. We found that heat shock protein 27 (HSP27), a cellular stress response protein, is activated by COL11A1. Furthermore, we observed that depletion and drug inhibition of HSP27 makes ovarian cancer cells grown on COL11A1 to be more susceptible to cisplatin treatment. We also discovered that ovarian cancer cells upregulate fatty acid oxidation (FAO), a metabolic process that breaks down fats to generate energy and biomolecules, to compensate for the loss of HSP27. Our findings have therapeutic implications for clinicians who wish to treat ovarian tumors that maintain high levels of COL11A1 and HSP27.Collagen type XI alpha 1 (COL11A1) is a novel biomarker associated with cisplatin resistance in ovarian cancer. We have previously reported that COL11A1 activates Src-Akt signaling through the collagen receptors discoidin domain receptor 2 (DDR2) and integrin α1β1 to confer cisplatin resistance to ovarian cancer cells. To identify the potential signaling molecules downstream of COL11A1 signaling, we performed protein kinase arrays and identified heat shock protein 27 (HSP27) as a potential mediator of COL11A1-induced cisplatin resistance. Through receptor knockdown and inhibitor experiments, we demonstrated that COL11A1 significantly upregulates HSP27 phosphorylation and expression via DDR2/integrin α1β1 and Src/Akt signaling in ovarian cancer cells. Furthermore, genetic knockdown and pharmacological inhibition of HSP27, via ivermectin treatment, significantly sensitizes ovarian cancer cells cultured on COL11A1 to cisplatin treatment. HSP27 knockdown or inhibition also decreases NFκB activity as well as the expression of inhibitors of apoptosis proteins (IAPs), which are known downstream effector molecules of COL11A1 that promote cisplatin resistance. Interestingly, HSP27 knockdown or inhibition stimulates ovarian cancer cells to upregulate fatty acid oxidation (FAO) for survival and cisplatin resistance, and dual inhibition of HSP27 and FAO synergistically kills ovarian cancer cells that are cultured on COL11A1. Collectively, this study identifies HSP27 as a novel and druggable COL11A1 downstream effector molecule that may be targeted to overcome cisplatin resistance in recurrent ovarian cancer, which often overexpress COL11A1.

Aurora kinase A inhibition reverses the Warburg effect and elicits unique metabolic vulnerabilities in glioblastoma

Aurora kinase A (AURKA) has emerged as a drug target for glioblastoma (GBM). However, resistance to therapy remains a critical issue. By integration of transcriptome, chromatin immunoprecipitation sequencing (CHIP-seq), Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq), proteomic and metabolite screening followed by carbon tracing and extracellular flux analyses we show that genetic and pharmacological AURKA inhibition elicits metabolic reprogramming mediated by inhibition of MYC targets and concomitant activation of Peroxisome Proliferator Activated Receptor Alpha (PPARA) signaling. While glycolysis is suppressed by AURKA inhibition, we note an increase in the oxygen consumption rate fueled by enhanced fatty acid oxidation (FAO), which was accompanied by an increase of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α). Combining AURKA inhibitors with inhibitors of FAO extends overall survival in orthotopic GBM PDX models. Taken together, these data suggest that simultaneous targeting of oxidative metabolism and AURKAi might be a potential novel therapy against recalcitrant malignancies.

Avocado-derived avocadyne is a potent inhibitor of fatty acid oxidation.

Avocatin-B (Avo-B), an avocado-derived 1:1 mixture of the polyhydroxylated alcohols avocadyne (AYNE) and avocadene, eliminated leukemia cells by suppressing fatty acid oxidation (FAO) in vivo and in vitro while sparing healthy blood cells. In this study, we identified AYNE as the most potent FAO inhibitor within the Avo-B mixture capable of inducing cell death in leukemia cells lines (IC50 : 3.10±0.14µM in TEX cells; 11.53±3.32µM in OCI-AML2) and patient-derived acute myeloid leukemia cells. When added individually, the two Avo-B constituents demonstrated antagonism (Combination Index values >1), highlighting the need for future studies to assess AYNE alone. Together, this work highlighted AYNE as the most potent FAO inhibitor within the Avo-B mixture. PRACTICAL APPLICATIONS: This work identifies which of the two molecules in avocatin B (Avo-B), an avocado-derived mixture of two molecules with demonstrated human safety, utility against leukemia, insulin resistance and diabetes, is most useful. Therefore, it provides the basis for future clinical studies that will focus on testing and developing the most active Avo-B constituent.

Inhibition of Fatty Acid Metabolism Increases EPA and DHA Levels and Protects against Myocardial Ischaemia-Reperfusion Injury in Zucker Rats.

Long-chain ω-3 polyunsaturated fatty acids (PUFAs) are known to induce cardiometabolic benefits, but the metabolic pathways of their biosynthesis ensuring sufficient bioavailability require further investigation. Here, we show that a pharmacological decrease in overall fatty acid utilization promotes an increase in the levels of PUFAs and attenuates cardiometabolic disturbances in a Zucker rat metabolic syndrome model. Metabolome analysis showed that inhibition of fatty acid utilization by methyl-GBB increased the concentration of PUFAs but not the total fatty acid levels in plasma. Insulin sensitivity was improved, and the plasma insulin concentration was decreased. Overall, pharmacological modulation of fatty acid handling preserved cardiac glucose and pyruvate oxidation, protected mitochondrial functionality by decreasing long-chain acylcarnitine levels, and decreased myocardial infarct size twofold. Our work shows that partial pharmacological inhibition of fatty acid oxidation is a novel approach to selectively increase the levels of PUFAs and modulate lipid handling to prevent cardiometabolic disturbances.

Effect of High Fat Diet on the Severity and Repair of Lung Fibrosis in Mice.

Lung fibrosis is a progressive fatal disease, and the underlying mechanisms remain unclear. These involve a combination of altered fibroblasts, excessive accumulation of extracellular matrix, inflammation, and aberrant activation of epithelial cells. Previously, we showed that high-fat diet (HFD) induces lung inflammation, aberrant activation of stem cells, and lung mitochondria impairment. Therefore, we hypothesized that HFD-induced changes would influence lung fibrosis. Mice were fed standard diet (SD) or HFD, administered bleomycin, then examined for fibrosis severity and the start of repair 3 weeks after injury, and for fibrosis repair/resolution 6-9 weeks after injury. At 3 weeks, no significant differences in inflammation and fibrosis severity were observed between SD- and HFD-fed mice. However, infiltration of alveolar type (AT)-2 cells and bronchioalveolar stem cells (BASCs) into the fibrotic areas (the start of repair) was impaired in HFD-fed mice. At 6 weeks, SD-fed mice showed near-complete resolution/repair of fibrosis and inflammation, while HFD-fed mice still showed residual fibrosis and inflammation. Infiltration of the fibrotic areas with AT2 cells was observed, but very few BASCs were detectable. At 9 weeks, mice from both groups showed complete resolution/repair of fibrosis and inflammation, indicating that HFD induced delayed, rather than failed, resolution of fibrosis and alveolar repair. To further confirm the direct role of enhanced fatty-acid oxidation (FAO) in delayed resolution/repair, we administered etomoxir, a FAO inhibitor, to HFD-fed mice for 3-6 weeks after bleomycin injury. Inhibition of FAO abolished the HFD-induced delay in alveolar repair and fibrosis resolution at both time points. In conclusion, after a fibrosis-inducing injury, HFD slows resolution of fibrosis/inflammation and delays alveolar repair by slowing the contribution of AT2 stem cells and abolishing the contribution of BASCs in the repair process. FAO activation appears to be involved in this delay mechanism; thus, inhibiting FAO may be useful in the treatment of lung injury and fibrosis.

Mechanisms underlying neonate-specific metabolic effects of volatile anesthetics.

Volatile anesthetics (VAs) are widely used in medicine, but the mechanisms underlying their effects remain ill-defined. Though routine anesthesia is safe in healthy individuals, instances of sensitivity are well documented, and there has been significant concern regarding the impact of VAs on neonatal brain development. Evidence indicates that VAs have multiple targets, with anesthetic and non-anesthetic effects mediated by neuroreceptors, ion channels, and the mitochondrial electron transport chain. Here, we characterize an unexpected metabolic effect of VAs in neonatal mice. Neonatal blood β-hydroxybutarate (β-HB) is rapidly depleted by VAs at concentrations well below those necessary for anesthesia. β-HB in adults, including animals in dietary ketosis, is unaffected. Depletion of β-HB is mediated by citrate accumulation, malonyl-CoA production by acetyl-CoA carboxylase, and inhibition of fatty acid oxidation. Adults show similar significant changes to citrate and malonyl-CoA, but are insensitive to malonyl-CoA, displaying reduced metabolic flexibility compared to younger animals.

Listeria monocytogenes infection rewires host metabolism with regulatory input from type I interferons.

Listeria monocytogenes (L. monocytogenes) is a food-borne bacterial pathogen. Innate immunity to L. monocytogenes is profoundly affected by type I interferons (IFN-I). Here we investigated host metabolism in L. monocytogenes-infected mice and its potential control by IFN-I. Accordingly, we used animals lacking either the IFN-I receptor (IFNAR) or IRF9, a subunit of ISGF3, the master regulator of IFN-I-induced genes. Transcriptomes and metabolite profiles showed that L. monocytogenes infection induces metabolic rewiring of the liver. This affects various metabolic pathways including fatty acid (FA) metabolism and oxidative phosphorylation and is partially dependent on IFN-I signaling. Livers and macrophages from Ifnar1-/- mice employ increased glutaminolysis in an IRF9-independent manner, possibly to readjust TCA metabolite levels due to reduced FA oxidation. Moreover, FA oxidation inhibition provides protection from L. monocytogenes infection, explaining part of the protection of Irf9-/- and Ifnar1-/- mice. Our findings define a role of IFN-I in metabolic regulation during L. monocytogenes infection. Metabolic differences between Irf9-/- and Ifnar1-/- mice may underlie the different susceptibility of these mice against lethal infection with L. monocytogenes.

CPT1A and fatty acid β-oxidation are essential for tumor cell growth and survival in hormone receptor-positive breast cancer.

Chromosome 11q13-14 amplification is a defining feature of high-risk hormone receptor-positive (HR+) breast cancer; however, the mechanism(s) by which this amplicon contributes to breast tumorigenesis remains unclear. In the current study, proteogenomic analyses of >3000 breast tumors from the TCGA, METABRIC and CPTAC studies demonstrated that carnitine palmitoyltransferase 1A (CPT1A), which is localized to this amplicon, is overexpressed at the mRNA and protein level in aggressive luminal tumors, strongly associated with indicators of tumor proliferation and a predictor of poor prognosis. In vitro genetic studies demonstrated that CPT1A is required for and can promote luminal breast cancer proliferation, survival, as well as colony and mammosphere formation. Since CPT1A is the rate-limiting enzyme during fatty acid oxidation (FAO), our data indicate that FAO may be essential for these tumors. Pharmacologic inhibition of FAO prevented in vitro and in vivo tumor growth and cell proliferation as well as promoted apoptosis in luminal breast cancer cells and orthotopic xenograft tumor models. Collectively, our data establish an oncogenic role for CPT1A and FAO in HR+ luminal tumors and provide preclinical evidence and rationale supporting further investigation of FAO as a potential therapeutic opportunity for the treatment of HR+ breast cancer.

Abstract 2751: Secreted fatty acids from head and neck cancer cell lines mediate M2-like macrophage polarization

Abstract Head and neck cancers (HNC) can persist despite infiltration of immune cells into the tumor microenvironment. One mechanism that may contribute to failed immune clearance of tumors stems from the conditioning of tumor infiltrating macrophages into a cell type that supports tumor survival rather than a cell with pro-inflammatory characteristics. To understand mechanisms that lead to conditioning of macrophages by tumors, we assessed the effects of HNC cell lines on macrophage maturation. Macrophages were matured by incubating purified human monocytes for 3-4 days in medium alone or in medium supplemented with supernatants from HNC cell lines (FADU and SCC9) or a human leukemia monocytic cell line (THP-1). Macrophages were recovered and examined by flow cytometry for markers of maturation (CD14, CD68), activation (HLA-DR, CD86, and IL-15R), scavenger receptor (CD36), toll-like receptor (TLR4), M2 marker (CD206), immune checkpoint (PD-L1) and intracellular chemokine expression (IP-10). Relative to unconditioned macrophages or cells exposed to THP-1 supernatants, cells incubated with FADU or SCC9 supernatants displayed enhanced survival, downregulation of cell surface HLA-DR, CD86, IL-15R, CD36 and intracellular IP-10 expression. In contrast, the expression of PD-L1, CD14, and CD206 were increased by HNC cell line supernatants. To assess function, matured macrophages were stimulated overnight with the TLR4 agonist, LPS. Induction of macrophage activation was measured by increased expression of HLA-DR and IL-15R. Despite sustained the expression of TLR4 and CD14, macrophages matured in HNC supernatants failed to upregulate HLA-DR and IL-15R expression when stimulated with the canonical TLR4 agonist, LPS. These phenotypic and functional changes were accompanied by a decrease in intracellular phospho-p38 expression and a robust induction of WIP-1 phosphatase. Next, we considered the hypothesis that fatty acids released from tumors might contribute to these phenotypes. To test this, we added Etomoxir, an inhibitor of fatty acid oxidation, into cell cultures during exposure to tumor supernatants. The addition of Etomoxir reversed a number of these phenotypic changes induced by tumor supernatants with the exception of PD-L1 induction. Moreover, depletion of fatty acids from tumor supernatants with lipid removal agent, mitigated the effects of supernatants on macrophage maturation. Additionally, we found that macrophages incubated with either palmitic acid or oleic acid for 3-4 days developed similar phenotypes as cells incubated in tumor supernatants. Together, our data suggest that fatty acids are both necessary in tumor supernatants and sufficient on their own to mediate maturation of macrophages into a cell type with limited pro-inflammatory characteristics (M2-like). Approaches that modify the expression or effects of fatty acids in tumor microenvironment may be useful in HNC. Citation Format: Marwah M. Albakri, Stanley C. Huang, Hammad Tashkandi, Scott F. Sieg. Secreted fatty acids from head and neck cancer cell lines mediate M2-like macrophage polarization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2751.

Abstract 2326: Differential lipid metabolism in mammary cancer cell progression

Abstract There is an association between increased lipid accumulation and aggressive breast cancer phenotypes, and therefore poorer clinical outcomes. The mechanism by which dysregulated lipid metabolism may promote metastasis remains incompletely understood. Several potential mechanisms by which the neutral lipid storage organelles, cytoplasmic lipid droplets, may promote metastasis include utilization of fatty acids (FAs) for oxidation, protection against lipotoxcity, and production of lipid signaling mediators. We have previously demonstrated that metastatic MCF10CA1a mammary cancer cells rely on fatty acid oxidation (FAO) compared to non-metastatic MCF10A-ras cells, using etomoxir to inhibit the rate-limiting enzyme of FAO, carnitine palmitoyltransferase 1 (CPT1). Previous literature indicates that there is an increase in various lipid metabolism pathways, including FA uptake, de novo lipogenesis, and FA storage in neutral lipid. We found that there is no significant difference between palmitic acid uptake between the non-metastatic and metastatic cells. In the current study, we show that MCF10CA1a cells have significantly higher levels of carbon incorporation into palmitic and oleic FAs from 13C-acetate (25% and 20% greater), 13C-glucose (21% and 11% greater), and 13C-lactate (29% and 22% greater) respectively, compared to the MCF10A-ras cells, as detected by LC-MS/MS. In order to determine the source of FAs that contributes to the migration-dependent FAO, we assessed MCF10CA1a cell migration following inhibition of lipolysis (adipose triglyceride lipase—ATGL; ATGListatin) alone or in combination with the FAO inhibitor, etomoxir. We determined that inhibition of ATGL, the initial enzyme of triglyceride lipolysis pathway, significantly reduces MCF10CA1a cell migration (20% decrease compared to vehicle). Inhibition of FAO and lipolysis did not induce additional reduction in cell migration. We also showed that MCF10CA1a cells are more dependent on FA metabolism than the non-metastatic MCF10A-ras cells, demonstrated by MCF10CA1a cells having a greater decrease in cell viability compared to the MCF10A-ras cells given each FA metabolism inhibition (p&amp;lt;0.05: 22% greater decrease for FAO inhibition and 16% greater decrease for dual FAO and lipolysis inhibition, p=0.05: 17% greater decrease for FA synthesis inhibition). These data suggest that metastatic MCF10CA1a cells rely on their increased FA metabolism to survive and migrate, and specifically that the catabolism of FAs from triglyceride supports the FAO-driven migration of metastatic cancer cells. Citation Format: Chaylen Andoline, Parik Pawar, Dorothy Teegarden. Differential lipid metabolism in mammary cancer cell progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2326.

Inhibition of FAO in AML Co-Cultured with BM Adipocytes: Mechanisms of Survival and Chemosensitization to Cytarabine

Adipocytes are the prevalent type of stromal cells in adult bone marrow (BM), and fatty acids produced by adipocytes modulate the activity of signaling molecules. Leukemia cells are forced to continuously adapt to deficiency of nutrients and acquire chemoresistant profiles in the BM environment. We have previously shown that fatty acid metabolism is a key energy pathway for survival of acute myeloid leukemia (AML) cells in the adipocyte-abundant BM microenvironment (Tabe, Cancer Res. 2017) .The novel fatty acid oxidation (FAO) inhibitor avocatin B is an odd-numbered carbon lipid derived from the avocado fruit, and avocatin B-induced apoptosis and growth inhibition in mono-cultured AML cells has been demonstrated previously (Lee, Cancer Res. 2015, Tabe, ASH. 2016) . However, in AML cells co-cultured with mesenchymal stem cells (MSC)-derived BM-adipocyte (BM adipocyte) mimicking BM microenvironment, the cytotoxic effects of avocatin B were significantly diminished compared to mono-cultured condition (decrease of specific cell death; THP1 14.4%, U937 25.0%, OCI-AML3 36.2%, 48 h, trypan blue cell count). We therefore investigated the adaptive molecular mechanisms against avocatin B cytotoxicity in AML cells co-cultured with BM adipocytes. Under BM adipocyte co-culture conditions, avocatin B induced increase in reactive oxygen species (ROS) (cellROX, flow cytometry) and activation of stress-induced ATF4 and AMPK signaling; increase in phospho- (p-) AMPK and repression of p-S6, p-4E-BP1 (immunoblotting) in AML cells (THP1, U937, OCI-AML3). On the other hand, free fatty acid (FFA) and glucose uptake was stimulated after avocatin B treatment (fluorometric assay), which was accompanied by upregulation of FABP4 and CPT1 mRNA (Q-RT-PCR) and lactate and alanine (CE-MS). These changes might reflect the compensatory response to a shortage of FFA supply to the mitochondria.AMPK is known to regulate energy metabolism including FAO and glycolysis dependent / independent gene transcription through mTOR inhibition. To access the role of AMPK in anti-leukemic effects of avocatin B, we utilized AMPK knockdown (sh AMPK ) OCI-AML3 cells. In mono-culture condition, sh AMPK OCI-AML3 cells were less sensitive to avocatin B, showing higher baseline level of glucose uptake than parental shC OCI-AML3 cells, which was further increased by avocatin B. Of note, co-culture with BM adipocytes induced significant increase of glucose uptake in sh AMPK cells, which was not affected by avocatin B. In parental OCI-AML3 cells, FFA uptake was repressed by avocatin B in mono-culture but increased in BM adipocyte co-culture conditions, concordant with other tested AML cells. However, avocatin B-stimulated FFA uptake was not observed in sh AMPK OCI-AML3 cells regardless of the absence or presence of BM adipocytes. These results indicate that AMPK plays a role in adaptive uptake regulation of glucose and FFA in response to avocatin B-induced FAO suppression. We next performed ATF4 knockdown experiments to determine the role of ATF4. Whereas no significant difference in apoptotic responses to avocatin B was observed between mono-cultured sh ATF4 - and shC-OCI-AML3 cells, sh ATF4 OCI-AML3 cells demonstrated lower sensitivity to avocatin B compared to shC OCI-AML3 cells under the BM adipocyte co-culture condition (p<0.05). These findings suggest that avocatin B induced ATF4 activation facilitates apoptosis of AML cells co-cultured with BM adipocytesWe further investigated the effects of combinational treatment of avocatin B and conventional anti-AML therapeutic agent cytarabine (AraC). AraC itself increased ROS, and avocatin B / AraC combination synergistically induced cell growth inhibition of THP1 cells under BM adipocyte co-culture condition (Combination Index; mono-culture 0.7, co-culture 0.4). Immunoblotting analysis revealed that avocatin B induced p-S6 repression was accelerated by co-treatment with AraC irrespective of BM adipocyte presence, suggesting the efficient inhibition of mTOR by avocatin B / AraC combination under BM adipocyte co-culture condition.Taken together, these findings highlight the potential of AraC and FAO inhibitor combination which abrogates the adaptive pro-survival mechanisms and chemoresistance of BM residual AML cells. [Display omitted] DisclosuresAndreeff:Daiichi Sankyo: Consultancy.

Pharmacological inhibition of fatty acid oxidation reduces atherosclerosis progression by suppression of macrophage NLRP3 inflammasome activation

BackgroundInflammation is a key process during atherosclerotic lesion development and propagation. Recent evidence showed clearly that especially the inhibition of interleukin (IL)-1β reduced atherosclerotic adverse events in human patients. Fatty acid oxidation (FAO) was previously demonstrated to interact with the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) pathway which is required for mature IL-1β secretion. To understand possible anti-inflammatory properties of FAO inhibition, we tested the effect of pharmacological FAO inhibition using the inhibitor for long-chain 3-ketoacyl coenzyme A thiolase trimetazidine on atherosclerotic plaque development and inflammation. Experimental ApproachThe effect of FAO inhibition was determined in LDL-R−/− male mice on a C57/BL6 background. In vitro effects of trimetazidine treatment were analyzed in human umbilical vein endothelial cells and human monocyte derived macrophages. Key ResultsWe were able to demonstrate that inhibition of FAO reduced atherosclerotic plaque growth. We did not find direct anti-inflammatory properties of trimetazidine in endothelial cells or macrophages in vitro. However, we found that the activation of the NLRP3 system and the secretion of IL-1β were significantly reduced in macrophages after FAO inhibition. These results were confirmed in atherosclerotic lesions of mice treated with trimetazidine as they showed a significant reduction of IL-1β and cleaved caspase-1 in the atherosclerotic lesion as well as of IL-1β and IL-18 in the circulation. ConclusionOverall, we therefore suggest that the main mechanism of reducing inflammation of trimetazidine and FAO inhibition is the reduction of the NLRP-3 activation leading to reduced levels of the proinflammatory cytokine IL-1β.

Targeting Adrenergic Receptors in Metabolic Therapies for Heart Failure.

The heart has a reduced capacity to generate sufficient energy when failing, resulting in an energy-starved condition with diminished functions. Studies have identified numerous changes in metabolic pathways in the failing heart that result in reduced oxidation of both glucose and fatty acid substrates, defects in mitochondrial functions and oxidative phosphorylation, and inefficient substrate utilization for the ATP that is produced. Recent early-phase clinical studies indicate that inhibitors of fatty acid oxidation and antioxidants that target the mitochondria may improve heart function during failure by increasing compensatory glucose oxidation. Adrenergic receptors (α1 and β) are a key sympathetic nervous system regulator that controls cardiac function. β-AR blockers are an established treatment for heart failure and α1A-AR agonists have potential therapeutic benefit. Besides regulating inotropy and chronotropy, α1- and β-adrenergic receptors also regulate metabolic functions in the heart that underlie many cardiac benefits. This review will highlight recent studies that describe how adrenergic receptor-mediated metabolic pathways may be able to restore cardiac energetics to non-failing levels that may offer promising therapeutic strategies.

Orphan nuclear receptor COUP-TFII enhances myofibroblast glycolysis leading to kidney fibrosis.

Recent studies demonstrate that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in human fibrotic kidneys, lungs, kidney organoids, and mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts also induced production of alpha-smooth muscle actin (αSMA) and collagen 1. Knockout of COUP-TFII decreased glycolysis and collagen 1 levels in fibroblasts. Chip-qPCR revealed the binding of COUP-TFII on the promoter of PGC1α. Overexpression of COUP-TFII reduced the cellular level of PGC1α. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potentially fibrosis in other organs.

Adipocyte-like signature in ovarian cancer minimal residual disease identifies metabolic vulnerabilities of tumor-initiating cells.

Similar to tumor-initiating cells (TICs), minimal residual disease (MRD) is capable of reinitiating tumors and causing recurrence. However, the molecular characteristics of solid tumor MRD cells and drivers of their survival have remained elusive. Here we performed dense multiregion transcriptomics analysis of paired biopsies from 17 ovarian cancer patients before and after chemotherapy. We reveal that while MRD cells share important molecular signatures with TICs, they are also characterized by an adipocyte-like gene expression signature and a portion of them had undergone epithelial-mesenchymal transition (EMT). In a cell culture MRD model, MRD-mimic cells showed the same phenotype and were dependent on fatty acid oxidation (FAO) for survival and resistance to cytotoxic agents. These findings identify EMT and FAO as attractive targets to eradicate MRD in ovarian cancer and make a compelling case for the further testing of FAO inhibitors in treating MRD.