Inhibition Of acetyl-CoA Carboxylase Research Articles

Herbicide resistance in China: a quantitative review

AbstractThe widespread, rapid evolution of herbicide-resistant weeds is a serious and escalating agronomic problem worldwide. During China’s economic boom, the country became one of the most important herbicide producers and consumers in the world, and herbicide resistance has dramatically increased in the past decade and has become a serious threat to agriculture. Here, following an evidence-based PRISMA (preferred reporting items for systematic reviews and meta-analyses) approach, we carried out a systematic review to quantitatively assess herbicide resistance in China. Multiple weed species, including 26, 18, 11, 9, 5, 5, 4, and 3 species in rice (Oryza sativa L.), wheat (Triticum aestivum L.), soybean [Glycine max (L.) Merr.], corn (Zea mays L.), canola (Brassica napus L.), cotton (Gossypium hirsutum L.)., orchards, and peanut (Arachis hypogaea L.) fields, respectively, have developed herbicide resistance. Acetolactate synthase inhibitors, acetyl-CoA carboxylase inhibitors, and synthetic auxin herbicides are the most resistance-prone herbicides and are the most frequently used mechanisms of action, followed by 5-enolpyruvylshikimate-3-phosphate synthase inhibitors and protoporphyrinogen oxidase inhibitors. The lack of alternative herbicides to manage weeds that exhibit cross-resistance or multiple resistance (or both) is an emerging issue and poses one of the greatest threats challenging the crop production and food safety both in China and globally.

Deployment of an attractive toxic sugar bait system (ATSB) with insecticide, for adult Tribolium castaneum (Coleoptera: Tenebrionidae)

Tribolium castaneum is a critical stored-grain pest of nuts and cereals. Insecticides have traditionally been used to control it; however, their overuse can result in the development of insecticide resistance. Effective bait pesticides could be useful tools for slowing down the rate of insecticide-resistance development and improving the management of stored grain pests. Attractive toxic sugar bait (ATSB) systems are used with mosquitoes and flies; they lure pests with liquid foods that contain sugars but also an oral toxin. It is unknown whether the ATSB system is applicable to T. castaneum adults who prefer dry food products with reduced water content. The objective of this study is to develop an optimal ATSB system for T. castaneum control. Thus far, it found that T. castaneum consumed a gypsum block. The dietary intake of T. castaneum adult beetles was facilitated with added mannitol into gypsum block. Combining insecticides that are oral toxins of the pests with these facilitating sugars results in the death of the pest. Spirotetramat causes a significant reduction in fatty acid biosynthesis by inhibiting acetyl-CoA carboxylase. In this investigation, we have demonstrated that spirotetramat, at low concentrations, was lethal to T. castaneum adults, when used with the ATSB system, combined with mannitol. The lethality of chlorfenapyr, an inhibitor of mitochondrial ATP synthesis, to adult T. castaneum did not increase when used in the ATSB system with mannitol. These results show that the active ingredient used in the ATSB system is integral for beetle control. Increasing the oral delivery rate of insecticides using carbohydrates leads to increased lethality rates of stored-grain pests.

WRINKLED1 Regulates BIOTIN ATTACHMENT DOMAIN-CONTAINING Proteins that Inhibit Fatty Acid Synthesis.

WRINKLED1 (WRI1) is a transcriptional activator that binds to a conserved sequence (designated as AW box) boxes in the promoters of many genes from central metabolism and fatty acid (FA) synthesis, resulting in their transcription. BIOTIN ATTACHMENT DOMAIN-CONTAINING (BADC) proteins lack a biotin-attachment domain and are therefore inactive, but in the presence of excess FA, BADC1 and BADC3 are primarily responsible for the observed long-term irreversible inhibition of ACETYL-COA CARBOXYLASE, and consequently FA synthesis. Here, we tested the interaction of WRI1 with BADC genes in Arabidopsis (Arabidopsis thaliana) and found purified WRI1 bound with high affinity to canonical AW boxes from the promoters of all three BADC genes. Consistent with this observation, both expression of BADC1, BADC2, and BADC3 genes and BADC1 protein levels were reduced in wri1-1 relative to the wild type, and elevated upon WRI1 overexpression. The double mutant badc1 badc2 phenocopied wri1-1 with respect to both reduction in root length and elevation of indole-3-acetic acid-Asp levels relative to the wild type. Overexpression of BADC1 in wri1-1 decreased indole-3-acetic acid-Asp content and partially rescued its short-root phenotype, demonstrating a role for BADCs in seedling establishment. That WRI1 positively regulates genes encoding both FA synthesis and BADC proteins (i.e. conditional inhibitors of FA synthesis), represents a coordinated mechanism to achieve lipid homeostasis in which plants couple the transcription of their FA synthetic capacity with their capacity to biochemically downregulate it.

1775-P: Impact of Acetyl-CoA Carboxylase 2 Inhibition on Skeletal Muscle Diacylglycerol and Ceramide Levels and Its Potential Contribution to Improved Glucose Metabolism

Intramyocellular lipid (IMCL) accumulation in skeletal muscle is closely associated with the development of insulin resistance. In particular, bioactive lipids such as diacylglycerol (DAG) and ceramide (Cer) have great impact on muscle insulin action. We have recently demonstrated that inhibition of acetyl-CoA carboxylase 2 (ACC2), which negatively modulates mitochondrial fatty acid oxidation in skeletal muscle, reduced IMCL levels and improved glucose metabolism in rodents. To further investigate the underlying mechanism, we examined the impact of pharmacological ACC2 inhibition on bioactive lipid composition and metabolic functions in skeletal muscle of rats, using an ACC2 selective inhibitor, compound A. As compared with vehicle treatment, chronic treatment with compound A significantly reduced both DAG and Cer levels in skeletal muscle of nondiabetic rats, which were strongly correlated with IMCL levels. Compound A also reduced the acyl-CoA pool along with enhanced acylcarnitine formation in skeletal muscle, while there were no differences in other bioactive lipid levels and DAG or Cer synthetic gene expression levels. Similarly, in Zucker diabetic fatty (ZDF) rats, compound A reduced both DAG and Cer levels in skeletal muscle without suppressing the synthetic gene expression levels. Treatment with compound A led to improved hyperinsulinemia and upregulated expression of key regulators in energy metabolism, such as Glut4 and Pgc1α, in skeletal muscle. Moreover, these positive effects on glucose metabolism were strongly correlated with diacylglycerol and ceramide reductions. Collectively, these findings suggest that ACC2 inhibition leads to preferential reductions of DAG and Cer by directly decreasing acyl-CoA pool via fatty acid oxidation in skeletal muscle, and these lipid reductions are closely linked to the improvement of glucose metabolism by ACC2 inhibition. Disclosure H. Takagi: None. T. Ikehara: None. K. Hashimoto: None. A. Shimazaki: None. Y. Kashiwagi: None. Y. Nishiura: None. M. Notoya: None. H. Yukioka: None.

Human sebum requires de novo lipogenesis, which is increased in acne vulgaris and suppressed by acetyl-CoA carboxylase inhibition.

Sebum plays important physiological roles in human skin. Excess sebum production contributes to the pathogenesis of acne vulgaris, and suppression of sebum production reduces acne incidence and severity. We demonstrate that sebum production in humans depends on local flux through the de novo lipogenesis (DNL) pathway within the sebocyte. About 80 to 85% of sebum palmitate (16:0) and sapienate (16:1n10) were derived from DNL, based on stable isotope labeling, much higher than the contribution of DNL to triglyceride palmitate in circulation (~20%), indicating a minor contribution by nonskin sources to sebum lipids. This dependence on local sebocyte DNL was not recapitulated in two widely used animal models of sebum production, Syrian hamsters and Göttingen minipigs. Confirming the importance of DNL for human sebum production, an acetyl-CoA carboxylase inhibitor, ACCi-1, dose-dependently suppressed DNL and blocked synthesis of fatty acids, triglycerides, and wax esters but not free sterols in human sebocytes in vitro. ACCi-1 dose-dependently suppressed facial sebum excretion by ~50% (placebo adjusted) in human individuals dosed orally for 2 weeks. Sebum triglycerides, wax esters, and free fatty acids were suppressed by ~66%, whereas non-DNL-dependent lipid species, cholesterol, and squalene were not reduced, confirming selective modulation of DNL-dependent lipids. Last, individuals with acne vulgaris exhibited increased sebum production rates relative to individuals with normal skin, with >80% of palmitate and sapienate derived from DNL. These findings highlight the importance of local sebocyte DNL for human skin sebaceous gland biology and illuminate a potentially exploitable therapeutic target for the treatment of acne vulgaris.

Trp-1999-Ser mutation of acetyl-CoA carboxylase and cytochrome P450s-involved metabolism confer resistance to fenoxaprop-P-ethyl in Polypogon fugax.

Asia minor bluegrass (Polypogon fugax Nees ex Steud.) is an invasive grass species severely infesting wheat and canola fields in China. In May 2017, a suspected resistant P. fugax population AHHY that survived fenoxaprop-P-ethyl applied at its field-recommended rate was collected from a wheat field in Huaiyuan County, Anhui Province, China. This study aimed to determine the resistance profile of AHHY to acetyl-CoA carboxylase (ACCase) inhibitors and to investigate its mechanisms of resistance to fenoxaprop. Single-dose testing indicated that the AHHY population had evolved resistance to fenoxaprop. The partial carboxyltransferase domain of ACCase in P. fugax was amplified and compared. Four loci encoding plastidic ACCase were isolated from both the resistant and sensitive individuals. Combining gene sequencing with the derived cleaved amplified polymorphic sequence assay, we found that 100% of the plants of AHHY carried Trp-1999-Ser mutation in their ACCase1,1-2 allele. Whole-plant dose-response bioassay indicated that AHHY was highly resistant to fenoxaprop and pinoxaden (resistance index (RI) ≥ 10) with low resistance to clodinafop-propargyl, sethoxydim, and clethodim (2 ≤ RI < 5). Pre-treatment with piperonyl butoxide largely reduced (55%) the weed's resistance to fenoxaprop. Both basal and fenoxaprop-induced glutathione S-transferases activities toward 1-chloro-2, 4-dinitrobenzene were significantly higher in resistant plants than in susceptible plants. This study revealed that P. fugax had multiple alleles encoding plastidic ACCase, and reported for the first time the occurrence of Trp-1999-Ser mutation and non-target-site resistance in this species. Fenoxaprop resistance in AHHY plants was conferred by target-site mutation and P450s-involved enhanced metabolism. © 2019 Society of Chemical Industry.

Enhanced metabolism causes reduced flufenacet sensitivity in black-grass (Alopecurus myosuroides Huds.) field populations.

Black-grass (Alopecurus myosuroides Huds.) is a frequent grass weed that commonly occurs in winter wheat in temperate Europe. Evolving resistance to post-emergence herbicides, e.g. acetyl CoA carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors requires more complex weed management strategies and ensuring good efficacy of pre-emergence treatments becomes increasingly important. Flufenacet, in particular, has become a key herbicide for the control of multiple-resistant A. myosuroides. However, in some of those populations, reduced flufenacet efficacy was already observed. In a screening of black-grass populations from several European countries, most populations were controlled with the registered field rate of flufenacet. However, differences in the level of flufenacet sensitivity were observed and correlated with glutathione S-transferase-mediated enhanced flufenacet metabolism. The efficacy of the pre-emergence herbicides pendimethalin, prosulfocarb, S-metolachlor and pethoxamid, was also significantly decreased in populations with reduced flufenacet sensitivity. The use of flufenacet in mixtures with diflufenican, particularly in combination with flurtamone or metribuzin, however, significantly improved efficacy in less susceptible black-grass populations. In several populations of different European origins, reduced efficacy of flufenacet was observed due to enhanced metabolism. Although differences between populations were relatively small, best weed management practices (e.g. application of full dose rates and herbicide mixtures and wide crop rotations) should be applied to reduce selection pressure and prevent flufenacet resistance from further evolving. This is particularly important as flufenacet is one of the few still-effective herbicides suitable for the control of multiple-resistant A. myosuroides genotypes in Europe, whereas alternative pre-emergence herbicides were less effective against multiple-resistant A. myosuroides populations. © 2019 Society of Chemical Industry.

Virtual Screening Strategy Combined Bayesian Classification Model, Molecular Docking for Acetyl-CoA Carboxylases Inhibitors.

Acetyl-CoA Carboxylases (ACC) have been an important target for the therapy of metabolic syndrome, such as obesity, hepatic steatosis, insulin resistance, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), type 2 diabetes (T2DM), and some other diseases. In this study, virtual screening strategy combined with Bayesian categorization modeling, molecular docking and binding site analysis with protein ligand interaction fingerprint (PLIF) was adopted to validate some potent ACC inhibitors. First, the best Bayesian model with an excellent value of Area Under Curve (AUC) value (training set AUC: 0.972, test set AUC: 0.955) was used to screen compounds of validation library. Then the compounds screened by best Bayesian model were further screened by molecule docking again. Finally, the hit compounds evaluated with four percentages (1%, 2%, 5%, 10%) were verified to reveal enrichment rates for the compounds. The combination of the ligandbased Bayesian model and structure-based virtual screening resulted in the identification of top four compounds which exhibited excellent IC 50 values against ACC in top 1% of the validation library. In summary, the whole strategy is of high efficiency, and would be helpful for the discovery of ACC inhibitors and some other target inhibitors.

PS-109-Partial inhibition of de novo lipogenesis with the acetyl-CoA carboxylase inhibitor PF-05221304 does not increase circulating triglycerides in humans and is sufficient to lower steatosis in rats

PS-109-Partial inhibition of de novo lipogenesis with the acetyl-CoA carboxylase inhibitor PF-05221304 does not increase circulating triglycerides in humans and is sufficient to lower steatosis in rats

FRI-284-A combination of fenofibrate with a liver-targeted acetyl-CoA carboxylase inhibitor enhances efficacy while reversing plasma triglyceride increases in a murine model of NASH

FRI-284-A combination of fenofibrate with a liver-targeted acetyl-CoA carboxylase inhibitor enhances efficacy while reversing plasma triglyceride increases in a murine model of NASH

PS-132-The acetyl-CoA carboxylase inhibitor PF-05221304 exerts direct effects on hepatic inflammation and fibrosis independent of benefits on steatosis

PS-132-The acetyl-CoA carboxylase inhibitor PF-05221304 exerts direct effects on hepatic inflammation and fibrosis independent of benefits on steatosis

Acetyl-CoA carboxylase inhibition regulates microtubule dynamics and intracellular transport in cystic fibrosis epithelial cells.

The use of high-dose ibuprofen as an anti-inflammatory therapy in cystic fibrosis (CF) has been shown to be an effective intervention although use is limited due to potential adverse events. Identifying the mechanism of ibuprofen efficacy would aid in the development of new therapies that avoid these adverse events. Previous findings demonstrated that ibuprofen treatment restores the regulation of microtubule dynamics in CF epithelial cells through a 5'-adenosine monophosphate-activated protein kinase (AMPK)-dependent mechanism. The goal of this study is to define the AMPK pathway that leads to microtubule regulation. Here, it is identified that inhibition of acetyl-CoA carboxylase (ACC) is the key step in mediating the AMPK effect. ACC inhibition with 5-(tetradecyloxy)-2-furoic acid (TOFA) increases microtubule reformation rates in cultured and primary CF epithelial cells to wild-type (WT) rates. TOFA treatment also restores microtubule-dependent distribution of cholesterol and Rab7-positive organelles, as well as reduces expression of the proinflammatory signaling molecule RhoA to WT levels. ACC activation with citrate replicates these CF phenotypes in WT cells further supporting the role of AMPK signaling through ACC as a key mediator in CF cell signaling. It is concluded that ACC inhibition is the key step in the efficacy of AMPK activation at the cellular level and could represent a novel site of therapeutic intervention to address inflammation in CF.

Long-Term Population Studies Uncover the Genome Structure and Genetic Basis of Xenobiotic and Host Plant Adaptation in the Herbivore Tetranychus urticae.

Pesticide resistance arises rapidly in arthropod herbivores, as can host plant adaptation, and both are significant problems in agriculture. These traits have been challenging to study as both are often polygenic and many arthropods are genetically intractable. Here, we examined the genetic architecture of pesticide resistance and host plant adaptation in the two-spotted spider mite, Tetranychus urticae, a global agricultural pest. We show that the short generation time and high fecundity of T. urticae can be readily exploited in experimental evolution designs for high-resolution mapping of quantitative traits. As revealed by selection with spirodiclofen, an acetyl-CoA carboxylase inhibitor, in populations from a cross between a spirodiclofen-resistant and a spirodiclofen-susceptible strain, and which also differed in performance on tomato, we found that a limited number of loci could explain quantitative resistance to this compound. These were resolved to narrow genomic intervals, suggesting specific candidate genes, including acetyl-CoA carboxylase itself, clustered and copy variable cytochrome P450 genes, and NADPH cytochrome P450 reductase, which encodes a redox partner for cytochrome P450s. For performance on tomato, candidate genomic regions for response to selection were distinct from those responding to the synthetic compound and were consistent with a more polygenic architecture. In accomplishing this work, we exploited the continuous nature of allele frequency changes across experimental populations to resolve the existing fragmented T. urticae draft genome to pseudochromosomes. This improved assembly was indispensable for our analyses, as it will be for future research with this model herbivore that is exceptionally amenable to genetic studies.

Salicylate enhances the response of prostate cancer to radiotherapy.

Radiotherapy (RT) is a key therapeutic modality for prostate cancer (PrCa), but RT resistance necessitates dose-escalation, often causing bladder and rectal toxicity. Aspirin, a prodrug of salicylate (SAL), has been associated with improved RT response in clinical PrCa cases, but the potential mechanism mediating this effect is unknown. SAL activates the metabolic stress sensor AMP-activated protein kinase (AMPK), which inhibits de novo lipogenesis, and protein synthesis via inhibition of Acetyl-CoA Carboxylase (ACC), and the mammalian Target of Rapamycin (mTOR), respectively. RT also activates AMPK through a mechanism distinctly different from SAL. Therefore, combining these two therapies may have synergistic effects on suppressing PrCa. Here, we examined the potential of SAL to enhance the response of human PrCa cells and tumors to RT. Androgen-insensitive (PC3) and -sensitive (LNCaP) PrCa cells were subjected to proliferation and clonogenic survival assays after treatment with clinically relevant doses of SAL and RT. Balb/c nude mice with PC3 xenografts were fed standard chow diet or chow diet supplemented with 2.5 g/kg salsalate (SAL pro-drug dimer) one week prior to a single dose of 0 or 10 Gy RT. Immunoblotting analysis of signaling events in the DNA repair and AMPK-mTOR pathways and lipogenesis were assessed in cells treated with SAL and RT. SAL inhibited proliferation and clonogenic survival in PrCa cells and enhanced the inhibition mediated by RT. Salsalate, added to diet, enhanced the anti-tumor effects of RT in PC3 tumor xenografts. RT activated genotoxic stress markers and the activity of mTOR pathway and AMPK and mediated inhibitory phosphorylation of ACC. Interestingly, SAL enhanced the effects of RT on AMPK and ACC but blocked markers of mTOR activation. Our results show that SAL can enhance RT responses in PrCa. Salsalate is a promising agent to investigate this concept in prospective clinical trials of PrCa in combination with RT.

The intriguing chemistry and biology of soraphens.

Covering: up to the end of 2018Soraphens are a class of polyketide natural products discovered from the myxobacterial strain Sorangium cellulosum. The review is intended to provide an overview on the biosynthesis, chemistry and biological properties of soraphens, that represent a prime example to showcase the value of natural products as tools to decipher cell biology, but also to open novel therapeutic options. The prototype soraphen A is an inhibitor of acetyl coenzyme A carboxylase (ACC1/2), an enzyme that converts acetyl-CoA to malonyl-CoA and thereby controls essential cellular metabolic processes like lipogenesis and fatty acid oxidation. Soraphens illustrate how the inhibition of a single target (ACC1/2) may be explored to treat various pathological conditions: initially developed as a fungicide, efforts in the past decade were directed towards human diseases, including diabetes/obesity, cancer, hepatitis C, HIV, and autoimmune disease - and led to a synthetic molecule, discovered by virtual screening of the allosteric binding site of soraphen in ACC, that is currently in phase 2 clinical trials. We will summarize how structural analogs of soraphen A have been generated through extensive isolation efforts, genetic engineering of the biosynthetic gene cluster, semisynthesis as well as partial and total synthesis.

Enterococcus faecalis extracts FK-23 modulates Propionibacterium acnes-induced lipogenesis in human sebocytes SZ-95 cells

It is generally accepted that Propionibacterium acnes (P. Acne) is involved in the development of acne, while the mechanisms of sebaceous lipogenesis and its control is unclear. Enterococcus faecalis FK-23 had shown to promote an anti-inflammatory action in several animal models. In the current study, we examined whether FK-23 modulate lipogenesis in sebocytes. FK-23 stimulated lipogenesis, while inhibited them in the presence of P. Acne. FK-23 acutely inhibited acetyl-CoA carboxylase (ACC) phosphorylation levels, while stimulated them with P. Acne. FK-23 stimulated PPARγ expression and activity, while inhibited them with P. Acne as pioglitazone did. These combined evidences demonstrated that the dual action by FK-23 on lipogenesis should reflect differentiation machinery prior to PPARγ, leading to producing adequate levels of sebum.

Effects of intravenous AICAR (5-aminoimidazole-4-carboximide riboside) administration on insulin signaling and resistance in premature baboons, Papio sp.

Premature baboons exhibit peripheral insulin resistance and impaired insulin signaling. 5’ AMP-activated protein kinase (AMPK) activation improves insulin sensitivity by enhancing glucose uptake (via increased glucose transporter type 4 [GLUT4] translocation and activation of the extracellular signal-regulated kinase [ERK]/ atypical protein kinase C [aPKC] pathway), and increasing fatty acid oxidation (via inhibition of acetyl-CoA carboxylase 1 [ACC]), while downregulating gluconeogenesis (via induction of small heterodimer partner [SHP] and subsequent downregulation of the gluconeogenic enzymes: phosphoenolpyruvate carboxykinase [PEPCK], glucose 6-phosphatase [G6PASE], fructose- 1,6-bisphosphatase 1 [FBP1], and forkhead box protein 1 [FOXO1]). The purpose of this study was to investigate whether pharmacologic activation of AMPK with AICAR (5-aminoimidazole-4-carboximide riboside) administration improves peripheral insulin sensitivity in preterm baboons. 11 baboons were delivered prematurely at 125±2 days (67%) gestation. 5 animals were randomized to receive 5 days of continuous AICAR infusion at a dose of 0.5 mg·g-1·day-1. 6 animals were in the placebo group. Euglycemic hyperinsulinemic clamps were performed at 5±2 and 14±2 days of life. Key molecules potentially altered by AICAR (AMPK, GLUT4, ACC, PEPCK, G6PASE, FBP1, and FOXO1), and the insulin signaling molecules: insulin receptor (INSR), insulin receptor substrate 1 (IRS-1), protein kinase B (AKT), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were measured using RT-PCR and western blotting. AICAR infusion did not improve whole body insulin-stimulated glucose disposal in preterm baboons (12.8±2.4 vs 12.4±2.0 mg/(kg·min), p = 0.8, placebo vs AICAR). One animal developed complications during treatment. In skeletal muscle, AICAR infusion did not increase phosphorylation of ACC, AKT, or AMPK whereas it increased mRNA expression of ACACA (ACC), AKT, and PPARGC1A (PGC1α). In the liver, INSR, IRS1, G6PC3, AKT, PCK1, FOXO1, and FBP1 were unchanged, whereas PPARGC1A mRNA expression increased after AICAR infusion. This study provides evidence that AICAR does not improve insulin sensitivity in premature euglycemic baboons, and may have adverse effects.

Glutamine Deprivation Enhances Acetyl-CoA Carboxylase Inhibitor-induced Death of Human Pancreatic Cancer Cells.

Acetyl-CoA carboxylase (ACC) is a rate-limiting enzyme in fatty acid synthesis. In this study, we investigated the effect of ACC inhibition on survival of pancreatic cancer cells. AsPC-1, BxPC-3 and PANC-1 were used as human pancreatic cancer cell lines. 5-(etradecyloxy)-2-furoic acid (TOFA) and bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide (BPTES) were used as inhibitors of ACC and glutaminase (GLS) respectively. Apoptotic and live cells were distinguished by annexin-V staining. The activity of caspase-3 was evaluated by measuring the fluorescence intensity of the degradation product of the substrate, N-acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin. TOFA increased the number of annexin V-positive cells and enhanced caspase-3 activity in AsPC-1 and BxPC-3, but not in PANC-1 cells. The number of PANC-1 cells increased after 48 h in Earle's balanced salt solution. Interestingly, proliferation of PANC-1 cells was drastically suppressed by glutamine deprivation, but not by inhibition of glycolysis. BPTES also induced cell death to the same extent as glutamine deprivation. In addition, TOFA induced cell death of PANC-1 cells, both in the presence of BPTES and with glutamine deprivation, suggesting that inhibition of glutaminolysis causes cell death and enhances the effect of TOFA in PANC-1 cells. These findings suggest that glutaminolysis is important for the survival of pancreatic cancer cells showing tolerance to nutrient starvation such as PANC-1 cells, and use of a combination of inhibitors of ACC and GLS may be a new strategy for treatment of pancreatic cancer.

Acetyl-CoA Carboxylase Inhibition Reverses NAFLD and Hepatic Insulin Resistance but Promotes Hypertriglyceridemia in Rodents.

Pharmacologic inhibition of acetyl-CoA carboxylase (ACC) enzymes, ACC1 and ACC2, offers an attractive therapeutic strategy for nonalcoholic fatty liver disease (NAFLD) through simultaneous inhibition of fatty acid synthesis and stimulation of fatty acid oxidation. However, the effects of ACC inhibition on hepatic mitochondrial oxidation, anaplerosis, and ketogenesis in vivo are unknown. Here, we evaluated the effect of a liver-directed allosteric inhibitor of ACC1 and ACC2 (Compound 1) on these parameters, as well as glucose and lipid metabolism, in control and diet-induced rodent models of NAFLD. Oral administration of Compound 1 preferentially inhibited ACC enzymatic activity in the liver, reduced hepatic malonyl-CoA levels, and enhanced hepatic ketogenesis by 50%. Furthermore, administration for 6 days to high-fructose-fed rats resulted in a 20% reduction in hepatic de novo lipogenesis. Importantly, long-term treatment (21 days) significantly reduced high-fat sucrose diet-induced hepatic steatosis, protein kinase C epsilon activation, and hepatic insulin resistance. ACCi treatment was associated with a significant increase in plasma triglycerides (approximately 30% to 130%, depending on the length of fasting). ACCi-mediated hypertriglyceridemia could be attributed to approximately a 15% increase in hepatic very low-density lipoprotein production and approximately a 20% reduction in triglyceride clearance by lipoprotein lipase (P ≤ 0.05). At the molecular level, these changes were associated with increases in liver X receptor/sterol response element-binding protein-1 and decreases in peroxisome proliferator-activated receptor-α target activation and could be reversed with fenofibrate co-treatment in a high-fat diet mouse model. Conclusion: Collectively, these studies warrant further investigation into the therapeutic utility of liver-directed ACC inhibition for the treatment of NAFLD and hepatic insulin resistance.

IDH1 Mutant AML Is Susceptible to Targeting De Novo Lipid Synthesis Independent of 2-Hydroxyglutarate and Has a Distinct Metabolic Profile from IDH2 Mutant AML

▪Introduction: Mutations in IDH1 and IDH2 are recurrent in AML and several other cancers, resulting in the aberrant production of the onco-metabolite, R-2-hydroxyglutarate (2-HG), as well as an inability of mutant IDH1 to convert cytoplasmic alpha-ketoglutarate to isocitrate via reductive carboxylation. Currently, inhibitors of the neomorphic enzymes that abrogate the production of 2-HG, such as AG-120, are FDA-approved, but are not curative. Using a novel computational method (MiSL), we identified acetyl CoA carboxylase (ACACA) as a potential druggable target specifically in IDH1-mutated AML. ACACA regulates the de novo synthesis of lipid precursors by converting acetyl CoA to malonyl CoA building blocks. We hypothesize that IDH1 mutant AML exhibits a defect in reductive carboxylation and de novo fatty acid synthesis conferring preferential susceptibility to ACACA inhibition. Here, we investigate this hypothesis by comprehensively quantifying the metabolic landscape, including non-polar lipid metabolites, conferred by IDH1 R132H mutation compared to IDH2 mutation in isogenic cell lines and primary samples. Moreover, we investigate the in vitro and in vivo effects of targeting de novo lipid synthesis on IDH1 and IDH2 mutant AML.Methods: Comprehensive metabolomic profiling of primary FACS-purified AML blasts was performed using an in-house protocol optimised for extraction of non-polar lipid metabolites from less than 1 million primary cells. CD33+CD45+ leukemic blasts were profiled from 17 patient samples with IDH1 mutation (n=6), IDH2 mutation (n=5), or IDH1/2 wildtype (n=6) after culturing in serum-free media. 6 independent cord-blood CD34+ cells were profiled as a negative control. For validation of IDH1-specific effects, isogenic THP-1 cells transduced with doxycycline-inducible wildtype and R132H mutant IDH1 or R140Q mutant and wildtype IDH2 were profiled. Molecules were identified according to their molecular weight and retention time using Mass Hunter software (Agilent) and the Human Metabolome Database. For in vivo studies, primary AML samples were engrafted in NSG mice that were subjected to dietary modification with low lipid diet and/or treatment with selective inhibitors of ACACA and mutant IDH1.Results: Principle component analysis of metabolite abundance of 1400 unique compounds revealed striking differences between IDH1 and IDH2 mutant AML. Both IDH1 and IDH2 mutant samples produced high levels of 2-HG compared to wildtype AML and CD34+ cells (50 fold increase, P=4.5E-05). A major perturbation in multiple phospholipid fatty acid species was conferred by IDH1 R132H, but not by IDH2 mutation. The same pattern was observed in cell lines with 49 lipid species decreased in the presence of mutant IDH1 compared to only 2 perturbed with mutant IDH2. Direct comparison of IDH1 vs IDH2 mutant primary samples revealed 54 lipid metabolites significantly down-regulated in IDH1 mutant blasts (adjusted P value <0.05).To investigate the effects of targeting de novo lipid synthesis on IDH1 mutant AML in vivo, we engrafted primary IDH1 mutant AML and tested growth with lipid-free compared to normal diet. At 12 weeks, IDH1 mutant AML showed reduced growth in the bone marrow of mice on lipid-free diet (SU389 11% vs 40%, n=10 mice, P=0.03 and SU372 21% vs 34%, n=10, P=0.02 Mann-Whitney U). IDH1 mutant AML was susceptible to ACACA inhibition with shRNA, CRISPR targeting, or selective nanomolar inhibitors. Knockdown of ACACA with independent shRNAs caused a defect in cell growth in the presence of IDH1 R132H, but not in its absence or with scrambled shRNA (p=0.009, shRNA #1 vs. scrambled; p=0.01, shRNA #2 vs. scrambled) in vitro and in xenografts. Primary IDH1 R132 mutated AML blasts were selectively sensitive to ACACA inhibitor treatment compared to IDH1 wildtype normal karyotype blasts (IC50 0.6 uM vs 6 uM, p=0.009). Notably, IDH1-mutant AML blasts pre-treated with 10mM AG-120 remained susceptible to ACACA inhibition, identifying a 2-HG independent vulnerability. Similar findings were observed in a solid tumor IDH1 mutant sarcoma model in vivo.Conclusion: These results support our hypothesis that IDH1 mutant AML exhibits a defect in de novo fatty acid synthesis conferring preferential susceptibility to ACACA inhibition, and suggests that pharmacologic inhibitors of ACACA may complement IDH1 mutation-specific inhibitors in the clinic. DisclosuresNo relevant conflicts of interest to declare.