Phosphorylation Of ATP-citrate Lyase Research Articles

Abstract P3029: The Role Of Fatty Acid Synthase In The Modulation Of Vascular Smooth Muscle Cells Into A Foam Cell Phenotype

Background: The contractile phenotype of differentiated VSMCs is essential for vascular function. When exposed to Cholesterol (CHO), VSMCs dedifferentiate and acquire characteristics of foam cells, lipid-laden macrophage-like cells that comprise the Atherosclerotic plaques. CHO uptake by VSMCs is either removed by ABCA1-mediated efflux or is stored in lipid droplets as cholesterol esters (CEs). CEs are formed by the reaction of CHO with fatty acids catalyzed by SOAT1. Thus, we hypothesize that fatty acid biosynthesis controls the formation of VSMC-derived foam cells. Results: In VSMCs, CHO treatment upregulates the lipogenic enzymes FASN (3.6±1 vs. 9.0±0.9 AU p<0.01) and acetyl-CoA carboxylase (ACC) (2.8±0.5 vs. 10.2±2.3 AU p=0.02) and induces the phosphorylation of ATP Citrate Lyase (ACLY) (1.4±0 vs. 6.2±1 AU). Consistent with the literature, these changes are accompanied by increased expression of the macrophage-related markers CD68 and KLF4. Significantly, downregulation of FASN inhibits the CHO-induced CD68 (9.1±0.8 vs. 4.6±1,9 p=0.002) and KLF4 (p=0.02) expression upregulation. FASN-deficient VSMCs failed to accumulate lipids and transition to a foam cell phenotype after CHO treatment measured by Oil Red O staining. Interestingly, FASN-deficient VSMCs have decreased expression of SOAT1 (p=0.04) and increased expression of the CHO efflux transporter ABCA1 (p=0.04). Conclusion: Our study shows lipogenic enzymes are upregulated during the VSMC to foam cell transition triggered by CHO treatment. FASN expression is required for VSMC-derived foam cell formation. Our data suggest that FASN participates in the esterification of CHO and foam cell formation and may contribute to atherosclerotic plaque accumulation. Thus, FASN may represent a novel target for therapeutic intervention.

Metformin attenuates cardiac deficiencies and repairs hepatic insulin signaling in a rat model of acute food insecurity followed by recovery

States of acute severe food restriction (sFR) can result voluntarily, e.g., anorexia nervosa (~1-4% of women in their lifetime) or involuntarily, e.g., very low food insecurity (~3.8% of U.S. households). This is often followed by periods of normal food intake, i.e., refeeding. We’ve shown increased propensity toward the development of metabolic syndrome (MetS) and cardiac injury in our sFR-Refed rat model. Rats receive 40% of their normal ad libitum grams of rat chow for 2-weeks followed by 3-months of recovery (ad libitum feeding). We hypothesized that treatment with metformin, a type 2 diabetic therapeutic, would attenuate features of MetS and potentially reverse or attenuate cardiac damage. Female Fischer rats (3-months old) were randomly assigned to one of four treatments: 1) Control (CT); 2) Metformin (MF); 3) sFR-Refed (FR); or 4) FR-MF (n = 9/group). MF (0.5% incorporated into the chow) was fed for 5-weeks (from week 8-12) of the study before euthanizing at week 14. Ultrasound was conducted to assess heart function just after food restriction ended (week 4, prior to beginning MF) and near the end of the study (week 13 after completion of MF). Cardiac output (CO) and heart rate (HR) were decreased (38 and 15%, respectively, p < 0.05) in the FR groups (week 4), relative to CT. In week 13, CO in the FR rats was still 11% lower than CT rats (p = 0.04); however, not in the FR-MF group (2% higher than CT). MF alone did not significantly alter CO or HR. There were no significant differences in final body weight; however, liver weight was increased by MF (p = 0.008, 2-way ANOVA). Fat pads were weighed and parametrial fat (visceral) was increased in the FR group and this rise attenuated by MF (g/185 g·bw): 5.7 ± 0.2 (CT); 5.5 ± 0.3 (MF); 7.0 ± 0.3 (FR); 6.1 ± 0.3 (FR-MT), (p < 0.0003 between CT and FR, unpaired t-test). To analyze the effects of MF on insulin signaling pathways in liver, hepatic cell suspensions were incubated ex vivo with insulin. Isolated proteins were biotinylated and incubated with antibody arrays (Phospho-antibody array, PIG-219, Full Moon Biosystems). Antibody spot densities were quantitated and normalized between chips. MF treatment altered the expression or phosphorylation of 24 distinct proteins (by 2-way ANOVA). FR in the presence or absence of MF altered 8. In addition, MF prevented the sFR-induced up-regulation of several proteins including the activating phosphorylation of ATP citrate lyase (ACLY), an enzyme involved in fatty acid biogenesis, and tuberous sclerosis complex (TSC2), a known inhibitor of insulin signaling. In sum, sFR-Refed rats had features of MetS including visceral adiposity, as well as, reduced cardiac function. Treatment in the post-sFR period (after body weight recovery) was beneficial in attenuating this phenotype. Thus, metformin may be therapeutic to reduce MetS and cardiac injury associated with periods of food insecurity. Funding included the Marriott Foundation fellowship to CE; and addition Georgetown University internal funding. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibiting glutamine utilization creates a synthetic lethality for suppression of ATP citrate lyase in KRas-driven cancer cells.

Metabolic reprogramming is now considered a hallmark of cancer cells. KRas-driven cancer cells use glutaminolysis to generate the tricarboxylic acid cycle intermediate α-ketoglutarate via a transamination reaction between glutamate and oxaloacetate. We reported previously that exogenously supplied unsaturated fatty acids could be used to synthesize phosphatidic acid–a lipid second messenger that activates both mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTOR complex 2 (mTORC2). A key target of mTORC2 is Akt–a kinase that promotes survival and regulates cell metabolism. We report here that mono-unsaturated oleic acid stimulates the phosphorylation of ATP citrate lyase (ACLY) at the Akt phosphorylation site at S455 in an mTORC2 dependent manner. Inhibition of ACLY in KRas-driven cancer cells in the absence of serum resulted in loss of cell viability. We examined the impact of glutamine (Gln) deprivation in combination with inhibition of ACLY on the viability of KRas-driven cancer cells. While Gln deprivation was somewhat toxic to KRas-driven cancer cells by itself, addition of the ACLY inhibitor SB-204990 increased the loss of cell viability. However, the transaminase inhibitor aminooxyacetate was minimally toxic and the combination of SB-204990 and aminooxtacetate led to significant loss of cell viability and strong cleavage of poly-ADP ribose polymerase–indicating apoptotic cell death. This effect was not observed in MCF7 breast cancer cells that do not have a KRas mutation or in BJ-hTERT human fibroblasts which have no oncogenic mutation. These data reveal a synthetic lethality between inhibition of glutamate oxaloacetate transaminase and ACLY inhibition that is specific for KRas-driven cancer cells and the apparent metabolic reprogramming induced by activating mutations to KRas.

53BP1-ACLY-SLBP-coordinated activation of replication-dependent histone biogenesis maintains genomic integrity.

p53-binding protein 1 (53BP1) regulates the DNA double-strand break (DSB) repair pathway and maintains genomic integrity. Here we found that 53BP1 functions as a molecular scaffold for the nucleoside diphosphate kinase-mediated phosphorylation of ATP-citrate lyase (ACLY) which enhances the ACLY activity. This functional association is critical for promoting global histone acetylation and subsequent transcriptome-wide alterations in gene expression. Specifically, expression of a replication-dependent histone biogenesis factor, stem-loop binding protein (SLBP), is dependent upon 53BP1-ACLY-controlled acetylation at the SLBP promoter. This chain of regulation events carried out by 53BP1, ACLY, and SLBP is crucial for both quantitative and qualitative histone biogenesis as well as for the preservation of genomic integrity. Collectively, our findings reveal a previously unknown role for 53BP1 in coordinating replication-dependent histone biogenesis and highlight a DNA repair-independent function in the maintenance of genomic stability through a regulatory network that includes ACLY and SLBP.

Nuclear-Translocation of ACLY Induced by Obesity Related Factors Enhances Pyrimidine Metabolism Through Regulating Histone Acetylation in Endometrial Cancer

Background: Endometrial cancer (EC) is a common female reproductive system cancer, and obesity is a major risk factor for the disease. ATP-citrate lyase (ACLY), a vital enzyme linking both glycolytic and lipid metabolism, plays important roles in cancer progression. However, there is little evidence of ACLY involvement in EC development. Methods: The ACLY expression in EC was evaluated by using TCGA and GEO databases and verified it using clinical samples by IHC and RT-PCR methods. The functions of ACLY was determined by both the vitro and vivo studies. Gene expression array, co-immunoprecipitation, TUNEL assay, EdU assay, chromatin immunoprecipitation and luciferase reporter assay were applied for mechanism dissection. Findings: ACLY was significantly upregulated in EC patients, particularly in the nucleus. Upregulated ACLY promoted EC cell proliferation and colony formation, and attenuated apoptosis. Mechanistically, cotreatment with obesity-related factors (estradiol, insulin and leptin) promoted nuclear translocation of ACLY through Akt-mediated phosphorylation of ACLY at Ser455 . Nuclear-localized ACLY increased histone acetylation levels, thus resulting in upregulation of pyrimidine metabolism genes, such as DHODH. Moreover, STAT3 altered the ACLY expression at the transcriptional level via directly binding to its promoter region. Interpretation: ACLY nuclear-translocation induced by obesity-related factors promotes EC progression by modulating histone acetylation levels, The ACLY axis could be served as potential therapeutic targets for the treatment of EC. Funding Statement: This research was supported by The National Natural Science Foundation of China (Grant number: 81974406) and The Sailing Program Foundation of Hunan Cancer Hospital. Declaration of Interests: The authors declare that they have no competing interests with the contents of this article. Ethics Approval Statement: The study was approved by the Research Ethics Committee of the Sixth People's Hospital, and informed consent was provided by all patients before this study.

Effects of PKB/Akt inhibitors on insulin-stimulated lipogenesis and phosphorylation state of lipogenic enzymes in white adipose tissue

We investigated acute effects of two allosteric protein kinase B (PKB) inhibitors, MK-2206 and Akti-1/2, on insulin-stimulated lipogenesis in rat epididymal adipocytes incubated with fructose as carbohydrate substrate. In parallel, the phosphorylation state of lipogenic enzymes in adipocytes and incubated epididymal fat pads was monitored by immunoblotting. Preincubation of rat epididymal adipocytes with PKB inhibitors dose-dependently inhibited the following: insulin-stimulated lipogenesis, increased PKB Ser473 phosphorylation, increased PKB activity and decreased acetyl-CoA carboxylase (ACC) Ser79 phosphorylation. In contrast, the effect of insulin to decrease the phosphorylation of pyruvate dehydrogenase (PDH) at Ser293 and Ser300 was not abolished by PKB inhibition. Insulin treatment also induced ATP-citrate lyase (ACL) Ser454 phosphorylation, but this effect was less sensitive to PKB inhibitors than ACC dephosphorylation by insulin. In incubated rat epididymal fat pads, Akti-1/2 treatment reversed insulin-induced ACC dephosphorylation, while ACL phosphorylation by insulin was maintained. ACL and ACC purified from white adipose tissue were poor substrates for PKBα in vitro. However, effects of wortmannin and torin, along with Akti-1/2 and MK-2206, on recognized PKB target phosphorylation by insulin were similar to their effects on insulin-induced ACL phosphorylation, suggesting that PKB could be the physiological kinase for ACL phosphorylation by insulin. In incubated epididymal fat pads from wild-type versus ACC1/2 S79A/S212A knockin mice, effects of insulin to increase lipogenesis from radioactive fructose or from radioactive acetate were reduced but not abolished. Together, the results support a key role for PKB in mediating insulin-stimulated lipogenesis by decreasing ACC phosphorylation, but not by decreasing PDH phosphorylation.

Integration of BCAA and Lipid Metabolism by the BCKDH Kinase and Phosphatase

Strong associations exist between branched chain amino acids (BCAA) and dysregulated glucose and lipid metabolism, but underlying mechanisms are not well understood. One factor contributing to elevated BCAA in obesity is inhibitory phosphorylation of the branched chain keto-acid dehydrogenase complex (BCKDH) in liver. We studied the impact of modulating the activity of the BCKDH kinase (BDK) and phosphatase (PPM1K) on amino acid, glucose and lipid metabolism in Zucker fatty rats (ZFR). Daily administration of a selective inhibitor of BDK, BT2 (20mg.kg-1, IP), for one week or expression of a recombinant adenovirus overexpressing PPM1K significantly lowered BCAA and branched chain keto acid (BCKA) levels in ZFR. Remarkably, these effects were accompanied by strong lowering of liver triacylglycerides and improved glucose tolerance in the absence of weight loss. Proteomics analysis of liver samples from these studies revealed that both inhibition of BDK with BT2 and adenoviral mediated overexpression of PPM1K results in reduced phosphorylation of the lipogenic enzyme ATP-citrate lyase (ACL) on its regulatory serine 454. Phosphorylation of ACL on this site is activating. Sequence analysis revealed the presence of a canonical BDK phosphorylation motif surrounding ser454 in ACL and incubation of ACL with purified BDK resulted in ACL phosphorylation in vitro. Furthermore, adenovirus-mediated overexpression of BDK increased ACL phosphorylation and activated de novo lipogenesis in liver of lean Wistar rats. Together these studies reveal a novel regulatory function for BDK and PPM1K in integration of BCAA and lipid metabolism. Moreover, we show that modulation of this node provides broad protection against metabolic abnormalities associated with obesity. Disclosure P.J. White: None. R.W. McGarrah: None. P.A. Grimsrud: None. W. Yang: None. G. Zhang: None. J.M. Haldeman: None. M. Arlotto: None. O. Ilkayeva: None. R.M. Wynn: None. D.T. Chuang: None. C.B. Newgard: Advisory Panel; Self; Eli Lilly and Company. Research Support; Self; Eli Lilly and Company.

The BCKDH Kinase and Phosphatase Integrate BCAA and Lipid Metabolism via Regulation of ATP-Citrate Lyase

Branched-chain amino acids (BCAA) are strongly associated with dysregulated glucose and lipid metabolism, but the underlying mechanisms are poorly understood. We report that inhibition of the kinase (BDK) or overexpression of the phosphatase (PPM1K) that regulates branched-chain ketoacid dehydrogenase (BCKDH), the committed step of BCAA catabolism, lowers circulating BCAA, reduces hepatic steatosis, and improves glucose tolerance in the absence of weight loss in Zucker fatty rats. Phosphoproteomics analysis identified ATP-citrate lyase (ACL) as an alternate substrate of BDK and PPM1K. Hepatic overexpression of BDK increased ACL phosphorylation and activated de novo lipogenesis. BDKand PPM1K transcript levels were increased and repressed, respectively, in response to fructose feeding or expression of the ChREBP-β transcription factor. These studies identify BDK and PPM1K as a ChREBP-regulated node that integrates BCAA and lipid metabolism. Moreover, manipulation of the BDK:PPM1K ratio relieves key metabolic disease phenotypes in a genetic model of severe obesity.

Nuclear Acetyl-CoA Production by ACLY Promotes Homologous Recombination

While maintaining the integrity of the genome and sustaining bioenergetics are both fundamental functions of the cell, potential crosstalk between metabolic and DNA repair pathways is poorly understood. Since histone acetylation plays important roles in DNA repair and is sensitive to the availability of acetyl coenzyme A (acetyl-CoA), we investigated a role for metabolic regulation of histone acetylation during the DNA damage response. In this study, we report that nuclear ATP-citrate lyase (ACLY) is phosphorylated at S455 downstream of ataxia telangiectasia mutated (ATM) and AKT following DNA damage. ACLY facilitates histone acetylation at double-strand break (DSB) sites, impairing 53BP1 localization and enabling BRCA1 recruitment and DNA repair by homologous recombination. ACLY phosphorylation and nuclear localization are necessary for its role inpromoting BRCA1 recruitment. Upon PARP inhibition, ACLY silencing promotes genomic instability and cell death. Thus, the spatial and temporal control of acetyl-CoA production by ACLY participates in the mechanism of DNA repair pathway choice.

MTOR complex-2 stimulates acetyl-CoA and de novo lipogenesis through ATP citrate lyase in HER2/PIK3CA-hyperactive breast cancer.

The mechanistic target of rapamycin (mTOR) is a major regulator of cell growth and is frequently dysregulated in cancer. While mTOR complex-1 (mTORC1) is a validated cancer target, the role of mTOR complex-2 (mTORC2) remains less defined. Here, we reveal mTORC2 as a critical regulator of breast cancer metabolism. We showed that hyperphosphorylation in ATP citrate lyase (ACL) occurs frequently in human breast tumors and correlates well with HER2+ and/or PIK3CA-mutant (HER2+/PIK3CAmut) status in breast tumor cell lines. In HER2+/PIK3CAmut cells, mTORC2 controls Ser-455 phosphorylation of ACL thereby promoting acetyl-CoA production, de novo lipogenesis and mitochondrial physiology, all of which were inhibited by an mTORC1/mTORC2 kinase inhibitor (mTOR-KI) or cellular depletion of mTORC2 or ACL. mTOR-KI but not rapamycin blocked the IGF-1-induced ACL phosphorylation and glucose to lipid conversion. Depletion of mTORC2 but not mTORC1 specifically inhibited the ACL-dependent acetyl-CoA production. In the HER2+/PIK3CAmut MDA361, MDA453, BT-474 and T47D cells, depletion of mTORC2 or ACL led to growth inhibition and mitochondrial hyperpolarization, which were partially rescued by an alternate source of acetyl-CoA. These same changes were not apparent in mTORC2- or ACL-depleted HER2-/PIK3CAwt MDA231 and HCC1806 cells, highlighting a differential dependence of mTORC2-ACL for survival in these two cell types. Moreover, ACL Ser-455 mutants S455E (phosphomimetic) and S455A (non-phosphorylatable) each increased or decreased, respectively, the acetyl-CoA production, mitochondrial homeostasis and survival in ACL-depleted MDA453 cells. These studies define a new and rapamycin-resistant mechanism of mTORC2-ACL in lipogenesis and acetyl-CoA biology and provide a rationale for targeting of mTORC1 and mTORC2 in HER2+/PIK3CAmut breast cancer.

Abstract PR03: Exploring the link between Kras and histone acetylation

Abstract KRAS mutations in pancreatic ductal adenocarcinoma (PDAC) have been shown to extensively rewire cellular metabolism to promote macromolecular biosynthesis and maintain redox homeostasis. Recently, compelling evidence has emerged demonstrating that many epigenetic modifications are sensitive to the availability of cellular metabolites. For example, acetyl-CoA is the donor substrate for lysine acetylation, and histone acetylation is responsive to levels of acetyl-CoA, which is produced largely from glucose metabolism. We thus hypothesized that oncogenic metabolic reprogramming alters metabolite levels in a way that impacts the epigenome and could thus contribute to tumorigenesis. Indeed, we find that oncogenic Akt activation promotes elevated histone acetylation, and that this effect is mediated by ATP-citrate-lyase (ACLY), a nucleocytoplasmic enzyme that converts glucose-derived citrate into acetyl-CoA. In order to understand what cellular processes are affected by such AKT-ACLY-mediated increase in histone acetylation, we employed a mouse model of pancreatic ductal adenocarcinoma, driven by oncogenic Kras (Pdx1-Cre; LSL-KrasG12D; TP53L/+). In this model, the PI3K/Akt pathway is activated downstream of Kras. By immunohistochemistry we found that, in mice with WT Kras, pancreatic acinar cells exhibit very low levels of histone H4 (K5/8/12/16) acetylation (AcH4), although AcH4 is clearly detectable in ductal epithelial cells and islets. By contrast, in mice expressing oncogenic Kras in the pancreas (LSL-KrasG12D; p53L/+; Pdx1-Cre), acinar H4 acetylation is dramatically increased. Remarkably, this overt increase in histone acetylation precedes the appearance of histological abnormalities and persists during several steps in tumor progression. Moreover, Kras and Akt-dependent reuglation of histone acetylation levels can be recapitulated in ex vivo acinar cell culture, suggesting that this is a primary effect of oncogene activation in these cells. Akt inhibition also significantly reduced glucose consumption and phosphorylation of ACLY and ultimately decreased acetyl-CoA levels. Akt inhibition in vitro reduces Kras-induced acinar cell hyperacetylation and block acinar-to-ductal metaplasia, an initiating event of pancreatic carcinoma. Our data indicate that Akt orchestrates a metabolic rewiring in pancreatic tumorigenesis, which promotes histone hyperacetylation, a phenotype evident before the manifestation of the disease and that might conceivably contribute to cell plasticity and tumor progression. Citation Format: Alessandro Carrer, Joyce V. Lee, Supriya Shah, Nicole M. Aiello, Nathaniel W. Snyder, Andrew J. Worth, Ian A. Blair, Ben Z. Stanger, Kathryn E. Wellen. Exploring the link between Kras and histone acetylation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr PR03.

Abstract B40: AKT-ACLY signaling preferentially maintains histone acetylation over lipid synthesis during nutrient limitation

Abstract Metabolic reprogramming by oncogenes is a mechanism to support biosynthesis and growth in cancer cells. Activation of the PI3K/AKT pathway increases glucose consumption and glycolysis, leading to higher acetyl-CoA production. Extra-mitochondrial acetyl-CoA is utilized in lipid synthesis and protein acetylation. We previously showed that tumor cell histone acetylation is regulated in a glucose-dependent manner, through ATP-citrate-lyase (ACLY) conversion of glucose-derived citrate into acetyl-CoA. In low glucose conditions, acetyl-CoA and histone acetylation levels decrease. However, AKT-dependent phosphorylation of ACLY at serine 455 promotes acetyl-CoA production to allow maintenance of global levels of histone acetylation under glucose limitation. It is unclear whether partitioning of acetyl-CoA between lipid synthesis and histone acetylation in the setting of nutrient limitation impacts cell growth or survival. In this study, we examined whether acetyl-CoA utilization for lipid synthesis is impacted by AKT-ACLY signaling during nutrient limitation. Using 13C-glucose labeling and mass spectrometry, we determined that de novo lipid synthesis is suppressed to a much greater extent than histone acetylation in low glucose conditions. Moreover, phosphorylation of ACLY does not sustain fatty acid synthesis under glucose limitation, in contrast to its effects on histone acetylation. These data indicate that the PI3K-AKT-ACLY axis preferentially maintains histone acetylation over lipid synthesis during nutrient limitation. This regulation is likely due to AMPK-dependent phosphorylation of acetyl-CoA carboxylase (p-ACC), blocking acetyl-CoA commitment to fatty acid synthesis. This study reveals that cancer cells employ robust mechanisms to maintain histone acetylation in nutrient limited environments. Citation Format: Joyce V. Lee, Zuo-Fei Yuan, Shichong Liu, Benjamin A. Garcia, Kathryn E. Wellen. AKT-ACLY signaling preferentially maintains histone acetylation over lipid synthesis during nutrient limitation. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B40.

NPM-ALK Mediated Tyrosine Phosphorylation of ATP Citrate Lyase Regulates Lipid Metabolism and Promotes Oncogenesis of Anaplastic Large Cell Lymphoma

Introduction: Anaplastic large cell lymphoma (ALCL) is characterized by recurrent translocations involving anaplastic lymphoma kinase (ALK) and resulting in oncogenic chimeric fusion tyrosine kinases such as NPM-ALK. We integrated mass-spectrometry-driven phosphoproteomics and metabolomics of ALK+ ALCL and discovered that ATP citrate lyase (ACLY) is a candidate substrate of NPM-ALK at residue Y682. ACLY is a critical enzyme responsible for synthesis of acetyl-CoA and connects vital biosynthetic pathways such as carbohydrate and lipid metabolism. Functional regulation of ACLY by tyrosine phosphorylation is unknown and the significance of tyrosine phosphorylation of ACLY on cancer cell growth and metabolism have never been described.Methods: To identify novel substrates of NPM-ALK, we performed phospho-proteomic analyses of 3 ALK+ALCL derived cell lines. The MS-phosphoproteomic data were subjected to custom bioinformatic algorithms to identify highly statistically correlated phosphotyrosine proteins and phosphosites regulated by ALK kinase activity. We generated a phosphospecific antibody against a highly-ranked candidate (pACLY Y682) and utilized it in western blot analyses on ALK+ and ALK- ALCL lymphoma cell lines. Immunohistochemistry (IHC) on well-characterized primary human ALK+ (n=20) and ALK-ALCL biopsies (n=28) were performed. Co-immunoprecipitations (Co-IP) with HA-tagged ACLY-WT and ACLY-Y682F and ALK antibodies were performed and analyzed by western blot using the pACLY Y682 antibody. In vitro kinase assays were performed to investigate the direct role of NPM-ALK in phosphorylation of ACLY at Y682 using GST-tagged ACLY-WT or ACLY-Y682F peptides in the presence or absence of immunocomplexes containing active NPM-ALK or the kinase-defective K210R mutant. To study the impact of Y682F mutant on lipid metabolism, we generated ALCL-derived cell lines stably transduced to express GFP-tagged ACLY-WT or ACLY-Y682F and subjected them to metabolomic analysis for flux, β-oxidation or lipid synthesis using liquid chromatography/tandem mass spectrometry or gas chromatography platforms. The Seahorse XF24 Flux analyzer was used to measure oxygen consumption rates (OCR) in DEL stably expressing ACLY-WT and ACLY-Y682F. Cells stably expressing ACLY-WT and ACLY-Y682F were evaluated for cell proliferation, colony formation and tumor formation in SCID-BEIGE mouse xenograft models.Results: Phosphoproteomic analyses yielded a dataset containing a total of 626 tyrosine phosphoproteins. Bioinformatic analysis identified pACLY Y682 as a novel candidate substrate of NPM-ALK. Western blot analysis corroborated phosphoproteomic studies and demonstrated that phosphorylation of ALCY at Y682 is regulated by NPM-ALK activity. Expression of active NPM-ALK increased the phosphorylation of ACLY at Y682 in HEK293T cells compared to the K210R mutant. In vitro kinase assays demonstrated that NPM-ALK phosphorylates ACLY at Y682. Immunoprecipitation (IP) of ALK and co-IP (HA) demonstrated interaction of NPM-ALK and ACLY in co-transfected HEK293T cell lysates. Stable expression of ACLY-Y682F or pharmacologic antagonism of ALK with crizotinib resulted in decreased ACLY activity, decreased lipid synthesis and increased fatty acid β-oxidation when compared to ACLY-WT. β-oxidation of 13 C-oleic acid-labeled fatty acid demonstrated increased labeling of +2-citrate (p<0.01) and +18-Oleyol carnitine (p<0.001) in ACLY-Y682F cells when compared to ACLY-WT. Similarly, the OCR was significantly increased in cells expressing ACLY-Y682F (p<0.001). IHC studies of ALCL biopsies (ALK+ and ALK-) demonstrated significant correlation between NPM-ALK and ACLY pY682- expression (p<0.01). Expression of ACLY-Y682F in the DEL cell line resulted in dramatically decreased cell proliferation, impaired clonogenic potential as observed by small colony size in colony formation assay and abrogated tumor growth in in vivo xenograft model when compared to ACLY-WT.Conclusion: We identify for the first time a direct tyrosine kinase-mediated mechanism for ACLY regulation. In this regard, our studies demonstrate a novel oncogenic mechanism whereby NPM-ALK controls lipid synthesis and b-oxidation by phosphorylation of ACLY at Y682 and this mechanism contributes to ALCL pathogenesis. Our findings have significant implications for novel therapies targeting tumor metabolism in ALCLs. DisclosuresNo relevant conflicts of interest to declare.

Diacylglycerol Kinase Delta Promotes Lipogenesis

We have studied the relationship between diacylglycerol kinase delta (DGKδ) and lipogenesis. There is a marked increase in the expression of DGKδ during the differentiation of 3T3-L1 cells to adipocytes, as well as in the synthesis of neutral and polar lipids. When 3T3-L1 undifferentiated fibroblasts are transfected to express DGKδ, there is increased triglyceride synthesis without differentiation to adipocytes. Hence, expression of DGKδ promotes lipogenesis. Lipid synthesis is decreased in DGKδ knockout mouse embryo fibroblasts, especially for lipids with shorter acyl chains and limited unsaturation. This reduction occurs for both neutral and polar lipids. These findings suggest reduced de novo lipid synthesis. This is confirmed by measuring the incorporation of glycerol into polar and neutral lipids, which is higher in the wild type cells than in the DGKδ knockouts. In comparison, there was no change in lipid synthesis in DGKε knockout mouse embryo fibroblasts. We also demonstrate that the DGKδ knockout cells had a lower expression of acetyl-CoA carboxylase and fatty acid synthase as well as a lower degree of activation by phosphorylation of ATP citrate lyase. These three enzymes are involved in the synthesis of long chain fatty acids. Our results demonstrate that DGKδ markedly increases lipid synthesis, at least in part as a result of promoting the de novo synthesis of fatty acids.

On the Catalytic Mechanism of Human ATP Citrate Lyase

ATP citrate lyase (ACL) catalyzes an ATP-dependent biosynthetic reaction which produces acetyl-coenzyme A and oxaloacetate from citrate and coenzyme A (CoA). Studies were performed with recombinant human ACL to ascertain the nature of the catalytic phosphorylation that initiates the ACL reaction and the identity of the active site residues involved. Inactivation of ACL by treatment with diethylpyrocarbonate suggested the catalytic role of an active site histidine (i.e., His760), which was proposed to form a phosphohistidine species during catalysis. The pH-dependence of the pre-steady-state phosphorylation of ACL with [γ-(33)P]-ATP revealed an ionizable group with a pK(a) value of ~7.5, which must be unprotonated for the catalytic phosphorylation of ACL to occur. Mutagenesis of His760 to an alanine results in inactivation of the biosynthetic reaction of ACL, in good agreement with the involvement of a catalytic histidine. The nature of the formation of the phospho-ACL was further investigated by positional isotope exchange using [γ-(18)O(4)]-ATP. The β,γ-bridge to nonbridge positional isotope exchange rate of [γ-(18)O(4)]-ATP achieved its maximal rate of 14 s(-1) in the absence of citrate and CoA. This rate decreased to 5 s(-1) when citrate was added, and was found to be 10 s(-1) when both citrate and CoA were present. The rapid positional isotope exchange rates indicated the presence of one or more catalytically relevant, highly reversible phosphorylated intermediates. Steady-state measurements in the absence of citrate and CoA showed that MgADP was produced by both wild type and H760A forms of ACL, with rates at three magnitudes lower than that of k(cat) for the full biosynthetic reaction. The ATPase activity of ACL, along with the small yet significant positional isotope exchange rate observed in H760A mutant ACL (~150 fold less than wild type), collectively suggested the presence of a second, albeit unproductive, phosphoryl transfer in ACL. Mathematical analysis and computational simulation suggested that the desorption of MgADP at a rate of ~7 s(-1) was the rate-limiting step in the biosynthesis of AcCoA and oxaloacetate.

Abstract B64: Development of a novel strategy to overcome drug resistance by targeting ATP citrate lyase and de novo lipogenesis in colorectal cancer cells

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer death in the United States. Although the response rate to current systemic therapies is ∼50%, drug resistance develops in nearly all patients leading to 50,000 deaths each year. Overcoming drug resistance involves understanding the mechanisms by which cancer cells adapt to the genotoxic stress. Metabolic changes at levels of mitochondria function, glucose metabolism, and de novo synthesis of fatty acids frequently occurs in malignant cells and impacts tumor development and growth. Our laboratory established in vitro drug-resistant models of CRC cells by chronic exposure of HT29 cells to increasing doses of chemotherapeutic agents (oxaliplatin, 5-FU and SN38) over a period of 4–6 months. We recently reported a metabolic switch to the glycolytic phenotype due to mitochondria defects in the oxaliplatin-resistant CRC cells (HT29-OXR) (Zhou et al, Cancer Research, 2012). In this study, we tested the hypothesis that deregulation of de novo lipogenesis pathways plays an important role in chemoresistance of CRC cells. Methods: A previous study (Bose et al. Br J Ca, 2011) using unbiased proteomic profiling by mass spectroscopy (MS) was used to determine the proteomic signature of defective metabolic pathways in the oxaliplatin-resistant CRC cells (OXR cells). ATP-citrate-lyase (ACLy), the key enzyme of de novo lipogenesis pathway, was examined for protein levels and activation. The lipid content of resistant cells was examined by transmission electron microscope (TEM) and Oil Red staining. Transient knockdown of the ACLy protein by siRNA was used to study re-chemosensitization of the resistant cells. A small molecule inhibitor of ACLy was studied in combination with chemotherapeutic agents in resistant CRC cells for growth inhibitory (MTT assay) and cytotoxic effects (PARP cleavage and Annexin V staining). A cell free assay was used to test the potency of the ACLy inhibitor on ACLy activation. Results: Activated ACLy protein level (phosphorylation on S454) was demonstrated by Western blot analysis in the HT29-OXR and SN38-resistant (HT29-SNR) cells, but not in 5-FU-resistant (HT29-FUR) cells. The OxR cells showed a 2–3 fold increase in lipid droplets numbers (by TEM examination) and fatty acid content (by Oil Red staining) than the parental cells. Furthermore, transient knockdown of the ACLy protein by siRNA demonstrated a return to chemosensitization when cells were treated with oxaliplatin. IC50 values of the ACLy inhibitor for parental HT29 and HT29-OXR, -SNR and —FUR cells were ∼30μM. As a single agent, the ACLy inhibitor blocked phosphorylation of ACLy and induced apoptosis in a concentration-dependent manner in parental HT29 cells, and its resistant derivatives -OXR and -SNR cells. Combination of the ACLy inhibitor at concentrations sufficient to block ACLy phosphorylation with oxaliplatin and SN38 showed enhanced effects on growth inhibition (MTT) and apoptosis induction (PARP cleavage and Annexin V assay) in HT29 cells-OXR and -SNR cells. Conclusions: Chemoresistant CRC cells demonstrated: 1) increased de novo lipogenesis, 2) elevated levels of key lipogenesis enzymes ACLy, 3) dependence on ACLy activity for cell survival under cytotoxic stress. This metabolic switch likely contributes to the chemoresistant phenotype of CRC cells. Targeting an early step of de novo lipogenesis such as blocking ACLy activity may provide a novel strategy to overcome drug-resistance in CRC cells.

The Identification of ATP-citrate Lyase as a Protein Kinase B (Akt) Substrate in Primary Adipocytes

Protein kinase B (Akt) plays a central role in cellular regulation, although many of the physiologically relevant substrates for the kinase remain to be identified. In this study, we have isolated a protein from primary epididymal adipocytes with an apparent molecular weight of 125,000. This protein exhibited immunoreactivity, in an insulin-dependent manner, with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) as well as a phosphospecific antibody that recognizes serine 21/9 of GSK-3alpha/beta. MALDI-TOF mass spectrometry revealed the protein to be ATP-citrate lyase, suggesting that the two phosphospecific antibodies recognize phosphoserine 454, a previously reported insulin- and isoproterenol-stimulated ATP-citrate lyase phosphorylation site. Indeed, both insulin and isoproterenol stimulated the phosphorylation of this protein on the site recognized by the phosphospecific antibodies in a wortmannin-sensitive and -insensitive manner, respectively. In addition, transient expression of a constitutively active protein kinase B in primary adipocytes mimicked the effect of insulin on ATP-citrate lyase phosphorylation. Furthermore, ATP-citrate lyase was phosphorylated in vitro by recombinant protein kinase B on the same site. Taken together, these results demonstrate that serine 454 of ATP-citrate lyase is a novel and major in vivo substrate for protein kinase B.

Two-component kinase-like activity of nm23 correlates with its motility-suppressing activity.

Nm23 genes, which encode nucleoside diphosphate kinases, have been implicated in suppressing tumor metastasis. The motility of human breast carcinoma cells can be suppressed by transfection with wild-type nm23-H1, but not by transfections with two nm23-H1 mutants, nm23-H1(S12OG) and nm23-H1(P96S). Here we report that nm23-H1 can transfer a phosphate from its catalytic histidine to aspartate or glutamate residues on 43-kDa membrane proteins. One of the 43-kDa membrane proteins was not phosphorylated by either nm23-H1(P96S) or nm23-H1(S120G), and another was phosphorylated much more slowly by nm23-H1(P96S) and by nm23-H1(S120G) than by wild-type nm23-H1. Nm23-H1 also can transfer phosphate from its catalytic histidine to histidines on ATP-citrate lyase and succinic thiokinase. The rates of phosphorylation of ATP-citrate lyase by nm23-H1(S120G) and nm23-H1(P96S) were similar to that by wild-type nm23-H1. The rate of phosphorylation of succinic thiokinase by nm23-H1(S120) was similar to that by wild-type nm23-H1, and the rate of phosphorylation of succinic thiokinase by nm23-H1(P96S) was about half that by wild-type nm23-H1. Thus, the transfer of phosphate from nm23-H1 to aspartates or glutamates on other proteins appears to correlate better with the suppression of motility than does the transfer to histidines.

Phosphorylation of ATP-Citrate Lyase by Nucleoside Diphosphate Kinase

Rat liver nucleoside diphosphate kinase (NDPK) and PC12 cell cytosol were used to determine whether NDPK could function as a protein kinase. NDPK was phosphorylated on its catalytic histidine using [gamma-32P]ATP, and the phosphorylated NDPK separated from [gamma-32P]ATP. The addition of phosphorylated NDPK to dialyzed PC12 cell cytosol resulted in the phosphorylation of a protein with a subunit molecular mass of about 120 kDa. This phosphorylation appeared to occur by a direct transfer of a phosphoryl group from the catalytic histidine of NDPK to a histidine on the 120-kDa protein. The 120-kDa protein was partially purified and shown by peptide sequencing to be ATP-citrate lyase. ATP-citrate lyase is the primary source of cytosolic acetyl-CoA. NDPK phosphorylated the histidine at the catalytic site of ATP-citrate lyase. This histidine can also be phosphorylated by ATP, and its phosphorylation is the first step in the conversion of citrate and CoA to oxaloacetate and acetyl-CoA by ATP-citrate lyase. The level of phosphorylation of PC12 cell ATP-citrate lyase by phosphorylated NDPK was comparable with that by ATP. Thus, in addition to its nucleoside diphosphate kinase activity, NDPK can function as a protein kinase.

Phospho-oligosaccharide dependent phosphorylation of ATP citrate lyase

The effect of insulin on ATP citrate lyase phosphorylation has been shown to be mimicked by a phospho-oligosaccharide in intact adipocytes (see ref. 4). We demonstrate that the addition of phospho-oligosaccharide to intact adipocytes enhances the phosphorylation of ATP citrate lyase in the same tryptic peptide as insulin does. The addition of phosphooligosaccharide to an adipocyte extract also results in an increase in ATP citrate lyase phosphorylation but in a different site than that observed in intact cells. The phospho-oligosaccharide-dependent incorporation of phosphate into ATP citrate lyase in intact cells is resistant to isopropanol and acetic acid, but the phosphoenzyme phosphorylated in cell extracts is acid labile. In cell extracts, the addition of phospho-oligosaccharide markedly inhibits ATP hydrolysis, which may explain the effect of this molecule on ATP citrate lyase phosphorylation in broken cells. These results support the hypothesis that this phospho-oligosaccharide mediates some of the effects of insulin on protein phosphorylation. They also indicate that caution should be exercised in interpreting the results obtained by adding phospho-oligosaccharide to broken cell preparations.