DCA Treatment Research Articles

Transcriptional and methylation outcomes of didehydro-cortistatin A use in HIV-1-infected CD4+ T cells.

Ongoing viral transcription from the reservoir of HIV-1 infected long-lived memory CD4+ T cells presents a barrier to cure and associates with poorer health outcomes for people living with HIV, including chronic immune activation and inflammation. We previously reported that didehydro-cortistatin A (dCA), an HIV-1 Tat inhibitor, blocks HIV-1 transcription. Here, we examine the impact of dCA on host immune CD4+ T-cell transcriptional and epigenetic states. We performed a comprehensive analysis of genome-wide transcriptomic and DNA methylation profiles upon long-term dCA treatment of primary human memory CD4+ T cells. dCA prompted specific transcriptional and DNA methylation changes in cell cycle, histone, interferon-response, and T-cell lineage transcription factor genes, through inhibition of both HIV-1 and Mediator kinases. These alterations establish a tolerogenic Treg/Th2 phenotype, reducing viral gene expression and mitigating inflammation in primary CD4+ T cells during HIV-1 infection. In addition, dCA suppresses the expression of lineage-defining transcription factors for Th17 and Th1 cells, critical HIV-1 targets, and reservoirs. dCA's benefits thus extend beyond viral transcription inhibition, modulating the immune cell landscape to limit HIV-1 acquisition and inflammatory environment linked to HIV infection.

#999 PDK4 triggers ferroptosis in diabetic kidney disease by promoting NRF2 ubiquitination

Abstract Background and Aims Diabetic kidney disease (DKD) is the principal cause of chronic kidney disease and end-stage renal disease. Despite this burden, the detailed understanding of the pathogenic mechanism that promote the initiation and progression of DKD remain to be fully elucidated. This study aims to investigate the role of pyruvate dehydrogenase kinase 4 (PDK4) in diabetic renal tubular cell death from the perspectives of mitochondrial injury, metabolic reprogramming and ferroptosis. Method Cellular transcriptomics was employed to screen the differential genes between normal glucose (as control) and 35 mM glucose combined with 120 uM palmitic acid (as HGL) groups. The vitro experimental groups include Ctl, HGL, HGL/DCA (5 mM PDK4 inhibitor, sodium dichloroacetate), HGL/DMSO, culturing for 48 h. C57BL/6J mice were used to perform the vivo experiments. A total of 40 C57BL/6J mice were randomly divided into 5 groups respectively labeled as control, DKD, DKD/DCA (25 mg/kg, daily), DKD/DMSO (1% DMSO, daily) and DKD/Fer-1 (1 mg/kg, daily) group. Western blot, qRT-PCR, Si-RNA transfection, transmission electron microscopy, ROS probe, FerroOrange/Green probes, liperfluo probe, immunofluorescence and Co-IP assay were used in this study to detect the indicators of metabolic reprogramming, mitochondrial function, oxidative stress and ferroptosis, as well as the NRF2 ubiquitination level. Results Based on the cellular transcriptomics, PDK4 mRNA was significantly elevated in the HK-2 cells treated with HGL. Likewise, mRNA and protein levels were also elevated in the kidneys of mice treated with streptozotocin and high fat diet. In kidneys with PDK4 inhibitor DCA treatment or PDK4 genetic knockdown, glycolipid toxicity-induced metabolic reprogramming and destruction of mitochondrial function were markedly ameliorated. In addition, mitochondrial reactive oxygen species and the oxidative stress markers NADPH oxidase 2 and 4 (NOX2, NOX4) were diminished in DCA-treated or PDK4 knockdown HK-2 cells, while superoxide dismutase 1 and 2 (SOD1, SOD2) and catalase (CAT) expression and glutathione (GSH) levels were restored. DCA intervention or PDK4 knockdown in DKD mice or HK-2 cells alleviated the signs of ferroptosis caused by glycolipid toxicity, demonstrated as up-regulation of SLCA11, GPX4, FSP1 and GSH to maintain the balance of oxidative stress, increase of FTH1 and FPN1 to promote Fe2+ storage and discharge, decrease of TFR1 to reduce Fe2+ intake, as well as reduction of lipid peroxidation products including 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). PDK4 promoted NRF2 ubiquitination under HGL condition, since inhibition or knockdown of PDK4 in HK-2 cells increased the protein level of NRF2, while the mRNA level was unchanged. Conclusion Collectively, we identified a new ferroptosis inducer gene, PDK4, which is commonly up-regulated in diabetes. PDK4-induced metabolic reprogramming, mitochondrial damage and oxidative stress promote ROS accumulation and ultimately lead to ferroptosis in DKD, this effect can be achieved in part by promoting NRF2 ubiquitination.

Regulation of Alternative Splicing of Lipid Metabolism Genes in Sepsis-Induced Liver Damage by RNA-Binding Proteins.

RNA binding proteins (RBPs) have the potential for transcriptional regulation in sepsis-induced liver injury, but precise functions remain unclear. Our aim is to conduct a genome-wide expression analysis of RBPs and illuminate changes in the regulation of alternative splicing in sepsis-induced liver injury. RNA-seq data on "sepsis and liver" from the publicly available NCBI data set was analyzed, and differentially expressed RBPs and alternative splicing events (ASEs) in the healthy and septic liver were identified. Co-expression analyses of sepsis-regulated RBPs and ASEs were performed. Models of sepsis were established to validate hepatic RBP gene expression patterns with different treatments. Pairwise analysis of gene expression profiles of sham, cecum ligation puncture (CLP), and CLP with dichloroacetate (CLPDCA) mice allowed 1208 differentially expressed genes (DEGs), of which 800 were up-regulated and 408 down-regulated, to be identified. DEGs were similar in both Sham and CLPDCA mice. TheKEGG analysis showed that up-regulated genes as being involved in cytokine-cytokine receptor interaction and IL-17 signaling pathway and down-regulated genes in metabolic pathways. Differences in lipid metabolism-related alternative splicing events, including A3SS, were also found in CLP and CLPDCA compared with sham mice. Thirty-seven RBPs, including S100a11, Ads2, Fndc3b, Fn1, Ddx28, Car2, Cisd1, and Ptms, were differentially expressed in CLP mice and the regulated alternative splicing genes(RASG) with the RBPshown to be enriched in lipid metabolic and oxidation-reduction-related processes by GO functional analysis.In KEEG analysis the RASG mainly enriched in metabolic pathway. The models of sepsis were constructed with different treatment groups, and S100a11 expression in the CLP group found to be higher than in the sham group, a change that was reversed by DCA. The alternative splicing ratio of Srebf1 and Cers2 decreased compared with the sham group increased after DCA treatment. Abnormal profiles of gene expression and alternative splicing were associated with sepsis-induced liver injury. Unusual expression of RBPs, such as S100a11, may regulate alternative splicing of lipid metabolism-associated genes, such as Srebf1 and Cers2, in the septic liver. RBPs may constitute potential treatment targets for sepsis-induced liver injury.

Stimulating myocardial pyruvate dehydrogenase activity fails to alleviate cardiac abnormalities in a mouse model of human Barth syndrome.

Barth syndrome (BTHS) is a rare genetic disorder due to mutations in the TAFAZZIN gene, leading to impaired maturation of cardiolipin and thereby adversely affecting mitochondrial function and energy metabolism, often resulting in cardiomyopathy. In a murine model of BTHS involving short-hairpin RNA mediated knockdown of Tafazzin (TazKD mice), myocardial glucose oxidation rates were markedly reduced, likely secondary to an impairment in the activity of pyruvate dehydrogenase (PDH), the rate-limiting enzyme of glucose oxidation. Furthermore, TazKD mice exhibited cardiac hypertrophy with minimal cardiac dysfunction. Because the stimulation of myocardial glucose oxidation has been shown to alleviate diabetic cardiomyopathy and heart failure, we hypothesized that stimulating PDH activity would alleviate the cardiac hypertrophy present in TazKD mice. In order to address our hypothesis, 6-week-old male TazKD mice and their wild-type (WT) littermates were treated with dichloroacetate (DCA; 70 mM in the drinking water), which stimulates PDH activity via inhibiting PDH kinase to prevent inhibitory phosphorylation of PDH. We utilized ultrasound echocardiography to assess cardiac function and left ventricular wall structure in all mice prior to and following 6-weeks of treatment. Consistent with systemic activation of PDH and glucose oxidation, DCA treatment improved glycemia in both TazKD mice and their WT littermates, and decreased PDH phosphorylation equivalently at all 3 of its inhibitory sites (serine 293/300/232). However, DCA treatment had no impact on left ventricular structure, or systolic and diastolic function in TazKD mice. Therefore, it is unlikely that stimulating glucose oxidation is a viable target to improve BTHS-related cardiomyopathy.

150 Changes in pyruvate metabolism alters the epigenetic and molecular maturation of bovine oocytes

During invitro maturation (IVM), bovine oocytes undergo important metabolic, epigenetic, and transcriptional changes for the acquisition of developmental competence. Particularly, metabolic changes that alter the availability of cytoplasmic acetyl-CoA, the main substrate for histones acetylation, may alter the epigenetic profile of the oocyte, with consequences for correct molecular maturation. To test this hypothesis, cumulus–oocyte complexes (COCs) were IVM in three experimental groups: Control [IVM medium (TCM-199-Bicarbonate, 10% fetal bovine serum, 1µg mL−1 oestradiol, 10µg mL−1 FSH, and 10µg mL−1 human chorionic gonadotrophin)], DCA (IVM medium supplemented with 1.5mM sodium dichloroacetate, a pyruvate to acetyl-CoA conversion stimulator) and IA (IVM medium supplemented with 5µM sodium iodoacetate, a glycolysis inhibitor). Cumulus cells (CC) and oocytes (Oo) were analysed separately at 24h (mitochondrial activity, MA; MitoTracker Red CMXRos, ThermoFisher Scientific] and at 0, 4, 8, 16, and 24h of IVM [lysine 9 histone 3 acetylation (H3K9ac immunofluorescence) and new transcript synthesis (only CC; Click-iT® RNA, ThermoFisher Scientific). The images were acquired using a fluorescence microscope and analysed by Image J software. The results from at least 3 replicates were compared by Student’s t-test (treatment vs. control) or by ANOVA followed by Tukey’s test (comparison within the same group in different time points) considering P<0.05. As expected, DCA treatment led to an increase in MA in CC and oocytes (CC control vs. DCA, P=0.003; Oo control vs. DCA, P=0.003). In CC, during the first 4h, H3K9ac increased significantly in the treated group and decreased in the control group. At 8, 16, and 24h, both groups presented similar tendencies, although H3K9ac levels remained higher in DCA compared with control at all time points (P<0.001). The synthesis of new transcripts in CC was stimulated by DCA compared with control at 8h (P=0.02) and particularly at 16h (P=0.002), when acetylation levels were at the lowest point. Interestingly, in oocytes, the initial trend was reversed. An increase was observed in the H3K9ac levels of the control group (P=0.014), whereas no difference was observed for DCA in the first 4h. Moreover, although acetylation levels followed a downward tendency with time in both groups, oocytes treated with DCA showed lower H3K9ac levels at 4 and 8h and a higher level at 24h (P=0.04) compared with control. Regarding IA, lower MA were verified in CC whereas oocytes had the opposite profile (CC control vs. IA: P=0.0035; Oc control vs. IA: P<0.001). In CC, this decrease in MA was not accompanied by a decrease in H3K9ac. In contrast, H3K9ac increased compared with the control group at 8 and 16h (control 8h vs. IA 8 h: P=0.019 and control 16h vs. IA 16 h: P=0.019). These changes were accompanied by an increase in the synthesis of new transcripts in the IA group over the time of IVM. Based on these data, we can conclude that changes in pyruvate metabolism caused by manipulation of the IVM system lead to epigenetic and molecular changes in both CC and oocytes.

Proteomics analysis of zebrafish larvae exposed to 3,4-dichloroaniline using the fish embryo acute toxicity test.

The zebrafish (Danio rerio) is a small teleost fish that is becoming increasingly popular in laboratories worldwide and several attributes have also placed the zebrafish under the spotlight of (eco)toxicological studies. Since the 1990s, international organizations such as ISO and OECD have published guidelines for the use of zebrafish in ecotoxicological assessment of environmental toxicants such as the Fish Embryo Acute Toxicity (FET) test, OECD n° 236 guideline. This protocol uses 3,4-dichloroaniline (DCA), an aniline pesticide whose toxicity to fish species at early life stages is well known, as a positive control. Despite its use, little is known about its molecular mechanisms, especially in the context of the FET test. Therefore, this study aimed to investigate such changes in zebrafish larvae exposed to DCA (4 mg/L) for 96 hours using gel-free proteomics. Twenty-four proteins detected in both groups were identified as significantly affected by DCA exposure, and, when considering group-specific entities, 48 proteins were exclusive to DCA (group-specific proteins) while 248 were only detected in the control group. Proteins modulated by DCA treatment were found to be involved in metabolic processes, especially lipids and hormone metabolism (eg, Apoa1 and Apoa1b and vitelogenins), as well as proteins important for developmental processes and organogenesis (eg, Myhc4, Acta2, Sncb, and Marcksb). The results presented here may therefore provide a better understanding of the relationships between molecular changes and phenotype in zebrafish larvae treated with DCA, the reference compound of the FET test.

Butyrate Inhibits Deoxycholic-Acid-Resistant Colonic Cell Proliferation via Cell Cycle Arrest and Apoptosis: A Potential Pathway Linking Dietary Fiber to Cancer Prevention.

Butyrate, an intestinal microbiota metabolite of dietary fiber, exhibits colon cancer preventive effects. In contrast, a high fat intake increases fecal secondary bile acids, such as deoxycholic acid (DCA, a potential cancer promoter), which selectively enrich mutant epithelial cells with an abnormally high resistance to DCA-induced apoptosis in the colon. This study is conducted to test the hypothesis that physiological concentrations of butyrate inhibit DCA-resistant colonic cell proliferation. With human HCT-116 cells as parental colonic cells, a human DCA-resistant colonic cell line (DCA-RCL) is developed. DCA treatment increases apoptosis and intracellular reactive oxygen species (an apoptotic trigger) at a rate threefold greater in HCT-116 cells than in DCA-RCL cells. Subsequently, 41 apoptosis related genes (including signaling pathways) with greater than onefold (mRNA) change in DCA-RCL cells are identified compared with HCT-116 cells. Moreover, butyrate treatment inhibits DCA-RCL cell proliferation with similar efficacy when compared with HCT116 cells via cellular myelocytomatosis oncogene (c-Myc)/p38 mitogen-activated protein kinase pathway. It is demonstrated that butyrate inhibits DCA-RCL cell proliferation at the cellular and molecular level. These data provide a proof of concept that butyrate can protect against colon carcinogenesis through a specific targeting of DCA-resistant colonic cells.

Inhibition of P65 and PDK2 Increases Cytotoxicity and Radiation Sensitivity of Pancreatic Cancer Cells

Background: Pancreatic ductal adenocarcinoma (PDAC) is currently the 4th leading cause of cancer death in the US because of late detection and resistance to chemotherapy and radiation. This tumor resistance is partially due to high activity of the transcription factor, NF-κB which promotes cell survival and pyruvate dehydrogenase kinase 2 (PDK2) which regulates aerobic glycolysis (Warburg Metabolism), cell proliferation, and inhibition of apoptosis. We have shown that simultaneous treatment with the chemical NF-κB inhibitor DMAPT and the Warburg inhibitor DCA is cytotoxic and enhances radiation-induced cell death in human PDAC cells. We have also shown that double gene knockdown of the P65 (RELA) subunit of NF-κB and PDK2 by siRNA increased cytotoxicity and radiation sensitivity of pancreatic cancer cells but to a lesser extent than DMAPT and DCA treatment.
 Experimental Design: Western blots suggested the siRNA did not completely suppress p65 and PDKS protein levels. To test the hypothesis that more complete inhibition of NF-κB and PDK2 is required to increase cytotoxicity in PDAC cells, we transfected the Mia PaCa-2 cell line with appropriate siRNA and exposed the cells to the chemical inhibitors DMAPT and DCA. The cells were plated and followed by irradiation with either 0, 2, 4, or 6 Gy of 160 kVp X-rays
 Results: Compared to previous experiments that utilized siRNA knockdown alone, the combination of dual drugs and dual siRNA shows an increase in both cytotoxicity and radiation-induced cell killing (p< 0.001, t test)
 Conclusion: The data suggest that a more definitive approach such as utilizing CRISPR-Cas9 may be required to ensure complete gene knockdown of p65 and PDK2 to elucidate the mechanism(s) by which simultaneous inhibition of NF-kB and PDK2 inhibition enhance cytotoxicity and radiation-induced cell killing in pancreatic cancer.

Inhibitory Effect of Decitabine-inhibeting Methylation of SHP-1 Gene on Proliferation and Apoptosis of MDS Cell Line Skm-1

To investigate the phenotype and molecular mechanism of DCA on MDS cell model, and to study the response of chemotherapeutic medicines to MDS cells through multiple dimensions, such as cell proliferation, invasion, migration and apoptosis, thus revealing the molecular mechanism of DCA treatment of MDS and its relationship with SHP-1 gene methylation. MTT assay was used to determine the survival rate of MDS cells after treated by different concentrations of DCA. The effect of DCA on the invasion and migration of MDS cells was detected by Transwell assay method. Apoplexin V-FITCPI was used to detect apoptosis, the MDS treatment on the mechanism of DCA was investigated by Western blot and Real-time PCR experiment. According to the experiment, it was found that tumor proliferation could be inhibited when MDS skm-1 cells was treated by DCA, and the absorbance was lower and the inhibitory effect was more obvious in the 2.0, 5.0 μmol/L DCA group than in the 0.5 μmol/L DCA group and the negative control group. Compared with the control group, the number of MDS skm-1 cells crossing through the transwell upper chamber was significantly decreased after DCA application. After treated with 0.5, 2.0 and 5.0 μmol/L DCA, the apoptosis rate of MDS cells was 4.54%, 9.31% and 16.58% respectively, while the apoptosis rate of the control group was 3.20%, which shows the apoptosis rate increased significantly with the concentration of DCA. After treatment of MDS cell lines with different concentration of DCA, the methylation status of SHP-1 gene was decreased with the increase of drug concentration, the expression of SHP-1 was increased, the expression of STAT3 was decreased and the level of phosphorylation was decreased. By analyzing the phenotypic response of DCA treatment on MDS cells, it was found that interfere with MDS can be performed by inhibiting proliferation, metastasis, and inducing apoptosis in a dose-dependent way. It revealed that the molecular mechanism by DCA treatment can improve the methylation of SHP-1 gene and inhibit the expression of p-STAT3.

Tat inhibition by didehydro-Cortistatin A promotes heterochromatin formation at the HIV-1 long terminal repeat

BackgroundTranscription from the integrated HIV-1 promoter is directly governed by its chromatin environment, and the nucleosome-1 downstream from the transcription start site directly impedes transcription from the HIV-1 promoter. The HIV-1 Tat protein regulates the passage from viral latency to active transcription by binding to the viral mRNA hairpin (TAR) and recruiting transcriptional factors to promote transcriptional elongation. The Tat inhibitor didehydro-Cortistatin A (dCA) inhibits transcription and overtime, the lack of low-grade transcriptional events, triggers epigenetic changes at the latent loci that “lock” HIV transcription in a latent state.ResultsHere we investigated those epigenetic changes using multiple cell line models of HIV-1 latency and active transcription. We demonstrated that dCA treatment does not alter the classic nucleosome positioning at the HIV-1 promoter, but promotes tighter nucleosome/DNA association correlating with increased deacetylated H3 occupancy at nucleosome-1. Recruitment of the SWI/SNF chromatin remodeling complex PBAF, necessary for Tat-mediated transactivation, is also inhibited, while recruitment of the repressive BAF complex is enhanced. These results were supported by loss of RNA polymerase II recruitment on the HIV genome, even during strong stimulation with latency-reversing agents. No epigenetic changes were detected in cell line models of latency with Tat-TAR incompetent proviruses confirming the specificity of dCA for Tat.ConclusionsWe characterized the dCA-mediated epigenetic signature on the HIV genome, which translates into potent blocking effects on HIV expression, further strengthening the potential of Tat inhibitors in “block-and-lock” functional cure approaches.

Modulating Glucose Metabolism and Lactate Synthesis in Injured Mouse Tendons: Treatment With Dichloroacetate, a Lactate Synthesis Inhibitor, Improves Tendon Healing

Background: Tendon injuries are common problems among athletes. Complete recovery of the mechanical structure and function of ruptured tendons is challenging. It has been demonstrated that upregulation of glycolysis and lactate production occurs in wounds, inflammation sites, and cancerous tumors, and these metabolic changes also control growth and differentiation of stem and progenitor cells. Similar metabolic changes have been reported in human healing tendons. In addition, lactate production has increased in progenitors isolated from injured tendons after treatment with IL-1β. It is thought that the metabolic changes play a role in tendon healing after injury. Hypothesis: Glucose metabolism is altered during tendon injury and healing, and modulation of this altered metabolism improves tendon repair. Study Design: Controlled laboratory study. Methods: The authors used the tendon injury model involving a complete incision of the Achilles tendon in C57BL/6J female mice and studied alterations of glucose metabolism in injured tendons with [U-13C]glucose and metabolomics analysis 1 and 4 weeks after surgery. They also examined the effects of dichloroacetate (DCA; an indirect lactate synthesis inhibitor) treatment on the recovery of structure and mechanical properties of injured tendons 4 weeks after surgery in the same mouse model. Results: Significant changes in glucose metabolism in tendons after injury surgery were detected. 13C enrichment of metabolites and intermediates, flux through glycolysis, and lactate synthesis, as well as tricarboxylic acid cycle activity, were acutely increased 1 week after injury. Increased glycolysis and lactate generation were also found 4 weeks after injury. DCA-treated injured tendons showed decreased cross-sectional area and higher values of modulus, maximum stress, and maximum force when compared with vehicle-treated injured tendons. Improved alignment of the collagen fibers was also observed in the DCA group. Furthermore, DCA treatment reduced mucoid accumulation and ectopic calcification in injured tendons. Conclusion: The findings indicate that injured tendons acutely increase glycolysis and lactate synthesis after injury and that the inhibition of lactate synthesis by DCA is beneficial for tendon healing. Clinical Relevance: Changing metabolism in injured tendons may be a therapeutic target for tendon repair.

The effect of dichloroacetate in mouse models of epilepsy

A recent report has found that glucose oxidation and the activity of pyruvate dehydrogenase (PDH) are reduced in the chronic stage of the pilocarpine mouse epilepsy model. This is likely caused by increased phosphorylation by PDH kinase of the E1α subunit of PDH, downregulating its activity. Inhibition of this phosphorylation has not yet been explored as a possible approach to treat epilepsy. Chronic dichloroacetate (DCA, 50 and 100 mg/kg/day) treatment was tested in acute seizure and the chronic pilocarpine models. We also determined the effects on phosphorylation state, activity and protein levels of PDH in the chronic stage of the pilocarpine model. DCA treatment did not increase latencies to seizures in the acute flurothyl seizure test and was slightly proconvulsant in the 6 Hz test. The latencies to seizures in a second-hit flurothyl test were decreased in SE vs. No SE mice in the chronic stage, but were not restored by DCA. In mice that had experienced pilocarpine-induced SE and were in the chronic “epileptic” stage of the model, PDH activity was reduced by 65% compared to “healthy” No SE mice. This was partially alleviated with DCA treatment. Also, PDH protein levels were decreased by 37% and phosphorylation at Ser300 of PDH was increased by 52% in SE mice, but were not significantly changed with DCA. Moreover DCA treatment decreased the amounts of total PDH by 23% in No SE mice, which may explain the proconvulsant effects in the 6 Hz test. The reduction in PDH protein levels during the chronic epileptic stage suggests increased degradation of the protein, which may contribute to the deficient glucose oxidation found in epilepsy. Taken together, DCA did not have any anti-convulsant effects in the tested models. Future studies utilising other PDH kinase inhibitors are required to determine whether this treatment approach is viable.

Abstract B56: Treatment-induced autophagy increases amino acid uptake and switches glucose addiction to amino acid catabolism in cancer

Abstract Background: Autophagy is a cellular catabolic degradation response to starvation or stress and the rerouting of cellular metabolism by autophagic cancer cells, to sustain cellular homeostasis during starvation or treatment-induced stress, remains poorly understood. We investigated the hypotheses that treatment-induced autophagy could prolong cancer cell survival, supported by adapted metabolism reversibly maintaining cellular homeostasis. Methods: We induced autophagy in human prostate (PC3) and colorectal (HT29, HCT116 WT (wild-type) and HCT116 Bax-ko (Bax-knock out)) cancer cells by 6 or 24 hours of amino-acid and serum deprivation (in Hank's balanced salt solution), or by 24 hours of treatment with PI103 (a phosphatidyl inositol-3 kinase inhibitor) or dichloroacetate (DCA, a pyruvate dehydrogenase kinase inhibitor). Cellular metabolism during starvation- or treatment-induced autophagy and subsequent recovery were examined by 1H- and 13C-magnetic resonance spectroscopy metabolomic studies. 13C-labeled glucose was used to assess metabolic flux. In vitro findings were verified in two corresponding colorectal xenograft models treated with PI103 or DCA. Results and Discussion: Increased expression of LC3II by western blots and the increased level of autophagosomes visualized by electron microscopy confirmed the induction of autophagy with minimal apoptosis and necrosis in HCT116 Bax-ko cells following starvation, DCA or PI103 treatment and in HCT116 WT, PC3 and HT29 cells following DCA treatment. PI103-induced autophagy prolonged cell survival, whereas starvation-induced autophagic cells eventually died. In PI103- or DCA-induced autophagy, metabolism was re-routed by i) reduced aerobic glycolysis with unchanged glucose uptake, increased cellular glucose levels (P<0.003) and reduced lactate excretion (P<0.0001); ii) increased uptake of branched-chain amino acids and glutamine (P<0.005), with a net accumulation of many intracellular amino acids and succinate (P<0.003), a TCA cycle intermediate. These metabolic alterations can prolong cancer cell survival during stress in a well-nourished environment by providing energy from amino-acid catabolism. Metabolic changes were reversed on recovery from treatment-induced autophagy. Increased levels of glutamine (P<0.01) and TCA-cycle intermediates (P<0.04) were also observed in DCA- and PI103-treated HT29 and HCT116-Bax-ko tumor xenografts, providing potential non-invasive biomarkers for treatment-induced autophagy. This work is supported by the CR-UK and EPSRC Cancer Imaging Centre in association with the MRC and Department of Health (England) grants C1060/A10334 and C1060/A16464, NHS funding to the NIHR Biomedical Research Centre. Chang Gung Medical Foundation (Taiwan) grant CMRPG370441 and MRC-funded studentship (MRC119X). MOL is an NIHR Senior Investigator. We thank Alice Warley at the Kings College London Centre for Ultrastructural Imaging (CUI) for providing facilities for electron microscopy. Citation Format: Gigin Lin, Helen Troy, Gabriela Andrejeva, Anne-Christine LF Wong Te Fong, Dow-Mu Koh, Simon P. Robinson, Ian R. Judson, John R. Griffiths, Martin O. Leach, Yuen-Li Chung. Treatment-induced autophagy increases amino acid uptake and switches glucose addiction to amino acid catabolism in cancer. [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 B56.

Abstract A62: Chemoresistance through the intratumoral metabolism of dichloroacetate

Abstract Dichloroacetate (DCA), a small molecule commonly used to treat metabolic disorders, is under investigation as an anti-cancer therapy due to its ability to reverse the Warburg effect and induce apoptosis. While DCA's mechanism of action is well-studied, other factors that influence its success as a cancer treatment have not been thoroughly studied. Here we present data showing that expression of glutathione transferase zeta 1 (GSTZ1), the enzyme responsible for the metabolism of DCA, is often misregulated in cancer, resulting in expression in tissues where there generally is none and a lack of expression in sites that have a high level of basal expression. Expression of GSTZ1 in a tumor would be expected to reduce intratumoral DCA concentrations. As we see a disconnect between GSTZ1 mRNA and protein expression in these tumors, control of expression is likely through miRNA. We also report that chloride concentrations, which strongly impact the stability of GSTZ1 in the presence of DCA, may be abnormal in tumors, further influencing DCA metabolism, concentration, and treatment success. Both two- and three-dimensional cell culture of cells expressing or not expressing GSTZ1 provide a proof of principle that GSTZ1 expression confers a level of resistance to DCA. These results have important clinical implications as GSTZ1 expression and chloride concentration could be used as markers to predict outcomes of DCA anticancer therapy. Citation Format: Stephan C. Jahn, Mohamed H. Solaymen, Ryan J. Lorenzo, Taimour Langaee, Peter W. Stacpoole, Margaret O. James. Chemoresistance through the intratumoral metabolism of dichloroacetate. [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 A62.

TH‐C‐12A‐05: Dynamic Couch Motion for Improvement of Radiation Therapy Trajectories in DCA and VMAT

Purpose:To investigate the potential improvement in dosimetric external beam radiation therapy plan quality using an optimized dynamic gantry and couch motion trajectory which minimizes exposure to the organs at risk.Methods:Patient‐specific anatomical information of head‐and‐neck and cranial cancer patients was used to quantify the geometric overlap between target volumes and organs‐at‐risk (OARs) based on their two‐dimensional projection from source to a plane at isocentre as a function of gantry and couch angle. QUANTEC dose constraints were then used as weighting factors for the OARs to generate a map of couch‐gantry coordinate space indicating degree of overlap at each point in space. A couch‐gantry collision space was generated by direct measurement on a Varian Truebeam linac using an anthropomorphic solid‐water phantom. A dynamic, fully customizable algorithm was written to generate a navigable ideal trajectory for the patient specific couch‐gantry space. The advanced algorithm includes weighting factors which can be used to balance the implementation of absolute minimum values of overlap, with the clinical practicality of largescale couch motion and delivery time. Optimized trajectories were calculated for cranial DCA treatments and for head‐and‐neck VMAT treatments and compared to conventional DCA and VMAT treatment trajectories.Results:Comparison of optimized treatment trajectories with conventional treatment trajectories indicates a decrease in dose to the organs‐at‐risk between 4.64% and 6.82% (2.39 and 3.52 Gy) of the prescription dose per patient per organ at risk.Conclusion:Using simultaneous couch and gantry motion during radiation therapy to minimize the geometrical overlap in the beams‐eye‐view target volumes and the organs‐at‐risk can have an appreciable dose reduction to organs‐at‐risk.

Sa1839 Atonal Homolog 1 (ATOH1) Upregulates Gastric-and Intestinal-Type Protein Expression in Esophageal Squamous Cells, and Increases Proliferation in Barrett's Cells: Potential Roles for ATOH1 in Barrett's Pathogenesis and Carcinogenesis

in response to DCA treatment. 14% of the K14-Cdx2 mice treated with DCA developed two nodules each at the SCJ, whereas none of the K14-Cdx2 mice receiving water alone developed nodules. In contrast, 70% of the IL-1β mice, and an equal number of Cdx2/IL1β mice developed nodular disease at the SCJ after DCA treatment. All DCA treatmentassociated nodules were histologically consistent with metaplasia. However overall the double-transgenic mice had half as many nodules as IL-1β mice. Cdx2 did not affect IL-1β mRNA levels in the double transgenic mice, nor was the inflammation associated with IL1β expression reduced in the Cdx2/IL-1β mice compared to IL-1β mice, as determined by CD45R+ cell infiltration in the esophagi and forestomach. Moreover, cell proliferation rates were not altered by Cdx2 expression, as EdU incorporation was not significantly different. Lastly, the morphologic appearance of the nodules in all three lines, K14-Cdx2, L2-IL-1β, and K14-Cdx2/L2-IL-1β mice was similar, with abundant alcian blue expressing goblet cells detected, along with expression of other markers of intestinalization. Further molecular characterization of the metaplastic nodules by cDNA microarray profiling is underway. Conclusion: Ectopic expression of Cdx2 in the IL-1Beta mouse model for BE reduces the nodular intestinal metaplasia disease burden. These studies raise new questions as to the specific role of inflammation and intestinal transcription factors in the development of Barrett's esophagus in humans.

Sa1842 In Esophageal Epithelial Cells From Barrett's Patients, Protein Kinase a Phosphorylates SOX9, Enhancing Its Binding to Target Gene Promoters: A Potential Role for SOX9 in Barrett's Pathogenesis

in response to DCA treatment. 14% of the K14-Cdx2 mice treated with DCA developed two nodules each at the SCJ, whereas none of the K14-Cdx2 mice receiving water alone developed nodules. In contrast, 70% of the IL-1β mice, and an equal number of Cdx2/IL1β mice developed nodular disease at the SCJ after DCA treatment. All DCA treatmentassociated nodules were histologically consistent with metaplasia. However overall the double-transgenic mice had half as many nodules as IL-1β mice. Cdx2 did not affect IL-1β mRNA levels in the double transgenic mice, nor was the inflammation associated with IL1β expression reduced in the Cdx2/IL-1β mice compared to IL-1β mice, as determined by CD45R+ cell infiltration in the esophagi and forestomach. Moreover, cell proliferation rates were not altered by Cdx2 expression, as EdU incorporation was not significantly different. Lastly, the morphologic appearance of the nodules in all three lines, K14-Cdx2, L2-IL-1β, and K14-Cdx2/L2-IL-1β mice was similar, with abundant alcian blue expressing goblet cells detected, along with expression of other markers of intestinalization. Further molecular characterization of the metaplastic nodules by cDNA microarray profiling is underway. Conclusion: Ectopic expression of Cdx2 in the IL-1Beta mouse model for BE reduces the nodular intestinal metaplasia disease burden. These studies raise new questions as to the specific role of inflammation and intestinal transcription factors in the development of Barrett's esophagus in humans.

Structural origin of anisotropic transport in electrically conducting dichloroacetic acid-treated polymers

The post-deposition treatment of water-processable, polymer acid-templated conducting polymers with dichloroacetic acid has recently been shown to enhance in-plane conductivity by orders of magnitude. Here, we evaluate both the in-plane and out-of-plane conductivities of dichloroacetic acid-treated conducting polymer films, and track the accompanying evolution of film structure and composition. Films of poly(ethylene dioxythiophene)–poly(styrene sulfonic acid), PEDOT–PSS, exhibit improvements in both in-plane and out-of-plane conductivities following dichloroacetic acid treatment, associated with the removal of excess PSS from the surface. Conversely, polyaniline–poly(2-acrylamido-2-methyl-1-propanesulfonic acid), PANI–PAAMPSA, films are characterized by high anisotropies in conductivity, with the in-plane conductivity being orders of magnitude higher following DCA treatment and the out-of-plane conductivity substantially lower after the same treatment. Our experiments indicate that this unusual electrical anisotropy results from a vertically inhomogeneous composition profile.

Case Report of Long Term Complete Remission of Metastatic Renal Squamous Cell Carcinoma after Palliative Radiotherapy and Adjuvant Dichloroacetate

Renal squamous cell carcinoma is a rare form of renal cancer which is considered incurable once metastases develop. Prognosis is poor and average survival of advanced stage disease is typically in the range of several months, despite all available conventional therapies. We describe the case of a 72 year old female with metastatic renal squamous cell carcinoma who had a radical nephrectomy with positive surgical margins, renal vein invasion and metastases to multiple abdominal lymph nodes. She received a course of palliative radiotherapy to the abdomen with 4500cGy in 25 fractions over 5 weeks. Following radiotherapy, she was treated with a cyclic regimen of oral sodium dichloroacetate (“DCA”). Treatment was discontinued after 3 months due to development of peripheral neuropathy. Follow-up imaging upon completion of DCA treatment revealed no sign of metastatic disease. The neuropathy gradually improved and computed tomography imaging four years later demonstrated no cancer recurrence. The patient continues to feel well with no clinical evidence of recurrence five years after completion of therapy, and is living a normal and active life.

Mutations of the Mitochondrial-tRNA Modifier MTO1 Cause Hypertrophic Cardiomyopathy and Lactic Acidosis

Dysfunction of mitochondrial respiration is an increasingly recognized cause of isolated hypertrophic cardiomyopathy. To gain insight into the genetic origin of this condition, we used next-generation exome sequencing to identify mutations in MTO1, which encodes mitochondrial translation optimization 1. Two affected siblings carried a maternal c.1858dup (p.Arg620Lysfs(∗)8) frameshift and a paternal c.1282G>A (p.Ala428Thr) missense mutation. A third unrelated individual was homozygous for the latter change. In both humans and yeast, MTO1 increases the accuracy and efficiency of mtDNA translation by catalyzing the 5-carboxymethylaminomethylation of the wobble uridine base in three mitochondrial tRNAs (mt-tRNAs). Accordingly, mutant muscle and fibroblasts showed variably combined reduction in mtDNA-dependent respiratory chain activities. Reduced respiration in mutant cells was corrected by expressing a wild-type MTO1 cDNA. Conversely, defective respiration of a yeast mto1Δ strain failed to be corrected by an Mto1(Pro622∗) variant, equivalent to human MTO1(Arg620Lysfs∗8), whereas incomplete correction was achieved by an Mto1(Ala431Thr) variant, corresponding to human MTO1(Ala428Thr). The respiratory yeast phenotype was dramatically worsened in stress conditions and in the presence of a paromomycin-resistant (P(R)) mitochondrial rRNA mutation. Lastly, in vivo mtDNA translation was impaired in the mutant yeast strains.