Publications by authors named "Niki M Zacharias"

18 Publications

  • Page 1 of 1

Mammalian Expression and Biotinylation of Extracellular Protein Targets for Directed Evolution.

ACS Omega 2020 Oct 22;5(39):25440-25455. Epub 2020 Sep 22.

Department of Cancer Systems Imaging, MD Anderson Cancer Center, Houston, Texas 77030, United States.

Directed evolution is a powerful tool for the selection of functional ligands from molecular libraries. Extracellular domains (ECDs) of cell surface receptors are common selection targets for therapeutic and imaging agent development. Unfortunately, these proteins are often post-translationally modified and are therefore unsuitable for expression in bacterial systems. Directional immobilization of these targets is further hampered by the absence of biorthogonal groups for site-specific chemical conjugation. We have developed a nonadherent mammalian expression system for rapid, high-yield expression of biotinylated ECDs. ECDs from EGFR, HER2, and HER3 were site-specifically biotinylated and recovered from the cell culture supernatant with yields of up to 10 mg/L at >90% purity. Biotinylated ECDs also contained a protease cleavage site for rapid and selective release of the ECD after immobilization on avidin/streptavidin resins and library binding. A model mRNA display selection round was carried out against the HER2 ECD with the HER2 affibody expressed as an mRNA-protein fusion. HER2 affibody-mRNA fusions were selectively released by thrombin and quantitative PCR revealed substantial improvements in the enrichment of functional affibody-mRNA fusions relative to direct PCR amplification of the resin-bound target. This methodology allows rapid purification of high-quality targets for directed evolution and selective elution of functional sequences at the conclusion of each selection round.
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http://dx.doi.org/10.1021/acsomega.0c03990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7542843PMC
October 2020

Hyperpolarized [1-C]pyruvate-to-[1-C]lactate conversion is rate-limited by monocarboxylate transporter-1 in the plasma membrane.

Proc Natl Acad Sci U S A 2020 09 24;117(36):22378-22389. Epub 2020 Aug 24.

Department of Cancer System Imaging, University of Texas MD Anderson Cancer Center, Houston, TX 77030;

Hyperpolarized [1-C]pyruvate magnetic resonance spectroscopic imaging (MRSI) is a noninvasive metabolic-imaging modality that probes carbon flux in tissues and infers the state of metabolic reprograming in tumors. Prevailing models attribute elevated hyperpolarized [1-C]pyruvate-to-[1-C]lactate conversion rates in aggressive tumors to enhanced glycolytic flux and lactate dehydrogenase A (LDHA) activity (Warburg effect). By contrast, we find by cross-sectional analysis using genetic and pharmacological tools in mechanistic studies applied to well-defined genetically engineered cell lines and tumors that initial hyperpolarized [1-C]pyruvate-to-[1-C]lactate conversion rates as well as global conversion were highly dependent on and critically rate-limited by the transmembrane influx of [1-C]pyruvate mediated predominately by monocarboxylate transporter-1 (MCT1). Specifically, in a cell-encapsulated alginate bead model, induced short hairpin (shRNA) knockdown or overexpression of MCT1 quantitatively inhibited or enhanced, respectively, unidirectional pyruvate influxes and [1-C]pyruvate-to-[1-C]lactate conversion rates, independent of glycolysis or LDHA activity. Similarly, in tumor models in vivo, hyperpolarized [1-C]pyruvate-to-[1-C]lactate conversion was highly dependent on and critically rate-limited by the induced transmembrane influx of [1-C]pyruvate mediated by MCT1. Thus, hyperpolarized [1-C]pyruvate MRSI measures primarily MCT1-mediated [1-C]pyruvate transmembrane influx in vivo, not glycolytic flux or LDHA activity, driving a reinterpretation of this maturing new technology during clinical translation. Indeed, Kaplan-Meier survival analysis for patients with pancreatic, renal, lung, and cervical cancers showed that high-level expression of MCT1 correlated with poor overall survival, and only in selected tumors, coincident with LDHA expression. Thus, hyperpolarized [1-C]pyruvate MRSI provides a noninvasive functional assessment primarily of MCT1 as a clinical biomarker in relevant patient populations.
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http://dx.doi.org/10.1073/pnas.2003537117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486767PMC
September 2020

Early Detection of Pancreatic Intraepithelial Neoplasias (PanINs) in Transgenic Mouse Model by Hyperpolarized C Metabolic Magnetic Resonance Spectroscopy.

Int J Mol Sci 2020 May 25;21(10). Epub 2020 May 25.

Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, Houston, TX 77054, USA.

While pancreatic cancer (PC) survival rates have recently shown modest improvement, the disease remains largely incurable. Early detection of pancreatic cancer may result in improved outcomes and therefore, methods for early detection of cancer, even premalignant lesions, may provide more favorable outcomes. Pancreatic intraepithelial neoplasias (PanINs) have been identified as premalignant precursor lesions to pancreatic cancer. However, conventional imaging methods used for screening high-risk populations do not have the sensitivity to detect PanINs. Here, we have employed hyperpolarized metabolic imaging in vivo and nuclear magnetic resonance (H-NMR) metabolomics ex vivo to identify and understand metabolic changes, towards enabling detection of early PanINs and progression to advanced PanINs lesions that precede pancreatic cancer formation. Progression of disease from tissue containing predominantly low-grade PanINs to tissue with high-grade PanINs showed a decreasing alanine/lactate ratio from high-resolution NMR metabolomics ex vivo. Hyperpolarized magnetic resonance spectroscopy (HP-MRS) allows over 10,000-fold sensitivity enhancement relative to conventional magnetic resonance. Real-time HP-MRS was employed to measure non-invasively changes of alanine and lactate metabolites with disease progression and in control mice in vivo, following injection of hyperpolarized [1-C] pyruvate. The alanine-to-lactate signal intensity ratio was found to decrease as the disease progressed from low-grade PanINs to high-grade PanINs. The biochemical changes of alanine transaminase (ALT) and lactate dehydrogenase (LDH) enzyme activity were assessed. These results demonstrate that there are significant alterations of ALT and LDH activities during the transformation from early to advanced PanINs lesions. Furthermore, we demonstrate that real-time conversion kinetic rate constants (k and k) can be used as metabolic imaging biomarkers of pancreatic premalignant lesions. Findings from this emerging HP-MRS technique can be translated to the clinic for detection of pancreatic premalignant lesion in high-risk populations.
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http://dx.doi.org/10.3390/ijms21103722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279395PMC
May 2020

6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase-2 Regulates TP53-Dependent Paclitaxel Sensitivity in Ovarian and Breast Cancers.

Clin Cancer Res 2019 09 7;25(18):5702-5716. Epub 2019 Aug 7.

Department of Experimental Therapeutics, University of Texas, MD Anderson Cancer Center, Houston, Texas.

Purpose: Paclitaxel is an integral component of primary therapy for breast and epithelial ovarian cancers, but less than half of these cancers respond to the drug. Enhancing the response to primary therapy with paclitaxel could improve outcomes for women with both diseases. Twelve kinases that regulate metabolism were depleted in multiple ovarian and breast cancer cell lines to determine whether they regulate sensitivity to paclitaxel in Sulforhodamine B assays. The effects of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 () depletion on cell metabolomics, extracellular acidification rate, nicotinamide adenine dinucleotide phosphate, reactive oxygen species (ROS), and apoptosis were studied in multiple ovarian and breast cancer cell lines. Four breast and ovarian human xenografts and a breast cancer patient-derived xenograft (PDX) were used to examine the knockdown effect of on tumor cell growth .

Results: Knockdown of inhibited clonogenic growth and enhanced paclitaxel sensitivity in ovarian and breast cancer cell lines with wild-type (wt). Silencing significantly inhibited tumor growth and enhanced paclitaxel sensitivity in four xenografts derived from two ovarian and two breast cancer cell lines, and prolonged survival in a triple-negative breast cancer PDX. Transfection of si increased the glycolysis rate, but decreased the flow of intermediates through the pentose-phosphate pathway in cancer cells with wt, decreasing NADPH. ROS accumulated after knockdown, which stimulated Jun N-terminal kinase and p53 phosphorylation, and induced apoptosis that depended upon upregulation of p21 and Puma.

Conclusions: PFKFB2 is a novel target whose inhibition can enhance the effect of paclitaxel-based primary chemotherapy upon ovarian and breast cancers retaining wt.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744996PMC
September 2019

Assessing Metabolic Intervention with a Glutaminase Inhibitor in Real-Time by Hyperpolarized Magnetic Resonance in Acute Myeloid Leukemia.

Mol Cancer Ther 2019 11 6;18(11):1937-1946. Epub 2019 Aug 6.

Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, Houston, Texas.

Acute myeloid leukemia (AML) is an aggressive hematopoietic disease characterized by glutamine-dependent metabolism. A novel glutaminase (GLS) inhibitor, CB-839, is currently under evaluation for treatment of hematopoietic malignancies and solid tumors. Our purpose was to measure cellular changes in AML associated with CB-839 treatment and to test the ability of hyperpolarized pyruvate for interrogating these changes to OCI-AML3 cells. Our results show that treatment with CB-839 interfered with the citric acid cycle, reduced the NADH/NAD ratio and ATP levels, reduced cell proliferation and viability, and reduced the basal and maximal respiratory capacities [oxygen consumption rate (OCR)]. We observed a reduction of the conversion of hyperpolarized pyruvate to lactate in cell lines and in a mouse AML model after CB-839 treatment. Our and results support the hypothesis that, in AML, glutamine is utilized to generate reducing equivalents (NADH, FADH) through the citric acid cycle and that reduction in redox state by GLS inhibition decreases the rate of pyruvate to lactate conversion catalyzed by lactate dehydrogenase. We propose hyperpolarized pyruvate/lactate measurement as a method for direct monitoring of metabolic changes occurring in AML patients receiving CB-839. With further optimization, this method may provide a noninvasive imaging tool to assess the early efficacy of therapeutic intervention with GLS inhibitors.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7080291PMC
November 2019

Combining Hyperpolarized Real-Time Metabolic Imaging and NMR Spectroscopy To Identify Metabolic Biomarkers in Pancreatic Cancer.

J Proteome Res 2019 07 4;18(7):2826-2834. Epub 2019 Jun 4.

Department of Gastrointestinal Oncology , H. Lee Moffitt Cancer Center , Tampa , Florida 33612 , United States.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer that progresses without any symptom, and oftentimes, it is detected at an advanced stage. The lack of prior symptoms and effective treatments have created a knowledge gap in the management of this lethal disease. This issue can be addressed by developing novel noninvasive imaging-based biomarkers in PDAC. We explored in vivo hyperpolarized (HP) C MRS of pyruvate to lactate conversion and ex vivo H NMR spectroscopy in a panel of well-annotated patient-derived PDAC xenograft (PDXs) model and investigated the correlation between aberrant glycolytic metabolism and aggressiveness of the tumor. Real-time metabolic imaging data demonstrate the immediate intracellular conversion of HP C pyruvate to lactate after intravenous injection interrogating upregulated lactate dehydrogenase (LDH) activity in aggressive PDXs. Total ex vivo lactate measurement by H NMR spectroscopy showed a direct correlation with in vivo dynamic pyruvate-to-lactate conversion and demonstrated the potential of dynamic metabolic flux as a biomarker of total lactate concentration and aggressiveness of the tumor. Furthermore, the metabolite concentrations were very distinct among all four tumor types analyzed in this study. Overexpression of LDH-A and hypoxia-inducible factor (HIF-1α) plays a significant role in the conversion kinetics of HP pyruvate-to-lactate in tumors. Collectively, these data identified aberrant metabolic characteristics of pancreatic cancer PDXs and could potentially delineate metabolic targets for therapeutic intervention. Metabolic imaging with HP pyruvate and NMR metabolomics may enable identification and classification of aggressive subtypes of patient-derived xenografts. Translation of this real-time metabolic technique to the clinic may have the potential to improve the management of patients at high risk of developing pancreatic diseases.
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http://dx.doi.org/10.1021/acs.jproteome.9b00132DOI Listing
July 2019

Assessing Therapeutic Efficacy in Real-time by Hyperpolarized Magnetic Resonance Metabolic Imaging.

Cells 2019 04 11;8(4). Epub 2019 Apr 11.

Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

Precisely measuring tumor-associated alterations in metabolism clinically will enable the efficient assessment of therapeutic responses. Advances in imaging technologies can exploit the differences in cancer-associated cell metabolism as compared to normal tissue metabolism, linking changes in target metabolism to therapeutic efficacy. Metabolic imaging by Positron Emission Tomography (PET) employing 2-fluoro-deoxy-glucose ([F]FDG) has been used as a routine diagnostic tool in the clinic. Recently developed hyperpolarized Magnetic Resonance (HP-MR), which radically increases the sensitivity of conventional MRI, has created a renewed interest in functional and metabolic imaging. The successful translation of this technique to the clinic was achieved recently with measurements of C-pyruvate metabolism. Here, we review the potential clinical roles for metabolic imaging with hyperpolarized MRI as applied in assessing therapeutic intervention in different cancer systems.
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http://dx.doi.org/10.3390/cells8040340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6523855PMC
April 2019

Real-Time Interrogation of Aspirin Reactivity, Biochemistry, and Biodistribution by Hyperpolarized Magnetic Resonance Spectroscopy.

Angew Chem Int Ed Engl 2019 03 20;58(13):4179-4183. Epub 2019 Feb 20.

Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX, 77030, USA.

Hyperpolarized magnetic resonance spectroscopy enables quantitative, non-radioactive, real-time measurement of imaging probe biodistribution and metabolism in vivo. Here, we investigate and report on the development and characterization of hyperpolarized acetylsalicylic acid (aspirin) and its use as a nuclear magnetic resonance (NMR) probe. Aspirin derivatives were synthesized with single- and double- C labels and hyperpolarized by dynamic nuclear polarization with 4.7 % and 3 % polarization, respectively. The longitudinal relaxation constants (T ) for the labeled acetyl and carboxyl carbonyls were approximately 30 seconds, supporting in vivo imaging and spectroscopy applications. In vitro hydrolysis, transacetylation, and albumin binding of hyperpolarized aspirin were readily monitored in real time by C-NMR spectroscopy. Hyperpolarized, double-labeled aspirin was well tolerated in mice and could be observed by both C-MR imaging and C-NMR spectroscopy in vivo.
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http://dx.doi.org/10.1002/anie.201812759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467058PMC
March 2019

Parahydrogen-Based Hyperpolarization for Biomedicine.

Angew Chem Int Ed Engl 2018 08 1;57(35):11140-11162. Epub 2018 Aug 1.

Russian Academy of Sciences (RAS), Leninskiy Prospekt 14, Moscow, 119991, Russia.

Magnetic resonance (MR) is one of the most versatile and useful physical effects used for human imaging, chemical analysis, and the elucidation of molecular structures. However, its full potential is rarely used, because only a small fraction of the nuclear spin ensemble is polarized, that is, aligned with the applied static magnetic field. Hyperpolarization methods seek other means to increase the polarization and thus the MR signal. A unique source of pure spin order is the entangled singlet spin state of dihydrogen, parahydrogen (pH ), which is inherently stable and long-lived. When brought into contact with another molecule, this "spin order on demand" allows the MR signal to be enhanced by several orders of magnitude. Considerable progress has been made in the past decade in the area of pH -based hyperpolarization techniques for biomedical applications. It is the goal of this Review to provide a selective overview of these developments, covering the areas of spin physics, catalysis, instrumentation, preparation of the contrast agents, and applications.
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http://dx.doi.org/10.1002/anie.201711842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105405PMC
August 2018

Induction of autophagy by ARHI (DIRAS3) alters fundamental metabolic pathways in ovarian cancer models.

BMC Cancer 2016 10 26;16(1):824. Epub 2016 Oct 26.

Department of Cancer Systems Imaging, the University of Texas M.D. Anderson Cancer Center, Houston, USA.

Background: Autophagy is a bulk catabolic process that modulates tumorigenesis, therapeutic resistance, and dormancy. The tumor suppressor ARHI (DIRAS3) is a potent inducer of autophagy and its expression results in necroptotic cell death in vitro and tumor dormancy in vivo. ARHI is down-regulated or lost in over 60 % of primary ovarian tumors yet is dramatically up-regulated in metastatic disease. The metabolic changes that occur during ARHI induction and their role in modulating death and dormancy are unknown.

Methods: We employed Nuclear Magnetic Resonance (NMR)-based metabolomic strategies to characterize changes in key metabolic pathways in both cell culture and xenograft models of ARHI expression and autophagy. These pathways were further interrogated by cell-based immunofluorescence imaging, tracer uptake studies, targeted metabolic inhibition, and in vivo PET/CT imaging.

Results: Induction of ARHI in cell culture models resulted in an autophagy-dependent increase in lactate production along with increased glucose uptake and enhanced sensitivity to glycolytic inhibitors. Increased uptake of glutamine was also dependent on autophagy and dramatically sensitized cultured ARHI-expressing ovarian cancer cell lines to glutaminase inhibition. Induction of ARHI resulted in a reduction in mitochondrial respiration, decreased mitochondrial membrane potential, and decreased Tom20 staining suggesting an ARHI-dependent loss of mitochondrial function. ARHI induction in mouse xenograft models resulted in an increase in free amino acids, a transient increase in [F]-FDG uptake, and significantly altered choline metabolism.

Conclusions: ARHI expression has previously been shown to trigger autophagy-associated necroptosis in cell culture. In this study, we have demonstrated that ARHI expression results in decreased cellular ATP/ADP, increased oxidative stress, and decreased mitochondrial function. While this bioenergetic shock is consistent with programmed necrosis, our data indicates that the accompanying up-regulation of glycolysis and glutaminolysis is autophagy-dependent and serves to support cell viability rather than facilitate necroptotic cell death. While the mechanistic basis for metabolic up-regulation following ARHI induction is unknown, our preliminary data suggest that decreased mitochondrial function and increased metabolic demand may play a role. These alterations in fundamental metabolic pathways during autophagy-associated necroptosis may provide the basis for new therapeutic strategies for the treatment of dormant ovarian tumors.
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http://dx.doi.org/10.1186/s12885-016-2850-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5080741PMC
October 2016

Developing hyperpolarized silicon particles for in vivo MRI targeting of ovarian cancer.

J Med Imaging (Bellingham) 2016 Jul 10;3(3):036001. Epub 2016 Aug 10.

University of Texas MD Anderson Cancer Center , Department of Cancer Systems Imaging, 1515 Holcombe Boulevard, Houston, Texas 77030, United States.

Silicon-based nanoparticles are ideally suited for use as biomedical imaging agents due to their biocompatibility, biodegradability, and simple surface chemistry that facilitates drug loading and targeting. A method of hyperpolarizing silicon particles using dynamic nuclear polarization, which increases magnetic resonance imaging signals by several orders-of-magnitude through enhanced nuclear spin alignment, has recently been developed to allow silicon particles to function as contrast agents for in vivo magnetic resonance imaging. The enhanced spin polarization of silicon lasts significantly longer than other hyperpolarized agents (tens of minutes, whereas [Formula: see text] for other species at room temperature), allowing a wide range of potential applications. We report our recent characterizations of hyperpolarized silicon particles, with the ultimate goal of targeted, noninvasive, and nonradioactive molecular imaging of various cancer systems. A variety of particle sizes (20 nm to [Formula: see text]) were found to have hyperpolarized relaxation times ranging from [Formula: see text] to 50 min. The addition of various functional groups to the particle surface had no effect on the hyperpolarization buildup or decay rates and allowed in vivo imaging over long time scales. Additional in vivo studies examined a variety of particle administration routes in mice, including intraperitoneal injection, rectal enema, and oral gavage.
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http://dx.doi.org/10.1117/1.JMI.3.3.036001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4979585PMC
July 2016

Towards Real-time Metabolic Profiling of Cancer with Hyperpolarized Succinate.

J Mol Imaging Dyn 2016 Jun 11;6(1). Epub 2016 Jan 11.

Department of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, USA.

Purpose: The energy-yielding mitochondrial Krebs cycle has been shown in many cancers and other diseases to be inhibited or mutated. In most cells, the Krebs cycle with oxidative phosphorylation generates approximately 90% of the adenosine triphosphate in the cell. We designed and hyperpolarized carbon-13 labeled succinate (SUC) and its derivative diethyl succinate (DES) to interrogate the Krebs cycle in real-time in cancer animal models.

Procedures: Using Parahydrogen Induced Polarization (PHIP), we generated hyperpolarized SUC and DES by hydrogenating their respective fumarate precursors. DES and SUC metabolism was studied in five cancer allograft animal models: breast (4T1), Renal Cell Carcinoma (RENCA), colon (CT26), lymphoma NSO, and lymphoma A20.

Results: The extent of hyperpolarization was 8 ± 2% for SUC and 2.1 ± 0.6% for DES. The metabolism of DES and SUC in the Krebs cycle could be followed in animals 5 s after tail vein injection. The biodistribution of the compounds was observed using C FISP imaging. We observed significant differences in uptake and conversion of both compounds in different cell types both and .

Conclusion: With hyperpolarized DES and SUC, we are able to meet many of the requirements for a useable metabolic imaging compound - high polarization, relatively long T values, low toxicity and high water solubility. However, succinate and its derivative DES are metabolized robustly by RENCA but not by the other cancer models. Our results underscore the heterogeneity of cancer cells and the role cellular uptake plays in hyperpolarized metabolic spectroscopy.
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http://dx.doi.org/10.4172/2155-9937.1000123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4989923PMC
June 2016

Role of Increased n-acetylaspartate Levels in Cancer.

J Natl Cancer Inst 2016 Jan 26;108(6):djv426. Epub 2016 Jan 26.

Departments of Gynecologic Oncology and Reproductive Medicine (BZ, RAP, RR, TM, ASN, HJD, WH, CI, SYW, KMG, SP, LSM, AMN, GAP, AKS), Cancer Systems Imaging (NMZ, CRM, PKB), Biostatistics (MG), Cancer Medicine (CVP), Center for RNA Interference and Non-Coding RNA (CI, LSM, CRA, GLB, AKS), Cancer Biology (YS, MBE, GLB, AKS), Experimental Therapeutics (CRA, GLB), Bioinformatics and Computational Biology (YW, KAB), Melanoma Medical Oncology (MAD), and Pathology (JL), University of Texas M. D. Anderson Cancer Center, Houston, TX; Department of Nanomedicine and Bioengineering, UT Health, Houston, TX (GLB, AKS); Departments of Psychology, Urology, and Obstetrics and Gynecology, the University of Iowa, Iowa City, IA (SKL); Laboratory for Systems Biology of Human Diseases (LY, JB, AA, DN), Department of Chemical and Biomolecular Engineering (LY, JB, AA, DN), and Department of Bioengineering (DN), Rice University, Houston, TX.

Background: The clinical and biological effects of metabolic alterations in cancer are not fully understood.

Methods: In high-grade serous ovarian cancer (HGSOC) samples (n = 101), over 170 metabolites were profiled and compared with normal ovarian tissues (n = 15). To determine NAT8L gene expression across different cancer types, we analyzed the RNA expression of cancer types using RNASeqV2 data available from the open access The Cancer Genome Atlas (TCGA) website (http://www.cbioportal.org/public-portal/). Using NAT8L siRNA, molecular techniques and histological analysis, we determined cancer cell viability, proliferation, apoptosis, and tumor growth in in vitro and in vivo (n = 6-10 mice/group) settings. Data were analyzed with the Student's t test and Kaplan-Meier analysis. Statistical tests were two-sided.

Results: Patients with high levels of tumoral NAA and its biosynthetic enzyme, aspartate N-acetyltransferase (NAT8L), had worse overall survival than patients with low levels of NAA and NAT8L. The overall survival duration of patients with higher-than-median NAA levels (3.6 years) was lower than that of patients with lower-than-median NAA levels (5.1 years, P = .03). High NAT8L gene expression in other cancers (melanoma, renal cell, breast, colon, and uterine cancers) was associated with worse overall survival. NAT8L silencing reduced cancer cell viability (HEYA8: control siRNA 90.61% ± 2.53, NAT8L siRNA 39.43% ± 3.00, P < .001; A2780: control siRNA 90.59% ± 2.53, NAT8L siRNA 7.44% ± 1.71, P < .001) and proliferation (HEYA8: control siRNA 74.83% ± 0.92, NAT8L siRNA 55.70% ± 1.54, P < .001; A2780: control siRNA 50.17% ± 4.13, NAT8L siRNA 26.52% ± 3.70, P < .001), which was rescued by addition of NAA. In orthotopic mouse models (ovarian cancer and melanoma), NAT8L silencing reduced tumor growth statistically significantly (A2780: control siRNA 0.52 g ± 0.15, NAT8L siRNA 0.08 g ± 0.17, P < .001; HEYA8: control siRNA 0.79 g ± 0.42, NAT8L siRNA 0.24 g ± 0.18, P = .008, A375-SM: control siRNA 0.55 g ± 0.22, NAT8L siRNA 0.21 g ± 0.17 g, P = .001). NAT8L silencing downregulated the anti-apoptotic pathway, which was mediated through FOXM1.

Conclusion: These findings indicate that the NAA pathway has a prominent role in promoting tumor growth and represents a valuable target for anticancer therapy.Altered energy metabolism is a hallmark of cancer (1). Proliferating cancer cells have much greater metabolic requirements than nonproliferating differentiated cells (2,3). Moreover, altered cancer metabolism elevates unique metabolic intermediates, which can promote cancer survival and progression (4,5). Furthermore, emerging evidence suggests that proliferating cancer cells exploit alternative metabolic pathways to meet their high demand for energy and to accumulate biomass (6-8).
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http://dx.doi.org/10.1093/jnci/djv426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4849357PMC
January 2016

Real-time molecular imaging of tricarboxylic acid cycle metabolism in vivo by hyperpolarized 1-(13)C diethyl succinate.

J Am Chem Soc 2012 Jan 22;134(2):934-43. Epub 2011 Dec 22.

Enhanced MR Laboratory, Huntington Medical Research Institutes, Pasadena, California, USA.

The Krebs tricarboxylic acid cycle (TCA) is central to metabolic energy production and is known to be altered in many disease states. Real-time molecular imaging of the TCA cycle in vivo will be important in understanding the metabolic basis of several diseases. Positron emission tomography (PET) with FDG-glucose (2-[(18)F]fluoro-2-deoxy-d-glucose) is already being used as a metabolic imaging agent in clinics. However, FDG-glucose does not reveal anything past glucose uptake and phosphorylation. We have developed a new metabolic imaging agent, hyperpolarized diethyl succinate-1-(13)C-2,3-d(2) , that allows for real-time in vivo imaging and spectroscopy of the TCA cycle. Diethyl succinate can be hyperpolarized via parahydrogen-induced polarization (PHIP) in an aqueous solution with signal enhancement of 5000 compared to Boltzmann polarization. (13)C magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) were achieved in vivo seconds after injection of 10-20 μmol of hyperpolarized diethyl succinate into normal mice. The downstream metabolites of hyperpolarized diethyl succinate were identified in vivo as malate, succinate, fumarate, and aspartate. The metabolism of diethyl succinate was altered after exposing the animal to 3-nitropropionate, a known irreversible inhibitor of succinate dehydrogenase. On the basis of our results, hyperpolarized diethyl succinate allows for real-time in vivo MRI and MRS with a high signal-to-noise ratio and with visualization of multiple steps of the TCA cycle. Hyperpolarization of diethyl succinate and its in vivo applications may reveal an entirely new regime wherein the local status of TCA cycle metabolism is interrogated on the time scale of seconds to minutes with unprecedented chemical specificity and MR sensitivity.
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http://dx.doi.org/10.1021/ja2040865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3262122PMC
January 2012

A selenide-based approach to photochemical cleavage of peptide and protein backbones at engineered backbone esters.

J Org Chem 2009 Dec;74(23):9241-4

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.

A strategy for photochemical cleavage of peptide and protein backbones is described, which is based on a selenide-mediated cleavage of a backbone ester moiety. Studies in model systems establish the viability of the chemistry and suggest the method could be a valuable tool for chemical biology studies of proteins.
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http://dx.doi.org/10.1021/jo901368gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2953381PMC
December 2009

Caging proteins through unnatural amino acid mutagenesis.

Methods Enzymol 2003 ;360:258-73

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125, USA.

The caging of specific residues of proteins is a powerful tool. This discussion attempts to alert the reader to the considerations that must be made in preparing and analyzing a caged protein through nonsense suppression. Although the suppression methodology is conceptually straightforward, it not possible to provide a failsafe "cook book" method for using caged unnaturals. We have emphasized the preparation of caged receptors expressed in Xenopus oocytes, but these approaches can clearly be adapted to many other systems.
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http://dx.doi.org/10.1016/s0076-6879(03)60114-xDOI Listing
May 2003

Cation-pi interactions in ligand recognition by serotonergic (5-HT3A) and nicotinic acetylcholine receptors: the anomalous binding properties of nicotine.

Biochemistry 2002 Aug;41(32):10262-9

Divisions of Chemistry and Chemical Engineering and Biology, California Institute of Technology, Pasadena, California 91125, USA.

A series of tryptophan analogues has been introduced into the binding site regions of two ion channels, the ligand-gated nicotinic acetylcholine and serotonin 5-HT(3A) receptors, using unnatural amino acid mutagenesis and heterologous expression in Xenopus oocytes. A cation-pi interaction between serotonin and Trp183 of the serotonin channel 5-HT(3A)R is identified for the first time, precisely locating the ligand-binding site of this receptor. The energetic contribution of the observed cation-pi interaction between a tryptophan and the primary ammonium ion of serotonin is estimated to be approximately 4 kcal/mol, while the comparable interaction with the quaternary ammonium of acetylcholine is approximately 2 kcal/mol. The binding mode of nicotine to the nicotinic receptor of mouse muscle is examined by the same technique and found to differ significantly from that of the natural agonist, acetylcholine.
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http://dx.doi.org/10.1021/bi020266dDOI Listing
August 2002

Improved Synthesis of the Boc and Fmoc Derivatives of 4-(2'-Aminoethyl)-6-dibenzofuranpropionic Acid: An Unnatural Amino Acid That Nucleates beta-Sheet Folding.

J Org Chem 1997 Apr;62(7):2259-2262

Department of Chemistry, Texas A&M University, College Station, Texas 77843-3255.

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http://dx.doi.org/10.1021/jo961063wDOI Listing
April 1997