Glycinamide Ribonucleotide Transformylase Research Articles

Competing Endogenous RNAs Crosstalk in Hippocampus: A Potential Mechanism for Neuronal Developing Defects in Down Syndrome.

Down syndrome (DS) is the most example of aneuploidy, resulting from an additional copy of all or part of chromosome 21. Competing endogenous RNAs (ceRNAs) play important roles in neuronal development and neurological defects. This study aimed to identify hub genes and synergistic crosstalk among ceRNAs in the DS fetal hippocampus as potential targets for the treatment of DS-related neurodegenerative diseases. We profiled differentially expressed long non-coding RNAs (DElncRNAs), differentially expressed circular RNAs (DEcircRNAs), differentially expressed microRNAs (DEmiRNAs), and differentially expressed messenger RNAs (DEmRNAs) in hippocampal samples from patients with or without DS. Functional enrichment analysis and gene set enrichment analysis were performed, and chromosome 21-related ceRNA and protein-protein interaction networks were constructed. Additionally, the correlations between lncRNA-mRNA and miRNA-mRNA expression in the samples and HEK293T cells were validated. Our finding of changes in the expression of some key genes and ncRNAs on chromosome 21 in DS might not fully conform to the gene dosage hypothesis. Moreover, we found that four lncRNAs (MIR99AHG, PLCB4, SNHG14, GIGYF2) and one circRNA (hsa_circ_0061697) may competitively bind with three miRNAs (hsa-miR-548b-5p, miR-730-5p, and hsa-miR-548i) and subsequently regulate five mRNAs (beta-1,3-galactosyltransferase 5 [B3GALT5], helicase lymphoid-specific [HELLS], thrombospondin-2 [THBS2], glycinamide ribonucleotide transformylase [GART], clathrin heavy chain like 1 [CLTCL1]). These RNAs, whether located on chromosome 21 or not, interact with each other and might activate the PI3K/Akt/mTOR and Wnt signaling pathways, which are involved in autophagosome formation and tau hyperphosphorylation, possibly leading to adverse consequences of trisomy 21. These findings provide researchers with a better understanding of the fundamental molecular mechanisms underlying DS-related progressive defects in neuronal development.

GART Functions as a Novel Methyltransferase in the RUVBL1/β-Catenin Signaling Pathway to Promote Tumor Stemness in Colorectal Cancer.

Tumor stemness is associated with the recurrence and incurability of colorectal cancer (CRC), which lacks effective therapeutic targets and drugs. Glycinamide ribonucleotide transformylase (GART) fulfills an important role in numerous types of malignancies. The present study aims to identify the underlying mechanism through which GART may promote CRC stemness, as to developing novel therapeutic methods. An elevated level of GART is associated with poor outcomes in CRC patients and promotes the proliferation and migration of CRC cells. CD133+ cells with increased GART expression possess higher tumorigenic and proliferative capabilities both in vitro and in vivo. GART is identified to have a novel methyltransferase function, whose enzymatic activity center is located at the E948 site. GART also enhances the stability of RuvB-like AAA ATPase 1 (RUVBL1) through methylating its K7 site, which consequently aberrantly activates the Wnt/β-catenin signaling pathway to induce tumor stemness. Pemetrexed (PEM), a compound targeting GART, combined with other chemotherapy drugs greatly suppresses tumor growth both in a PDX model and in CRC patients. The present study demonstrates a novel methyltransferase function of GART and the role of the GART/RUVBL1/β-catenin signaling axis in promoting CRC stemness. PEM may be a promising therapeutic agent for the treatment of CRC.

Dietary intake of fructose increases purine de novo synthesis: A crucial mechanism for hyperuricemia.

Fructose consumption is a potential risk factor for hyperuricemia because uric acid (UA) is a byproduct of fructose metabolism caused by the rapid consumption of adenosine triphosphate and accumulation of adenosine monophosphate (AMP) and other purine nucleotides. Additionally, a clinical experiment with four gout patients demonstrated that intravenous infusion of fructose increased the purine de novo synthesis rate, which implied fructose-induced hyperuricemia might be related to purine nucleotide synthesis. Moreover, the mechanistic (mammalian) target of rapamycin (mTOR) is a key protein both involved in fructose metabolism and purine de novo synthesis. The present study was conducted to elucidate how fructose influences mTOR and purine de novo synthesis in a hepatic cell line and livers of mice. RNA-sequencing in NCTC 1469 cells treated with 0- and 25-mM fructose for 24 h and metabolomics analysis on the livers of mice fed with 0- and 30-g/kg fructose for 2 weeks were assessed. Gene and protein expression of phosphoribosyl pyrophosphate synthase (PRPSAP1), Glutamine PRPP aminotransferase (PPAT), adenyl succinate lyase (ADSL), adenyl succinate synthetase isozyme-1 (Adss1), inosine-5'-monophosphate dehydrogenase (IMPDH), and guanine monophosphate synthetase (GMPS) was measured. The location of PRPSAP1 and PPAT in the liver was assessed by an immunofluorescence assay. Metabolite profiling showed that the level of AMP, adenine, adenosine, hypoxanthine, and guanine was increased significantly. RNA-sequencing showed that gene expression of phosphoribosyl pyrophosphate synthase (PRPS2), phosphoribosyl glycinamide formyl transferase (GART), AICAR transformylase (ATIC), ADSL, Adss1, and IMPDH were raised, and gene expression of adenosine monophosphate deaminase 3 (AMPD3), adenosine deaminase (ADA), 5',3'-nucleotidase, cytosolic (NT5C), and xanthine oxidoreductase (XOR) was also increased significantly. Fructose increased the gene expression, protein expression, and fluorescence intensity of PRPSAP1 and PPAT in mice livers by increasing mTOR expression. Fructose increased the expression and activity of XOR, decreased the expression of uricase, and increased the serum level of UA. This study demonstrated that the increased purine de novo synthesis may be a crucial mechanism for fructose-induced hyperuricemia.

PurN Is Involved in Antibiotic Tolerance and Virulence in Staphylococcus aureus.

Staphylococcus aureus can cause chronic infections which are closely related to persister formation. Purine metabolism is involved in S. aureus persister formation, and purN, encoding phosphoribosylglycinamide formyltransferase, is an important gene in the purine metabolism process. In this study, we generated a ΔpurN mutant of the S. aureus Newman strain and assessed its roles in antibiotic tolerance and virulence. The ΔpurN in the late exponential phase had a significant defect in persistence to antibiotics. Complementation of the ΔpurN restored its tolerance to different antibiotics. PurN significantly affected virulence gene expression, hemolytic ability, and biofilm formation in S. aureus. Moreover, the LD50 (3.28 × 1010 CFU/mL) of the ΔpurN for BALB/c mice was significantly higher than that of the parental strain (2.81 × 109 CFU/mL). Transcriptome analysis revealed that 58 genes that were involved in purine metabolism, alanine, aspartate, glutamate metabolism, and 2-oxocarboxylic acid metabolism, etc., were downregulated, while 24 genes involved in ABC transporter and transferase activity were upregulated in ΔpurN vs. parental strain. Protein-protein interaction network showed that there was a close relationship between PurN and GltB, and SaeRS. The study demonstrated that PurN participates in the formation of the late exponential phase S. aureus persisters via GltB and regulates its virulence by activating the SaeRS two-component system.

De Novo Purine Nucleotide Biosynthesis Pathway Is Required for Development and Pathogenicity in Magnaporthe oryzae.

Purine nucleotides are indispensable compounds for many organisms and participate in basic vital activities such as heredity, development, and growth. Blocking of purine nucleotide biosynthesis may inhibit proliferation and development and is commonly used in cancer therapy. However, the function of the purine nucleotide biosynthesis pathway in the pathogenic fungus Magnaporthe oryzae is not clear. In this study, we focused on the de novo purine biosynthesis (DNPB) pathway and characterized MoAde8, a phosphoribosylglycinamide formyltransferase, catalyzing the third step of the DNPB pathway in M. oryzae. MoAde8 was knocked out, and the mutant (∆Moade8) exhibited purine auxotroph, defects in aerial hyphal growth, conidiation, and pathogenicity, and was more sensitive to hyperosmotic stress and oxidative stress. Moreover, ∆Moade8 caused decreased activity of MoTor kinase due to blocked purine nucleotide synthesis. The autophagy level was also impaired in ∆Moade8. Additionally, MoAde5, 7, 6, and 12, which are involved in de novo purine nucleotide biosynthesis, were also analyzed, and the mutants showed defects similar to the defects of ∆Moade8. In summary, de novo purine nucleotide biosynthesis is essential for conidiation, development, and pathogenicity in M. oryzae.

Rational Proteomic Analysis of a New Domesticated Klebsiella pneumoniae x546 Producing 1,3-Propanediol.

In order to improve the capability of Klebsiella pneumoniae to produce an important chemical raw material, 1,3-propanediol (1,3-PDO), a new type of K. pneumoniae x546 was obtained by glycerol acclimation and subsequently was used to produce 1,3-PDO. Under the control of pH value using Na+ pH neutralizer, the 1,3-PDO yield of K. pneumoniae x546 in a 7.5-L fermenter was 69.35 g/L, which was 1.5-fold higher than the original strain (45.91 g/L). After the addition of betaine, the yield of 1,3-PDO reached up to 74.44 g/L at 24 h, which was 40% shorter than the original fermentation time of 40 h. To study the potential mechanism of the production improvement of 1,3-PDO, the Tandem Mass Tags (TMT) technology was applied to investigate the production of 1,3-PDO in K. pneumoniae. Compared with the control group, 170 up-regulated proteins and 291 down-regulated proteins were identified. Through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, it was found that some proteins [such as homoserine kinase (ThrB), phosphoribosylglycinamide formyltransferase (PurT), phosphoribosylaminoimidazolesuccinocarboxamide synthase (PurC), etc.] were involved in the fermentation process, whereas some other proteins (such as ProX, ProW, ProV, etc.) played a significant role after the addition of betaine. Moreover, combined with the metabolic network of K. pneumoniae during 1,3-PDO, the proteins in the biosynthesis of 1,3-PDO [such as DhaD, DhaK, lactate dehydrogenase (LDH), BudC, etc.] were analyzed. The process of 1,3-PDO production in K. pneumoniae was explained from the perspective of proteome for the first time, which provided a theoretical basis for genetic engineering modification to improve the yield of 1,3-PDO. Because of the use of Na+ pH neutralizer in the fermentation, the subsequent environmental pollution treatment cost was greatly reduced, showing high potential for industry application in the future.

Structural features of Cryptococcus neoformans bifunctional GAR/AIR synthetase may present novel antifungal drug targets

Cryptococcus neoformans is a fungus that causes life-threatening systemic mycoses. During infection of the human host, this pathogen experiences a major change in the availability of purines; the fungus can scavenge the abundant purines in its environmental niche of pigeon excrement, but must employ de novo biosynthesis in the purine-poor human CNS. Eleven sequential enzymatic steps are required to form the first purine base, IMP, an intermediate in the formation of ATP and GTP. Over the course of evolution, several gene fusion events led to the formation of multifunctional purine biosynthetic enzymes in most organisms, particularly the higher eukaryotes. In C. neoformans, phosphoribosyl-glycinamide synthetase (GARs) and phosphoribosyl-aminoimidazole synthetase (AIRs) are fused into a bifunctional enzyme, while the human ortholog is a trifunctional enzyme that also includes GAR transformylase. Here we functionally, biochemically, and structurally characterized C. neoformans GARs and AIRs to identify drug targetable features. GARs/AIRs are essential for de novo purine production and virulence in a murine inhalation infection model. Characterization of GARs enzymatic functional parameters showed that C. neoformans GARs/AIRs have lower affinity for substrates glycine and PRA compared with the trifunctional metazoan enzyme. The crystal structure of C. neoformans GARs revealed differences in the glycine- and ATP-binding sites compared with the Homo sapiens enzyme, while the crystal structure of AIRs shows high structural similarity compared with its H. sapiens ortholog as a monomer but differences as a dimer. The alterations in functional and structural characteristics between fungal and human enzymes could potentially be exploited for antifungal development.

PH Effects and Cooperativity among Key Titratable Residues for Escherichia coli Glycinamide Ribonucleotide Transformylase.

Human glycinamide ribonucleotide transformylase (GAR Tfase) is a regulatory enzyme in the de novo purine biosynthesis pathway that has been extensively studied as an anticancer target. To some extent, inhibition of GAR Tfase selectively targets cancer cells over normal cells and inhibits purine formation and DNA replication. In this study, we investigated E. coli GAR Tfase, which shares high sequence similarity with the human GAR Tfase, and most functional residues are conserved. Herein, we aim to predict the pH-activity curve through a computational approach. We carried out pH-replica exchange molecular dynamics (pH-REMD) simulations to investigate pH-dependent functions such as structural changes, ligand binding, and catalytic activity. To compute the pH-activity curve, we identified the catalytic residues in specific protonation states, referred to as the catalytic competent protonation states (CCPS), which maintain the structure, keep ligands bound, and facilitate catalysis. Our computed population of CCPS with respect to pH matches well with the experimental pH-activity curve. To compute the microscopic pKa values in the catalytically active conformation, we devised a thermodynamic model that considers the coupling between protonation states of CCPS residues and conformational states. These results allow us to correctly identify the general acid and base catalysts and interpret the pH-activity curve at an atomistic level.

Transcriptome and metabolome analysis of crGART, a novel cell model of de novo purine synthesis deficiency: Alterations in CD36 expression and activity.

In humans, GART [phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) / phosphoribosylglycinamide synthetase (EC 6.3.4.13) / phosphoribosylaminoimidazole synthetase (EC 6.3.3.1)] is a trifunctional protein which catalyzes the second, third, and fifth reactions of the ten step de novo purine synthesis (DNPS) pathway. The second step of DNPS is conversion of phosphoribosylamine (5-PRA) to glycineamide ribonucleotide (GAR). 5-PRA is extremely unstable under physiological conditions and is unlikely to accumulate in the absence of GART activity. Recently, a HeLa cell line null mutant for GART was constructed via CRISPR-Cas9 mutagenesis. This cell line, crGART, is an important cellular model of DNPS inactivation that does not accumulate DNPS pathway intermediates. In the current study, we characterized the crGART versus HeLa transcriptomes in purine-supplemented and purine-depleted growth conditions. We observed multiple transcriptome changes and discuss pathways and ontologies particularly relevant to Alzheimer disease and Down syndrome. We selected the Cluster of Differentiation (CD36) gene for initial analysis based on its elevated expression in crGART versus HeLa as well as its high basal expression, high log2 value, and minimal P-value.

Abstract PR05: A metabolomics discovery profiling approach identifies an E2F1-associated purine synthesis pathway as a major component of the MET-DNA damage response network

Abstract Various lines of investigation support a signaling interphase shared by various receptor tyrosine kinases and the DNA damage response. However, the underlying driving network nodes and their contribution to the maintenance of DNA integrity remain unknown. We used discovery metabolomics combined with transcriptomics to identify changes in global metabolic pathways that are relevant to DNA repair following MET inhibition (METi). METi was associated with formation of γH2AX foci together with significant alterations in major metabolic circuits. Remarkably, 5′-phosphoribosyl-N-formylglycinamide (FGAR), a de novo purine synthesis pathway metabolite, was decreased in MET-dependent cancer models and a METi-related depletion of dNTPs was observed. Likewise, METi instigated the downregulation of critical purine synthesis enzymes including phosphoribosylglycinamide formyltransferase (GART), which is responsible for FGAR synthesis. Genes encoding these enzymes are regulated through the E2F1 transcription factor, whose levels decreased upon METi in MET-driven cells. To validate those findings, a transient E2F1 overexpression was sufficient to prevent MET targeting-dependent dNTPs depletion and the concomitant METi-associated DNA damage in MET-driven tumor cells. These novel observations support the notion that METi-mediated sensitization to DNA-damaging agents commonly used in cancer therapeutics is largely linked to the depletion of critical metabolites used in DNA repair due to downregulation of key metabolic enzymes whose expression is regulated through MET signaling. This abstract is also being presented as Poster A51. Citation Format: Michaela Poliaková, Matúš Medo, Jonas Koch, SiAna Coggins, Baek Kim, Daniel M. Aebersold, Nicola Zamboni, Michaela Medová, Yitzhak Zimmer. A metabolomics discovery profiling approach identifies an E2F1-associated purine synthesis pathway as a major component of the MET-DNA damage response network [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR05.

Molecular mechanism of c-Myc and PRPS1/2 against thiopurine resistance in Burkitt's lymphoma.

Patients with relapsed/refractory Burkitt's lymphoma (BL) have a dismal prognosis. Current research efforts aim to increase cure rates by identifying high‐risk patients in need of more intensive or novel therapy. The 8q24 chromosomal translocation of the c‐Myc gene, a main molecular marker of BL, is related to the metabolism by regulating phosphoribosyl pyrophosphate synthetase 2 (PRPS2). In our study, BL showed significant resistance to thiopurines. PRPS2 homologous isoenzyme, PRPS1, was demonstrated to play the main role in thiopurine resistance. c‐Myc did not have direct effects on thiopurine resistance in BL for only driving PRPS2. PRPS1 wild type (WT) showed different resistance to 6‐mercaptopurine (6‐mp) in different metabolic cells because it could be inhibited by adenosine diphosphate or guanosine diphosphate negative feedback. PRPS1 A190T mutant could dramatically increase thiopurine resistance in BL. The interim analysis of the Treatment Regimen for Children or Adolescent with mature B cell non‐Hodgkin's lymphoma in China (CCCG‐B‐NHL‐2015 study) confirms the value of high‐dose methotrexate (MTX) and cytarabine (ARA‐C) in high‐risk paediatric patients with BL. However, there remains a subgroup of patients with lactate dehydrogenase higher than four times of the normal value (4N) for whom novel treatments are needed. Notably, we found that the combination of thiopurines and the phosphoribosylglycinamide formyltransferase (GART) inhibitor lometrexol could serve as a therapeutic strategy to overcome thiopurine resistance in BL.

Retraction of "Rationalizing the pH-Activity Response of Escherichia coli Glycinamide Ribonucleotide Transformylase through Computational Methods".

Human Glycinamide ribonucleotide transformylase (GAR Tfase) is a regulatory enzyme in the de novo purine biosynthesis pathway that has been extensively studied as an anti-cancer target. To some ext...

Nwd1 Regulates Neuronal Differentiation and Migration through Purinosome Formation in the Developing Cerebral Cortex.

SummaryEngagement of neural stem/progenitor cells (NSPCs) into proper neuronal differentiation requires the spatiotemporally regulated generation of metabolites. Purines are essential building blocks for many signaling molecules. Enzymes that catalyze de novo purine synthesis are assembled as a huge multienzyme complex called “purinosome.” However, there is no evidence of the formation or physiological function of the purinosome in the brain. Here, we showed that a signal transduction ATPases with numerous domains (STAND) protein, NACHT and WD repeat domain-containing 1 (Nwd1), interacted with Paics, a purine-synthesizing enzyme, to regulate purinosome assembly in NSPCs. Altered Nwd1 expression affected purinosome formation and induced the mitotic exit and premature differentiation of NSPCs, repressing neuronal migration and periventricular heterotopia. Overexpression/knockdown of Paics or Fgams, other purinosome enzymes, in the developing brain resulted in a phenocopy of Nwd1 defects. These findings indicate that strict regulation of purinosome assembly/disassembly is crucial for maintaining NSPCs and corticogenesis.

Electrostatic and Chemical Properties in Natural and Designed Enzymes

Advances have been made in understanding how enzymes achieve their exquisite catalytic power. However there are still gaps in our understanding, as computationally driven enzyme design efforts are still in their infancy and must be followed by years of directed evolution in order to achieve reasonable turnover rates. The goal of this work is to understand the intrinsic properties that give natural enzymes their catalytic capabilities and to learn how to build these properties in silico, for the design and expression of artificial enzymes that catalyze reactions that do not occur in nature. Partial Order Optimum Likelihood (POOL) is a machine learning method developed by us to predict residues important for function, using the 3D structure of the query protein. The input features to POOL are based on computed electrostatic and chemical properties from THEMATICS. These input features are effectively measures of the strength of coupling between protonation events. POOL is used to characterize the properties of natural enzymes that are necessary for efficient catalysis. Catalytic sites in proteins are characterized in part by networks of strongly coupled protonation states; these networks impart the necessary electrostatic and proton‐transfer properties to the active residues in the first layer around the reacting substrate molecule(s). Typically these networks include first‐, second‐, and sometimes third‐layer residues. POOL‐predicted, multi‐layer active sites with significant participation by distal residues have been verified experimentally by single‐point site‐directed mutagenesis and kinetics assays for Ps. putida nitrile hydratase, human phosphoglucose isomerase, E. coli replicative DNA polymerase Pol III, E. coli Y family DNA polymerase DinB, ornithine transcarbamoylase, and glycinamide ribonucleotide transformylase (GART). In designed enzymes, such as retroaldolases, the residue‐specific input features to POOL – measures of the strength of coupling between protonation equilibria – rise as the enzymes evolve to higher rates of catalytic turnover. These concepts can enhance current enzyme design protocols.Support or Funding InformationSupported by NSF MCB‐1517290.

232. Genomic Evidence for Dissemination of Mycobacterium marinum in an HIV Patient with Multifocal Cutaneous Disease

BackgroundHematogenous dissemination has been proposed to explain multifocal cutaneous disease caused by Mycobacterium marinum in certain patients. Treatment duration for disseminated disease is often months longer than for skin infection alone. However, distinguishing multiple independent inoculation events from dissemination has relied primarily on clinical judgement. Additionally, whether temperature-sensitive non-tuberculous mycobacteria such as M. marinum are indeed capable of invading the vascular space at core body temperature is debated. Here we used whole-genome sequencing (WGS) of serial isolates from a single patient with multifocal cutaneous M. marinum infection to distinguish dissemination of a clonal strain from multiple inoculation events.MethodsA 35-year-old male with HIV (CD4 of 66 cells/µL) presented with a two-month history of a non-healing M. marinum wound on his left elbow (isolate MM0). This was followed a month later after initiation of antiretroviral therapy by a second M. marinum lesion on the right heel (MM1) without history of repeat inoculation, and increased swelling and erythema of the wound on the left arm (MM2) consistent with paradoxical immune reconstitution inflammatory syndrome. A PacBio genome was generated for MM0 and short read Illumina genomes were generated for MM1 and MM2.ResultsAll isolates were found to be closely related, with MM1 and MM2 distinguished from MM0 by one and five single-nucleotide variants (SNVs), respectively. Given the substantial genetic heterogeneity among environmental M. marinum strains, such close relatedness of these isolates suggests common origin, and provides strong evidence for dissemination of a clonal strain in this patient. The SNVs included a frameshift mutation in the purT gene, which encodes a formate-dependent phosphoribosylglycinamide formyltransferase involved in de novo purine synthesis, and missense mutations in atsA and the DNA methylase hsdM. All isolates grew at 35°C, compared with the optimal growth temperature of 30°C typically observed for M. marinum, suggesting thermotolerance permissive for dissemination.ConclusionThese results demonstrate the potential role of WGS for providing supportive evidence of disseminated infection with M. marinum.Disclosures All authors: No reported disclosures.

PvdF of pyoverdin biosynthesis is a structurally unique N10-formyltetrahydrofolate-dependent formyltransferase

The hydroxyornithine transformylase from Pseudomonas aeruginosa is known by the gene name pvdF, and has been hypothesized to use N10-formyltetrahydrofolate (N10-fTHF) as a co-substrate formyl donor to convert N5-hydroxyornithine (OHOrn) to N5-formyl- N5-hydroxyornithine (fOHOrn). PvdF is in the biosynthetic pathway for pyoverdin biosynthesis, a siderophore generated under iron-limiting conditions that has been linked to virulence, quorum sensing and biofilm formation. The structure of PvdF was determined by X-ray crystallography to 2.3 Å, revealing a formyltransferase fold consistent with N10-formyltetrahydrofolate dependent enzymes, such as the glycinamide ribonucleotide transformylases, N-sugar transformylases and methionyl-tRNA transformylases. Whereas the core structure, including the catalytic triad, is conserved, PvdF has three insertions of 18 or more amino acids, which we hypothesize are key to binding the OHOrn substrate. Steady state kinetics revealed a non-hyperbolic rate curve, promoting the hypothesis that PvdF uses a random-sequential mechanism, and favors folate binding over OHOrn.

Abstract 348: Comparison of the response of the NCI60 NSCLC panel with the response of patient-derived NSCLC lines to approved and investigational agents

Abstract An 800 compound screen with the NCI60 cell lines and 5 patient-derived NSCLC lines (PD NSCLC), was conducted at 9 concentrations and included the FDA approved oncology agents and an investigational agents library. The screen was conducted using 384-well monolayer cultures, an exposure time of 72 hrs and compound concentrations from 1 nM to 10 uM. CellTiter-Glo was used to measure viability as an endpoint. The NCI60 NSCLC panel consists of 9 cell lines: A549, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460 and NCI-H522. The response of these lines was compared with the response of 5 PD NSCLC lines and 60 SCLC lines. While both sets of NSCLC lines had similar responses to the majority of compounds, there were some marked differences. Differences in response of the NCI60 NSCLC lines and the PD NSCLC lines were notable upon exposure to tubulin fragmenters, KSP/EG5 inhibitors, GAR transformylase inhibitors, Polo-like kinase-1 inhibitors (PLK1), MEK inhibitors, and IAP inhibitors. The NCI60 NSCLC lines were more sensitive to the tubulin fragmenters than were the PD NSCLC and the SCLC lines. Verubulin had a mean GI50 in NCI60 NSCLC of 0.0037 uM and 0.0042 uM in SCLC versus 0.047uM in the PD NSCLC lines. The NCI60 NSCLC lines were more sensitive to the KSP inhibitor, ARRY-520 (mean GI50 0.0049 uM) than the SCLC lines (0.028 uM) and the PD NSCLC lines (0.26 uM). The NCI60 NSCLC lines (mean GI50 0.019 uM) and the SCLC lines (mean GI50 0.04 uM) were more sensitive to PLK1 inhibitors such as BI-2536 than the PD NSCLC lines (0.13 uM). However, the NCI60 NSCLC lines (mean GI50 0.8 uM) and the SCLC lines (mean GI50 0.134 uM) were less sensitive than the PD NSCLC lines (mean GI50 0.025uM) to GAR transformylase inhibitors such as pelitrexol. The NCI60 NSCLC lines (mean GI50 6.3 uM) and SCLC lines (mean GI50 10 uM) were also less responsive to IAP inhibitors, than were PD NSCLC lines (mean GI50 1.6 uM). A heterogeneous response to the MEK inhibitors such as cobimetinib (GDC-0973) was observed with the SCLC lines (mean GI50 8.9 uM) versus 1.15 uM in the NCI60 NSCLC lines and 0.56 uM in the PD NSCLC lines. PD NSCLC lines exhibit some interesting differences in response from established lung cancer lines upon in vitro exposure to anticancer agents and thus add to our knowledge and understanding of NSCLC and help inform discovery efforts and/or clinical development of therapeutics in this disease. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261200800001E. Citation Format: Beverly A. Teicher, David Evans, Thomas Silvers, Michael Selby, Rene Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Joel Morris, Gurmeet Kaur, James Doroshow. Comparison of the response of the NCI60 NSCLC panel with the response of patient-derived NSCLC lines to approved and investigational agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 348. doi:10.1158/1538-7445.AM2017-348

Screening of potential biomarkers for prenatal diagnosis of trisomy 21

We aimed to identify key genes located on chromosome 21 as potential biomarkers for prenatal diagnosis of trisomy 21 (Ts21). The microarray data of GSE48051, including 10 cultivated amniocyte samples with Ts21 and 9 controls with normal euploid constitution, was obtained from Gene Expression Omnibus database. The differentially expressed genes (DEGs) in cultivated amniocyte samples with Ts21 compared to normal controls were screened using limma package. Then, we performed GO enrichment analysis using DAVID and chromosomal location of DEGs based on the information of the University of California Santa Cruz (UCSC) Genome Browser Database. Finally, protein–protein interaction (PPI) network analysis was performed using STRING. Total 155 DEGs in cultivated amniocyte samples with Ts21 were identified, including 89 up- and 66 down-regulated DEGs. The over-represented GO terms of DEGs were mainly related with apoptosis, programmed cell death and cell death. In total, 13 DEGs were located on chromosome 21, thereinto, only 6 DEGs were included into the PPI network, including superoxide dismutase 1 (SOD1), phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase (GART), downstream neighbour of SON (DONSON), ATP synthase, H + transporting, mitochondrial F1 complex, O subunit (ATP5O), chromatin assembly factor 1, subunit B (p60) (CHAF1B) and proteasome (prosome, macropain) assembly chaperone 1 (PSMG1). Our results suggest that SOD1, GART, DONSON, ATP5O, CHAF1B and PSMG1 may play important roles in the pathogenesis of Down syndrome and may serve as potential biomarkers for prenatal diagnosis of Ts21.

Comparative Genomic Analysis of Haemophilus haemolyticus and Nontypeable Haemophilus influenzae and a New Testing Scheme for Their Discrimination.

Haemophilus haemolyticus has been recently discovered to have the potential to cause invasive disease. It is closely related to nontypeable Haemophilus influenzae (NT H. influenzae). NT H. influenzae and H. haemolyticus are often misidentified because none of the existing tests targeting the known phenotypes of H. haemolyticus are able to specifically identify H. haemolyticus Through comparative genomic analysis of H. haemolyticus and NT H. influenzae, we identified genes unique to H. haemolyticus that can be used as targets for the identification of H. haemolyticus A real-time PCR targeting purT (encoding phosphoribosylglycinamide formyltransferase 2 in the purine synthesis pathway) was developed and evaluated. The lower limit of detection was 40 genomes/PCR; the sensitivity and specificity in detecting H. haemolyticus were 98.9% and 97%, respectively. To improve the discrimination of H. haemolyticus and NT H. influenzae, a testing scheme combining two targets (H. haemolyticus purT and H. influenzae hpd, encoding protein D lipoprotein) was also evaluated and showed 96.7% sensitivity and 98.2% specificity for the identification of H. haemolyticus and 92.8% sensitivity and 100% specificity for the identification of H. influenzae, respectively. The dual-target testing scheme can be used for the diagnosis and surveillance of infection and disease caused by H. haemolyticus and NT H. influenzae.

Folic Acid-Metabolizing Enzymes Regulate the Antitumor Effect of 5-Fluoro-2'-Deoxyuridine in Colorectal Cancer Cell Lines.

In colorectal cancer chemotherapy, the current standard of care includes combination therapy with 5-fluorouracil (5-FU) and leucovorin (LV). However, the factors that determine the LV-mediated enhancement of 5-FU antitumor activity are not fully understood. Therefore, we investigated the roles of thymidine synthase (TYMS), folate receptor 1 (FOLR1), dihydrofolate reductase (DHFR), phosphoribosylglycinamide formyltransferase (GART), methylenetetrahydrofolate dehydrogenase (MTHFD1), and methylenetetrahydrofolate reductase (MTHFR) in LV-mediated enhancement of 5-fluoro-2′-deoxyuridine (FdUrd) cytotoxicity in vitro as a model of 5-FU antitumor activity. These genes were downregulated in DLD-1 and HCT116 human colorectal cancer cells by using small-interfering RNA. Reduced expression of TYMS mRNA significantly increased FdUrd cytotoxicity by 100- and 8.3-fold in DLD-1 and HCT116 cells, respectively. In contrast, reducing the expression of FOLR1, DHFR, GART, MTHFD1, and MTHFR decreased FdUrd cytotoxicity by 2.13- to 12.91-fold in DLD-1 cells and by 3.52- to 10.36-fold in HCT116 cells. These results demonstrate that folate metabolism is important for the efficacy of FdUrd. Overall, the results indicate that it is important to clarify the relationship between folate metabolism-related molecules and 5-FU treatment in order to improve predictions of the effectiveness of 5-FU and LV combination therapy.