In addition to their essential catalytic role in protein biosynthesis, aminoacyl-tRNA synthetases participate in numerous other functions, including regulation of gene expression and amino acid biosynthesis via transamidation pathways. Herein, we describe a class of aminoacyl-tRNA synthetase-like (HisZ) proteins based on the catalytic core of the contemporary class II histidyl-tRNA synthetase whose members lack aminoacylation activity but are instead essential components of the first enzyme in histidine biosynthesis ATP phosphoribosyltransferase ( Protein synthesis requires the association of amino acids with the nucleotide triplets of the genetic code, a reaction mediated by tRNA adapters and their specific aminoacyl-tRNA synthetases (aaRSs). As reflected in the absence of some of the canonical 20 aaRSs in contemporary organisms and the duplication and truncation of aaRS in others, some variation in components involved in proteins synthesis has persisted over evolution (1-3). For example, contemporary archaebacterial and bacterial species possess transamidation pathways that use glutamyl-tRNA Gln and aspartyl-tRNA Asn (produced by GluRS and AspRS, respectively) as substrates (4-6). These transamidation pathways account for the absence of GlnRS and AsnRS in these species. The aaRSs also regulate the biosynthetic operons responsible for tryptophan, branched-chain amino acids, and histidine (7-9) by attenuation mechanisms that couple transcription of the operon to translation of leader peptides rich in codons specific for the amino acids in question. Notably, both the transamidation pathways and the regulation by attenuation are dependent on the same aminoacylation reactions that generate aminoacylated tRNA for protein synthesis.The further involvement of aaRS or aaRS-like proteins in amino acid biosynthesis is also suggested by the existence of proteins that are based on the catalytic domains of an aaRS yet do not catalyze the aminoacylation reaction. A striking illustration is the asparagine synthetase A (AsnA), whose recently solved structure contains a class II aaRS catalytic domain (closely related to AspRS and AsnRS). The role of AsnA is to convert aspartate to asparagine via an amidation reaction involving a transient aspartyl-adenylate (10). The high degree of structural homology between AsnA and AspRS and the observation that truncated aaRS catalytic domains retain residual catalytic activity [e.g., MetRS (11), AlaRS (12), and HisRS (13)] lend support to proposals that synthetases arose by fusion of specialized tRNA interaction and editing domains to primordial catalytic domains capable of amino acid activation (14). Obtaining corroborating evidence for this theory is hindered by the difficulty in distinguishing between homologous proteins that might have been antecedents to the aaRS and proteins that might have started as functional aaRS but lost aminoacylation capacity over evolution.We therefore sought to address this model by identifying and characterizing proteins in contemporary organisms th...

Comparative oncology is a developing research discipline that is being used to assist our understanding of human neoplastic diseases. Companion canines are a preferred animal oncology model due to spontaneous tumor development and similarity to human disease at the pathophysiological level. We use a paired RNA sequencing (RNA-Seq)/microarray analysis of a set of four normal canine lymph nodes and ten canine lymphoma fine needle aspirates to identify technical biases and variation between the technologies and convergence on biological disease pathways. Surrogate Variable Analysis (SVA) provides a formal multivariate analysis of the combined RNA-Seq/microarray data set. Applying SVA to the data allows us to decompose variation into contributions associated with transcript abundance, differences between the technology, and latent variation within each technology. A substantial and highly statistically significant component of the variation reflects transcript abundance, and RNA-Seq appeared more sensitive for detection of transcripts expressed at low levels. Latent random variation among RNA-Seq samples is also distinct in character from that impacting microarray samples. In particular, we observed variation between RNA-Seq samples that reflects transcript GC content. Platform-independent variable decomposition without a priori knowledge of the sources of variation using SVA represents a generalizable method for accomplishing cross-platform data analysis. We identified genes differentially expressed between normal lymph nodes of disease free dogs and a subset of the diseased dogs diagnosed with B-cell lymphoma using each technology. There is statistically significant overlap between the RNA-Seq and microarray sets of differentially expressed genes. Analysis of overlapping genes in the context of biological systems suggests elevated expression and activity of PI3K signaling in B-cell lymphoma biopsies compared with normal biopsies, consistent with literature describing successful use of drugs targeting this pathway in lymphomas.

Abstract. Medulloblastoma, a neuroectodermal tumor arising in the cerebellum, is the most common brain tumor found in children. We recently showed that nifurtimox induces production of reactive oxygen species (ROS) and subsequent apoptosis in neuroblastoma cells both in vitro and in vivo. Tetrathiomolybdate (TM) has been shown to decrease cell proliferation by inhibition of superoxide dismutase-1 (SOD1). Since both nifurtimox and TM increase ROS levels in cells, we investigated whether the combination of nifurtimox and TM would act synergistically in medulloblastoma cell lines (D283, DAOY). Genome-wide transcriptional analysis, by hybridizing RNA isolated from nifurtimox and TM alone or in combination treated and control cells (D283) on Affymetrix exon array gene chips was carried out to further confirm synergy. We show that nifurtimox and TM alone and in combination decreased cell viability and increased ROS levels synergistically. Examination of cell morphology following drug treatment (nifurtimox + TM) and detection of caspase-3 activation via Western blotting indicated that cell death was primarily due to apoptosis. Microarray data from cells treated with nifurtimox and TM validated the induction of oxidative stress, as many Nrf2 target genes (HMOX1, GCLM, SLC7A11 and SRXN1) (p<10 -5 ) were upregulated. Other genes related to apoptosis, oxidative stress, DNA damage, protein folding and nucleosome formation were differentially involved in cells following treatment with nifurtimox + TM. Taken together, our results suggest nifurtimox and TM act synergistically in medulloblastoma cells in vitro, and that this combination warrants further studies as a new treatment for medulloblastoma.

Background: Pediatric cancer is the leading cause of death by disease in children in the US. Significant advances have been made in survival in the past 30 years and genomic understanding of tumors is underway. Gains in the identification of biomarkers, drug targets, and the molecular characterization of cancer are due to improved technology including gene sequencing, proteomics, and epigenetics. Still, this remains limited primarily to large academic centers and patients with high risk/metastatic disease experience &lt; 30% survival. Phase 1/2 trials not based on precision medicine result in poor response rates (&lt;10%). Therefore, genomic understanding of tumors and molecular targeted therapies with aims of reaching all children and reducing toxicity while improving efficacy is needed. Methods: The Signature Study is an IRB-approved biology study that seeks to perform genomic analysis, high throughput (HTP) drug testing, and creation of patient derived xenograft (PDX) models of all pediatric cancer patients diagnosed/relapsed at Helen DeVos Children’s Hospital. Patients are consented, clinical history is collected in RedCap, and tumors are collected flash frozen and in cell culture media. Blood is collected for germline analysis. Tumors are analyzed through gene expression arrays, DNA panels and exomes and RNA sequencing. Tumors in cell culture are used for generation of primary patient cell lines (confirmed by IHC and STR) and immediate injection into NSG mice for PDX models. Cell lines undergo HTP drug testing using the Prestwick and NCI drug libraries, novel therapeutics and combinations. Results: Enrollment has increased since inception in 2011 with now &gt;50 patients/year; a total of 284 pediatric tumors collected representing over 30 tumor types. The most common diagnosis is neuroblastoma, followed by medulloblastoma, osteosarcoma, and rhabdomyosarcoma. Sequencing for genomic analysis has been performed on 166 tumors to identify mutations, fusions, CNV, and deletions. To date 184 have been grown as primary patient cell lines and 75 as PDX models. Over 60 samples have been evaluated in HTP drug testing. Genomic analysis of cultured tumor cells has been correlated with response to drug libraries to establish correlative predictive markers for therapeutic decision making to be tested in clinical trials. The study stores remaining tissue, cell lines, and PDX models for additional future research. Conclusion: This study shows it is feasible for a mid-sized hospital system to coordinate and collect tumors for genomic analysis in real time for clinical decision making in the future. This resource is an integrated TransMed database system which is a powerful resource which correlates clinical outcomes, therapies, genomic sequencing, PDX models and HTP drug testing used to answer research questions of biomarkers, biological characterization, drug sensitivity, and driver pathways within and between pediatric cancers. Citation Format: Elizabeth VanSickle, Ping Zhao, Deanna Mitchell, Jessica Foley, Julie Steinbrecher, Maria Rich, Abhinav Nagulapally, Jeff Bond, William Hendricks, Giselle Saulnier Sholler. The Signature Study: Molecular analysis of pediatric tumors with establishment of tumor models in a biology study [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 4869. doi:10.1158/1538-7445.AM2017-4869

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