hiPathDB: a human-integrated pathway database with facile visualization.

One of the biggest challenges in the study of biological regulatory networks is the systematic organization and integration of complex interactions taking place within various biological pathways. Currently, the information of the biological pathways is dispersed in multiple databases in various formats. hiPathDB is an integrated pathway database that combines the curated human pathway data of NCI-Nature PID, Reactome, BioCarta and KEGG. In total, it includes 1661 pathways consisting of 8976 distinct physical entities. hiPathDB provides two different types of integration. The pathway-level integration, conceptually a simple collection of individual pathways, was achieved by devising an elaborate model that takes distinct features of four databases into account and subsequently reformatting all pathways in accordance with our model. The entity-level integration creates a single unified pathway that encompasses all pathways by merging common components. Even though the detailed molecular-level information such as complex formation or post-translational modifications tends to be lost, such integration makes it possible to investigate signaling network over the entire pathways and allows identification of pathway cross-talks. Another strong merit of hiPathDB is the built-in pathway visualization module that supports explorative studies of complex networks in an interactive fashion. The layout algorithm is optimized for virtually automatic visualization of the pathways. hiPathDB is available at http://hiPathDB.kobic.re.kr.

Performance of a Machine Learning-Based Methicillin Resistance of Staphylococcus aureus Identification System Using MALDI-TOF MS and Comparison of the Accuracy according to SCCmec Types.

The prompt presumptive identification of methicillin-resistant Staphylococcus aureus (MRSA) using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can aid in early clinical management and infection control during routine bacterial identification procedures. This study applied a machine learning approach to MALDI-TOF peaks for the presumptive identification of MRSA and compared the accuracy according to staphylococcal cassette chromosome mec (SCCmec) types. We analyzed 194 S. aureus clinical isolates to evaluate the machine learning-based identification system (AMRQuest software, v.2.1, ASTA: Suwon, Korea), which was constructed with 359 S. aureus clinical isolates for the learning dataset. This system showed a sensitivity of 91.8%, specificity of 83.3%, and accuracy of 87.6% in distinguishing MRSA. For SCCmec II and IVA types, common MRSA types in a hospital context, the accuracy was 95.4% and 96.1%, respectively, while for the SCCmec IV type, it was 21.4%. The accuracy was 90.9% for methicillin-susceptible S. aureus. This presumptive MRSA identification system may be helpful for the management of patients before the performance of routine antimicrobial resistance testing. Further optimization of the machine learning model with more datasets could help achieve rapid identification of MRSA with less effort in routine clinical procedures using MALDI-TOF MS as an identification method.

GARNET--gene set analysis with exploration of annotation relations.

BACKGROUND: Gene set analysis is a powerful method of deducing biological meaning for an a priori defined set of genes. Numerous tools have been developed to test statistical enrichment or depletion in specific pathways or gene ontology (GO) terms. Major difficulties towards biological interpretation are integrating diverse types of annotation categories and exploring the relationships between annotation terms of similar information. RESULTS: GARNET (Gene Annotation Relationship NEtwork Tools) is an integrative platform for gene set analysis with many novel features. It includes tools for retrieval of genes from annotation database, statistical analysis & visualization of annotation relationships, and managing gene sets. In an effort to allow access to a full spectrum of amassed biological knowledge, we have integrated a variety of annotation data that include the GO, domain, disease, drug, chromosomal location, and custom-defined annotations. Diverse types of molecular networks (pathways, transcription and microRNA regulations, protein-protein interaction) are also included. The pair-wise relationship between annotation gene sets was calculated using kappa statistics. GARNET consists of three modules--gene set manager, gene set analysis and gene set retrieval, which are tightly integrated to provide virtually automatic analysis for gene sets. A dedicated viewer for annotation network has been developed to facilitate exploration of the related annotations. CONCLUSIONS: GARNET (gene annotation relationship network tools) is an integrative platform for diverse types of gene set analysis, where complex relationships among gene annotations can be easily explored with an intuitive network visualization tool (http://garnet.isysbio.org/ or http://ercsb.ewha.ac.kr/garnet/).

Genomic Analysis of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Strains from Different Clonal Lineages in South Korea.

Recent genomic studies of methicillin-resistant Staphylococcus aureus (MRSA) have revealed genetic diversity in the various clonal lineages. Along with clinical concerns of MRSA infection, infection with heterogeneous vancomycin-intermediate S. aureus (hVISA) is closely associated with treatment failure. In this study, we investigated the magnitude of genetic variation and features at the genomic level of hVISA strains isolated in South Korea. Four hVISA strains were analyzed by molecular epidemiology, antimicrobial susceptibility, and whole-genome sequencing methods, and they were compared with the reference VISA and vancomycin-susceptible S. aureus strains in the same clonal lineage. The epidemiologic features of hVISA strains were closely related to the ST5 and ST239 clones. Comparative analysis of the whole genome showed genetic mutations, particularly in two-component systems (TCSs) and transcriptional regulators. Genetic mutations in walK were commonly found in both ST5- (F545L, E378K, T500K) and ST239-related (E424D, T492R) hVISA strains. hVISA strains in the ST5 clonal lineage contained mutations in TCS genes, including the walK, vraR, and agr loci, whereas ST239-related strains harbored different genetic variations in walK, lytR, and saeR. This study suggests that the diverse genetic variation of TCSs and transcriptional regulators are involved in reduced vancomycin susceptibility through different mechanisms in each clonal lineage.

Aberrant CDK4 amplification in refractory rhabdomyosarcoma as identified by genomic profiling.

Rhabdomyosarcoma (RMS) is the most commonly occurring type of soft tissue tumor in children. However, it is rare in adults, and therefore, very little is known about the most appropriate treatment strategy for adult RMS patients. We performed genomic analysis of RMS cells derived from a 27-year-old male patient whose disease was refractory to treatment. A peritoneal seeding nodule from the primary tumor, pleural metastases, malignant pleural effusion, and ascites obtained during disease progression, were analyzed. Whole exome sequencing revealed 23 candidate variants, and 10 of 23 mutations were validated by Sanger sequencing. Three of 10 mutations were present in both primary and metastatic tumors, and 3 mutations were detected only in metastatic specimens. Comparative genomic hybridization array analysis revealed prominent amplification in the 12q13-14 region, and more specifically, the CDK4 proto-oncogene was highly amplified. ALK overexpression was observed at both protein and RNA levels. However, an ALK fusion assay using NanoString technology failed to show any ALK rearrangements. Little genetic heterogeneity was observed between primary and metastatic RMS cells. We propose that CDK4, located at 12q14, is a potential target for drug development for RMS treatment.

Translational validation of personalized treatment strategy based on genetic characteristics of glioblastoma.

Glioblastoma (GBM) heterogeneity in the genomic and phenotypic properties has potentiated personalized approach against specific therapeutic targets of each GBM patient. The Cancer Genome Atlas (TCGA) Research Network has been established the comprehensive genomic abnormalities of GBM, which sub-classified GBMs into 4 different molecular subtypes. The molecular subtypes could be utilized to develop personalized treatment strategy for each subtype. We applied a classifying method, NTP (Nearest Template Prediction) method to determine molecular subtype of each GBM patient and corresponding orthotopic xenograft animal model. The models were derived from GBM cells dissociated from patient's surgical sample. Specific drug candidates for each subtype were selected using an integrated pharmacological network database (PharmDB), which link drugs with subtype specific genes. Treatment effects of the drug candidates were determined by in vitro limiting dilution assay using patient-derived GBM cells primarily cultured from orthotopic xenograft tumors. The consistent identification of molecular subtype by the NTP method was validated using TCGA database. When subtypes were determined by the NTP method, orthotopic xenograft animal models faithfully maintained the molecular subtypes of parental tumors. Subtype specific drugs not only showed significant inhibition effects on the in vitro clonogenicity of patient-derived GBM cells but also synergistically reversed temozolomide resistance of MGMT-unmethylated patient-derived GBM cells. However, inhibitory effects on the clonogenicity were not totally subtype-specific. Personalized treatment approach based on genetic characteristics of each GBM could make better treatment outcomes of GBMs, although more sophisticated classifying techniques and subtype specific drugs need to be further elucidated.

A recurrent inactivating mutation in RHOA GTPase in angioimmunoblastic T cell lymphoma.

The molecular mechanisms underlying angioimmunoblastic T cell lymphoma (AITL), a common type of mature T cell lymphoma of poor prognosis, are largely unknown. Here we report a frequent somatic mutation in RHOA (encoding p.Gly17Val) using exome and transcriptome sequencing of samples from individuals with AITL. Further examination of the RHOA mutation encoding p.Gly17Val in 239 lymphoma samples showed that the mutation was specific to T cell lymphoma and was absent from B cell lymphoma. We demonstrate that the RHOA mutation encoding p.Gly17Val, which was found in 53.3% (24 of 45) of the AITL cases examined, is oncogenic in nature using multiple molecular assays. Molecular modeling and docking simulations provided a structural basis for the loss of GTPase activity in the RHOA Gly17Val mutant. Our experimental data and modeling results suggest that the RHOA mutation encoding p.Gly17Val is a driver mutation in AITL. On the basis of these data and through integrated pathway analysis, we build a comprehensive signaling network for AITL oncogenesis.

Identification of upstream regulators for prognostic expression signature genes in colorectal cancer.

BACKGROUND: Gene expression signatures have been commonly used as diagnostic and prognostic markers for cancer subtyping. However, expression signatures frequently include many passengers, which are not directly related to cancer progression. Their upstream regulators such as transcription factors (TFs) may take a more critical role as drivers or master regulators to provide better clues on the underlying regulatory mechanisms and therapeutic applications. RESULTS: In order to identify prognostic master regulators, we took the known 85 prognostic signature genes for colorectal cancer and inferred their upstream TFs. To this end, a global transcriptional regulatory network was constructed with total >200,000 TF-target links using the ARACNE algorithm. We selected the top 10 TFs as candidate master regulators to show the highest coverage of the signature genes among the total 846 TF-target sub-networks or regulons. The selected TFs showed a comparable or slightly better prognostic performance than the original 85 signature genes in spite of greatly reduced number of marker genes from 85 to 10. Notably, these TFs were selected solely from inferred regulatory links using gene expression profiles and included many TFs regulating tumorigenic processes such as proliferation, metastasis, and differentiation. CONCLUSIONS: Our network approach leads to the identification of the upstream transcription factors for prognostic signature genes to provide leads to their regulatory mechanisms. We demonstrate that our approach could identify upstream biomarkers for a given set of signature genes with markedly smaller size and comparable performances. The utility of our method may be expandable to other types of signatures such as diagnosis and drug response.

A high-dimensional, deep-sequencing study of lung adenocarcinoma in female never-smokers.

BACKGROUND: Deep sequencing techniques provide a remarkable opportunity for comprehensive understanding of tumorigenesis at the molecular level. As omics studies become popular, integrative approaches need to be developed to move from a simple cataloguing of mutations and changes in gene expression to dissecting the molecular nature of carcinogenesis at the systemic level and understanding the complex networks that lead to cancer development. RESULTS: Here, we describe a high-throughput, multi-dimensional sequencing study of primary lung adenocarcinoma tumors and adjacent normal tissues of six Korean female never-smoker patients. Our data encompass results from exome-seq, RNA-seq, small RNA-seq, and MeDIP-seq. We identified and validated novel genetic aberrations, including 47 somatic mutations and 19 fusion transcripts. One of the fusions involves the c-RET gene, which was recently reported to form fusion genes that may function as drivers of carcinogenesis in lung cancer patients. We also characterized gene expression profiles, which we integrated with genomic aberrations and gene regulations into functional networks. The most prominent gene network module that emerged indicates that disturbances in G2/M transition and mitotic progression are causally linked to tumorigenesis in these patients. Also, results from the analysis strongly suggest that several novel microRNA-target interactions represent key regulatory elements of the gene network. CONCLUSIONS: Our study not only provides an overview of the alterations occurring in lung adenocarcinoma at multiple levels from genome to transcriptome and epigenome, but also offers a model for integrative genomics analysis and proposes potential target pathways for the control of lung adenocarcinoma.

Selecting SNPs for pharmacogenomic association study.

SNP genotyping device is an essential tool in the upcoming era ofpersonal genome and personalised medicine. Human genome has more than 10 million SNPs whereas conventional SNP genotyping device can only hold 1 million SNPs. Thus, intelligent SNP contents selection is required to maximise the value of SNP genotyping device. Here, we developed a new selection algorithm and applied this method to design genotyping contents for cancerand pharmacogenomic association study. This approach significantly increased the product value when compared with contents of competitive SNP genotyping product.

CDA: combinatorial drug discovery using transcriptional response modules.

BACKGROUND: Anticancer therapies that target single signal transduction pathways often fail to prevent proliferation of cancer cells because of overlapping functions and cross-talk between different signaling pathways. Recent research has identified that balanced multi-component therapies might be more efficacious than highly specific single component therapies in certain cases. Ideally, synergistic combinations can provide 1) increased efficacy of the therapeutic effect 2) reduced toxicity as a result of decreased dosage providing equivalent or increased efficacy 3) the avoidance or delayed onset of drug resistance. Therefore, the interest in combinatorial drug discovery based on systems-oriented approaches has been increasing steadily in recent years. METHODOLOGY: Here we describe the development of Combinatorial Drug Assembler (CDA), a genomics and bioinformatics system, whereby using gene expression profiling, multiple signaling pathways are targeted for combinatorial drug discovery. CDA performs expression pattern matching of signaling pathway components to compare genes expressed in an input cell line (or patient sample data), with expression patterns in cell lines treated with different small molecules. Then it detects best pattern matching combinatorial drug pairs across the input gene set-related signaling pathways to detect where gene expression patterns overlap and those predicted drug pairs could likely be applied as combination therapy. We carried out in vitro validations on non-small cell lung cancer cells and triple-negative breast cancer (TNBC) cells. We found two combinatorial drug pairs that showed synergistic effect on lung cancer cells. Furthermore, we also observed that halofantrine and vinblastine were synergistic on TNBC cells. CONCLUSIONS: CDA provides a new way for rational drug combination. Together with phExplorer, CDA also provides functional insights into combinatorial drugs. CDA is freely available at http://cda.i-pharm.org.

Rational drug repositioning guided by an integrated pharmacological network of protein, disease and drug.

BACKGROUND: The process of drug discovery and development is time-consuming and costly, and the probability of success is low. Therefore, there is rising interest in repositioning existing drugs for new medical indications. When successful, this process reduces the risk of failure and costs associated with de novo drug development. However, in many cases, new indications of existing drugs have been found serendipitously. Thus there is a clear need for establishment of rational methods for drug repositioning. RESULTS: In this study, we have established a database we call "PharmDB" which integrates data associated with disease indications, drug development, and associated proteins, and known interactions extracted from various established databases. To explore linkages of known drugs to diseases of interest from within PharmDB, we designed the Shared Neighborhood Scoring (SNS) algorithm. And to facilitate exploration of tripartite (Drug-Protein-Disease) network, we developed a graphical data visualization software program called phExplorer, which allows us to browse PharmDB data in an interactive and dynamic manner. We validated this knowledge-based tool kit, by identifying a potential application of a hypertension drug, benzthiazide (TBZT), to induce lung cancer cell death. CONCLUSIONS: By combining PharmDB, an integrated tripartite database, with Shared Neighborhood Scoring (SNS) algorithm, we developed a knowledge platform to rationally identify new indications for known FDA approved drugs, which can be customized to specific projects using manual curation. The data in PharmDB is open access and can be easily explored with phExplorer and accessed via BioMart web service (http://www.i-pharm.org/, http://biomart.i-pharm.org/).

A theoretical model for p53 dynamics: identifying optimal therapeutic strategy for its activation and stabilization.

The activation and stabilization of tumor suppressor p53 are very important in preventing cells from becoming cancerous. Hence, many experimental works have been carried out to investigate p53's dynamics through its interactions with other proteins and its therapeutic applications for the treatment of cancers. In this work, by analyzing a theoretical model, we attempt to search for an optimal therapeutic strategy that guarantees the activation and stabilization of p53. For this purpose, we introduce a new mathematical model including oncogene activation and ARF, which are recognized as crucial for tumor suppression but have not yet been considered in most theoretical works. Through mathematical modeling and numerical simulations, we confirm several important properties of p53 dynamics: the role of the oncogene-mediated activation of ARF as an important factor for the activation and stabilization of p53, the necessity of time delays in negative feedback loops to guarantee sustained p53 oscillations, and the digital behavior of p53 pulses. Furthermore, we propose that the binding of ARF to Mdm2 and enhancing the degradation of Mdm2 is an efficient strategy for therapeutic targeting, which may assure the activation and stabilization of p53.