Boosting signal-to-noise in complex biology: prior knowledge is power.

A major difficulty in the analysis of complex biological systems is dealing with the low signal-to-noise inherent to nearly all large biological datasets. We discuss powerful bioinformatic concepts for boosting signal-to-noise through external knowledge incorporated in processing units we call filters and integrators. These concepts are illustrated in four landmark studies that have provided model implementations of filters, integrators, or both.

A global network of transcription factors, involving E2A, EBF1 and Foxo1, that orchestrates B cell fate.

It is now established that the transcription factors E2A, EBF1 and Foxo1 have critical roles in B cell development. Here we show that E2A and EBF1 bound regulatory elements present in the Foxo1 locus. E2A and EBF1, as well as E2A and Foxo1, in turn, were wired together by a vast spectrum of cis-regulatory sequences. These associations were dynamic during developmental progression. Occupancy by the E2A isoform E47 directly resulted in greater abundance, as well as a pattern of monomethylation of histone H3 at lysine 4 (H3K4) across putative enhancer regions. Finally, we divided the pro-B cell epigenome into clusters of loci with occupancy by E2A, EBF and Foxo1. From this analysis we constructed a global network consisting of transcriptional regulators, signaling and survival factors that we propose orchestrates B cell fate.

Multilineage priming of enhancer repertoires precedes commitment to the B and myeloid cell lineages in hematopoietic progenitors.

Recent studies have documented genome-wide binding patterns of transcriptional regulators and their associated epigenetic marks in hematopoietic cell lineages. In order to determine how epigenetic marks are established and maintained during developmental progression, we have generated long-term cultures of hematopoietic progenitors by enforcing the expression of the E-protein antagonist Id2. Hematopoietic progenitors that express Id2 are multipotent and readily differentiate upon withdrawal of Id2 expression into committed B lineage cells, thus indicating a causative role for E2A (Tcf3) in promoting the B cell fate. Genome-wide analyses revealed that a substantial fraction of lymphoid and myeloid enhancers are premarked by the poised or active enhancer mark H3K4me1 in multipotent progenitors. Thus, in hematopoietic progenitors, multilineage priming of enhancer elements precedes commitment to the lymphoid or myeloid cell lineages.

A Distributed Network for Intensive Longitudinal Monitoring in Metastatic Triple-Negative Breast Cancer.

Accelerating cancer research is expected to require new types of clinical trials. This report describes the Intensive Trial of OMics in Cancer (ITOMIC) and a participant with triple-negative breast cancer metastatic to bone, who had markedly elevated circulating tumor cells (CTCs) that were monitored 48 times over 9 months. A total of 32 researchers from 14 institutions were engaged in the patient's evaluation; 20 researchers had no prior involvement in patient care and 18 were recruited specifically for this patient. Whole-exome sequencing of 3 bone marrow samples demonstrated a novel ROS1 variant that was estimated to be present in most or all tumor cells. After an initial response to cisplatin, a hypothesis of crizotinib sensitivity was disproven. Leukapheresis followed by partial CTC enrichment allowed for the development of a differential high-throughput drug screen and demonstrated sensitivity to investigational BH3-mimetic inhibitors of BCL-2 that could not be tested in the patient because requests to the pharmaceutical sponsors were denied. The number and size of CTC clusters correlated with clinical status and eventually death. Focusing the expertise of a distributed network of investigators on an intensively monitored patient with cancer can generate high-resolution views of the natural history of cancer and suggest new opportunities for therapy. Optimization requires access to investigational drugs.

NeXO Web: the NeXO ontology database and visualization platform.

The Network-extracted Ontology (NeXO) is a gene ontology inferred directly from large-scale molecular networks. While most ontologies are constructed through manual expert curation, NeXO uses a principled computational approach which integrates evidence from hundreds of thousands of individual gene and protein interactions to construct a global hierarchy of cellular components and processes. Here, we describe the development of the NeXO Web platform (http://www.nexontology.org)-an online database and graphical user interface for visualizing, browsing and performing term enrichment analysis using NeXO and the gene ontology. The platform applies state-of-the-art web technology and visualization techniques to provide an intuitive framework for investigating biological machinery captured by both data-driven and manually curated ontologies.

Inferring gene ontologies from pairwise similarity data.

MOTIVATION: While the manually curated Gene Ontology (GO) is widely used, inferring a GO directly from -omics data is a compelling new problem. Recognizing that ontologies are a directed acyclic graph (DAG) of terms and hierarchical relations, algorithms are needed that: analyze a full matrix of gene-gene pairwise similarities from -omics data; infer true hierarchical structure in these data rather than enforcing hierarchy as a computational artifact; and respect biological pleiotropy, by which a term in the hierarchy can relate to multiple higher level terms. Methods addressing these requirements are just beginning to emerge-none has been evaluated for GO inference. METHODS: We consider two algorithms [Clique Extracted Ontology (CliXO), LocalFitness] that uniquely satisfy these requirements, compared with methods including standard clustering. CliXO is a new approach that finds maximal cliques in a network induced by progressive thresholding of a similarity matrix. We evaluate each method's ability to reconstruct the GO biological process ontology from a similarity matrix based on (a) semantic similarities for GO itself or (b) three -omics datasets for yeast. RESULTS: For task (a) using semantic similarity, CliXO accurately reconstructs GO (>99% precision, recall) and outperforms other approaches (<20% precision, <20% recall). For task (b) using -omics data, CliXO outperforms other methods using two -omics datasets and achieves ∼30% precision and recall using YeastNet v3, similar to an earlier approach (Network Extracted Ontology) and better than LocalFitness or standard clustering (20-25% precision, recall). CONCLUSION: This study provides algorithmic foundation for building gene ontologies by capturing hierarchical and pleiotropic structure embedded in biomolecular data.

Novel data-mining approach identifies biomarkers for diagnosis of Kawasaki disease.

BACKGROUND: As Kawasaki disease (KD) shares many clinical features with other more common febrile illnesses and misdiagnosis, leading to a delay in treatment, increases the risk of coronary artery damage, a diagnostic test for KD is urgently needed. We sought to develop a panel of biomarkers that could distinguish between acute KD patients and febrile controls (FC) with sufficient accuracy to be clinically useful. METHODS: Plasma samples were collected from three independent cohorts of FC and acute KD patients who met the American Heart Association definition for KD and presented within the first 10 d of fever. The levels of 88 biomarkers associated with inflammation were assessed by Luminex bead technology. Unsupervised clustering followed by supervised clustering using a Random Forest model was used to find a panel of candidate biomarkers. RESULTS: A panel of biomarkers commonly available in the hospital laboratory (absolute neutrophil count, erythrocyte sedimentation rate, alanine aminotransferase, γ-glutamyl transferase, concentrations of α-1-antitrypsin, C-reactive protein, and fibrinogen, and platelet count) accurately diagnosed 81-96% of KD patients in a series of three independent cohorts. CONCLUSION: After prospective validation, this eight-biomarker panel may improve the recognition of KD.

Improving breast cancer survival analysis through competition-based multidimensional modeling.

Breast cancer is the most common malignancy in women and is responsible for hundreds of thousands of deaths annually. As with most cancers, it is a heterogeneous disease and different breast cancer subtypes are treated differently. Understanding the difference in prognosis for breast cancer based on its molecular and phenotypic features is one avenue for improving treatment by matching the proper treatment with molecular subtypes of the disease. In this work, we employed a competition-based approach to modeling breast cancer prognosis using large datasets containing genomic and clinical information and an online real-time leaderboard program used to speed feedback to the modeling team and to encourage each modeler to work towards achieving a higher ranked submission. We find that machine learning methods combined with molecular features selected based on expert prior knowledge can improve survival predictions compared to current best-in-class methodologies and that ensemble models trained across multiple user submissions systematically outperform individual models within the ensemble. We also find that model scores are highly consistent across multiple independent evaluations. This study serves as the pilot phase of a much larger competition open to the whole research community, with the goal of understanding general strategies for model optimization using clinical and molecular profiling data and providing an objective, transparent system for assessing prognostic models.

Protein networks as logic functions in development and cancer.

Many biological and clinical outcomes are based not on single proteins, but on modules of proteins embedded in protein networks. A fundamental question is how the proteins within each module contribute to the overall module activity. Here, we study the modules underlying three representative biological programs related to tissue development, breast cancer metastasis, or progression of brain cancer, respectively. For each case we apply a new method, called Network-Guided Forests, to identify predictive modules together with logic functions which tie the activity of each module to the activity of its component genes. The resulting modules implement a diverse repertoire of decision logic which cannot be captured using the simple approximations suggested in previous work such as gene summation or subtraction. We show that in cancer, certain combinations of oncogenes and tumor suppressors exert competing forces on the system, suggesting that medical genetics should move beyond cataloguing individual cancer genes to cataloguing their combinatorial logic.

Systematic Analysis of Genetic and Pathway Determinants of Eribulin Sensitivity across 100 Human Cancer Cell Lines from the Cancer Cell Line Encyclopedia (CCLE).

Eribulin, a natural product-based microtubule targeting agent with cytotoxic and noncytotoxic mechanisms, is FDA approved for certain patients with advanced breast cancer and liposarcoma. To investigate the feasibility of developing drug-specific predictive biomarkers, we quantified antiproliferative activities of eribulin versus paclitaxel and vinorelbine against 100 human cancer cell lines from the Cancer Cell Line Encyclopedia, and correlated results with publicly available databases to identify genes and pathways associated with eribulin response, either uniquely or shared with paclitaxel or vinorelbine. Mean expression ratios of 11,985 genes between the most and least sensitive cell line quartiles were sorted by p-values and drug overlaps, yielding 52, 29 and 80 genes uniquely associated with eribulin, paclitaxel and vinorelbine, respectively. Further restriction to minimum 2-fold ratios followed by reintroducing data from the middle two quartiles identified 9 and 13 drug-specific unique fingerprint genes for eribulin and vinorelbine, respectively; surprisingly, no gene met all criteria for paclitaxel. Interactome and Reactome pathway analyses showed that unique fingerprint genes of both drugs were primarily associated with cellular signaling, not microtubule-related pathways, although considerable differences existed in individual pathways identified. Finally, four-gene (C5ORF38, DAAM1, IRX2, CD70) and five-gene (EPHA2, NGEF, SEPTIN10, TRIP10, VSIG10) multivariate regression models for eribulin and vinorelbine showed high statistical correlation with drug-specific responses across the 100 cell lines and accurately calculated predicted mean IC50s for the most and least sensitive cell line quartiles as surrogates for responders and nonresponders, respectively. Collectively, these results provide a foundation for developing drug-specific predictive biomarkers for eribulin and vinorelbine.

Pathway-level mutation analysis in primary high-grade serous ovarian cancer and matched brain metastases.

Brain metastases (BMs) in ovarian cancer (OC) are a rare event. BMs occur most frequently in high-grade serous (HGS) OC. The molecular features of BMs in HGSOC are poorly understood. We performed a whole-exome sequencing analysis of ten matched pairs of formalin-fixed paraffin-embedded samples from primary HGSOC and corresponding BMs. Enrichment significance (p value; false discovery rate) was computed using the Reactome, the Kyoto Encyclopedia of Genes and Genomes pathway collections, and the Gene Ontology Biological Processes. Germline DNA damage repair variants were found in seven cases (70%) and involved the BRCA1, BRCA2, ATM, RAD50, ERCC4, RPA1, MLHI, and ATR genes. Somatic mutations of TP53 were found in nine cases (90%) and were the only stable mutations between the primary tumor and BMs. Disturbed pathways in BMs versus primary HGSOC constituted a complex network and included the cell cycle, the degradation of the extracellular matrix, cell junction organization, nucleotide metabolism, lipid metabolism, the immune system, G-protein-coupled receptors, intracellular vesicular transport, and reaction to chemical stimuli (Golgi vesicle transport and olfactory signaling). Pathway analysis approaches allow for a more intuitive interpretation of the data as compared to considering single-gene aberrations and provide an opportunity to identify clinically informative alterations in HGSOC BM.

Safety, Feasibility, and Merits of Longitudinal Molecular Testing of Multiple Metastatic Sites to Inform mTNBC Patient Treatment in the Intensive Trial of Omics in Cancer.

PURPOSE: Patients with metastatic triple-negative breast cancer (mTNBC) have poor outcomes. The Intensive Trial of Omics in Cancer (ITOMIC) sought to determine the feasibility and potential efficacy of informing treatment decisions through multiple biopsies of mTNBC deposits longitudinally over time, accompanied by analysis using a distributed network of experts. METHODS: Thirty-one subjects were enrolled and 432 postenrollment biopsies performed (clinical and study-directed) of which 332 were study-directed. Molecular profiling included whole-genome sequencing or whole-exome sequencing, cancer-associated gene panel sequencing, RNA-sequencing, and immunohistochemistry. To afford time for analysis, subjects were initially treated with cisplatin (19 subjects), or another treatment they had not received previously. The results were discussed at a multi-institutional ITOMIC Tumor Board, and a report transmitted to the subject's oncologist who arrived at the final treatment decision in conjunction with the subject. Assistance was provided to access treatments that were predicted to be effective. RESULTS: Multiple biopsies in single settings and over time were safe, and comprehensive analysis was feasible. Two subjects were found to have lung cancer, one had carcinoma of unknown primary site, tumor samples from three subjects were estrogen receptor-positive and from two others, human epidermal growth factor receptor 2-positive. Two subjects withdrew. Thirty-four of 112 recommended treatments were accessed using approved drugs, clinical trials, and single-patient investigational new drugs. After excluding the three subjects with nonbreast cancers and the two subjects who withdrew, 22 of 26 subjects (84.6%) received at least one ITOMIC Tumor Board-recommended treatment. CONCLUSION: Further exploration of this approach in patients with mTNBC is merited.

MODEVO: exploring modularity and evolution of protein interaction networks.

SUMMARY: Interrogating protein complexes and pathways in an evolutionary context provides insights into the formation of the basic functional components of the cell. We developed two independent Cytoscape plugins that can be cooperatively used to map evolving protein interaction networks at the module level. The APCluster plugin implements a recent affinity propagation (AP) algorithm for graph clustering and can be applied to decompose networks into coherent modules. The NetworkEvolution plugin provides the capability to visualize selected modules in consecutive evolutionary stages. AVAILABILITY: The plugins, input data and usage scenarios are freely available from the project web site: http://bioputer.mimuw.edu.pl/modevo. The plugins are also available from the Cytoscape plugin repository.

Phylogeny-guided interaction mapping in seven eukaryotes.

BACKGROUND: The assembly of reliable and complete protein-protein interaction (PPI) maps remains one of the significant challenges in systems biology. Computational methods which integrate and prioritize interaction data can greatly aid in approaching this goal. RESULTS: We developed a Bayesian inference framework which uses phylogenetic relationships to guide the integration of PPI evidence across multiple datasets and species, providing more accurate predictions. We apply our framework to reconcile seven eukaryotic interactomes: H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. cerevisiae and A. thaliana. Comprehensive GO-based quality assessment indicates a 5% to 44% score increase in predicted interactomes compared to the input data. Further support is provided by gold-standard MIPS, CYC2008 and HPRD datasets. We demonstrate the ability to recover known PPIs in well-characterized yeast and human complexes (26S proteasome, endosome and exosome) and suggest possible new partners interacting with the putative SWI/SNF chromatin remodeling complex in A. thaliana. CONCLUSION: Our phylogeny-guided approach compares favorably to two standard methods for mapping PPIs across species. Detailed analysis of predictions in selected functional modules uncovers specific PPI profiles among homologous proteins, establishing interaction-based partitioning of protein families. Provided evidence also suggests that interactions within core complex subunits are in general more conserved and easier to transfer accurately to other organisms, than interactions between these subunits.

Identification of functional modules from conserved ancestral protein-protein interactions.

MOTIVATION: The increasing availability of large-scale protein-protein interaction (PPI) data has fueled the efforts to elucidate the building blocks and organization of cellular machinery. Previous studies have shown cross-species comparison to be an effective approach in uncovering functional modules in protein networks. This has in turn driven the research for new network alignment methods with a more solid grounding in network evolution models and better scalability, to allow multiple network comparison. RESULTS: We develop a new framework for protein network alignment, based on reconstruction of an ancestral PPI network. The reconstruction algorithm is built upon a proposed model of protein network evolution, which takes into account phylogenetic history of the proteins and the evolution of their interactions. The application of our methodology to the PPI networks of yeast, worm and fly reveals that the most probable conserved ancestral interactions are often related to known protein complexes. By projecting the conserved ancestral interactions back onto the input networks we are able to identify the corresponding conserved protein modules in the considered species. In contrast to most of the previous methods, our algorithm is able to compare many networks simultaneously. The performed experiments demonstrate the ability of our method to uncover many functional modules with high specificity. AVAILABILITY: Information for obtaining software and supplementary results are available at http://bioputer.mimuw.edu.pl/papers/cappi.

On consensus biomarker selection.

BACKGROUND: Recent development of mass spectrometry technology enabled the analysis of complex peptide mixtures. A lot of effort is currently devoted to the identification of biomarkers in human body fluids like serum or plasma, based on which new diagnostic tests for different diseases could be constructed. Various biomarker selection procedures have been exploited in recent studies. It has been noted that they often lead to different biomarker lists and as a consequence, the patient classification may also vary. RESULTS: Here we propose a new approach to the biomarker selection problem: to apply several competing feature ranking procedures and compute a consensus list of features based on their outcomes. We validate our methods on two proteomic datasets for the diagnosis of ovarian and prostate cancer. CONCLUSION: The proposed methodology can improve the classification results and at the same time provide a unified biomarker list for further biological examinations and interpretation.

Translation of Genotype to Phenotype by a Hierarchy of Cell Subsystems.

Accurately translating genotype to phenotype requires accounting for the functional impact of genetic variation at many biological scales. Here we present a strategy for genotype-phenotype reasoning based on existing knowledge of cellular subsystems. These subsystems and their hierarchical organization are defined by the Gene Ontology or a complementary ontology inferred directly from previously published datasets. Guided by the ontology's hierarchical structure, we organize genotype data into an "ontotype," that is, a hierarchy of perturbations representing the effects of genetic variation at multiple cellular scales. The ontotype is then interpreted using logical rules generated by machine learning to predict phenotype. This approach substantially outperforms previous, non-hierarchical methods for translating yeast genotype to cell growth phenotype, and it accurately predicts the growth outcomes of two new screens of 2,503 double gene knockouts impacting DNA repair or nuclear lumen. Ontotypes also generalize to larger knockout combinations, setting the stage for interpreting the complex genetics of disease.

Rac2 controls tumor growth, metastasis and M1-M2 macrophage differentiation in vivo.

Although it is well-established that the macrophage M1 to M2 transition plays a role in tumor progression, the molecular basis for this process remains incompletely understood. Herein, we demonstrate that the small GTPase, Rac2 controls macrophage M1 to M2 differentiation and the metastatic phenotype in vivo. Using a genetic approach, combined with syngeneic and orthotopic tumor models we demonstrate that Rac2-/- mice display a marked defect in tumor growth, angiogenesis and metastasis. Microarray, RT-PCR and metabolomic analysis on bone marrow derived macrophages isolated from the Rac2-/- mice identify an important role for Rac2 in M2 macrophage differentiation. Furthermore, we define a novel molecular mechanism by which signals transmitted from the extracellular matrix via the α4ß1 integrin and MCSF receptor lead to the activation of Rac2 and potentially regulate macrophage M2 differentiation. Collectively, our findings demonstrate a macrophage autonomous process by which the Rac2 GTPase is activated downstream of the α4ß1 integrin and the MCSF receptor to control tumor growth, metastasis and macrophage differentiation into the M2 phenotype. Finally, using gene expression and metabolomic data from our Rac2-/- model, and information related to M1-M2 macrophage differentiation curated from the literature we executed a systems biologic analysis of hierarchical protein-protein interaction networks in an effort to develop an iterative interactome map which will predict additional mechanisms by which Rac2 may coordinately control macrophage M1 to M2 differentiation and metastasis.

A probabilistic model of neutral and selective dynamics of protein network evolution.

Comparative approaches in genomics have long relied on rigorous mathematical models of sequence evolution. Such models provide the basis for formulating and solving well-defined computational problems, in turn yielding key insights into the evolutionary processes acting on the genome. Analogous model-based approaches for analyzing biological networks are still under development. Here we describe a model-based approach for estimating the probability of network rewiring events during evolution. Our method builds on the standard duplication-and-divergence model and incorporates phylogenetic analysis to guide the comparison of protein networks across species. We apply our algorithm to study the evolution of functional modules and unconstrained network regions in seven available eukaryotic interactomes. Based on this analysis we identify a map of co-functioning protein families whose members participate in strongly conserved interactions and form major complexes and pathways in the eukaryotic cell. The proposed approach provides principled means for inferring the probability of network rewiring events, enabling insights into the conservation and divergence of protein interactions and the formation of functional modules in protein networks.

A gene ontology inferred from molecular networks.

Ontologies have proven very useful for capturing knowledge as a hierarchy of terms and their interrelationships. In biology a major challenge has been to construct ontologies of gene function given incomplete biological knowledge and inconsistencies in how this knowledge is manually curated. Here we show that large networks of gene and protein interactions in Saccharomyces cerevisiae can be used to infer an ontology whose coverage and power are equivalent to those of the manually curated Gene Ontology (GO). The network-extracted ontology (NeXO) contains 4,123 biological terms and 5,766 term-term relations, capturing 58% of known cellular components. We also explore robust NeXO terms and term relations that were initially not cataloged in GO, a number of which have now been added based on our analysis. Using quantitative genetic interaction profiling and chemogenomics, we find further support for many of the uncharacterized terms identified by NeXO, including multisubunit structures related to protein trafficking or mitochondrial function. This work enables a shift from using ontologies to evaluate data to using data to construct and evaluate ontologies.