WebGestalt is a popular tool for the interpretation of gene lists derived from large scale -omics studies. In the 2019 update, WebGestalt supports 12 organisms, 342 gene identifiers and 155 175 functional categories, as well as user-uploaded functional databases. To address the growing and unique need for phosphoproteomics data interpretation, we have implemented phosphosite set analysis to identify important kinases from phosphoproteomics data. We have completely redesigned result visualizations and user interfaces to improve user-friendliness and to provide multiple types of interactive and publication-ready figures. To facilitate comprehension of the enrichment results, we have implemented two methods to reduce redundancy between enriched gene sets. We introduced a web API for other applications to get data programmatically from the WebGestalt server or pass data to WebGestalt for analysis. We also wrapped the core computation into an R package called WebGestaltR for users to perform analysis locally or in third party workflows. WebGestalt can be freely accessed at http://www.webgestalt.org.

Although cellular behaviors are dynamic, the networks that govern these behaviors have been mapped primarily as static snapshots. Using an approach called differential epistasis mapping, we have discovered widespread changes in genetic interaction among yeast kinases, phosphatases, and transcription factors as the cell responds to DNA damage. Differential interactions uncover many gene functions that go undetected in static conditions. They are very effective at identifying DNA repair pathways, highlighting new damage-dependent roles for the Slt2 kinase, Pph3 phosphatase, and histone variant Htz1. The data also reveal that protein complexes are generally stable in response to perturbation, but the functional relations between these complexes are substantially reorganized. Differential networks chart a new type of genetic landscape that is invaluable for mapping cellular responses to stimuli.One of the most basic approaches to understanding gene function relies on the identification of genetic interactions, which occur when the phenotypic effects of one gene depend on the presence of a second. Recently, a number of technologies have been developed to systematically map genetic interaction networks over large sets of genes in budding yeast (1-3) and other model organisms (4,5 To gain insight into how genetic networks are altered by stress, we assembled a large genetic interactome with and without perturbation by the DNA-damaging agent methyl methane-sulfonate (MMS). Using the technique of epistatic miniarray profiles (E-MAP) (8), genetic interactions were interrogated among a set of 418 yeast genes selected to provide broad coverage of the cellular signaling and transcriptional machinery, including nearly all yeast kinases, phosphatases, and transcription factors, as well as known DNA repair factors ( fig. S1 and table S1). About 80,000 double-mutant strains were generated from all pairwise mutant combinations of the 418 genes, in which mutations were complete gene deletions (nonessential genes) or hypomorphic alleles (essential genes) as appropriate. Double-mutant combinations were grown with or without 0.02% MMS, and their colony sizes were analyzed statistically to compute a genetic interaction score (S score) in each condition (9), which indicates whether the strain was healthier or sicker than expected (positive or negative S, respectively) (10).From established score thresholds for positive and negative interactions (S ≥ +2.0, S ≤ −2.5) (9) we identified two genetic networks: a set of 1905 interactions for the untreated condition, and a set of 2297 interactions under MMS. Analysis of these "static" genetic maps showed strong associations with physical interaction networks of various kinds. For example, gene pairs with either positive or negative genetic interactions were highly enriched for proteins known to physically interact. In addition, both maps were enriched for known kinase-and phosphatase-substrate pairs, as well as transcription factor-target pairs ( fig. S2). The correspondence to physical...

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SUMMARY The integration of mass spectrometry-based proteomics with next-generation DNA and RNA sequencing profiles tumors more comprehensively. Here this “proteogenomics” approach was applied to 122 treatment-naive primary breast cancers accrued to preserve post-translational modifications, including protein phosphorylation and acetylation. Proteogenomics challenged standard breast cancer diagnoses, provided detailed analysis of the ERBB2 amplicon, defined tumor subsets that could benefit from immune checkpoint therapy, and allowed more accurate assessment of Rb status for prediction of CDK4/6 inhibitor responsiveness. Phosphoproteomics profiles uncovered novel associations between tumor suppressor loss and targetable kinases.Acetylproteome analysis highlighted acetylation on key nuclear proteins involved in the DNA damage response and revealed cross-talk between cytoplasmic and mitochondrial acetylation and metabolism. Our results underscore the potential of proteogenomics for clinical investigation of breast cancer through more accurate annotation of targetable pathways and biological features of this remarkably heterogeneous malignancy.

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Highlights d A systematic inventory of HNSCC-associated proteins, phosphosites, and pathways d Three multi-omic subtypes linked to targeted treatment approaches and immunotherapy d Widespread deletion of immune modulatory genes accounts for loss of immunogenicity d Two modes of EGFR activation inform response to anti-EGFR monoclonal antibodies

Highlights d Unsupervised clustering revealed subtype with EMT and phosphoprotein signatures d Potential therapeutic vulnerabilities included survivin, NSD3, LSD1, and EZH2 d Rb phosphorylation nominated as a biomarker for trials with CDK4/6 inhibitors d Detailed immune landscape analysis highlighted targetable points of immuneregulation

SUMMARY DNA damage activates checkpoint kinases that induce several downstream events, including widespread changes in transcription. However, the specific connections between the checkpoint kinases and downstream transcription factors (TFs) are not well understood. Here, we integrate kinase mutant expression profiles, transcriptional regulatory interactions, and phosphoproteomics to map kinases and downstream TFs to transcriptional regulatory networks. Specifically, we investigate the role of the Saccharomyces cerevisiae checkpoint kinases (Mec1, Tel1, Chk1, Rad53, and Dun1) in the transcriptional response to DNA damage caused by methyl methanesulfonate. The result is a global kinase-TF regulatory network in which Mec1 and Tel1 signal through Rad53 to synergistically regulate the expression of more than 600 genes. This network involves at least nine TFs, many of which have Rad53-dependent phosphorylation sites, as regulators of checkpoint-kinase-dependent genes. We also identify a major DNA damage-induced transcriptional network that regulates stress response genes independently of the checkpoint kinases.