Clinical associations and genetic interactions of oncogenic BRAF alleles.

BRAF is a serine/threonine-specific protein kinase that regulates the MAPK/ERK signaling pathway, and mutations in the BRAF gene are considered oncogenic drivers in diverse types of cancer. Based on the signaling mechanism, oncogenic BRAF mutations can be assigned to three different classes: class 1 mutations constitutively activate the kinase domain and lead to RAS-independent signaling, class 2 mutations induce artificial dimerization of BRAF and RAS-independent signaling and class 3 mutations display reduced or abolished kinase function and require upstream signals. Despite the importance of BRAF mutations in cancer, the clinical associations, genetic interactions and therapeutic implications of non-V600 BRAF mutations have not been explored comprehensively yet.

Substrate spectrum of PPM1D in the cellular response to DNA double-strand breaks.

PPM1D is a p53-regulated protein phosphatase that modulates the DNA damage response (DDR) and is frequently altered in cancer. Here, we employed chemical inhibition of PPM1D and quantitative mass spectrometry-based phosphoproteomics to identify the substrates of PPM1D upon induction of DNA double-strand breaks (DSBs) by etoposide. We identified 73 putative PPM1D substrates that are involved in DNA repair, regulation of transcription, and RNA processing.

VCP inhibition induces an unfolded protein response and apoptosis in human acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a heterogeneous malignancy characterized by the accumulation of undifferentiated white blood cells (blasts) in the bone marrow. Valosin-containing protein (VCP) is an abundant molecular chaperone that extracts ubiquitylated substrates from protein complexes and cellular compartments prior to their degradation by the proteasome. We found that treatment of AML cell lines with the VCP inhibitor CB-5083 leads to an accumulation of ubiquitylated proteins, activation of unfolded protein response (UPR) and apoptosis.

Cancer Risks Associated With and Pathogenic Variants.

Purpose: To provide precise age-specific risk estimates of cancers other than female breast and ovarian cancers associated with pathogenic variants (PVs) in and for effective cancer risk management.

Methods: We used data from 3,184 and 2,157 families in the Consortium of Investigators of Modifiers of to estimate age-specific relative (RR) and absolute risks for 22 first primary cancer types adjusting for family ascertainment.

Results: PVs were associated with risks of male breast (RR = 4.

Proteomic analysis of tyrosine phosphorylation induced by exogenous expression of oncogenic kinase fusions identified in lung adenocarcinoma.

Kinase fusions are considered oncogenic drivers in numerous types of cancer. In lung adenocarcinoma 5-10% of patients harbor kinase fusions. The most frequently detected kinase fusion involves the Anaplastic Lymphoma Kinase (ALK) and Echinoderm Microtubule-associated protein-Like 4 (EML4).

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification.

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions.

Correction: LSD1 inhibition by tranylcypromine derivatives interferes with GFI1-mediated repression of PU.1 target genes and induces differentiation in AML.

The original version of this Article contained an error in the Acknowledgements section.

LSD1 inhibition by tranylcypromine derivatives interferes with GFI1-mediated repression of PU.1 target genes and induces differentiation in AML.

LSD1 has emerged as a promising epigenetic target in the treatment of acute myeloid leukemia (AML). We used two murine AML models based on retroviral overexpression of Hoxa9/Meis1 (H9M) or MN1 to study LSD1 loss of function in AML. The conditional knockout of Lsd1 resulted in differentiation with both granulocytic and monocytic features and increased ATRA sensitivity and extended the survival of mice with H9M-driven AML.

p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage.

Ultraviolet (UV) light radiation induces the formation of bulky photoproducts in the DNA that globally affect transcription and splicing. However, the signaling pathways and mechanisms that link UV-light-induced DNA damage to changes in RNA metabolism remain poorly understood. Here we employ quantitative phosphoproteomics and protein kinase inhibition to provide a systems view on protein phosphorylation patterns induced by UV light and uncover the dependencies of phosphorylation events on the canonical DNA damage signaling by ATM/ATR and the p38 MAP kinase pathway.

Proteomic profiling of VCP substrates links VCP to K6-linked ubiquitylation and c-Myc function.

Valosin-containing protein (VCP) is an evolutionarily conserved ubiquitin-dependent ATPase that mediates the degradation of proteins through the ubiquitin-proteasome pathway. Despite the central role of VCP in the regulation of protein homeostasis, identity and nature of its cellular substrates remain poorly defined. Here, we combined chemical inhibition of VCP and quantitative ubiquitin remnant profiling to assess the effect of VCP inhibition on the ubiquitin-modified proteome and to probe the substrate spectrum of VCP in human cells.

The experience of physicians in pharmacogenomic clinical decision support within eight German university hospitals.

Aim: The aim of this study was to assess the physicians' attitude, their knowledge and their experience in pharmacogenomic clinical decision support in German hospitals.

Materials & Methods: We conducted an online survey to address physicians of 13 different medical specialties across eight German university hospitals. In total, 564 returned questionnaires were analyzed.

Integrating clinical decision support systems for pharmacogenomic testing into clinical routine - a scoping review of designs of user-system interactions in recent system development.

Background: Pharmacogenomic clinical decision support systems (CDSS) have the potential to help overcome some of the barriers for translating pharmacogenomic knowledge into clinical routine. Before developing a prototype it is crucial for developers to know which pharmacogenomic CDSS features and user-system interactions have yet been developed, implemented and tested in previous pharmacogenomic CDSS efforts and if they have been successfully applied. We address this issue by providing an overview of the designs of user-system interactions of recently developed pharmacogenomic CDSS.

Mass Spectrometry-Based Proteomics for Quantifying DNA Damage-Induced Phosphorylation.

Protein phosphorylation plays central regulatory roles in DNA damage repair and signaling. Protein kinases of the phosphatidylinositol 3-kinase-related kinase family ATM, ATR, and DNA-PKcs mediate phosphorylation of hundreds of substrates after DNA damage and thereby orchestrate the cellular response to DNA damage. Protein phosphorylation can be studied using antibodies that specifically recognize phosphorylated protein species; however, this approach is limited by existing antibodies and does not permit unbiased discovery of phosphorylation sites or analyzing phosphorylation sites in a high-throughput manner.

Mass Spectrometry-Based Proteomics for Investigating DNA Damage-Associated Protein Ubiquitylation.

Modification of proteins with the 76 amino acid protein ubiquitin plays essential roles in cellular signaling. Development of methods for specific enrichment of ubiquitin remnant peptides and advances in high-resolution mass spectrometry have enabled proteome-wide identification of endogenous ubiquitylation sites. Moreover, ubiquitin remnant profiling has emerged as a powerful approach for investigating changes in protein ubiquitylation in response to cellular perturbations, such as DNA damage, as well as for identification of substrates of ubiquitin-modifying enzymes.

SPATA2 links CYLD to the TNF-α receptor signaling complex and modulates the receptor signaling outcomes.

TNF-α is a key regulator of innate immune and proinflammatory responses. However, the composition of the TNF-α receptor-associated signaling complexes (TNF-RSC) and the architecture of the downstream signaling networks are incompletely understood. We employed quantitative mass spectrometry to demonstrate that TNF-α stimulation induces widespread protein phosphorylation and that the scope of phosphorylation expands in a temporal manner.

Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria.

Selective autophagy of damaged mitochondria requires autophagy receptors optineurin (OPTN), NDP52 (CALCOCO2), TAX1BP1, and p62 (SQSTM1) linking ubiquitinated cargo to autophagic membranes. By using quantitative proteomics, we show that Tank-binding kinase 1 (TBK1) phosphorylates all four receptors on several autophagy-relevant sites, including the ubiquitin- and LC3-binding domains of OPTN and p62/SQSTM1 as well as the SKICH domains of NDP52 and TAX1BP1. Constitutive interaction of TBK1 with OPTN and the ability of OPTN to bind to ubiquitin chains are essential for TBK1 recruitment and kinase activation on mitochondria.

Illuminating Spatial and Temporal Organization of Protein Interaction Networks by Mass Spectrometry-Based Proteomics.

Protein-protein interactions are at the core of all cellular functions and dynamic alterations in protein interactions regulate cellular signaling. In the last decade, mass spectrometry (MS)-based proteomics has delivered unprecedented insights into human protein interaction networks. Affinity purification-MS (AP-MS) has been extensively employed for focused and high-throughput studies of steady state protein-protein interactions.

p38- and MK2-dependent signalling promotes stress-induced centriolar satellite remodelling via 14-3-3-dependent sequestration of CEP131/AZI1.

Centriolar satellites (CS) are small granular structures that cluster in the vicinity of centrosomes. CS are highly susceptible to stress stimuli, triggering abrupt displacement of key CS factors. Here we discover a linear p38-MK2-14-3-3 signalling pathway that specifically targets CEP131 to trigger CS remodelling after cell stress.

ATR inhibition rewires cellular signaling networks induced by replication stress.

The slowing down or stalling of replication forks is commonly known as replication stress and arises from multiple causes such as DNA lesions, nucleotide depletion, RNA-DNA hybrids, and oncogene activation. The ataxia telangiectasia and Rad3-related kinase (ATR) plays an essential role in the cellular response to replication stress and inhibition of ATR has emerged as therapeutic strategy for the treatment of cancers that exhibit high levels of replication stress. However, the cellular signaling induced by replication stress and the substrate spectrum of ATR has not been systematically investigated.

Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation.

B-cell receptor (BCR) signaling is essential for the development and function of B cells; however, the spectrum of proteins involved in BCR signaling is not fully known. Here we used quantitative mass spectrometry-based proteomics to monitor the dynamics of BCR signaling complexes (signalosomes) and to investigate the dynamics of downstream phosphorylation and ubiquitylation signaling. We identify most of the previously known components of BCR signaling, as well as many proteins that have not yet been implicated in this system.

Acetylation site specificities of lysine deacetylase inhibitors in human cells.

Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acetylation signatures for 19 different KDACIs, covering all 18 human lysine deacetylases.

Proteome-wide analysis of SUMO2 targets in response to pathological DNA replication stress in human cells.

SUMOylation is a form of post-translational modification involving covalent attachment of SUMO (Small Ubiquitin-like Modifier) polypeptides to specific lysine residues in the target protein. In human cells, there are four SUMO proteins, SUMO1-4, with SUMO2 and SUMO3 forming a closely related subfamily. SUMO2/3, in contrast to SUMO1, are predominantly involved in the cellular response to certain stresses, including heat shock.

A quantitative 14-3-3 interaction screen connects the nuclear exosome targeting complex to the DNA damage response.

RNA metabolism is altered following DNA damage, but the underlying mechanisms are not well understood. Through a 14-3-3 interaction screen for DNA damage-induced protein interactions in human cells, we identified protein complexes connected to RNA biology. These include the nuclear exosome targeting (NEXT) complex that regulates turnover of noncoding RNAs termed promoter upstream transcripts (PROMPTs).

Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae.

Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation accumulated in growth-arrested cells in a manner that depended on acetyl-CoA generation in distinct subcellular compartments.