Understanding large scale sequencing datasets through changes to protein folding.

The expansion of high-quality, low-cost sequencing has created an enormous opportunity to understand how genetic variants alter cellular behaviour in disease. The high diversity of mutations observed has however drawn a spotlight onto the need for predictive modelling of mutational effects on phenotype from variants of uncertain significance. This is particularly important in the clinic due to the potential value in guiding clinical diagnosis and patient treatment. Recent computational modelling has highlighted the importance of mutation induced protein misfolding as a common mechanism for loss of protein or domain function, aided by developments in methods that make large computational screens tractable. Here we review recent applications of this approach to different genes, and how they have enabled and supported subsequent studies. We further discuss developments in the approach and the role for the approach in light of increasingly high throughput experimental approaches.

Interpreting the effect of mutations to protein binding sites from large-scale genomic screens.

Predicting the functionality of missense mutations is extremely difficult. Large-scale genomic screens are commonly performed to identify mutational correlates or drivers of disease and treatment resistance, but interpretation of how these mutations impact protein function is limited. One such consequence of mutations to a protein is to impact its ability to bind and interact with partners or small molecules such as ATP, thereby modulating its function. Multiple methods exist for predicting the impact of a single mutation on protein-protein binding energy, but it is difficult in the context of a genomic screen to understand if these mutations with large impacts on binding are more common than statistically expected. We present a methodology for taking mutational data from large-scale genomic screens and generating functional and statistical insights into their role in the binding of proteins both with each other and their small molecule ligands. This allows a quantitative and statistical analysis to determine whether mutations impacting protein binding or ligand interactions are occurring more or less frequently than expected by chance. We achieve this by calculating the potential impact of any possible mutation and comparing an expected distribution to the observed mutations. This method is applied to examples demonstrating its ability to interpret mutations involved in protein-protein binding, protein-DNA interactions, and the evolution of therapeutic resistance.

SMAD4 and KCNQ3 alterations are associated with lymph node metastases in oesophageal adenocarcinoma.

Metastasis in oesophageal adenocarcinoma (OAC) is an important predictor of survival. Radiological staging is used to stage metastases in patients, and guide treatment selection, but is limited by the accuracy of the approach. Improvements in staging will lead to improved clinical decision making and patient outcomes. Sequencing studies on primary tumours and pre-cancerous tissue have revealed the mutational landscape of OAC, and increasingly cheap and widespread sequencing approaches offer the potential to improve staging assessment. In this work we present an analysis of lymph node metastases found by radiological and pathological sampling, identifying new roles of the genes SMAD4 and KCNQ3 in metastasis. Through transcriptomic analysis we find that both genes are associated with canonical Wnt pathway activity, but KCNQ3 is uniquely associated with changes in planar cell polaritiy associated with non-canonical Wnt signalling. We go on to validate our observations in KCNQ3 in cell line and xenograph systems, showing that overexpression of KCNQ3 reduces wound closure and the number of metastases observed. Our results suggest both genes as novel biomarkers of metastatic risk and offer new potential routes to drug targeting.

KCNQ potassium channels modulate Wnt activity in gastro-oesophageal adenocarcinomas.

Voltage-sensitive potassium channels play an important role in controlling membrane potential and ionic homeostasis in the gut and have been implicated in gastrointestinal (GI) cancers. Through large-scale analysis of 897 patients with gastro-oesophageal adenocarcinomas (GOAs) coupled with in vitro models, we find KCNQ family genes are mutated in ∼30% of patients, and play therapeutically targetable roles in GOA cancer growth. KCNQ1 and KCNQ3 mediate the WNT pathway and MYC to increase proliferation through resultant effects on cadherin junctions. This also highlights novel roles of KCNQ3 in non-excitable tissues. We also discover that activity of KCNQ3 sensitises cancer cells to existing potassium channel inhibitors and that inhibition of KCNQ activity reduces proliferation of GOA cancer cells. These findings reveal a novel and exploitable role of potassium channels in the advancement of human cancer, and highlight that supplemental treatments for GOAs may exist through KCNQ inhibitors.

Mutations observed in somatic evolution reveal underlying gene mechanisms.

Highly sensitive DNA sequencing techniques have allowed the discovery of large numbers of somatic mutations in normal tissues. Some mutations confer a competitive advantage over wild-type cells, generating expanding clones that spread through the tissue. Competition between mutant clones leads to selection. This process can be considered a large scale, in vivo screen for mutations increasing cell fitness. It follows that somatic missense mutations may offer new insights into the relationship between protein structure, function and cell fitness. We present a flexible statistical method for exploring the selection of structural features in data sets of somatic mutants. We show how this approach can evidence selection of specific structural features in key drivers in aged tissues. Finally, we show how drivers may be classified as fitness-enhancing and fitness-suppressing through different patterns of mutation enrichment. This method offers a route to understanding the mechanism of protein function through in vivo mutant selection.

Preferential MGMT hypermethylation in SDH-deficient wild-type GIST.

AIMS: Wild-type gastrointestinal stromal tumours (wtGIST) are frequently caused by inherited pathogenic variants, or somatic alterations in the succinate dehydrogenase subunit genes (SDHx). Succinate dehydrogenase is a key enzyme in the citric acid cycle. SDH deficiency caused by SDHx inactivation leads to an accumulation of succinate, which inhibits DNA and histone demethylase enzymes, resulting in global hypermethylation. Epigenetic silencing of the DNA repair gene MGMT has proven utility as a positive predictor of the therapeutic efficacy of the alklyating drug temozolomide (TMZ) in tumours such as glioblastoma multiforme. The aim of this study was to examine MGMT promoter methylation status in a large cohort of GIST. METHODS: MGMT methylation analysis was performed on 65 tumour samples including 47 wtGIST (33 SDH-deficient wtGIST and 11 SDH preserved wtGIST) and 21 tyrosine kinase (TK) mutant GIST. RESULTS: MGMT promoter methylation was detected in 8 cases of SDH-deficient (dSDH) GIST but in none of the 14 SDH preserved wild-type GIST or 21 TK mutant GIST samples analysed. Mean MGMT methylation was significantly higher (p 0.0449) and MGMT expression significantly lower (p<0.0001) in dSDH wtGIST compared with TK mutant or SDH preserved GIST. No correlation was identified between SDHx subunit gene mutations or SDHC epimutation status and mean MGMT methylation levels. CONCLUSION: MGMT promoter hypermethylation occurs exclusively in a subset of dSDH wtGIST. Data from this study support testing of tumour MGMT promoter methylation in patients with dSDH wtGIST to identify those patients who may benefit from most from TMZ therapy.

Heterogeneity of the cancer cell line metabolic landscape.

The unravelling of the complexity of cellular metabolism is in its infancy. Cancer-associated genetic alterations may result in changes to cellular metabolism that aid in understanding phenotypic changes, reveal detectable metabolic signatures, or elucidate vulnerabilities to particular drugs. To understand cancer-associated metabolic transformation, we performed untargeted metabolite analysis of 173 different cancer cell lines from 11 different tissues under constant conditions for 1,099 different species using mass spectrometry (MS). We correlate known cancer-associated mutations and gene expression programs with metabolic signatures, generating novel associations of known metabolic pathways with known cancer drivers. We show that metabolic activity correlates with drug sensitivity and use metabolic activity to predict drug response and synergy. Finally, we study the metabolic heterogeneity of cancer mutations across tissues, and find that genes exhibit a range of context specific, and more general metabolic control.

HOXA9 has the hallmarks of a biological switch with implications in blood cancers.

Blood malignancies arise from the dysregulation of haematopoiesis. The type of blood cell and the specific order of oncogenic events initiating abnormal growth ultimately determine the cancer subtype and subsequent clinical outcome. HOXA9 plays an important role in acute myeloid leukaemia (AML) prognosis by promoting blood cell expansion and altering differentiation; however, the function of HOXA9 in other blood malignancies is still unclear. Here, we highlight the biological switch and prognosis marker properties of HOXA9 in AML and chronic myeloproliferative neoplasms (MPN). First, we establish the ability of HOXA9 to stratify AML patients with distinct cellular and clinical outcomes. Then, through the use of a computational network model of MPN, we show that the self-activation of HOXA9 and its relationship to JAK2 and TET2 can explain the branching progression of JAK2/TET2 mutant MPN patients towards divergent clinical characteristics. Finally, we predict a connection between the RUNX1 and MYB genes and a suppressive role for the NOTCH pathway in MPN diseases.

The NALCN channel regulates metastasis and nonmalignant cell dissemination.

We identify the sodium leak channel non-selective protein (NALCN) as a key regulator of cancer metastasis and nonmalignant cell dissemination. Among 10,022 human cancers, NALCN loss-of-function mutations were enriched in gastric and colorectal cancers. Deletion of Nalcn from gastric, intestinal or pancreatic adenocarcinomas in mice did not alter tumor incidence, but markedly increased the number of circulating tumor cells (CTCs) and metastases. Treatment of these mice with gadolinium-a NALCN channel blocker-similarly increased CTCs and metastases. Deletion of Nalcn from mice that lacked oncogenic mutations and never developed cancer caused shedding of epithelial cells into the blood at levels equivalent to those seen in tumor-bearing animals. These cells trafficked to distant organs to form normal structures including lung epithelium, and kidney glomeruli and tubules. Thus, NALCN regulates cell shedding from solid tissues independent of cancer, divorcing this process from tumorigenesis and unmasking a potential new target for antimetastatic therapies.

Tumor-Derived Lactic Acid Modulates Activation and Metabolic Status of Draining Lymph Node Stroma.

Communication between tumors and the stroma of tumor-draining lymph nodes (TDLN) exists before metastasis arises, altering the structure and function of the TDLN niche. Transcriptional profiling of fibroblastic reticular cells (FRC), the dominant stromal population of lymph nodes, has revealed that FRCs in TDLNs are reprogrammed. However, the tumor-derived factors driving the changes in FRCs remain to be identified. Taking an unbiased approach, we have shown herein that lactic acid (LA), a metabolite released by cancer cells, was not only secreted by B16.F10 and 4T1 tumors in high amounts, but also that it was enriched in TDLNs. LA supported an upregulation of Podoplanin (Pdpn) and Thy1 and downregulation of IL7 in FRCs of TDLNs, making them akin to activated fibroblasts found at the primary tumor site. Furthermore, we found that tumor-derived LA altered mitochondrial function of FRCs in TDLNs. Thus, our results demonstrate a mechanism by which a tumor-derived metabolite connected with a low pH environment modulates the function of fibroblasts in TDLNs. How lymph node function is perturbed to support cancer metastases remains unclear. The authors show that tumor-derived LA drains to lymph nodes where it modulates the function of lymph node stromal cells, prior to metastatic colonization.

The disambiguation of people names in biological collections.

Scientific collections have been built by people. For hundreds of years, people have collected, studied, identified, preserved, documented and curated collection specimens. Understanding who those people are is of interest to historians, but much more can be made of these data by other stakeholders once they have been linked to the people's identities and their biographies. Knowing who people are helps us attribute work correctly, validate data and understand the scientific contribution of people and institutions. We can evaluate the work they have done, the interests they have, the places they have worked and what they have created from the specimens they have collected. The problem is that all we know about most of the people associated with collections are their names written on specimens. Disambiguating these people is the challenge that this paper addresses. Disambiguation of people often proves difficult in isolation and can result in staff or researchers independently trying to determine the identity of specific individuals over and over again. By sharing biographical data and building an open, collectively maintained dataset with shared knowledge, expertise and resources, it is possible to collectively deduce the identities of individuals, aggregate biographical information for each person, reduce duplication of effort and share the information locally and globally. The authors of this paper aspire to disambiguate all person names efficiently and fully in all their variations across the entirety of the biological sciences, starting with collections. Towards that vision, this paper has three key aims: to improve the linking, validation, enhancement and valorisation of person-related information within and between collections, databases and publications; to suggest good practice for identifying people involved in biological collections; and to promote coordination amongst all stakeholders, including individuals, natural history collections, institutions, learned societies, government agencies and data aggregators.

A botanical demonstration of the potential of linking data using unique identifiers for people.

Natural history collection data available digitally on the web have so far only made limited use of the potential of semantic links among themselves and with cross-disciplinary resources. In a pilot study, botanical collections of the Consortium of European Taxonomic Facilities (CETAF) have therefore begun to semantically annotate their collection data, starting with data on people, and to link them via a central index system. As a result, it is now possible to query data on collectors across different collections and automatically link them to a variety of external resources. The system is being continuously developed and is already in production use in an international collection portal.

SARS-CoV-2 Variants Are Selecting for Spike Protein Mutations That Increase Protein Stability.

The emergence of variants of SARS-CoV-2 with mutations in their spike protein are a major cause for concern for the efficacy of vaccines and control of the pandemic. We show that mutations in the spike protein of SARS-CoV-2 are selecting for amino acid changes that result in a more thermodynamically stable protein than expected from background. We suggest that the computationally efficient analysis of mutational stability may aid in early screening of variants.

Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance.

Cancer-causing missense mutations in the 3418 amino acid BRCA2 breast and ovarian cancer suppressor protein frequently affect a short (∼340 residue) segment in its carboxyl-terminal domain (DBD). Here, we identify a shared molecular mechanism underlying their pathogenicity. Pathogenic BRCA2 missense mutations cluster in the DBD's helical domain (HD) and OB1-fold motifs, which engage the partner protein DSS1. Pathogenic - but not benign - DBD mutations weaken or abolish DSS1-BRCA2 assembly, provoking mutant BRCA2 oligomers that are excluded from the cell nucleus, and disable DNA repair by homologous DNA recombination (HDR). DSS1 inhibits the intracellular oligomerization of wildtype, but not mutant, forms of BRCA2. Remarkably, DSS1 expression corrects defective HDR in cells bearing pathogenic BRCA2 missense mutants with weakened, but not absent, DSS1 binding. Our findings identify a DSS1-mediated intracellular protein assembly mechanism that is disrupted by cancer-causing BRCA2 missense mutations, and suggest an approach for its therapeutic correction.

Computational Saturation Screen Reveals the Landscape of Mutations in Human Fumarate Hydratase.

Single amino acid substitutions within protein structures often manifest with clinical conditions in humans. The mutation of a single amino can significantly alter protein folding and stability, or change protein dynamics to influence function. The chemical engineering field has developed a large toolset for predicting the influence of point mutations with the aim of guiding the design of improved and more stable proteins. Here, we reverse this general protocol and adapt these tools for the prediction of damaging mutations within proteins. Mutations to fumarate hydratase (FH), an enzyme of the citric acid cycle, can lead to human diseases. The inactivation of FH by mutation causes leiomyomas and renal cell carcinoma by subsequent fumarate buildup and reduction in available malate. We present a scheme for accurately predicting the clinical effects of every possible mutation in FH by adaptation to a database of characterized damaging and benign mutations. Using energy prediction tools Rosetta and FoldX coupled with molecular dynamics simulations, we accurately predict individual mutations as well as mutational hotspots with a high disruptive capability in FH. Furthermore, through dynamic analysis, we find that hinge regions of the protein can be stabilized or destabilized by mutations, with mechanistic implications for the functional ability of the enzyme. Finally, we categorize all potential mutations in FH into functional groups, predicting which known mutations in the human population are loss of function, therefore having clinical implications, and validate our findings through metabolomics data of characterized human cell lines.

Selection of Oncogenic Mutant Clones in Normal Human Skin Varies with Body Site.

Skin cancer risk varies substantially across the body, yet how this relates to the mutations found in normal skin is unknown. Here we mapped mutant clones in skin from high- and low-risk sites. The density of mutations varied by location. The prevalence of NOTCH1 and FAT1 mutations in forearm, trunk, and leg skin was similar to that in keratinocyte cancers. Most mutations were caused by ultraviolet light, but mutational signature analysis suggested differences in DNA-repair processes between sites. Eleven mutant genes were under positive selection, with TP53 preferentially selected in the head and FAT1 in the leg. Fine-scale mapping revealed 10% of clones had copy-number alterations. Analysis of hair follicles showed mutations in the upper follicle resembled adjacent skin, but the lower follicle was sparsely mutated. Normal skin is a dense patchwork of mutant clones arising from competitive selection that varies by location. SIGNIFICANCE: Mapping mutant clones across the body reveals normal skin is a dense patchwork of mutant cells. The variation in cancer risk between sites substantially exceeds that in mutant clone density. More generally, mutant genes cannot be assigned as cancer drivers until their prevalence in normal tissue is known.See related commentary by De Dominici and DeGregori, p. 227.This article is highlighted in the In This Issue feature, p. 211.

People are essential to linking biodiversity data.

People are one of the best known and most stable entities in the biodiversity knowledge graph. The wealth of public information associated with people and the ability to identify them uniquely open up the possibility to make more use of these data in biodiversity science. Person data are almost always associated with entities such as specimens, molecular sequences, taxonomic names, observations, images, traits and publications. For example, the digitization and the aggregation of specimen data from museums and herbaria allow us to view a scientist's specimen collecting in conjunction with the whole corpus of their works. However, the metadata of these entities are also useful in validating data, integrating data across collections and institutional databases and can be the basis of future research into biodiversity and science. In addition, the ability to reliably credit collectors for their work has the potential to change the incentive structure to promote improved curation and maintenance of natural history collections.

Exploring the role of stromal osmoregulation in cancer and disease using executable modelling.

Osmotic regulation is a vital homoeostatic process in all cells and tissues. Cells initially respond to osmotic stresses by activating transmembrane transport proteins to move osmotically active ions. Disruption of ion and water transport is frequently observed in cellular transformations such as cancer. We report that genes involved in membrane transport are significantly deregulated in many cancers, and that their expression can distinguish cancer cells from normal cells with a high degree of accuracy. We present an executable model of osmotic regulation and membrane transport in mammalian cells, providing a mechanistic explanation for phenotype change in varied disease states, and accurately predicting behaviour from single cell expression data. We also predict key proteins involved in cellular transformation, SLC4A3 (AE3), and SLC9A1 (NHE1). Furthermore, we predict and verify a synergistic drug combination in vitro, of sodium and chloride channel inhibitors, which target the osmoregulatory network to reduce cancer-associated phenotypes in fibroblasts.

A toolbox for discrete modelling of cell signalling dynamics.

In an age where the volume of data regarding biological systems exceeds our ability to analyse it, many researchers are looking towards systems biology and computational modelling to help unravel the complexities of gene and protein regulatory networks. In particular, the use of discrete modelling allows generation of signalling networks in the absence of full quantitative descriptions of systems, which are necessary for ordinary differential equation (ODE) models. In order to make such techniques more accessible to mainstream researchers, tools such as the BioModelAnalyzer (BMA) have been developed to provide a user-friendly graphical interface for discrete modelling of biological systems. Here we use the BMA to build a library of discrete target functions of known canonical molecular interactions, translated from ordinary differential equations (ODEs). We then show that these BMA target functions can be used to reconstruct complex networks, which can correctly predict many known genetic perturbations. This new library supports the accessibility ethos behind the creation of BMA, providing a toolbox for the construction of complex cell signalling models without the need for extensive experience in computer programming or mathematical modelling, and allows for construction and simulation of complex biological systems with only small amounts of quantitative data.

Lung tumors with distinct p53 mutations respond similarly to p53 targeted therapy but exhibit genotype-specific statin sensitivity.

Lung adenocarcinoma accounts for ∼40% of lung cancers, the leading cause of cancer-related death worldwide, and current therapies provide only limited survival benefit. Approximately half of lung adenocarcinomas harbor mutations in TP53 (p53), making these mutants appealing targets for lung cancer therapy. As mutant p53 remains untargetable, mutant p53-dependent phenotypes represent alternative targeting opportunities, but the prevalence and therapeutic relevance of such effects (gain of function and dominant-negative activity) in lung adenocarcinoma are unclear. Through transcriptional and functional analysis of murine KrasG12D -p53null , -p53R172H (conformational), and -p53R270H (contact) mutant lung tumors, we identified genotype-independent and genotype-dependent therapeutic sensitivities. Unexpectedly, we found that wild-type p53 exerts a dominant tumor-suppressive effect on mutant tumors, as all genotypes were similarly sensitive to its restoration in vivo. These data show that the potential of p53 targeted therapies is comparable across all p53-deficient genotypes and may explain the high incidence of p53 loss of heterozygosity in mutant tumors. In contrast, mutant p53 gain of function and their associated vulnerabilities can vary according to mutation type. Notably, we identified a p53R270H -specific sensitivity to simvastatin in lung tumors, and the transcriptional signature that underlies this sensitivity was also present in human lung tumors, indicating that this therapeutic approach may be clinically relevant.