Publication highlights

In collaboration with GSK and the Crick-GSK LInkLabs we selected single-domain antibodies (dAbs) based on a human scaffold that recognise the catalytic domain of HOIP, a subunit of the multi-component E3 ligase LUBAC. We used these dAbs to interrogate the ubiquitin transfer mechanism of HOIP, and as crystallisation chaperones to crystallise a HOIP RBR/dAb complex. This complex now serves as a robust platform for soaking of ligands that target the active site cysteine of HOIP, thereby providing easy access to structure-based ligand design for this important class of E3 ligases.

The influenza HA is one of two glycoproteins on the surface of influenza virus and mediates receptor binding and membrane fusion during viral entry.In order to understand the function of HA in influenza infectivity it is necessary to understand the mechanism of endocytosis. It has previously been established that endocytosis involves a large conformational rearrangement of the HA protein that can be triggered by a change in pH, revealed by structures of initial and final states. Here, we directly image structural transformations in the HA at the pH of membrane fusion and solve the structure of three structural intermediates including a 150 Å-long triple-helical coiled coil of the HA2 transmembrane subunit. This was a long sought-after result and showed new, surprising concerted conformational rearrangements important to the membrane fusion mechanism.

Our previous work showed how stromal fibroblasts lead the collective invasion of cancer cells, and documented how remodelling of the extracellular matrix was important for this behaviour. Following our observation of direct cell-cell contacts between cancer cells and fibroblasts, we hypothesised that the two cells might be mechanically coupled; therefore, we began collaborating with Xavi Trepat (IBEC Barcelona), who is a world leader in the mechanics of multi-cellular systems. By biophysical measurements and a range of conventional cell and molecular biology manipulations, we demonstrated that fibroblasts actively ‘pull’ cancer cells into the surrounding extracellular matrix.

In this study, we used our bank of patient-dervied stromal fibroblasts to ask why some fibroblasts generate highly aligned extra-cellular matrices and other do not. We were able to show how cell migration and cell-cell collisions can dictate the patterns formed by fibroblasts, and that furthermore, the higher order organisation of fibroblasts and matrix is associated with millimetre scale contraction of reconstituted tissues and cancer invasion. The quantitative tool developed during the course of this work and a related study is now being tested for its prognostic value in simple histological stains of breast and prostate cancers.

We set up a complex model for lung alveoli by co-culturing lung fibroblasts and alveolar epithelial type I and type II cells on a gas permeable support, with the expectation that the fibroblasts would strongly influence the behaviour of cancer cells introduced into the system. However, we discovered that the largest effect came from the alveolar epithelial cells, and we then used a range of approaches to delineate the signalling mechanisms involved. This work, together with a concomitant study from the Malanchi group, established the role of epithelial cells in the tumour microenvironment of indolent and micro-metastases.

This work shows why stromal fibroblasts are an important source of inflammatory modulators in tumours. We show fibroblasts can respond to cGAMP produced by cancer cells, but only when the two cell types are in direct contact. This in turn promotes the STING and IRF3 dependent expression of interferon beta and various chemokines. The subsequent up-regulation of interferon-stimulated gene expression undermines the efficacy of oncolytic viruses. We propose the requirement for direct contact represents a ‘tissue level’ mechanism for triggering this response specifically in the context of tissue damage, as in healthy tissue, the basement membrane precludes such interactions.

This manuscript was the first demonstration that irreversible commitment to cellular differentiation during early development happens unexpectedly early. The paper reflects our interdisciplinary work, combining single cell imaging, mathematical modelling and -omics approaches. We discovered a new class of genes which we termed early commitment genes (ECG) that are responsible for the pluripotency-to-differentiation transition. It was also our first manuscript in developmental biology, a new field outside of our lab’s expertise.

Neuroprosthetics and neuroscience research alike are limited by the bandwidth of neural recording. At the same time, ever-more powerful silicon-based technology is ubiquitous in our phones, tablets and computers. Here we showed that progress in neural recording can be coupled to this by fusing bundles of microwires to pixel array chips, lifting silicon technology to the third dimension for deep brain recording.

The first publication from our group at the Crick comprises the development of a precision tool to understand O-GalNAc glycosylation, one of the most abundant and disease-relevant types of glycans. We apply the technique to run state-of-the-art methods of biology, including chemical glycoproteomics with the Proteomics STP and a genome-wise CRISPR screen with collaborators from Stanford. We also collaborate in-house with Vivian Li to apply the probe to imaging intestinal organoids.

In this paper, led by the Sanger Institute, we characterised global human genetic diversity in whole-genomes at a greater scale and with broader diversity than before. We documented complex divergence between modern humans and archaic groups such as Neanderthals. Lead author Anders Bergström started work on the project during PhD studies at the Sanger, and continued it with my input as a Crick postdoc from April 2018 until publication in 2020.

This first research paper led by our lab presents the first large-scale study of ancient genomes from early domestic dogs. We show that dogs were domesticated prior to the agricultural transition, with a dynamic history that includes collapse of early genetic diversity of dogs in Europe, and a complex evolution of genetic adaptation to starch rich diets.

This paper established the AHR induced feedback system of cytochrome P4501 metabolising enzymes as critical regulators of AHR signalling. Excessive ligand degradation via Cytochrome P4501 phenocopies AHR deficiency and has detrimental consequences for intestinal health which can be counterbalanced by increasing the intake of AHR ligands in the diet. The intestinal epithelium acts as gatekeeper for the supply of ligands throughout the body emphasising the importance of the gut barrier for whole body physiology.

A detailed, genome-wide study of the transcriptional response to DNA damage, including the identification of genes that start expressing short, alternative splice isoforms after genomic insult. One of these genes, ASCC3, normally expresses a protein-coding mRNA, but after UV-irradiation shifts expression to a short non-coding RNA isoform, which is important for survival after DNA damage. The protein-coding ASCC3 isoform counteracts the function of the non-coding isoform, indicating crosstalk between them.

This TRACERx work shows that bespoke patient-specific panels to analyse ctDNA can be used to monitor MRD recurrence and tumour branched evolution in the adjuvant setting in the absence of macroscopic disease, and that tumour Ki67 index, necrosis, squamous histology and FDG-PET avidity are closely associated with ctDNA release. We further demonstrate the limitations of ctDNA approaches for early detection as a function of tumour volume and cancer cell number, and show that the subclone identified in ctDNA prior to disease recurrence is identical to the tumour subclone identified at metastatic sites, permitting adjuvant MRD studies to prevent recurrence.

This work evaluates the relationship between intratumour heterogeneity of single nucleotide variants and somatic copy number aberrations and recurrence free survival in non-small cell lung cancer. Diversity of chromosome number or structure rather than single nucleotide variants is associated with poorer recurrence free survival, independent of tumour stage in multivariable analyses. Through subclonal copy number analyses, mirrored subclonal allelic imbalance is found, driving parallel evolution of chromosome copy number gains or losses on either the maternal or paternal chromosome in different regions of the same tumour.

Through an analysis of TRACERx, extended from our haplotyping analysis, we developed an algorithm called LOHHLA which infers allele specific copy number aberrations in HLA. We find HLA loss occurs in 40% of early stage lung cancers, usually as a subclonal event, and is permissive for branched evolution associated with expansion of mutations predicted to bind the lost HLA allele.

We demonstrated that mechanical forces and not enzymatic liberation are the physiological mechanism for acquisition of antigens by B cells from live presenting cells. Using DNA-based nanosensors we showed that B cell affinity discrimination is regulated by physical properties of the antigen-presenting cells and identified follicular dendritic cells as a stiff antigen presenting subset that promotes B cell affinity discrimination in germinal centres.

We demonstrate species-specific remodelling of red blood cells by the most virulent malaria-causing parasite, mediated by a species-specific expansion of an exported kinase family. Systematic deletion of all 20 members and quantitative phosphoproteomics identifies the kinase targets and supports their role in pathogenesis.

Unlike kinases, PPP-family phosphatases such as PP1 have little intrinsic specificity. PP1 acts in partnership with over 200 different PP1-interacting proteins, but it has remained unclear how they might confer sequence-specificity on PP1. We used proteomics to identify dozens of candidate Phactr1/PP1 substrates, and used structural and biochemical approaches to show that the Phactr1/PP1 holoenzyme is sequence-specific. Phactr1 binding reshapes the PP1 hydrophobic groove, thereby creating a novel composite hydrophobic surface for substrate recognition. This study explains how cofactors can enhance the reactivity of PP1 toward specific substrates, and suggests a way forward for the development of PP1 holoenzyme-specific inhibitors.

Fs-indels that escape the nonsense-mediated decay (NMD) pathway, can elicit anti-tumor immune responses, especially those the highly elongated neo open reading frames. NMD-escape fs-indels represent an attractive target for biomarker optimisation and immunotherapy design.