Publication highlights

This work reveals that amino acids, rather than fructose 1,6-bisphosphate, are the relevant cellular regulators of pyruvate kinase M2 (PKM2), a critical node in cancer metabolism. It further elucidates the molecular mechanism of PKM2 regulation by amino acids with a new algorithm that predicts allosteric pathways in proteins, a major and difficult problem in structural biology.

Evidence suggested that the telomere adopts a lasso-like t-loop configuration, which safeguards chromosome ends from being recognised as DNA double strand breaks. However, the regulation and physiological importance of t-loops in end-protection was uncertain. This study uncovered a phospho-switch in TRF2 that coordinates the timely assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response. These results were the first to definitively establish the t-loop as a physiologically important structure required to suppress checkpoint activation at telomere ends.

Oesophageal adenocarcinoma shows high genetic heterogeneity making the identification of cancer drivers challenging. We developed a machine learning algorithm to identify cancer drivers in 261 oesophageal adenocarcinomas. Although most predicted drivers were rare or patient-specific, they all perturbed well-known cancer pathways. Using the recurrence of the same pathway perturbations rather than individual genes, we stratified patients into six groups different for their clinical features. We validated experimentally the contribution of these genes to disease progression and revealed acquired dependencies exploitable in therapy. This study described a new way to identify cancer drivers that we have recently further developed for application in precision oncology.

The MCM replicative helicase is loaded onto duplex DNA as a double hexamer. Here we use time-resolved cryo-EM to show that ORC binds to its high affinity binding site to load the first MCM hexamer. ORC then releases this site and it, or another ORC molecule then binds the B2 element, which contains a degenerate ORC binding site. This binding is stabilised by a novel interaction between the Orc6 subunit of ORC and the N-terminus of the MCM hexamer. ORC then recruits and loads the second hexamer by the same mechanism as the first hexamer. We employed newly developed in silico reconstitution approaches to describe the full context of the helicase loading reaction, studied on a near-native, chromatinised origin of replication. This study radically changes our approach to investigating chromosome replication with cryo-EM.

Metastatic melanoma is a lethal disease, in part because of rapid acquisition of resistance to therapy. Using genetically engineered mouse models, we demonstrate that the activating RAC1 P29S mutation, present in up to 5% of melanoma patients, cooperates with BRAF as a driver of melanoma initiation and promotes BRAF inhibitor resistance. The critical RAC1 effector pathway in melanoma is shown to be the transcription factor complex SRF/MRTF, which initiates a switch to a mesenchymal-like state characterized by therapy resistance. Therapeutic targeting of SRF/MRTF may have potential to reverse BRAF inhibitor resistance in melanoma patients bearing the oncogenic RAC1 P29S mutation

KRAS is the most commonly mutated oncogene in human lung cancer, but direct targeting of RAS proteins has proved difficult. A recently developed inhibitor of G12C mutant KRAS protein inhibits lung cancer progression in mouse models but does not provide durable regressions. By studying signalling pathways required for survival of KRAS mutant cells, we demonstrate a strong and selective potentiation of the effects of G12C KRAS inhibitors when mTOR and/or IGF1R are also inhibited. Using mutant specific G12C KRAS inhibitors rather than MEK inhibitors in these combinations is associated with greater specificity and lower toxicity. We propose that adding IGF1R and mTOR inhibitors will increase the impact of G12C KRAS inhibitors in clinical trials.

A key requirement for patterning networks is that the scale of pattern be appropriately matched to the size of the system to be patterned. Through a combination of theory and experiment, we show that failure of the PAR network to scale with cell size restricts stable cell polarity to a specific size range and imposes a minimum cell size threshold for polarity. Experimental alteration of cell size indicates that embryos are sensitive to this size threshold. We thus propose a general strategy by which cells can use intrinsic length scales of patterning networks to enable size-dependent decision making.

Improving chemotherapies against intracellular pathogens requires an understanding of how antibiotic distribution within infected cells affects efficacy. In this work, we developed an approach to visualise antibiotics in human macrophages infected with the tubercle bacillus. We showed that the antitubercular (anti-TB) drug bedaquiline accumulated in host lipid droplets, which seemed to act as an antibiotic reservoir that could be transferred to bacteria during host lipid consumption. Indeed, alterations in host lipid droplet content affected the anti-TB activity of bedaquiline against intracellular bacilli.

Here we describe our discovery that preservation of specific cellular geometry across a range of cell sizes is essential for correct division site positioning and survival, demonstrating the organismal-level function for scaling.

We report a new and powerful metabolic anti-stress mechanism, ‘Lysine harvesting’, that protects microbial cells in stress situations. We noticed that extracellular lysine is taken up to reach concentrations up to 100x higher than those required for growth. Uptake is dependent on the polyamine pathway, connected via promiscuous metabolic reactions, and triggers a reprogramming of redox metabolism: NADPH is channelled into glutathione metabolism, leading to a large increase in glutathione, lower levels of reactive oxygen species and increased oxidant tolerance. Therefore, nutrient uptake occurs not only to enable cell growth, but allows cells to reconfigure their metabolism to preventatively mount stress protection.

Conventional dendritic cells (cDCs) originate from a committed precursor in bone marrow (BM) that exits via the blood as a pre-cDC to seed tissues with the cDC1 and cDC2 subsets. We used a multi-colour genetic tracing mouse model to analyse colonisation of tissues by pre-cDC. We found that cDCs in tissues comprise clones mostly composed of a single cDC subset and that ‘flu infection causes an efflux of pre-cDCs from BM and influx into the lungs. The latter finding indicates that cDCpoiesis is responsive to emergency need, which suggests previously undiscovered communication between tissues and cDC progenitors in BM.

Protein ubiquitination is a key regulatory mechanism and E3 ubiquitin ligases are the key mediators of ubiquitination providing specificity to the process. In this study we describe the application of fragment-based covalent ligand screening to target the active site of an E3 ubiquitin ligase (HOIP) for which previously no specific inhibitors were known. Combining chemical biology, X-ray crystallography, chemoproteomics and cell biology we were able to identify a covalent binder for HOIP that now forms the basis for further inhibitor development. This study illustrates more generally the power of fragment-based covalent ligand screening to identify lead compounds against challenging targets.

Here we determined the structure by single particle cryo-EM of capsid assembly in an endogenous retrovirus. This is the first atomic resolution structure of a closed capsid shell, which in retroviruses packages and protects the genome. By studying 4 different types of symmetric assemblies, we discovered how the underlying Fullerene geometry is achieved by the CA protein forming both pentamers and hexamers and found structural rules by which invariant pentamers and structurally plastic hexamers associate to form the unique polyhedral structures.

Taking advantage of our sgRNA library, we performed a large-scale characterisation of CRISPR-induced in/del patterns and discovered that Cas9-induced double strand breaks are repaired in a predictable or unpredictable way, depending on the target site. These findings provided the broad scientific community with guidelines for a more effective and safer use of CRISPR technology, with important implications for clinical applications. They also revealed a striking influence of DNA sequence in dictating DSB repair outcomes and laid the foundation for future mechanistic studies that can increase our understanding of end-joining processes in human cells.

This paper, and another published the same year (Gentsch GE et al. (2019) iScience, 16, 485-498), analysed gene activation in the early Xenopus embryo and asked what causes cells to become competent to respond to Wnt, Nodal and BMP signalling. We identified regulatory DNA sequences associated with early-expressed genes, and deduced from them that the maternal pluripotency factors Pou5f3 and Sox3 remodel compacted chromatin before the onset of inductive signalling. This remodelling includes the opening and marking of thousands of regulatory elements, extensive chromatin looping, and the co-recruitment of signal-mediating transcription factors. Our work informs our understanding of how pluripotent stem cells interpret inductive signals.

SCAF4 and SCAF8 were isolated in the mid-90s as proteins that bind the RNAPII C-terminal repeat domain (CTD) but little or nothing was known about their cellular function. This paper describes their function as the first eukaryotic mRNA anti-terminator proteins. Together, SCAF4 and SCAF8 coordinate the transition between elongation and termination, ensuring correct polyA site selection and RNAPII transcriptional termination in human cells.