Researchers at the Crick are tackling the big questions about human health and disease, and new findings are published every week. Our faculty have picked some of the most significant papers published by Crick scientists, all of which are freely available thanks to our open science policy.
Functional cross-talk between allosteric effects of activating and inhibiting ligands underlies PKM2 regulation
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.
CD9 identifies pancreatic cancer stem cells and modulates glutamine metabolism to fuel tumour growth
This work identifies a cancer stem cell (CSC) population in pancreatic ductal adenocarcinoma (PDAC) marked by the tetraspanin CD9. We showed that CD9Hi CSCs are required for the epithelial and mesenchymal cellular heterogeneity seen in PDAC. We found that CD9 assembles a protein complex involved in regulating PDAC metabolism on the cell surface. CD9 depletion dramatically inhibited PDAC growth, identifying CD9 as a therapeutic PDAC target. These findings suggest that the cellular composition of pancreatic cancer is controlled by a CSC population.
Tissue curvature and apicobasal mechanical tension imbalance instruct cancer morphogenesis
This study introduces a new technique, FLASH, which enables immunostaining of whole organs for imaging and opens up the possibility of analysing a plethora of antigens and tissues that were previously impossible to study in 3D. By achieving this feat, we were able to study epithelial deformation from the moment of transformation within the intact pancreas, to show that early tumours adopt different shapes depending on tissue curvature, due to the distribution of intracellular forces. The work connects cell mechanics with the biology of tumour development in an unprecedented manner.
CDK phosphorylation of TRF2 controls t-loop dynamics during the cell cycle
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.
Patient-specific cancer genes contribute to recurrently perturbed pathways and establish therapeutic vulnerabilities in esophageal adenocarcinoma
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.
Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM
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.
RAC1P29S induces a mesenchymal phenotypic switch via serum response factor to promote melanoma development and therapy resistance
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
Development of combination therapies to maximize the impact of KRAS-G12C inhibitors in lung cancer
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 cell-size threshold limits cell polarity and asymmetric division potential
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.
Subcellular antibiotic visualization reveals a dynamic drug reservoir in infected macrophages
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.
LTR retroelement expansion of the human cancer transcriptome and immunopeptidome revealed by de novo transcript assembly
We assembled and disseminated the most complete, to date, transcriptome with a focus on transcripts initiated by or overlapping with endogenous retroelements. This assembly doubles the number of known transcripts and forms the basis for in-depth analysis of retroelement studies in health and disease, particularly in cancer. It also provided unconventional targets for novel cancer vaccines that are being developed by Enara Bio.
Transcriptional profiling unveils type I and II interferon networks in blood and tissues across diseases
Using advanced bioinformatics approaches, we deciphered the global transcriptional response in the lungs of mice infected or challenged with a broad spectrum of infectious pathogens, including parasites, bacteria, viruses, fungi, or allergens; we also determined to what extent each of these responses is preserved in the blood. We demonstrated a unique global transcriptional signature for each of the different diseases in both lung and blood. The lung transcriptional signatures showed a gradation, ranging from IFN-inducible gene clusters, to those associated with granulocyte/neutrophil/IL-17 dominated genes, to responses dominated by expression of genes encoding TH2 cytokines, mast cells and B cells.
Cellular geometry scaling ensures robust division site positioning
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.
Lysine harvesting is an antioxidant strategy and triggers underground polyamine metabolism
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.
Tissue clonality of dendritic cell subsets and emergency DCpoiesis revealed by multicolor fate mapping of DC progenitors
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.
Fragment-based covalent ligand screening enables rapid discovery of inhibitors for the RBR E3 ubiquitin ligase HOIP
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.
Published in Journal of the American Chemical Society
Structural basis for Fullerene geometry in a human endogenous retrovirus capsid
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.
Target-specific precision of CRISPR-mediated genome editing
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.
Maternal pluripotency factors initiate extensive chromatin remodelling to predefine first response to inductive signals
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.