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The rapid cell turnover of the intestinal epithelium is achieved from small numbers of stem cells located in the base of glandular crypts. These stem cells have been variously described as rapidly cycling or quiescent. A functional arrangement of stem cells that reconciles both of these behaviours has so far been difficult to obtain. Alternative explanations for quiescent cells have been that they act as a parallel or reserve population that replace rapidly cycling stem cells periodically or after injury; their exact nature remains unknown. Here we show mouse intestinal quiescent cells to be precursors that are committed to mature into differentiated secretory cells of the Paneth and enteroendocrine lineage. However, crucially we find that after intestinal injury they are capable of extensive proliferation and can give rise to clones comprising the main epithelial cell types. Thus, quiescent cells can be recalled to the stem-cell state. These findings establish quiescent cells as an effective clonogenic reserve and provide a motivation for investigating their role in pathologies such as colorectal cancers and intestinal inflammation. A new method to trace the lineage of slow cycling label-retaining cells (LRCs) in vivo identifies a population of LRCs that have features of committed Paneth cells but still express stem-cell markers such as Lgr5; the slow cycling cells differentiate into Paneth cells without cell division, but after injury can also repopulate the stem-cell niche and contribute to the regeneration of all intestinal lineages. Quiescent cells in intestinal epithelium Whether slowly cycling or quiescent stem cells co-exist with more rapidly cycling stem cells in the intestinal crypt is a matter of intense debate. Using a new method to trace the lineage of slowly cycling label-retaining cells (LRCs) in vivo , Douglas Winton and colleagues have identified a population of LRCs that has features of committed Paneth cells but still expresses stem cell markers such as Lgr5. The slowly cycling cells differentiate into Paneth cells without cell division, but in response to injury, they can also repopulate the stem cell niche and contribute to the regeneration of the all intestinal lineages. This work suggests that quiescent cells do act as a clonogenic reserve that could play a part in the pathology of intestinal cancers and inflammation.

Lynch Syndrome (LS) is an autosomal dominant disease conferring a high risk of colorectal cancer due to germline heterozygous mutations in a DNA mismatch repair (MMR) gene. Although cancers in LS patients show elevated somatic mutation burdens, information on mutation rates in normal tissues and understanding of the trajectory from normal to cancer cell is limited. Here we whole genome sequence 152 crypts from normal and neoplastic epithelial tissues from 10 LS patients. In normal tissues the repertoire of mutational processes and mutation rates is similar to that found in wild type individuals. A morphologically normal colonic crypt with an increased mutation burden and MMR deficiency-associated mutational signatures is identified, which may represent a very early stage of LS pathogenesis. Phylogenetic trees of tumour crypts indicate that the most recent ancestor cell of each tumour is already MMR deficient and has experienced multiple cycles of clonal evolution. This study demonstrates the genomic stability of epithelial cells with heterozygous germline MMR gene mutations and highlights important differences in the pathogenesis of LS from other colorectal cancer predisposition syndromes. It is unclear whether somatic mutation rates are elevated in Lynch Syndrome (LS), which is the most common cause of hereditary colorectal cancer. Here, the authors use whole-genome sequencing and organoid cultures to show that normal tissues in LS patients are genomically stable, while ancestor cells of neoplastic tissues undergo multiple cycles of clonal evolution.

Cytosine hydroxymethylation (5hmC) in mammalian DNA is the product of oxidation of methylated cytosines (5mC) by Ten-Eleven-Translocation (TET) enzymes. While it has been shown that the TETs influence 5mC metabolism, pluripotency and differentiation during early embryonic development, the functional relationship between gene expression and 5hmC in adult (somatic) stem cell differentiation is still unknown. Here we report that 5hmC levels undergo highly dynamic changes during adult stem cell differentiation from intestinal progenitors to differentiated intestinal epithelium. We profiled 5hmC and gene activity in purified mouse intestinal progenitors and differentiated progeny to identify 43425 differentially hydroxymethylated regions and 5325 differentially expressed genes. These differentially marked regions showed both losses and gains of 5hmC after differentiation, despite lower global levels of 5hmC in progenitor cells. In progenitors, 5hmC did not correlate with gene transcript levels, however, upon differentiation the global increase in 5hmC content showed an overall positive correlation with gene expression level as well as prominent associations with histone modifications that typify active genes and enhancer elements. Our data support a gene regulatory role for 5hmC that is predominant over its role in controlling DNA methylation states.

Cellular DNA damage caused by reactive oxygen species is repaired by the base excision repair (BER) pathway which includes the DNA glycosylase MUTYH. Inherited biallelic MUTYH mutations cause predisposition to colorectal adenomas and carcinoma. However, the mechanistic progression from germline MUTYH mutations to MUTYH-Associated Polyposis (MAP) is incompletely understood. Here, we sequence normal tissue DNAs from 10 individuals with MAP. Somatic base substitution mutation rates in intestinal epithelial cells were elevated 2 to 4-fold in all individuals, except for one showing a 31-fold increase, and were also increased in other tissues. The increased mutation burdens were of multiple mutational signatures characterised by C > A changes. Different mutation rates and signatures between individuals are likely due to different MUTYH mutations or additional inherited mutations in other BER pathway genes. The elevated base substitution rate in normal cells likely accounts for the predisposition to neoplasia in MAP. Despite ubiquitously elevated mutation rates, individuals with MAP do not display overt evidence of premature ageing. Thus, accumulation of somatic mutations may not be sufficient to cause the global organismal functional decline of ageing. Inherited mutations in MUTYH have been shown to predispose patients to colorectal cancers. Here, the authors show that MUTYH mutations lead to an increased somatic base substitution mutation rate in normal intestinal epithelial cells, which is the likely cause for the increased cancer risk.

An immunosuppressive microenvironment causes poor tumor T cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumors is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identified complement receptor C5aR1 as a druggable target, which when inhibited improved radiotherapy, even in tumors displaying immunosuppressive features and poor CD8+ T cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we found that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumor cell-specific functions. C5aR1 targeting resulted in increased NF-κB-dependent apoptosis specifically in tumors and not normal tissues, indicating that, in malignant cells, C5aR1 primarily regulated cell fate. Collectively, these data revealed that increased complement gene expression is part of the stress response mounted by irradiated tumors and that targeting C5aR1 could improve radiotherapy, even in tumors displaying immunosuppressive features.

Prevention of SARS-CoV-2 entry in cells through the modulation of viral host receptors, such as ACE2, could represent a new therapeutic approach complementing vaccination. However, the mechanisms controlling ACE2 expression remain elusive. Here, we identify the farnesoid X receptor (FXR) as a direct regulator of ACE2 transcription in multiple COVID19-affected tissues, including the gastrointestinal and respiratory systems. We demonstrate that FXR antagonists, including the over-the-counter compound z-guggulsterone (ZGG) and the off-patent drug ursodeoxycholic acid (UDCA), downregulate ACE2 levels, and reduce susceptibility to SARS-CoV-2 infection in lung, cholangiocyte and gut organoids. We then show that therapeutic levels of UDCA downregulate ACE2 in human organs perfused ex situ and reduce SARS-CoV-2 infection ex vivo. Finally, we perform a retrospective study using registry data and identify a correlation between UDCA treatment and positive clinical outcomes following SARS-CoV-2 infection, including hospitalisation, ICU admission and death. In conclusion, we identify a novel function of FXR in controlling ACE2 expression and provide evidence that this approach could be beneficial for reducing SARS-CoV-2 infection, thereby paving the road for future clinical trials. Competing Interest Statement F.S., L.V. and K.S.-P. are founders and shareholders of Bilitech LTD. L.V. is a founder and shareholder of DEFINIGEN. The remaining authors have no competing interests to disclose.

An immunosuppressive microenvironment causes poor tumour T-cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumours is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identify complement receptor C5aR1 as a druggable target which when inhibited improves radiotherapy even in tumours displaying immunosuppressive features and poor CD8+ T-cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we find that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumour-cell specific functions. C5aR1 targeting results in increased NF-κB-dependent apoptosis specifically in tumours and not normal tissues; indicating that in malignant cells, C5aR1 primarily regulates cell fate. Collectively, these data reveal that increased complement gene expression is part of the stress response mounted by irradiated tumours and that targeting C5aR1 can improve radiotherapy even in tumours displaying immunosuppressive features.Competing Interest StatementProf Woodruff has previously consulted to Alsonex Pty Ltd, who are commercially developing PMX205. He holds no stocks, shares or other commercial interest in this company.

Inflammatory intestinal injury, which may occur following cytotoxic cancer treatments such as radiotherapy, compromises homeostasis when intestinal stem cells undergo apoptosis and regeneration programmes are not effectively engaged. Recent evidence indicates the importance of macrophages in regulating stem regeneration following injury, but how to therapeutically promote specific macrophage subtypes which may tip the balance away from apoptosis and towards regeneration is still unclear. Here we show that targeting complement C5a receptor 1 (C5aR1) reduces radiation-induced intestinal injury by promoting macrophage phenotypes which reduce intestinal stem cell apoptosis. Specifically, C5aR1 signalling attenuates a macrophage maturation response characterised by increased IL10 and CX3CR1 expression. Our data indicate that increased IL10 signalling, and macrophage maturation induced following C5aR1 targeting are important for regulating crypt cell loss via radiotherapy-induced apoptosis. Interestingly, IL10-signalling is also critical for the improved tumour radiation response observed following C5aR1 targeting.

Cancer is a disease in which cells accumulate genetic aberrations that are believed to confer a clonal advantage over cells in the surrounding tissue. However, the quantitative benefit of frequently occurring mutations during tumor development remains unknown. We quantified the competitive advantage of Ape loss, Kras activation, and P53 mutations in the mouse intestine. Our findings indicate that the fate conferred by these mutations is not deterministic, and many mutated stem cells are replaced by wild-type stem cells after biased, but still stochastic events. Furthermore, P53 mutations display a condition-dependent advantage, and especially in colitis-affected intestines, clones harboring mutations in this gene are favored. Our work confirms the previously theoretical notion that the tissue architecture of the intestine suppresses the accumulation of mutated lineages.