Discovery of the Potent and Selective ATR Inhibitor Camonsertib (RP-3500).

ATR is a key kinase in the DNA-damage response (DDR) that is synthetic lethal with several other DDR proteins, making it an attractive target for the treatment of genetically selected solid tumors. Herein we describe the discovery of a novel ATR inhibitor guided by a pharmacophore model to position a key hydrogen bond. Optimization was driven by potency and selectivity over the related kinase mTOR, resulting in the identification of camonsertib (RP-3500) with high potency and excellent ADME properties. Preclinical evaluation focused on the impact of camonsertib on myelosuppression, and an exploration of intermittent dosing schedules to allow recovery of the erythroid compartment and mitigate anemia. Camonsertib is currently undergoing clinical evaluation both as a single agent and in combination with talazoparib, olaparib, niraparib, lunresertib, or gemcitabine (NCT04497116, NCT04972110, NCT04855656). A preliminary recommended phase 2 dose for monotherapy was identified as 160 mg QD given 3 days/week.

Iron chelation improves ineffective erythropoiesis and iron overload in myelodysplastic syndrome mice.

Myelodysplastic syndrome (MDS) is a heterogeneous group of bone marrow stem cell disorders characterized by ineffective hematopoiesis and cytopenias, most commonly anemia. Red cell transfusion therapy for anemia in MDS results in iron overload, correlating with reduced overall survival. Whether the treatment of iron overload benefits MDS patients remains controversial. We evaluate underlying iron-related pathophysiology and the effect of iron chelation using deferiprone on erythropoiesis in NUP98-HOXD13 transgenic mice, a highly penetrant well-established MDS mouse model. Our results characterize an iron overload phenotype with aberrant erythropoiesis in these mice which was reversed by deferiprone-treatment. Serum erythropoietin levels decreased while erythroblast erythropoietin receptor expression increased in deferiprone-treated MDS mice. We demonstrate, for the first time, normalized expression of the iron chaperones Pcbp1 and Ncoa4 and increased ferritin stores in late-stage erythroblasts from deferiprone-treated MDS mice, evidence of aberrant iron trafficking in MDS erythroblasts. Importantly, erythroblast ferritin is increased in response to deferiprone, correlating with decreased erythroblast ROS. Finally, we confirmed increased expression of genes involved in iron uptake, sensing, and trafficking in stem and progenitor cells from MDS patients. Taken together, our findings provide evidence that erythroblast-specific iron metabolism is a novel potential therapeutic target to reverse ineffective erythropoiesis in MDS.

A subpopulation of lipogenic brown adipocytes drives thermogenic memory.

Sustained responses to transient environmental stimuli are important for survival. The mechanisms underlying long-term adaptations to temporary shifts in abiotic factors remain incompletely understood. Here, we find that transient cold exposure leads to sustained transcriptional and metabolic adaptations in brown adipose tissue, which improve thermogenic responses to secondary cold encounter. Primary thermogenic challenge triggers the delayed induction of a lipid biosynthesis programme even after cessation of the original stimulus, which protects from subsequent exposures. Single-nucleus RNA sequencing and spatial transcriptomics reveal that this response is driven by a lipogenic subpopulation of brown adipocytes localized along the perimeter of Ucp1hi adipocytes. This lipogenic programme is associated with the production of acylcarnitines, and supplementation of acylcarnitines is sufficient to recapitulate improved secondary cold responses. Overall, our data highlight the importance of heterogenous brown adipocyte populations for 'thermogenic memory', which may have therapeutic implications for leveraging short-term thermogenesis to counteract obesity.

Single-cell analysis and spatial resolution of the gut microbiome.

Over the past decade it has become clear that various aspects of host physiology, metabolism, and immunity are intimately associated with the microbiome and its interactions with the host. Specifically, the gut microbiome composition and function has been shown to play a critical role in the etiology of different intestinal and extra-intestinal diseases. While attempts to identify a common pattern of microbial dysbiosis linked with these diseases have failed, multiple studies show that bacterial communities in the gut are spatially organized and that disrupted spatial organization of the gut microbiome is often a common underlying feature of disease pathogenesis. As a result, focus over the last few years has shifted from analyzing the diversity of gut microbiome by sequencing of the entire microbial community, towards understanding the gut microbiome in spatial context. Defining the composition and spatial heterogeneity of the microbiome is critical to facilitate further understanding of the gut microbiome ecology. Development in single cell genomics approach has advanced our understanding of microbial community structure, however, limitations in approaches exist. Single cell genomics is a very powerful and rapidly growing field, primarily used to identify the genetic composition of microbes. A major challenge is to isolate single cells for genomic analyses. This review summarizes the different approaches to study microbial genomes at single-cell resolution. We will review new techniques for microbial single cell sequencing and summarize how these techniques can be applied broadly to answer many questions related to the microbiome composition and spatial heterogeneity. These methods can be used to fill the gaps in our understanding of microbial communities.

Hierarchical contribution of individual lifestyle factors and their interactions on adenomatous and serrated polyp risk.

BACKGROUND: Individual colorectal polyp risk factors are well characterized; however, insights into their pathway-specific interactions are scarce. We aimed to identify the impact of individual risk factors and their joint effects on adenomatous (AP) and serrated polyp (SP) risk. METHODS: We collected information on 363 lifestyle and metabolic parameters from 1597 colonoscopy participants, resulting in over 521,000 data points. We used multivariate statistics and machine-learning approaches to assess associations of single variables and their interactions with AP and SP risk. RESULTS: Individual factors and their interactions showed common and polyp subtype-specific effects. Abdominal obesity, high body mass index (BMI), metabolic syndrome, and red meat consumption globally increased polyp risk. Age, gender, and western diet associated with AP risk, while smoking was associated with SP risk. CRC family history was associated with advanced adenomas and diabetes with sessile serrated lesions. Regarding lifestyle factor interactions, no lifestyle or dietary adjustments mitigated the adverse smoking effect on SP risk, whereas its negative effect was exacerbated by alcohol in the conventional pathway. The adverse effect of red meat on SP risk was not ameliorated by any factor, but was further exacerbated by western diet along the conventional pathway. No modification of any factor reduced the negative impact of metabolic syndrome on AP risk, whereas increased fatless fish or meat substitutes' intake mitigated its effect on SP risk. CONCLUSIONS: Individual risk factors and their interactions for polyp formation along the adenomatous and serrated pathways are strongly heterogeneous. Our findings may facilitate tailored lifestyle recommendations and contribute to a better understanding of how risk factor combinations impact colorectal carcinogenesis.

The enteric nervous system relays psychological stress to intestinal inflammation.

Mental health profoundly impacts inflammatory responses in the body. This is particularly apparent in inflammatory bowel disease (IBD), in which psychological stress is associated with exacerbated disease flares. Here, we discover a critical role for the enteric nervous system (ENS) in mediating the aggravating effect of chronic stress on intestinal inflammation. We find that chronically elevated levels of glucocorticoids drive the generation of an inflammatory subset of enteric glia that promotes monocyte- and TNF-mediated inflammation via CSF1. Additionally, glucocorticoids cause transcriptional immaturity in enteric neurons, acetylcholine deficiency, and dysmotility via TGF-ß2. We verify the connection between the psychological state, intestinal inflammation, and dysmotility in three cohorts of IBD patients. Together, these findings offer a mechanistic explanation for the impact of the brain on peripheral inflammation, define the ENS as a relay between psychological stress and gut inflammation, and suggest that stress management could serve as a valuable component of IBD care.

Preterm birth is associated with xenobiotics and predicted by the vaginal metabolome.

Spontaneous preterm birth (sPTB) is a leading cause of maternal and neonatal morbidity and mortality, yet its prevention and early risk stratification are limited. Previous investigations have suggested that vaginal microbes and metabolites may be implicated in sPTB. Here we performed untargeted metabolomics on 232 second-trimester vaginal samples, 80 from pregnancies ending preterm. We find multiple associations between vaginal metabolites and subsequent preterm birth, and propose that several of these metabolites, including diethanolamine and ethyl glucoside, are exogenous. We observe associations between the metabolome and microbiome profiles previously obtained using 16S ribosomal RNA amplicon sequencing, including correlations between bacteria considered suboptimal, such as Gardnerella vaginalis, and metabolites enriched in term pregnancies, such as tyramine. We investigate these associations using metabolic models. We use machine learning models to predict sPTB risk from metabolite levels, weeks to months before birth, with good accuracy (area under receiver operating characteristic curve of 0.78). These models, which we validate using two external cohorts, are more accurate than microbiome-based and maternal covariates-based models (area under receiver operating characteristic curve of 0.55-0.59). Our results demonstrate the potential of vaginal metabolites as early biomarkers of sPTB and highlight exogenous exposures as potential risk factors for prematurity.

A microbiome-dependent gut-brain pathway regulates motivation for exercise.

Exercise exerts a wide range of beneficial effects for healthy physiology1. However, the mechanisms regulating an individual's motivation to engage in physical activity remain incompletely understood. An important factor stimulating the engagement in both competitive and recreational exercise is the motivating pleasure derived from prolonged physical activity, which is triggered by exercise-induced neurochemical changes in the brain. Here, we report on the discovery of a gut-brain connection in mice that enhances exercise performance by augmenting dopamine signalling during physical activity. We find that microbiome-dependent production of endocannabinoid metabolites in the gut stimulates the activity of TRPV1-expressing sensory neurons and thereby elevates dopamine levels in the ventral striatum during exercise. Stimulation of this pathway improves running performance, whereas microbiome depletion, peripheral endocannabinoid receptor inhibition, ablation of spinal afferent neurons or dopamine blockade abrogate exercise capacity. These findings indicate that the rewarding properties of exercise are influenced by gut-derived interoceptive circuits and provide a microbiome-dependent explanation for interindividual variability in exercise performance. Our study also suggests that interoceptomimetic molecules that stimulate the transmission of gut-derived signals to the brain may enhance the motivation for exercise.

Ketogenic Diet and Beta-Hydroxybutyrate in Colorectal Cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death in the United States. Although certain genetic predispositions may contribute to one's risk for developing CRC, dietary and lifestyle factors may play an important role as well. In a recent study in Nature, Dmitrieva-Posocco et al, reveal a potential protective role of the ketogenic diet in colorectal cancer growth and progression. Administration of a ketogenic diet to CRC-bearing mice demonstrated a tumor-suppressive effect. Specifically, the ketone body ß-hydroxybutyrate (BHB) exhibited the ability to suppress epithelial cell proliferation and inhibit tumor growth. BHB acts on cancer cells through regulation of homeodomain-only protein Hopx, known regulator of CRC. Furthermore, BHB requires a surface receptor Hcar to induce Hopx expression and suppress proliferation of intestinal epithelial cells. Taken together, these results describe a new therapeutic approach of using dietary intervention for the prevention and treatment of colorectal cancer.

Sequential addition of neuronal stem cell temporal cohorts generates a feed-forward circuit in the Drosophila larval nerve cord.

How circuits self-assemble starting from neuronal stem cells is a fundamental question in developmental neurobiology. Here, we addressed how neurons from different stem cell lineages wire with each other to form a specific circuit motif. In Drosophila larvae, we combined developmental genetics (twin-spot mosaic analysis with a repressible cell marker, multi-color flip out, permanent labeling) with circuit analysis (calcium imaging, connectomics, network science). For many lineages, neuronal progeny are organized into subunits called temporal cohorts. Temporal cohorts are subsets of neurons born within a tight time window that have shared circuit-level function. We find sharp transitions in patterns of input connectivity at temporal cohort boundaries. In addition, we identify a feed-forward circuit that encodes the onset of vibration stimuli. This feed-forward circuit is assembled by preferential connectivity between temporal cohorts from different lineages. Connectivity does not follow the often-cited early-to-early, late-to-late model. Instead, the circuit is formed by sequential addition of temporal cohorts from different lineages, with circuit output neurons born before circuit input neurons. Further, we generate new tools for the fly community. Our data raise the possibility that sequential addition of neurons (with outputs oldest and inputs youngest) could be one fundamental strategy for assembling feed-forward circuits.

The nervous system of an animal consists of complex arrangements of nerve cells or neurons. These arrangements are called neuronal circuits, and they contain both input and output neurons. Input neurons sense signals, such as external cues, and output neurons pass these signals on to the brain, for example. The nerve cells in a circuit connect to each other through so-called synapses in specific patterns. Neuronal circuits first assemble during the development of an animal. The assembly process starts when a nerve stem cell divides and gives rise to more specialized neurons, its progeny. A lot of what we know about neuronal circuit assembly comes from studying the nerve cord of fruit fly larva, which shares many features with the spinal cord of vertebrates. Previous studies had used experimental techniques to trace, or follow, the fate of the progeny of specific nerve stem cells. These approaches provided information about which nerve stem cells contribute to which neuronal circuits. However, major questions in developmental neurobiology remain about how exactly these neuronal circuits assemble. For example, it was not clear in what order input and output neurons build a circuit. Here Wang, Wreden et al. took a different approach by starting with a specific circuit in the fruit fly nerve cord ­ a circuit that detects vibrations ­ and looking for the stem cells contributing to that circuit. Using a number of techniques, Wang, Wreden et al. determined when particular nerve cells were 'born', what they looked like, and what other nerve cells they formed synapses with. Although nerve stem cells gave rise to many different neurons during development, those neurons did not change gradually over time. Instead, neurons were born in short bursts, and those in the same 'temporal cohort' were similar to each other, while neurons in different cohorts were different. The neuronal circuit that detects vibrations assembled itself from three temporal cohorts of neurons coming from different stem cells. The output neurons, which send information from the nerve cord to the brain, were born before the input neurons, which detect vibrations in the surroundings. All in all, these experiments offer more detailed insights into how neuronal circuits assemble during development. The study also provides experimental resources for other scientists working with fruit flies, and poses new research questions for developmental biologists studying vertebrates.

Second trimester short cervix is associated with decreased abundance of cervicovaginal lipid metabolites.

BACKGROUND: A short cervix is a risk factor for preterm birth. The molecular drivers of a short cervix remain elusive. Metabolites may function as mediators of pathologic processes. OBJECTIVE: We sought to determine if a distinct cervicovaginal metabolomic profile is associated with a short cervix (<25 mm) to unveil the potential mechanisms by which premature cervical remodeling leads to a short cervix. STUDY DESIGN: This was a secondary analysis of a completed prospective pregnancy cohort. Cervicovaginal fluid was obtained between 20 and 24 weeks' gestation. The participants selected for metabolomic profiling were frequency-matched by birth outcome and cervicovaginal microbiota profile. This analysis included 222 participants with cervical length measured. A short cervix was defined as one having length <25 mm, as measured by transvaginal ultrasound. Unpaired t-tests were performed with a Bonferroni correction for multiple comparisons. RESULTS: There were 27 participants with a short cervix, and 195 with normal cervical length. Of the 637 metabolites detected, 26 differed between those with a short cervix and those with normal cervical lengths; 22 were decreased, of which 21 belonged to the lipid metabolism pathway (all P<.000079). Diethanolamine, erythritol, progesterone, and mannitol or sorbitol were increased in the cases of short cervix. Among participants with Lactobacillus-deficient microbiota, only diethanolamine and mannitol or sorbitol differed between short cervix (n=17) and normal cervical length (n=75), both increased. CONCLUSION: A short cervix is associated with decreased cervicovaginal lipid metabolites, particularly sphingolipids. This class of lipids stabilizes cell membranes and protects against environmental exposures. Increased diethanolamine-an immunostimulatory xenobiotic-is associated with a short cervix. These observations begin to identify the potential mechanisms by which modifiable environmental factors may invoke cell damage in the setting of biological vulnerability, thus promoting premature cervical remodeling in spontaneous preterm birth.

ß-Hydroxybutyrate suppresses colorectal cancer.

Colorectal cancer (CRC) is among the most frequent forms of cancer, and new strategies for its prevention and therapy are urgently needed1. Here we identify a metabolite signalling pathway that provides actionable insights towards this goal. We perform a dietary screen in autochthonous animal models of CRC and find that ketogenic diets exhibit a strong tumour-inhibitory effect. These properties of ketogenic diets are recapitulated by the ketone body ß-hydroxybutyrate (BHB), which reduces the proliferation of colonic crypt cells and potently suppresses intestinal tumour growth. We find that BHB acts through the surface receptor Hcar2 and induces the transcriptional regulator Hopx, thereby altering gene expression and inhibiting cell proliferation. Cancer organoid assays and single-cell RNA sequencing of biopsies from patients with CRC provide evidence that elevated BHB levels and active HOPX are associated with reduced intestinal epithelial proliferation in humans. This study thus identifies a BHB-triggered pathway regulating intestinal tumorigenesis and indicates that oral or systemic interventions with a single metabolite may complement current prevention and treatment strategies for CRC.

A non-optimal cervicovaginal microbiota in pregnancy is associated with a distinct metabolomic signature among non-Hispanic Black individuals.

Biomechanical and molecular processes of premature cervical remodeling preceding spontaneous preterm birth (sPTB) likely result from interactions between the cervicovaginal microbiota and host immune responses. A non-optimal cervicovaginal microbiota confers increased risk of sPTB. The cervicovaginal space is metabolically active in pregancy; microbiota can produce, modify, and degrade metabolites within this ecosystem. We establish that cervicovaginal metabolomic output clusters by microbial community in pregnancy among Black individuals, revealing increased metabolism within the amino acid and dipeptide pathways as hallmarks of a non-optimal microbiota. Few differences were detected in metabolomic profiles when stratified by birth outcome. The study raises the possibility that metabolites could distinguish women with greater risk of sPTB among those with similar cervicovaginal microbiota, and that metabolites within the amino acid and carbohydrate pathways may play a role in this distinction.

Microbial memories.

The microbiota impedes pathogen invasion of the intestinal ecosystem, a phenomenon termed colonization resistance. In an upcoming issue of Cell, Stacy et al. describe host-initiated metabolite pathways that functionally alter the microbiota after primary infection, thereby augmenting colonization resistance to subsequent infection.

Weak Microbial Metabolites: a Treasure Trove for Using Biomimicry to Discover and Optimize Drugs.

For decades, traditional drug discovery has used natural product and synthetic chemistry approaches to generate libraries of compounds, with some ending as promising drug candidates. A complementary approach has been to adopt the concept of biomimicry of natural products and metabolites so as to improve multiple drug-like features of the parent molecule. In this effort, promiscuous and weak interactions between ligands and receptors are often ignored in a drug discovery process. In this Emerging Concepts article, we highlight microbial metabolite mimicry, whereby parent metabolites have weak interactions with their receptors that then have led to discrete examples of more potent and effective drug-like molecules. We show specific examples of parent-metabolite mimics with potent effects in vitro and in vivo. Furthermore, we show examples of emerging microbial ligand-receptor interactions and provide a context in which these ligands could be improved as potential drugs. A balanced conceptual advance is provided in which we also acknowledge potential pitfalls-hyperstimulation of finely balanced receptor-ligand interactions could also be detrimental. However, with balance, we provide examples of where this emerging concept needs to be tested. SIGNIFICANCE STATEMENT: Microbial metabolite mimicry is a novel way to expand on the chemical repertoire of future drugs. The emerging concept is now explained using specific examples of the discovery of therapeutic leads from microbial metabolites.

Agreement Between Ti-RADS Classification and Bethesda Cytopathological Findings from Thyroid Nodules in Young Adults.

INTRODUCTION: Thyroid nodules are common in young adults. We sought to compare the sonographic characteristics (thyroid imaging reporting and data system [Ti-RADS] classification) with the reported cytological results (Bethesda categories) from thyroid nodules in young recruits and examine the efficiency of our unique multidisciplinary clinic. MATERIALS AND METHODS: In this retrospective cohort, we identified young recruits (18-25 years) who underwent needle biopsies for asymptomatic thyroid nodule(s), with cytology reports of Bethesda categories II-V, performed in our "Rapid Diagnostic Service" at a large Israeli Defense Forces Medical Corps healthcare facility, between 2013 and 2018. We studied the concordance rates between their Ti-RADS and Bethesda grades, and the preoperative Bethesda grades accuracy versus final pathology results, and the time period needed for their thyroid nodules workup. RESULTS: A total of 81 patients were included who contributed 91 nodules. A fair agreement was found between the Ti-RADS classifications and the Bethesda grades (Cohen's κ = 0.238) that was more noticeable in males than in females. The agreement rate was 87.5% in males (21 of 24), but only 77.6% in females (59 of 67) [P = .029]. Of the 5 operated benign cases, all had low Ti-RADS, and 2 had low Bethesda. Of the 8 operated malignant cases, 6 had high Ti-RADS, and 5 had high Bethesda. On average, 37.64 days elapsed between the first visit to the ultrasound study, and 24.2 days elapsed until biopsy was performed. CONCLUSIONS: Despite an overall fair agreement between the sonographic features and cytological findings in young adults, we recommend a more aggressive approach and repeated biopsies despite reported benign pathology, because of a high false-negative rate.

High-Throughput Screen Identifies Host and Microbiota Regulators of Intestinal Barrier Function.

BACKGROUND & AIMS: The intestinal barrier protects intestinal cells from microbes and antigens in the lumen-breaches can alter the composition of the intestinal microbiota, the enteric immune system, and metabolism. We performed a screen to identify molecules that disrupt and support the intestinal epithelial barrier and tested their effects in mice. METHODS: We performed an imaging-based, quantitative, high-throughput screen (using CaCo-2 and T84 cells incubated with lipopolysaccharide; tumor necrosis factor; histamine; receptor antagonists; and libraries of secreted proteins, microbial metabolites, and drugs) to identify molecules that altered epithelial tight junction (TJ) and focal adhesion morphology. We then tested the effects of TJ stabilizers on these changes. Molecules we found to disrupt or stabilize TJs were administered mice with dextran sodium sulfate-induced colitis or Citrobacter rodentium-induced intestinal inflammation. Colon tissues were collected and analyzed by histology, fluorescence microscopy, and RNA sequencing. RESULTS: The screen identified numerous compounds that disrupted or stabilized (after disruption) TJs and monolayers of epithelial cells. We associated distinct morphologic alterations with changes in barrier function, and identified a variety of cytokines, metabolites, and drugs (including inhibitors of actomyosin contractility) that prevent disruption of TJs and restore TJ integrity. One of these disruptors (putrescine) disrupted TJ integrity in ex vivo mouse colon tissues; administration to mice exacerbated colon inflammation, increased gut permeability, reduced colon transepithelial electrical resistance, increased pattern recognition receptor ligands in mesenteric lymph nodes, and decreased colon length and survival times. Putrescine also increased intestine levels and fecal shedding of viable C rodentium, increased bacterial attachment to the colonic epithelium, and increased levels of inflammatory cytokines in colon tissues. Colonic epithelial cells from mice given putrescine increased expression of genes that regulate metal binding, oxidative stress, and cytoskeletal organization and contractility. Co-administration of taurine with putrescine blocked disruption of TJs and the exacerbated inflammation. CONCLUSIONS: We identified molecules that disrupt and stabilize intestinal epithelial TJs and barrier function and affect development of colon inflammation in mice. These agents might be developed for treatment of barrier intestinal impairment-associated and inflammatory disorders in patients, or avoided to prevent inflammation.

The bidirectional nature of microbiome-epithelial cell interactions.

The biogeography of the mammalian intestine is remarkable in that a vast microbial consortium exists inside the organism, surrounded by intestinal epithelial cells. The microbiome and the intestinal epithelium have developed a complex network of interactions that maintain intestinal homeostasis. We now recognize that functions of the epithelium are compartmentalized in specific intestinal epithelial cell subtypes. Furthermore, we are beginning to understand the ways in which microbes and their metabolic products impact the specific epithelial subsets. Here, we survey the mechanisms utilized by the microbiome to regulate intestinal epithelial function, and inversely, how different epithelial cell subtypes cooperate in regulating the microbiome.