Vitamin B12 is a limiting factor for induced cellular plasticity and tissue repair.

Transient reprogramming by the expression of OCT4, SOX2, KLF4 and MYC (OSKM) is a therapeutic strategy for tissue regeneration and rejuvenation, but little is known about its metabolic requirements. Here we show that OSKM reprogramming in mice causes a global depletion of vitamin B12 and molecular hallmarks of methionine starvation. Supplementation with vitamin B12 increases the efficiency of reprogramming both in mice and in cultured cells, the latter indicating a cell-intrinsic effect. We show that the epigenetic mark H3K36me3, which prevents illegitimate initiation of transcription outside promoters (cryptic transcription), is sensitive to vitamin B12 levels, providing evidence for a link between B12 levels, H3K36 methylation, transcriptional fidelity and efficient reprogramming. Vitamin B12 supplementation also accelerates tissue repair in a model of ulcerative colitis. We conclude that vitamin B12, through its key role in one-carbon metabolism and epigenetic dynamics, improves the efficiency of in vivo reprogramming and tissue repair.

Serum metabolomic adaptations following a 12-week high-intensity interval training combined to citrulline supplementation in obese older adults.

Physical activity and nutrition play important roles in preventing adverse health outcomes that accompany aging. It has been shown that high-intensity interval training (HIIT) combined with citrulline (CIT) supplementation can improve physical and functional capacities. The aim of this study was to evaluate serum metabolites following a 12-week HIIT combined or not with CIT in obese older adults, and to correlate the metabolic changes with clinico-biological parameters changes. Eighty-six obese older adults completed a 12-week HIIT program combined with a 10 g daily supplementation of either CIT or placebo (PLA) during a double-blinded randomized interventional trial. Only participants with blood samples at T0 (before the intervention) and/or T12 (after the intervention) were included in our sub-analysis (HIIT-PLA-T0: n = 44 and HIIT-PLA-T12: n = 28; HIIT-CIT-T0: n = 39 and HIIT-CIT-T12: n = 42). Serum samples were analyzed by different liquid or gas phase chromatography methods coupled to mass spectrometry. Among the identified metabolites, 44 changed significantly following the 12-week intervention (Time effect), and 10 of them were more affected when HIIT was combined with CIT (Time × Supp effect). Arginine increased significantly due to the 12-week intervention. Correlation analyses demonstrated that decreased triglyceride (TG) (16:1/18:1/16:0) and aspartic acid significantly correlated with a reduction of adiposity-related parameters (fat mass, leg lean mass, leptin, total triglycerides and low-density lipoprotein). Arginine, TG (16:1/18:1/16:0) and aspartic acid might constitute biomarkers of cardiometabolic health and adiposity. Further studies are needed to confirm these associations and understand the underlying mechanisms.Highlights A 12-week intervention involving high-intensity interval training (HIIT) with or without citrulline (CIT) supplementation induced adaptations in the serum metabolome of obese older adults through significant changes in 44 metabolites.Changes in 23 metabolites were observed when a CIT supplementation was administered along with a 12-week HIIT intervention.TG (16:1/18:1/16:0) correlated with several adiposity parameters including leptin, triglycerides, legs lean mass.Aspartic acid correlated with several adiposity parameters including leptin, LDL cholesterol as well as android, arms and trunk fat mass.

Autophagy-inducing nutritional interventions in experimental and clinical oncology.

Numerous pro-autophagic dietary interventions are being investigated for their potential cancer-preventive or therapeutic effects. This applies to different fasting regimens, methionine restriction and ketogenic diets. In addition, the supplementation of specific micronutrients such as nicotinamide (vitamin B3) or spermidine induces autophagy. In humans, leanness, plant-based diets (that may lead to partial methionine restriction) and high dietary uptake of spermidine are associated with a low incidence of cancers. Moreover, clinical trials have demonstrated the capacity of nicotinamide to prevent non-melanoma skin carcinogenesis. Multiple interventional trials are evaluating the capacity of autophagy-inducing regimens to improve the outcome of chemotherapy and immunotherapy. Here, we discuss the mechanistic underpinnings of autophagy induction by nutritional interventions, as well as the mechanisms through which autophagy induction in malignant or immune cells improves anticancer immunosurveillance.

Science-Driven Nutritional Interventions for the Prevention and Treatment of Cancer.

In population studies, dietary patterns clearly influence the development, progression, and therapeutic response of cancers. Nonetheless, interventional dietary trials have had relatively little impact on the prevention and treatment of malignant disease. Standardization of nutritional interventions combined with high-level mode-of-action studies holds the promise of identifying specific entities and pathways endowed with antineoplastic properties. Here, we critically review the effects of caloric restriction and more specific interventions on macro- and micronutrients in preclinical models as well as in clinical studies. We place special emphasis on the prospect of using defined nutrition-relevant molecules to enhance the efficacy of established anticancer treatments. SIGNIFICANCE: The avoidance of intrinsically hypercaloric and toxic diets contributes to the prevention and cure of cancer. In addition, specific diet-induced molecules such as ketone bodies and micronutrients, including specific vitamins, have drug-like effects that are clearly demonstrable in preclinical models, mostly in the context of immunotherapies. Multiple trials are underway to determine the clinical utility of such molecules.

Modulation of the Bile Acid Enterohepatic Cycle by Intestinal Microbiota Alleviates Alcohol Liver Disease.

Reshaping the intestinal microbiota by the ingestion of fiber, such as pectin, improves alcohol-induced liver lesions in mice by modulating bacterial metabolites, including indoles, as well as bile acids (BAs). In this context, we aimed to elucidate how oral supplementation of pectin affects BA metabolism in alcohol-challenged mice receiving feces from patients with alcoholic hepatitis. Pectin reduced alcohol liver disease. This beneficial effect correlated with lower BA levels in the plasma and liver but higher levels in the caecum, suggesting that pectin stimulated BA excretion. Pectin modified the overall BA composition, favoring an augmentation in the proportion of hydrophilic forms in the liver, plasma, and gut. This effect was linked to an imbalance between hydrophobic and hydrophilic (less toxic) BAs in the gut. Pectin induced the enrichment of intestinal bacteria harboring genes that encode BA-metabolizing enzymes. The modulation of BA content by pectin inhibited farnesoid X receptor signaling in the ileum and the subsequent upregulation of Cyp7a1 in the liver. Despite an increase in BA synthesis, pectin reduced BA serum levels by promoting their intestinal excretion. In conclusion, pectin alleviates alcohol liver disease by modifying the BA cycle through effects on the intestinal microbiota and enhanced BA excretion.

Immunostimulatory effects of vitamin B5 improve anticancer immunotherapy.

Vitamin B5 (panthotenic acid), the precursor of coenzyme A (CoA), is contained in most food items and is produced by the intestinal microbiota. A recent study published in Cell Metabolism reports that vitamin B5 and CoA favor the differentiation of CD8+ cytotoxic T cells into interleukin-22 (IL-22)-producing Tc22 cells, likely through fueling mitochondrial metabolism. Importantly, in a small cohort of melanoma patients, the plasma levels of vitamin B5 positively correlate with responses to PD-1-targeted immunotherapy. Moreover, in mice, supplementation with vitamin B5 increases the efficacy of PD-L1-targeted cancer immunotherapy, and in vitro culture of T cells with CoA enhances their antitumor activity upon adoptive transfer into mice. These finding suggest that vitamin B5 is yet another B vitamin that stimulates anti-cancer immunosurveillance.

High throughput screening for autophagy.

Robotized high throughput screening allows for the assessment of autophagy in a large number of samples. Here, we describe a drug discovery platform for the phenotypic identification of novel autophagy inducers by means of automated cell biology workflows employing robotized cell culture, sample preparation and data acquisition. In this setting, fluorescent biosensor cells that express microtubule-associated proteins 1A/1B light chain 3B (best known as LC3) conjugated to green fluorescent protein (GFP), are utilized together with automated high content microscopy for the image-based assessment of autophagy. In sum, we detail a drug discovery screening workflow from high throughput sample preparation and processing to data acquisition and analysis.

Dietary spermidine improves cognitive function.

Decreased cognitive performance is a hallmark of brain aging, but the underlying mechanisms and potential therapeutic avenues remain poorly understood. Recent studies have revealed health-protective and lifespan-extending effects of dietary spermidine, a natural autophagy-promoting polyamine. Here, we show that dietary spermidine passes the blood-brain barrier in mice and increases hippocampal eIF5A hypusination and mitochondrial function. Spermidine feeding in aged mice affects behavior in homecage environment tasks, improves spatial learning, and increases hippocampal respiratory competence. In a Drosophila aging model, spermidine boosts mitochondrial respiratory capacity, an effect that requires the autophagy regulator Atg7 and the mitophagy mediators Parkin and Pink1. Neuron-specific Pink1 knockdown abolishes spermidine-induced improvement of olfactory associative learning. This suggests that the maintenance of mitochondrial and autophagic function is essential for enhanced cognition by spermidine feeding. Finally, we show large-scale prospective data linking higher dietary spermidine intake with a reduced risk for cognitive impairment in humans.

Old Paradoxes and New Opportunities for Appetite Control in Obesity.

Human obesity is accompanied by alterations in the blood concentrations of multiple circulating appetite regulators. Paradoxically, most of the appetite-inhibitory hormones are elevated in nonsyndromic obesity, while most of the appetite stimulatory hormones are reduced, perhaps reflecting vain attempts of regulation by inefficient feedback circuitries. In this context, it is important to understand which appetite regulators exhibit a convergent rather than paradoxical behavior and hence are likely to contribute to the maintenance of the obese state. Pharmacological interventions in obesity should preferentially consist of the supplementation of deficient appetite inhibitors or the neutralization of excessive appetite stimulators. Here, we critically analyze the current literature on appetite-regulatory peptide hormones. We propose a short-list of appetite modulators that may constitute the best candidates for therapeutic interventions.

Nicotinamide for the treatment of heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent and intractable form of cardiac decompensation commonly associated with diastolic dysfunction. Here, we show that diastolic dysfunction in patients with HFpEF is associated with a cardiac deficit in nicotinamide adenine dinucleotide (NAD+). Elevating NAD+ by oral supplementation of its precursor, nicotinamide, improved diastolic dysfunction induced by aging (in 2-year-old C57BL/6J mice), hypertension (in Dahl salt-sensitive rats), or cardiometabolic syndrome (in ZSF1 obese rats). This effect was mediated partly through alleviated systemic comorbidities and enhanced myocardial bioenergetics. Simultaneously, nicotinamide directly improved cardiomyocyte passive stiffness and calcium-dependent active relaxation through increased deacetylation of titin and the sarcoplasmic reticulum calcium adenosine triphosphatase 2a, respectively. In a long-term human cohort study, high dietary intake of naturally occurring NAD+ precursors was associated with lower blood pressure and reduced risk of cardiac mortality. Collectively, these results suggest NAD+ precursors, and especially nicotinamide, as potential therapeutic agents to treat diastolic dysfunction and HFpEF in humans.

Possible mechanisms of cancer prevention by nicotinamide.

Nicotinamide (NAM) is a precursor of vitamin B3 commonly sold over the counter as a nutritional supplement with anti-aging properties. Accumulating preclinical evidence indicates that NAM also mediates oncopreventive effects against a variety of neoplasms. Supporting the translational relevance of dietary NAM supplementation, results from a Phase 3 randomized clinical trial have demonstrated that oral NAM was safe and efficiently reduced the incidence of new non-melanoma skin cancers and actinic keratosis amongst high-risk individuals. However, the molecular and cellular mechanisms that underlie this ability of NAM to delay carcinogenesis remain to be clarified, as discussed in this short review. LINKED ARTICLES: This article is part of a themed issue on Cellular metabolism and diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.10/issuetoc.

Microbiota tryptophan metabolism induces aryl hydrocarbon receptor activation and improves alcohol-induced liver injury.

OBJECTIVE: Chronic alcohol consumption is an important cause of liver-related deaths. Specific intestinal microbiota profiles are associated with susceptibility or resistance to alcoholic liver disease in both mice and humans. We aimed to identify the mechanisms by which targeting intestinal microbiota can improve alcohol-induced liver lesions. DESIGN: We used human associated mice, a mouse model of alcoholic liver disease transplanted with the intestinal microbiota of alcoholic patients and used the prebiotic, pectin, to modulate the intestinal microbiota. Based on metabolomic analyses, we focused on microbiota tryptophan metabolites, which are ligands of the aryl hydrocarbon receptor (AhR). Involvement of the AhR pathway was assessed using both a pharmacological approach and AhR-deficient mice. RESULTS: Pectin treatment modified the microbiome and metabolome in human microbiota-associated alcohol-fed mice, leading to a specific faecal signature. High production of bacterial tryptophan metabolites was associated with an improvement of liver injury. The AhR agonist Ficz (6-formylindolo (3,2-b) carbazole) reduced liver lesions, similarly to prebiotic treatment. Conversely, inactivation of the ahr gene in alcohol-fed AhR knock-out mice abrogated the beneficial effects of the prebiotic. Importantly, patients with severe alcoholic hepatitis have low levels of bacterial tryptophan derivatives that are AhR agonists. CONCLUSIONS: Improvement of alcoholic liver disease by targeting the intestinal microbiota involves the AhR pathway, which should be considered as a new therapeutic target.

Regulatory approval of photoimmunotherapy: photodynamic therapy that induces immunogenic cell death.

In September 2020, the Japanese government approved cetuximab saratolacan (previously known as RM-1929, commercial name: Akalux) for the treatment of unresectable locally advanced or recurrent head and neck cancer. Cetuximab saratolacan is a chemical conjugate of the photosensitizer IR700 with cetuximab, which targets EGFR. The treatment consists in the intravenous injection of cetuximab saratolacan, which binds to head and neck cancer cells expressing high levels of EGFR, followed by illumination of the tumor with red light (690 nm) for photodynamic therapy. This approach causes immunogenic cell death in malignant tissues, thus triggering a potent anticancer immune response.

Combination treatments with hydroxychloroquine and azithromycin are compatible with the therapeutic induction of anticancer immune responses.

Amid controversial reports that COVID-19 can be treated with a combination of the antimalarial drug hydroxychloroquine (HCQ) and the antibiotic azithromycin (AZI), a clinical trial (ONCOCOVID, NCT04341207) was launched at Gustave Roussy Cancer Campus to investigate the utility of this combination therapy in cancer patients. In this preclinical study, we investigated whether the combination of HCQ+AZI would be compatible with the therapeutic induction of anticancer immune responses. For this, we used doses of HCQ and AZI that affect whole-body physiology (as indicated by a partial blockade in cardiac and hepatic autophagic flux for HCQ and a reduction in body weight for AZI), showing that their combined administration did not interfere with tumor growth control induced by the immunogenic cell death inducer oxaliplatin. Moreover, the HCQ+AZI combination did not affect the capacity of a curative regimen (cisplatin + crizotinib + PD-1 blockade) to eradicate established orthotopic lung cancers in mice. In conclusion, it appears that HCQ+AZI does not interfere with the therapeutic induction of therapeutic anticancer immune responses.

On-target versus off-target effects of drugs inhibiting the replication of SARS-CoV-2.

The current epidemic of coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for the development of inhibitors of viral replication. Here, we performed a bioinformatic analysis of published and purported SARS-CoV-2 antivirals including imatinib mesylate that we found to suppress SARS-CoV-2 replication on Vero E6 cells and that, according to the published literature on other coronaviruses is likely to act on-target, as a tyrosine kinase inhibitor. We identified a cluster of SARS-CoV-2 antivirals with characteristics of lysosomotropic agents, meaning that they are lipophilic weak bases capable of penetrating into cells. These agents include cepharentine, chloroquine, chlorpromazine, clemastine, cloperastine, emetine, hydroxychloroquine, haloperidol, ML240, PB28, ponatinib, siramesine, and zotatifin (eFT226) all of which are likely to inhibit SARS-CoV-2 replication by non-specific (off-target) effects, meaning that they probably do not act on their 'official' pharmacological targets, but rather interfere with viral replication through non-specific effects on acidophilic organelles including autophagosomes, endosomes, and lysosomes. Imatinib mesylate did not fall into this cluster. In conclusion, we propose a tentative classification of SARS-CoV-2 antivirals into specific (on-target) versus non-specific (off-target) agents based on their physicochemical characteristics.

Immunoprophylactic and immunotherapeutic control of hormone receptor-positive breast cancer.

Hormone receptor (HR)+ breast cancer (BC) causes most BC-related deaths, calling for improved therapeutic approaches. Despite expectations, immune checkpoint blockers (ICBs) are poorly active in patients with HR+ BC, in part reflecting the lack of preclinical models that recapitulate disease progression in immunocompetent hosts. We demonstrate that mammary tumors driven by medroxyprogesterone acetate (M) and 7,12-dimethylbenz[a]anthracene (D) recapitulate several key features of human luminal B HR+HER2- BC, including limited immune infiltration and poor sensitivity to ICBs. M/D-driven oncogenesis is accelerated by immune defects, demonstrating that M/D-driven tumors are under immunosurveillance. Safe nutritional measures including nicotinamide (NAM) supplementation efficiently delay M/D-driven oncogenesis by reactivating immunosurveillance. NAM also mediates immunotherapeutic effects against established M/D-driven and transplantable BC, largely reflecting increased type I interferon secretion by malignant cells and direct stimulation of immune effector cells. Our findings identify NAM as a potential strategy for the prevention and treatment of HR+ BC.

Artificial tethering of LC3 or p62 to organelles is not sufficient to trigger autophagy.

The retention using selective hooks (RUSH) system allows to retain a target protein fused to green fluorescent protein (GFP) and a streptavidin-binding peptide (SBP) due to the interaction with a molar excess of streptavidin molecules ("hooks") targeted to selected subcellular compartments. Supplementation of biotin competitively disrupts the interaction between the SBP moiety and streptavidin, liberating the chimeric target protein from its hooks, while addition of avidin causes the removal of biotin from the system and reestablishes the interaction. Based on this principle, we engineered two chimeric proteins involved in autophagy, namely microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B, best known as LC3) and sequestosome-1 (SQSTM1, best known as p62) to move them as SBP-GFP-LC3 and p62-SBP-GFP at will between the cytosol and two different organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. Although both proteins were functional in thus far that SBP-GFP-LC3 and p62-SBP-GFP could recruit their endogenous binding partners, p62 and LC3, respectively, their enforced relocation to the ER or Golgi failed to induce organelle-specific autophagy. Hence, artificial tethering of LC3 or p62 to the surface of the ER and the Golgi is not sufficient to trigger autophagy.

Crizotinib - a tyrosine kinase inhibitor that stimulates immunogenic cell death.

Crizotinib is a tyrosine kinase inhibitor (TKI) approved for the treatment of non-small cell lung cancers (NSLCL) and lymphomas expressing activating translocations or mutations of oncogenic tyrosine kinases (in particular ALK and ROS1). We recently observed that high-dose (final concentration in vivo: ~10 µM) crizotinib can induce immunogenic cell death (ICD) in cancer cells lacking ALK/ROS1 activation through off-target effects that require the inhibition of several other tyrosine kinases. When combined with cisplatin (which alone does not induce ICD), crizotinib sensitizes NSCLC models to subsequent immunotherapy with PD-1 blockade, allowing to cure more than 90% of established orthotopic cancers. Of note, simultaneous treatment of mice with cisplatin, crizotinib and PD-1 blocking antibodies causes acute hepatotoxicity that can be avoided by a sequential regimen involving initial treatment with cisplatin plus crizotinib, followed by PD-1 blockade one week later. It will be important to translate these results obtained in mice into a clinical trial in NSCLC patients.

Genistein antagonizes gliadin-induced CFTR malfunction in models of celiac disease.

In celiac disease (CD), an intolerance to dietary gluten/gliadin, antigenic gliadin peptides trigger an HLA-DQ2/DQ8-restricted adaptive Th1 immune response. Epithelial stress, induced by other non-antigenic gliadin peptides, is required for gliadin to become fully immunogenic. We found that cystic-fibrosis-transmembrane-conductance-regulator (CFTR) acts as membrane receptor for gliadin-derived peptide P31-43, as it binds to CFTR and impairs its channel function. P31-43-induced CFTR malfunction generates epithelial stress and intestinal inflammation. Maintaining CFTR in an active open conformation by the CFTR potentiators VX-770 (Ivacaftor) or Vrx-532, prevents P31-43 binding to CFTR and controls gliadin-induced manifestations. Here, we evaluated the possibility that the over-the-counter nutraceutical genistein, known to potentiate CFTR function, would allow to control gliadin-induced alterations. We demonstrated that pre-treatment with genistein prevented P31-43-induced CFTR malfunction and an epithelial stress response in Caco-2 cells. These effects were abrogated when the CFTR gene was knocked out by CRISP/Cas9 technology, indicating that genistein protects intestinal epithelial cells by potentiating CFTR function. Notably, genistein protected gliadin-sensitive mice from intestinal CFTR malfunction and gliadin-induced inflammation as it prevented gliadin-induced IFN-γ production by celiac peripheral-blood-mononuclear-cells (PBMC) cultured ex-vivo in the presence of P31-43-challenged Caco-2 cells. Our results indicate that natural compounds capable to increase CFTR channel gating might be used for the treatment of CD.

The flavonoid 4,4'-dimethoxychalcone promotes autophagy-dependent longevity across species.

Ageing constitutes the most important risk factor for all major chronic ailments, including malignant, cardiovascular and neurodegenerative diseases. However, behavioural and pharmacological interventions with feasible potential to promote health upon ageing remain rare. Here we report the identification of the flavonoid 4,4'-dimethoxychalcone (DMC) as a natural compound with anti-ageing properties. External DMC administration extends the lifespan of yeast, worms and flies, decelerates senescence of human cell cultures, and protects mice from prolonged myocardial ischaemia. Concomitantly, DMC induces autophagy, which is essential for its cytoprotective effects from yeast to mice. This pro-autophagic response induces a conserved systemic change in metabolism, operates independently of TORC1 signalling and depends on specific GATA transcription factors. Notably, we identify DMC in the plant Angelica keiskei koidzumi, to which longevity- and health-promoting effects are ascribed in Asian traditional medicine. In summary, we have identified and mechanistically characterised the conserved longevity-promoting effects of a natural anti-ageing drug.