Imperial College London


Faculty of MedicineDepartment of Surgery & Cancer

Professor of Biochemistry



+44 (0)20 7594 3161h.keun




officesInstitute of Reproductive and Developmental BiologyHammersmith Campus





Publication Type

178 results found

Rothwell JA, Bešević J, Dimou N, Breeur M, Murphy N, Jenab M, Wedekind R, Viallon V, Ferrari P, Achaintre D, Gicquiau A, Rinaldi S, Scalbert A, Huybrechts I, Prehn C, Adamski J, Cross AJ, Keun H, Chadeau-Hyam M, Boutron-Ruault M-C, Overvad K, Dahm CC, Nøst TH, Sandanger TM, Skeie G, Zamora-Ros R, Tsilidis KK, Eichelmann F, Schulze MB, van Guelpen B, Vidman L, Sánchez M-J, Amiano P, Ardanaz E, Smith-Byrne K, Travis R, Katzke V, Kaaks R, Derksen JWG, Colorado-Yohar S, Tumino R, Bueno-de-Mesquita B, Vineis P, Palli D, Pasanisi F, Eriksen AK, Tjønneland A, Severi G, Gunter MJet al., 2023, Circulating amino acid levels and colorectal cancer risk in the European Prospective Investigation into Cancer and Nutrition and UK Biobank cohorts, BMC Medicine, Vol: 21, Pages: 1-13, ISSN: 1741-7015

BACKGROUND: Amino acid metabolism is dysregulated in colorectal cancer patients; however, it is not clear whether pre-diagnostic levels of amino acids are associated with subsequent risk of colorectal cancer. We investigated circulating levels of amino acids in relation to colorectal cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC) and UK Biobank cohorts. METHODS: Concentrations of 13-21 amino acids were determined in baseline fasting plasma or serum samples in 654 incident colorectal cancer cases and 654 matched controls in EPIC. Amino acids associated with colorectal cancer risk following adjustment for the false discovery rate (FDR) were then tested for associations in the UK Biobank, for which measurements of 9 amino acids were available in 111,323 participants, of which 1221 were incident colorectal cancer cases. RESULTS: Histidine levels were inversely associated with colorectal cancer risk in EPIC (odds ratio [OR] 0.80 per standard deviation [SD], 95% confidence interval [CI] 0.69-0.92, FDR P-value=0.03) and in UK Biobank (HR 0.93 per SD, 95% CI 0.87-0.99, P-value=0.03). Glutamine levels were borderline inversely associated with colorectal cancer risk in EPIC (OR 0.85 per SD, 95% CI 0.75-0.97, FDR P-value=0.08) and similarly in UK Biobank (HR 0.95, 95% CI 0.89-1.01, P=0.09) In both cohorts, associations changed only minimally when cases diagnosed within 2 or 5 years of follow-up were excluded. CONCLUSIONS: Higher circulating levels of histidine were associated with a lower risk of colorectal cancer in two large prospective cohorts. Further research to ascertain the role of histidine metabolism and potentially that of glutamine in colorectal cancer development is warranted.

Journal article

Fabbri L, Garlantézec R, Audouze K, Bustamante M, Carracedo Á, Chatzi L, Ramón González J, Gražulevičienė R, Keun H, Lau C-HE, Sabidó E, Siskos AP, Slama R, Thomsen C, Wright J, Lun Yuan W, Casas M, Vrijheid M, Maitre Let al., 2023, Childhood exposure to non-persistent endocrine disrupting chemicals and multi-omic profiles: A panel study., Environ Int, Vol: 173

BACKGROUND: Individuals are exposed to environmental pollutants with endocrine disrupting activity (endocrine disruptors, EDCs) and the early stages of life are particularly susceptible to these exposures. Previous studies have focused on identifying molecular signatures associated with EDCs, but none have used repeated sampling strategy and integrated multiple omics. We aimed to identify multi-omic signatures associated with childhood exposure to non-persistent EDCs. METHODS: We used data from the HELIX Child Panel Study, which included 156 children aged 6 to 11. Children were followed for one week, in two time periods. Twenty-two non-persistent EDCs (10 phthalate, 7 phenol, and 5 organophosphate pesticide metabolites) were measured in two weekly pools of 15 urine samples each. Multi-omic profiles (methylome, serum and urinary metabolome, proteome) were measured in blood and in a pool urine samples. We developed visit-specific Gaussian Graphical Models based on pairwise partial correlations. The visit-specific networks were then merged to identify reproducible associations. Independent biological evidence was systematically sought to confirm some of these associations and assess their potential health implications. RESULTS: 950 reproducible associations were found among which 23 were direct associations between EDCs and omics. For 9 of them, we were able to find corroborating evidence from previous literature: DEP - serotonin, OXBE - cg27466129, OXBE - dimethylamine, triclosan - leptin, triclosan - serotonin, MBzP - Neu5AC, MEHP - cg20080548, oh-MiNP - kynurenine, oxo-MiNP - 5-oxoproline. We used these associations to explore possible mechanisms between EDCs and health outcomes, and found links to health outcomes for 3 analytes: serotonin and kynurenine in relation to neuro-behavioural development, and leptin in relation to obesity and insulin resistance. CONCLUSIONS: This multi-omics network analysis at two time points identified biologically relevant molecular si

Journal article

Maitre L, Bustamante M, Hernandez-Ferrer C, Thiel D, Lau C-HE, Siskos A, Vives-Usano M, Ruiz-Arenas C, Pelegri-Siso D, Robinson O, Mason D, Wright J, Cadiou S, Slama R, Heude B, Casas M, Sunyer J, Papadopoulou EZ, Gutzkow KB, Andrusaityte S, Grazuleviciene R, Vafeiadi M, Chatzi L, Sakhi AK, Thomsen C, Tamayo I, Nieuwenhuijsen M, Urquiza J, Borras E, Sabido E, Quintela I, Carracedo A, Estivill X, Coen M, Gonzalez JR, Keun HC, Vrijheid Met al., 2022, Multi-omics signatures of the human early life exposome, Nature Communications, Vol: 13, Pages: 1-18, ISSN: 2041-1723

Environmental exposures during early life play a critical role in life-course health, yet the molecular phenotypes underlying environmental effects on health are poorly understood. In the Human Early Life Exposome (HELIX) project, a multi-centre cohort of 1301 mother-child pairs, we associate individual exposomes consisting of >100 chemical, outdoor, social and lifestyle exposures assessed in pregnancy and childhood, with multi-omics profiles (methylome, transcriptome, proteins and metabolites) in childhood. We identify 1170 associations, 249 in pregnancy and 921 in childhood, which reveal potential biological responses and sources of exposure. Pregnancy exposures, including maternal smoking, cadmium and molybdenum, are predominantly associated with child DNA methylation changes. In contrast, childhood exposures are associated with features across all omics layers, most frequently the serum metabolome, revealing signatures for diet, toxic chemical compounds, essential trace elements, and weather conditions, among others. Our comprehensive and unique resource of all associations ( will serve to guide future investigation into the biological imprints of the early life exposome.

Journal article

Breeur M, Ferrari P, Dossus L, Jenab M, Johansson M, Rinaldi S, Travis RC, His M, Key TJ, Schmidt JA, Overvad K, Tjønneland A, Kyrø C, Rothwell JA, Laouali N, Severi G, Kaaks R, Katzke V, Schulze MB, Eichelmann F, Palli D, Grioni S, Panico S, Tumino R, Sacerdote C, Bueno-de-Mesquita B, Olsen KS, Sandanger TM, Nøst TH, Quirós JR, Bonet C, Barranco MR, Chirlaque M-D, Ardanaz E, Sandsveden M, Manjer J, Vidman L, Rentoft M, Muller D, Tsilidis K, Heath AK, Keun H, Adamski J, Keski-Rahkonen P, Scalbert A, Gunter MJ, Viallon Vet al., 2022, Pan-cancer analysis of pre-diagnostic blood metabolite concentrations in the European Prospective Investigation into Cancer and Nutrition, BMC Medicine, Vol: 20, ISSN: 1741-7015

BACKGROUND: Epidemiological studies of associations between metabolites and cancer risk have typically focused on specific cancer types separately. Here, we designed a multivariate pan-cancer analysis to identify metabolites potentially associated with multiple cancer types, while also allowing the investigation of cancer type-specific associations. METHODS: We analysed targeted metabolomics data available for 5828 matched case-control pairs from cancer-specific case-control studies on breast, colorectal, endometrial, gallbladder, kidney, localized and advanced prostate cancer, and hepatocellular carcinoma nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. From pre-diagnostic blood levels of an initial set of 117 metabolites, 33 cluster representatives of strongly correlated metabolites and 17 single metabolites were derived by hierarchical clustering. The mutually adjusted associations of the resulting 50 metabolites with cancer risk were examined in penalized conditional logistic regression models adjusted for body mass index, using the data-shared lasso penalty. RESULTS: Out of the 50 studied metabolites, (i) six were inversely associated with the risk of most cancer types: glutamine, butyrylcarnitine, lysophosphatidylcholine a C18:2, and three clusters of phosphatidylcholines (PCs); (ii) three were positively associated with most cancer types: proline, decanoylcarnitine, and one cluster of PCs; and (iii) 10 were specifically associated with particular cancer types, including histidine that was inversely associated with colorectal cancer risk and one cluster of sphingomyelins that was inversely associated with risk of hepatocellular carcinoma and positively with endometrial cancer risk. CONCLUSIONS: These results could provide novel insights for the identification of pathways for cancer development, in particular those shared across different cancer types.

Journal article

Breeur M, Ferrari P, Dossus L, Jenab M, Johansson M, Rinaldi S, Travis RC, His M, Key TJ, Schmidt JA, Overvad K, Tjønneland A, Kyrø C, Rothwell JA, Laouali N, Severi G, Kaaks R, Katzke V, Schulze MB, Eichelmann F, Palli D, Grioni S, Panico S, Tumino R, Sacerdote C, Bueno-de-Mesquita B, Olsen KS, Sandanger TM, Nøst TH, Quirós JR, Bonet C, Barranco MR, Chirlaque M-D, Ardanaz E, Sandsveden M, Manjer J, Vidman L, Rentoft M, Muller D, Tsilidis K, Heath AK, Keun H, Adamski J, Keski-Rahkonen P, Scalbert A, Gunter MJ, Viallon Vet al., 2022, Pan-cancer analysis of pre-diagnostic blood metabolite concentrations in the European Prospective Investigation into Cancer and Nutrition, Publisher: Cold Spring Harbor Laboratory

<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Epidemiological studies of associations between metabolites and cancer risk have typically focused on specific cancer types separately. Here, we designed a multivariate pan-cancer analysis to identify metabolites potentially associated with multiple cancer types, while also allowing the investigation of cancer type-specific associations.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>We analyzed targeted metabolomics data available for 5,828 matched case-control pairs from cancer-specific case-control studies on breast, colorectal, endometrial, gallbladder, kidney, localized and advanced prostate cancer, and hepatocellular carcinoma nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. From pre-diagnostic blood levels of an initial set of 117 metabolites, 33 cluster representatives of strongly correlated metabolites, and 17 single metabolites were derived by hierarchical clustering. The mutually adjusted associations of the resulting 50 metabolites with cancer risk were examined in penalized conditional logistic regression models adjusted for body mass index, using the data shared lasso penalty.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Out of the 50 studied metabolites, <jats:italic>(i)</jats:italic> six were inversely associated with risk of most cancer types: glutamine, butyrylcarnitine, lysophosphatidylcholine a C18:2 and three clusters of phosphatidylcholines (PCs); <jats:italic>(ii)</jats:italic> three were positively associated with most cancer types: proline, decanoylcarnitine and one cluster of PCs; and <jats:italic>(iii)</jats:italic> 10 were specifically associated with particular cancer types, including histidine that was inversely associate

Working paper

Xu Y, Nijhuis A, Keun HC, 2022, RNA-binding motif protein 39 (RBM39): an emerging cancer target, British Journal of Pharmacology, Vol: 179, Pages: 2795-2812, ISSN: 0007-1188

RNA-binding motif protein 39 (RBM39) is an RNA-binding protein involved in transcriptional co-regulation and alternative RNA splicing. Recent studies have revealed that RBM39 is the unexpected target of aryl sulphonamides, which act as molecular glues between RBM39 and the DCAF15-associated E3 ubiquitin ligase complex leading to selective degradation of the target. Loss of RBM39 leads to aberrant splicing events and differential gene expression, thereby inhibiting cell cycle progression and causing tumour regression in a number of preclinical models. Many clinical studies have shown that aryl sulphonamides were well tolerated, but their clinical performance was limited due to an insufficient understanding of the target, RBM39 biology and a lack of predictive biomarkers. This review summarises the current knowledge of RBM39 function and discusses the therapeutic potential of this spliceosome target in cancer therapy.

Journal article

Noble RA, Thomas H, Zhao Y, Herendi L, Howarth R, Dragoni I, Keun HC, Vellano CP, Marszalek JR, Wedge SRet al., 2022, Simultaneous targeting of glycolysis and oxidative phosphorylation as a therapeutic strategy to treat diffuse large B-cell lymphoma, British Journal of Cancer, Vol: 127, Pages: 937-947, ISSN: 0007-0920

BackgroundWe evaluated the therapeutic potential of combining the monocarboxylate transporter 1 (MCT1) inhibitor AZD3965 with the mitochondrial respiratory Complex I inhibitor IACS-010759, for the treatment of diffuse large B-cell lymphoma (DLBCL), a potential clinically actionable strategy to target tumour metabolism.MethodsAZD3965 and IACS-010759 sensitivity were determined in DLBCL cell lines and tumour xenograft models. Lactate concentrations, oxygen consumption rate and metabolomics were examined as mechanistic endpoints. In vivo plasma concentrations of IACS-010759 in mice were determined by LC-MS to select a dose that reflected clinically attainable concentrations.ResultsIn vitro, the combination of AZD3965 and IACS-010759 is synergistic and induces DLBCL cell death, whereas monotherapy treatments induce a cytostatic response. Significant anti-tumour activity was evident in Toledo and Farage models when the two inhibitors were administered concurrently despite limited or no effect on the growth of DLBCL xenografts as monotherapies.ConclusionsThis is the first study to examine a combination of two distinct approaches to targeting tumour metabolism in DLBCL xenografts. Whilst nanomolar concentrations of either AZD3965 or IACS-010759 monotherapy demonstrate anti-proliferative activity against DLBCL cell lines in vitro, appreciable clinical activity in DLBCL patients may only be realised through their combined use.

Journal article

Garcia-Dominguez DJ, Hajji N, Lopez-Alemany R, Sanchez-Molina S, Figuerola-Bou E, Civanto FJM, Rello-Varona S, Andres-Leon E, Benito A, Keun HC, Mora J, Tirado OM, de Alava E, Hontecillas-Prieto Let al., 2022, Selective histone methyltransferase G9a inhibition reduces metastatic development of Ewing sarcoma through the epigenetic regulation of NEU1, Oncogene, Vol: 41, Pages: 2638-2650, ISSN: 0950-9232

Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor with high susceptibility to metastasize. The underlying molecular mechanisms leading to EWS metastases remain poorly understood. Epigenetic changes have been implicated in EWS tumor growth and progression. Linking epigenetics and metastases may provide insight into novel molecular targets in EWS and improve its treatment. Here, we evaluated the effects of a selective G9a histone methyltransferase inhibitor (BIX01294) on EWS metastatic process. Our results showed that overexpression of G9a in tumors from EWS patients correlates with poor prognosis. Moreover, we observe a significantly higher expression of G9a in metastatic EWS tumor as compared to either primary or recurrent tumor. Using functional assays, we demonstrate that pharmacological G9a inhibition using BIX01294 disrupts several metastatic steps in vitro, such as migration, invasion, adhesion, colony formation and vasculogenic mimicry. Moreover, BIX01294 reduces tumor growth and metastases in two spontaneous metastases mouse models. We further identified the sialidase NEU1 as a direct target and effector of G9a in the metastatic process in EWS. NEU1 overexpression impairs migration, invasion and clonogenic capacity of EWS cell lines. Overall, G9a inhibition impairs metastases in vitro and in vivo through the overexpression of NEU1. G9a has strong potential as a prognostic marker and may be a promising therapeutic target for EWS patients.

Journal article

Nijhuis A, Sikka A, Yogev O, Herendi L, Balcells C, Ma Y, Poon E, Valbuena G, Xu Y, Liu Y, Martins da Costa B, Gruet M, Wickremesinghe C, Benito A, Kramer H, Montoya A, Carling D, Want E, Jamin Y, Chesler L, Keun Het al., 2022, Indisulam targets RNA splicing and metabolism to serve as a therapeutic strategy for high-risk neuroblastoma, Nature Communications, Vol: 13, ISSN: 2041-1723

Neuroblastoma is the most common paediatric solid tumour and prognosis remains poor for high-risk cases despite the use of multimodal treatment. Analysis of public drug sensitivity data showed neuroblastoma lines to be sensitive to indisulam, a molecular glue that selectively targets RNA splicing factor RBM39 for proteosomal degradation via DCAF15-E3-ubiquitin ligase. In neuroblastoma models, indisulam induces rapid loss of RBM39, accumulation of splicing errors and growth inhibition in a DCAF15-dependent manner. Integrative analysis of RNAseq and proteomics data highlight a distinct disruption to cell cycle and metabolism. Metabolic profiling demonstrates metabolome perturbations and mitochondrial dysfunction resulting from indisulam. Complete tumour regression without relapse was observed in both xenograft and the Th-MYCN transgenic model of neuroblastoma after indisulam treatment, with RBM39 loss, RNA splicing and metabolic changes confirmed in vivo. Our data show that dual-targeting of metabolism and RNA splicing with anticancer indisulam is a promising therapeutic approach for high-risk neuroblastoma.

Journal article

Stratakis N, Siskos AP, Papadopoulou E, Nguyen AN, Zhao Y, Margetaki K, Lau C-HE, Coen M, Maitre L, Fernández-Barrés S, Agier L, Andrusaityte S, Basagaña X, Brantsaeter AL, Casas M, Fossati S, Grazuleviciene R, Heude B, McEachan RRC, Meltzer HM, Millett C, Rauber F, Robinson O, Roumeliotaki T, Borras E, Sabidó E, Urquiza J, Vafeiadi M, Vineis P, Voortman T, Wright J, Conti DV, Vrijheid M, Keun HC, Chatzi Let al., 2022, Urinary metabolic biomarkers of diet quality in European children are associated with metabolic health, eLife, Vol: 11, Pages: 1-20, ISSN: 2050-084X

Urinary metabolic profiling is a promising powerful tool to reflect dietary intake and can help understand metabolic alterations in response to diet quality. Here, we used 1H NMR spectroscopy in a multicountry study in European children (1147 children from 6 different cohorts) and identified a common panel of 4 urinary metabolites (hippurate, N-methylnicotinic acid, urea, and sucrose) that was predictive of Mediterranean diet adherence (KIDMED) and ultra-processed food consumption and also had higher capacity in discriminating children’s diet quality than that of established sociodemographic determinants. Further, we showed that the identified metabolite panel also reflected the associations of these diet quality indicators with C-peptide, a stable and accurate marker of insulin resistance and future risk of metabolic disease. This methodology enables objective assessment of dietary patterns in European child populations, complementary to traditional questionary methods, and can be used in future studies to evaluate diet quality. Moreover, this knowledge can provide mechanistic evidence of common biological pathways that characterize healthy and unhealthy dietary patterns, and diet-related molecular alterations that could associate to metabolic disease.

Journal article

Rana S, Valbuena GN, Curry E, Bevan CL, Keun HCet al., 2022, MicroRNAs as biomarkers for prostate cancer prognosis: a systematic review and a systematic reanalysis of public data, British Journal of Cancer, Vol: 126, Pages: 502-513, ISSN: 0007-0920

BackgroundReliable prognostic biomarkers to distinguish indolent from aggressive prostate cancer (PCa) are lacking. Many studies investigated microRNAs (miRs) as PCa prognostic biomarkers, often reporting inconsistent findings. We present a systematic review of these; also systematic reanalysis of public miR-profile datasets to identify tissue-derived miRs prognostic of biochemical recurrence (BCR) in patients undergoing radical prostatectomy.MethodsIndependent PubMed searches were performed for relevant articles from January 2007 to December 2019. For the review, 128 studies were included. Pooled-hazard-ratios (HRs) for miRs in multiple studies were calculated using a random-effects model (REM). For the reanalysis, five studies were included and Cox proportional-hazard models, testing miR association with BCR, performed for miRs profiled in all.ResultsSystematic review identified 120 miRs as prognostic. Five (let-7b-5p, miR-145-5p, miR152-3p, miR-195-5p, miR-224-5p) were consistently associated with progression in multiple cohorts/studies. In the reanalysis, ten (let-7a-5p, miR-148a-3p, miR-203a-3p, miR-26b-5p, miR30a-3p, miR-30c-5p, miR-30e-3p, miR-374a-5p, miR-425-3p, miR-582-5p) were significantly prognostic of BCR. Of these, miR-148a-3p (HR = 0.80/95% CI = 0.68-0.94) and miR-582-5p (HR = 0.73/95% CI = 0.61-0.87) were also reported in prior publication(s) in the review.ConclusionsFifteen miRs were consistently associated with disease progression in multiple publications or datasets. Further research into their biological roles is warranted to support investigations into their performance as prognostic PCa biomarkers.

Journal article

Varshavi D, Varshavi D, McCarthy N, Veselkov K, Keun HC, Everett JRet al., 2021, Metabonomics study of the effects of single copy mutant KRAS in the presence or absence of WT allele using human HCT116 isogenic cell lines, Metabolomics, Vol: 17, Pages: 1-12, ISSN: 1573-3882

IntroductionKRAS was one of the earliest human oncogenes to be described and is one of the most commonly mutated genes in different human cancers, including colorectal cancer. Despite KRAS mutants being known driver mutations, KRAS has proved difficult to target therapeutically, necessitating a comprehensive understanding of the molecular mechanisms underlying KRAS-driven cellular transformation.ObjectivesTo investigate the metabolic signatures associated with single copy mutant KRAS in isogenic human colorectal cancer cells and to determine what metabolic pathways are affected.MethodsUsing NMR-based metabonomics, we compared wildtype (WT)-KRAS and mutant KRAS effects on cancer cell metabolism using metabolic profiling of the parental KRAS G13D/+ HCT116 cell line and its isogenic, derivative cell lines KRAS +/– and KRAS G13D/–.ResultsMutation in the KRAS oncogene leads to a general metabolic remodelling to sustain growth and counter stress, including alterations in the metabolism of amino acids and enhanced glutathione biosynthesis. Additionally, we show that KRASG13D/+ and KRASG13D/− cells have a distinct metabolic profile characterized by dysregulation of TCA cycle, up-regulation of glycolysis and glutathione metabolism pathway as well as increased glutamine uptake and acetate utilization.ConclusionsOur study showed the effect of a single point mutation in one KRAS allele and KRAS allele loss in an isogenic genetic background, hence avoiding confounding genetic factors. Metabolic differences among different KRAS mutations might play a role in their different responses to anticancer treatments and hence could be exploited as novel metabolic vulnerabilities to develop more effective therapies against oncogenic KRAS.

Journal article

de Prado-Bert P, Ruiz-Arenas C, Vives-Usano M, Andrusaityte S, Cadiou S, Carracedo Á, Casas M, Chatzi L, Dadvand P, González JR, Grazuleviciene R, Gutzkow KB, Haug LS, Hernandez-Ferrer C, Keun HC, Lepeule J, Maitre L, McEachan R, Nieuwenhuijsen MJ, Pelegrí D, Robinson O, Slama R, Vafeiadi M, Sunyer J, Vrijheid M, Bustamante Met al., 2021, The early-life exposome and epigenetic age acceleration in children, Environment International, Vol: 155, ISSN: 0160-4120

The early-life exposome influences future health and accelerated biological aging has been proposed as one of the underlying biological mechanisms. We investigated the association between more than 100 exposures assessed during pregnancy and in childhood (including indoor and outdoor air pollutants, built environment, green environments, tobacco smoking, lifestyle exposures, and biomarkers of chemical pollutants), and epigenetic age acceleration in 1,173 children aged 7 years old from the Human Early-Life Exposome project. Age acceleration was calculated based on Horvath’s Skin and Blood clock using child blood DNA methylation measured by Infinium HumanMethylation450 BeadChips. We performed an exposure-wide association study between prenatal and childhood exposome and age acceleration. Maternal tobacco smoking during pregnancy was nominally associated with increased age acceleration. For childhood exposures, indoor particulate matter absorbance (PMabs) and parental smoking were nominally associated with an increase in age acceleration. Exposure to the organic pesticide dimethyl dithiophosphate and the persistent pollutant polychlorinated biphenyl-138 (inversely associated with child body mass index) were protective for age acceleration. None of the associations remained significant after multiple-testing correction. Pregnancy and childhood exposure to tobacco smoke and childhood exposure to indoor PMabs may accelerate epigenetic aging from an early age.

Journal article

Vrijheid M, Basagaña X, Gonzalez JR, Jaddoe VWV, Jensen G, Keun HC, McEachan RRC, Porcel J, Siroux V, Swertz MA, Thomsen C, Aasvang GM, Andrušaitytė S, Angeli K, Avraam D, Ballester F, Burton P, Bustamante M, Casas M, Chatzi L, Chevrier C, Cingotti N, Conti D, Crépet A, Dadvand P, Duijts L, van Enckevort E, Esplugues A, Fossati S, Garlantezec R, Gómez Roig MD, Grazuleviciene R, Gützkow KB, Guxens M, Haakma S, Hessel EVS, Hoyles L, Hyde E, Klanova J, van Klaveren JD, Kortenkamp A, Le Brusquet L, Leenen I, Lertxundi A, Lertxundi N, Lionis C, Llop S, Lopez-Espinosa M-J, Lyon-Caen S, Maitre L, Mason D, Mathy S, Mazarico E, Nawrot T, Nieuwenhuijsen M, Ortiz R, Pedersen M, Perelló J, Pérez-Cruz M, Philippat C, Piler P, Pizzi C, Quentin J, Richiardi L, Rodriguez A, Roumeliotaki T, Sabin Capote JM, Santiago L, Santos S, Siskos AP, Strandberg-Larsen K, Stratakis N, Sunyer J, Tenenhaus A, Vafeiadi M, Wilson RC, Wright J, Yang T, Slama Ret al., 2021, Advancing tools for human early lifecourse exposome research and translation (ATHLETE). Project overview, Environmental Epidemiology, Vol: 5, ISSN: 2474-7882

Early life stages are vulnerable to environmental hazards and present important windows of opportunity for lifelong disease prevention. This makes early life a relevant starting point for exposome studies. The Advancing Tools for Human Early Lifecourse Exposome Research and Translation (ATHLETE) project aims to develop a toolbox of exposome tools and a Europe-wide exposome cohort that will be used to systematically quantify the effects of a wide range of community- and individual-level environmental risk factors on mental, cardiometabolic, and respiratory health outcomes and associated biological pathways, longitudinally from early pregnancy through to adolescence. Exposome tool and data development include as follows: (1) a findable, accessible, interoperable, reusable (FAIR) data infrastructure for early life exposome cohort data, including 16 prospective birth cohorts in 11 European countries; (2) targeted and nontargeted approaches to measure a wide range of environmental exposures (urban, chemical, physical, behavioral, social); (3) advanced statistical and toxicological strategies to analyze complex multidimensional exposome data; (4) estimation of associations between the exposome and early organ development, health trajectories, and biological (metagenomic, metabolomic, epigenetic, aging, and stress) pathways; (5) intervention strategies to improve early life urban and chemical exposomes, co-produced with local communities; and (6) child health impacts and associated costs related to the exposome. Data, tools, and results will be assembled in an openly accessible toolbox, which will provide great opportunities for researchers, policymakers, and other stakeholders, beyond the duration of the project. ATHLETE's results will help to better understand and prevent health damage from environmental exposures and their mixtures from the earliest parts of the life course onward.

Journal article

Dossus L, Kouloura E, Biessy C, Viallon V, Siskos AP, Dimou N, Rinaldi S, Merritt MA, Allen N, Fortner R, Kaaks R, Weiderpass E, Gram IT, Rothwell JA, Lécuyer L, Severi G, Schulze MB, Nøst TH, Crous-Bou M, Sánchez M-J, Amiano P, Colorado-Yohar SM, Gurrea AB, Schmidt JA, Palli D, Agnoli C, Tumino R, Sacerdote C, Mattiello A, Vermeulen R, Heath AK, Christakoudi S, Tsilidis KK, Travis RC, Gunter MJ, Keun HCet al., 2021, Prospective analysis of circulating metabolites and endometrial cancer risk, Gynecologic Oncology, Vol: 162, Pages: 475-481, ISSN: 0090-8258

AbstractBackgroundEndometrial cancer is strongly associated with obesity and dysregulation of metabolic factors such as estrogen and insulin signaling are causal risk factors for this malignancy. To identify additional novel metabolic pathways associated with endometrial cancer we performed metabolomic analyses on pre-diagnostic plasma samples from 853 case-control pairs from the European Prospective Investigation into Cancer and Nutrition (EPIC).MethodsA total of 129 metabolites (acylcarnitines, amino acids, biogenic amines, glycerophospholipids, hexoses, and sphingolipids) were measured by liquid chromatography-mass spectrometry. Conditional logistic regression estimated the associations of metabolites with endometrial cancer risk. An analysis focusing on clusters of metabolites using the bootstrap lasso method was also employed.ResultsAfter adjustment for body mass index, sphingomyelin [SM] C18:0 was positively (OR1SD: 1.18, 95% CI: 1.05–1.33), and glycine, serine, and free carnitine (C0) were inversely (OR1SD: 0.89, 95% CI: 0.80–0.99; OR1SD: 0.89, 95% CI: 0.79–1.00 and OR1SD: 0.91, 95% CI: 0.81–1.00, respectively) associated with endometrial cancer risk. Serine, C0 and two sphingomyelins were selected by the lasso method in >90% of the bootstrap samples. The ratio of esterified to free carnitine (OR1SD: 1.14, 95% CI: 1.02–1.28) and that of short chain to free acylcarnitines (OR1SD: 1.12, 95% CI: 1.00–1.25) were positively associated with endometrial cancer risk. Further adjustment for C-peptide or other endometrial cancer risk factors only minimally altered the results.ConclusionThese findings suggest that variation in levels of glycine, serine, SM C18:0 and free carnitine may represent specific pathways linked to endometrial cancer development. If causal, these pathways may offer novel targets for endometrial cancer prevention.

Journal article

Gallego-Paüls M, Hernández-Ferrer C, Bustamante M, Basagaña X, Barrera-Gómez J, Lau C-HE, Siskos AP, Vives-Usano M, Ruiz-Arenas C, Wright J, Slama R, Heude B, Casas M, Grazuleviciene R, Chatzi L, Borràs E, Sabidó E, Carracedo Á, Estivill X, Urquiza J, Coen M, Keun HC, González JR, Vrijheid M, Maitre Let al., 2021, Variability of multi-omics profiles in a population-based child cohort., BMC Medicine, Vol: 19, Pages: 1-16, ISSN: 1741-7015

BACKGROUND: Multiple omics technologies are increasingly applied to detect early, subtle molecular responses to environmental stressors for future disease risk prevention. However, there is an urgent need for further evaluation of stability and variability of omics profiles in healthy individuals, especially during childhood. METHODS: We aimed to estimate intra-, inter-individual and cohort variability of multi-omics profiles (blood DNA methylation, gene expression, miRNA, proteins and serum and urine metabolites) measured 6 months apart in 156 healthy children from five European countries. We further performed a multi-omics network analysis to establish clusters of co-varying omics features and assessed the contribution of key variables (including biological traits and sample collection parameters) to omics variability. RESULTS: All omics displayed a large range of intra- and inter-individual variability depending on each omics feature, although all presented a highest median intra-individual variability. DNA methylation was the most stable profile (median 37.6% inter-individual variability) while gene expression was the least stable (6.6%). Among the least stable features, we identified 1% cross-omics co-variation between CpGs and metabolites (e.g. glucose and CpGs related to obesity and type 2 diabetes). Explanatory variables, including age and body mass index (BMI), explained up to 9% of serum metabolite variability. CONCLUSIONS: Methylation and targeted serum metabolomics are the most reliable omics to implement in single time-point measurements in large cross-sectional studies. In the case of metabolomics, sample collection and individual traits (e.g. BMI) are important parameters to control for improved comparability, at the study design or analysis stage. This study will be valuable for the design and interpretation of epidemiological studies that aim to link omics signatures to disease, environmental exposures, or both.

Journal article

Halford SER, Walter H, McKay P, Townsend W, Linton K, Heinzmann K, Dragoni I, Brotherton L, Veal G, Siskos A, Keun HC, Bacon C, Wedge S, Dyer MJS, Plummer ERet al., 2021, Phase I expansion study of the first-in-class monocarboxylate transporter 1 (MCT1) inhibitor AZD3965 in patients with diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL)., ASCO Annual Meeting I, Publisher: American Society of Clinical Oncology, Pages: 3115-3115, ISSN: 0732-183X

Background: Tumours rely on lactate transporters (MCT1-4) to maintain glycolytic flux and avoid intracellular acidification. In haematological tumours the MCT1 transporter acts as a lactate and pyruvate exporter. AZD3965 is a potent and specific inhibitor of MCT1 causing intracellular lactate accumulation. In vivo efficacy was observed in xenografts of DLBCL and BL, expressing high levels of MCT1 and no or low levels of MCT4. In the AZD3965 phase I (NCT01791595) dose-escalation an oral recommended phase 2 dose (RP2D) of 10mg twice-daily (bd) was determined. Pharmacokinetic (PK) showed exposure estimated to produce a minimum MCT1 occupancy of 90% (based on modelling). DLTs were primarily on-target dose-dependent, reversible, asymptomatic alterations in retinal function seen on ERG. Methods: This expansion cohort enrolled patients with relapsed/refractory DLBCL and BL. Expression of MCT1/MCT4 was assessed by immunohistochemistry. Pharmacokinetic (PK) sampling was performed and pharmacodynamic assessments included [18F]FDG-PET/CT imaging and plasma/urine metabolomics. Results: 11 DLBCL patients were treated with AZD3965 10mg bd. PK showed exposure to be broadly in line with the escalation cohort. No significant ERG changes were observed. One patient experienced a dose-limiting SUSAR of Troponin I increase. MCT1 is expressed in erythrocytes, however no serious events of anaemia were seen, with one non-clinically significant episode of grade 3 anaemia reported. Urine analysis showed increased excretion of lactate and ketone bodies post AZD3965 treatment consistent with renal target engagement. No alteration was detected in plasma. Ongoing stable disease at cycle 5 was observed in one patient and an additional patient had a confirmed complete response (CR) lasting 15 months, with no significant toxicity. In the patient showing CR a reduction in tumour FDG uptake was observed on day 3 of cycle 1. The other four patients who consented to research imaging did not have a clin

Conference paper

Kliemann N, Viallon V, Murphy N, Beeken RJ, Rothwell JA, Rinaldi S, Assi N, van Roekel EH, Schmidt JA, Borch KB, Agnoli C, Rosendahl AH, Sartor H, Huerta JM, Tjonneland A, Halkjaer J, Bueno-de-Mesquita B, Gicquiau A, Achaintre D, Aleksandrova K, Schulze MB, Heath AK, Tsilidis KK, Masala G, Panico S, Kaaks R, Fortner RT, Van Guelpen B, Dossus L, Scalbert A, Keun HC, Travis RC, Jenab M, Johansson M, Ferrari P, Gunter MJet al., 2021, Metabolic signatures of greater body size and their associations with risk of colorectal and endometrial cancers in the European Prospective Investigation into Cancer and Nutrition, BMC Medicine, Vol: 19, ISSN: 1741-7015

Background:The mechanisms underlying the obesity-cancer relationship are incompletely understood. This study aimed to characterise metabolic signatures of greater body size and to investigate their association with two obesity-related malignancies, endometrial and colorectal cancers, and with weight loss within the context of an intervention study.Methods:Targeted mass spectrometry metabolomics data from 4326 participants enrolled in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort and 17 individuals from a single-arm pilot weight loss intervention (Intercept) were used in this analysis. Metabolic signatures of body size were first determined in discovery (N = 3029) and replication (N = 1297) sets among EPIC participants by testing the associations between 129 metabolites and body mass index (BMI), waist circumference (WC), and waist-to-hip ratio (WHR) using linear regression models followed by partial least squares analyses. Conditional logistic regression models assessed the associations between the metabolic signatures with endometrial (N = 635 cases and 648 controls) and colorectal (N = 423 cases and 423 controls) cancer risk using nested case-control studies in EPIC. Pearson correlation between changes in the metabolic signatures and weight loss was tested among Intercept participants.Results:After adjustment for multiple comparisons, greater BMI, WC, and WHR were associated with higher levels of valine, isoleucine, glutamate, PC aa C38:3, and PC aa C38:4 and with lower levels of asparagine, glutamine, glycine, serine, lysoPC C17:0, lysoPC C18:1, lysoPC C18:2, PC aa C42:0, PC ae C34:3, PC ae C40:5, and PC ae C42:5. The metabolic signature of BMI (OR1-sd 1.50, 95% CI 1.30–1.74), WC (OR1-sd 1.46, 95% CI 1.27–1.69), and WHR (OR1-sd 1.54, 95% CI 1.33–1.79) were each associated with endometrial cancer risk. Risk of colorectal cancer was positively associated with

Journal article

Saavedra-Garcia P, Roman-Trufero M, Al-Sadah HA, Blighe K, Lopez-Jimenez E, Christoforou M, Penfold L, Capece D, Xiong X, Miao Y, Parzych K, Caputo V, Siskos AP, Encheva V, Liu Z, Thiel D, Kaiser MF, Piazza P, Chaidos A, Karadimitris A, Franzoso G, Snijder AP, Keun HC, Oyarzún DA, Barahona M, Auner Het al., 2021, Systems level profiling of chemotherapy-induced stress resolution in cancer cells reveals druggable trade-offs, Proceedings of the National Academy of Sciences of USA, Vol: 118, ISSN: 0027-8424

Cancer cells can survive chemotherapy-induced stress, but how they recover from it is not known.Using a temporal multiomics approach, we delineate the global mechanisms of proteotoxic stressresolution in multiple myeloma cells recovering from proteasome inhibition. Our observations definelayered and protracted programmes for stress resolution that encompass extensive changes acrossthe transcriptome, proteome, and metabolome. Cellular recovery from proteasome inhibitioninvolved protracted and dynamic changes of glucose and lipid metabolism and suppression ofmitochondrial function. We demonstrate that recovering cells are more vulnerable to specific insultsthan acutely stressed cells and identify the general control nonderepressable 2 (GCN2)-driven cellularresponse to amino acid scarcity as a key recovery-associated vulnerability. Using a transcriptomeanalysis pipeline, we further show that GCN2 is also a stress-independent bona fide target intranscriptional signature-defined subsets of solid cancers that share molecular characteristics. Thus,identifying cellular trade-offs tied to the resolution of chemotherapy-induced stress in tumour cellsmay reveal new therapeutic targets and routes for cancer therapy optimisation.

Journal article

Capece D, D'Andrea D, Begalli F, Goracci L, Tornatore L, Alexander JL, Di Veroli A, Leow S-C, Vaiyapuri TS, Ellis JK, Verzella D, Bennett J, Savino L, Ma Y, McKenzie JS, Doria ML, Mason SE, Chng KR, Keun HC, Frost G, Tergaonkar V, Broniowska K, Stunkel W, Takats Z, Kinross JM, Cruciani G, Franzoso Get al., 2021, Enhanced triacylglycerol catabolism by Carboxylesterase 1 promotes aggressive colorectal carcinoma., Journal of Clinical Investigation, ISSN: 0021-9738

The ability to adapt to low-nutrient microenvironments is essential for tumor-cell survival and progression in solid cancers, such as colorectal carcinoma (CRC). Signaling by the NF-κB transcription-factor pathway associates with advanced disease stages and shorter survival in CRC patients. NF-κB has been shown to drive tumor-promoting inflammation, cancer-cell survival and intestinal epithelial cell (IEC) dedifferentiation in mouse models of CRC. However, whether NF-κB affects the metabolic adaptations that fuel aggressive disease in CRC patients is unknown. Here, we identified carboxylesterase 1 (CES1) as an essential NF-κB-regulated lipase linking obesity-associated inflammation with fat metabolism and adaptation to energy stress in aggressive CRC. CES1 promoted CRC-cell survival via cell-autonomous mechanisms that fuel fatty-acid oxidation (FAO) and prevent the toxic build-up of triacylglycerols. We found that elevated CES1 expression correlated with worse outcomes in overweight CRC patients. Accordingly, NF-κB drove CES1 expression in CRC consensus molecular subtype (CMS)4, associated with obesity, stemness and inflammation. CES1 was also upregulated by gene amplifications of its transcriptional regulator, HNF4A, in CMS2 tumors, reinforcing its clinical relevance as a driver of CRC. This subtype-based distribution and unfavourable prognostic correlation distinguished CES1 from other intracellular triacylglycerol lipases and suggest CES1 could provide a route to treat aggressive CRC.

Journal article

Dusad V, Thiel D, Barahona M, Keun H, Oyarzun Det al., 2021, Opportunities at the interface of network science and metabolic modelling, Frontiers in Bioengineering and Biotechnology, Vol: 8, ISSN: 2296-4185

Metabolism plays a central role in cell physiology because it provides the molecular machinery for growth. At the genome-scale, metabolism is made up of thousands of reactions interacting with one another. Untangling this complexity is key to understand how cells respond to genetic, environmental, or therapeutic perturbations. Here we discuss the roles of two complementary strategies for the analysis of genome-scale metabolic models: Flux Balance Analysis (FBA) and network science. While FBA estimates metabolic flux on the basis of an optimization principle, network approaches reveal emergent properties of the global metabolic connectivity. We highlight how the integration of both approaches promises to deliver insights on the structure and function of metabolic systems with wide-ranging implications in discovery science, precision medicine and industrial biotechnology.

Journal article

Calvo-Serra B, Maitre L, Lau C-HE, Siskos AP, Gutzkow KB, Andrusaityte S, Casas M, Cadiou S, Chatzi L, Gonzalez JR, Grazuleviciene R, McEachan R, Slama R, Vafeiadi M, Wright J, Coen M, Vrijheid M, Keun HC, Escaramis G, Bustamante Met al., 2020, Urinary metabolite quantitative trait loci in children and their interaction with dietary factors, Human Molecular Genetics, Vol: 29, Pages: 3830-3844, ISSN: 0964-6906

Human metabolism is influenced by genetic and environmental factors. Previous studies have identified over 23 loci associated with more than 26 urine metabolites levels in adults, which are known as urinary metabolite quantitative trait loci (metabQTLs). The aim of the present study is the identification for the first time of urinary metabQTLs in children and their interaction with dietary patterns. Association between genome-wide genotyping data and 44 urine metabolite levels measured by proton nuclear magnetic resonance spectroscopy was tested in 996 children from the Human Early Life Exposome project. Twelve statistically significant urine metabQTLs were identified, involving 11 unique loci and 10 different metabolites. Comparison with previous findings in adults revealed that six metabQTLs were already known, and one had been described in serum and three were involved the same locus as other reported metabQTLs but had different urinary metabolites. The remaining two metabQTLs represent novel urine metabolite-locus associations, which are reported for the first time in this study [single nucleotide polymorphism (SNP) rs12575496 for taurine, and the missense SNP rs2274870 for 3-hydroxyisobutyrate]. Moreover, it was found that urinary taurine levels were affected by the combined action of genetic variation and dietary patterns of meat intake as well as by the interaction of this SNP with beverage intake dietary patterns. Overall, we identified 12 urinary metabQTLs in children, including two novel associations. While a substantial part of the identified loci affected urinary metabolite levels both in children and in adults, the metabQTL for taurine seemed to be specific to children and interacted with dietary patterns.

Journal article

Robinson O, 2020, Prenatal exposure to perfluoroalkyl substances associated with increased susceptibility to liver Injury in children, Hepatology, Vol: 72, Pages: 1758-1770, ISSN: 0270-9139

Background and AimsPer‐ and polyfluoroalkyl substances (PFAS) are widespread and persistent pollutants that have been shown to have hepatotoxic effects in animal models. However, human evidence is scarce. We evaluated how prenatal exposure to PFAS associates with established serum biomarkers of liver injury and alterations in serum metabolome in children.Approach and ResultsWe used data from 1,105 mothers and their children (median age, 8.2 years; interquartile range, 6.6‐9.1) from the European Human Early‐Life Exposome cohort (consisting of six existing population‐based birth cohorts in France, Greece, Lithuania, Norway, Spain, and the United Kingdom). We measured concentrations of perfluorooctane sulfonate, perfluorooctanoate, perfluorononanoate, perfluorohexane sulfonate, and perfluoroundecanoate in maternal blood. We assessed concentrations of alanine aminotransferase, aspartate aminotransferase, and gamma‐glutamyltransferase in child serum. Using Bayesian kernel machine regression, we found that higher exposure to PFAS during pregnancy was associated with higher liver enzyme levels in children. We also measured child serum metabolomics through a targeted assay and found significant perturbations in amino acid and glycerophospholipid metabolism associated with prenatal PFAS. A latent variable analysis identified a profile of children at high risk of liver injury (odds ratio, 1.56; 95% confidence interval, 1.21‐1.92) that was characterized by high prenatal exposure to PFAS and increased serum levels of branched‐chain amino acids (valine, leucine, and isoleucine), aromatic amino acids (tryptophan and phenylalanine), and glycerophospholipids (phosphatidylcholine [PC] aa C36:1 and Lyso‐PC a C18:1).ConclusionsDevelopmental exposure to PFAS can contribute to pediatric liver injury.

Journal article

Benito A, Hajji N, O'Neill K, Keun HC, Syed Net al., 2020, β-Hydroxybutyrate oxidation promotes the accumulation of immunometabolites in activated microglia cells, Metabolites, Vol: 10, ISSN: 2218-1989

Metabolic regulation of immune cells has arisen as a critical set of processes required for appropriate response to immunological signals. While our knowledge in this area has rapidly expanded in leukocytes, much less is known about the metabolic regulation of brain-resident microglia. In particular, the role of alternative nutrients to glucose remains poorly understood. Here, we use stable-isotope (13C) tracing strategies and metabolomics to characterize the oxidative metabolism of β-hydroxybutyrate (BHB) in human (HMC3) and murine (BV2) microglia cells and the interplay with glucose in resting and LPS-activated BV2 cells. We found that BHB is imported and oxidised in the TCA cycle in both cell lines with a subsequent increase in the cytosolic NADH:NAD+ ratio. In BV2 cells, stimulation with LPS upregulated the glycolytic flux, increased the cytosolic NADH:NAD+ ratio and promoted the accumulation of the glycolytic intermediate dihydroxyacetone phosphate (DHAP). The addition of BHB enhanced LPS-induced accumulation of DHAP and promoted glucose-derived lactate export. BHB also synergistically increased LPS-induced accumulation of succinate and other key immunometabolites, such as α-ketoglutarate and fumarate generated by the TCA cycle. Finally, BHB upregulated the expression of a key pro-inflammatory (M1 polarisation) marker gene, NOS2, in BV2 cells activated with LPS. In conclusion, we identify BHB as a potentially immunomodulatory metabolic substrate for microglia that promotes metabolic reprogramming during pro-inflammatory response.

Journal article

Robinson O, 2020, In utero and childhood exposure to tobacco smoke and multi-layer molecular signatures in children, BMC Medicine, Vol: 18, Pages: 1-19, ISSN: 1741-7015

BackgroundThe adverse health effects of early life exposure to tobacco smoking have been widely reported. In spite of this, the underlying molecular mechanisms of in utero and postnatal exposure to tobacco smoke are only partially understood. Here, we aimed to identify multi-layer molecular signatures associated with exposure to tobacco smoke in these two exposure windows.MethodsWe investigated the associations of maternal smoking during pregnancy and childhood secondhand smoke (SHS) exposure with molecular features measured in 1203 European children (mean age 8.1 years) from the Human Early Life Exposome (HELIX) project. Molecular features, covering 4 layers, included blood DNA methylation and gene and miRNA transcription, plasma proteins, and sera and urinary metabolites.ResultsMaternal smoking during pregnancy was associated with DNA methylation changes at 18 loci in child blood. DNA methylation at 5 of these loci was related to expression of the nearby genes. However, the expression of these genes themselves was only weakly associated with maternal smoking. Conversely, childhood SHS was not associated with blood DNA methylation or transcription patterns, but with reduced levels of several serum metabolites and with increased plasma PAI1 (plasminogen activator inhibitor-1), a protein that inhibits fibrinolysis. Some of the in utero and childhood smoking-related molecular marks showed dose-response trends, with stronger effects with higher dose or longer duration of the exposure.ConclusionIn this first study covering multi-layer molecular features, pregnancy and childhood exposure to tobacco smoke were associated with distinct molecular phenotypes in children. The persistent and dose-dependent changes in the methylome make CpGs good candidates to develop biomarkers of past exposure. Moreover, compared to methylation, the weak association of maternal smoking in pregnancy with gene expression suggests different reversal rates and a methylation-based memory to

Journal article

Braga M, Kaliszczak M, Carroll L, Schug ZT, Heinzmann K, Baxan N, Benito A, Valbuena GN, Stribbling S, Beckley A, Mackay G, Mauri F, Latigo J, Barnes C, Keun H, Gottlieb E, Aboagye EOet al., 2020, Tracing nutrient flux following monocarboxylate transporter-1 inhibition with AZD3965., Cancers (Basel), Vol: 12, ISSN: 2072-6694

The monocarboxylate transporter 1 (MCT1) is a key element in tumor cell metabolism and inhibition of MCT1 with AZD3965 is undergoing clinical trials. We aimed to investigate nutrient fluxes associated with MCT1 inhibition by AZD3965 to identify possible biomarkers of drug action. We synthesized an 18F-labeled lactate analogue, [18F]-S-fluorolactate ([18F]-S-FL), that was used alongside [18F]fluorodeoxyglucose ([18F]FDG), and 13C-labeled glucose and lactate, to investigate the modulation of metabolism with AZD3965 in diffuse large B-cell lymphoma models in NOD/SCID mice. Comparative analysis of glucose and lactate-based probes showed a preference for glycolytic metabolism in vitro, whereas in vivo, both glucose and lactate were used as metabolic fuel. While intratumoral L-[1-13C]lactate and [18F]-S-FL were unchanged or lower at early (5 or 30 min) timepoints, these variables were higher compared to vehicle controls at 4 h following treatment with AZD3965, which indicates that inhibition of MCT1-mediated lactate import is reversed over time. Nonetheless, AZD3965 treatment impaired DLBCL tumor growth in mice. This was hypothesized to be a consequence of metabolic strain, as AZD3965 treatment showed a reduction in glycolytic intermediates and inhibition of the TCA cycle likely due to downregulated PDH activity. Glucose ([18F]FDG and D-[13C6]glucose) and lactate-based probes ([18F]-S-FL and L-[1-13C]lactate) can be successfully used as biomarkers for AZD3965 treatment.

Journal article

Varshavi D, Varshavi D, McCarthy N, Veselkov K, Keun HC, Everett JRet al., 2020, Metabolic characterization of colorectal cancer cells harbouring different KRAS mutations in codon 12, 13, 61 and 146 using human SW48 isogenic cell lines, Metabolomics, Vol: 16, Pages: 1-13, ISSN: 1573-3882

IntroductionKirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) mutations occur in approximately one-third of colorectal (CRC) tumours and have been associated with poor prognosis and resistance to some therapeutics. In addition to the well-documented pro-tumorigenic role of mutant Ras alleles, there is some evidence suggesting that not all KRAS mutations are equal and the position and type of amino acid substitutions regulate biochemical activity and transforming capacity of KRAS mutations.ObjectivesTo investigate the metabolic signatures associated with different KRAS mutations in codons 12, 13, 61 and 146 and to determine what metabolic pathways are affected by different KRAS mutations.MethodsWe applied an NMR-based metabonomics approach to compare the metabolic profiles of the intracellular extracts and the extracellular media from isogenic human SW48 CRC cell lines with different KRAS mutations in codons 12 (G12D, G12A, G12C, G12S, G12R, G12V), 13 (G13D), 61 (Q61H) and 146 (A146T) with their wild-type counterpart. We used false discovery rate (FDR)-corrected analysis of variance (ANOVA) to determine metabolites that were statistically significantly different in concentration between the different mutants.ResultsCRC cells carrying distinct KRAS mutations exhibited differential metabolic remodelling, including differences in glycolysis, glutamine utilization and in amino acid, nucleotide and hexosamine metabolism.ConclusionsMetabolic differences among different KRAS mutations might play a role in their different responses to anticancer treatments and hence could be exploited as novel metabolic vulnerabilities to develop more effective therapies against oncogenic KRAS.

Journal article

McNeillis R, Greystoke A, Walton J, Bacon C, Keun H, Sitkos A, Petrides G, Leech N, Jenkinson F, Bowron A, Halford S, Plummer Ret al., 2020, A case of malignant hyperlactaemic acidosis appearing upon treatment with the mono-carboxylase transporter 1 inhibitor AZD3965, British Journal of Cancer, Vol: 122, Pages: 1141-1145, ISSN: 0007-0920

A 47-year-old man with metastatic melanoma presented with refractory hyperlactaemic acidosis following the first dose of the mono-carboxylase transporter 1 inhibitor AZD3965 within a "first time in man" clinical trial. The mechanism of the agent and the temporal relationship suggested that this event was potentially drug related and recruitment was suspended. However, urinary metabolomics showed extensive abnormalities even prior to drug administration, leading to investigations for an underlying metabolic disorder. The lack of clinical symptoms from the elevated lactate and low blood glucose suggested a diagnosis of "hyper-Warburgism", where the high tumour burden was associated with extensive glucose uptake and lactate efflux from malignant cells, and the subsequent impact on blood biochemistry. This was supported by an FDG-PET scan showing extensive glucose uptake in numerous metastases and lack of uptake in the brain. A review of the literature showed 16 case reports of "hyper-Warburgism" in non-haematological malignancies, none of them with melanoma, with most associated with a poor outcome. The patient was treated symptomatically, but died 2 months later. The development of AZD3965 continues with the exclusion of patients with elevated plasma lactate at screening added to the protocol as a safety measure. Trial identification number ClinicalTrials.Gov. NCT01791595.

Journal article

Barnes EME, Xu Y, Benito A, Herendi L, Siskos AP, Aboagye EO, Keun H, Nijhuis Aet al., 2020, Lactic acidosis induces resistance to the pan-Akt inhibitor uprosertib in colon cancer cells, British Journal of Cancer, Vol: 122, Pages: 1298-1308, ISSN: 0007-0920

BackgroundAkt signalling regulates glycolysis and drives the Warburg effect in cancer, thus decreased glucose utilisation is a pharmacodynamic marker of Akt inhibition. However, cancer cells can utilise alternative nutrients to glucose for energy such as lactate, which is often elevated in tumours together with increased acidity. We therefore hypothesised that lactic acidosis may confer resistance to Akt inhibition.MethodsThe effect of the pan-Akt inhibitor uprosertib (GSK2141795), on HCT116 and LS174T colon cancer cells was evaluated in the presence and absence of lactic acid in vitro. Expression of downstream Akt signalling proteins was determined using a phosphokinase array and immunoblotting. Metabolism was assessed using 1H nuclear magnetic resonance spectroscopy, stable isotope labelling and gas chromatography-mass spectrometry.ResultsLactic acid-induced resistance to uprosertib was characterised by increased cell survival and reduced apoptosis. Uprosertib treatment reduced Akt signalling and glucose uptake irrespective of lactic acid supplementation. However, incorporation of lactate carbon and enhanced respiration was maintained in the presence of uprosertib and lactic acid. Inhibiting lactate transport or oxidative phosphorylation was sufficient to potentiate apoptosis in the presence of uprosertib.ConclusionsLactic acidosis confers resistance to uprosertib, which can be reversed by inhibiting lactate transport or oxidative metabolism.

Journal article

Valbuena G, Keun H, Bendotti C, Cantoni L, Tortarolo Met al., 2019, Spinal cord metabolic signatures in models of fast- and slow-progressing SOD1G93A Amyotrophic Lateral Sclerosis, Frontiers in Neuroscience, Vol: 13, Pages: 1-16, ISSN: 1662-453X

The rate of disease progression in amyotrophic lateral sclerosis (ALS) is highly variable, even between patients with the same genetic mutations. Metabolic alterations may affect disease course variability in ALS patients, but challenges in identifying the preclinical and early phases of the disease limit our understanding of molecular mechanisms underlying differences in the rate of disease progression. We examined effects of SOD1G93A on thoracic and lumbar spinal cord metabolites in two mouse ALS models with different rates of disease progression: the transgenic SOD1G93A-C57BL/6JOlaHsd (C57-G93A, slow progression) and transgenic SOD1G93A-129SvHsd (129S-G93A, fast progression) strains. Samples from three timepoints (presymptomatic, disease onset, and late stage disease) were analyzed using Gas Chromatography-Mass Spectrometry metabolomics. Tissue metabolome differences in the lumbar spinal cord were driven primarily by mouse genetic background, although larger responses were observed in metabolic trajectories after the onset of symptoms. The significantly affected lumbar spinal cord metabolites were involved in energy and lipid metabolism. In the thoracic spinal cord, metabolic differences related to genetic background, background-SOD1 genotype interactions, and longitudinal SOD1G93A effects. The largest responses in thoracic spinal cord metabolic trajectories related to SOD1G93A effects before onset of visible symptoms. More metabolites were significantly affected in the thoracic segment, which were involved in energy homeostasis, neurotransmitter synthesis and utilization, and the oxidative stress response. We find evidence that initial metabolic alterations in SOD1G93A mice confer disadvantages for maintaining neuronal viability under ALS-related stressors, with slow-progressing C57-G93A mice potentially having more favorable spinal cord bioenergetic profiles than 129S-G93A. These genetic background-associated metabolic differences together with the different ea

Journal article

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