Natural killer (NK) cells are a population of innate effector lymphocytes, involved in host-defences against viral infections and cancer. Upon activation, NK cells can produce a milieu of cytotoxic molecules and cytokines, which can directly target infected and transformed cells, but also amplify an immune response. Metabolic rewiring underpins NK cell effector functionality, providing the required signals, energy and biointermediates to support their immune responses. Obesity is associated with significant defects in the functionality of human NK cells, especially in the periphery. Dysregulated cellular metabolism has been demonstrated to be a major mechanistic driver of the reported defects. However, how obesity links to defective NK cell metabolism and functionality remains unclear. Iron deficiency is a common co-morbidity in people living with obesity (PWO). Recent studies have highlighted the importance for iron in host immunity, with murine models of iron deficiency resulting in defective cellular metabolism and function. We hypothesized that obesity-driven iron deficiency might underpin the reported defects in NK cells. Our data demonstrates that in response to cytokine stimulation, healthy human NK cells utilize iron to support their metabolic activity and cytokine responses. In a cohort of PWO, we demonstrate alterations in NK cell metabolism, mitochondrial fitness and cytokine production. Furthermore, upon stratification into PWO with normal iron status versus low iron status, we show the observed obesity-related defects in NK cell metabolism, mitochondrial fitness and cytokine production are concentrated in the PWO with low-iron status. Collectively, our data highlights the importance of iron for human NK cell responses and provides evidence that obesity-driven defects in NK cell metabolism and function are linked in part to altered iron availability.### Competing Interest StatementThe authors have declared no competing interest.

The pyruvate transporter MPC1 (mitochondrial pyruvate carrier 1) acts as a tumour-suppressor, loss of which correlates with a pro-tumorigenic phenotype and poor survival in several tumour types. In high-grade serous ovarian cancers (HGSOC), patients display copy number loss of MPC1 in around 78% of cases and reduced MPC1 mRNA expression. To explore the metabolic effect of reduced expression, we demonstrate that depleting MPC1 in HGSOC cell lines drives expression of key proline biosynthetic genes; PYCR1, PYCR2 and PYCR3, and biosynthesis of proline. We show that altered proline metabolism underpins cancer cell proliferation, reactive oxygen species (ROS) production, and type I and type VI collagen formation in ovarian cancer cells. Furthermore, exploring The Cancer Genome Atlas, we discovered the PYCR3 isozyme to be highly expressed in a third of HGSOC patients, which was associated with more aggressive disease and diagnosis at a younger age. Taken together, our study highlights that targeting proline metabolism is a potential therapeutic avenue for the treatment of HGSOC.

Summary statistics for all FAP and MAP patient-derived intestinal samples analysed in this study. Data sets are from two centres, Cardiff University (CU) and MD Anderson Cancer Center (MDACC). Statistically significant P-values < 0.05 are in bold.

Colorectal cancer (CRC) is a multi-stage process initiated through the formation of a benign adenoma, progressing to an invasive carcinoma and finally metastatic spread. Tumour cells must adapt their metabolism to support the energetic and biosynthetic demands associated with disease progression. As such, targeting cancer cell metabolism is a promising therapeutic avenue in CRC. However, to identify tractable nodes of metabolic vulnerability specific to CRC stage, we must understand how metabolism changes during CRC development. Here, we use a unique model system – comprising human early adenoma to late adenocarcinoma. We show that adenoma cells transition to elevated glycolysis at the early stages of tumour progression but maintain oxidative metabolism. Progressed adenocarcinoma cells rely more on glutamine-derived carbon to fuel the TCA cycle, whereas glycolysis and TCA cycle activity remain tightly coupled in early adenoma cells. Adenocarcinoma cells are more flexible with respect to fuel source, enabling them to proliferate in nutrient-poor environments. Despite this plasticity, we identify asparagine (ASN) synthesis as a node of metabolic vulnerability in late-stage adenocarcinoma cells. We show that loss of asparagine synthetase (ASNS) blocks their proliferation, whereas early adenoma cells are largely resistant to ASN deprivation. Mechanistically, we show that late-stage adenocarcinoma cells are dependent on ASNS to support mTORC1 signalling and maximal glycolytic and oxidative capacity. Resistance to ASNS loss in early adenoma cells is likely due to a feedback loop, absent in late-stage cells, allowing them to sense and regulate ASN levels and supplement ASN by autophagy. Together, our study defines metabolic changes during CRC development and highlights ASN synthesis as a targetable metabolic vulnerability in later stage disease.

A VCF file containing putatively pathogenic variants found in 1142 intestinal cancer associated genes in 21 of the duodenal adenomas studied here (see supplementary table 1 for sample details). Full genomic datasets for these samples are available through EGA under accession: EGAD00001009332.

About 50% of poor prognosis neuroblastoma arises due to MYCN over-expression. We previously demonstrated that MYCN and PRMT5 proteins interact and PRMT5 knockdown led to apoptosis of MYCN amplified (MNA) neuroblastoma. Here we evaluate PRMT5 inhibitors GSK3203591/GSK3326593 as targeted therapeutics for MNA neuroblastoma and show MYCN-dependent growth inhibition and apoptosis. RNAseq revealed dysregulated MYCN transcriptional programmes and altered mRNA splicing, converging on key regulatory pathways such as DNA damage response, epitranscriptomics and cellular metabolism. Metabolic tracing showed glutamine metabolism was impeded following GSK3203591 treatment, which disrupted the MLX/Mondo nutrient sensors via intron retention of MLX mRNA. Glutaminase (GLS) protein was decreased by GSK3203591 despite unchanged transcript levels, suggesting post-transcriptional regulation. We demonstrate the RNA methyltransferase METTL3 and cognate reader YTHDF3 proteins are lowered following splicing alterations; accordingly, we observed hypomethylation of GLS mRNA and decreased GLS following YTHDF3 knockdown. In vivo efficacy of GSK3326593 was confirmed by increased survival of Th-MYCN mice together with splicing events and protein decreases consistent with in vitro data. Our study supports the spliceosome as a key vulnerability of MNA neuroblastoma and rationalises PRMT5 inhibition as a targeted therapy. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

Mucosal-Associated Invariant T (MAIT) cells are a population of innate T cells that play a critical role in host protection against bacterial and viral pathogens. Upon activation, MAIT cells can rapidly respond via both TCR-dependent and -independent mechanisms, resulting in robust cytokine production. The metabolic and nutritional requirements for optimal MAIT cell effector responses are still emerging. Iron is an important micronutrient and is essential for cellular fitness, in particular cellular metabolism. Iron is also critical for many pathogenic microbes, including those that activate MAIT cells. However, iron has not been investigated with respect to MAIT cell metabolic or functional responses. In this study, we show that human MAIT cells require exogenous iron, transported via CD71 for optimal metabolic activity in MAIT cells, including their production of ATP. We demonstrate that restricting iron availability by either chelating environmental iron or blocking CD71 on MAIT cells results in impaired cytokine production and proliferation. These data collectively highlight the importance of a CD71-iron axis for human MAIT cell metabolism and functionality, an axis that may have implications in conditions where iron availability is limited.

Approximately 50% of poor prognosis neuroblastomas arise due to MYCN over-expression. We previously demonstrated that MYCN and PRMT5 proteins interact and PRMT5 knockdown led to apoptosis of MYCN amplified (MNA) neuroblastoma. Here we evaluate the highly selective first-in-class PRMT5 inhibitor GSK3203591 and its in vivo analogue GSK3326593 as targeted therapeutics for MNA neuroblastoma. Cell-line analyses show MYCN-dependent growth inhibition and apoptosis, with approximately 200-fold greater sensitivity of MNA neuroblastoma lines. RNA sequencing of three MNA neuroblastoma lines treated with GSK3203591 reveal deregulated MYCN transcriptional programmes and altered mRNA splicing, converging on key regulatory pathways such as DNA damage response, epitranscriptomics and cellular metabolism. Stable isotope labelling experiments in the same cell lines demonstrate that glutamine metabolism is impeded following GSK3203591 treatment, linking with disruption of the MLX/Mondo nutrient sensors via intron retention of MLX mRNA. Interestingly, glutaminase (GLS) protein decreases after GSK3203591 treatment despite unchanged transcript levels. We demonstrate that the RNA methyltransferase METTL3 and cognate reader YTHDF3 proteins are lowered following their mRNAs undergoing GSK3203591-induced splicing alterations, indicating epitranscriptomic regulation of GLS; accordingly, we observe decreases of GLS mRNA m6A methylation following GSK3203591 treatment, and decreased GLS protein following YTHDF3 knockdown. In vivo efficacy of GSK3326593 is confirmed by increased survival of Th-MYCN mice, with drug treatment triggering splicing events and protein decreases consistent with in vitro data. Together our study demonstrates the PRMT5-dependent spliceosomal vulnerability of MNA neuroblastoma and identifies the epitranscriptome and glutamine metabolism as critical determinants of this sensitivity.

RNASeq WTS pipeline validation by quantitative RT-PCR (qRT-PCR) and IHC A. Comparison of normalised RNA-Seq read counts (orange) to relative qRT-PCR expression levels (purple) for four top DEGs. Δ CT values are shown, with low values indicative of low mRNA abundances. High concordance between the two methods can be observed. qRT-PCR confirms increased levels of S100P and KRT7 and decreased levels of FCER2 and CA4 mRNA in duodenal adenomas in comparison to normal duodenal mucosa samples (purple bars). B. IHC results for S100 and CK7 expression in duodenal normal mucosa or duodenal adenoma tissue sections. Moderate and relatively strong positive staining for CK7 and S100, respectively, was also confirmed in duodenal adenomas compared to corresponding normal duodenal mucosa samples. Representative samples that had undergone RNA-Seq are shown. Scale bar: 50μm. COL=Colorectal samples; DUO=Duodenal samples

A VCF file containing putatively pathogenic variants found in 1142 intestinal cancer associated genes in 27 of the duodenal adenomas studied here (see supplementary table 1 for sample details). Full genomic datasets for these samples are available through EGA under accession: EGAD00001009332.

The pathogenesis of duodenal tumors in the inherited tumor syndromes familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP) is poorly understood. This study aimed to identify genes that are significantly mutated in these tumors and to explore the effects of these mutations. Whole exome and whole transcriptome sequencing identified recurrent somatic coding variants of phosphatidylinositol N-acetylglucosaminyltransferase subunit A (PIGA) in 19/70 (27%) FAP and MAP duodenal adenomas, and further confirmed the established driver roles for APC and KRAS. PIGA catalyzes the first step in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Flow cytometry of PIGA-mutant adenoma-derived and CRISPR-edited duodenal organoids confirmed loss of GPI anchors in duodenal epithelial cells and transcriptional profiling of duodenal adenomas revealed transcriptional signatures associated with loss of PIGA.

Overview of all intestinal samples used in this study. *Sequenced previously (Thomas et al, 2017) and reanalysed here. Somatic mutations in APC, KRAS and PIGA as identified by WES and WTS analysis of CU and MDACC duodenal adenomas. Samples where matched WTS data was available are indicated with * and those where the variant was present in both WES and WTS data are denoted with **. 1All protein changes refer to the respective canonical isoforms. 2CADD is a meta-estimator for the deleteriousness of SNVs and indels in the human genome. The score given here ranks a variant relative to all possible substitutions of the genome on a logarithmic scale, i.e. a variant with a score >10 is predicted to be amongst the 10% most deleterious mutations possible, a variant with a score >20 indicates the 1% most deleterious. The developers suggest a cut-off of 15 to identify potentially pathogenic variants (Kircher et al, 2014). C=Colectomy (exact surgical procedure unspecified); Can=Cancer; CRC=Colorectal cancer; DUO=Duodenum; H=Homozygous; IPAA=Ileoanal pouch anastomosis; IRA=Ileo-rectal anastomosis; IS=Ileostomy; PC=Proctocolectomy; NA=Not available, CADD=Combined Annotation Dependent Depletion; DEL=Deletion; FS=Frameshift, INS=Insertion; SNV=Single nucleotide variant; VAF=Variant allele frequency. WES. Whole exome sequencing only. WES/WTS. Whole exome and whole transcriptome sequencing. WTS. Whole transcriptome sequencing only

The gating tree was set as follows, shown for an organoid line without a PIGA somatic variant. A: FSC/SSC to isolate cells (represents the distribution of cells in the light scatter based on size and intracellular composition, respectively) to B: FSC-A/FSC-H (selects for single cells) to C: live gate (DRAQ7 negative, which represents the fraction of viable cells within the sample analysed) to D: SSC-A/FITC-A positive (selecting for FLAER positive cells). SSC-A/FITC-A gates of an unstained sample (E) and PIGA- sample (F) is included for comparison.

Germline and somatic mutations in FAP patient derived duodenal organoids

Background To support proliferation and survival within a challenging microenvironment, cancer cells must reprogramme their metabolism. As such, targeting cancer cell metabolism is a promising therapeutic avenue. However, identifying tractable nodes of metabolic vulnerability in cancer cells is challenging due to their metabolic plasticity. Identification of effective treatment combinations to counter this is an active area of research. Aspirin has a well-established role in cancer prevention, particularly in colorectal cancer (CRC), although the mechanisms are not fully understood. Methods We generated a model to investigate the impact of long-term (52 weeks) aspirin exposure on CRC cells, which has allowed us comprehensively characterise the metabolic impact of long-term aspirin exposure (2–4mM for 52 weeks) using proteomics, Seahorse Extracellular Flux Analysis and Stable Isotope Labelling (SIL). Using this information, we were able to identify nodes of metabolic vulnerability for further targeting, investigating the impact of combining aspirin with metabolic inhibitors in vitro and in vivo. Results We show that aspirin regulates several enzymes and transporters of central carbon metabolism and results in a reduction in glutaminolysis and a concomitant increase in glucose metabolism, demonstrating reprogramming of nutrient utilisation. We show that aspirin causes likely compensatory changes that render the cells sensitive to the glutaminase 1 (GLS1) inhibitor—CB-839. Of note given the clinical interest, treatment with CB-839 alone had little effect on CRC cell growth or survival. However, in combination with aspirin, CB-839 inhibited CRC cell proliferation and induced apoptosis in vitro and, importantly, reduced crypt proliferation in Apc fl/fl mice in vivo . Conclusions Together, these results show that aspirin leads to significant metabolic reprogramming in colorectal cancer cells and raises the possibility that aspirin could significantly increase the efficacy of metabolic cancer therapies in CRC.

Mucosal-associated invariant T (MAIT) cells are an abundant population of innate T cells that recognize bacterial ligands and play a key role in host protection against bacterial and viral pathogens. Upon activation, MAIT cells undergo proliferative expansion and increase their production of effector molecules such as cytokines. In this study, we found that both mRNA and protein abundance of the key metabolism regulator and transcription factor MYC was increased in stimulated MAIT cells. Using quantitative mass spectrometry, we identified the activation of two MYC-controlled metabolic pathways, amino acid transport and glycolysis, both of which were necessary for MAIT cell proliferation. Last, we showed that MAIT cells isolated from people with obesity showed decreased MYC mRNA abundance upon activation, which was associated with defective MAIT cell proliferation and functional responses. Collectively, our data uncover the importance of MYC-regulated metabolism for MAIT cell proliferation and provide additional insight into the molecular basis for the functional defects of MAIT cells in obesity.

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells which recognize a limited repertoire of ligands presented by the MHC class -I like molecule MR1. In addition to their key role in host protection against bacterial and viral pathogens, MAIT cells are emerging as potent anti -cancer effectors. With their abundance in human, unrestricted properties, and rapid effector functions MAIT cells are emerging as attractive candidates for immunotherapy. In the current study, we demonstrate that MAIT cells are potent cytotoxic cells, rapidly degranulating and inducing target cell death. Previous work from our group and others has highlighted glucose metabolism as a critical process for MAIT cell cytokine responses at 18 h. However, the metabolic processes supporting rapid MAIT cell cytotoxic responses are currently unknown. Here, we show that glucose metabolism is dispensable for both MAIT cell cytotoxicity and early (<3 h) cytokine production, as is oxidative phosphor ylation. We show that MAIT cells have the machinery required to make (GYS-1) and metabolize (PYGB) glycogen and further demonstrate that that MAIT cell cytotoxicity and rapid cytokine responses are dependent on glycogen metabolism. In summary, we show that glycogen-fueled metabolism supports rapid MAIT cell effector functions (cytotoxicity and cytokine production) which may have implications for their use as an immunotherapeutic agent.

Pregnancy is characterised by metabolic changes that occur to support the growth and development of the fetus over the course of gestation. These metabolic changes can be classified into two distinct phases: an initial anabolic phase to prepare an adequate store of substrates and energy which are then broken down and used during a catabolic phase to meet the energetic demands of the mother, placenta and fetus. Dynamic readjustment of immune homeostasis is also a feature of pregnancy and is likely linked to the changes in energy substrate utilisation at this time. As cellular metabolism is increasingly recognised as a key determinant of immune cell phenotype and function, we consider how changes in maternal metabolism might contribute to T cell plasticity during pregnancy.

Glutamate decarboxylase 1 (GAD1) is best known for its role in producing the neurotransmitter γ-amino butyric acid (GABA) as part of the “GABA shunt” metabolic pathway, an alternative mechanism of glutamine anaplerosis for TCA cycle metabolism (Yogeeswari et al., 2005). However, understanding of the metabolic function of GAD1 in non-neuronal tissues has remained limited. Here, we show that GAD1 supports cancer cell proliferation independent of the GABA shunt. Despite its elevated expression in lung cancer tissue, GAD1 is not engaged in the GABA shunt in proliferating non-small cell lung cancer (NSCLC) cells, but rather is required for regulating amino acid homeostasis. Silencing GAD1 promotes a broad deficiency in amino acid uptake, leading to reduced glutamine-dependent TCA cycle metabolism and defects in serum- and amino acid-stimulated mTORC1 activation. Mechanistically, GAD1 regulates amino acid uptake through ATF4-dependent amino acid transporter expression including SLC7A5 (LAT1), an amino acid transporter required for branched chain amino acid (BCAA) uptake. Overexpression of LAT1 rescues the proliferative and mTORC1 signalling defects of GAD1-deficient tumor cells. Our results, therefore, define a non-canonical role for GAD1, independent of its characterised role in GABA metabolism, whereby GAD1 regulates amino acid homeostasis to maintain tumor cell proliferation.