Targeting the autophagy-NAD axis protects against cell death in Niemann-Pick type C1 disease models.

Impairment of autophagy leads to an accumulation of misfolded proteins and damaged organelles and has been implicated in plethora of human diseases. Loss of autophagy in actively respiring cells has also been shown to trigger metabolic collapse mediated by the depletion of nicotinamide adenine dinucleotide (NAD) pools, resulting in cell death. Here we found that the deficit in the autophagy-NAD axis underpins the loss of viability in cell models of a neurodegenerative lysosomal storage disorder, Niemann-Pick type C1 (NPC1) disease. Defective autophagic flux in NPC1 cells resulted in mitochondrial dysfunction due to impairment of mitophagy, leading to the depletion of both the reduced and oxidised forms of NAD as identified via metabolic profiling. Consequently, exhaustion of the NAD pools triggered mitochondrial depolarisation and apoptotic cell death. Our chemical screening identified two FDA-approved drugs, celecoxib and memantine, as autophagy activators which effectively restored autophagic flux, NAD levels, and cell viability of NPC1 cells. Of biomedical relevance, either pharmacological rescue of the autophagy deficiency or NAD precursor supplementation restored NAD levels and improved the viability of NPC1 patient fibroblasts and induced pluripotent stem cell (iPSC)-derived cortical neurons. Together, our findings identify the autophagy-NAD axis as a mechanism of cell death and a target for therapeutic interventions in NPC1 disease, with a potential relevance to other neurodegenerative disorders.

Phenotypic profiling of solute carriers characterizes serine transport in cancer.

Serine is a vital amino acid in tumorigenesis. While cells can perform de novo serine synthesis, most transformed cells rely on serine uptake to meet their increased biosynthetic requirements. Solute carriers (SLCs), a family of transmembrane nutrient transport proteins, are the gatekeepers of amino acid acquisition and exchange in mammalian cells and are emerging as anticancer therapeutic targets; however, the SLCs that mediate serine transport in cancer cells remain unknown. Here we perform an arrayed RNAi screen of SLC-encoding genes while monitoring amino acid consumption and cell proliferation in colorectal cancer cells using metabolomics and high-throughput imaging. We identify SLC6A14 and SLC25A15 as major cytoplasmic and mitochondrial serine transporters, respectively. We also observe that SLC12A4 facilitates serine uptake. Dual targeting of SLC6A14 and either SLC25A15 or SLC12A4 diminishes serine uptake and growth of colorectal cancer cells in vitro and in vivo, particularly in cells with compromised de novo serine biosynthesis. Our results provide insight into the mechanisms that contribute to serine uptake and intracellular handling.

Metabolic profiling stratifies colorectal cancer and reveals adenosylhomocysteinase as a therapeutic target.

The genomic landscape of colorectal cancer (CRC) is shaped by inactivating mutations in tumour suppressors such as APC, and oncogenic mutations such as mutant KRAS. Here we used genetically engineered mouse models, and multimodal mass spectrometry-based metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine. We show that untargeted metabolic profiling can be applied to stratify intestinal tissues according to underlying genetic alterations, and use mass spectrometry imaging to identify tumour, stromal and normal adjacent tissues. By identifying ions that drive variation between normal and transformed tissues, we found dysregulation of the methionine cycle to be a hallmark of APC-deficient CRC. Loss of Apc in the mouse intestine was found to be sufficient to drive expression of one of its enzymes, adenosylhomocysteinase (AHCY), which was also found to be transcriptionally upregulated in human CRC. Targeting of AHCY function impaired growth of APC-deficient organoids in vitro, and prevented the characteristic hyperproliferative/crypt progenitor phenotype driven by acute deletion of Apc in vivo, even in the context of mutant Kras. Finally, pharmacological inhibition of AHCY reduced intestinal tumour burden in ApcMin/+ mice indicating its potential as a metabolic drug target in CRC.

NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency.

Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5-/-) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5-/- neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5-/- neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5-/- neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect.

Autophagy promotes cell survival by maintaining NAD levels.

Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components. Loss of autophagy in age-related human pathologies contributes to tissue degeneration through a poorly understood mechanism. Here, we identify an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD) levels, which are critical for cell survival. In respiring mouse fibroblasts with autophagy deficiency, loss of mitochondrial quality control was found to trigger hyperactivation of stress responses mediated by NADases of PARP and Sirtuin families. Uncontrolled depletion of the NAD(H) pool by these enzymes ultimately contributed to mitochondrial membrane depolarization and cell death. Pharmacological and genetic interventions targeting several key elements of this cascade improved the survival of autophagy-deficient yeast, mouse fibroblasts, and human neurons. Our study provides a mechanistic link between autophagy and NAD metabolism and identifies targets for interventions in human diseases associated with autophagic, lysosomal, and mitochondrial dysfunction.

Sensitisation of cancer cells to radiotherapy by serine and glycine starvation.

BACKGROUND: Cellular metabolism is an integral component of cellular adaptation to stress, playing a pivotal role in the resistance of cancer cells to various treatment modalities, including radiotherapy. In response to radiotherapy, cancer cells engage antioxidant and DNA repair mechanisms which mitigate and remove DNA damage, facilitating cancer cell survival. Given the reliance of these resistance mechanisms on amino acid metabolism, we hypothesised that controlling the exogenous availability of the non-essential amino acids serine and glycine would radiosensitise cancer cells. METHODS: We exposed colorectal, breast and pancreatic cancer cell lines/organoids to radiation in vitro and in vivo in the presence and absence of exogenous serine and glycine. We performed phenotypic assays for DNA damage, cell cycle, ROS levels and cell death, combined with a high-resolution untargeted LCMS metabolomics and RNA-Seq. RESULTS: Serine and glycine restriction sensitised a range of cancer cell lines, patient-derived organoids and syngeneic mouse tumour models to radiotherapy. Comprehensive metabolomic and transcriptomic analysis of central carbon metabolism revealed that amino acid restriction impacted not only antioxidant response and nucleotide synthesis but had a marked inhibitory effect on the TCA cycle. CONCLUSION: Dietary restriction of serine and glycine is a viable radio-sensitisation strategy in cancer.

SHMT2-mediated mitochondrial serine metabolism drives 5-FU resistance by fueling nucleotide biosynthesis.

5-Fluorouracil (5-FU) is a key component of chemotherapy for colorectal cancer (CRC). 5-FU efficacy is established by intracellular levels of folate cofactors and DNA damage repair strategies. However, drug resistance still represents a major challenge. Here, we report that alterations in serine metabolism affect 5-FU sensitivity in in vitro and in vivo CRC models. In particular, 5-FU-resistant CRC cells display a strong serine dependency achieved either by upregulating endogenous serine synthesis or increasing exogenous serine uptake. Importantly, regardless of the serine feeder strategy, serine hydroxymethyltransferase-2 (SHMT2)-driven compartmentalization of one-carbon metabolism inside the mitochondria represents a specific adaptation of resistant cells to support purine biosynthesis and potentiate DNA damage response. Interfering with serine availability or affecting its mitochondrial metabolism revert 5-FU resistance. These data disclose a relevant mechanism of mitochondrial serine use supporting 5-FU resistance in CRC and provide perspectives for therapeutic approaches.

Global metabolic alterations in colorectal cancer cells during irinotecan-induced DNA replication stress.

BACKGROUND: Metabolic adaptations can allow cancer cells to survive DNA-damaging chemotherapy. This unmet clinical challenge is a potential vulnerability of cancer. Accordingly, there is an intense search for mechanisms that modulate cell metabolism during anti-tumor therapy. We set out to define how colorectal cancer CRC cells alter their metabolism upon DNA replication stress and whether this provides opportunities to eliminate such cells more efficiently. METHODS: We incubated p53-positive and p53-negative permanent CRC cells and short-term cultured primary CRC cells with the topoisomerase-1 inhibitor irinotecan and other drugs that cause DNA replication stress and consequently DNA damage. We analyzed pro-apoptotic mitochondrial membrane depolarization and cell death with flow cytometry. We evaluated cellular metabolism with immunoblotting of electron transport chain (ETC) complex subunits, analysis of mitochondrial mRNA expression by qPCR, MTT assay, measurements of oxygen consumption and reactive oxygen species (ROS), and metabolic flux analysis with the Seahorse platform. Global metabolic alterations were assessed using targeted mass spectrometric analysis of extra- and intracellular metabolites. RESULTS: Chemotherapeutics that cause DNA replication stress induce metabolic changes in p53-positive and p53-negative CRC cells. Irinotecan enhances glycolysis, oxygen consumption, mitochondrial ETC activation, and ROS production in CRC cells. This is connected to increased levels of electron transport chain complexes involving mitochondrial translation. Mass spectrometric analysis reveals global metabolic adaptations of CRC cells to irinotecan, including the glycolysis, tricarboxylic acid cycle, and pentose phosphate pathways. P53-proficient CRC cells, however, have a more active metabolism upon DNA replication stress than their p53-deficient counterparts. This metabolic switch is a vulnerability of p53-positive cells to irinotecan-induced apoptosis under glucose-restricted conditions. CONCLUSION: Drugs that cause DNA replication stress increase the metabolism of CRC cells. Glucose restriction might improve the effectiveness of classical chemotherapy against p53-positive CRC cells. The topoisomerase-1 inhibitor irinotecan and other chemotherapeutics that cause DNA damage induce metabolic adaptations in colorectal cancer (CRC) cells irrespective of their p53 status. Irinotecan enhances the glycolysis and oxygen consumption in CRC cells to deliver energy and biomolecules necessary for DNA repair and their survival. Compared to p53-deficient cells, p53-proficient CRC cells have a more active metabolism and use their intracellular metabolites more extensively. This metabolic switch creates a vulnerability to chemotherapy under glucose-restricted conditions for p53-positive cells.

Mitochondrial ROS signalling requires uninterrupted electron flow and is lost during ageing in flies.

Mitochondrial reactive oxygen species (mtROS) are cellular messengers essential for cellular homeostasis. In response to stress, reverse electron transport (RET) through respiratory complex I generates high levels of mtROS. Suppression of ROS production via RET (ROS-RET) reduces survival under stress, while activation of ROS-RET extends lifespan in basal conditions. Here, we demonstrate that ROS-RET signalling requires increased electron entry and uninterrupted electron flow through the electron transport chain (ETC). We find that in old fruit flies, ROS-RET is abolished when electron flux is decreased and that their mitochondria produce consistently high levels of mtROS. Finally, we demonstrate that in young flies, limiting electron exit, but not entry, from the ETC phenocopies mtROS generation observed in old individuals. Our results elucidate the mechanism by which ROS signalling is lost during ageing.

Supply and demand: Cellular nutrient uptake and exchange in cancer.

Nutrient supply and demand delineate cell behavior in health and disease. Mammalian cells have developed multiple strategies to secure the necessary nutrients that fuel their metabolic needs. This is more evident upon disruption of homeostasis in conditions such as cancer, when cells display high proliferation rates in energetically challenging conditions where nutritional sources may be scarce. Here, we summarize the main routes of nutrient acquisition that fuel mammalian cells and their implications in tumorigenesis. We argue that the molecular mechanisms of nutrient acquisition not only tip the balance between nutrient supply and demand but also determine cell behavior upon nutrient limitation and energetic stress and contribute to nutrient partitioning and metabolic coordination between different cell types in inflamed or tumorigenic environments.

Mechanistic insights into p53-regulated cytotoxicity of combined entinostat and irinotecan against colorectal cancer cells.

Late-stage colorectal cancer (CRC) is still a clinically challenging problem. The activity of the tumor suppressor p53 is regulated via post-translational modifications (PTMs). While the relevance of p53 C-terminal acetylation for transcriptional regulation is well defined, it is unknown whether this PTM controls mitochondrially mediated apoptosis directly. We used wild-type p53 or p53-negative human CRC cells, cells with acetylation-defective p53, transformation assays, CRC organoids, and xenograft mouse models to assess how p53 acetylation determines cellular stress responses. The topoisomerase-1 inhibitor irinotecan induces acetylation of several lysine residues within p53. Inhibition of histone deacetylases (HDACs) with the class I HDAC inhibitor entinostat synergistically triggers mitochondrial damage and apoptosis in irinotecan-treated p53-positive CRC cells. This specifically relies on the C-terminal acetylation of p53 by CREB-binding protein/p300 and the presence of C-terminally acetylated p53 in complex with the proapoptotic BCL2 antagonist/killer protein. This control of C-terminal acetylation by HDACs can mechanistically explain why combinations of irinotecan and entinostat represent clinically tractable agents for the therapy of p53-proficient CRC.

mTORC1 activity is supported by spatial association with focal adhesions.

The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogenic and stress signals to control growth and metabolism. Activation of mTORC1 by amino acids and growth factors involves recruitment of the complex to the lysosomal membrane and is further supported by lysosome distribution to the cell periphery. Here, we show that translocation of lysosomes toward the cell periphery brings mTORC1 into proximity with focal adhesions (FAs). We demonstrate that FAs constitute discrete plasma membrane hubs mediating growth factor signaling and amino acid input into the cell. FAs, as well as the translocation of lysosome-bound mTORC1 to their vicinity, contribute to both peripheral and intracellular mTORC1 activity. Conversely, lysosomal distribution to the cell periphery is dispensable for the activation of mTORC1 constitutively targeted to FAs. This study advances our understanding of spatial mTORC1 regulation by demonstrating that the localization of mTORC1 to FAs is both necessary and sufficient for its activation by growth-promoting stimuli.

Serine synthesis pathway inhibition cooperates with dietary serine and glycine limitation for cancer therapy.

Many tumour cells show dependence on exogenous serine and dietary serine and glycine starvation can inhibit the growth of these cancers and extend survival in mice. However, numerous mechanisms promote resistance to this therapeutic approach, including enhanced expression of the de novo serine synthesis pathway (SSP) enzymes or activation of oncogenes that drive enhanced serine synthesis. Here we show that inhibition of PHGDH, the first step in the SSP, cooperates with serine and glycine depletion to inhibit one-carbon metabolism and cancer growth. In vitro, inhibition of PHGDH combined with serine starvation leads to a defect in global protein synthesis, which blocks the activation of an ATF-4 response and more broadly impacts the protective stress response to amino acid depletion. In vivo, the combination of diet and inhibitor shows therapeutic efficacy against tumours that are resistant to diet or drug alone, with evidence of reduced one-carbon availability. However, the defect in ATF4-response seen in vitro following complete depletion of available serine is not seen in mice, where dietary serine and glycine depletion and treatment with the PHGDH inhibitor lower but do not eliminate serine. Our results indicate that inhibition of PHGDH will augment the therapeutic efficacy of a serine depleted diet.

Metabolic cross-feeding in imbalanced diets allows gut microbes to improve reproduction and alter host behaviour.

The impact of commensal bacteria on the host arises from complex microbial-diet-host interactions. Mapping metabolic interactions in gut microbial communities is therefore key to understand how the microbiome influences the host. Here we use an interdisciplinary approach including isotope-resolved metabolomics to show that in Drosophila melanogaster, Acetobacter pomorum (Ap) and Lactobacillus plantarum (Lp) a syntrophic relationship is established to overcome detrimental host diets and identify Ap as the bacterium altering the host's feeding decisions. Specifically, we show that Ap uses the lactate produced by Lp to supply amino acids that are essential to Lp, allowing it to grow in imbalanced diets. Lactate is also necessary and sufficient for Ap to alter the fly's protein appetite. Our data show that gut bacterial communities use metabolic interactions to become resilient to detrimental host diets. These interactions also ensure the constant flow of metabolites used by the microbiome to alter reproduction and host behaviour.

Polyamine pathway activity promotes cysteine essentiality in cancer cells.

Cancer cells have high demands for non-essential amino acids (NEAAs), which are precursors for anabolic and antioxidant pathways that support cell survival and proliferation. It is well-established that cancer cells consume the NEAA cysteine, and that cysteine deprivation can induce cell death; however, the specific factors governing acute sensitivity to cysteine starvation are poorly characterized. Here, we show that that neither expression of enzymes for cysteine synthesis nor availability of the primary precursor methionine correlated with acute sensitivity to cysteine starvation. We observed a strong correlation between efflux of the methionine-derived metabolite methylthioadenosine (MTA) and sensitivity to cysteine starvation. MTA efflux results from genetic deletion of methylthioadenosine phosphorylase (MTAP), which is frequently deleted in cancers. We show that MTAP loss upregulates polyamine metabolism which, concurrently with cysteine withdrawal, promotes elevated reactive oxygen species and prevents cell survival. Our results reveal an unexplored metabolic weakness at the intersection of polyamine and cysteine metabolism.

The creatine-phosphagen system is mechanoresponsive in pancreatic adenocarcinoma and fuels invasion and metastasis.

Pancreatic ductal adenocarcinoma is particularly metastatic, with dismal survival rates and few treatment options. Stiff fibrotic stroma is a hallmark of pancreatic tumours, but how stromal mechanosensing affects metastasis is still unclear. Here, we show that mechanical changes in the pancreatic cancer cell environment affect not only adhesion and migration, but also ATP/ADP and ATP/AMP ratios. Unbiased metabolomic analysis reveals that the creatine-phosphagen ATP-recycling system is a major mechanosensitive target. This system depends on arginine flux through the urea cycle, which is reflected by the increased incorporation of carbon and nitrogen from L-arginine into creatine and phosphocreatine on stiff matrix. We identify that CKB is a mechanosensitive transcriptional target of YAP, and thus it increases phosphocreatine production. We further demonstrate that the creatine-phosphagen system has a role in invasive migration, chemotaxis and liver metastasis of cancer cells.

Use of 13C315N1-Serine or 13C515N1-Methionine for Studying Methylation Dynamics in Cancer Cell Metabolism and Epigenetics.

Tracing the fate of carbon-13 (13C) labeled metabolites within cells by liquid chromatography mass spectrometry (LCMS) is a powerful analytical technique used for many years in the study of cell metabolism. Conventional experiments using LCMS and labeled nutrients tend to track the incorporation of 13C from exogenous nutrients (such as amino acids) into other, relatively proximal, cellular metabolites. Several labs have extended this technique to track transfer of 13C from the metabolite pool onto macromolecules, such as DNA, where methylation acts as an important functional modification. Here we describe a complete method that integrates previously established techniques to simultaneously track the use of 13C-serine or 13C-methionine into metabolite pools of the methionine cycle and into methylation of DNA and RNA. Given the ability to track methyl-transfer in a time-dependent way, this technique can provide temporal information about active methyl-transfer as well as quantification of total DNA/RNA methylation levels.

Measurement of Tumor Antioxidant Capacity and Prediction of Chemotherapy Resistance in Preclinical Models of Ovarian Cancer by Positron Emission Tomography.

PURPOSE: Drug resistance is a major obstacle for the effective treatment of patients with high-grade serous ovarian cancer (HGSOC). Currently, there is no satisfactory way to identify patients with HGSOC that are refractive to the standard of care. Here, we propose the system xc - radiotracer (4S)-4-(3-[18F]fluoropropyl)-l-glutamate ([18F]FSPG) as a non-invasive method to measure upregulated antioxidant pathways present in drug-resistant HGSOC. EXPERIMENTAL DESIGN: Using matched chemotherapy sensitive and resistant ovarian cancer cell lines, we assessed their antioxidant capacity and its relation to [18F]FSPG uptake, both in cells and in animal models of human ovarian cancer. We identified the mechanisms driving differential [18F]FSPG cell accumulation and evaluated [18F]FSPG tumor uptake as predictive marker of treatment response in drug-resistant tumors. RESULTS: High intracellular glutathione (GSH) and low reactive oxygen species corresponded to decreased [18F]FSPG cell accumulation in drug-resistant versus drug-sensitive cells. Decreased [18F]FSPG uptake in drug-resistant cells was a consequence of changes in intracellular cystine, a key precursor in GSH biosynthesis. In vivo, [18F]FSPG uptake was decreased nearly 80% in chemotherapy-resistant A2780 tumors compared with parental drug-sensitive tumors, with nonresponding tumors displaying high levels of oxidized-to-reduced GSH. Treatment of drug-resistant A2780 tumors with doxorubicin resulted in no detectable change in tumor volume, GSH, or [18F]FSPG uptake. CONCLUSIONS: This study demonstrates the ability of [18F]FSPG to detect upregulated antioxidant pathways present in drug-resistant cancer. [18F]FSPG may therefore enable the identification of patients with HGSOC that are refractory to standard of care, allowing the transferal of drug-resistant patients to alternative therapies, thereby improving outcomes in this disease.