Further delineation of the phenotypic and metabolomic profile of ALDH1L2-related neurodevelopmental disorder.

ALDH1L2, a mitochondrial enzyme in folate metabolism, converts 10-formyl-THF (10-formyltetrahydrofolate) to THF (tetrahydrofolate) and CO2. At the cellular level, deficiency of this NADP+-dependent reaction results in marked reduction in NADPH/NADP+ ratio and reduced mitochondrial ATP. Thus far, a single patient with biallelic ALDH1L2 variants and the phenotype of a neurodevelopmental disorder has been reported. Here, we describe another patient with a neurodevelopmental disorder associated with a novel homozygous missense variant in ALDH1L2, Pro133His. The variant caused marked reduction in the ALDH1L2 enzyme activity in skin fibroblasts derived from the patient as probed by 10-FDDF, a stable synthetic analog of 10-formyl-THF. Additional associated abnormalities in these fibroblasts include reduced NADPH/NADP+ ratio and pool of mitochondrial ATP, upregulated autophagy and dramatically altered metabolomic profile. Overall, our study further supports a link between ALDH1L2 deficiency and abnormal neurodevelopment in humans.

Exploratory Metabolomics Underscores the Folate Enzyme ALDH1L1 as a Regulator of Glycine and Methylation Reactions.

Folate (vitamin B9) is involved in one-carbon transfer reactions and plays a significant role in nucleic acid synthesis and control of cellular proliferation, among other key cellular processes. It is now recognized that the role of folates in different stages of carcinogenesis is complex, and more research is needed to understand how folate reactions become dysregulated in cancers and the metabolic consequences that occur as a result. ALDH1L1 (cytosolic 10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism expressed in many tissues, is ubiquitously downregulated in cancers and is not expressed in cancer cell lines. The RT4 cell line (derived from papillary bladder cancer) which expresses high levels of ALDH1L1 represents an exception, providing an opportunity to explore the metabolic consequences of the loss of this enzyme. We have downregulated this protein in RT4 cells (shRNA driven knockdown or CRISPR driven knockout) and compared metabolomes of ALDH1L1-expressing and -deficient cells to determine if metabolic changes linked to the loss of this enzyme might provide proliferative and/or survival advantages for cancer cells. In this study, cell extracts were analyzed using Ultra High Performance Liquid Chromatography High Resolution Mass Spectrometry (UHPLC-HR-MS). A total of 13,339 signals were identified or annotated using an in-house library and public databases. Supervised and unsupervised multivariate analysis revealed metabolic differences between RT4 cells and ALDH1L1-deficient clones. Glycine (8-fold decrease) and metabolites derived from S-adenosylmethionine utilizing pathways were significantly decreased in the ALDH1L1-deficient clones, compared with RT4 cells. Other changes linked to ALDH1L1 downregulation include decreased levels of amino acids, Krebs cycle intermediates, and ribose-5-phosphate, and increased nicotinic acid. While the ALDH1L1-catalyzed reaction is directly linked to glycine biosynthesis and methyl group flux, its overall effect on cellular metabolism extends beyond immediate metabolic pathways controlled by this enzyme.

Aldh1l2 knockout mouse metabolomics links the loss of the mitochondrial folate enzyme to deregulation of a lipid metabolism observed in rare human disorder.

BACKGROUND: Mitochondrial folate enzyme ALDH1L2 (aldehyde dehydrogenase 1 family member L2) converts 10-formyltetrahydrofolate to tetrahydrofolate and CO2 simultaneously producing NADPH. We have recently reported that the lack of the enzyme due to compound heterozygous mutations was associated with neuro-ichthyotic syndrome in a male patient. Here, we address the role of ALDH1L2 in cellular metabolism and highlight the mechanism by which the enzyme regulates lipid oxidation. METHODS: We generated Aldh1l2 knockout (KO) mouse model, characterized its phenotype, tissue histology, and levels of reduced folate pools and applied untargeted metabolomics to determine metabolic changes in the liver, pancreas, and plasma caused by the enzyme loss. We have also used NanoString Mouse Inflammation V2 Code Set to analyze inflammatory gene expression and evaluate the role of ALDH1L2 in the regulation of inflammatory pathways. RESULTS: Both male and female Aldh1l2 KO mice were viable and did not show an apparent phenotype. However, H&E and Oil Red O staining revealed the accumulation of lipid vesicles localized between the central veins and portal triads in the liver of Aldh1l2-/- male mice indicating abnormal lipid metabolism. The metabolomic analysis showed vastly changed metabotypes in the liver and plasma in these mice suggesting channeling of fatty acids away from ß-oxidation. Specifically, drastically increased plasma acylcarnitine and acylglycine conjugates were indicative of impaired ß-oxidation in the liver. Our metabolomics data further showed that mechanistically, the regulation of lipid metabolism by ALDH1L2 is linked to coenzyme A biosynthesis through the following steps. ALDH1L2 enables sufficient NADPH production in mitochondria to maintain high levels of glutathione, which in turn is required to support high levels of cysteine, the coenzyme A precursor. As the final outcome, the deregulation of lipid metabolism due to ALDH1L2 loss led to decreased ATP levels in mitochondria. CONCLUSIONS: The ALDH1L2 function is important for CoA-dependent pathways including ß-oxidation, TCA cycle, and bile acid biosynthesis. The role of ALDH1L2 in the lipid metabolism explains why the loss of this enzyme is associated with neuro-cutaneous diseases. On a broader scale, our study links folate metabolism to the regulation of lipid homeostasis and the energy balance in the cell.

ALDH1L1 and ALDH1L2 Folate Regulatory Enzymes in Cancer.

Epidemiological studies implicate excess ethanol ingestion as a risk factor for several cancers and support the concept of a synergistic effect of chronic alcohol consumption and folate deficiency on carcinogenesis. Alcohol consumption affects folate-related genes and enzymes including two major folate-metabolizing enzymes, ALDH1L1 and ALDH1L2. ALDH1L1 (cytosolic 10-formyltetrahydrofolate dehydrogenase) is a regulatory enzyme in folate metabolism that controls the overall flux of one-carbon groups in folate-dependent biosynthetic pathways. It is strongly and ubiquitously down-regulated in malignant tumors via promoter methylation, and recent studies underscored this enzyme as a candidate tumor suppressor and potential marker of aggressive cancers. A related enzyme, ALDH1L2, is the mitochondrial homolog of ALDH1L1 encoded by a separate gene. In contrast to its cytosolic counterpart, ALDH1L2 is expressed in malignant tumors and cancer cell lines and was implicated in metastasis regulation. This review discusses the link between folate and cancer, modifying effects of alcohol consumption on folate-associated carcinogenesis, and putative roles of ALDH1L1 and ALDH1L2 in this process.

CerS6 Is a Novel Transcriptional Target of p53 Protein Activated by Non-genotoxic Stress.

Our previous study suggested that ceramide synthase 6 (CerS6), an enzyme in sphingolipid biosynthesis, is regulated by p53: CerS6 was elevated in several cell lines in response to transient expression of p53 or in response to folate stress, which is known to activate p53. It was not clear, however, whether CerS6 gene is a direct transcriptional target of p53 or whether this was an indirect effect through additional regulatory factors. In the present study, we have shown that the CerS6 promoter is activated by p53 in luciferase assays, whereas transcriptionally inactive R175H p53 mutant failed to induce the luciferase expression from this promoter. In vitro immunoprecipitation assays and gel shift analyses have further demonstrated that purified p53 binds within the CerS6 promoter sequence spanning 91 bp upstream and 60 bp downstream of the transcription start site. The Promo 3.0.2 online tool for the prediction of transcription factor binding sites indicated the presence of numerous putative non-canonical p53 binding motifs in the CerS6 promoter. Luciferase assays and gel shift analysis have identified a single motif upstream of the transcription start as a key p53 response element. Treatment of cells with Nutlin-3 or low concentrations of actinomycin D resulted in a strong elevation of CerS6 mRNA and protein, thus demonstrating that CerS6 is a component of the non-genotoxic p53-dependent cellular stress response. This study has shown that by direct transcriptional activation of CerS6, p53 can regulate specific ceramide biosynthesis, which contributes to the pro-apoptotic cellular response.

Rho GTPases RhoA and Rac1 mediate effects of dietary folate on metastatic potential of A549 cancer cells through the control of cofilin phosphorylation.

Folate, an important nutrient in the human diet, has been implicated in cancer, but its role in metastasis is not established. We have shown previously that the withdrawal of medium folate leads to the inhibition of migration and invasion of A549 lung carcinoma cells. Here we have demonstrated that medium folate regulates the function of Rho GTPases by enabling their carboxyl methylation and translocation to plasma membrane. Conversely, the lack of folate leads to the retention of these proteins in endoplasmic reticulum. Folate also promoted the switch from inactive (GDP-bound) to active (GTP-bound) GTPases, resulting in the activation of downstream kinases p21-activated kinase and LIM kinase and phosphorylation of the actin-depolymerizing factor cofilin. We have further demonstrated that in A549 cells two GTPases, RhoA and Rac1, but not Cdc42, are immediate sensors of folate status: the siRNA silencing of RhoA or Rac1 blocked effects of folate on cofilin phosphorylation and cellular migration and invasion. The finding that folate modulates metastatic potential of cancer cells was confirmed in an animal model of lung cancer using tail vein injection of A549 cells in SCID mice. A folate-rich diet enhanced lung colonization and distant metastasis to lymph nodes and decreased overall survival (35 versus 63 days for mice on a folate-restricted diet). High folate also promoted epithelial-mesenchymal transition in cancer cells and experimental mouse tumors. Our study provides experimental evidence for a mechanism of metastasis promotion by dietary folate and highlights the interaction between nutrients and metastasis-related signaling.

Folate stress induces apoptosis via p53-dependent de novo ceramide synthesis and up-regulation of ceramide synthase 6.

We have investigated the role of ceramide in the cellular adaptation to folate stress induced by Aldh1l1, the enzyme involved in the regulation of folate metabolism. Our previous studies demonstrated that Aldh1l1, similar to folate deficiency, evokes metabolic stress and causes apoptosis in cancer cells. Here we report that the expression of Aldh1l1 in A549 or HCT116 cells results in the elevation of C16-ceramide and a transient up-regulation of ceramide synthase 6 (CerS6) mRNA and protein. Pretreatment with ceramide synthesis inhibitors myriocin and fumonisin B1 or siRNA silencing of CerS6 prevented C16-ceramide accumulation and rescued cells supporting the role of CerS6/C16-ceramide as effectors of Aldh1l1-induced apoptosis. The CerS6 activation by Aldh1l1 and increased ceramide generation were p53-dependent; this effect was ablated in p53-null cells. Furthermore, the expression of wild type p53 but not transcriptionally inactive R175H p53 mutant strongly elevated CerS6. Also, this dominant negative mutant prevented accumulation of CerS6 in response to Aldh1l1, indicating that CerS6 is a transcriptional target of p53. In support of this mechanism, bioinformatics analysis revealed the p53 binding site 3 kb downstream of the CerS6 transcription start. Interestingly, ceramide elevation in response to Aldh1l1 was inhibited by silencing of PUMA, a proapoptotic downstream effector of p53 whereas the transient expression of CerS6 elevated PUMA in a p53-dependent manner indicating reciprocal relationships between ceramide and p53/PUMA pathways. Importantly, folate withdrawal also induced CerS6/C16-ceramide elevation accompanied by p53 accumulation. Overall, these novel findings link folate and de novo ceramide pathways in cellular stress response.

Molecular mechanisms underlying the potentially adverse effects of folate.

The importance of proper consumption of dietary folate for human health has been highlighted by an extensive number of publications over several decades. Fortification of grain products with folic acid was initiated with the specific intent to prevent neural tube defects, and the scope of this endeavor is unique in that its target population (women of the periconceptional period) is many times smaller than the population it affects (everyone who ingests fortified grain products). Folate fortification has been wildly successful in terms of its goal; since its inception, the incidence of neural tube defects has markedly decreased. In the wake of this public health triumph, it is important to catalog both the serendipitous benefits and potential side effects of folic acid supplementation. The vitamin is generally regarded as a harmless nutrient based on studies evaluating the safe upper limits of folate intake. In recent years, however, a concern has been raised with respect to a potential downside to folate supplementation; namely, its proposed ability to enhance proliferation of malignant tumors. The current review summarizes the available literature on the effects of folate supplementation and the molecular mechanisms by which high doses of folate may have negative consequences on human health, especially with regard to cancer.

Distinct inflammatory Th17 subsets emerge in autoimmunity and infection.

Th17 cells play a critical role in both tissue homeostasis and inflammation during clearance of infections as well as autoimmune and inflammatory disorders. Despite numerous efforts to distinguish the homeostatic and inflammatory roles of Th17 cells, the mechanism underlying the divergent functions of inflammatory Th17 cells remains poorly understood. In this study, we demonstrate that the inflammatory Th17 cells involved in autoimmune colitis and those activated during colitogenic infection are distinguishable populations characterized by their differential responses to the pharmacological molecule, clofazimine (CLF). Unlike existing Th17 inhibitors, CLF selectively inhibits proautoimmune Th17 cells while preserving the functional state of infection-elicited Th17 cells partially by reducing the enzyme ALDH1L2. Overall, our study identifies two distinct subsets within the inflammatory Th17 compartment with distinct regulatory mechanisms. Furthermore, we highlight the feasibility to develop disease-promoting Th17 selective inhibitor for treating autoimmune diseases.

Pancreatic islet protection at the expense of secretory function involves serine-linked mitochondrial one-carbon metabolism.

Type 2 diabetes is characterized by insulin hypersecretion followed by reduced glucose-stimulated insulin secretion (GSIS). Here we show that acute stimulation of pancreatic islets with the insulin secretagogue dextrorphan (DXO) or glibenclamide enhances GSIS, whereas chronic treatment with high concentrations of these drugs reduce GSIS but protect islets from cell death. Bulk RNA sequencing of islets shows increased expression of genes for serine-linked mitochondrial one-carbon metabolism (OCM) after chronic, but not acute, stimulation. In chronically stimulated islets, more glucose is metabolized to serine than to citrate, and the mitochondrial ATP/ADP ratio decreases, whereas the NADPH/NADP+ ratio increases. Activating transcription factor-4 (Atf4) is required and sufficient to activate serine-linked mitochondrial OCM genes in islets, with gain- and loss-of-function experiments showing that Atf4 reduces GSIS and is required, but not sufficient, for full DXO-mediated islet protection. In sum, we identify a reversible metabolic pathway that provides islet protection at the expense of secretory function.

Conserved catalytic residues of the ALDH1L1 aldehyde dehydrogenase domain control binding and discharging of the coenzyme.

The C-terminal domain (C(t)-FDH) of 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an NADP(+)-dependent oxidoreductase and a structural and functional homolog of aldehyde dehydrogenases. Here we report the crystal structures of several C(t)-FDH mutants in which two essential catalytic residues adjacent to the nicotinamide ring of bound NADP(+), Cys-707 and Glu-673, were replaced separately or simultaneously. The replacement of the glutamate with an alanine causes irreversible binding of the coenzyme without any noticeable conformational changes in the vicinity of the nicotinamide ring. Additional replacement of cysteine 707 with an alanine (E673A/C707A double mutant) did not affect this irreversible binding indicating that the lack of the glutamate is solely responsible for the enhanced interaction between the enzyme and the coenzyme. The substitution of the cysteine with an alanine did not affect binding of NADP(+) but resulted in the enzyme lacking the ability to differentiate between the oxidized and reduced coenzyme: unlike the wild-type C(t)-FDH/NADPH complex, in the C707A mutant the position of NADPH is identical to the position of NADP(+) with the nicotinamide ring well ordered within the catalytic center. Thus, whereas the glutamate restricts the affinity for the coenzyme, the cysteine is the sensor of the coenzyme redox state. These conclusions were confirmed by coenzyme binding experiments. Our study further suggests that the binding of the coenzyme is additionally controlled by a long-range communication between the catalytic center and the coenzyme-binding domain and points toward an α-helix involved in the adenine moiety binding as a participant of this communication.

Structure of putative tumor suppressor ALDH1L1.

Putative tumor suppressor ALDH1L1, the product of natural fusion of three unrelated genes, regulates folate metabolism by catalyzing NADP+-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Cryo-EM structures of tetrameric rat ALDH1L1 revealed the architecture and functional domain interactions of this complex enzyme. Highly mobile N-terminal domains, which remove formyl from 10-formyltetrahydrofolate, undergo multiple transient inter-domain interactions. The C-terminal aldehyde dehydrogenase domains, which convert formyl to CO2, form unusually large interfaces with the intermediate domains, homologs of acyl/peptidyl carrier proteins (A/PCPs), which transfer the formyl group between the catalytic domains. The 4'-phosphopantetheine arm of the intermediate domain is fully extended and reaches deep into the catalytic pocket of the C-terminal domain. Remarkably, the tetrameric state of ALDH1L1 is indispensable for catalysis because the intermediate domain transfers formyl between the catalytic domains of different protomers. These findings emphasize the versatility of A/PCPs in complex, highly dynamic enzymatic systems.

Sex-Specific Metabolic Effects of Dietary Folate Withdrawal in Wild-Type and Aldh1l1 Knockout Mice.

ALDH1L1 (10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism, is highly expressed in the liver. It regulates the overall flux of folate-bound one-carbon groups by converting 10-formyltetrahydrofolate to tetrahydrofolate and CO2 in a NADP+-dependent reaction. Our previous study revealed that Aldh1l1 knockout (KO) mice have an altered liver metabotype with metabolic symptoms of folate deficiency when fed a standard chow diet containing 2 ppm folic acid. Here we performed untargeted metabolomic analysis of liver and plasma of KO and wild-type (WT) male and female mice fed for 16 weeks either standard or folate-deficient diet. OPLS-DA, a supervised multivariate technique that was applied to 6595 and 10,678 features for the liver and plasma datasets, respectively, indicated that genotype and diet, alone or in combination, gave distinct metabolic profiles in both types of biospecimens. A more detailed analysis of affected metabolic pathways based on most confidently identified metabolites in the liver and plasma (OL1 and OL2a ontology level) indicated that the dietary folate restriction itself does not fully recapitulate the metabolic effect of the KO. Of note, dietary folate withdrawal enhanced the metabolic perturbations linked to the ALDH1L1 loss only for a subset of metabolites. Importantly, both the ALDH1L1 loss and dietary folate deficiency produced sex-specific metabolic effects.

Genetic variants in ALDH1L1 and GLDC influence the serine-to-glycine ratio in Hispanic children.

BACKGROUND: Glycine is a proteogenic amino acid that is required for numerous metabolic pathways, including purine, creatine, heme, and glutathione biosynthesis. Glycine formation from serine, catalyzed by serine hydroxy methyltransferase, is the major source of this amino acid in humans. Our previous studies in a mouse model have shown a crucial role for the 10-formyltetrahydrofolate dehydrogenase enzyme in serine-to-glycine conversion. OBJECTIVES: We sought to determine the genomic influence on the serine-glycine ratio in 803 Hispanic children from 319 families of the Viva La Familia cohort. METHODS: We performed a genome-wide association analysis for plasma serine, glycine, and the serine-glycine ratio in Sequential Oligogenic Linkage Analysis Routines while accounting for relationships among family members. RESULTS: All 3 parameters were significantly heritable (h2 = 0.22-0.78; P < 0.004). The strongest associations for the serine-glycine ratio were with single nucleotide polymorphisms (SNPs) in aldehyde dehydrogenase 1 family member L1 (ALDH1L1) and glycine decarboxylase (GLDC) and for glycine with GLDC (P < 3.5 × 10-8; effect sizes, 0.03-0.07). No significant associations were found for serine. We also conducted a targeted genetic analysis with ALDH1L1 exonic SNPs and found significant associations between the serine-glycine ratio and rs2886059 (ß = 0.68; SE, 0.25; P = 0.006) and rs3796191 (ß = 0.25; SE, 0.08; P = 0.003) and between glycine and rs3796191 (ß = -0.08; SE, 0.02; P = 0.0004). These exonic SNPs were further associated with metabolic disease risk factors, mainly adiposity measures (P < 0.006). Significant genetic and phenotypic correlations were found for glycine and the serine-glycine ratio with metabolic disease risk factors, including adiposity, insulin sensitivity, and inflammation-related phenotypes [estimate of genetic correlation = -0.37 to 0.35 (P < 0.03); estimate of phenotypic correlation = -0.19 to 0.13 (P < 0.006)]. The significant genetic correlations indicate shared genetic effects among glycine, the serine-glycine ratio, and adiposity and insulin sensitivity phenotypes. CONCLUSIONS: Our study suggests that ALDH1L1 and GLDC SNPs influence the serine-to-glycine ratio and metabolic disease risk.

Acyl carrier protein-specific 4'-phosphopantetheinyl transferase activates 10-formyltetrahydrofolate dehydrogenase.

4'-Phosphopantetheinyl transferases (PPTs) catalyze the transfer of 4'-phosphopantetheine (4-PP) from coenzyme A to a conserved serine residue of their protein substrates. In humans, the number of pathways utilizing the 4-PP post-translational modification is limited and may only require a single broad specificity PPT for all phosphopantetheinylation reactions. Recently, we have shown that one of the enzymes of folate metabolism, 10-formyltetrahydrofolate dehydrogenase (FDH), requires a 4-PP prosthetic group for catalysis. This moiety acts as a swinging arm to couple the activities of the two catalytic domains of FDH and allows the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. In the current study, we demonstrate that the broad specificity human PPT converts apo-FDH to holoenzyme and thus activates FDH catalysis. Silencing PPT by small interfering RNA in A549 cells prevents FDH modification, indicating the lack of alternative enzymes capable of accomplishing this transferase reaction. Interestingly, PPT-silenced cells demonstrate significantly reduced proliferation and undergo strong G(1) arrest, suggesting that the enzymatic function of PPT is essential and nonredundant. Our study identifies human PPT as the FDH-modifying enzyme and supports the hypothesis that mammals utilize a single enzyme for all phosphopantetheinylation reactions.

ALDH1L2 is the mitochondrial homolog of 10-formyltetrahydrofolate dehydrogenase.

Cytosolic 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an abundant enzyme of folate metabolism. It converts 10-formyltetrahydrofolate to tetrahydrofolate and CO(2) in an NADP(+)-dependent reaction. We have identified a gene at chromosome locus 12q24.11 of the human genome, the product of which has 74% sequence similarity with cytosolic FDH. This protein has an extra N-terminal sequence of 22 amino acid residues, predicted to be a mitochondrial translocation signal. Transfection of COS-7 or A549 cell lines with a construct in which green fluorescent protein was introduced between the leader sequence and the rest of the putative mitochondrial FDH (mtFDH) has demonstrated mitochondrial localization of the fusion protein, suggesting that the identified gene encodes a mitochondrial enzyme. Purified pig liver mtFDH displayed dehydrogenase/hydrolase activities similar to cytosolic FDH. Real-time PCR performed on an array of human tissues has shown that although cytosolic FDH mRNA is highest in liver, kidney, and pancreas, mtFDH mRNA is most highly expressed in pancreas, heart, and brain. In contrast to the cytosolic enzyme, which is not detectable in cancer cells, the presence of mtFDH was demonstrated in several human cancer cell lines by conventional and real-time PCR and by Western blot. Analysis of genomes of different species indicates that the mitochondrial enzyme is a later evolutionary product when compared with the cytosolic enzyme. We propose that this novel mitochondrial enzyme is a likely source of CO(2) production from 10-formyltetrahydrofolate in mitochondria and plays an essential role in the distribution of one-carbon groups between the cytosolic and mitochondrial compartments of the cell.

Is ALDH1L1 Elevated in Lung Cancer? Comment on: Lee, S.-H.; et al. "The Combination of Loss of ALDH1L1 Function and Phenformin Treatment Decreases Tumor Growth in KRAS-Driven Lung Cancer" Cancers 2020, 12, 1382.

We read with interest the article by Lee et al [...].

Metabolic Response of Triple-Negative Breast Cancer to Folate Restriction.

BACKGROUND: Triple-negative breast cancers (TNBCs), accounting for approximately 15% of breast cancers, lack targeted therapy. A hallmark of cancer is metabolic reprogramming, with one-carbon metabolism essential to many processes altered in tumor cells, including nucleotide biosynthesis and antioxidant defenses. We reported that folate deficiency via folic acid (FA) withdrawal in several TNBC cell lines results in heterogenous effects on cell growth, metabolic reprogramming, and mitochondrial impairment. To elucidate underlying drivers of TNBC sensitivity to folate stress, we characterized in vivo and in vitro responses to FA restriction in two TNBC models differing in metastatic potential and innate mitochondrial dysfunction. METHODS: Metastatic MDA-MB-231 cells (high mitochondrial dysfunction) and nonmetastatic M-Wnt cells (low mitochondrial dysfunction) were orthotopically injected into mice fed diets with either 2 ppm FA (control), 0 ppm FA, or 12 ppm FA (supplementation; in MDA-MB-231 only). Tumor growth, metabolomics, and metabolic gene expression were assessed. MDA-MB-231 and M-Wnt cells were also grown in media with 0 or 2.2 µM FA; metabolic alterations were assessed by extracellular flux analysis, flow cytometry, and qPCR. RESULTS: Relative to control, dietary FA restriction decreased MDA-MB-231 tumor weight and volume, while FA supplementation minimally increased MDA-MB-231 tumor weight. Metabolic studies in vivo and in vitro using MDA-MB-231 cells showed FA restriction remodeled one-carbon metabolism, nucleotide biosynthesis, and glucose metabolism. In contrast to findings in the MDA-MB-231 model, FA restriction in the M-Wnt model, relative to control, led to accelerated tumor growth, minimal metabolic changes, and modest mitochondrial dysfunction. Increased mitochondrial dysfunction in M-Wnt cells, induced via chloramphenicol, significantly enhanced responsiveness to the cytotoxic effects of FA restriction. CONCLUSIONS: Given the lack of targeted treatment options for TNBC, uncovering metabolic vulnerabilities that can be exploited as therapeutic targets is an important goal. Our findings suggest that a major driver of TNBC sensitivity to folate restriction is a high innate level of mitochondrial dysfunction, which can increase dependence on one-carbon metabolism. Thus, folate deprivation or antifolate therapy for TNBCs with metabolic inflexibility due to their elevated levels of mitochondrial dysfunction may represent a novel precision-medicine strategy.

Knockout of Putative Tumor Suppressor Aldh1l1 in Mice Reprograms Metabolism to Accelerate Growth of Tumors in a Diethylnitrosamine (DEN) Model of Liver Carcinogenesis.

Cytosolic 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) is commonly downregulated in human cancers through promoter methylation. We proposed that ALDH1L1 loss promotes malignant tumor growth. Here, we investigated the effect of the Aldh1l1 mouse knockout (Aldh1l1-/-) on hepatocellular carcinoma using a chemical carcinogenesis model. Fifteen-day-old male Aldh1l1 knockout mice and their wild-type littermate controls (Aldh1l1+/+) were injected intraperitoneally with 20 µg/g body weight of DEN (diethylnitrosamine). Mice were sacrificed 10, 20, 28, and 36 weeks post-DEN injection, and livers were examined for tumor multiplicity and size. We observed that while tumor multiplicity did not differ between Aldh1l1-/- and Aldh1l1+/+ animals, larger tumors grew in Aldh1l1-/- compared to Aldh1l1+/+ mice at 28 and 36 weeks. Profound differences between Aldh1l1-/- and Aldh1l1+/+ mice in the expression of inflammation-related genes were seen at 10 and 20 weeks. Of note, large tumors from wild-type mice showed a strong decrease of ALDH1L1 protein at 36 weeks. Metabolomic analysis of liver tissues at 20 weeks showed stronger differences in Aldh1l1+/+ versus Aldh1l1-/- metabotypes than at 10 weeks, which underscores metabolic pathways that respond to DEN in an ALDH1L1-dependent manner. Our study indicates that Aldh1l1 knockout promoted liver tumor growth without affecting tumor initiation or multiplicity.

10-formyltetrahydrofolate dehydrogenase-induced c-Jun-NH2-kinase pathways diverge at the c-Jun-NH2-kinase substrate level in cells with different p53 status.

10-Formyltetrahydrofolate dehydrogenase (FDH) suppresses cancer cell proliferation through p53-dependent apoptosis but also induces strong cytotoxicity in p53-deficient prostate cells. In the present study, we have shown that FDH induces apoptosis in PC-3 prostate cells through simultaneous activation of the c-Jun-NH(2)-kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways with JNK phosphorylating c-Jun and ERK1/2 phosphorylating Elk-1. The JNK1/2 inhibitor SP600125 or ERK1/2 inhibitor PD98059 prevented phosphorylation of c-Jun and Elk-1, correspondingly and partially protected PC-3 cells from FDH-induced cytotoxicity. Combination of the two inhibitors produced an additive effect. The contribution from the JNK cascade to FDH-induced apoptosis was significantly stronger than from the ERK pathway. siRNA knockdown of JNK1/2 or "turning off" the downstream target c-Jun by either siRNA or expression of the dominant-negative c-Jun mutant, TAM67, rescued PC-3 cells from FDH-induced apoptosis. The pull-down assays on immobilized c-Jun showed that c-Jun is directly phosphorylated by JNK2 in FDH-expressing cells. Interestingly, the FDH-induced apoptosis in p53-proficient A549 cells also proceeds through activation of JNK1/2, but the down-stream target for JNK2 is p53 instead of c-Jun. Furthermore, in A549 cells, FDH activates caspase 9, whereas in PC-3 cells, it activates caspase 8. Our studies indicate that the JNK pathways are common downstream mechanisms of FDH-induced cytotoxicity in different cell types, whereas the end point target in the cascade is cell type specific. JNK activation in response to FDH was inhibited by high supplementation of reduced folate leucovorin, further indicating a functional connection between folate metabolism and mitogen-activated protein kinase pathways.