Tracing the Diverse Paths of One-Carbon Metabolism in Cancer and Beyond

Abstract

One-carbon (1C) metabolism is a network of biochemical reactions distributed across organelles that delivers folate-activated 1C units to support macromolecule synthesis, methylation, and reductive homeostasis. Fluxes through these pathways are up-regulated in highly proliferative cancer cells, and anti-folates, which target enzymes within the 1C pathway, have long been used in the treatment of cancer. In this work, we review fundamental aspects of 1C metabolism and place it in context with other biosynthetic and redox pathways, such that 1C metabolism acts to bridge pathways across compartments. We further discuss the importance of stable-isotope-tracing techniques combined with mass spectrometry analysis to study 1C metabolism and conclude by highlighting therapeutic approaches that could exploit cancer cells’ dependency on 1C metabolism.

Metabolic reprogramming is essential for tumorigenesis (Pavlova and Thompson 2016; Faubert et al. 2020). The altered metabolic state of glycolysis and mitochondrial metabolism tumors and proliferating cells was first described almost a century ago by Warburg et al. (1927). Since then, many new biochemical mechanisms that drive the growth and survival of tumors have been discovered (DeBerardinis and Chandel 2020). Many of these alterations are needed to support the increased bioenergetic and biosynthetic demand for survival and proliferation. As such, these metabolic alterations often include up-regulated nutrient acquisition (e.g., glucose, lactate, amino acids), increased need for electron acceptors, expanded biosynthesis pathways, and changes in gene regulation (Pavlova and Thompson 2016). One such metabolic pathway that supports many of these needs is one-carbon (1C) metabolism.

1C metabolism consists of a broad range of biochemical reactions that are essential for maintaining cellular homeostasis. In cancer, genes in this pathway are often transcriptionally up-regulated. These include dihydrofolate reductase (DHFR) (Guo et al. 1999; Yang et al. 2003), thymidylate synthase (TYMS) (Burdelski et al. 2015; Sun et al. 2015; Fu et al. 2019), mitochondrial serine hydroxymethyl transferase (SHMT2) (Jain et al. 2012; Lee et al. 2014), and mitochondrial methylenetetrahydrofolate dehydrogenase (MTHFD2) (Fig. 1; Nilsson et al. 2014). In fact, patients with high expression of these genes such as SHMT2, MTHFD2, and ALDH1L2 have a shorter survival rate compared to patients with low expression of these genes (Koseki et al. 2018). The gene for the first enzyme for serine synthesis, phosphoglycerate dehydrogenase (PHGDH), is often up-regulated in cancer as well (Locasale et al. 2011; Possemato et al. 2011) and this is important because serine is one of the inputs for 1C metabolism. The reliance on 1C metabolism in cancer cells is also reflected in the use of antifolates for the treatment of cancer (Fig. 1), although these therapies have harmful side effects due to the importance of 1C metabolism in nontransformed cells as well.

Figure 1.

Overview of 1C metabolism and established therapeutics that target this pathway and their effects on cancer. Genes in bold encode enzymes in the 1C metabolic pathway that are transcriptionally up-regulated in some cancers. These include dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), mitochondrial serine hydroxymethyl transferase (SHMT2), and mitochondrial methylenetetrahydrofolate dehydrogenase (MTHFD2). Chemotherapeutics such as methotrexate, pemetrexed, and 5-Fluorouracil inhibit annotated enzymes in the pathway. (Figure generated with BioRender; https://biorender.com.)

Here, we review 1C metabolism in the context of other biosynthetic and redox pathways and discuss how this reaction network acts as a dynamic system to bridge pathways across compartments such as the nucleus, cytosol, and mitochondria. Additionally, we highlight the utility of stable-isotope tracing techniques combined with mass spectrometry measurement to study 1C metabolism and then discuss therapeutic approaches that could exploit cancer cells’ dependency on 1C metabolism.

1C METABOLISM IN RELATION TO THE TRICARBOXYLIC ACID CYCLE

The Warburg effect, which describes the high rate of glucose uptake and lactate secretion even in the presence of oxygen, is well known. The observation that cancer cells actively take up glucose has led to the routine use of 2-[18F]-fluor-2-deoxy-D-glucose position emission tomography/computed tomography (FDG-PET/CT) to diagnose many different cancers in the clinic (Otsuka et al. 2004; Rohren et al. 2004). Although Warburg postulated that cancer cells have defective mitochondria, many cancer cells use these glucose-derived carbons to feed into the Krebs or tricarboxylic acid (TCA) cycle in the mitochondria to support multiple metabolic processes. The TCA cycle produces reducing equivalents in the form of NADH and FADH₂, which are used to generate ATP though oxidative phosphorylation. Additionally, the TCA cycle provides precursors for fatty acid and steroid biosynthesis and fuels other anabolic pathways including amino acid biosynthesis and gluconeogenesis. Beyond these anabolic pathways, TCA cycle intermediates can act as effector molecules to modulate signaling pathways and gene expression (Martínez-Reyes and Chandel 2020).

In many ways, 1C metabolism is like the TCA cycle (Fig. 2), where both pathways include a cyclic network of reactions to generate precursors for the synthesis of other metabolites. Additionally, both 1C and TCA cycles consume and regenerate reducing equivalents as cofactors to support bioenergetics and maintain redox homeostasis (with production of reducing equivalents favored in the matrix). Like the TCA cycle, several enzymatic reactions of 1C metabolism such as MTHFD2/2L and ALDH1L2 produce NAD(P)H in the mitochondrial matrix. As such, many of the reactions in 1C metabolism are linked to changes in redox status. For example, metastatic melanoma cells reversibly increase the expression of NADPH-regenerating enzyme ALDH1L2 to increase their capacity to withstand oxidative stress (Piskounova et al. 2015). Notably, folate-dependent serine catabolism within mitochondria can generate NADPH under hypoxia (Ye et al. 2014) and NADH when respiration is impaired (Yang et al. 2020), indicating the critical role of 1C metabolism in regulating redox homeostasis. On the other hand, both pathways are fueled by substrates carried by vitamin cofactors in the form of tetrahydrofolate and coenzyme A.

Figure 2.

Comparison between 1C and 2C cycle. It can be helpful to think of 1C metabolism in the context of the Krebs or tricarboxylic acid (TCA) cycle. (2-PG) 2-phoshoglycerate, (3-PG) 3-phoshoglycerate, (PEP) phosphoenolpyruvate, (3PHP) 3-phosphohydroxypyruvate, (3PS) 3-phosphoserine, (THF) tetrahydrofolate. (Figure generated with BioRender; https://biorender.com.)

1C metabolism encompasses a complex metabolic network that supports multiple biological processes through the generation of 1C units. These pathways include the interconversion of serine to glycine and 1C units, the glycine cleavage system (GCS), betaine and choline metabolism, and the catabolism of histidine as well as other amino acids. Among these inputs, serine is a major source of 1C units (Labuschagne et al. 2014; Maddocks et al. 2016). Serine is metabolized to/from formate by two complementary pathways in the cytosol and mitochondria (Fig. 3), which consist of similar biochemical reactions: (1) transfer of 1C unit to tetrahydrofolate (THF) by forming 5,10-methylene-THF and glycine, and (2) oxidation of 5,10-methylene-THF to10-formyl-THF. In the cytosol, these steps are catalyzed by SHMT1 and MTHFD1, whereas in the mitochondrial matrix these steps are catalyzed by SHMT2, MTHFD2 or MTHFD2L, and MTHFD1L. In the cytosol, CH₂-THF is also reduced to 5-methyl-THF to support methyl group synthesis. Through these intermediates, the 1C metabolism is associated and interlinked with other metabolic pathways such as the folate cycle, methionine cycle, and indirectly the TCA cycle through redox status (Fig. 2). Folate molecules chemically activate 1C units and must first be reduced from folic acid to dihydrofolate (DHF) by DHF reductase and subsequently to tetrahydrofolate (THF). Once bound to a folate molecule, 1C units exist at different oxidation states: 5, 10-methylene THF, 5-methyl-THF, and 10-formyl-THF with each supporting a different biosynthetic function.

Figure 3.

1C metabolism network across organelles. 1C pathways are distributed across the cytosol, mitochondria, and nucleus. (DHF) Dihydrofolate, (THF) tetrahydrofolate, (DMG) dimethylglycine, (SAM) S-adenosylmethionine, (SAH) S-adenosylhomocysteine, (dcSAM) decarboxylated SAM, (AMD1) S-adenosylmethionine decarboxylase, (MTAP) methylthioadenosine phosphorylase, (PE) phosphatidylethanolamine, (PC) phosphatidylcholine, (PEMT) phosphatidylethanolamine N-methyltransferase, (DHFR) dihydrofolate reductase, (SHMT1/2) serine hydroxymethyl transferase, cytosolic(1)/mitochondrial(2), (MTHFD1) methylenetetrahydrofolate dehydrogenase, cyclohydrolase, and formyltetrahydrofolate synthetase 1, (MTHFD2/2L) methylenetetrahydrofolate dehydrogenase 2/2-like, (MTHFD1L) monofunctional tetrahydrofolate synthase, (ALDH1L1/2) cytosolic(1)/mitochondrial (2) 10-formyltetrahydrofolate dehydrogenase, (GLDC) glycine decarboxylase, (TYMS) thymidylate synthase, (GNMT) glycine N-methyltransferase, (SARDH) sarcosine dehydrogenase, (DMGDH) dimethylglycine dehydrogenase, (MTFMT) mitochondrial methionyl-tRNA formyltransferase, (MTHFR) methylenetetrahydrofolate reductase, (MS) methionine synthase, (BHMT) betaine-homocysteine S-methyltransferase, (MAT) methionine adenosyltransferase, (AHCY) S-adenosyl-L-homocysteine hydrolase, (CBS) cystathionine beta synthase, (CSE) cystathionine γ lyase, (GART) phosphoribosylglycinamide formyltransferase, (ATIC) 5-aminoimidazole-4-carboxamideribonucleotide formyltransferase/IMP cyclohydrolase. (Figure generated with BioRender; https://biorender.com.)

1C metabolism contributes to the methionine cycle by reduction of CH₂-THF to 5-methyl-THF. The methionine cycle produces S-adenosylmethionine (SAM), a ubiquitous methyl group donor that is used by SAM-dependent methyltransferases for the methylation of DNA, RNA, proteins, and lipids. 5,10-methylene-THF derived from the folate cycle is irreversibly converted to 5-methyl-THF by MTHFR. 5-methyl-THF then donates its methyl group to homocysteine in a B12-dependent reaction catalyzed by methionine synthase to produce methionine and THF. THF can then reenter the folate cycle. The remethylation of homocysteine to form methionine is also driven by choline through the generation of betaine via betaine-homocysteine S-methyltransferase (BHMT). A product of that reaction, dimethylglycine (DMG) can further contribute 1C units to the mitochondrial folate cycle. Methionine is then converted into SAM by S-adenosylmethionine synthase in an ATP-dependent process. The folate cycle helps contribute to this reaction as well by maintaining ATP levels through de novo purine synthesis in cancer cells (Maddocks et al. 2016). SAM can also be diverted to the polyamine synthesis pathway after decarboxylation by adenosylmethionine decarboxylase 1 (AMD1). Decarboxylated SAM along with putrescine are precursors for polyamines, spermidine, and spermine, and generate 5-methylthioadenosine as a byproduct that can be recycled back to methionine by 5-methylthioadenosine phosphorylase (MTAP).

The methionine cycle is also involved in the biosynthesis of phospholipids such as phosphatidylcholine through SAM (Zatz et al. 1981). The head group of phosphatidylcholines is synthesized from choline through the adenylation of methionine to SAM, such that alterations in 1C metabolism may influence the balance of phosphatidylcholine and phosphoethanolamine. These glycerophospholipids are abundant in tissues and are important components of the plasma membrane that facilitate transport and signaling across cells (Szlasa et al. 2020; Saito et al. 2022).

Besides choline, glycine is another potential source of 1C units through the GCS, which catalyzes the breakdown of glycine to CO₂, ammonia, and a 1C unit in the form of 5,10-methylene-THF. As such, the GCS is coupled to 1C metabolism via the activity of MTHFD2/2L and MTHFD1L. Glycine is subsequently catabolized or converted to serine by SHMT1, as patients lacking GCS function accumulate glycine (Kralik et al. 2023). While highly active in liver, kidney, and select cell types (Handzlik and Metallo 2023), immortalized cancer cell lines lack a significant contribution of GCS activity to the 1C pool (Labuschagne et al. 2014; Meiser et al. 2016).

The 1C cycle is also influenced by redox homeostasis. Tetrahydrofolate reductase reduces THF, which enters the folate-mediated 1C cycle, and this reaction consumes one molecule of NADPH for each turn of the cycle. The reactions catalyzed by MTHFD1 or MTHFD2/L either consume or produce NAD(P)H depending on the direction of the reaction. While many of these reactions are reversible, the oxidation of 10-formyl-THF to CO₂ is not and generates NADPH in the mitochondrial matrix. In addition to supporting redox maintenance, NAPDH also supports lipid synthesis and NADPH oxidase (NOX) signaling to support cancer cell proliferation. In some cancers, the mitochondrial 1C pathway is a contributor to the NADPH pool (Fan et al. 2014; Ye et al. 2014; Piskounova et al. 2015). Finally, the transsulfuration pathway connects to the methionine and folate cycle through homocysteine, such that the condensation of serine and homocysteine yields cystathionine and subsequently cysteine, a precursor for glutathione. Glutathione is an important antioxidant that helps maintain redox homeostasis as levels are maintained at mM concentrations in cells and tissues.

Overall, 1C metabolism is highly interconnected and dependent on multiple inputs that fuel the folate-mediated 1C cycle. It may therefore be helpful to think about the 1C cycle in relation to the 2-carbon TCA cycle. Both pathways are amphibolic in that they are involved in catabolic and anabolic processes, they are both linked directly by electron flow to/from reducing equivalents, and they also influence epigenetic regulation. In the same way, reactions in both of these pathways occur in the cytosol, mitochondrial matrix, and nucleus.

COMPARTMENTALIZATION OF 1C METABOLISM

The wide network of metabolic reactions in 1C metabolism and associated pathways occur in various organelles, such that specific reactions are compartmentalized across the cytoplasm, mitochondria, and nucleus (Fig. 3; Tibbetts and Appling 2010). While MTHFD1 catalyzes the conversion of formate to 10-formyl-THF, 5,10-methenyl-THF, and 5,10-methylene-THF in the cytosol, these reactions are catalyzed separately by MTHFD2/2L and MTHFD1L in the mitochondria. 1C-activated folates are not known to travel across intracellular membranes, so to enable the transfer of 1C units between compartments, 10-formyl-THF is converted to formate. The hydrolysis of 10-formyl-THF is coupled to ATP production and is reversible. Transport of 1C units between organelles can also occur through serine and glycine. In the mitochondria, dimethylglycine and sarcosine can also serve as 1C donors via dimethylglycine and sarcosine dehydrogenase reactions, highlighting the fact that different sources exist for 1C units in distinct organelles.

The reversibility and compartmentation of enzymes in the cytosol and mitochondria allow pathway flexibility. In most cells, serine is catabolized in the mitochondria (Lewis et al. 2014) to generate 1C units in the cytosol, which end up on thymidylate, formate (Meiser et al. 2016), or methionine (Maddocks et al. 2016). This unidirectional flow of 1C units is thermodynamically driven by the higher NADPH/NADP⁺ ratio in the cytosol through MTHFD1 reaction (Yang and MacKenzie 1993). In contrast, there is more oxidative redox potential in the mitochondria, which would favor the MTHFD2/2L reaction to use NAD(P)⁺ as a cofactor to drive the reaction. These redox-dependent reactions in the 1C cycle can influence the mitochondrial state through redox homeostasis. Mitochondrial enzymes, ALDH1L2 and MTHFD2/2L can produce NADPH through their reactions and the NADPH/NADP⁺ ratio can help maintain the flux of 1C units through the cytosol. Although mitochondrial serine catabolism dominates in most cells, there are cells that show mixed cytosolic and mitochondrial 1C generation since some of these enzymes are differentially expressed across tissues (Girgis et al. 1998). Furthermore, reversal of the MTHFD1 reaction can occur when formyl-THF is depleted (Ducker et al. 2016).

The connection between mitochondrial function and 1C metabolism is further supported by studies that show the remodeling of 1C metabolism upon mitochondrial respiratory chain deficiency. Disruption of the electron transport chain reduces mitochondrial formate production, although the expression of serine synthesis genes is increased through ATF4 (Bao et al. 2016). Similarly, mtDNA replication dysfunction reduces formate and 10-formyl-THF levels while increasing flux to through the de novo serine synthesis pathway in mitochondrial myopathy mice (Nikkanen et al. 2016). The 1C pool can also impact mitochondrial function through production of SAM. While SAM is synthesized in the cytosol, it is transported into the matrix through the mitochondrial SAM carrier (Agrimi et al. 2004) and influences mitochondrial energy metabolism though protein methylation in complex I and iron–sulfur cluster biosynthesis (Schober et al. 2021), highlighting other biosynthetic fates for 1C metabolites.

1C metabolic reactions also exist in the nucleus (Anderson et al. 2007; Woeller et al. 2007). Nuclear folate metabolism occurs through the small ubiquitin-related modifier (SUMO)-dependent import of the enzymes that support de novo thymidylate synthesis from the cytosol to the nucleus (Anderson et al. 2007; Woeller et al. 2007). Upon sumoylation, SHMT, thymidylate synthase, and DHFR can be translocated to the nucleus during the S and G₂/M phase of cell replication (Anderson et al. 2007; Woeller et al. 2007). Using 5,10-methylene-THF as a 1C donor, thymidylate synthase catalyzes the methylation of deoxyuridylate (dUMP) to form thymidylate (dTMP). To reenter the 1C pool, DHF is reduced back to THF and eventually back to 5,10-methylene-THF through Sumo-SHMT1. Other subnetworks in this pathway are localized to distinct organelles as well. While the GCS occurs only in mitochondria (Kikuchi and Hiraga 1982), thymidylate synthesis takes place in the cytosol, mitochondria, and nucleus to support DNA synthesis (MacFarlane et al. 2011). On the other hand, purine synthesis occurs in the cytosol (An et al. 2008). Similarly, the methionine cycle operates in the cytosol although its downstream products are transported into mitochondria (Schober et al. 2021).

In addition to being highly compartmentalized intracellularly, 1C metabolism is differentially active across tissues and cell types. While many nonproliferative adult tissues express 1C enzymes such as SHMT1 and SHMT2, they are highly expressed in the liver and kidney (Girgis et al. 1998). On the other hand, the expression of SHMT2 is greater than that of SHMT1 in tissues like the brain, skeletal muscle, and heart (Girgis et al. 1998). As SHMT1 and SHMT2 operate in different directions and support distinct metabolic pathways, this specification suggests that different functions exist for 1C metabolism across tissues. For example, SHMT1 in mammalian cells has been shown to behave like a switch diverting 1C units for SAM or dTMP synthesis (Herbig et al. 2002). Perturbing 1C metabolism in the liver usually results in a fatty liver phenotype and is thought to be associated with impaired phosphatidylcholine synthesis (Pogribny et al. 2013). Furthermore, the knockout of methionine adenosyltransferase has been shown to induce a fatty liver phenotype, reduce 1C-related metabolites, and lead to hepatocellular carcinoma (Lu et al. 2001). Beyond 1C enzymes, genes encoding enzymes in the GCS are also distinctly expressed in the liver (Yoshida and Kikuchi 1973), which suggests a differential preference for sources of 1C units across tissues. Similarly, transporters for serine and choline, inputs to the 1C cycle, vary according to tissue type (Hediger et al. 2013).

QUANTIFYING 1C METABOLISM FLUX AND METABOLITE LEVELS

The interconnected pathways and compartmentalization of these reactions make studying 1C metabolism challenging. However, by combining stable isotope tracers with mass spectrometry analysis researchers have made great insights into the topology of this pathway. Serine contributes to the 1C pool and stable isotope tracers such as [2,3,3-²H]serine and [3,3-²H]serine will generate ²H-labeled 1C folate intermediates downstream of SHMT (Gregory et al. 2000; Herbig et al. 2002). Tracing with [2,3,3-²H]serine, however, provides compartment-specific information on this pathway (detailed in Fig. 4A). If serine is metabolized in the mitochondria, one deuterium on the third carbon eventually ends up on NADPH and the other on formate. The incorporation of this 1C unit from formate into DNA will result in formation of a singly deuterated thymidine monophosphate (TMP M + 1). In contrast, if serine is metabolized in the cytosol by SHMT1, both deuterons on serine will be transferred to 5,10-methylene THF that will eventually result in doubly labeled TMP M + 2 (Fig. 4A). In most cells and tissues analyzed, TMP M + 1 is the predominant product from this tracer, suggesting that mitochondrial serine catabolism occurs along with cytosolic reduction of formate by MTHFD1.

Figure 4.

Tracing 1C metabolism. (A) [2,3,3-²H]serine can be used to distinguish compartment-specific metabolism of serine since ²H from serine is incorporated differently into 1C intermediates and dTMP. (B) [3-¹³C] serine allows us to exclude the carbon contribution from glycine cleavage because only the ¹³C carbon on serine transfers to 5,10-me-THF (5,10-methylene-THF) via SHMT. (THF) Tetrahydrofolate, (SHMT1/2) serine hydroxymethyl transferase, cytosolic(1)/mitochondrial(2), (MTHFD1) methylenetetrahydrofolate dehydrogenase, cyclohydrolase, and formyltetrahydrofolate synthetase 1, (MTHFD2/2L), methylenetetrahydrofolate dehydrogenase 2/2-like, (MTHFD1L) monofunctional tetrahydrofolate synthase, (TYMS) thymidylate synthase. (Figure generated with BioRender; https://biorender.com.)

Another serine tracer that may effectively be used to investigate 1C metabolism is [3-¹³C]serine (Fig. 4B). This serine tracer enables one to exclude the carbon contribution from glycine cleavage because the ¹³C carbon on serine transfers to 5,10-methylene-THF only via SHMT (Fig. 4B). This tracer has been used to observe serine catabolism to formate in vivo (Meiser et al. 2016). Similarly, ¹³C-MeOH may be employed to quantify serine catabolism to formate in vivo because methanol is metabolized to formate in the liver (Meiser et al. 2018). Fully ¹³C- and ¹⁵N-labeled serine or methionine can track flux into intermediates of the methionine cycle and methylation of DNA and RNA, which is helpful to study methylation dynamics (Newman et al. 2019). Multiple stable-isotope tracers can also be used in parallel to perform dynamic flux analysis. For example, parallel use of [4-²H]glucose and [²H]formate was performed to demonstrate that MTHFD2 has a noncanonical oxidative function to provide mitochondrial NAD⁺ (Achreja et al. 2022). This experiment is possible because [4-²H]glucose labels NADH and if the MTHFD2 reaction operates oxidatively rather than reductively, the ²H label appears on serine. Isotope from [²H]formate appears on serine through the folate cycle, and this helped Achreja et al. to deconvolute the noncanonical mitochondrial MTHFD2 activity.

As our knowledge of 1C metabolism advances, alternative sources for 1C units in specific cell and tissue contexts are being discovered. One example of a nontraditional source for 1C units is tryptophan in pancreatic ductal adenocarcinoma (PDAC) (Newman et al. 2021). Indoleamine 2,3-dioxygenase (IDO) expression in cancer cells promotes the generation of 1C units from tryptophan to support de novo purine synthesis, observed using ¹³C-tryptophan (Newman et al. 2021). Similarly, ¹³C-formaldehyde was used to show that formaldehyde detoxification by alcohol dehydrogenase 5 supplies 1C units for nucleotide synthesis in cells (Burgos-Barragan et al. 2017). Overall, these examples show the utility of stable isotope tracing in exploring the many pathways fueling 1C metabolism.

To better characterize organelle-specific directionality of 1C metabolism, Lewis et al. (2014) leveraged [2,3,3-²H]serine labeling of NAD(P)H and a compartment-specific reporter system. Specifically, cells were engineered to inducibly express either cytosolic IDH1 or mitochondrial IDH2 harboring neomorphic mutations that result in production of (D)2-hydroxyglutarate (2-HG) from α-ketoglutarate (Lewis et al. 2014). As this reaction reduces αKG by transferring a hydride from NADPH to form 2HG, the label on NADPH is transferred to 2HG. 2HG is typically present at low levels (Matsunaga et al. 2012) and the labeled 2HG can be used as a readout of NADPH metabolism. By using [2,3,3-²H]serine or other tracers in this reporter system, the authors have characterized redox reaction directionality of 1C (Lewis et al. 2014) and TCA cycle (Jiang et al. 2016; Badur et al. 2018) reactions in both the cytosol and mitochondria.

Quantitation of metabolites over time is the most direct approach to measure flux. Absolute quantification of formate by GC-MS has enabled measurements of key exchange fluxes between formate, serine, and glycine (Meiser et al. 2016). Quantifying rates of purine synthesis and formate release revealed the latter as the dominant fate of 1C metabolites (Meiser et al. 2016, 2018). These results when paired with metabolic flux analysis indicate that formate efflux exceeds anabolic 1C demands (Meiser et al. 2016, 2018). Similarly, measuring the flux of CO₂ released from [1-¹³C]glycine has demonstrated that the GCS is a major contributor for entry of 5,10-methylene-THF into 1C metabolism at a rate that exceeds the demand for methyl groups (Lamers et al. 2009). The ability to quantitatively assess and model these reactions greatly improves our ability to target 1C metabolism in specific tissues or contexts.

DYNAMIC REGULATION OF 1C METABOLISM

As described above, 1C metabolism functions as a central metabolic hub that connects metabolites and reactions across organelles to not only produce critical macromolecules but also for maintenance of redox homeostasis and epigenetic status. The many inputs into the cycle make it a dynamic system that is responsive to multiple perturbations and regulation. In fact, many of the reactions in the 1C cycle are reversible, highlighting the flexibility ingrained in the system (Ducker et al. 2016). The existence of multiple isoforms for most of the 1C enzymes enables such specialization and redundancy such that different isoforms to compensate for one if the other is comprised (Ducker et al. 2016). Again, this metabolic architecture and behavior are manifested in the TCA cycle, where NADP⁺-dependent isocitrate dehydrogenases afford flexibility under hypoxic or lipid stress. Although mitochondrial 1C metabolism is dominant in most cells, reversal of cytosolic 1C reactions can compensate for the loss of the mitochondrial folate pathway (Ducker et al. 2016). Additionally, when the cytosolic pathway is compromised, alternative sources of 1C units such as endogenous formaldehyde feeds the cycle, further emphasizing the adaptability of this critical pathway (Burgos-Barragan et al. 2017).

At the same time, the demand and role for 1C metabolism may be different across developmental stages. For example, consumption rates for 1C units are highest during fetal development, and folate deficiency is a known driver of neural tube and congenital heart defects (Bailey and Berry 2005; Beaudin et al. 2011). Additionally, genes encoding 1C enzymes are differentially expressed during embryonic development in the liver and brain, highlighting the dynamic regulation of this pathway as tissue morphogenesis and specialization occur (Imbard et al. 2021). Highly proliferative cells such as hematopoietic, immune, and gastrointestinal lineages are highly dependent on 1C metabolism (Ponziani et al. 2012; Henry et al. 2017; Kurniawan et al. 2021). Activated T cells, which likely exhibit the highest rates of nucleotide synthesis in the body, up-regulate 1C metabolism in the mitochondria to promote thymidylate and purine synthesis, while genetic targeting of mitochondrial 1C metabolism enzymes impairs T-cell survival (Ron-Harel et al. 2016). Recent research also suggests that MTHFD2 regulates both effector and regulatory T-cell fate and function through various mechanisms (Sugiura et al. 2022).

Given its contributions to diverse proliferating (and nonproliferating) normal cell types, it is not surprising that expression of 1C enzymes is altered in the context of cancer. MTHFD2 is mostly absent in healthy adult tissues, yet its expression is frequently up-regulated in many cancer types (Nilsson et al. 2014) and is associated with a poor prognosis in breast cancer (Liu et al. 2014). Other 1C enzymes that are highly expressed in various cancer types are TYMS (Burdelski et al. 2015; Sun et al. 2015; Fu et al. 2019), SHMT (Jain et al. 2012; Lee et al. 2014), and DHFR (Guo et al. 1999; Yang et al. 2003). In addition, the robust up-regulation of 1C metabolic enzymes has been observed in response to cancers associated with cancers associated with viral infection (Wang et al. 2019; Zhang et al. 2021). Epstein–Barr virus (EBV) is a driver of B-cell lymphomas (Epstein et al. 1964). Upon infection, EBV remodels B-cell mitochondrial 1C metabolism along with serine uptake and catabolism, and these were among the most highly induced pathways (Wang et al. 2019). Functionally, EBV-induced mitochondrial 1C metabolism generates NADPH, fuels nucleotide synthesis through formate, and generates glutathione (Wang et al. 2019). Outside of cancer, the 1C metabolism pathway is remodeled by viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Zhang et al. 2021). These findings also highlight the potential for inhibition of 1C metabolism as a therapeutic for viral infections, as practiced in cancer therapy.

1C metabolism is also responsive to perturbations in redox homeostasis. Formate production and efflux are sensitive to cellular redox state (Meiser et al. 2018). Specifically, shifting cells toward a more oxidative state induces increased formate efflux and vice versa (Meiser et al. 2018). During hypoxia, MYC-transformed cells induce the expression of SHMT2 to maintain mitochondrial redox balance, specifically cellular NADPH:NADP⁺ ratio (Ye et al. 2014). This is clinically relevant as hypoxia develops during tumor progression when there is insufficient vasculature to supply oxygen to tumor cells. During metastasis, some melanoma cells reversibly increase their expression of ALDH1L2, an NAPDH-regenerating enzyme, and knockdown of MTHFD1 or ALDH1L2 inhibits metastasis to distant organs (Piskounova et al. 2015). As discussed previously, 1C metabolism is linked to mitochondrial function through cofactors and intermediates such as NAD(P)H and SAM, providing various mechanisms for cross-regulation upon perturbation (Schober et al. 2021). Additionally, mitochondrial 10-formyl-THF is used for N-formylation of the initiator methionine in mitochondrial methionyl-tRNA^met, which is important for translation of mitochondrial-encoded proteins (Tucker et al. 2011).

Evidence indicates that 1C metabolism is tightly regulated by mammalian target of rapamycin (mTORC1), a critical nutrient sensor (Ben-Sahra et al. 2016; Rathore et al. 2021). A known downstream target of mTOR is ATF4, which regulates the expression of serine biosynthesis enzymes including PHGDH (Ye et al. 2012). When serine levels are low, ATF4 is activated to sustain glucose uptake, serine synthesis, and flux through the 1C pathway, as ATF4 also regulates expression of MTHFD2 (Ben-Sahra et al. 2016). mTORC1 also regulates the expression of other 1C enzymes, including SHMT2 and MTHFD1L through FOXK1 (He et al. 2018). Unsurprisingly, mTORC1 signaling is often hyperactivated in various cancers (Zhang et al. 2017). An upstream effector of mTORC1, LKB1, is a known tumor suppressor (Hemminki et al. 1998; Sanchez-Cespedes 2007). LKB1 loss results in up-regulation of enzymes involved in 1C metabolism and SAM biosynthesis, further connecting mTOR signaling to changes in DNA methylation and tumorigenesis (Kottakis et al. 2016). The activation of the energy sensor and LKB1 targeted AMP-activated kinase (AMPK), also down-regulates MTHFD1, MTHFD1L, and MTHFD2 expression through the PGC1a/ERRa axis (Audet-Walsh et al. 2016). Notably, expression of PGC1a is reduced in some cancers relative to healthy tissue (Deblois et al. 2013). Overall, these observations suggest that cells tune 1C pathway flux based on their energetic status and biosynthetic demands such that they can respond to perturbations to support the dynamic metabolic needs associated with tumor cell growth and survival. Redundancy and parallelization across compartments in enzymes within this pathway highlight the metabolic flexibility necessary to respond to the stresses, which is critical for embryonic cells and tumors.

TARGETING 1C METABOLISM

As tumor cells commonly overexpress key 1C enzymes, numerous therapeutics direct targeted key critical nodes in the pathway (Fig. 1). For example, methotrexate, pemetrexed, and 5-Fluoruoacil are commonly used chemotherapeutics approved for treatment of lymphomas, osteosarcomas, breast cancer, and lung cancer (Huennekens 1994; Longley et al. 2003; Vander Heiden 2011). Both methotrexate and pemetrexed target dihydrofolate reductase (DHFR), with pemetrexed targeting thymidylate synthase (TYMS) and serine hydroxymethyltransferases (SHMTs) as well (Shih et al. 1997; Goodsell 1999). While these drugs are effective against tumors, the benefits of these drugs are hampered due to toxicities caused by inhibition of 1C metabolism in nontransformed cells, including those in the bone marrow and intestinal epithelium resulting in various complications.

The challenge with targeting components of 1C metabolism more specifically is that tumors may rewire their metabolism to compensate for the intervention and eventually resist treatment. As such, combination therapies to target 1C metabolism have been explored in preclinical models. Combination treatment with SHMT1/2 inhibitor (e.g., SHIN2) and methotrexate showed a synergistic response in a patient-derived xenograft (PDX) T-cell acute lymphoblastic leukemia mouse model (García-Cañaveras et al. 2021). This result is likely due to methotrexate-induced depletion of cellular THF, resulting in decreased competition for SHIN2 that effectively inhibits SHMT. An alternate strategy is to design multitargeted inhibitors of 1C cycle enzymes, as they are traditionally targeted using antimetabolites (García-Cañaveras et al. 2021). Small molecules that possess structural features to inhibit de novo purine biosynthesis, SHMT1, and SHMT2 demonstrated significant in vivo antitumor efficacy in early- and late-stage pancreatic adenocarcinoma xenografts in mice (Dekhne et al. 2019).

Recent efforts to uncover collateral deletion of essential genes in chromosomal proximity during inactivation of tumor suppressor genes can provide potential targets for combination therapies. For example, MTHFD2 has recently been identified as a collateral gene in UQCR11-deleted ovarian tumors, and inhibiting MTHFD2 in UQCR11-null cancers dramatically decreased xenograft tumor growth, showing the essentiality of MTHFD2 in these cancers (Achreja et al. 2022). Inhibitors against 1C metabolism combined with inhibitors of other targets such as PHGDH or mTOR could be effective in such cases. Indeed, the rewiring of the serine synthesis pathway has been identified as a mechanism for resistance in certain cancer cells (Ross et al. 2017; Rathore et al. 2021).

Nutrient availability has a significant impact on the progression of some tumors. Special diets such as the ketogenic diet can sensitize some cancers to standard treatment by exploiting the reprogrammed metabolism of cancer cells (Hopkins et al. 2018; Tajan and Vousden 2020; Ferrere et al. 2021). Studies have shown that dietary removal of amino acids including serine/glycine (Maddocks et al. 2013; Muthusamy et al. 2020), methionine (Gao et al. 2019), and asparagine (Krall et al. 2016) can modulate cancer growth in animal models. In some tumors, this effect was further improved when combining serine restriction with biguanides (i.e., metformin) that disrupt mitochondrial oxidative phosphorylation (Maddocks et al. 2017). The response to dietary serine/glycine restriction, however, is limited in tumors that can effectively up-regulate expression of PHGDH or serine transporters, which can occur downstream of activated KRAS (Maddocks et al. 2017). To address this issue, a combination of dietary serine restriction with PHGDH or PSAT inhibitors has been tested in animal models and demonstrated some effect against tumors that are resistant to diet or inhibitor alone (Méndez-Lucas et al. 2020; Tajan et al. 2021). Similarly, dietary methionine restriction has been shown to have anticancer effects mediated through changes to 1C metabolism (Gao et al. 2019). In addition to reducing tumor growth in colorectal PDX models, methionine restriction also sensitized these models to chemotherapy 5-FU and radiation, providing evidence that targeted dietary manipulation can affect specific tumor metabolism and impact cancer progression in mice (Gao et al. 2019). On the other hand, dietary supplementation of histidine enhances the sensitivity of cancer cells to methotrexate by up-regulating the histidine degradation pathway to deplete THF (Kanarek et al. 2018), highlighting the interconnected nature and complexity of targeting 1C metabolism.

BYSTANDER EFFECTS

Antifolates have been the standard of care for cancer patients since the 1950s, and yet patients still must endure the deleterious side effects during treatment due to the importance of 1C metabolism in healthy proliferating, nontransformed cells. Toxicities caused by these drugs tend to cause anemia, gastric complications, and immune deficiency. In fact, the immunosuppressant impact from methotrexate has made it useful to treat chronic inflammatory conditions such as rheumatoid arthritis, psoriasis, and Crohn's disease (Willkens et al. 1984; Weinblatt et al. 1985; Kozarek et al. 1989; Feagan et al. 1995; Esteitie et al. 2005). However, immunosuppression can be devastating to cancer patients as the protective role of immune cells such as T-cell lymphocytes are instrumental in the body's defense against tumors. The efficacy of cell-based therapies and vaccinations are dependent on the generation of robust and stable populations of antigen-specific T-cell populations (Waldman et al. 2020). As such, the disruption of T-cell metabolism through cancer therapy is deleterious to immune function and patient outcomes in the long run.

CONCLUDING REMARKS

Advances in analytical chemistry, molecular biology techniques, and genetically engineered mouse models have improved our understanding of 1C metabolism in various contexts relevant to cancer. Nonetheless, the complexity and compartmentalization of 1C cycle reactions across organelles are yet to be fully deciphered. While some mitochondrial transporters for 1C metabolites have been identified (Porter et al. 1992; Titus and Moran 2000; Agrimi et al. 2004; Kory et al. 2018), genes encoding other transporters remain unknown. Additionally, metabolic cross talk between organelles such as the mitochondria and nucleus is not well understood, yet this communication between organelles is likely to have important implications for tumor growth and metastasis. New disciplines such as functional genomics and organelle-specific biology are rapidly enabling discoveries in this area. The challenge then remains to translate such mechanistic and biological insights into physiologically relevant therapies that have improved precision or efficacy in cancer patients.