Therapeutic Targeting of Mitochondrial One-Carbon Metabolism in Cancer a C Aamod S

Published OnlineFirst September 2, 2020; DOI: 10.1158/1535-7163.MCT-20-0423

MOLECULAR CANCER THERAPEUTICS | REVIEW

Therapeutic Targeting of Mitochondrial One-Carbon Metabolism in Cancer A C Aamod S. Dekhne1, Zhanjun Hou1, Aleem Gangjee2, and Larry H. Matherly1

ABSTRACT ◥ One-carbon (1C) metabolism encompasses folate-mediated 1C of 1C metabolism in cancer cells, including the critical role of the transfer reactions and related processes, including nucleotide and mitochondrial 1C pathway as a source of 1C units, glycine, reducing amino acid biosynthesis, antioxidant regeneration, and epigenetic equivalents, and ATP, have spurred the discovery of novel com- regulation. 1C pathways are compartmentalized in the cytosol, pounds that target these reactions, with particular focus on 5,10- mitochondria, and nucleus. 1C metabolism in the cytosol has methylene tetrahydrofolate dehydrogenase 2 and serine hydroxy- been an important therapeutic target for cancer since the inception methyltransferase 2. In this review, we discuss key aspects of of modern chemotherapy, and “antifolates” targeting cytosolic 1C 1C metabolism, with emphasis on the importance of mitochondrial pathways continue to be a mainstay of the chemotherapy arma- 1C metabolism to metabolic homeostasis, its relationship with the mentarium for cancer. Recent insights into the complexities oncogenic phenotype, and its therapeutic potential for cancer.

Introduction synthesize folates de novo, mammals cannot (4, 16). Accordingly, folate cofactors must be acquired through the diet (e.g., leafy green Metabolic reprogramming is a hallmark of cancer (1). Of the vegetables) as reduced forms or as folic acid in fortified foods. altered metabolisms in cancer, one-carbon (1C) metabolism is Reflecting their hydrophilic nature, circulating folates have limited especially noteworthy. While 1C metabolism in the cytosol has capacities to diffuse across plasma membranes. Accordingly, mam- been an important therapeutic target for cancer since the inception malian cells have evolved sophisticated uptake systems (Fig. 1)to of modern chemotherapy (typified by aminopterin, methotrexate, facilitate folate transport across plasma membranes, most notably the and 5-fluorouracil; refs. 2, 3), increasing attention has been focused reduced folate carrier (RFC; SLC19A1; refs. 17–19) and the proton- on mitochondrial 1C metabolism and its importance to the malig- coupled folate transporter (PCFT; SLC46A1; refs. 18, 19). The ubiq- nant phenotype as a critical source of 1C units, glycine, reducing uitously expressed RFC is the major uptake mechanism for folates into equivalents, and ATP (4–8). Indeed, growing evidence suggests that tissues and tumors from the systemic circulation (17–19). RFC is a serine hydroxymethyltransferase 2 (SHMT2) and 5,10-methylene folate–anion antiporter and exchanges reduced folates for organic tetrahydrofolate dehydrogenase 2 (MTHFD2), the first and second anions such as organic phosphates (17–19). PCFT is a proton–folate enzymes in the serine catabolic pathway in mitochondria, are symporter that facilitates absorption of dietary folates at the acidic pH independent prognostic factors and potential therapeutic targets ( pH 6) of the upper gastrointestinal (GI) tract (19). While PCFT is for a number of cancers (9–14). In this review, we discuss key also detected in the kidney, liver, placenta, and spleen (20, 21), it is not a aspects of 1C metabolism with particular emphasis on the impor- major folate transporter in most normal tissues as its activity is very tance of mitochondrial 1C metabolism to metabolic homeostasis, its low in tissues outside the upper GI tract secondary to bicarbonate relationship with the oncogenic phenotype, and its therapeutic inhibition (at neutral pH; ref. 22). PCFT is optimally active at acidic pH potential for cancer. (pH 5–5.5), with detectable activity up to pH 6.5–7 (23). PCFT is expressed in tumors including non–small cell lung cancer (24), malig- Folate Homeostasis and nant pleural mesothelioma (25), epithelial ovarian cancer (26), and Compartmentation of Cellular 1C pancreatic cancer (27), where it functions in the cellular uptake of folates and related compounds at the acidic pH characterizing the Metabolism microenvironments of many tumors (20, 23). Folates encompass a group of water soluble compounds within the Following internalization, folates are compartmentalized in the vitamin B9 family composed of pteridine, p-aminobenzoic acid, and cytosol and the mitochondria (28), with a smaller pool in the nucleus L-glutamate moieties (15). While many species from bacteria to plants (Fig. 1; ref. 29). In the cytosol, folate cofactors participate in 1C- dependent metabolism, leading to the synthesis of thymidylate, purine nucleotides, serine, and methionine (15). Cytosolic and mitochondrial 1Department of Oncology, Wayne State University School of Medicine, and the 1C pathways are interconnected by an interchange between serine, 2 Barbara Ann Karmanos Cancer Institute, Detroit, Michigan. Division of Medicinal glycine, and formate (Fig. 1; refs. 4, 5, 28), with uptake of folates from Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, the cytosol into mitochondria via a “mitochondrial folate transporter” Pittsburgh, Pennsylvania. (MFT; SLC25A32; refs. 30, 31). MFT is the only known transporter of Corresponding Author: Larry H. Matherly, Wayne State University School of folates from the cytosol into the mitochondrial matrix (31) and is a Medicine and the Barbara Ann Karmanos Cancer Institute, 4100 John R, Detroit, MI 48201. Phone: 313-578-4280; Fax: 313-578-4287; E-mail: member of the mitochondrial carrier family, which includes the [email protected] ATP/ADP exchange carrier and the phosphate carrier (32). In the cytosol and mitochondria, folates are substrates for alternate Mol Cancer Ther 2020;19:2245–55 isoforms of folylpoly-g-glutamate synthetase (FPGS), representing doi: 10.1158/1535-7163.MCT-20-0423 splice variants encoded by a single gene (33). FPGS catalyzes the 2020 American Association for Cancer Research. conjugation of up to eight additional glutamate residues to the

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+ ( )+ ( )

MTHFD2/L MTFMT

PKM2

SHMT

+ 1 MTHFD1 MTHFD1

TS MTHFD1

MTR SAMS

RFC PCFT

Figure 1. 1C metabolism reactions, intermediates, and products. 1C metabolism is compartmentalized into the mitochondria and cytosol with a smaller pool in the nucleus. Folates are transported across the plasma membrane by the RFC (SLC19A1) and PCFT (SLC46A1). Serine is generated from glycolysis intermediates. Serine then enters the mitochondria and provides 1C units for the serine cycle (noted with bold blue arrows). Formate is exported to the cytosol where it serves as a source of 1C units for biosynthetic reactions. Nuclear 1C metabolism generates dTMP from dUMP to eliminate uracil misincorporation in DNA. Nuclear enzymes, in light blue, undergo SUMOylation in the cytosol and are imported into the nucleus during S-phase. Folate uptake into the mitochondria is mediated by the MFT (SLC25A32). 2-PG, 2-phosphoglycerate; FAICAR, 5-formamidoimidazole-4-carboxamide ribonucleotide; FGAR, formyl glycinamide ribonucleotide; f-Met, formyl methionine; GAR, glycinamide ribonucleotide; MTFMT, methionyl tRNA formyl transferase; PEP, phosphoenolpyruvate; THF, tetrahydrofolate.

g-carboxyl of the terminal glutamate of folate substrates (34). Poly- the cytosol (4–7). Cells deﬁcient in mitochondrial 1C metabolism or glutamyl folates are the preferred substrates for C1 transfer reac- MFT are glycine auxotrophs and can require exogenous formate for tions (34). Furthermore, cytosolic folate polyglutamates are retained in survival (31, 35, 36). Mitochondrial 1C metabolism is also an impor- cells (34), and mitochondrial folate polyglutamates do not exchange tant source of NAD(P)H and glycine for glutathione (GSH) synthesis with cytosolic forms (33). and ATP (refs. 4, 6, 7, 37; see below). In mitochondria, folates are required for 1C metabolism originating In the cytosol, 10-CHO-THF is resynthesized from formate and from serine (Fig. 1). Serine catabolism involves three primary steps, tetrahydrofolate by MTHFD1 (Fig. 1), a trifunctional enzyme that catalyzed by SHMT2, MTHFD2 (or methylene tetrahydrofolate dehy- includes dehydrogenase, cyclohydrolase, and 10-formyl tetrahydro- drogenase 2-like, MTHFD2L), and methylene tetrahydrofolate dehy- folate synthetase activities (38). This provides 10-CHO-THF substrate drogenase 1-like (MTHFD1L; ref. 4). The net result is generation of for de novo purine biosynthesis [by glycinamide ribonucleotide glycine and 1C units, with MTHFD1L catalysis resulting in conversion formyl transferase (GARFTase) and 5-aminoimidazole-4-carboxa- of 10-formyl tetrahydrofolate (10-CHO-THF) to formate, which mide ribonucleotide (AICAR) formyltransferase (AICARFTase)] and passes to the cytosol. Serine catabolism in mitochondria serves as the 5,10-methylene tetrahydrofolate (5,10-CH2-THF) for thymidylate principal source of 1C units and glycine for cellular biosynthesis, synthase (TS) and other reactions including SHMT1 (Fig. 1). De novo including de novo synthesis of purine nucleotides and thymidylate in purine biosynthesis includes 10 sequential reactions from

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phosphoribosyl pyrophosphate (PRPP) to IMP (Fig. 1; ref. 39). To Serine Biosynthesis, Mitochondrial 1C facilitate an efficient flux of pathway intermediates, these enzymes Metabolism, and Cancer assemble into a structure termed the “purinosome” (39), which sequesters pathway intermediates and reduced folates, and colocalizes Synthesis of serine is upregulated in cancer (53, 54). Serine is with mitochondria to consume five moles of ATP per mole of IMP synthesized from 3-phosphoglycerate (3-PG) with 3-phosphoglycer- synthesized. SHMT1 converts glycine to serine with 1C units from ate dehydrogenase (PGDH) as the first committed step (Fig. 1; ref. 54). 5,10-CH -THF (Fig. 1). Thymidylate is synthesized from dUMP and PGDH is overexpressed in breast cancers and melanomas, in part, due 2 fi 5,10-CH2-THF by TS, generating dihydrofolate (DHF) which, in turn, to gene ampli cation (53, 55). The 3-phosphopyruvate product is is reduced to the active tetrahydrofolate form by dihydrofolate reduc- transaminated by phosphoserine aminotransferase 1 (PSAT1) into tase (DHFR; ref. 15). Interestingly, TS and DHFR (as DHFR-like 1 or 3-phosphoserine (3PS), which is converted into serine by phospho- DHFRL1) are also expressed in mitochondria to protect the integrity of serine phosphatase (PSPH; ref. 54). Serine is an allosteric inhibitor of mitochondrial DNA (40). While the impact of noncytosolic thymi- PGDH (56) and regulates its own synthesis. Furthermore, serine is a dylate biosynthesis on therapeutic targeting with antifolate drugs ligand and allosteric activator of pyruvate kinase M2 (PKM2), which is (below) is not entirely clear, evidence suggests a role in response to expressed in proliferating cancer cells. Thus, under conditions of serine TS inhibitors (41). deprivation, PKM2 activity is reduced and more glucose-derived 5,10-CH2-THF is metabolized in the cytosol to 5-methyl tetra- carbon is channeled into serine biosynthesis to support cell prolifer- hydrofolate by 5,10-methylene tetrahydrofolate reductase ation (57). Serine is actively transported from the cytosol into mito- (MTHFR; Fig. 1;ref.42).5-Methyltetrahydrofolateisamethyl chondria by sideroflexin 1/3 (SFXN1/SFXN3; Fig. 1; ref. 58). donor in the conversion of homocysteine to methionine by the Serine catabolism is often activated in cancer, with the genes vitamin B12–dependent enzyme, methionine synthase (MTR; encoding SHMT2 and MTHFD2 among the most overexpressed ref. 43). Furthermore, methionine is converted by methionine metabolic genes in all human cancers as compared with normal adenosyltransferase (S-adenosyl methionine synthetase, SAMS) tissues (59). Metabolomics analyses of 219 extracellular metabolites into S-adenosyl methionine (SAM), which is required for methyl- from the NCI-60 cancer cell lines showed that glycine consumption ation of DNA, phospholipids, and proteins (44). Thus, serine and serine catabolism including SHMT2, MTHFD2, and MTHFD1L metabolism in the mitochondria supports cellular methylation closely correlated with cancer cell proliferation (60). High levels of reactions via 5-methyl tetrahydrofolate–dependent methylation of expression of these enzymes in cancer cells may, in part, reflect their homocysteine and de novo synthesis of ATP. regulation by MYC as MYC binds to the promoters for SHMT2 and The 1C reactions depicted in Fig. 1 (beginning with serine in MTHFD2, as well as for MTHFD1L (61, 62). MTHFD2 is a tumor- mitochondria) can be viewed as proceeding in a clockwise direction. selective target which is not significantly expressed in differentiated þ This reflects the high NAD(P) /NAD(P)H ratios in mitochondria that adult cells (63). Thus, targeting mitochondrial 1C metabolism at a þ favor serine oxidation to formate, and low NADP /NADPH ratios in number of levels would likely afford selective tumor inhibition, sparing the cytosol that favor conversion of formate to 10-CHO-THF by normal tissues. MTHFD1 and eventually to serine (via SHMT1; ref. 28). When the In MDA-MB-231 breast cancer cells and tissue-tropic metastatic mitochondrial 1C pathway is lost (e.g., deletion of SHMT2 or subclones, serine catabolic enzymes in mitochondria are further MTHFD2), rapid depletion of cytosolic 10-CHO-THF reverses the upregulated, suggesting their critical roles as drivers of proliferation thermodynamic favorability of the MTHFD1 reaction, resulting in a of a subset of metastatic breast cancers (64). Overexpression of SHMT2 compensatory reversal of the cytosolic 1C flux (serine ! formate) to and/or MTHFD2 has been associated with poor prognosis for a meet cellular 1C demand (45). However, this compensation is incom- number of cancers including breast cancer (12, 59, 64, 65), non– plete as these cells exhibit signs of 1C stress (reflected in increased small cell lung cancer (66), pancreatic cancer (11), gliomas (67), cellular AICAR) and glycine auxotrophy (4, 35, 45). Interestingly, cholangiocarcinoma (10), and GI cancers (including esophageal, whereas SHMT1 and MTHFD1 reversal compensates for the loss of gastric, and colon cancers; ref. 9). SHMT2 expression is also increased the mitochondrial 1C pathway, SHMT1 regulates translation of in invasive breast cancer, adrenocortical carcinoma, chromophobe SHMT2 via direct binding to SHMT2 mRNAs when cellular glycine renal cell carcinoma, and papillary renal cell carcinoma, including late- and folate levels are high (46). stage tumors (64). This suggests that targeting SHMT2 and/or In addition to SHMT1 and SHMT2, an alternatively transcribed MTHFD2 could be promising for treating late-stage tumors. SHMT2 isoform (SHMT2a), lacking a mitochondrial targeting Serine catabolism in mitochondria serves as the principal source of sequence (47), is transcribed from a distinct gene (48) and is at 1C units for cellular biosynthesis, including de novo synthesis of purine least partly localized to the nucleus along with TS, DHFR, SHMT1, nucleotides and thymidylate in the cytosol, and SHMT2 provides and MTHFD1 (29). These proteins undergo posttranslational mod- >85% of glycine for proteins, purines, and GSH in tumor cells (4–8). ifications involving the small ubiquitin-like modifier (SUMO) and Mitochondrial 1C metabolism is a major source of NAD(P)H for are translocated from the cytosol to the nucleus at the onset of S- synthesis of macromolecules and protection against oxidative phase where they associate with the DNA repair/replication stress (6, 37, 68). In mitochondria, glycine is required for heme machinery (“replitase;” ref. 49) to generate nuclear thymidylate, biosynthesis (69). which limits uracil misincorporation into DNA (29) during repair SHMT2 catalyzes the conversion of serine to glycine with the and replication. MTHFD2 was reported (50) to promote tumor cell generation of 5,10-CH2-THF (Fig. 1). SHMT2 is essential for cell proliferation by localizing to the nucleus, suggesting a noncanonical survival under hypoxic (6) or ischemic (14) conditions and is upre- role in tumor progression. MTHFD2 has also been reported to gulated by HIF1a in a MYC-dependent manner (6). As MYC- interact with RNA processing proteins to regulate DNA repair and transformed cells rely on SHMT2 to sustain 1C and glycine pools, regulation (51) and to play a role in controlling global N6-methy- and on NAD(P)H for cell survival under hypoxic conditions (6), ladenosine methylation, including hypoxia-inducible factor (HIF) therapeutic targeting of SHMT2 (below) should be selective against 2a mRNA (52). hypoxic MYC-transformed tumors that are resistant to other

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modalities such as radiation. Knockout of SHMT2 (in HCT116 and Mitochondrial NADPH can also be generated by the catabolism of fl Jurkat cells) was accompanied by increased glycolytic ux, suggesting 10-CHO-THF to CO2 and tetrahydrofolate by aldehyde dehydroge- defects in oxidative phosphorylation (70, 71). SHMT2 knockout nase 1 family member L2 (ALDH1L2; Fig. 1; ref. 90). ALDH1L2 is impairs formylation of the initiating methionine tRNA (formyl- approximately 72% homologous to its cytosolic counterpart, aldehyde Met-tRNA), resulting in decreased translation of mitochondria- dehydrogenase 1 family member L1 (ALDH1L1). Although ALDH1L1 encoded complex I and IV subunits, leading to reduced basal and is involved in regulating cell proliferation through its control of maximal respiratory capacities (71). 5,10-CH2-THF is also important tetrahydrofolate pools, ALDH1L2 is a major source of NADPH in for synthesis of the taurinomethyluridine base of other tRNAs (e.g., mitochondria (68, 90). lysine and leucine) in mitochondria (70). Serine is converted to glycine via SHMT2 and is a precursor of SHMT2 is a substrate of histone deacetylase (HDAC) enzymes, cysteine (via the transsulfuration pathway), both of which are impor- which modulates enzyme activity. Desuccinylation of SHMT2 by tant for synthesis of GSH (7). MTHFD2 with ALDH1L2 (Fig. 1) SIRT5, a class III HDAC and member of the sirtuin family (72), provide reducing equivalents (such as NADH and NADPH), which are promotes carcinogenesis through activation of SHMT2 catalytic activ- essential for redox homeostasis and resistance to oxidative ity (73). While the enzymatically active SHMT2 tetramer is stabilized stress (63, 68). Knockdown of SHMT2, MTHFD2, or ALDH1L2 by pyridoxal phosphate (74), the inactive dimer binds the deubiqui- increased reactive oxygen species, with concomitant decrease in þ tinating BRCC36 isopeptidase complex (BRISC), preventing degra- cellular ratios of NAD(P)H to NAD(P) and reduced to oxidized dation of plasma membrane type I IFN receptors and promoting GSH (68). This increased redox stress results in increased cell death inflammatory signaling (75). HDAC11 deacylates SHMT2 and pre- that could be rescued by N-acetylcysteine (6). ALDH1L2 levels are vents its association with BRISC; this permits ubiquitination and elevated in many tumor types (90) and ALDH1L2 was implicated in sequestration of type I IFN receptors (76). As HDAC11 is over- metastasis in a mouse melanoma model, associated with its role in expressed in multiple cancers, HDAC11-mediated SHMT2 deacyla- counteracting oxidative stress (91). Thus, mitochondrial 1C metabo- tion could enable broad spectrum suppression of immune response by lism is important for redox homeostasis and oxidative stress. Further- cancer cells (77). more, NRF2, a key regulator of antioxidant response (37, 92–94), MTHFD2 is a bifunctional enzyme with dehydrogenase and cyclo- regulates expression of genes involved in serine synthesis (PDGH and hydrolase activities that converts 5,10-CH2-THF to 10-CHO-THF PSAT1) and catabolism (SHMT2) via ATF4 (66, 95). This, in turn, þ with synthesis of NADH from NAD (Fig. 1; ref. 78). MTHFD2 can regulates cellular responses to amino acid starvation. þ also use NADP as a cofactor to generate mitochondrial NADPH (63). p53 is associated with the capacity of cancer cells to adapt to MTHFD2 was originally reported to be expressed in transformed, serine starvation and oxidative stress, and, indeed, p53 is activated embryonic, and undifferentiated adult cells, whereas MTHFD2L is upon serine deficiency, resulting in p21 cell-cycle arrest (96). This expressed in differentiated adult cells and at all stages of embryogen- diverts metabolism toward GSH synthesis to combat oxidative esis (79). While recent studies found that normal and cancer cells stress. The protective effects of p53 under serine-depleted condi- express both enzymes, MTHFD2L is unlikely to have an important role tions suggest that p53-null tumors could be particularly vulnerable in cancer (80). For MTHFD2, regulation by MYC and mTOR (81, 82) to serine depletion (96). is consistent with its essential role in supporting the increased bio- The unique demands of tumor cells for 1C units and glycine, and for synthetic demands of rapidly proliferating cells (78). In lung cancer, redox balance under hypoxia (4–6, 8), combined with the high levels of MTHFD2 was implicated in gefitinib resistance and cancer stem–like expression of serine biosynthetic and catabolic pathways in cancers properties by depleting cellular AICAR (83). versus normal tissues (53, 54, 59, 60), suggest that SHMT2 and Downstream from MTHFD2 is the enzyme MTHFD1L (Fig. 1; MTHFD2 could be important therapeutic targets for cancer. Thus, ref. 78), which catalyzes the reverse 10-formyl tetrahydrofolate syn- targeting mitochondrial 1C metabolism would likely afford selective thetase reaction by which 10-CHO-THF is converted to tetrahydro- tumor inhibition, sparing normal tissues. In the following sections, we folate, formate, and ATP. Generation of ATP in this step is significant describe progress toward developing therapeutics for direct targeting such that when combined with that from NADH and oxidative of SHMT2 and MTHFD2 in cancer. phosphorylation, each formate generated from serine yields 3.5 ATPs (84). In proliferating cancer cells, synthesis of 1C units exceeds Discovery of MTHFD2 Inhibitors for the 1C demand for purine biosynthesis (84). Indeed, formate “overflow” is a characteristic of oxidative cancers and is associated with Cancer tumor invasion (85). Formate generated in the mitochondria and In acute myeloid leukemia (AML), downregulation of MTHFD2 is exported to the cytosol serves an important “protective” role in commonly associated with several treatments that cause AML-related limiting loss of unsubstituted tetrahydrofolate by oxidative stress (86). death and differentiation (i.e., 1,25-dihydroxy vitamin D3, PMA, all Glycine is the product of SHMT2 catalysis and itself can be a source trans retinoic acid, JQ1, and EPZ004777; ref. 81). This is particularly of 1C units via the mitochondrial glycine cleavage system (GCS; pronounced in FLT3-ITD AMLs with poor prognoses to standard ref. 87). The GCS including glycine decarboxylase (GDC) converts therapies (61). In breast cancer, MTHFD2 overexpression correlated glycine to CO2,NH3, and 5,10-CH2-THF (Fig. 1). While the impor- with metastasis and invasion (97), and with poor prognosis (13). tance of the GCS as a source of reducing potential is uncertain, Knockdown of MTHFD2 in breast cancer cells (98) increased the overexpression of GDC was reported to drive tumor formation in dependence on extracellular glycine that could not be rescued by lung adenocarcinomas (88). Furthermore, in gliomas with high levels formate supplementation. Metabolomics analyses in these MTHFD2- of SHMT2, the GCS is important for clearing glycine because loss of knockdown cells revealed decreased mitochondrial 1C metabolism, activity results in accumulation of the toxic glycine metabolites, coupled with increased glycolytic and glutaminolytic fluxes (98). aminoacetone and methylglyoxal (14). Increased glycine can also MTHFD2 has 40% sequence identity with MTHFD1, the cyto- impair cell growth and decrease NAD(P)H and 5,10-CH2-THF, solic isoform (78). Although early crystallization of MTHFD1 in possibly due to a reversal of SHMT2 catalysis (89). complex with inhibitors (99) identified critical catalytic residues,

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such as Lys56, Ser49, and Cys147, the development of small- against triple-negative breast cancer MDA-MB-231 xenografts in nude molecule inhibitors of MTHFD2 has remained challenging. Initial mice with minimal treatment-related toxicity (105). DS18561882 efforts to identify MTHFD2 inhibitors focused on the antibacterial would seem to be a promising candidate for future clinical evaluation. benefits from inhibiting the bacterial MTHFD2 ortholog, FolD (100).Ahigh-throughputscreeningassaytoidentifyinhibitorsof Discovery of SHMT2-targeted FolD in Pseudomonas aeruginosa identified several compound leads; however, these showed modest enzyme inhibition and could Therapeutics for Cancer not provide scaffolds for further development as MTHFD2 inhi- SHMT2 is 60% homologous to SHMT1 (35), suggesting that bitors. The macrolide keto-carboxylic acid, carolacton (Fig. 2), inhibitors of SHMT2 would likely target both enzymes. Knockdown produced by the myxobacterium Sorangium cellulosum inhibited of SHMT1 in HCT116 colon cancer xenografts in immunocompro- FolD, as well as MTHFD2, in the low nanomolar range (101). mised mice had no impact on tumor proliferation, whereas SHMT2 However, carolacton was a poor inhibitor of tumor cell prolifera- knockdown slightly suppressed cell proliferation (36). Knockdown of tion, as the EC50 values against human cancer cell lines (i.e., both SHMT1 and SHMT2 exerted profound inhibition of tumor HCT116, KB, and U937) in vitro were in excess of 10 mmol/L, progression (36), suggesting that dual inhibition of both SHMT1 and with drug export cited as the main hurdle (101). SHMT2 is essential (45). Studies with LY345899 (Eli Lilly; Fig. 2) yielded the first crystal Classic antifolates, including lometrexol, pemetrexed, and metho- þ structure of an inhibitor complexed with MTHFD2, NAD , and trexate, were tested as inhibitors of SHMT1 in vitro (106, 107), all with inorganic phosphate (102). While LY345899 inhibited human modest activity. Lometrexol showed a Ki of 20 mmol/L (106); the Ki for ¼ MTHFD2 (IC50 663 nmol/L), it was a more potent inhibitor of pemetrexed was 19.1 mmol/L (107), approximately 500-fold less potent ¼ MTHFD1 (IC50 96 nmol/L; ref. 102); however, there was no loss of than that for TS (109 nmol/L; ref. 108). Toward human SHMT2, an viability for tumor cells (U2OS and Hs-587T) treated with LY345899 IC50 value of approximately 100 mmol/L was reported for lome- in vitro (102). Although LY345899 was reported to suppress growth of trexol (109). Analogous results were reported for pemetrexed with a SW620 colorectal cancer xenograft and a colorectal cancer patient- SHMT2 (35). While inhibitions may be greater for polyglutamyl drug derived xenograft (PDX) in vivo (103), it is unclear whether this forms, these results, nonetheless, suggest that any biological effects of response was due to inhibition of MTHFD1 or MTHFD2. classic antifolates resulting from direct targeting of SHMT1 and A novel isozyme-selective MTHFD2 inhibitor, DS44960156 (Fig. 2), SHMT2 are likely to be minor. with a tricyclic coumarin scaffold, was initially discovered via high- Early efforts to generate targeted small inhibitors of SHMT proteins throughput screening, and then modified by structure-based drug initially focused on herbicidal pyrazolopyran compounds originally design (104). DS44960156 was >18-fold more selective toward human described as inhibitors of plant SHMT (Fig. 3A; ref. 110). Optimiza- ¼ MTHFD2 (IC50 1.6 mmol/L) than MTHFD1 (IC50 > 30 mmol/L; tion of these compounds (Fig. 3B) yielded molecules with antimalarial ref. 104). Further optimization of this scaffold yielded DS18561882 activity (111). Although these were inhibitors of Plasmodium falci- ¼ (Fig. 2), with potent inhibition of MTHFD2 (IC50 6.3 nmol/L) and parum and P. vivax SHMTs in vitro (submicromolar IC50s), they were 90-fold selectivity for MTHFD2 over MTHFD1 (105). When admin- poorly active against rat L6 myeloblasts or HepG2 human hepatoma istered orally, DS18561882 demonstrated remarkable in vivo efficacy cells (111). Additional pyrazolopyran compounds were tested against

Figure 2. OH H MTHFD2 inhibitors. MTHFD2 inhibitors NH2 N O OH include the macrolide keto-carboxylic O acid, carolacton, produced by the N OHO N myxobacterium Sorangium cellulo- O O O OCH3O N O sum (101) and the Eli Lilly compound HN HN LY345899 (102). Newer agents OH include the Daiichi Sankyo compounds DS44960156 (104) and LY-345899 OH DS18561882 (105). Carolacton O

N N O O O O

DS18561882 N O N O DS44960156 F F F O O HN S O OH O

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O Figure 3. O HO SHMT2 inhibitors. A, SHMT2 inhibi- AB CD tors include the early-generation pyrazolopyran compounds original- S ly designed as inhibitors of plant Cl OH SHMT2 (110).The pyrazolopyran scaffold was later optimized for inhibition OH of Plasmodium SHMT (ref. 111; B) Cl CN CN and human SHMT1/2, with RZ-2994 CN CN CN or SHIN1 (ref. 36; C)andSHIN2 (ref. 114; D). N N N N NH2 N N O N O NH2 N O NH2 O NH2 H H H H RZ-2994/SHIN1 SHIN2

A549 and H1299 human lung cancer cells (112) and a lead compound metabolic derangements consistent with inhibition of SHMT1 and (compound 2.12) was reported to preferentially inhibit SHMT1 over SHMT2. SHIN2 showed potent in vivo inhibition of NOTCH1- SHMT2 and effect apoptosis with an LD50 of 34 mmol/L. induced T-cell acute lymphoblastic leukemia (T-ALL) xenografts in The pyrazolopyran series was further optimized by Rabinowitz and a mouse model at comparatively high doses (200 mg/kg twice a day, colleagues for inhibition against human SHMT1 and SHMT2. The first 11 days), with efficacy comparable with that of the standard-of-care study using these optimized compounds (113) showed potent (nano- treatment methotrexate. Moreover, SHIN2 demonstrated efficacy in a molar) inhibition of isolated human SHMT1 and SHMT2. The IC50 methotrexate-resistant PDX T-ALL model. Combination treatment value for the lead compound RZ-2994 (later renamed SHIN1 for with SHIN2 and methotrexate in the PDX T-ALL model showed a “SHMT inhibitor 1”; Fig. 3C) toward human T effector cells was in the synergistic response, possibly because of methotrexate-induced deple- low micromolar range, suggesting its therapeutic potential. Although tion of cellular tetrahydrofolates, resulting in decreased competition not an analog of folic acid, a crystal structure of SHIN1 in complex with for SHIN2 binding and greater inhibition of SHMT1 and SHMT2. SHMT2 (36) revealed its binding at the folate binding site of SHMT2. Ducker and colleagues further assessed these optimized pyrazolopyran SHMT inhibitors against human tumor cell lines (36). Against Discovery of Multi-targeted Inhibitors wild-type HCT116 colon cancer cells in vitro, SHIN1 inhibited pro- of SHMT2 and Cytosolic 1C Metabolism ¼ liferation with a submicromolar potency (IC50 870 nmol/L), with at SHMT1 and de novo Purine complete pharmacologic rescue by formate and glycine, suggesting on- Biosynthesis target inhibition of SHMT2 (36). The IC50 value for SHIN1 in SHMT2- knockout HCT116 cells decreased nearly two orders of magnitude to As mitochondrial 1C metabolism from SHMT2 is the major source approximately 10 nmol/L, reflecting potent inhibition of SHMT1 in of 1C units for de novo purine biosynthesis in the cytosol, molecules addition to SHMT2, whereas the IC50 value in SHMT1-knockout cells targeting SHMT2, along with de novo purine biosynthesis at GARF- was indistinguishable from that in wild-type cells (36). This confirmed Tase and/or AICARFTase, should afford especially potent antitumor that efficacy toward wild-type HCT116 cells was primarily due to its agents (35). Primary inhibition of mitochondrial 1C metabolism at inhibition of SHMT2 rather than SHMT1. However, inhibition of both SHMT2 would deplete cytosolic formate (e.g., 10-CHO-THF) pools enzymes is essential, as this prevents metabolic compensation by required for nucleotide biosynthesis, potentiating drug efficacy by reversal of SHMT1 catalysis (serine ! glycine) and synthesis of glycine reducing competition for inhibitor binding at these cytosolic enzyme in response to loss of SHMT2 (45). targets. Moreover, concurrent inhibition of SHMT1 would augment Interestingly, enhanced potency of SHIN1 toward 8988T pancreatic inhibition at SHMT2 by preventing metabolic compensation involving cancer cells and diffuse large B-cell lymphoma cells revealed distinct reversal (serine ! glycine) of the SHMT1 reaction, analogous to metabolic vulnerabilities of these cancer types that could be exploited SHIN1 and SHIN2 (36, 114). by SHMT1 and SHMT2 inhibition (36). 8988T cells exhibit defects in To achieve this, Matherly, Gangjee, Dann, and colleagues combined mitochondrial 1C metabolism with an overreliance on SHMT1, structural features from 5-substituted pyrrolo[2,3-d]pyrimidine inhi- whereas B-cell lymphomas have intrinsic defects in glycine uptake bitors of de novo purine biosynthesis (115) with those from 5-formyl that render these cells overly reliant on glycine synthesis from serine by tetrahydrofolate (a SHMT inhibitor; ref. 116) and 5,10-CH2-THF SHMT2 (36). Addition of formate did not rescue B-cell lymphomas (SHMT2 product), to generate novel 5-substituted pyrrolo[3,2- from the effects of SHIN1 as with HCT116 cells, but rather paradox- d]pyrimidine benzoyl and thienoyl analogs (35). The lead compounds ically potentiated SHIN1 effects. Cytotoxicity was not due to 1C of this series, AGF291, AGF320, and AGF347 (Fig. 4), demonstrated depletion, but instead was due to glycine deficiency being exacerbated broad range in vitro efficacy toward human tumor cell lines expressing by formate excess, which drives SHMT catalysis in the glycine- PCFT, including non–small cell lung cancer (H460), colon cancer consuming (glycine ! serine) direction. Despite these promising (HCT116), and pancreatic adenocarcinoma (MIA PaCa2, AsPC1, in vitro results, SHIN1 showed a disappointing lack of in vivo anti- BxPC3, CFPAC, and HPAC; refs. 27, 35) cells. SHMT2 inhibition tumor efficacy (36), likely due to its poor pharmacokinetics and/or was confirmed by targeted metabolomics and flux analysis with metabolic instability. [2,3,3-2H]serine, accompanied by direct inhibition of GARFTase and To improve on these shortcomings of SHIN1, Rabinowitz AICARFTase in de novo purine biosynthesis and reduced purine and colleagues synthesized a next-generation pyrazolopyran com- nucleotide pools (35). In vivo antitumor efficacy with curative potential pound, SHIN2 (Fig. 3D; ref. 114). Like SHIN1, SHIN2 induced was confirmed with AGF347 in early- and late-stage MIA PaCa2

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H O N H2N N O OHO N H O N S OH OH N O O H AGF291 OHO O HN N OHO H N O 2 N AGF320 H O OH N N H2N H O N N F

AGF347

Figure 4. Multi-targeted inhibitors of SHMT2 and cytosolic 1C metabolism. Structures are shown for 5-subsituted pyrrolo[3,2-d]pyrimidine compounds AGF291, AGF320, and AGF347 (35). pancreatic adenocarcinoma xenografts in SCID mice at modest dosing localize exclusively in the cytosol are now recognized to be expressed (120 mg/kg total, every 2 days 8; ref. 35). Toxicity was modest and as distinct, but functionally homologous, forms in the mitochondria consisted of limited weight loss that was completely reversible upon and nucleus, including SHMT1, SHMT2, and SHMT2a, and completion of therapy. MTHFD1, MTHFD1L, and MTHFD2 (4). In some cases, these A follow-up study demonstratedthatAGF347accumulatedin enzymes appear to perform noncanonical functions in the nucleus both the cytosol and mitochondria nearly exclusively (>98%) as or mitochondria (117), although this is controversial. Even within polyglutamate conjugates (27). In mitochondria, AGF347 accumu- mitochondria, functional redundancies exist between normal tissues lation was mediated, at least in part, by MFT, albeit seemingly less (MTHFD2L) and tumors (MTFHD2; refs. 78, 79). than for folates (27). Treatment of HCT116 cells with AGF347 It is of further interest that the mitochondrial 1C enzymes, under hypoxic (0.5% O2) conditions resulted in elevated reactive MTHFD2 and SHMT2, are among the most differentially expressed oxygen species (ROS), accompanied by decreased reduced and total metabolic enzymes between tumors and normal tissues (59). Serine GSH, analogous to HCT116 SHMT2-knockout cells (27). The catabolism in mitochondria ensures that the unique metabolic require- extent of ROS induction with AGF347 varied with different tumor ments of hypoxic tumors are met for 1C units, glycine, reducing cell lines (27) However, unlike knockout of SHMT2, treatment of equivalents, and ATP (4–6, 8), while also ensuring that proteins HCT116 cells with AGF347 did not suppress mitochondrial respi- required for mitochondrial respiration are efficiently translat- ration (27), likely due to incomplete inhibition of this mitochon- ed (70, 71). Although the evolutionary rationale for compartmental- drial target (71). ization of 1C metabolism continues to emerge, studies suggest its importance for preserving labile tetrahydrofolates from oxidation (86). Furthermore, compartmentalization of 1C metabolism Conclusions and Future Outlook between cytosol and mitochondrial ensures a directional flux of 1C þ In spite of recent advances in targeted therapies for cancer, the units, in relation to the high NAD(P) /NAD(P)H ratios in mito- classic antifolates, typified by methotrexate and pemetrexed, remain chondria that favor serine oxidation to formate. In the cytosol, þ vital components of the therapeutic armamentarium (2). These agents low NADP /NADPH ratios favor synthesis of 10-CHO-THF from have found important clinical applications for cancer in the United formate and tetrahydrofolate (via MTHFD1), and synthesis of States and abroad. Most recently, PCFT-targeted pyrrolo[2,3- serine from glycine and 5,10-CH2-THF (via SHMT1; refs. 28, 86). d]pyrimidine antifolate inhibitors were described, with tumor target- Thus, MTHFD2 and SHMT2 are important biomarkers for cancer, ing based on their selective membrane transport by PCFT under acidic the levels of which correlate with tumor aggressiveness and disease conditions of the tumor microenvironment (20, 23). Notably, all these progression (9–12, 59, 64–67). Although these enzymes limit 1C units agents inhibit 1C metabolism at cytosolic enzymes involved in nucle- and glycine for cellular biosynthesis in the cytosol, for HCT116 colon otide biosynthesis (2, 20, 23). cancer cells, SHMT2 knockout results in GSH depletion as the most It is now recognized that 1C metabolism involves compartmental- significant metabolic change, because supplementation with GSH or ization of folate cofactors and 1C pathways between the cytosol and N-acetylcysteine completely rescues cells from cytotoxicity (45). This mitochondria (4, 7, 8, 28), which extends to the nucleus (29). Even suggests that antioxidant synthesis, rather than synthesis of proteins or within a particular cellular compartment, regulatory networks and purine nucleotides, is the major driver of demand for 1C metabolism in protein associations (e.g., purinosome; ref. 39) can occur, designed to mitochondria, at least in HCT116 cells. Indeed, as serum glycine can be ensure an efficient flow of 1C units for biosynthesis of nucleotides and abundant, therapeutic efforts to target SHMT2 (or MTHFD2) seem key amino acids, and/or for methylation of critical genes and/or ideally suited for intrinsically glycine-deficient tumors (e.g., diffuse proteins. It is interesting that enzymes previously considered to large B-cell lymphoma; ref. 36).

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Promising lead inhibitors have been described for SHMT2 (SHIN1, approaches for targeting MYC (bromodomain and extra-terminal SHIN2, and AGF347; refs. 35, 36, 114) or MTHDF2 (DS18561882; motif inhibitors; ref. 124), as MYC regulates both SHMT2 and ref. 105). As compensatory metabolic changes in cytosolic fluxes (e.g., MTHFD2 (6, 62, 81). SHMT1 reversal) accompany loss of SHMT2 or MTHFD2 and result In conclusion, the pervasive importance of mitochondrial 1C in viable and tumorigenic cells (35, 45), it will be important to combine metabolism to the malignant phenotype demonstrates the immense targeting mitochondrial 1C metabolism by small molecules with value of the therapeutic targeting of this critical pathway at SHMT2 inhibition of other targets (e.g., SHMT1), as seen with SHIN1/ and/or MTHFD2, along with other enzymes. By any measure, these SHIN2 (36, 114) and AGF347 (35). For AGF347, in addition to new agents afford a valuable and exciting platform for future anti- SHMT1, additional direct inhibition of de novo purine biosynthesis cancer drug development. at GARFTase and AICARFTase offers targets for which inhibition is independent of wild-type/mutant p53 status (118) and results in Disclosure of Potential Conflicts of Interest suppression of mTOR signaling (119). Selectivity for de novo purine L.H. Matherly and A. Gangjee report collaboration with Flag Therapeutics, Inc., biosynthesis in tumors is further augmented by loss of methylthioa- an early-stage oncology company focused on the development of novel therapies. fl denosine phosphorylase (MTAP) in purine salvage (120). While the No potential con icts of interest were disclosed by the other authors. mechanisms of cellular uptake of SHIN1/SHIN2 and DS18461882 Acknowledgments have not been established, AGF347 is transported into cells by both This work was supported, in part, by grants R01 CA53535 (to L.H. Matherly RFC and PCFT (27). If second-generation analogs of this series with and Z. Hou), R01 CA152316 (to L.H. Matherly and A. Gangjee), and R01 preferential uptake via PCFT were identified, these would afford even CA166711 (to A. Gangjee and L.H. Matherly) from the NIH, a Strategic Initiative greater tumor selectivity (23). Grant from the Barbara Ann Karmanos Cancer Institute (to Z. Hou and As cancer cells depend on serine biosynthesis from glycolysis in L.H. Matherly), the Eunice and Milton Ring Endowed Chair for Cancer Research the cytosol, and high serine concentrations were reported in the (to L.H. Matherly), and the Duquesne University Adrian Van Kaam Chair in Scholarly Excellence (to A. Gangjee). This work was also supported by grants T32 tumor microenvironment (121), it might be possible to combine CA009531 (to L.H. Matherly) and F30 CA228221 (to A.S. Dekhne). inhibitors of SHMT2 or MTHFD2 with inhibitors of upstream targets including DHFR (114), PDGH (122), NRF2 (66), or ATF4 (123). Received May 18, 2020; revised July 6, 2020; accepted August 25, 2020; Another possibility is to combine SHMT2 or MTHFD2 inhibitors with published first September 2, 2020.

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Therapeutic Targeting of Mitochondrial One-Carbon Metabolism in Cancer

Aamod S. Dekhne, Zhanjun Hou, Aleem Gangjee, et al.

Mol Cancer Ther 2020;19:2245-2255. Published OnlineFirst September 2, 2020.

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