This table shows purity of Subcellular Fractions.

This table shows IC50 values for antiproliferative activities toward EOC versus normal ovary cell lines by 5-substituted pyrrolo[3,2-d]pyrimidine inhibitors.

This table shows primer sequences for quantitative real-time RT-PCR.

This table shows information of ovarian cancer tissue cDNA array for real-time RT-PCR.

This figure shows generation of SKOV3 SHMT1 and SHMT2 knockdown (KD) cell lines.

This figure shows results of apoptosis assays with SKOV3 cells treated with AGF347.

This figure shows immunohistochemical staining of tumor microarray (TMA) for SHMT2 protein.

This figure shows purine nucleotides in SKOV3 EOC cells treated with pyrrolo[3,2-d]pyrimidine antifolates.

Metabolic reprogramming is a defining feature of cancer, and folate-mediated one-carbon (C1) metabolism occupies a central role in supporting malignant growth, survival, and immune evasion. Although cytosolic C1 enzymes have long been exploited for the therapy of a host of cancers through the use of “classical” antifolates, growing evidence suggests that mitochondrial C1 metabolism is a critical driver of tumor progression, metastasis, and redox homeostasis. Mitochondrial serine catabolism via serine hydroxymethyltransferase 2 and 5,10-methylene tetrahydrofolate dehydrogenase 2 provides formate, glycine, and reducing equivalents to sustain nucleotide and amino acid biosynthesis, biological methylation reactions, and antioxidant defenses. Dysregulation of mitochondrial C1 metabolism promotes “formate overflow”, alters tumor–immune interactions, and supports metastatic signaling. This review synthesizes recent key advances in folate biology, and compartmentalized C1 metabolism, leading to the development of an exciting new generation of cytosolic- and mitochondrial-targeted therapies. We examine emerging links between C1 metabolism and the tumor microenvironment, including relationships between C1 metabolism and immune suppression, T cell infiltration, and macrophage polarization. Finally, we highlight critical unresolved questions and vexing future challenges in implementing the next generation of C1-targeted anticancer agents. Collectively, these insights underscore the remarkable biology of C1 metabolism, and mitochondrial C1 metabolism in paticular, and its potential as a multifaceted and therapeutically actionable vulnerability in cancer. Significance Statement This minireview summarizes recent advances in folate biology and one-carbon (C1) metabolism. Topical areas include mechanisms of membrane transport, compartmentalization of C1 metabolism and its role in tumor biology, and the relationship between C1 biology and the tumor microenvironment. The goal of this study is to provide an evolving framework for discovery of tumor-specific vulnerabilities in C1 metabolism that can be exploited for therapy.

This table shows information of epithelial ovarian cancer tissue microarray (TMA) for immunohistochemistry.

This table shows Kis for inhibition of one-carbon enzymes for 5-substituted pyrrolo[3,2- d]pyrimidine antifolates.

This figure shows purification of the recombinant human thymidylate synthase (TS) and in vitro inhibition by pyrrolo[3,2-d]pyrimidine antifolates.

This figure shows FRα, PCFT and RFC expression in EOC cell line models and EOC patient cDNAs.

Nuclear and cytoplasmic green fluorescence were measured in all cells from cultures represented in Figure 3C using image J. Nuclear (left panel) and cytoplasmic (right panel) quantifications are reported separately for each cell line. Statistical significance between control and treated cultures was not achieved.

One-carbon (C1) metabolism includes cytosolic and mitochondrial pathways connected by interchange between serine, glycine, and formate. Mitochondrial C1 metabolism through serine hydroxymethyltransferase (SHMT) 2 generates glycine and C1 units for de novo nucleotide biosynthesis in the cytosol, whereas cytosolic SHMT1 consumes C1 units and glycine. Folates and classical antifolates are transported into tumors by facilitative folate transporters (reduced folate carrier [RFC] and proton-coupled folate transporter [PCFT]) and are metabolized to polyglutamates by folylpolyglutamate synthetase (FPGS). Folate transporter-null HeLa cells were engineered to express RFC under the control of a tetracycline-inducible promoter. Constitutive expression of PCFT and/or FPGS increased cytosolic and mitochondrial folates over that of RFC alone. By targeted metabolomics, the C1 flux in mitochondria through SHMT2 paralleled RFC transport and folate accumulation in mitochondria and cytosol, whereas the SHMT1 flux was constant. Expression of PCFT resulted in further increased C1 flux through SHMT2, in excess of SHMT1. In vitro inhibition of cell proliferation by targeting SHMT1/2 with pyrrolo[3,2-d]pyrimidine antifolates (eg, AGF347) decreased with increasing RFC and with PCFT. Inhibition by AGF347 (not SHIN1/2) was stimulated with ectopic FPGS, accompanying increased AGF347 polyglutamates; decreased sensitivities were seen for nonclassical SHMT1/2 inhibitors (SHIN1/2), which are neither substrates for facilitative transport nor polyglutamylation. Our results document the complex interrelationships among (anti)folate membrane transport, polyglutamylation, and C1 fluxes through SHMT1 and SHMT2. They also demonstrate the profound impact of physiologic folates on antitumor activities and the extraordinary promise of multitargeted pyrrolo[3,2-d]pyrimidine antifolates for cancer therapy. SIGNIFICANCE STATEMENT: Novel pyrrolo[3,2-d]pyrimidine antifolates typified by AGF347 target serine hydroxymethyltransferase (SHMT) 2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. This manuscript documents the complex interrelationships among (anti)folate membrane transport, polyglutamylation, and one-carbon fluxes through SHMT1 and SHMT2 in the context of physiologic folate levels. The results document the therapeutic promise of classical multitargeted pyrrolo[3,2-d]pyrimidine antifolates typified by AGF347. These novel compounds offer an exciting new platform for one-carbon-targeted drug development for cancer.

Chord diagrams of top 10 differentially regulated Pathways and involved DEGs in BRCA1mt vs HRwt

Chord diagrams of top 10 differentially regulated Pathways and involved DEGs in BRCA2mt vs BRCA1mt

Epithelial ovarian cancer (EOC) accounts for ∼90% of ovarian cancers and continues to be a deadly disease. High grade serous ovarian cancer (HGSOC) is the most common subtype of EOC. PARP inhibitors (PARPis) are important drugs for treating recurrent ovarian cancer post-platinum-based chemotherapy and recently this has been applied to first-line maintenance treatment after chemotherapy. Meta-analysis suggested that PARPis are most effective for patients with Brca mutations. Studies also suggested better efficacy of PARPis in HGSOC patients with Brca2 mutations than patients with Brca1 mutations. However, only ∼15-20% of HGSOC tumors carry germline Brca1 or Brca2 mutations and somatic mutations are found in ∼8% of cases. In addition, the increasing use of PARPis for treating HGSOC has raised concerns about PARPi resistance in clinical practice. Clearly, there is an urgent need to identify new metabolic vulnerabilities in HGSOC in general, and in Brca1/Brca2 mutant subtypes in particular, that can be targeted alone, or in combination with existing medications including PARPis. The ID8 HGSOC model is the most widely used transplantable mouse syngeneic model of ovarian cancer. ID8 variants including single (Trp53-/-) or double (Trp53-/-/Brca1-/- or Trp53-/-/Brca2-/-) gene deletions recapulate characteristics of human HGSOC subtypes including the expected patterns of sensitivity to cisplatin and PARPis (i.e., olaparib). Our previous studies showed that in primary patient specimens [including normal ovary (n=8) and HGSOC (n=40)], transcripts for one-carbon (C1) genes in mitochondria (SLC25A32, SHMT2, MTHFD2, MTHFD1L) and in cytosol (GARFTase, AICARFTase) increased significantly over normal ovaries. We performed untargeted metabolomics studies (∼2000 metabolites) with the aforementioned ID8 sublines. The results showed significant quantitative differences between wild-type ID8 and ID8/Trp53-/-, ID8/Trp53-/-/Brca1-/- or ID8/Trp53-/-/Brca2-/- cells, as well as between ID8/Trp53-/-/Brca1-/- and ID8/Trp53-/-/Brca2-/- cells, involving C1 metabolism including metabolites of glycine, serine, threonine, cysteine, methionine, pyrimidines and purines. Targeted metabolomics and flux analysis with [13C (U)]serine and LC-MS/MS revealed enhanced mitochondrial C1 flux in the ID8 variants, reflected in levels of total serine and serine isotopomers (ID8 > ID8/Trp53-/- = ID8/Trp53-/-/Brca1-/- > ID8/Trp53-/-/Brca2-/-). C1 flux from [13C (U)]serine in the cytosol was also significantly elevated in ID8 variants, reflected in a dramatic decrease of glycinamide ribonucleotide (GAR) (M+0, M+2) in de novo purine biosynthesis compared to wild-type ID8 cells. Collectively, our results suggest that C1 metabolism in both the mitochondria and cytosol is a bona fide metabolic vulnerability in HGSOC that can be therapeutically exploited. Zhanjun Hou, Madelyn Brzezinski, Carrie O’Connor, Xun Bao, Ayesha B. Alvero, Radhika Gogoi, Ramandeep Rattan, Gil Mor, Jing Li, Aleem Gangjee, Larry H. Matherly. Unraveling biological differences of high grade serous ovarian cancer subtypes by metabolomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 279.

A UHPLC-MS/MS method for the detection and quantification of the 6-substituted pyrrolo[2,3-d] pyrimidine AGF94, a novel antifolate, in mouse plasma and tissue was developed and validated. The developed method relies on a simple protein precipitation with methanol, followed by separation on a C18 column using a gradient solvent system of acetonitrile and water with 0.1 % formic acid in both. Detection and quantification of AGF94 were achieved by multiple reaction monitoring using a Sciex QTRAP 5500 mass spectrometer operated in positive electrospray ionization mode. The transitions for AGF94 and the internal standard were m/z 448 to 137 and 442 to 295, respectively. The calibration curve ranged from 5 to 500 ng/mL in mouse plasma with a linearity of R2 = 0.99611 ± 0.00280 across multiple days. Accuracy of the assay ranged from -6.22 to 5.56 % and precision was less than 11.58 % off from nominal concentrations. Benchtop, freeze/thaw cycling, and autosampler stabilities did not indicate any substantial changes in concentrations during processing. The overall process efficiency was greater than 96 % for both the analyte and internal standard. The precision and accuracy of the assay were established, and the assay was utilized to analyze preclinical samples from a pharmacokinetic study using AGF94 in a murine pancreatic cancer model. Pharmacokinetic parameters from a noncompartmental analysis of AGF94 in multiple matrices were reported utilizing the validated method.