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September 10, 2025

Pharmacodynamic Determinants of Mitochondrial One-Carbon Flux and Serine Hydroxymethyltransferase Inhibition in Human Tumors

Key Points

Targeting serine hydroxymethyltransferase (SHMT) 2 inhibits tumor cell proliferation through altered one-carbon metabolism.
Pyrrolo[3,2-d]pyrimidine antifolates, such as AGF347, affect folate transport and show efficacy in promoting C1 flux in tumors.
Increased expression of folate transporters correlated with enhanced folate accumulation and mitochondrial C1 flux in engineered HeLa cells.
The study highlights the interplay between (anti)folate transport, polyglutamylation, and SHMT activity, indicating rewarding avenues for drug development.

Abstract

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 pyrrolo3,2-dpyrimidine 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 pyrrolo3,2-dpyrimidine antifolates for cancer therapy. SIGNIFICANCE STATEMENT: Novel pyrrolo3,2-dpyrimidine 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 pyrrolo3,2-dpyrimidine antifolates typified by AGF347. These novel compounds offer an exciting new platform for one-carbon-targeted drug development for cancer.

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