Abstract
Folate-dependent one-carbon (C1) metabolism encompasses distinct cytosolic and mitochondrial pathways connected by an interchange among serine, glycine, and formate. In both the cytosol and mitochondria, folates exist as polyglutamates, with polyglutamylation catalyzed by folylpolyglutamate synthetase (FPGS), including cytosolic and mitochondrial isoforms. Serine is metabolized by serine hydroxymethyltransferase (SHMT)2 in the mitochondria and generates glycine and C1 units for cellular biosynthesis in the cytosol. AGF347 is a novel pyrrolo[3,2-day]pyrimidine antifolate that targets SHMT2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. FPGS is expressed in primary pancreatic cancer specimens, and FPGS levels correlate with in vitro efficacies of AGF347 toward human pancreatic cancer cells. MIA PaCa-2 pancreatic cancer cells with CRISPR knockout of FPGS were engineered to express doxycycline-inducible FPGS exclusively in the cytosol (cFPGS) or in both the cytosol and mitochondria (mFPGS). Folate and AGF347 accumulations increased in both the cytosol and mitochondria with increased mFPGS but were restricted to the cytosol with cFPGS. AGF347-Glu5 inhibited SHMT2 ∼19-fold greater than AGF347. By metabolomics analysis, mFPGS stimulated the C1 flux from serine in the mitochondria and de novo purine and dTTP synthesis far greater than cFPGS. mFPGS enhanced in vitro inhibition of MIA PaCa-2 cell proliferation by AGF347 (∼30-fold) more than cFPGS (∼4.9-fold). Similar results were seen with other pyrrolo[3,2-d]pyrimidine antifolates (AGF291, AGF320); however, elevated mFPGS adversely impacted inhibition by the nonclassical SHMT2/SHMT1 inhibitor SHIN1. These results suggest a critical role of mFPGS levels in determining antitumor efficacies of mitochondrial-targeted pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer.
SIGNIFICANCE STATEMENT AGF347 is a novel pyrrolo[3,2-d]pyrimidine antifolate that targets serine hydroxymethyltransferase (SHMT)2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. AGF347 accumulation increases with folylpolyglutamate synthetase (FPGS) levels in both the cytosol and mitochondria. Increased mitochondrial FPGS stimulated one-carbon metabolic fluxes in the cytosol and mitochondria and substantially enhanced in vitro inhibition of pancreatic cancer cells by AGF347. Mitochondrial FPGS levels play important roles in determining the antitumor efficacies of pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer.
Footnotes
- Received March 14, 2024.
- Accepted July 11, 2024.
This work was supported in part by grants from National Institutes of Health National Cancer Institute [Grant R01 CA53535] (to L.H.M. and Z.H.) and [Grant R01 CA250469] (to L.H.M., A.G., and C.E.D.), the Eunice and Milton Ring Endowed Chair for Cancer Research (to L.H.M.), and the Duquesne University Adrian Van Kaam Chair in Scholarly Excellence (to A.G.). M.S. was supported by a training grant from the National Cancer Institute [Grant T32 CA009531] (to L.H.M.). The Animal Models and Therapeutics Evaluation Core; the Biostatistics and Bioinformatics Core; the Microscopy, Imaging and Cytometry Resources Core; and the Pharmacology and Metabolomics Core at the Barbara Ann Karmanos Cancer Institute were supported in part by the National Cancer Institute Center [Grant P30CA22453].
No author has an actual or perceived conflict of interest with the contents of this article.
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- Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics
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