Targeting DHFR to suppress one-carbon metabolism and enhance radiation sensitivity in patients with NRF2-active head and neck squamous cell carcinoma

Abstract Purpose: NRF2 hyperactivity is associated with radiation resistance in head and neck squamous cell carcinoma (HNSCC). We evaluated the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and its potent analog, WCDD115, as radiosensitizing agents through disruption of one-carbon metabolism. Patients and Methods: Preclinical studies used NRF2-competent and deficient HNSCC models, clonogenic assays, transcriptomics, metabolomics, pharmacokinetics/toxicity testing, and syngeneic tumor grafts to assess WCDD115 as a novel radiosensitizing agent. In parallel, we conducted an open-label, single-center, window-of-opportunity early phase I clinical trial (NCT05678348) of previously untreated patients with HPV(-) stage III/IV HNSCC to receive pyrimethamine 50mg prior to surgery. Correlative analysis included mass spectrometry, western blotting, and pathway analysis. Results: WCDD115 enhanced radiation-induced DNA damage and suppressed one-carbon metabolism and nucleotide biosynthesis. Parallel mouse tumor studies demonstrated that WCDD115 enhanced the efficacy of radiation in NRF2-competent but not NRF2-deficient tumors. In a clinical trial of neoadjuvant pyrimethamine, DHFR protein expression was enriched post-treatment by a mean fold change of 9.91 (95% confidence interval 8.1 to 12.2), consistent with a strong on-target effect. The drug was well tolerated with no drug-related grade 3-5 adverse events or surgical delays. Conclusions: NRF2 hyperactivity is associated with enhanced one-carbon metabolism which can be therapeutically targeted with DHFR inhibitors to promote radiation sensitization. In HNSCC patients, pyrimethamine achieves potent DHFR inhibition and a favorable safety profile supporting a future phase 2 trial integrating pyrimethamine with definitive radiation-based therapy for NRF2-active HNSCCs.