Deoxythymidylate Kinase (DTYMK)-Driven Pyrimidine Hypermetabolism Is Associated with a Folate-Dependent Macrophage Programme in Triple-Negative Breast Cancer.

Immune checkpoint blockade benefits only a minority of patients with triple-negative breast cancer (TNBC), and the tumour-intrinsic programmes that establish its immunosuppressive microenvironment remain poorly defined. Pyrimidine metabolism supplies nucleotides and one-carbon units in malignant cells, but whether it shapes the immune microenvironment is unknown. Integrating single-cell, bulk breast cancer transcriptomics with Xenium spatial transcriptomics, DTYMK knockdown, tumour-conditioned-medium co-culture and folate rescue, we found that pyrimidine pathway activity was elevated in the malignant epithelium of TNBC, within which we identified Epi4, a high-potency subpopulation coupling pyrimidine and folate metabolism and predicted to export folate. Furthermore, this folate was predicted to be taken up by FOLR2+ tumour-associated macrophages (TAMs) and was associated with S-adenosylmethionine(SAM)-dependent methylation signatures and MAFB activity. Spatially, Epi4 and FOLR2+ TAMs co-localised at the tumour-stroma margin, a region of reduced T-cell density. DTYMK depletion reduced tumour fitness and attenuated folate-mediated macrophage reprogramming, which was largely restored by exogenous folate. Here, we therefore identify a DTYMK-folate-MAFB axis in which tumour metabolic rewiring is associated with epigenetic reprogramming of macrophages.