Cancer cells rely on nucleotide synthesis for proliferation, and antimetabolites targeting nucleotide metabolism remain a cornerstone of cancer therapy. Despite their clinical success, these agents often face high rates of resistance, potentially due to interactions with stromal cells within the tumor microenvironment.

To investigate the impact of altered nucleotide metabolism in the tumor microenvironment, we suppressed de novo pyrimidine synthesis in mice by generating a whole-body inducible knockout of dihydroorotate dehydrogenase (DHODH), the essential enzyme in this pathway.

Unexpectedly, systemic DHODH deficiency led to accelerated growth of orthotopic lung tumors. Single-cell transcriptomics of tumor-bearing lungs revealed that DHODH loss significantly affected stromal cells, including immune cells and, notably, endothelial cells. To dissect the specific role of endothelial metabolism, we generated a mouse model with inducible, endothelium-specific DHODH deletion. Strikingly, selective loss of de novo pyrimidine synthesis in endothelial cells recapitulated the whole-body knockout phenotype, creating a more permissive environment for tumor growth. Transcriptomic analysis of the endothelial-specific model identified alterations in the immune composition of the tumor microenvironment, particularly in monocytes, which was further validated by spectral flow cytometry. Ongoing studies aim to elucidate the precise mechanisms by which endothelial pyrimidine deficiency promotes tumor progression.

Our findings reveal an unexpected pro-tumorigenic effect of systemic pyrimidine synthesis inhibition, driven by changes in the endothelial compartment. These results highlight a previously unrecognized role of endothelial pyrimidine synthesis in shaping the tumor microenvironment and raise concerns about the unintended consequences of targeting this pathway in cancer therapy.