Tryptophan and serine metabolic axis reveals novel therapeutic vulnerabilities in triple-negative breast cancer

Tryptophan (Trp) metabolism is recognised to have immunomodulatory effects in cancer, with downstream metabolites such as kynurenine suppressing T cell responses. However, the role of Trp catabolism in cancer cell biosynthesis and tumor growth is poorly understood. Herein, we investigated for the first time the role of Trp as a source of one-carbon metabolism through formate production in triple-negative breast cancer (TNBC) and its effect on serine synthesis via phosphoglycerate dehydrogenase (PHGDH), a rate-limiting enzyme for de novo serine synthesis. CRISPR-Cas9 system was used to generate tryptophan 2,3-dioxygenase (TDO2) and/or indoleamine 2,3-dioxygenase (IDO1) knockout TNBC cell lines. Multi-omics approach using LCMS Q-TOF system and RNA-seq was employed to integrate targeted and untargeted metabolomics, 13 C fluxomics, and transcriptomics in TNBC cell lines. For fluxomics studies, cells were cultured with 13 C-labeled tryptophan to trace the metabolic flux of carbon atoms through Trp metabolism into kynurenine pathway. While serine is recognised as a major source of one-carbon metabolism, our fluxomics data revealed that one-carbon units are released during the conversion of N-formylkynurenine to kynurenine. Labelled metabolites derived from tryptophan that give rise to tetrahydrofolate and methionine cycles were identified and these were subsequently incorporated to generate de novo purines. Integrated transcriptome and metabolomic analysis of tryptophan dioxygenase KO versus WT, as well as formylkynurenine and kynurenine treated cells, revealed a link between Trp catabolism and serine anabolism via alteration in PHGDH expression. Moreover, KO cells deprived of one-carbon units derived from Trp metabolism showed upregulation of PHGDH gene expression. Interestingly, KO cells showed a significantly lower growth rate in serine-glycine free media and also when PHGDH was inhibited with 2.5 μM NCT-503. This study provides evidence that Trp metabolism contributes to one-carbon metabolism in TNBC beyond its immunosuppressive functions. We also show that disruption of Trp metabolism upregulates PHGDH as a compensatory mechanism. This study identifies simultaneous inhibition of both pathways as a promising dual-targeting therapeutic strategy for TNBC treatment.