Abstract Metabolic reprogramming is a well-established hallmark of cancer cells, which supports tumor growth, survival, and chemotherapy resistance. In this respect, a subset of cancers gets addicted to intracellular serine/glycine synthesis via overexpression of the enzymes PHGDH, PSAT1, PSPH or SHMT1/2. This cancer-specific dependency highlights a novel therapeutic opportunity, as normal cells solely rely on serine and glycine uptake from their environment. Well established examples of serine/glycine-synthesis addicted tumors are triple-negative breast cancer and T-cell leukemia (1, 2), which are both currently treated with toxic intensive chemotherapy regimens. In the past decade, researchers have attempted to repurpose approved drugs for cancer treatment, with the aim to develop less toxic low cost therapies that can rapidly enter the clinical practice. In this work, we made use of Candida albicans biofilms as a lower eukaryotic screening system that specifically upregulates expression of serine/glycine synthesis enzymes in response to sub-lethal stress. Using this platform, we discovered two repurposed compounds, sertraline and thimerosal, that show selective toxicity to serine/glycine synthesis dependent cancer cell lines, while they had no effect on cancer and normal lymphoid cell lines that take up serine and glycine from their environment. By using a combination of a PHGDH enzymatic activity assay, molecular docking and labeled serine tracing experiments, we discovered that sertraline and thimerosal inhibit serine/glycine synthesis enzymes SHMT and PHGDH respectively. In contrast to thimerosal, for which clinical applications are limited because of a toxic mercury group in its structure, sertraline is a widely used anti-depressant for which synergy with established anticancer drugs in breast cancer had already been described. Most notably, combining sertraline with a mitochondrial inhibitor, such as the clinically used antimalarial drug artemether, resulted in an even more pronounced inhibition of serine/glycine synthesis dependent proliferation, both in cultured cells and in an in vivo breast cancer xenograft model. Collectively, our study provides molecular insights into the repurposed mode-of-action of sertraline and allows to delineate a hitherto unidentified group of cancers that are particularly sensitive to treatment with sertraline, including breast cancers and leukemias. References: 1. R. Possemato, et al., Functional genomics reveal that the serine synthesis pathway is essential in breast cancer. Nature 476, 346–350 (2011). 2. K. R. Kampen, et al., Translatome analysis reveals altered serine and glycine metabolism in T-cell acute lymphoblastic leukemia cells. Nature Communications 10, 2542 (2019). Citation Format: Shauni L. Geeraerts, Kim R. Kampen, Gianmarco Rinaldi, Purvi Gupta, Mélanie Planque, Kaat De Cremer, Katrijn De Brucker, Stijn Vereecke, Benno Verbelen, Pieter Vermeersch, David Cassiman, Sarah-Maria Fendt, Arnout Voet, Bruno P.A. Cammue, Karin Thevissen, Kim De Keersmaecker. Repurposing the anti-depressant sertraline to target serine/glycine synthesis addicted cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1789.