SHMT2-mediated mitochondrial serine metabolism drives 5-FU resistance by fueling nucleotide biosynthesis

5-Fluorouracil (5-FU) is a key component of chemotherapy for colorectal cancer (CRC). 5-FU efficacy is established by intracellular levels of folate cofactors and DNA damage repair strategies. However, drug resistance still represents a major challenge. Here, we report that alterations in serine metabolism affect 5-FU sensitivity in in vitro and in vivo CRC models. In particular, 5-FU-resistant CRC cells display a strong serine dependency achieved either by upregulating endogenous serine synthesis or increasing exogenous serine uptake. Importantly, regardless of the serine feeder strategy, serine hydroxymethyltransferase-2 (SHMT2)-driven compartmentalization of one-carbon metabolism inside the mitochondria represents a specific adaptation of resistant cells to support purine biosynthesis and potentiate DNA damage response. Interfering with serine availability or affecting its mitochondrial metabolism revert 5-FU resistance. These data disclose a relevant mechanism of mitochondrial serine use supporting 5-FU resistance in CRC and provide perspectives for therapeutic approaches. [Display omitted] •Serine metabolism is a key driver of 5-FU acquired resistance in CRC•Affecting serine availability impairs DNA damage response in 5-FU-resistant cells•Mitochondrial compartmentalization of serine metabolism sustains 5-FU resistance•Targeting mitochondrial serine metabolism is effective in overcoming 5-FU resistance Pranzini et al. show that 5-FU resistance is supported by increased serine consumption and its mitochondrial compartmentalization to support purine biosynthesis and potentiate DNA damage repair to overcome drug toxicity. Interfering with serine accessibility and targeting mitochondrial serine metabolism overcome 5-FU resistance.