Abstract 540: Cancer cells are sensitive to methionine cycle perturbation in low-lipid environments.

Abstract Nutrient availability varies across tissues and shapes how cancer cells use metabolism to proliferate and survive, creating context-specific vulnerabilities that might be targeted for improved therapy. Because access to lipids is constrained in some tissue environments, we sought to identify metabolic pathways required for proliferation under lipid-depleted conditions. Specifically, we performed a CRISPR/Cas9 loss-of-function screen targeting metabolic synthesis genes in human cancer cells cultured in lipid-replete versus lipid-depleted media. The screen identified methionine synthase (MTR), an enzyme linking the folate and methionine cycles, as a top hit required for proliferation in lipid-depleted conditions. Genetic validation confirmed that MTR knockout (KO) cell proliferation is impaired in lipid-depleted compared to lipid-replete conditions. Follow-up pharmacologic studies revealed that inhibition of methionine adenosyltransferase 2A (MAT2A), an enzyme in the methionine cycle that generates S-adenosylmethionine (SAM), also reduced proliferation in lipid-depleted conditions, suggesting that cells become broadly dependent on the methionine/folate cycle when lipids are scarce.To determine how methionine/folate cycle perturbations alter intracellular metabolism in lipid-depleted conditions, we performed LC-MS-based metabolomics. MTR KO in lipid-depleted media caused depletion of multiple nucleotide species, and supplementation with purine nucleotides or folinic acid fully rescued proliferation, consistent with impaired folate-dependent nucleotide synthesis. MTR KO cells also exhibited elevated markers of DNA damage, supporting a model in which MTR loss limits nucleotide availability under lipid-depleted conditions, leading to DNA damage. In contrast, MAT2A-inhibited cells were not rescued by nucleotides or folinic acid. Instead, supplementation with phosphatidylcholine, a major membrane phospholipid synthesized in a SAM-dependent manner, restored proliferation in lipid-depleted media. These results suggest that perturbing different nodes of the methionine cycle triggers distinct metabolic liabilities: MTR loss primarily limits nucleotide availability, whereas MAT2A inhibition restricts SAM-dependent phospholipid synthesis. Together, these findings reveal that cancer cells rely more heavily on methionine and folate metabolism when lipids are scarce and uncover two mechanistically distinct vulnerabilities that emerge in low-lipid environments with potential relevance for treating cancer. Citation Format: Diya Lakshmi Ramesh, Keene L. Abbott, Ryan Elbashir, Edrees H. Rashan, Raphael Ferreira, Matthew G. Vander Heiden. Cancer cells are sensitive to methionine cycle perturbation in low-lipid environments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 540.