Cancer cell metabolism is extensively reprogrammed to support rapid growth, with glucose and glutamine serving as key nutrients for biosynthesis. Within tumors, cancer cells often experience glutamine deprivation, triggering adaptive responses to maintain homeostasis. One such response involves the compartmentalization and polymerization of CTP synthase (CTPS), a crucial enzyme that catalyzes the rate-limiting step of pyrimidine biosynthesis, essential for DNA, RNA, and phospholipid production. Notably, elevated CTPS expression is linked to the progression of multiple cancers. Recent studies have revealed that CTPS can assemble into filamentous structures under various physiological conditions across multiple species, including human cancer cells, bacteria, yeast, and Drosophila. Our laboratory has demonstrated that CTPS filaments protect the enzyme from degradation under glutamine deprivation, thereby providing cancer cells with a survival advantage upon stress relief. Additionally, we have identified histidine-mediated methylation and ubiquitination as key regulators of CTPS assembly, and we have shown that the cytokeratin network functions as a storage compartment for CTPS filaments in cancer cells. Beyond cancer, CTPS assembly plays a crucial role in Drosophila oogenesis, where it is required for endoreplication in follicle cells and for border cell migration, a model of collective directional cell movement. Using APEX proximity labeling, we have identified several components involved in CTPS filament assembly, though the precise molecular mechanisms remain under investigation. In this grant proposal, we aim to elucidate the roles of these components in CTPS filament formation through knockdown assays and immunostaining approaches. We will also investigate the molecular mechanisms governing CTPS polymerization and assess how disrupting CTPS assembly impacts cancer metabolism using metabolomics analysis. These studies will provide fundamental insights into the regulation of CTPS dynamics and its implications in cancer biology.