Divergent Role of ULK1 to Balance Mitochondrial Homeostasis and Bioenergetics in Ovarian Cancer Spheroids.

Epithelial ovarian cancer (EOC) is the deadliest gynaecologic malignancy, largely due to late-stage diagnosis and ineffective therapy. EOC commonly spreads through the peritoneal cavity as multicellular spheroids, which are metastatic structures that enhance survival under detachment stress, promote dissemination, and contribute to therapeutic resistance. We previously showed that ULK1, a serine/threonine kinase classically linked to macroautophagy initiation, supports EOC progression, suggesting non-canonical roles in spheroid biology and pathogenesis. CRISPR/Cas9 ULK1 knockout (ULK1KO) models were generated in OVCAR8, HEYA8, and ES2 cells. Mitochondrial degradation phenotypes were assessed in spheroids by immunoblotting and fluorescence microscopy. Label-free proteomics with bioinformatic pathway analysis identified ULK1-associated programs in EOC spheroids. Bioenergetic consequences were quantified using Seahorse ATP-Rate assays. Therapeutic interactions were evaluated using multi-dose combination matrices testing the ULK1 inhibitor DCC-3116 with metformin. ULK1 modulated mitochondrial degradation in a cell-line-specific manner, either promoting or protecting against mitochondrial loss through mechanisms that were uncoupled from canonical autophagy machinery. Proteomic and bioinformatic analyses revealed significant alterations in mitochondria-related processes, aligning with emerging ULK1 functions in mitochondrial homeostasis. ULK1 loss broadly reduced OXPHOS complex proteins in EOC spheroids and consistently decreased hexokinase 2 (HK2), indicating coordinated metabolic remodeling. Seahorse profiling mirrored these shifts: OVCAR8 ULK1KO spheroids showed reduced OCR and ATP production, whereas HEYA8 and ES2 ULK1KO spheroids exhibited increased mitochondrial ATP production. Combination matrices showed potential synergy between DCC-3116 and metformin. These data show that ULK1 differentially regulates mitochondrial degradation across EOC spheroid models through potential mechanisms alternative to canonical autophagy machinery, while reshaping spheroid metabolism and revealing potential therapeutic vulnerabilities in advanced EOC.