Background/Objectives: Epithelial ovarian cancer (EOC) is often diagnosed at advanced stages, with metastasis driven by spheroid dissemination within the peritoneal cavity. We previously demonstrated that autophagy supports spheroid cell survival and suggest that it contributes to chemoresistance. Unc-51-like autophagy activating kinase 1 (ULK1), a key regulator of autophagy, has emerged as a promising therapeutic target. Here, we evaluated the effects of ULK1 inhibition via MRT68921, alone and in combination with afatinib—a tyrosine kinase inhibitor (TKI) known to induce pro-survival autophagy—in EOC. Methods: High-grade serous (HGSOC) and ovarian clear cell carcinoma (OCCC) cell lines were cultured under adherent and spheroid conditions. Immunoblotting confirmed on-target effects and modulation of autophagy. Autophagic flux was assessed using mCherry-eGFP-LC3 reporter assays. We assessed 96 dose combinations of MRT68921 and afatinib using drug combination matrices, with synergy evaluated via Synergy Finder. Promising combinations were evaluated across multiple EOC spheroid models and patient ascites-derived organoids. Results: MRT68921 inhibited ULK1 activity and reduced autophagic flux in a context-dependent manner while afatinib alone induced autophagy. Their combination produced synergistic effects at select concentrations, impairing spheroid reattachment and viability. However, MRT68921 alone significantly reduced viability across multiple EOC models, including patient ascites-derived organoids. Conclusions: This study is the first to evaluate the combined effects of MRT68921 and afatinib in epithelial ovarian cancer. Our findings demonstrate that ULK1 inhibition via MRT68921 consistently reduces cell viability across multiple ovarian cancer models, supporting ULK1 as a promising therapeutic target. In contrast, combination with afatinib produced limited and context-dependent effects, indicating that further investigation is needed to identify optimal combination strategies for ULK1-targeted therapies.

Macroautophagy (herein referred to as autophagy) is a highly conserved intracellular degradation system that maintains cell homeostasis when activated by cellular stress. 1 These cellular stresses ...

Background/objectives: 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. Methods: 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. Results: 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. Conclusions: 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.