ULK1 promotes metastatic progression in experimental models of epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is a leading cause of gynecological cancer mortality, driven largely by late diagnosis and chemoresistance. While autophagy is critical for EOC spheroid survival during metastasis, the role of ULK1, a key regulator of autophagy, in EOC progression remains unclear. To investigate this, we utilized CRISPR/Cas9 technology to delete ULK1 in EOC cell lines OVCAR8, HEYA8, ES2 and the fallopian tube epithelial cell line FT190. ULK1 loss and autophagy disruption were confirmed in EOC spheroids, with reduced Beclin-1 phosphorylation, impaired LC3 processing, and p62 accumulation. ULK1 knockout decreased EOC spheroid cell viability via increased apoptosis, and impaired matrix-bound organoid growth, offering new insights into ULK1 activity in affecting EOC tumor growth and spread. These findings were supported by in vivo xenograft models, in which ULK1 loss significantly reduced tumor burden and metastatic potential. ULK1 requirement during metastasis was supported by diminished invasive capacity of ULK1 knockout spheroid cells in mesothelial clearance assays. To investigate ULK1 mechanisms contributing to EOC tumor progression and metastasis, we conducted proteomic analyses of OVCAR8 spheroids, which revealed ULK1 loss disrupted critical pathways, including MEK-MAPK, PI3K-AKT-mTOR, and apoptosis regulation. Although ULK1 knockout failed to synergize with standard-of-care chemotherapeutics, it significantly enhanced sensitivity to MEK and mTOR inhibition. Analysis of ovarian cancer datasets demonstrates that high ULK1 mRNA correlates with a poorer 10-year overall and progression-free survival; in fact, its expression is further elevated in metastases as compared with primary tumors and normal tissue. Treatment of metastatic patient-derived organoids with the clinical ULK1 inhibitor DCC-3116, MEK inhibitor trametinib, or mTORC1/2 inhibitor AZD-8055 reduced viability in a subset of these samples, reflecting inter-patient heterogeneity and need for biomarker-guided selection. Overall, this study highlights ULK1 as a critical regulator of multiple steps of EOC disease progression, underscoring its potential as a therapeutic target in advanced ovarian cancer.