SAT-321 Investigating The Effect of Two TAK1 Inhibitors on an Ovarian Granulosa Cell Tumor-Derived Cell Line

Abstract Disclosure: T.M. Tran: None. M. Alexiadis: None. T. Nguyen: None. P.J. Fuller: None. S. Chu: None. Granulosa cell tumors (GCT) are a rare type of ovarian cancer, comprising ∼5% of all ovarian cancers, of which 95% are adult (a)GCT. These endocrine-related neoplasms are characterized by the heterozygous FOXL2 C134W mutation, and are associated with late and invasive recurrences, contributing to a high mortality rate (approximately 80%) with limited treatment options. Preliminary research from our laboratory has identified constitutive activation of the NF-κB and AP-1 signaling pathways, and upregulation of X-linked inhibitor of apoptosis protein (XIAP) in aGCT. XIAP, regulated by NF-κB, forms a positive feed-forward loop by activating transforming growth factor beta-activated kinase 1 (TAK1). This study investigates the effects of TAK1 inhibition using two TAK1 inhibitors, takinib and 5Z-7 oxozeaenol (5Z-7), in the KGN (an aGCT-derived) cell line.Cell proliferation and viability were assessed in the hGrC1 (transformed normal granulosa cells) and the KGN cell lines following treatment with varying concentrations of takinib and 5Z-7 for 48 hours. Proliferation was assessed using MTS assays and xCELLigence® RTCA, while cell viability and morphology were monitored with the Incucyte Live-Cell Analysis System. Caspase-3/7 activity was quantified to assess apoptosis, and NF-κB and AP-1 activity was evaluated using transactivation assays. RNA sequencing (RNA-seq) was performed to analyze transcriptomic changes.Both inhibitors significantly reduced cell proliferation in a dose-dependent manner, with IC50 values of 6.9µM (takinib) and 4.3µM (5Z-7) in hGrC1 cells, and 4.5µM (takinib) and 3.8µM (5Z-7) for KGN cells. Cell viability was significantly reduced in both cell lines, but no change in cell morphology was observed. Caspase 3/7 activity was markedly increased in KGN cells treated with 5Z-7, but not with takinib, suggesting differential mechanisms of cell death. The inhibition of TAK1 led to substantial reductions in NF-κB and AP-1 transactivation in KGN cells. RNA-seq analysis revealed significant differential gene expression: 141 genes (64 upregulated, 77 downregulated) in KGN-treated cells versus 8 genes in hGrC1-treated cells when treated with takinib (fold changes >=2, Q value <0.05). Pathway analysis highlighted the enrichment of the MAPK, TNF, and NF-κB signaling pathways. Broader differential expression patterns (2869 DEGs in KGN; 594 DEGs in hGrC1) were also identified when treated with 5Z-7 compared to controls.TAK1 inhibitors effectively reduced cell proliferation and viability in KGN cells by targeting NF-κB and AP-1 signaling pathways, with 5Z-7 inducing apoptosis. Transcriptomic analysis suggests that takinib exhibits greater specificity for TAK1. These findings underscore the potential of TAK1 inhibition as a therapeutic approach for aGCT. Presentation: Saturday, July 12, 2025