抑制WEE1激酶增强高级别浆液性卵巢癌细胞对新型KIF18A抑制剂ATX020的敏感性

High-grade serous ovarian cancer (HGSOC) is the most common (≈80%) and lethal subtype of ovarian cancer in the United States. HGSOC is characterized by universal TP53 mutations, genomic instability, and defects in DNA damage repair (DDR) pathways, which often lead to chromosomal instability (CIN). The cytoskeletal motor protein KIF18A is essential for chromosomal congression and segregation during metaphase in CIN+ cancer cells but not necessary in normal cells. We recently demonstrated that a novel KIF18A inhibitor, ATX020 (tool compound), specifically inhibits cell growth in HGSOC cells with higher ploidy and aneuploidy scores (AS), which are markers of CIN (Nair et al, Cancers, 2025). It does this by blocking the plus-end movement of KIF18A from spindle poles and disrupting chromosomal congression and segregation. In that study, we also observed increased WEE1 activity in resistant cells treated with ATX020, as indicated by higher levels of its phosphorylated substrate CDK1 (pCDK1-Y15), a key component of the G2/M cell cycle checkpoint. WEE1 inhibitors can make cells more sensitive to DNA-damaging agents by overriding the G2/M checkpoint and forcing cells with unrepaired DNA damage into premature mitosis, leading to increased DNA damage and CIN. Furthermore, recent RNAseq analysis shows enrichment of pathways involved in regulating the G2/M checkpoint and DNA damage repair in cells treated with ATX020 cells. We hypothesized that targeting WEE1 could enhance sensitivity of both resistant and sensitive cells to ATX020. We used ATX020 resistant (A2780, PEO4, and OVCAR5) and sensitive (OVCAR3, OVCAR8, and PEO1) cell lines to examine the effects of an WEE1 inhibitor, AZD1775 (Adavosertib), on inducing CIN and thus sensitivity to ATX020. Using multi-well growth inhibition assays, immunofluorescent microscopy, live cell imaging, westerns and flow cytometry, we show that WEE1 inhibition made both resistant and sensitive HGSOC cells more responsive to ATX020, while also increasing DNA damage, prolonging/disrupting mitotic process and causing proliferative arrest. Transcriptomic analysis (bulk RNAseq) and differential gene expression (DEG) analysis show consistent upregulation of target genes and associated pro-inflammatory pathways, TNF signaling, and epithelial-mesenchymal transition in both sensitive and resistant cells when treated with ATX020. This uncovers potential targets that could improve the efficacy of ATX020 in preclinical models. Furthermore, murine models of both resistant and sensitive HGSOC cells treated with ATX020 exhibit tumor growth inhibition that is consistent with the in vitro results. These findings suggest new research approaches that could potentially enhance the impact of KIF18A inhibition in a clinical setting.