Identifying and overcoming a mechanism of resistance to WEE1 kinase inhibitor AZD1775 in high grade serous ovarian cancer cells

Objective: As a result of TP53 gene mutation high grade serous ovarian cancer (HGSOC) is dependent on the G2 checkpoint for the repair of DNA damage and survival. The key role of WEE1 kinase at this checkpoint makes inhibition of WEE1 kinase in combination with DNA damaging agents an attractive therapeutic strategy for HGSOC. Our aim was to characterise resistance mechanisms to WEE1 inhibitor AZD1775 and identify ways to overcome resistance ready for use in the clinic. Methods: AZD1775-resistant HGSOC cell clones were isolated and western blotting, cell cycle analysis, growth assays, RNA-Seq and gene expression analysis were used to characterise resistance mechanisms and investigate a way to overcome resistance. Results: A resistance mechanism previously reported in small cell lung cancer did not operate in HGSOC. Instead, resistance resulted from different cell cycle control pathway changes that slow AZD1775-induced cell cycle progression and reduce accumulation of replication associated DNA damage. One major change was reduced levels of CDK1, the substrate for WEE1 kinase inhibition; another was increased levels of PKMYT1, which can also inhibit CDK1. Increased expression of TGF$\beta$ signalling to slow cell cycle progression occurred in resistant clones. A TGF$\beta$R1 inhibitor overcame resistance in a clone with the highest TGF$\beta$R1 receptor expression. Conclusions: Although overexpression of the membrane glycoprotein MDR1 is a common mechanism of drug resistance, it was not involved in our HGSOC cells. Instead AZD1775 resistance resulted from cell cycle control pathway changes that combine to slow AZD1775-induced cell cycle progression and so reduce accumulation of replication-associated DNA damage.

ovarian cancer; cell cycle control; WEE1 kinase inhibition; resistance mechanism; DNA repair

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