Investigating resistance mechanisms to ATR inhibitors

The kinase activity of Ataxia-Telangiectasia and Rad3 related (ATR) protein plays a key role in controlling the DNA damage response (DDR) by acting through downstream targets to stabilise DNA replication forks, induce a transient cell cycle arrest to facilitate the repair of DNA, and thereby prevent premature mitosis in cells with unrepaired or unreplicated DNA. ATR is activated when single stranded DNA arises, for example, from stalled DNA replication forks or during the intermediate steps of double strand DNA break repair, and it is thought to mediate most of its responses through the downstream checkpoint kinase 1, CHK1. As ATR plays an important role in intra-S phase and G2/M cell cycle checkpoints, it has been proposed that inhibition of ATR activity selectively targets tumour cells that have defects in DNA damage-induced G1 cell cycle checkpoints by causing the accumulation of DNA damage and cell death. AZD6738 is a selective ATR kinase inhibitor that is currently in early clinical development (phase 1). Since development of resistance has been universally observed with long-term use of kinase inhibitors in the clinic, we aimed to identify potential mechanisms of acquired resistance to AZD6738. In this study, we used an in vitro assay system using PC-9 human non-small cell lung cancer cells to generate independent clonal cell lines (SO25, SO45, and SO46) that were resistant to AZD6738. The resistant cells have increased relative cell viability in the presence of AZD6738 compared to the parental cells and, surprisingly, grow better in the presence of the inhibitor than in its absence. AZD6738 resistant cells are also cross-resistant to other chemically unrelated ATR inhibitors (VE-821 and VE-822) as well as CHK1/2 and WEE1 inhibitors, but not ataxia telangiectasia mutated (ATM) inhibitors. Together, cell cycle, DNA fibre, and RNA-sequencing analyses all point towards differential cell cycle regulation in the resistant clones while western blot analysis revealed activation of ATM signalling in the presence of AZD6738 in parental PC-9, but not in the resistant cells. Western blot and RNA-sequencing analyses demonstrated that cyclins A2 and E1 are downregulated in the resistant cells. Knockdown of cyclin A2 and/or cyclin E1 using siRNA in parental PC-9 cells increased cell viability in the presence of AZD6738. We conclude that downregulation of cyclins A2 and E1 can confer resistance to ATR kinase inhibitors, and may also confer cross-resistance to other inhibitors targeting certain pathways downstream of ATR signalling (CHK1, WEE1).