Targeting ATR for selective cancer therapy

• Main Author: Middleton, Fiona Kay
• Format: Dissertation
• Language: English
• Published: University of Newcastle upon Tyne 2014

The DNA damage response (DDR), comprising repair and cell cycle checkpoints, maintains genomic stability. DDR dysfunction, particularly loss of the G1 checkpoint (e.g. p53 mutation), is common in cancer increasing reliance on S/G2 checkpoints. ATR, a kinase activated by ssDNA-dsDNA junctions arising following DNA damage (e.g. from chemo/radiotherapy), signals to homologous recombination repair (HRR) and S/G2 checkpoints. ATR inhibitors (ATRi) may therefore be tumour-selective chemo/radiosensitisers. Vertex Pharmaceuticals developed two novel, potent and selective ATRi, VE-821 and VE-822/VX-970. VX-970 is currently in clinical trial. ATR activity/inhibition was explored using a proprietary enzyme assay at Vertex Pharmaceuticals (Europe) Ltd. VE-821, although less potent than VE-822, was more selective for ATR over ATM. VE-821 and VE-822 inhibited ATR in breast cancer cells with wild type or mutant p53, which correlated with reduced proliferation and survival independently of p53 status. VE-821 was more cytotoxic to cancer than non-cancer breast cells. VE-821 sensitisation to gemcitabine/ionising radiation (IR) was p53-independent but tumour cell-specific in these unmatched cells. Radiosensitisation was not attributed to cell cycle effects of VE-821. In isogenic p53 WT or null/mutant cells dysfunctional p53 was associated with greater chemo/radiopotentiation by VE-821 (but not sensitivity to VE-821 alone) but not consistently with reduction of IR-induced G2 arrest. Defects in ATM, BRCA2 or XRCC3 (HRR), XRCC1 (base excision repair) and Ku80 (non- homologous end joining - NHEJ) conferred sensitivity to VE-821. Sensitivity to VE-821 was dependent on the expression of DNA-PKcs (NHEJ) but not its activity or NHEJ per se. High DNA-PKcs was associated with higher cMyc levels but cMyc was not a determinant of VE-821. To support clinical trials of ATRi a pharmacodynamic (PD) biomarker was developed. After UV/4NQ0 exposure pChklSer345 was most ATR-specific but unreliable in surrogate tissue (human PBMCs). Phosphorylation of H2AX was robust and ATR-specific at <1 hour and therefore the recommended PD biomarker.