Abstract 835: Sensitivity of PTEN deficient non-small cell lung cancer to ionising radiation through inhibition of ataxia terangiectasia related 3 kinase (ATR)
Authors: Dunne Victoria Louise , Redmond Kelly , Coffey Caroline , Butterworth Karl , Prise Kevin , Hanna Gerard 
Published: July 2017

This study sought to determine the therapeutic efficacy of AZD6738, a small molecule inhibitor of ataxia telangiectasia related 3 kinase (ATR), in combination with ionising radiation (IR) for the treatment of PTEN deficient non-small cell lung cancer (NSCLC).

An isogenic PTEN deficient cell model were generated in H460 and A549 cell lines by HuSH PTEN shRNA constructs in pGFP-V-RS vectors from Origene. Radiosensitivity was determined at various concentrations of AZD6738 by clonogenic assay following IR with 225 kV X-rays. Target validation and mechanistic evaluation of the ATR radiation treatments was performed by western blotting. Cell cycle distribution was obtained using propidium iodine flow cytometry analysis which was performed 48 hours following treatment with AZD6738 alone or in combination with IR. In vivo efficacy was determined using cell line derived xenografts in female SCID mice treated with AZD6738 at a concentration of 25 mg/kg per day by oral gavage for 28 days. Animals were exposed to size fraction and hypofractionated radiation dose under CBCT image guidance using the small animal radiotherapy research platform (SARRP). Radiation induced toxicity in normal lung tissue effects was investigated in a model of radiation induced fibrosis in C57BL6/J mice.

In-vitro, combined treatment with AZD6738 and IR selectively targets PTEN-deficient tumour cells causing a significant reduction in clonogenic survival for both H460- and A549-PTEN KO cell models (p= &lt;0.05 at 8Gy). Following 2 Gy IR exposure there were significantly higher mean foci per cell in both H460-KO and A549-KO cells in comparison to WT cells (p= &lt;0.05 at 2, 4 and 8 hours following treatment). Both PTEN-KO cell lines exhibited increased sub G0/G1 and G2/M populations in comparison to PTEN-WT cell lines when treated with AZD6738 and IR. Minimal toxicity was observed in both normal HBE and BEAS-2B cell lines.

Currently in vivo tumour response experiments are underway to investigate IR and ATR kinase inhibitor AZD6738 using H460-WT and H460-PTEN KO cell models as well as determining the effects of AZD6738 in a radiation induced model of lung fibrosis.

Approximately 10% of NSCLC patients have mutations in PTEN. This research therefore elucidates PTEN loss as a therapeutic target for combined IR with pharmacological inhibition of ATR in NSCLC. Targeting ATR inhibition in PTEN-deficient NSCLC may serve to increase radiation sensitivity in an in vivo model thus highlighting its clinical relevance as a potential personalised medicine approach for patients with PTEN deficient NSCLC.

Citation Format: Victoria Louise Dunne, Kelly Redmond, Caroline Coffey, Karl Butterworth, Kevin Prise, Gerard Hanna. Sensitivity of PTEN deficient non-small cell lung cancer to ionising radiation through inhibition of ataxia terangiectasia related 3 kinase (ATR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 835. doi:10.1158/1538-7445.AM2017-835