# ATR Blockade Potentiates the Effects of Genotoxic Agents In Vitro and Promotes Antitumor Immunity in a Mouse Model of Non-Small Cell Lung Cancer

## TL;DR

Blocking ATR in lung cancer cells boosts the effects of DNA-damaging drugs and improves immune response in mice, suggesting a new treatment approach.

## Contribution

Combining ATR inhibitors, genotoxic drugs, and immune checkpoint inhibitors enhances antitumor effects in non-small cell lung cancer.

## Key findings

- ATR inhibition with AZD6738 increases the cytotoxicity of genotoxic agents in NSCLC cells.
- Combining ATR inhibitors, cisplatin, and anti-PD1 boosts T cell infiltration and antitumor efficacy in mice.
- Triple therapy with AZD6738, cisplatin, and anti-PD1 achieves the strongest antitumor effect.

## Abstract

The ataxia-telangiectasia mutated and Rad3-related (ATR) kinase plays a crucial role in sensing and responding to DNA damage and replication stress by coordinating cell cycle arrest, promoting DNA repair, and stabilizing replication forks. Previous studies have shown that ATR blockade can sensitize cancer cells to genotoxic agents by disrupting the DNA damage response network and by inducing immunogenicity. In this study, we found that the combination of the ATR inhibitor AZD6738 and genotoxic agents decreased the viability of non-small cell lung cancer (NSCLC) cells in vitro and increased the antitumor efficacy of an immune checkpoint inhibitor in a mouse NSCLC model. These preclinical results suggest that combining ATR inhibitors, genotoxic drugs, and immune checkpoint inhibitors may lead to the development of a new cancer treatment strategy for NSCLC.

Background/Objectives: Non-small cell lung cancer (NSCLC) is the most frequent type of lung cancer, and its main treatments include chemotherapy with genotoxic drugs and immunotherapy. Central to the cellular response to genotoxic stress is the DNA damage response (DDR) network, regulated by key kinases such as ataxia-telangiectasia mutated and Rad3-related (ATR). Herein, we tested the hypothesis that inhibition of ATR enhances the cytotoxicity of genotoxic agents and the antitumor immune response. Methods: DDR-related parameters and redox status, expressed as GSH/GSSG ratio, and apurinic/apyrimidinic lesions, were evaluated in human (A549, H1299) and murine (LLC) NSCLC cell lines after co-exposure to ATR inhibitor (AZD6738) and ultraviolet C (UVC) irradiation or cisplatin. Using a syngeneic LLC model, treatments of AZD6738 alone or in combination with cisplatin and/or anti-programmed cell death 1 antibody (anti-PD1) were examined. Results: In all cell lines, combined treatment with AZD6738 and cisplatin or UVC irradiation markedly decreased cell viability, DNA repair efficiency, and GSH/GSSG ratios; increased drug-induced DNA damage; and augmented apurinic/apyrimidinic lesions. In vivo, following treatment with AZD6738 and cisplatin, flow cytometry analysis performed in tumor cells revealed an increased infiltration of CD3+ and CD8+ T cells, with the triple combination of AZD6738, cisplatin, and anti-PD1 achieving the strongest antitumor effect. The CD3+CD4−CD8− double-negative (DN) T cell population in tumor samples also emerged as a contributing factor in this context. Conclusions: These results demonstrate that ATR blockade concurrently enhances the efficacy of genotoxic agents and immune checkpoint inhibitors, thus paving the way for combination therapies in NSCLC.