Abstract 1486: Early biomarker dynamics are associated with treatment efficacy in a preclinical model of radiotherapy combined with a DNA damage response agent

Abstract Introduction: Radiotherapy is an essential treatment for many types of cancer, with almost half of all cancer patients undergoing it as part of their treatment regimen. It can initiate cytotoxic signaling pathways to induce cancer cell death, but it can also activate cytoprotective mechanisms that respond to cellular damage. These protective mechanisms can be targeted by anticancer agents like DNA damage response (DDR) agents, indicating that radiotherapy might boost the effectiveness of these drugs. Early response biomarkers can be utilized to support preclinical studies to assess the pharmacodynamic effects of a drug, providing insights into its mechanism of action. RNA sequencing technologies provide an opportunity to elucidate gene expression profiles across the breadth of the transcriptome, uncovering disease-specific gene signatures and biomarker candidates. Methods and Experimental Procedures: The A549 lung cancer cell line was utilized to establish in vitro assays and the in vivo preclinical model for the study. An inhibitor targeting DNA PK (AZD7648) was used as it targets pathways crucial for the repair of radiation induced DNA double strand breaks. The effects on cell viability and DDR biomarkers were assessed after radiotherapy, compound or in combination both in vitro and in vivo. Targeted X rays were delivered to the tumor only using an Xstrahl CIX3 cabinet irradiator. Total RNA was isolated from excised tumors, and RNAseq analysis was performed (Illumina NovaSeq). Differential gene expression and pathway analysis was performed using Qiagen Ingenuity Pathway Analysis software. Protein biomarkers were assessed using the Jess Simple Western platform. Data: In vitro assays showed increased sensitivity to the AZD7648 inhibitor when combined with radiotherapy, compared to standalone treatments. This combination also resulted in delayed tumor growth in vivo, with radiotherapy being well tolerated. The induction of biomarkers by radiotherapy, either as a monotherapy or in combination with the inhibitor, was assessed. Changes in biomarker expression were consistent with target engagement and treatment efficacy. Notably, γH2AX upregulation was evident in the irradiation monotherapy group but was delayed when combined with the DNA PK inhibitor, demonstrating target engagement as DNA PK inhibition alters the kinetics of γH2AX. Conclusion: In summary, both in vitro assays and in vivo models have been developed to explore the synergistic effects of combining radiotherapy with DDR agents. The combination of radiotherapy with the AZD7648 inhibitor resulted in delayed tumor growth in vivo. Early changes in protein and gene biomarkers were consistent with target engagement and treatment efficacy. These assays can serve as preclinical tools to investigate compounds or mechanisms that can be effectively combined with radiotherapy. Citation Format: Pablo Binder, Theoni (Tania) Katopodi, Lyndsey Hanson, Charlotte Bell, Eimear Flanagan, Paul Farrington, Nick Moore, Emily Wright, Tobias Bunday, Stewart Brown, Amy Cantrell, Jon Greenall, Shannon Sharman, John Woolley, Lorraine Mooney, Jane Kendrew. Early biomarker dynamics are associated with treatment efficacy in a preclinical model of radiotherapy combined with a DNA damage response agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1486.