S2519
Radiobiology – DNA damage repair
ESTRO 2026
Simon Lam 1 , Yaron Galanty 1 , Stephen Jackson 1 , Gregory Hannon 1 , Ultan McDermott 2 , Saif S Ahmad 1 1 CRUK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom. 2 Functional Genomics, AstraZeneca, Cambridge, United Kingdom. 3 Functional Genomics, Cancer Research Horizons, Cambridge, United Kingdom. 4 Biosciences, AstraZeneca, Cambridge, United Kingdom. 5 School of Medicine, Queen's University of Belfast, Belfast, United Kingdom Purpose/Objective: Radiation therapy (RT) is a core component of lung cancer treatment, however KRAS mutant non-small cell lung cancer (NSCLC) is associated with poor responses to radiation, and the mechanism driving resistance remains poorly understood. To address this question, a large-scale, multidimensional approach using CRISPR-based screening was used to identify and validate genes/pathways associated with radiation response across a range of in silico, in vitro (2D and 3D) and in vivo KRAS mutant NSCLC models. Material/Methods: First, we performed a genome-wide pooled CRISPR/Cas9 screen under radiation across a panel of 6 KRAS mutant NSCLC cell lines. We built a robust workflow for optimising radiation delivery to CRISPR edited cells to deliver one of the largest radiation screens to date. A dose of 2Gy in 2 fractions over 2 days was used. This identified a network of targets primarily clustering around DNA damage response pathways as sensitizers to RT treatment, including positive controls such as ATM and PRKDC. We validated the identified radiosensitisers using an arrayed CRISPR screening platform, coupled with high- content imaging, using a panel of NSCLC KRAS mutant cell lines, with TP53 isogenic lines. We selected specific imaging endpoints to assess the impact of each gene knockout on (i) ionising radiation-induced DNA damage, (ii) repair kinetics at 1h, 24h and 48h post irradiation and (iii) cell cycle profile, and these were integrated using novel statistical methods. Thirdly, a bespoke CRISPR library (~200 genes) was used for further validation in additional 2D lines, normal lung tissue organoids, and in vivo models. SW1573 (TP53 isogenic) NSCLC models were used for in vivo studies with tumours treated on the SARRP at 10 GY in 5 fractions over 5 days. Alongside, this an in silico model for radiation response in NSCLC was used to prioritise and validate hits. Results: Our data has identified novel drug targets in NSCLC showing highly preferential radiosensitisation in tumour tissue, which mechanistically appear to be driven by P53 function. Moreover, it provides a comprehensive dataset in NSCLC indicating that distinct DDR pathway genes demonstrate greater RT- sensitisation effects, within genetically similar
tumours. *Gene targets to be shared at meeting as currently under consideration for patenting.
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