ESTRO 2025 - Abstract Book

S4006

Radiobiology - Tumour radiobiology

ESTRO 2025

Material/Methods: Identification of relevant radioresistance genes by a siRNA screen, based on colony forming ability in 3D cultures of MDA-MB-231 cells. Verification was done in further TNBC cell models and confirmed by specific, clinically relevant inhibitors. Determination of DNA damage by the detection of yH2AX, RAD51, or RPA foci. Analysis of replication processes using the DNA fiber assay. Results: Significant radiosensitization up to 35% was observed for 19 of the 44 genes analyzed after siRNA knockdown. Most of the top targets were associated with theta-mediated end joining (TMEJ) or translesion synthesis (TLS), both error prone signaling pathways that are crucial for increased tolerance to replication stress. Among the top hits for radiosensitization, polymerase theta (Polθ, encoded by POLQ) was most important (p<0.01), avoiding replication stress by participating in both TMEJ and TLS. Confirming the radiosensitizing potential, the sensitivity enhancement ratio (SER 50 ) after Polθ inhibition ranged from 1,1 to 1,4 (p<0.05) across several TNBC cell lines. Supporting this, increased DNA damage was observed (p<0,001). Moreover, enhanced replication stress was indicated by an increased formation of RAD51 foci (p<0.001) and altered replication fork progression. Polθ inhibition alone had no effect on replication processes. In contrast, irradiation in combination with Polθ inhibition had a significant effect on replication speed (p<0.0001), with a 50% reduction in actively replicating DNA strands compared to irradiation alone. Specific inhibitors are currently being used to investigate which of the functional Polθ domains, helicase or polymerase, are responsible for the observed radiosensitizing effect. Conclusion: Inhibition of Polθ resulted in significant radiosensitization due to enhanced DNA damage and DNA replication stress. Although the effect of Polθ inhibition on tumor control is currently being investigated in several clinical trials further knowledge of the underlying mechanisms is required for an effective combination with radiotherapy. Proffered Paper SHLD2-deficient tumours display high sensitivity to DNA polymerase-theta (Polθ) inhibition combined with radiotherapy Gonzalo Rodriguez-Berriguete 1 , Marco Ranzani 2 , Purusotha Thambiayah 1 , Nicole Machado 1 , Celia Gotorbe 1 , David Nderitu 1 , Alessandro Cicconi 2 , Aurora Cerutti 2 , Vera Grinkevich 2 , Katjusa Koler 2 , Jayesh Majithiya 2 , Suraj Menon 2 , Joana Neves 2 , Marina Roy-Luzarraga 2 , Nan Shao 2 , Asmita Thapa 2 , Graeme C. M. Smith 2 , Helen M. R. Robinson 2 , Geoff S. Higgins 1 1 Department of Oncology, University of Oxford, Oxford, United Kingdom. 2 Artios Pharma, Babraham Research Campus, Cambridge, United Kingdom Purpose/Objective: DNA polymerase theta (Polθ, encoded by the POLQ gene) is crucial for repairing double-stranded DNA breaks through microhomology-mediated end joining (MMEJ). Its selective overexpression in cancer cells makes Polθ an ideal target for tumour-specific radiosensitisation. Indeed, our previous work has demonstrated that targeting Polθ polymerase domain with small-molecule inhibitors is an effective approach to increase tumour radiosensitivity without enhancing side-effects in preclinical models 1 . As a single agent therapy, Polθ inhibition has shown promising synthetically lethal effects in tumour models with deficiencies in homologous recombination (HR) repair 2,3 . In contrast, no biomarkers have been established yet to predict which patients may benefit the most from the combination of Polθ inhibition with radiotherapy. Keywords: Breast cancer, Radioresistance, Replication stress 2821