Zupan, Nezka; (2025) Mitigating radiation induced normal tissue toxicity. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The mechanistic target of rapamycin (mTOR) plays a central role in regulating cell growth, replication, and DNA damage responses. While mTOR inhibition and radiation effects have been extensively studied independently, their interplay in DNA repair and chromatin dynamics remains unclear. This study investigates rapamycin’s potential as a selective radioprotector, protecting normal colorectal organoids while not conferring the same benefit to cancerous models. Using a combination of immunofluorescence, western blotting, and phosphoproteomics, this study explored the effects of mTOR inhibition on DNA damage repair pathways, replication stress responses, and chromatin remodelling. The findings reveal that rapamycin reduces total protein abundance and phosphorylation of RAD18, a key regulator of translesion synthesis (TLS). This reduction likely disrupts TLS polymerase recruitment, affecting replication fork progression and genomic stability. Furthermore, rapamycin modulates replication origin licensing and firing by downregulating MCM2 and PCNA, slowing replication to allow for enhanced repair mechanisms. Concurrently, rapamycin-induced changes in Chk1 signalling suggest an intricate relationship between mTOR inhibition, replication stress, and DNA damage response regulation. Additionally, rapamycin promotes chromatin compaction, increasing heterochromatin-associated histone modifications (H3K9me3, H3K27me3, pKAP1 Ser824), which may contribute to enhanced DNA repair efficiency and radioprotection. Our findings suggest that chromatin condensation might act as a protective mechanism, shielding DNA from radiation-induced damage while optimizing repair processes. These results highlight mTOR as a key coordinator of DNA replication, repair, and chromatin dynamics, with significant implications for radiotherapy. The selective radioprotection observed in organoid models suggests potential therapeutic applications for mTOR inhibition in protecting healthy tissues while maintaining tumour sensitivity to radiation.

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