Abstract P04: TARGETING THYMIDYLATE SYNTHASE (HTS) SUPPRESSES COLORECTAL CANCER CELL GROWTH BY INDUCING DNA DAMAGE AND APOPTOSIS

Thymidylate synthase (hTS) is considered an important target for cancer therapy. Novel TS-dimer disruptors, Ddis, inhibit hTS with a different mechanism of action compared to drugs currently used in chemotherapy strategies. Ddis inhibits the activity of hTS, favours its intracellular degradation and inhibits cancer growth. In the present study, we demonstrate the effect of Ddis in HCT116 colon cancer cell line and the role of hTS in DNA damage and DNA damage response. Ddis, unlike other drugs currently in clinical use (e.g. 5-Fluorouracil, 5-FU) reduced hTS protein level, induced replication stress and DNA damage (γ-H2AX) followed by G2/M-phase arrest and apoptosis of HCT116 cell lines. Our immunofluorescence experiment demonstrated that Ddis caused the tumor suppressor P53-binding protein 1 (53BP1) accumulation in the nuclear foci in HCT116 cells after treatment with Ddis with respect to untreated control cells. The 53BP1 is one of the main mediators of DNA Damage Repair (DDR) and plays a pivotal role in orchestrating the choice of double-strand break (DSB) repair pathway. Furthermore, we observed the activation of Checkpoint kinase 1, Chk1 (pChk1-ser345). Chk1 is a crucial DNA damage checkpoint kinase and its activation, which requires ATR serine/threonine kinase, leads to inhibition of DNA replication and cell cycle progression. It is noteworthy that through Flow cytometry analysis, we observed a significant increase in the percentage of the G2/M phase of the cell cycle after 24 hours of Ddis treatment with respect to untreated cells. This result was confirmed through the inactivation of nuclear cyclin B/CDK1 activity that halted mitotic entry, allowing time for DNA damage repair. It is well known that the G2/M block prevents cells from entering mitosis when DNA damage has occurred. Overall these results demonstrate a functional link between hTS inhibition and DNA damage that can be exploited therapeutically in a clinical setting. Citation Format: Maria Gaetana Moschella, Michele Lamesta, Daniele Aiello, Gaetano Marverti, Alberto Venturelli, Maria PaolaCosti, Domenico D’Arca. TARGETING THYMIDYLATE SYNTHASE (HTS) SUPPRESSES COLORECTAL CANCER CELL GROWTH BY INDUCING DNA DAMAGE AND APOPTOSIS [abstract]. In: Proceedings of Frontiers in Cancer Science 2024; 2024 Nov 13-15; Singapore. Philadelphia (PA): AACR; Cancer Res 2025;85(15_Suppl):Abstract nr P04.