Gonzalo Rodriguez-Berriguete, Marco Ranzani, Remko Prevo, Rathi Puliyadi, Nicole Machado, Hannah R. Bolland, Val Millar, Daniel Ebner, Marie Boursier, Aurora Cerutti, Alessandro Cicconi, Alessandro Galbiati, Diego Grande, Vera Grinkevich, Jayesh Majithiya, Desiree Piscitello, Eeson Rajendra, Martin Stockley, Simon J. Boulton, Ester M. Hammond, Robert Heald, Graeme C.M. Smith, Helen Robinson, Geoff S. Higgins; Small-molecule Polθ inhibitors provide safe and effective tumor radiosensitization in preclinical models. Clin Cancer Res 2023; https://doi.org/10.1158/1078-0432.CCR-22-2977
Purpose: DNA polymerase theta (Polθ) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Polθ has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. In the present study we evaluate whether targeting Polθ with novel small-molecule inhibitors is a feasible strategy to improve the efficacy of radiotherapy. Experimental design: We characterized the response to Polθ inhibition in combination with ionizing radiation in different cancer cell models in vitro and in vivo. Results: Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Polθ DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, non-cancerous cells were not radiosensitized by Polθ inhibition. Mechanistically, we show that the radiosensitization caused by Polθ inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Polθ inhibitor with improved metabolic stability. In vivo, the combination of Polθ inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared to radiation alone. Conclusion: These results pave the way for future clinical trials of Polθ inhibitors in combination with radiotherapy.
Distribution of POLQ mRNA levels across cell lines from CCLE
Luciferase-based MMEJ assay in a panel of cancer cell lines.
Cell cycle-dependent effects of ART558 in combination with IR
Accompanies Figure 2 (ART558 treatment leads to increased residual IR-induced DNA damage foci)
Accompanies Figure 3 (Effect of ART558 under hypoxic conditions)
Accompanies Figure 4 (Characterization of ART899 as a specific and potent Polθ inhibitor with improved stability)
Accompanies Figure 5 (ART899 combined with radiation causes significant tumor growth delay in vivo and is well tolerated)
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