Figure 4 from Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models

https://doi.org/10.1158/1078-0432.30707638

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posted on 2025-11-25, 13:07 authored by 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 B. Majithiya, Desiree Piscitello, Eeson Rajendra, Martin L. Stockley, Simon J. Boulton, Ester M. Hammond, Robert A. Heald, Graeme C.M. Smith, Helen M.R. Robinson, Geoff S. Higgins

Characterization of ART899 as a specific and potent Polθ inhibitor with improved stability. A, Chemical structures of the Polθ inhibitors ART558 and ART899. The table shows the in vitro intrinsic clearance values of ART558 and ART899 after exposure to rat and mouse liver microsomes. B, Nano-luciferase MMEJ assay showing ART899-mediated inhibition of MMEJ activity in HEK-293 cells. The nano-luciferase readings were normalized to control luciferase (firefly) readings, and these were then normalized to DMSO. Data points show the mean ± SEM of four technical replicates; representative of two independent experiments. C, Confirmation of MMEJ assay specificity. Same experiment described in B but showing both the nanoluc and firefly readings normalized to their own DMSO reading, confirming negligible inhibition by ART899 of the control firefly luciferase signal. D, Clonogenic survival of HCT116 and H460 cells treated with ART899. Graphs show the surviving fraction after 5 × 2 Gy IR. E, Confirmation of ART899 specificity in U2OS WT and Polθ KO cells. Cells were treated as described in D. F, Effect of ART899 in noncancerous cells. MRC-5 and AG01552 fibroblasts were treated as described in D. The effect of ART899 in unirradiated cells from D to F is shown in Supplementary Fig. S5A. Graphs shown in D to F correspond to average ± SD from triplicate wells (representative from three separate experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). G, Viability of HIEC-6 cells treated with ART899 and irradiated with 5 × 2 Gy, as determined by the alamar blue assay 8 days after the first IR fraction. Graph shows the viability normalized to unirradiated controls (Supplementary Fig. S5C); representative from three independent experiments.

Funding

Cancer Research UK Radnet Oxford Centre

Howat Foundation (The Howat Foundation)

HORIZON EUROPE European Research Council (ERC)

Wellcome Trust (WT)

Cancer Research UK (CRUK)

Engineering and Physical Sciences Research Council (EPSRC)

Brain Tumour Charity (The Brain Tumour Charity)

Cancer Research UK Clinician Scientist

History

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DOI - Is supplement to Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models

ARTICLE ABSTRACT

DNA polymerase theta (Polθ, encoded by the POLQ gene) 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 this study we evaluate whether targeting Polθ with novel small-molecule inhibitors is a feasible strategy to improve the efficacy of radiotherapy. We characterized the response to Polθ inhibition in combination with ionizing radiation in different cancer cell models in vitro and in vivo. 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, noncancerous 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 with radiation alone. These results pave the way for future clinical trials of Polθ inhibitors in combination with radiotherapy.

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Keywords

DNA Damage and Repair DNA repair Radiation Oncology Molecular targets of radiation response Small Molecule Agents

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CC BY