Small-Molecule Polu Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models

Purpose: DNA polymerase theta (Pol theta, encoded by the POLQ gene) is a DNA repair enzyme critical for microhomology medi-ated end joining (MMEJ). Pol theta has limited expression in normal tissues but is frequently overexpressed in cancer cells and, there-fore, represents an ideal target for tumor-specific radiosensitiza-tion. In this study we evaluate whether targeting Pol theta with novel small-molecule inhibitors is a feasible strategy to improve the efficacy of radiotherapy.Experimental Design: We characterized the response to Pol theta 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 Pol theta DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, noncancerous cells were not radiosensitized by Pol theta inhibition. Mechanistically, we show that the radiosensitization caused by Pol theta inhibition is most effec-tive 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 Pol theta inhibitor with improved metabolic stability. In vivo, the combination of Pol theta inhibition using ART899 with fractionated radiation is well toler-ated and results in a significant reduction in tumor growth com-pared with radiation alone.Conclusions: These results pave the way for future clinical trials of Pol theta inhibitors in combination with radiotherapy.