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. 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, normal fibroblasts are 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. These results pave the way for future clinical trials of Polθ inhibitors in combination with radiotherapy.

中文翻译：

---

小分子 Polθ 抑制剂在临床前模型中提供安全有效的肿瘤放射增敏作用

DNA 聚合酶 theta (Polθ) 是一种对微同源介导的末端连接 (MMEJ) 至关重要的 DNA 修复酶。Polθ 在正常组织中的表达有限，但在癌细胞中经常过度表达，因此是肿瘤特异性放射增敏的理想靶标。在这里，我们表明 ART558 和 ART899 是 Polθ DNA 聚合酶结构域的两种新型特异性变构抑制剂，可有效地使肿瘤细胞放射增敏，尤其是与分次放射相结合时。重要的是，正常成纤维细胞不会因 Polθ 抑制而放射致敏。从机制上讲，我们表明由 Polθ 抑制引起的放射增敏在复制细胞中最有效，并且是由于 DNA 损伤修复受损。我们还表明放射增敏在缺氧条件下仍然有效，表明这些抑制剂可能有助于克服缺氧诱导的放射抗性。此外，我们首次描述了 ART899，并将其描述为一种有效且特异性的 Polθ 抑制剂，具有改善的代谢稳定性。在体内，使用 ART899 的 Polθ 抑制与分次辐射的组合具有良好的耐受性，与单独的辐射相比，肿瘤生长显着减少。这些结果为 Polθ 抑制剂联合放疗的未来临床试验铺平了道路。使用 ART899 的 Polθ 抑制与分次辐射的组合具有良好的耐受性，与单独的辐射相比，肿瘤生长显着减少。这些结果为 Polθ 抑制剂联合放疗的未来临床试验铺平了道路。使用 ART899 的 Polθ 抑制与分次辐射的组合具有良好的耐受性，与单独的辐射相比，肿瘤生长显着减少。这些结果为 Polθ 抑制剂联合放疗的未来临床试验铺平了道路。