Division of Experimental Radiation Biology, Department of Radiation Therapy, University Hospital Essen, University of Duisburg-Essen, 45147, Essen, Germany; Institute of Medical Radiation Biology, University Hospital Essen, University of Duisburg-Essen, 45147, Essen, Germany.
Division of Experimental Radiation Biology, Department of Radiation Therapy, University Hospital Essen, University of Duisburg-Essen, 45147, Essen, Germany; Institute of Medical Radiation Biology, University Hospital Essen, University of Duisburg-Essen, 45147, Essen, Germany.
Radiother Oncol. 2024 Nov;200:110475. doi: 10.1016/j.radonc.2024.110475. Epub 2024 Aug 13.
The PARP inhibitor (PARPi), Talazoparib (BMN673), effectively and specifically radiosensitizes cancer cells. Radiosensitization is mediated by a shift in the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) toward PARP1-independent, alternative end-joining (alt-EJ). DNA polymerase theta (Polθ) is a key component of this PARP1-independent alt-EJ pathway and we show here that its inhibition can further radiosensitize talazoparib-treated cells. The purpose of the present work is to explore mechanisms and dynamics underpinning enhanced talazoparib radiosensitization by Polθ inhibitors in HR-proficient cancer cells.
Radiosensitization to PARPis, talazoparib, olaparib, rucaparib and veliparib was assessed by clonogenic survival. Polθ-proficient and -deficient cells were treated with PARPis and/or with the Polθ inhibitors ART558 or novobiocin. The role of DNA end-resection was studied by down-regulating CtIP and MRE11 expression using siRNAs. DSB repair was assessed by scoring γH2AX foci. The formation of chromosomal abnormalities was assessed as evidence of alt-EJ function using G-specific cytogenetic analysis.
Talazoparib exerted pronounced radiosensitization that varied among the tested cancer cell lines; however, radiosensitization was undetectable in normal cells. Other commonly used PARPis, olaparib, veliparib, or rucaparib were ineffective radiosensitizers under our experimental conditions. Although genetic ablation or pharmacological inhibition of Polθ only mildly radiosensitized cancer cells, talazoparib-treated cells were markedly further radiosensitized. Mechanistically, talazoparib shunted DSBs to Polθ-dependent alt-EJ by enhancing DNA end-resection in a CtIP- and MRE11-dependent manner - an effect detectable at low, but not high IR doses. Chromosomal translocation analysis in talazoparib-treated cells exposed to Polθ inhibitors suggested that PARP1- and Polθ-dependent alt-EJ pathways may complement, but also back up each other.
We propose that talazoparib promotes low-dose, CtIP/MRE11-dependent resection and increases the reliance of irradiated HR-proficient cancer cells, on Polθ-mediated alt-EJ. The combination of Polθ inhibitors with talazoparib suppresses this option and causes further radiosensitization. The results suggest that Polθ inhibition may be exploited to maximize talazoparib radiosensitization of HR-proficient tumors in the clinic.
聚腺苷二磷酸核糖聚合酶抑制剂（PARPi）他拉唑帕尼（BMN673）能有效且特异性地增强癌细胞的放射敏感性。放射增敏作用是通过将电离辐射（IR）诱导的 DNA 双链断裂（DSB）的修复转向 PARP1 非依赖性的替代末端连接（alt-EJ）来介导的。DNA 聚合酶θ（Polθ）是这种 PARP1 非依赖性 alt-EJ 途径的关键组成部分，我们在这里表明，其抑制作用可以进一步增强他拉唑帕尼处理的细胞的放射敏感性。本研究的目的是探索 HR 有效的癌细胞中，Polθ 抑制剂增强他拉唑帕尼放射敏感性的机制和动力学。
通过集落形成存活能力评估 PARPi、他拉唑帕尼、奥拉帕利、鲁卡帕利和 veliparib 的放射增敏作用。用 PARPi 和/或 Polθ 抑制剂 ART558 或新生霉素处理 Polθ 功能正常和缺陷的细胞。通过用 siRNA 下调 CtIP 和 MRE11 的表达来研究 DNA 末端切除的作用。通过评分 γH2AX 焦点来评估 DSB 修复。使用 G 特异性细胞遗传学分析作为替代末端连接功能的证据来评估染色体异常的形成。
他拉唑帕尼表现出明显的放射增敏作用，在测试的癌细胞系中有所不同；然而，在正常细胞中检测不到放射增敏作用。在我们的实验条件下，其他常用的 PARPi，奥拉帕利、veliparib 或 rucaparib 均不能作为有效的放射增敏剂。尽管 Polθ 的遗传缺失或药理学抑制仅轻度放射增敏癌细胞，但他拉唑帕尼处理的细胞则显著进一步放射增敏。从机制上讲，他拉唑帕尼通过增强 CtIP 和 MRE11 依赖性的 DNA 末端切除，将 DSB 转移到 Polθ 依赖性的 alt-EJ，这种作用可在低剂量但不能在高剂量 IR 下检测到。在暴露于 Polθ 抑制剂的他拉唑帕尼处理的细胞中进行的染色体易位分析表明，PARP1 和 Polθ 依赖性的替代末端连接途径可能互补，但也互为备份。
我们提出，他拉唑帕尼促进低剂量、CtIP/MRE11 依赖性的切除，并增加了受照射的 HR 有效的癌细胞对 Polθ 介导的替代末端连接的依赖。Polθ 抑制剂与他拉唑帕尼的联合使用抑制了这种选择，并导致进一步的放射增敏。结果表明，Polθ 抑制可能被利用来最大限度地提高 HR 有效的肿瘤中他拉唑帕尼的放射敏感性。
