Abstract

Purpose

Head and neck cancers (HNSCC) are routinely treated with radiotherapy; however, normal tissue toxicity remains a concern. Therefore, it is important to validate treatment modalities combining molecularly targeted agents with radiotherapy to improve the therapeutic ratio. The aim of this study was to assess the ability of the PARP inhibitor niraparib (MK-4827) alone, or in combination with cell cycle checkpoint abrogating drugs targeting Chk1 (MK-8776) or Wee1 (MK-1775), to radiosensitize HNSCCs in the context of HPV status.

Materials and methods

PARP1, PARP2, Chk1 or Wee1 shRNA constructs were analyzed from an in vivo shRNA screen of HNSCC xenografts comparing radiosensitization differences between HPV(+) and HPV(−) tumors. Radiosensitization by niraparib alone or in combination with MK-8776 or MK-1775 was assessed by clonogenic survival in HPV(−) and HPV(+) cells; and the role of p16 in determining response was explored. Relative expressions of DNA repair genes were compared by PCR array in HPV(+) and HPV(−) cells, and following siRNA-mediated knockdown of TRIP12 in HPV(−) cells.

Results

In vivo shRNA screening showed a modest preferential radiosensitization by Wee1 and PARP2 in HPV(−) and Chk1 in HPV(+) tumor models. Niraparib alone enhanced the radiosensitivity of all HNSCC cell lines tested. However, HPV(−) cells were sensitized to a greater degree, as suggested by the shRNA screen. When combined with MK-8776 or MK-1775, radiosensitization was further enhanced in an HPV dependent manner with HPV(+) cells enhanced by MK-8776 and HPV(−) cells enhanced by MK-1775. A PCR array for DNA repair genes showed PARP and HR proteins BRCA1 and RAD51 were much lower in HPV(+) cells than in HPV(−). Similarly, directly knocking down p16-dependent TRIP12 decreased expression of these same genes. Overexpressing p16 decreased TRIP12 expression and increased radiosensitivity in HPV(−) HN5. However, while PARP inhibition led to significant radiosensitization in the control, it led to no further significant radiosensitization in p16 overexpressing cells. Forced p16 expression in HPV(−) HN5 increased accumulation in G1 and subG1 and limited progression to S phase, thus reducing effectiveness of PARP inhibition.

Conclusions

Niraparib effectively radiosensitizes HNSCCs with a greater benefit seen in HPV(−). HPV status also plays a role in response to MK-8776 or MK-1775 when combined with niraparib due to differences in DNA repair mechanisms. This study suggests that using cell cycle abrogators in combination with PARP inhibitors may be a beneficial treatment option in HNSCC, but also emphasizes the importance of HPV status when considering effective treatment strategies.

中文翻译：

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通过废除细胞周期检查点来靶向头颈癌中的 DNA 损伤反应

 抽象的

 目的

头颈癌 (HNSCC) 通常采用放射疗法治疗；然而，正常组织毒性仍然是一个问题。因此，验证分子靶向药物与放射治疗相结合的治疗方式以提高治疗率非常重要。本研究的目的是评估 PARP 抑制剂 niraparib (MK-4827) 单独使用或与针对 Chk1 (MK-8776) 或 Wee1 (MK-1775) 的细胞周期检查点废除药物联合使用，使 HNSCC 放射增敏的能力。 HPV 状态的背景。

 材料和方法

通过 HNSCC 异种移植物的体内 shRNA 筛选分析 PARP1、PARP2、Chk1 或 Wee1 shRNA 构建体，比较 HPV(+) 和 HPV(-) 肿瘤之间的放射增敏差异。通过 HPV(-) 和 HPV(+) 细胞的克隆存活率评估尼拉帕尼单独使用或与 MK-8776 或 MK-1775 联合使用的放射增敏作用；并探讨了 p16 在决定反应中的作用。通过 PCR 阵列比较 HPV(+) 和 HPV(-) 细胞中 DNA 修复基因的相对表达，并在 HPV(-) 细胞中 siRNA 介导的 TRIP12 敲低后进行比较。

 结果

体内 shRNA 筛查显示，Wee1 和 PARP2 对 HPV(-) 肿瘤模型具有适度的优先放射增敏作用，而 Chk1 对 HPV(+) 肿瘤模型具有适度的放射增敏作用。 Niraparib 单独增强了所有测试的 HNSCC 细胞系的放射敏感性。然而，正如 shRNA 筛选所示，HPV(-) 细胞的敏感性更高。当与 MK-8776 或 MK-1775 联合使用时，放射增敏作用以 HPV 依赖性方式进一步增强，其中 MK-8776 增强 HPV(+) 细胞，MK-1775 增强 HPV(-) 细胞。 DNA修复基因的PCR阵列显示，HPV(+)细胞中的PARP和HR蛋白BRCA1和RAD51比HPV(-)细胞中低得多。同样，直接敲低 p16 依赖性 TRIP12 会降低这些相同基因的表达。过度表达 p16 会降低 TRIP12 的表达并增加 HPV(−) HN5 的放射敏感性。然而，虽然 PARP 抑制导致对照中显着的放射增敏，但它在 p16 过表达细胞中没有导致进一步显着的放射增敏。 HPV(−) HN5 中 p16 的强制表达增加了 G1 和 subG1 中的积累，并限制了向 S 期的进展，从而降低了 PARP 抑制的有效性。

 结论

Niraparib 有效地使 HNSCC 放射增敏，在 HPV(-) 中观察到更大的益处。由于 DNA 修复机制的差异，当与 niraparib 联合使用时，HPV 状态也在对 MK-8776 或 MK-1775 的反应中发挥作用。这项研究表明，细胞周期废除剂与 PARP 抑制剂联合使用可能是 HNSCC 的一种有益治疗选择，但也强调了在考虑有效治疗策略时 HPV 状态的重要性。