Dual Inhibition of PARP and the Intra-S/G2 Cell Cycle Checkpoints Results in Highly Effective Radiosensitization of HPV-Positive HNSCC Cells

Abstract

In head and neck squamous cell carcinoma (HNSCC), tumors positive for human papillomavirus (HPV) represent a distinct biological entity with favorable prognosis. An enhanced radiation sensitivity of these tumors is evident in the clinic and on the cellular level when comparing HPV-positive and HPV-negative HNSCC cell lines. We could show that the underlying mechanism is a defect in DNA double-strand break repair associated with a profound and sustained G2 arrest. This defect can be exploited by molecular targeting approaches additionally compromising the DNA damage response to further enhance their radiation sensitivity, which may offer new opportunities in the setting of future de-intensified regimes. Against this background, we tested combined targeting of PARP and the DNA damage-induced intra-S/G2 cell cycle checkpoints to achieve effective radiosensitization. Enhancing CDK1/2 activity through the Wee1 inhibitor adavosertib or a combination of Wee1 and Chk1 inhibition resulted in an abrogation of the radiation-induced G2 cell cycle arrest and induction of replication stress as assessed by γH2AX and chromatin-bound RPA levels in S phase cells. Addition of the PARP inhibitor olaparib had little influence on these endpoints, irrespective of checkpoint inhibition. Combined PARP/Wee1 targeting did not result in an enhancement in the absolute number of residual, radiation induced 53BP1 foci as markers of DNA double-strand breaks but it induced a shift in foci numbers from S/G2 to G1 phase cells. Most importantly, while sole checkpoint or PARP inhibition induced moderate radiosensitization, their combination was clearly more effective, while exerting little effect in p53/G1 arrest proficient normal human fibroblasts, thus indicating tumor specificity. We conclude that the combined inhibition of PARP and the intra-S/G2 checkpoint is a highly effective approach for the radiosensitization of HPV-positive HNSCC cells and may represent a viable alternative for the current standard of concomitant cisplatin-based chemotherapy. In vivo studies to further evaluate the translational potential are highly warranted.

Replication Stress: A Review of Novel Targets to Enhance Radiosensitivity-From Bench to Clinic

“…The cell cycle distribution of cancer cells affects radio sensitization, especially for some cancer types that depend more on other DDR pathways rather than homologous repair (HR) (41). In different cell cycles, the differences in chromosome structure lead to unequal radiosensitivity.…”

A Lack of Effectiveness in the ATM-Orchestrated DNA Damage Response Contributes to the DNA Repair Defect of HPV-Positive Head and Neck Cancer Cells

“…Exponentially growing HNSCC cells containing stably integrated copies of the previously described GFP-based NHEJ or HR reporter plasmids pGC or pEJ (25,26) were transfected with an I-SceI expression vector for targeted DSB induction using Fugene HD (Promega). Six hours post-transfection, the medium was exchanged and supplemented with ATM inhibitor or solvent (DMSO), followed by another exchange plus supplementation 24 h posttransfection.…”

“…We further tested the impact of ATM on the main DSB pathways, NHEJ and HR, using established I-SceI-based plasmid reconstruction assays (25,26). As a master regulator of DSB detection and processing, ATM interacts with critical components of both pathways.…”

PD-1 Inhibitor Maintenance Therapy Combined Iodine-125 Seed Implantation Successfully Salvage Recurrent Cervical Cancer after CCRT: A Case Report

“…As previously mentioned, PARP is an intracellular protein involved in the repair of single and double-stranded DNA breaks [ 7 , 8 ], and radiotherapy exerts its cytotoxic mitotic effects on tumor cells through DNA damage [ 58 , 59 ], so the combination with a PARP inhibitor would sensitize the effect to DNA-damaging induced by radiation. This sensitizing effect has been demonstrated in in vitro studies for both fraction radiotherapy and continuous LDR radiotherapy [ 60 , 61 , 62 , 63 ]; meanwhile, some clinical studies have investigated it [ 64 , 65 ].…”