Poly ADP Ribose Polymerase Inhibitors Potentiate Proton Therapy End-of-Range Effects by Accelerating Replication Forks and Promoting Transcription Conflict.

Poly ADP ribose polymerase inhibitors (PARPi) are being combined with photon and proton radiation therapy in clinical trials. We sought to investigate mechanisms of PARPi radiosensitization at varying linear energy transfer (LET) levels after observing an extreme normal tissue response in an 18-year-old with high-grade glioma, without a germline alteration predictive of heightened radiosensitivity, treated with veliparib and proton therapy. BRCA1/2 wild-type noncancerous and cancerous cells were treated with PARPi plus photons or protons at the entrance (dose-averaged LET 2.2 keV/µm) or the Bragg peak (BP, dose-averaged LET 7.0 keV/µm) of the proton beam profile. DNA fiber, immunofluorescence, and other DNA damage signaling assays were used to evaluate replication fork progression, gap formation, transcription-replication conflicts, and DNA damage signaling and repair. PARPi modestly sensitized cells to photons and low LET protons; however, PARPi-treated cells were hypersensitive to high LET protons administered at the BP. Unexpectedly, cells treated with PARPi plus BP protons displayed accelerated replication fork progression, enhanced single-stranded DNA gap formation, and greater transcription-replication conflict-induced DNA double-strand breaks. Despite evidence of more single-stranded DNA and cells arrested in G2/M following PARPi plus proton BP, PARPi decreased RAD51 recruitment to break sites and enhanced cytotoxic error-prone repair by nonhomologous end joining. PARPi renders cells hypersensitive to end-of-range high LET proton irradiation. The potential enhanced proton radiosensitization should be considered during clinical trial design with efforts to limit high physical dose and high LET overlap within normal tissues. Planning techniques that increase the LET within tumors warrant further investigation in combination with PARPi as a novel strategy to overcome therapeutic resistance.