Radiotherapy is one of the key treatment modalities for cancer, responsible for ∼ 50% of cancer cures. DNA damage response (DDR), a network of DNA repair and cell cycle control mechanisms, has been shown in the past decades to have significant influence on cell fate in response to radiation, and has recently been reviewed by Groelly et al. [1]. A large proportion of cancers contain DDR defects relative to their derivative normal tissues [2]. Cancer cells with compromised DDR may show reliance on specific DNA repair pathways causing additional interventions to be lethal. This concept, termed synthetic lethality, represents potential clinical benefit in radiotherapy such that DDR inhibitors can be applied as radiosensitizers to specifically target the mutated tumour cells while sparing normal tissues also inside the radiation field, creating a maximized therapeutic index.

DNA double strand breaks (DSBs) are the most cytotoxic form of DNA damage induced by ionizing radiation. Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two major DSB repair pathways. HR only takes places during S/G2 phases of the cell cycle, while NHEJ has been shown to be the predominant DSB repair pathway in mammalian cells, with a particularly critical role for cells in non-proliferating stages of the cell cycle [3]. The enzyme DNA-dependent protein kinase (DNA-PK) is a core component of NHEJ, and early evidence showed that DNA-PK knock-out mice (SCID) are hypersensitive to ionizing radiation, indicating a critical role of DNA-PK in radiation-induced DNA damage reparation [4]. Despite abundant preclinical evidence demonstrating efficient radiosensitization by DNA-PK, there is limited success for DNA-PK inhibitors in combination with radiotherapy in clinical trials, and only one of the current DNA-PK inhibitors, Merck’s peposertib (M3814), has progressed to clinical trials in combination with radiotherapy alone (NCT02316197: peposertib + radiotherapy in advanced solid tumours or chronic lymphocytic leukemia). Very recently in 2023, two phase I trials combining peposertib with chemoradiotherapy (NCT03770689: peposertib + capecitabine-based chemoradiotherapy in rectal cancer; NCT02516813: peposertib + radiotherapy in advanced solid tumours) were terminated early due to dose-limiting toxicities [5], [6]. AZD7648 is another highly potent and selective DNA-PK inhibitor that has demonstrated very effective sensitizing effects to radiotherapy both in vitro and in vivo in various types of cancers [7], [8], [9], [10], [11], though clinical trials at this point have been limited to combinations with chemotherapy (NCT03907969: AZD7648 + pegylated liposomal doxorubicin in patients with advanced cancers).

Poly-ADP-ribose polymerase (PARP) inhibition has attracted substantial clinical investigations since discovery of the synthetic lethal relationship between PARP inhibition and HR deficiency (BRCA1 or BRCA2 mutation) [12], [13]. Four PARP inhibitors, Olaparib, Rucaparib, Niraparib, and Talazoparib, have been approved by the U.S. Food and Drug Administration (FDA) and by the European Medicines Agency (EMA) for treatment of ovarian and breast cancer patients with germline or somatic mutations in BRCA1 or BRCA2 (BRCA1/2) genes [14]. PARP inhibitors (PARPi) block the base excision repair (BER) pathway, impeding repair of DNA single-strand breaks (SSB) then leading to the formation of much more lethal DSBs during subsequent DNA replication. Cells competent for homologous recombination (HR) pathways can repair PARPi-induced DSBs during replication, while those cells with HR defects are much more likely to die with PARPi treatment. Recent investigations have indicated PARP trapping as a major cytotoxicity source for some specific PARPi such as Niraparib and Olaparib. These agents can bind PARP enzymes to DNA at sites of damage, forming PARP-DNA complexes, which effectively 'traps' the PARP enzyme on the DNA, preventing it from participating in the repair process and leading to DNA DSB formation when colliding with replication forks during cell replication [15], [16]. In addition to its synthetical lethal effects in genetically vulnerable cancers, accumulating evidence suggests PARP inhibitors may also be useful as radiosensitizers regardless of tumour HR status potentially due to their natural targeting effect on proliferating cells [17], [18], [19], [20], [21]. Recent studies in glioblastoma have also indicated a new role of PARP outside DDR, mediating the secretion of inflammatory mediators, and thus PARP inhibitors may have potential protective effect in radiation-induced neuroinflammation [22]. Multiple clinical trials have been launched to evaluate the potential benefit of combining PARP inhibitors with radiotherapy in various cancers (NCT03212742: Olaparib and Temozolomide + radiotherapy in high grade gliomas patients; Durvalumab and Olaparib + radiotherapy in pancreatic cancer; NCT03598257: Olaparib + radiotherapy in inflammatory breast cancer).

The radiation sensitization mechanisms of PARP and DNA-PK inhibition strategies are distinct for the two drug classes. DNA-PK inhibitors directly inhibit the repair of DNA DSBs generated by radiation, leading to cell death. PARP inhibitors are more indirect, amplifying the extent of radiation-induced DNA damage by converting SSBs into the more lethal DSBs during cell replication. Despite extensive explorations of synthetic lethality between PARP inhibitors and HR-defective tumours, few investigations have evaluated the radiosensitizing effects of DNA-PK inhibitors in HR defective tumours.

In this study we evaluated in vitro and in vivo radiosensitizing effects of DNA-PK inhibitor AZD7648 and PARP inhibitor Olaparib in HR-competent and defective tumour cells, further examining the mechanistic activity of these effects by comparing response in quiescent and proliferating cells. We report data showing a notable advantage of AZD7648 over Olaparib as a potent radiosensitizer in both HR-competent and defective cells lines and tumour xenografts. However, non-proliferating tumour cells are also targeted by the DNA-PK inhibitor, and normal skin toxicity was observed to increase proportionally to the increased tumour control effects in mice treated with AZD7648 + radiation.