The Effect of Oxygen Levels on the Biological Response of DNA Damage Inhibitors
Overcoming the Hypoxic Barrier to Enhance DNA Damage
Approximately 50% of ovarian cancers and 10-20% of breast, metastatic prostate, or pancreatic cancers harbour mutations in homologous recombination (HR) factors, making these tumours candidates for PARP inhibitor (PARPi) therapy. However, despite the enthusiasm for the addition of PARP inhibitors to the clinical landscape of HR deficient tumours, not all patients benefit and many of whom initially respond to therapy develop resistance. Deciphering the mechanisms of resistance to PARPi is therefore essential in improving their clinical efficacy. While past studies have identified cancer cell intrinsic mechanisms driving PARPi resistance, this program will focus on the mechanisms by which the hypoxic tumour microenvironment promotes PARPi resistance in both HR deficient and proficient tumour cells (1), how it affects resistance to other DNA damage inhibitors, and the identification of small molecules that can reverse this resistance and promote PARPi efficacy. In addition, we will explore the role of PARP in promoting HIF activity. This is a new aspect of HIF biology at the level of chromatin regulation that can affect the adaptation of tumour cells to a hypoxic microenvironment.
Background AND Past Research:
Previous work has demonstrated that the HR pathway is compromised in hypoxia, resulting in contextual synthetic lethality with PARP inhibition (2). More recently, we determined that the hypoxia-dependent repression of HR is oxygen dependent and is suppressed only under severe hypoxia (<0.5% oxygen) in HR proficient cells. However, our work identifies a different effect under moderate hypoxia (1-2% oxygen), where both HR proficient and HR deficient cancer cells are resistant to PARPi therapy, suggesting a model where low levels of reactive oxygen species (ROS) under hypoxia results in reduced DNA damage induced by PARPi (1).
Significantly, we found that HR deficient tumours were not only resistant to PARPi in hypoxia (1-2% oxygen) but that hypoxia also promotes resistance to inhibitors of alt-NHEJ repair in HR deficient tumours and combining them with PARPi had no additional benefit. In contrast, PARPi in combination with inhibitors of alt-NHEJ results in increased cytotoxic DSBs and cell death under normoxic conditions (1). Therefore, our data suggest that PARPi in combination with POLQi or other inhibitors of the alt-NHEJ pathway are only beneficial under normoxic conditions.
Since PARPi toxicity is dependent on accumulated DNA damage in HR deficient tumours, one could hypothesise that inhibition of other DNA repair pathways would result in enhanced cell killing when combined with PARPi. However, this does not appear to be the case as previous studies have shown that inhibition of the C-NHEJ pathway in fact reduced lethality after exposure to PARP inhibitors and chemotherapeutic agents such as platinum crosslinking agents. We also found that combining PARPi with inhibitors of the C-NHEJ pathway, such as DNA-PKi, reduced PARPi induced lethality under both under normoxic and hypoxic conditions. Therefore, PARPi toxicity seems rather unique to HR defective tumour cells, and suppression the C-NHEJ pathway, such as through inhibition of DNA-PK does not increase toxicity to hypoxic cells or to normoxic cells, supporting the concept that it is not the DNA DSB itself that is important, but the consequence of the DSB on replication.
Since hypoxic cells are not inherently resistant to DNA damage, we hypothesised that agents capable of inducing single strand DNA damage in these cells would improve their sensitivity to PARPi. We show that combining PARPi with the hypoxia activated prodrug TPZ confers antitumour activity in HR deficient xenografts (1). TPZ undergoes an initial one-electron reduction to generate a TPZ radical. In the absence of oxygen, the radical undergoes spontaneous conversion to generate the toxic benzotriazinyl radical, causing increased DNA damage. In the presence of oxygen, however, the unstable radical anion is back oxidized to the parental TPZ resulting in the production of superoxide, which although is not known to be highly reactive itself but can be converted to the more toxic hydrogen peroxide and hydroxyl radicals. Our data indicates that the lesions caused by these toxic molecules are not efficiently repaired in PARP inhibited HR deficient cells and that this damage accounts for the relatively high doses of TPZ needed to synergise with PARPi in normoxia. Therefore, TPZ can be considered a double edge sword in elevating the concentration of ROS in normoxia and directly damaging DNA in hypoxic cells. This increased damage is further enhanced through inhibition of efficient repair by PARPi.
Despite early promise in Phase II clinical trials, TPZ did not show a statistically demonstrable therapeutic benefit compared to standard chemoradiotherapy or chemotherapy alone in a pivotal Phase III trial. The failure has been attributed to poor stratification of patients based on their tumour hypoxia status. Therefore, proper stratification of patients will be necessary to ensure exploitation of the full potential of TPZ and PARPi combination, to follow on from the success story of Nimorazole, a hypoxia targeting agent, that is currently the standard treatment for patients in Denmark receiving radiation therapy for head and neck cancer.
Our past work demonstrates that hypoxic HR deficient cancer cells are resistant to PARPi, but that synthetic lethality can be restored through the addition of a ROS-generating hypoxia activated cytotoxin. Importantly, the hypoxic cytotoxin, TPZ, significantly improves PARPi efficacy without enhancing PARPi normal tissue toxicity. Therefore, we propose the evaluation of TPZ and other hypoxic cytotoxins in combination with PARPi-based therapy in future clinical trials for hypoxic tumours that are not responsive to PARPi therapy alone. Prof. Ester Hammond at Oxford and an affiliate member of the MRC Unit also has significant experience with hypoxia activated prodrugs/cytotoxins which will prove beneficial to this programme.
In addition to the strong translational aspect of this program, there is also an important functional role for PARP in regulating the activity of HIF. Collaborative studies carried out in Prof. Ester Hammond’s lab have shown that inhibition of PARP either with small molecules or genetically with small hairpin RNAs reduces HIF-1 alpha stabilisation, recruitment to HIF-1 target genes as well as cellular adaptation to hypoxic conditions (3). Interestingly, PARP activation under hypoxia was proposed to be through non-mitochondrial derived free radicals. The role of hypoxia-induced ROS and their role in the response of cells to low oxygen conditions has not clearly delineated how ROS impacts HIF-1 stability regulated by prolyl hydroxylases and the VHL oxygen signalling machinery. We propose the hypothesis that in fact both processes are needed, the PHD/VHL machinery acting as a direct sensor of oxygen levels and ROS/PARP acting to regulate HIF-1 at the level of chromatin. We will explore the role of ROS and PARP in HIF-1 regulation and the cellular response to hypoxia.
1. Eliminating Hypoxic Tumor Cells Improves Response to PARP inhibitors in Homologous Recombination Deficient Cancer Models. Mehibel, M. et al., In press Journal of Clinical Investigation 2021.
2. Contextual Synthetic Lethality of Cancer Cell Kill Based on the Tumor Microenvironment. Chan, N. et al, Cancer Res. 2010; 70:8045-54
3. Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions. Martí J.M. et al, Redox Biol. 2021; 41:101885.