Oxidative base damage to telomeres sensitizes cancer cells to ATR inhibition

Targeted inhibition of DNA damage response proteins has received significant clinical attention owing to the success of PARP inhibitors. Due to the loss of G1/S checkpoint proteins like p16 and p53, cancer cells are reliant on the G2/M checkpoint to cope with elevated DNA replication stress, making them vulnerable to ATR and Chk1 inhibition. Cancer cells also experience elevated levels of oxidative stress, and targeting this is another therapeutic strategy, especially in photodynamic therapy. Our group has developed a chemoptogenetic tool (FAP-TRF1) which allows for targeted induction of singlet oxygen mediated 8-oxo-guianine (8oxoG) at telomeres. We previously demonstrated a single induction of 8oxoG at telomeres in cancer cells was sufficient to induce replication stress but was well tolerated at the cellular level. This led us to hypothesize that oxidative base damage to telomeres may sensitize cancer cells to low dose ATR inhibition. In this study we found inhibition of ATR, Chk1, or Wee1 after induction of telomeric 8oxoG significantly induced genome instability and reduced viability of cancer cells. This occurred at doses markedly less than those required to increase instability in non-cancer cells, and at levels in the cancer cells which the drug alone had no effect. We found p53 was critically protective of the non-cancerous cells in our model. We determined genome instability after 8oxoG induction and ATR inhibition was enhanced when cells were damaged in S-phase and when Mre11 was inhibited, while CDC7 inhibition restored stability. This phenotype resulted in an increased amount of time required to complete mitosis suggesting cells entered mitosis with unresolved replication intermediates. Indeed, pharmacologic prevention of mitotic entry during ATR inhibition rescued the genome instability caused by telomere oxidation. Together our findings indicate that manipulating the levels of reactive oxygen species in cancer cells may be an effective therapeutic alongside ATR inhibitors, allowing for tumor destruction while mitigating off-target effects to non-diseased tissue.