Non-canonical cytoplasmic ATR activity controls autophagy and chemoresistance in hypoxic cancer cells

Abstract Tumour hypoxia is a major barrier to effective cancer therapy, as it promotes survival and adaptive mechanisms that lead to chemoresistance. Although the DNA damage response (DDR) kinase ATR has been shown to promote cancer cell survival under hypoxic stress, its role in hypoxia-induced autophagy, a major adaptive survival mechanism of hypoxic cancer cells, remains unclear. Here, we uncover a non-canonical, DDR-independent role for ATR in regulating adaptive autophagy in hypoxic cancer cells. We show that hypoxia selectively induces cytoplasmic ATR kinase activity, which is key for phosphorylation of the selective autophagy adaptor p62 at Ser403, supports the polyubiquitination of proteins required for autophagy and activates NRF2 signaling in hypoxic breast and ovarian cancers. Pharmacological inhibition or genetic depletion of ATR was observed to block p62 phosphorylation, disrupt autophagic flux, induce cell death and selectively sensitize hypoxic cancer cells to cisplatin. Notably, ATR inhibition overcomes cisplatin resistance in hypoxic ovarian and breast cancer cells, highlighting its therapeutic potential. Mechanistically, we demonstrate that cytoplasmic ATR regulates p62 and NRF2 localisation and chromatin association in response to hypoxia, thereby linking non-canonical ATR signaling to stress adaptation independently of the canonical DDR. These findings reveal a previously unrecognized ATR-p62-NFR2 axis that supports survival and chemoresistance in hypoxic tumours and position ATR as a promising target to overcome hypoxia-driven therapy resistance in cancer treatment.