Biphasic DNA damage and non-canonical replication stress response govern radiation-induced senescence in glioblastoma

Therapy-induced senescence (TIS) in glioblastoma (GBM) residual disease and escape from TIS account for resistance and recurrence, but the mechanism of TIS manifestation remains obscure. Here, we demonstrate that replication stress (RS) is critical for the induction of TIS in residual cells by employing an in vitro GBM therapy-resistance cellular model. Interestingly, we found a 'biphasic' mode of DNA damage after radiation treatment and reveal that the second phase of DNA damage arises majorly in the S phase of residual cells due to RS. Mechanistically, we show that persistent phosphorylated ATR is a safeguard for radiation resilience, whereas the other canonical RS molecules remain unaltered during the second phase of DNA damage. Importantly, RS preceded the induction of senescence, and ATR inhibition resulted in TIS reduction, leading to apoptosis. Moreover, ATR inhibition sensitized PARP-1 inhibitor-induced enhanced TIS-mediated resistance, leading to cell death. Our study demonstrates the crucial role of RS in TIS induction and maintenance in GBM residual cells, and targeting ATR alone or in combination with a PARP-1 inhibitor will be an effective strategy to eliminate TIS for better treatment outcomes.