Radiotherapy results in tumour cell lethality through direct induction of DNA damage and genome instability. Technological advances in radiotherapy, such as Ablative Intensity Radiotherapy (ART; also called stereotactic ablative radiotherapy), enable treatment in a limited number of high-dose radiation fractions. While ART improves patient outcomes, how it specifically induces cell death remains unclear.
To address this, we employed long-duration live imaging of 3-colour FUCCI (cell cycle reporter) expressing human cancer cells treated with a single ablative dose of radiation (8 – 20 Gy). We found that ART induces two distinct waves of cell death regulated through cell cycle-dependent double-strand break (DSB) repair pathway choice. The first wave of cell death occurred in the S/G2-phase irradiated cells in the immediately following mitosis. Mitotic death was driven through WAPL- and spindle assembly checkpoint-dependent mitotic arrest resulting in cohesion fatigue and BAX/BAK-mediated intrinsic apoptosis. Inhibiting canonical homology-directed repair (HDR) through RAD51 depletion or ATR inhibition reduced mitotic death, as did suppression of the by CHK1 inhibition. The second wave of cell death occurred in the G1-phase irradiated cells following at least one cell division with chromosome segregation errors. This was driven through cell line-specific variations in cGAS/STING and/or MDA5/RIG-I/MAVS nucleic acid sensing pathways, concomitant with paracrine interferon signalling and STAT1-dependent extrinsic apoptosis. Targeting classical non-homologous end joining (NHEJ) through genetic and pharmacological inhibition of DNA-PKcs or LIG4 depletion, microhomology-mediated end joining (MMEJ) through POLθ depletion, or single strand annealing (SSA) through RAD52 depletion all promoted mitotic demise of the G1-phase irradiated cells. Additionally, RAD52 depletion promoted RAD51 foci in 2N cells within ART-treated cultures, consistent with HR engagement in G1 or very early S-phase. Altering the spectrum of cell death outcomes corresponded to reduced interferon signalling when mitotic death was promoted. Conversely, interferon signalling increased when mitotic death was rescued with one notable exception; targeting RAD51 promoted mitotic survival of ART-treated cells, but corresponding enhanced autophagy suppressed the immune response.
Cumulatively, ART lethality results from distinct cell death mechanisms dictated by the cell cycle-dependent DSB repair pathway engagement. NHEJ, MMEJ, and SSA promote mitotic survival of the G1-phase irradiated cells . Conversely, toxic HDR drives mitotic death during the first cell division after ART. Further, we have demonstrated that ART lethality is tuneable through pharmacological manipulation, suggesting the possibility to modulate cancer cell death and potentiate immunotherapy effectiveness for patient benefit.