Objective
Ablative Dose Radiotherapy (ART) is increasingly used worldwide, both as SABR/SBRT, and as SRS, but, molecular mechanisms of cell death after ART remain almost unknown.
Methods
A range of cell lines including immortalised normal and cancer cell lines were subjected to single fractions of radiation ranging from 2-20Gy in vitro. Outcomes were measured using long-duration live cell imaging, clonogenic assays, and standard biochemical assays. The Fluorescence Ubiquitin Cell Cycle Indicator system was used to monitor phase of the cell cycle. Key pathways in the DNA damage response (DDR) were inhibited genetically or pharmacologically.
Results
The first cell death wave occurred 24-48 hours after ART in cells irradiated in S- or G2-phase with a threshold ~8Gy, coinciding with prolonged mitotic duration. This wave was dependent on intrinsic apoptosis. Homologous recombination (HR) promoted the first wave of cell death, and restoration of BRCA2 (a key component of HR) restored mitotic cell death after ART in a patient-derived cell line which had spontaneously lost and subsequently re-gained BRCA2. The second wave, delayed lethality, occurred days after ART, in the G1-phase irradiated cells, following at least one cell division with chromosome segregation errors due to engagement of error-prone repair pathways. These activated the innate immune response via DNA and RNA sensing pathways and promoted extrinsic apoptosis. ART induced senescence in cells with intact TP53. Altering the spectrum of cell death outcomes through inhibition of specific DNA repair pathways corresponded to reduced interferon signalling when mitotic death was promoted, with the potential to decrease synergy with immunotherapy.
Conclusion
ART of p53-compromised cancer cells induced two distinct waves of cell death regulated through cell cycle-dependent DSB repair, with only the second wave expected to synergise with immunotherapy.