Poster Presentation 44th Lorne Genome Conference 2023

Genome-wide unresolved double-strand breaks underpin mitotic lethality following ablative radiotherapy (#205)

Sienna Casolin 1 2 , Lucy French 1 2 , Radoslaw Szmyd 1 2 , Anna Gonzalez Manjon 1 2 , Christopher Nelson 1 , Eric Hau 2 3 4 , Hilda A Pickett 1 , Harriet E Gee 1 2 3 , Tony J Cesare 1
  1. Children's Medical Research Institute (CMRI), University of Sydney, Westmead, NSW, Australia
  2. Sydney West Radiation Oncology Network (SWRON), Sydney, NSW, Australia
  3. Sydney Medical School, C24 - Westmead Hospital, University of Sydney, Sydney, NSW, Australia
  4. The Westmead Institute for Medical Research (WIMR), Sydney, NSW, Australia

Ablative Intensity Radiotherapy (ART) utilises high-dose radiation to cure tumours in limited treatments. While ART has increased clinical efficacy over conventional radiotherapy, improved understanding of ART-induced lethality may allow pharmacological intervention to further enhance cell death and immunogenicity for patient benefit. We have previously characterised a double-strand break (DSB) repair-dependent response to ART in which homologous recombination (HR) drives mitotic cell death in TP53-inactivated cancer cells. However, the mechanisms governing HR-related toxicity remain unclear. We set out to determine whether aberrant HR-mediated repair at repetitive centromeric sequences underpins ART lethality.

Single radiation treatments (2-14 Gy) were delivered in vitro to HeLa cells and outcomes were measured using biochemical assays including immunofluorescent fixed cell microscopy and metaphase spreads. 

Radiation-induced DSBs were dose-dependent and randomly distributed throughout the genome. Non-ablative doses (2-4 Gy) induced γH2AX and RAD51 foci which were resolved by 24h post-radiation. However, in 14 Gy-irradiated cells, γH2AX foci persisted 48h after ART and elevated γH2AX foci levels were evident in metaphase spreads, indicating that unrepaired breaks were transferred into the mitosis immediately following ART. Additionally, RAD51 foci formation and maturation had delayed kinetics following ablative radiation. RAD51 foci persisted 48h after 14 Gy, indicating possible de novo HR engagement in the 2nd cell cycle following ART. Though centromeric DSBs increased in a dose-dependent manner, the proportion of active HR localised to centromeric regions was independent of dosage and time after ART. Further, in the mitosis immediately following irradiation, γH2AX was not concentrated at centromeric regions; it was instead randomly distributed along the entire length of chromosomes.

Here we show that ART induces extensive DSBs which likely overwhelm DNA repair pathways, causing unrepaired breaks to persist into mitosis. Furthermore, we have demonstrated that HR-mediated repair of centromeric breaks is unlikely to be the cause of mitotic death following ART.