FLASH-RT does not affect chromosome translocations and junction structures beyond that of CONV-RT dose-rates

The molecular and cellular mechanisms driving the enhanced therapeutic ratio of ultra-high dose-rate radiotherapy (FLASH-RT) over slower conventional (CONV-RT) radiotherapy dose-rate remain to be elucidated. However, attenuated DNA damage and transient oxygen depletion are among several proposed models. Here, we tested whether FLASH-RT under physioxic (4% O2) and hypoxic conditions (≤2% O2) reduces genome-wide translocations relative to CONV-RT and whether any differences identified revert under normoxic (21% O2) conditions. We employed high-throughput rejoin and genome-wide translocation sequencing ( HTGTS-JoinT-seq ), using S. aureus and S. pyogenes Cas9 “bait” DNA double strand breaks (DSBs), to measure differences in bait-proximal repair and their genome-wide translocations to “prey” DSBs generated by electron beam CONV-RT (0.08-0.13Gy/s) and FLASH-RT (1×102-5×106 Gy/s), under varying ionizing radiation (IR) doses and oxygen tensions. Normoxic and physioxic irradiation of HEK293T cells increased translocations at the cost of decreasing bait-proximal repair but were indistinguishable between CONV-RT and FLASH-RT. Although no apparent increase in chromosome translocations was observed with hypoxia-induced apoptosis, the combined decrease in oxygen tension with IR dose-rate modulation did not reveal significant differences in the level of translocations nor in their junction structures. Thus, Irrespective of oxygen tension, FLASH-RT produces translocations and junction structures at levels and proportions that are indistinguishable from CONV-RT. ### Competing Interest Statement BWL Jr. has received research support outside this work from Varian Medical Systems, is a co-founder and board member of TibaRay, and is a consultant on a clinical trial steering committee for BeiGene. PGM is a co-founder of TibaRay. The other authors of this manuscript declare no conflict of interest.