Defining the cellular response to complex DNA damage induced by proton beam therapy

Abstract 

Ionising radiation (IR) is the cornerstone for treatment of a number of human cancers, the therapeutic effect of which is mostly attributable to generation of DNA damage, including DNA double strand breaks (DSBs) and complex DNA damage (CDD) where several DNA lesions are induced in close proximity by a single radiation track. CDD formation is particularly relevant for proton beam therapy (PBT), which is increasingly being utilised therapeutically in the UK with the establishment of new NHS facilities in Manchester and London operational in late 2018 and 2020, respectively. This is due to the fact that PBT is not monoenergetic and the energy is deposited via the Bragg peak, leading to high energy protons at the Bragg peak itself, and low energy protons at the distal end which are considered to have contrasting effects on DNA and on cells overall. Despite the established importance of CDD in the therapeutic effect of IR, the precise mechanism of recognition and repair of CDD in cells, particularly within chromatin, is lacking. This is in contrast to a large focus on examining the repair of DSBs. Using currently the only clinically operating PBT facility in the UK at the Clatterbridge Cancer Centre (CCC), we have demonstrated that low energy protons generate higher quantities of CDD than high energy protons and x-rays, which significantly promotes cancer cell death. Using a panel of antibodies targeting site-specific histone post-translational modifications, we have also identified that CDD triggers significant elevations in the levels of histone H2B ubiquitylation on lysine 120 (H2B_ub) mediated by two E3 ubiquitin ligases RNF20/40 and MSL2, several hours post-irradiation where CDD persists. This induction of H2B_ub promotes CDD repair and cell survival post-irradiation. Furthermore utilising an siRNA screen targeting deubiquitylation enzymes (DUBs), we have identified USP6 and USP9X that play a major role in the cellular response to low energy protons, albeit via different cellular mechanisms. Further work is ongoing to thoroughly investigate the DNA repair proteins, pathways and epigenetic mechanisms that are responsive to PBT, particularly at low energies that generate significant levels of CDD.