Characterisation of deubiquitylating enzymes in the cellular response to high-LET ionising radiation and complex DNA damage

Purpose: Ionising radiation, particular high linear energy transfer (LET) radiation, can induce complex DNA damage (CDD) where two or more DNA lesions are induced in close proximity which contributes significantly to the cell killing effects. However knowledge of the enzymes and mechanisms involved in co-ordinating the recognition and processing of CDD in cellular DNA are currently lacking. Methods and Materials: An siRNA screen of deubiquitylation enzymes was conducted in HeLa cells irradiated with high-LET α-particles or protons, versus low-LET protons and x-rays, and cell survival monitored by clonogenic assays. Candidates whose depletion led to decreased cell survival specifically in response to high-LET radiation were validated in both HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A) cells, and the association with CDD repair was confirmed by using an enzyme modified neutral comet assay. Results: Depletion of USP6 decreased cell survival specifically following high-LET α-particles and protons, but not by low-LET protons or x-rays. USP6 depletion caused cell cycle arrest and a deficiency in CDD repair mediated through instability of poly(ADP-ribose) polymerase-1 (PARP-1). This phenotype was mimicked using the PARP inhibitor olaparib. Conclusion: USP6 controls cell survival in response to high-LET radiation by stabilising PARP-1 protein levels which is essential for CDD repair. We also describe synergy between CDD induced by high-LET protons and PARP inhibition in effective cancer cell killing.