Ablating putative Ku70 phosphorylation sites results in defective DNA damage repair and spontaneous induction of hepatocellular carcinoma

Multiple pathways mediate the repair of DNA double-strand breaks (DSBs), with numerous mechanisms responsible for driving choice between the pathways. Previously, we reported that mutating five putative phosphorylation sites on the non-homologous end joining (NHEJ) factor, Ku70, results in sustained retention of human Ku70/80 at DSB ends and attenuation of DSB repair via homologous recombination (HR). In this study, we generated a knock-in mouse, in which the three conserved putative phosphorylation sites of Ku70 were mutated to alanine to ablate potential phosphorylation (Ku70(3A/3A)), in order to examine if disrupting DSB repair pathway choice by modulating Ku70/80 dynamics at DSB ends results in enhanced genomic instability and tumorigenesis. The Ku70(3A/3A) mice developed spontaneous and have accelerated chemical-induced hepatocellular carcinoma (HCC) compared to wild-type (Ku70(+/+)) littermates. The HCC tumors from the Ku70(3A/3A) mice have increased γH2AX and 8-oxo-G staining, suggesting decreased DNA repair. Spontaneous transformed cell lines from Ku70(3A/3A) mice are more radiosensitive, have a significant decrease in DNA end resection, and are more sensitive to the DNA cross-linking agent mitomycin C compared to cells from Ku70(+/+) littermates. Collectively, these findings demonstrate that mutating the putative Ku70 phosphorylation sites results in defective DNA damage repair and disruption of this process drives genomic instability and accelerated development of HCC.