SNS-595 causes selective double strand breaks during S-phase which are solely repaired through non-homologous end joining

2074

SNS-595, currently in phase II clinical trials, is a novel cell cycle modulator with potent activity against various tumor models. SNS-595 is an S-phase specific compound that induces p53 and p73 pathway activation, rapidly followed by caspase-3 cleavage and apoptosis. Recent work has sought to define and characterize the DNA damage effects occurring due to SNS-595 treatment. DNA damage triggers activation of one or more DNA-damage sensing kinases ATM, ATR, and DNAPK. These sensors then phosphorylate histone H2AX, forming large protein-DNA complexes called nuclear foci that activate one or more DNA repair pathways. The importance of these pathways in repairing a given lesion can be analyzed by selectively inhibiting the sensing kinases with small molecules or by genetic knockouts. Caffeine treatment inhibits the activities of ATM and ATR, leading to defects in homologous recombination, nucleotide excision repair, and mismatch repair. Repair by nonhomologous end-joining (NHEJ) is selectively inhibited by chemical inhibitors of DNAPK or by knocking out the kinase genetically. This work revealed that, during S phase, SNS-595 causes rapid phosphorylation and subsequent foci formation of H2AX and ATM, indicative of double strand breaks. Caffeine treated cells display no change in cytotoxicity caused by SNS-595, and no defects in resolving damage foci after removal of SNS-595. All other DNA damage agents tested rely on one or more of the ATM/ATR mediated repair systems for damage repair, as seen by sensitization after caffeine treatment. In contrast, cells are sensitized by greater than 10-fold to SNS-595 treatment when NHEJ is disrupted by inhibition or knock-out of DNAPK. Furthermore, these cells are unable to resolve DNA damage foci after removal of SNS-595; Etoposide mediated DNA damage, while sensitized by loss of DNAPK, shows no such defect in DNA repair. Thus, unlike other known double strand break damage agents, SNS-595 mediated damage is solely repaired through the DNAPK NHEJ pathway. These data suggest that SNS-595 is a novel DNA double strand break agent, causing selective double strand breaks during DNA synthesis, with unprecedented selectivity for double-strand break repair through NHEJ. The dependence on DNAPK suggests the use of this DNA damage sensor as a biomarker for patient selection.