Abstract 3581: How to deal with DNA damage during mitosis: a genetic interaction map of the PICH DNA translocase

Abstract

Abstract Genomic instability is one of the enabling characteristics of cancer, and is thought to be driven in large part by replication stress. It is generally believed that damaged DNA needs to be repaired before entry into mitosis. Recent evidence showed that DNA damage occasionally is present in mitosis, and can be repaired either through break-induced replication during early mitosis or during anaphase at so-called ultrafine DNA bridges (UFBs). These UBFs are characterized by the presence of the PICH DNA translocase, and Rif1, which we recently identified to be involved in the resolution of UFBs (Hengeveld et al, Dev Cell, 2015). However, detailed molecular composition of UFBs associated proteins and their mechanism of action are incompletely understood. Also, whether specific cancer types are dependent on this DNA repair pathway for their survival is not known. We hypothesise that cancer cells with high levels of replication stress may increasingly depend on these mechanisms for their survival. Here, we aimed to identify the genetic make-off of cancer cells that depend for their survival on repair of damage lesion during anaphase. We used inactivation PICH or Rif1 as a model for defective resolution of anaphase bridges. To elucidate the consequences of loss of DNA repair at UFBs, we analysed the sensitivity to genotoxic agents and assessed genomic instability in HAP1 cells, in which the ERCC6L gene, which encodes the PICH DNA translocase, or the Rif1 gene were inactivated using Cas9/CRISPR-mediated mutation. ERCC6L as well as RIF1 knock-out cells showed increased levels of intrinsic DNA damage, as judged by elevated levels of H2AX phosphorylation and increased frequencies of 53BP1 bodies, known to represent unrepaired replication damage. ERCC6L as well as RIF1 knock-out cells also display increased sensitive to the topoisomerase II inhibitor ICRF193. Amplifications and loss of genomic areas was assessed using single strand sequencing of single cells. Subsequently, we wanted to determine which genetic mutations are not allowed or show competitive outgrowth in the absence of UFB resolution during anaphase. To this end, we exploited the haploid nature of the HAP1 cells, which allows insertional mutagenesis using retroviruses carrying a strong spice-acceptor to create random instantaneous knock-outs. The list of candidate genes of which depletion leads to a lethal phenotype specifically in cells lacking PICH is currently being analysed. In conclusion, our studies have shown that the inactivation of PICH or RIF1 results in a marked increase in unresolved UFBs which ultimately lead to elevated numbers of 53BP1 bodies, and increased sensitivity to topoisomerase inhibitors. These data makes both RIF1 and PICH interesting therapeutic targets in tumours with high levels of replication stress, or targets to potentiate the effects of topoisomerases. Citation Format: Inês Teles Alves, Anne-Magriet Heijink, Floris Foijer, Marcel van Vugt. How to deal with DNA damage during mitosis: a genetic interaction map of the PICH DNA translocase. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3581.