Oncogenic Ras and p53 loss-of-function mutations are common in many advanced sporadic malignancies and together predict a limited responsiveness to conventional chemotherapy. Notably, studies in cultured cells have indicated that each of these genetic alterations creates a selective sensitivity to ataxia telangiectasia and Rad3-related (ATR) pathway inhibition. Here, we describe a genetic system to conditionally reduce ATR expression to 10% of normal levels in adult mice to compare the impact of this suppression on normal tissues and cancers in vivo. Hypomorphic suppression of ATR minimally affected normal bone marrow and intestinal homeostasis, indicating that this level of ATR expression was sufficient for highly proliferative adult tissues. In contrast, hypomorphic ATR reduction potently inhibited the growth of both p53-deficient fibrosarcomas expressing H-ras G12V and acute myeloid leukemias (AMLs) driven by MLL-ENL and N-ras G12D . Notably, DNA damage increased in a greater-than-additive fashion upon combining ATR suppression with oncogenic stress (H-ras G12V , K-ras G12D , or c-Myc overexpression), indicating that this cooperative genome-destabilizing interaction may contribute to tumor selectivity in vivo. This toxic interaction between ATR suppression and oncogenic stress occurred without regard to p53 status. These studies define a level of ATR pathway inhibition in which the growth of malignancies harboring oncogenic mutations can be suppressed with minimal impact on normal tissue homeostasis, highlighting ATR inhibition as a promising therapeutic strategy.

Summary DNA polymerase stalling activates the ATR checkpoint kinase, which in turn suppresses fork collapse and breakage. Herein, we describe use of ATR inhibition (ATRi) as a means to identify genomic sites of problematic DNA replication in murine and human cells. Over 500 high-resolution ATR-dependent sites were ascertained using two distinct methods (RPA-ChIP and BrITL). The genomic feature most strongly associated with ATR dependence was repetitive DNA that exhibited high structure-forming potential. Repeats most reliant on ATR for stability included structure-forming microsatellites, inverted retroelement repeats, and quasi-palindromic AT-rich repeats. Notably, these categories of repeats differed both in structure formation and in their ability to stimulate RPA accumulation and breakage, implying that the causes and character of replication fork collapse under ATR inhibition can vary in a DNA structure-specific manner. Collectively, these studies identify key sources of endogenous replication stress that rely on ATR for stability.

Previous studies have defined sequences that are difficult to replicate and, consequently, are more vulnerable to replication-associated breaks and recombination events. However, many of these sequences have been identified through indirect and biased approaches. To identify genomic sequences that contribute to replication-associated breakpoints, we have performed genome-wide screens to determine the location, sequence, and frequency of replication perturbations within the mammalian genome. To convert these transient pause sites in S phase into longer-lived intermediates, asynchronous ATR-inhibited cells were treated with a low dose of polymerase inhibitor (0.2 μM aphidicolin) to promote selective replication fork collapse at difficult-to-replicate sequences. Affected regions were then isolated by chromatin immunoprecipitation of RPA (RPA-ChIP) and by a recently developed novel DNA breakpoint isolation method. Following deep sequencing, these sites were mapped within 1-5 kb regions and sequences at the center of these peaks were binned to quantify the overall frequency of replication fork collapse at such sites. Surprisingly, sequences that have long been thought to be among the most difficult to replicate, such as CGG repeats, were overshadowed by distinct repetitive sequences that are relatively uncharacterized. These sites were greater than 50-fold enriched over background, and were more common as replication-troubled regions than other well characterized sequences (e.g. triplet repeats, fragile sites, etc). In total, over 100 independently confirmed sites were identified, with most sequences observed in multiple locations across the genome. Notably, common fragile sites, which have been speculated to harbor such replication slow zones, were not observed to be particularly enriched in RPA-ChIP samples, consistent with recent observations that these sites are fragile for reasons other than impeded DNA replication rates. These findings and others indicating a larger role of chromatin structure in common fragile site breakage will be discussed. Citation Format: Yu-Chen Tsai, Nishita Shastri, Kevin D. Smith, Jonathan Schug, Rafael Casellas, Eric J. Brown. Genome-wide identification of replication-associated breakpoints. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1777. doi:10.1158/1538-7445.AM2013-1777 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.