Introduction Certain tumor subgroups, including triple negative breast cancers (TNBCs), are characterized by high levels of genomic instability (GI). These tumors are typically very aggressive and difficult to threat. Increasingly, we realize that GI is fueled by replication stress (RS). RS can be induced by expression of oncogenes, including CCNE1, MYC and CDC25A, which perturb initiation, elongation and/or termination phases of DNA replication. RS poses a threat to cellular viability, and tumor cells with high levels of RS will therefore increasingly depend for their survival on DNA damage checkpoint and repair pathways. Possibly, this increased dependence creates therapeutic opportunities. Unfortunately, it is currently difficult to determine levels of RS in tumors, a requirement to efficiently select patients for novel treatments. The aim of this work is to develop an RNA expression-based signature for oncogene-induced replication-stress.
Material & Methods A panel of cell lines (RPE-1, RPE-1-P53-/-, MDA-MB-231, BT-549 and HCC-1806) was engineered to doxycycline-inducibly express a set of oncogenes (CDC25A, CCNE1 or MYC). Single DNA fiber replication analysis in combination with immunoblot analysis of RS markers was used to validate the cell line models. MTT assays were performed to assess sensitivity to ATR and Wee1 inhibitors. RNA-sequencing was used to uncover expression changes upon RS induction.
Results Treatment with doxycycline resulted in high expression levels of CDC25A, CYCLINE or MYC. Expression was heterogeneous but present in the far majority of RPE-1 cells (40% CDC25A, 88% CCNE1 and 93% MYC). DNA fiber analysis was subsequently used to study the alteration of DNA replication dynamics. Severe reduction on ongoing DNA synthesis velocity was shown in engineered cells (RPE-1 and MDA-MB231) under doxycycline treatment, confirming prominent RS. In line with elevated levels of RS, oncogene induction resulted in elevated levels of ATR activity, as judged by phospho-CHK1 and phospho-RPA levels. Importantly, oncogene-expression enhanced sensitivity to Wee1 and ATR checkpoint inhibitors. Subsequently, RNA samples were harvested under the presence and absence of doxycycline. In parallel to oncogene-expression, hydroxyurea, a chemical agent known to increase stalled replication forks was applied as a DNA damage control. RNA-sequencing analysis will be performed to identify differentially expressed genes upon oncogene expression. Commonly identified genes over multiple cell lines and shared between the different oncogenes will be used to build a ‘RS’ profile.
Conclusion Our results indicate that oncogene expression provokes RS in multiple cell line models and induces impaired replication fork stability and sensitivity to cell cycle checkpoint inhibitors. These models are therefore valid to develop an RNA expression-based qualifier in order to facilitate patient selection towards novel therapeutics.
Citation Format: Sergi Guerrero, Rudolf Fehrmann, Marcel ATM van Vugt. Towards an RNA expression-based signature for oncogene-induced replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1406. doi:10.1158/1538-7445.AM2017-1406