Impact of DNA repeat silencing on ATR inhibitor-driven genomic breaks
Rahul Shubhra Mandal1*, Nishita Shastri2*, Muzaffer Kassab1*, Chandan Das1, Piyush Borole1, Jonathan Schug3, Irfan A. Asangani1 and Eric J. Brown1 1Abramson Family Cancer Research Institute and Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104 2BreakSight, Inc., 7540 Windsor Drive, Suite 210, Allentown, PA, 18106 3Functional Genomics Core, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104
Poster # 5
Stalling of DNA replication activates the ATR checkpoint kinase, which in turn suppresses replication fork collapse and breakage. Previously, we reported the identification of over 500 ATR inhibitor (ATRi)-driven breakage sites in the mouse and human genomes. The vast majority of these breakage sites overlap significantly with repetitive DNA sequences predicted to form stable non-B form structures (Shastri, Tsai et al., Mol Cell, 2018). These repeats include inverted retroelement repeats (SINE, LINE, LTR) and microsatellite and minisatellite repeats. Herein, we report that chromatin derepression by SUV39H1 inhibition (SUV39H1i) potently increases ATRi-driven breakage at inverted retroelement repeats (IRERs). Indeed, the combination of ATRi with SUV39H1i increases genomic breakage over 80-fold in comparison to that induced by ATRi or SUV39H1i alone and 9-fold greater than that observed with ATRi combined with low-dose DNA polymerase inhibitor treatment (aphidicolin). This increase in breakage is more strongly associated with IRERs than microsatellite and minisatellite repeats. To determine if increased transcription of retroelements is required for breakage, we tested the effect of short-term inhibition of RNA POL III, which regulates SINE transcription. Notably, RNA POL III inhibition causes a near complete inhibition of ATRi-SUV39H1i-driven breakage at inverted SINE elements. Because increased DNA breaks is thought to be the mechanism by which ATRi suppresses cancer cell proliferation, we examined the impact of ATRi and SUV39H1i treatment on the viability of human prostate and breast cancer cell lines. The combination of ATRi and SUV39H1i demonstrated significant synergy as determined by colony formation and other viability assays. Collectively, these studies indicate that transcription of structure-forming repetitive elements contributes to genomic breakage and cancer cell killing caused by ATRi treatment.