PROJECT SUMMARY Inhibition of the DNA replication checkpoint regulator ATR is a new and promising cancer treatment. ATR inhibitors (ATRi) function as cancer treatments by causing double-stranded breaks (DSBs) at sites of problematic DNA replication. Indeed, we have recently demonstrated that structure-forming repetitive DNA sequences strongly influence on ATRi-driven breakage. However, our recent preliminary studies indicate that abnormal DNA structure formation is not the sole determinant of vulnerability at these sites. We have now shown that ATRi- driven breakage at inverted retroelement repeats is strongly stimulated by treatments that promote their transcription. Moreover, because the transcription of retroelements is silenced at most genomic locations, their derepression substantially increases breakage caused by ATRi. We hypothesize that cancer-associated alterations and silencing inhibitors that foster the transcription of inverted retroelements will increase sensitivity to ATRi treatment. Importantly, advanced prostate cancer, most notably castration-resistant prostate cancer (CRPC), exhibits many features expected to cause increased transcription of inverted retroelements. These alterations include the hypomethylation of retroelements, the loss of RB1 and p53-mediated repeat silencing, and the abnormal processing of RNA-DNA hybrids due to RNASEH2 deficiency. Herein, we propose to determine how each of these prostate cancer-associated changes affect the localization and number of DNA breaks induced by ATRi. Furthermore, we will explore the molecular mechanism by which inverted retroelement transcription increases ATRi-driven breakage at select sites and determine if further inhibition of retroelement silencing by clinically approved drugs synergizes with ATRi to suppress the growth of tumors in mouse models of CRPC. Finally, we will determine if this combination treatment is more effective in the context of prostate cancer-associated mutation of ATM, BRCA2 and RB1. Collectively, these studies will characterize new mechanisms by which cancer cells are sensitized to ATRi as well as identify novel combination treatments for CRPC.