Summary ATR is a master regulator of the DNA damage response in human cells. Inhibition of ATR sensitizes cancer cells to radiation and DNA-damaging drugs, providing an attractive strategy to improve radiotherapy and chemotherapy. Furthermore, in cancer cells with significant intrinsic genomic instability, inhibition of ATR results in massive DNA damage, thereby killing cancer cells by exploiting the vulnerability of their own genomes. Identification of the intrinsic liabilities of cancer cells that render them reliant on ATR for survival will greatly facilitate the use of ATR inhibitors in targeted therapy and radiotherapy. Several ATR inhibitors are already on clinical trials and showing promising efficacy. However, despite the success of initial trials, it is still largely unknown whether and how ATR inhibitors can be broadly used to target different types of genomic vulnerabilities of cancer cells. Recent studies suggested that R loops, a group of transcription intermediates containing DNA:RNA hybrids and displaced single-stranded DNA, are a source of genomic instability in cancer cells. In the preliminary studies leading to this application, we find that ATR is activated by R loops and it plays a key role in suppressing R loop-associated DNA damage. Furthermore, we show that cancer-associated RNA splicing factor mutations promote R loop formation and render cells sensitive to ATR inhibition. These exciting findings lead us to hypothesize that aberrant R loop accumulation is a new targetable liability of cancer cells. Furthermore, ATR is a key sensor of R loops and a critical suppressor of R loop-associated DNA damage, making ATR inhibition an attractive way to target the R-loop liability of cancer cells. To these hypotheses, in Aim 1, we will elucidate the mechanisms by which ATR is activated by R loops through a previously unknown pathway. In Aim 2, we will determine how ATR protects the genome against R loops through three novel mechanisms. In Aim 3, we will test whether ATR inhibitors can selectively eliminate cancer cells harboring high levels of R loops in vitro and in vivo, and identify markers to predict the R-loop liability of tumors. In addition, we will investigate whether radiation can be used to induce R loops in cancer cells and sensitize R loop-high tumors to ATR inhibitors. Together, these studies will mechanistically explain how R loops are sensed by ATR in cells, reveal how ATR enables cells to cope with R loop-associated genomic instability, and address whether the R-loop liability of cancer cells can be effectively exploited by ATR inhibitors in therapy. These studies will not only significantly advance our understanding of the fundamental biology of R loops and ATR signaling, but also transform the use of ATR inhibitors in targeted therapy and radiotherapy.