Project Summary DNA replication problems collectively known as replication stress are a major sources of genomic instability in cancer cells and also a vulnerability of cancer that can be targeted therapeutically. The recent success of PARP inhibitors in the treatment of BRCA mutant tumors provided an exciting example of targeting cancer cells by exploiting replication stress. However, our current understanding of the replication stress in cancer cells is still very limited. Although we know that many different oncogenic events in cancer cells can cause replication problems, we still don't fully understand whether these oncogenic events affect DNA replication in similar or distinct ways. Furthermore, we also know little about how DNA replication is altered by different oncogenic events, and whether altered replication can give rise to distinct cellular vulnerabilities. Understanding the basic molecular features of replication stress, the major causes of replication stress in cancer cells, and the different vulnerabilities resulting from altered replication will greatly enhance our ability to detect and exploit replication stress in cancer therapy. I have a longstanding interest in understanding the replication stress response in human cells. In particular, my lab has extensively studied the functions and regulation of the ATR checkpoint pathway, the master regulator of replication stress response in human cells. Our work has contributed significantly to the current models of stress sensing and signaling during DNA replication. From recent studies by us and others, it has become gradually clear that different oncogenic events in cancer cells can generate distinct problems in DNA replication. Furthermore, RNA transcripts, the products of transcription, have both positive and negative impacts on DNA replication and repair. Our studies also revealed that replication stress not only exerts cell autonomous effects on the genome, but also cell non-autonomous effects in cell populations. Based on these new findings, we propose to systematically define and characterize different types of replication stress in cancer cells, understand how RNA affects replication and repair in the genome, and explore the cell non-autonomous effects of replication stress in tumor microenvironments and cancer therapy. These studies may provide us a much more comprehensive understanding of the molecular underpinnings of replication stress in cancer cells, their impacts on the genome of cancer cells and cell populations in tumor microenvironments, and the cancer cell-specific vulnerabilities that they give rise to. The new concepts and findings from these studies could have transformative impacts on the research of cancer and cancer therapy.