ABSTRACT One of the grand challenges in cancer research is the vast heterogeneity in responsiveness to treatment for different cancer types. We have made great strides in treating some cancers, while the prognosis for others remains dismal. Large scale whole genome sequence (WGS) analyses have identified “breakpoint signatures” of different cancer types that presumably reflect heterogeneity in their underlying disrupted pathways but, drug- gable targets have emerged only in cases where the breaks create oncogenic fusion proteins. The ability to predict disrupted pathways using WGS would be highly impactful for cancer diagnosis and treatment. Chromo- some fragile sites (FSs) manifest as gaps and breaks in metaphase chromosomes when cultured cells experi- ence replication stress. However, many FSs are not cancer-type specific and many breakpoint hotspots in cancer are not known FSs, which has severely limited the impact of the FS field. Since cultured cells offer the potential to prospectively dissect mechanisms initiating chromosome breaks and to track their expansion into complex structural variation, there is a critical need to make these in vitro systems more cancer relevant. Our longterm goal is to establish a sustained research strategy that can predict which known pathways are disrupted in a given cancer type from their breakpoint patterns. The overall objective of this proposal is to reveal mechanisms by which perturbation of cancer-relevant cellular pathways produce pathway-specific patterns of FSs in cell culture and determine whether those patterns can be identified in cancers. Our central hypothesis is that the power of in vitro systems to dissect mechanisms leading to cancer breakpoint signatures will become evident only when shifted away from chemically-induced FSs towards FSs induced by perturbing known cancer-relevant pathways. We have shown that overexpression of different oncogenes leads to oncogene-specific spectra of FSs and pre- liminary data suggest some of the mechanisms by which this occurs. Our rationale is that understanding cancer- relevant mechanisms that specify FSs will fill the gap in linking in vitro FSs to cancer breakpoints. This would be a major step toward a strategy to predict disrupted pathways from cancer WGS data, thereby suggesting treat- ments for previously intractable cancers. Aim1 will use high throughput/resolution replication and Oligopaints assays to identify downstream mechanisms distinguishing which of many sites of oncogene-induced replication delay manifest as FSs. Aim2 will elucidate upstream mechanisms causing replication delays and FS at specific sites. Aim3 will map FSs at unprededented resolution and mine tumor sequencing databases for signatures that match those of oncogene-specific FSs. This contribution will be significant because the ability to identify affected pathways solely from breakpoint signatures would expose tumor-specific vulnerabilities for precision cancer medicine. The propos...