Project Summary/Abstract The clinical activities of oncogene targeted therapies are limited by the fact that a subset of residual cancer cells often survive treatment, eventually giving rise to progressive, resistant disease that is very difficult to treat. To date, relatively little is known about residual disease cells, in part because obtaining and analyzing tumor specimens at this stage has been logistically challenging. We recently discovered that residual cells surviving treatment with diverse, oncogene-matched targeted therapies exhibit DNA double strand breaks (DSBs) and consequent, ATM-dependent DSB repair. In mechanistic studies performed in EGFR mutant non-small cell lung cancer (NSCLC), this DNA damage was observed to be driven by the counterintuitive “sublethal” activation of executioner caspases 3 and 7, which drive DNA damage through their direct activation of caspase-activated DNase (CAD). As a consequence, residual cancer cells that survive upfront EGFR inhibitor treatment require ATM activity to resolve DSBs, and combining EGFR inhibitors with ATM inhibitors eradicates residual cells, leading to highly penetrant and durable therapeutic responses in cellular and animal models. These findings are further supported by our clinical observations that residual EGFR mutant lung tumors display marked upregulation of ATM activity and that rare NSCLC patients whose tumors harbor loss-of- function mutations in ATM exhibit increased progression-free survival on EGFR inhibitor therapy. In this proposal, we describe studies to define the mechanisms driving sublethal caspase activation in residual tumor cells and their consequent, ATM-dependent survival. Further, we propose to define the broad, functional implications of sublethal caspase and ATM activation in residual tumor cells, revealing consequent mechanistic vulnerabilities of residual tumors. Finally, we describe studies that integrate cellular, patient-derived xenograft, and genetically engineered mouse models with highly credentialed, longitudinally sampled clinical specimens to evaluate the therapeutic targeting of residual tumor cell survival. Together, these studies will define key, distinguishing features of residual tumor cells, advancing our basic understanding of this critically important but poorly understood aspect of tumor biology while defining a new class of mechanism-based strategies with the potential to minimize residual disease.