Project Summary Activating programmed cell death such as apoptosis is one of the most important therapeutic strategies for cancer therapy. Many cancers undergo dramatic initial responses to such therapy. However, cancer relapse with the development of drug resistance is a major problem in the clinical management of most types of cancer, leading to treatment failure. The classical view of cell death has long assumed that, once initiated, the apoptotic dying process is irreversible. However, we discovered that dying cancer cells and primary cells can reverse the apoptotic process even at late execution stages, and named this cell recovery mechanism Anastasis (Greek for “rising to life”). The discovery of anastasis suggests an unexpected tactic that cancer cells could use to escape cell death-inducing cancer therapy. Central to this proposal, we have demonstrated that some cells acquired permanent genetic changes during anastasis and undergo oncogenic transformation at a higher frequency than controls. Moreover, anastatic cells display an increased resistance to apoptotic stimuli such as anticancer drugs. These observations lead to an intriguing question: Can anastasis contribute to the development of drug resistance in recurrent tumors? If so, anastasis would be a potential and novel therapeutic target for suppressing cancer progression and recurrence. Since mutations contribute to the development of resistance, we propose that cancer cell survival by anastasis occurring at the interval between cycles of anticancer therapy could allow apoptosis-caused DNA damage to be perpetuated and lead to mutagenesis, progression, and evolution of drug resistance in recurrent cancers. In Specific Aim 1, we propose to test for and identify such mutations if they occur, and determine whether mutational hotspots exist in anastatic cells. We will then compare any mutations identified with the known mutational signatures that have been mapped in recurrent clinical tumors. This will directly test for connections between anastasis and mutagenesis in cancer cells. In Specific Aim 2, we propose to compare the sensitivity of anastatic cancer cells to different cell death inducers. Anastatic cells that have recovered from repeated inductions of apoptosis often develop resistance to the same cell death stimulus. However, it is not known whether these anastatic cells might also be resistant to other cell death inducers. We will test for this possibility by determining whether anastasis increases resistance of cancer cells to higher doses of the same inducer, and may also result in “cross-resistance” to other pathways of cell death, e.g., autophagy, ferroptosis, and necroptosis. Overall, these studies seek to elucidate connections between anastasis and development of drug resistance in cancer cells that can be targeted and useful in new approaches to anticancer therapy.