Acquired resistance to cancer therapy is a formidable obstacle preventing cancer

treatments from fully curing patients. Across multiple cancer types, analysis of tumor shrinkage in response to treatment reveals a residual, quiescent, and drug tolerant cancer cell population termed cancer persister cells. Persister cells initially survive drug treatment through reversible, non-genetic mechanisms but subsequently acquire resistance-conferring mutations and regrow to seed the emergence of drug resistant tumors. The mechanisms by which persister cells acquire mutations are unknown. Here, we investigate our hypothesis that persister cells experience drug stress-induced sublethal apoptotic signaling which results in activation of apoptotic DNases. These DNases, which normally serve to fragment chromosomal DNA during apoptosis, instead promote DNA damage and mutagenesis within surviving persister cells allowing for regrowth into drug-tolerant expanded persister cell (DTEP) colonies. We observed that during extended treatment with targeted therapies, persister cells require apoptotic DNase (DFFB) to regrow into drug-resistant proliferating cells. Consistent with the hypothesis that persister cells are mutationally active, we also observed that persister cells exhibit elevated levels of DNA damage. This DNA damage was absent in DFFB KO persister cells, suggesting that DFFB activation is required to induce DNA damage in persister cells. These findings reveal that sublethal apoptotic signaling and activation of DFFB may play a role in persister cell mutagenesis and tumor relapse.