Tumors frequently acquire resistance to therapy resulting in disease relapse and patient mortality. Therapeutic approaches to overcome acquired resistance are hindered by limited treatment options and the presence of multiple parallel resistance mechanisms. Rather than treating resistance after it emerges, it may possible to prevent it by inhibiting the adaptive processes which initiate resistance. However, these processes and the genes which control them are poorly understood. Here we report that drug-tolerant cancer persister cells, which constitute residual disease and seed relapse, undergo drug stress-induced sublethal apoptotic signaling resulting in activation of apoptotic DNase DFFB without cell death. DFFB induces DNA damage and mutagenesis in persister cells, revealing DFFB as a key regulator of stress-induced mutagenesis in persister cells. Furthermore, we found in multiple tumor types that DFFB is required for acquired resistance to targeted therapies. Mechanistically, DFFB induces multiple putative resistance mutations and may also promote epigenetic changes which result in resistance. These results reveal DFFB as a promising potential therapeutic target to prevent acquired resistance. In addition, this stress-sensing mutagenic mechanism may promote mutagenesis in other contexts including normal tissues because sublethal apoptotic signaling may result from a variety of physiological stresses. Citation Format: August Finley Williams, David Gervasio, Claire Turkal, Anna Stuhlfire, Michael Wang, Brandon Mauch, Ariel West, Michelle Paw, Mehrshad Hairani, Cooper Lathrop, Sophie Harris, Jennifer Page, Matthew Hangauer. Apoptotic DNase DFFB mediates cancer persister cell mutagenesis and acquired drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 99.

It was recently discovered that actively proliferating and post-mitotic normal human tissues acquire somatic mutations at the same rate. This indicates that somatic mutagenesis is primarily independent of cell proliferation and therefore cannot be based on DNA replication errors. Furthermore, the mutagenic mechanisms which contribute to ubiquitous mutational signatures such as SBS5 remain unknown. It has been hypothesized that tissues accumulate mutations through unknown physiologic processes which contribute to DNA damage but the molecular details remain unknown. These observations have revealed our lack of understanding of the fundamental processes of mutagenesis. Stress-induced mutagenesis has been previously characterized in bacteria which respond to stress by transiently increasing the rate of mutagenesis to promote the emergence of resistance mutations. This phenomenon was recently discovered in human cancer cells but the mechanism is unknown. Here, we present our discovery that cancer cells under drug stress undergo sublethal apoptotic signaling resulting in activation of apoptotic DNase DFFB. DFFB, which canonically acts to fragment chromosomal DNA during apoptosis, induces DNA strand breaks in surviving cancer cells resulting in error prone repair and mutagenesis. In the context of targeted therapy-treated cancer, we found that DFFB is required for acquired resistance to emerge in multiple tumor types. We also found that CD8 T cells induce a similar process in targeted tumor cells which survive. Therefore, sublethal apoptotic signaling may be a general phenomenon which promotes mutagenesis in human cancer. Based on these observations and prior literature showing sublethal apoptotic signaling occurs in normal cells in response to various stress stimuli, we propose that this mechanism may be a previously unknown fundamental mutagenic process which contributes not only to acquired resistance in cancer but also to tumor initiation, aging and other conditions. Citation Format: August F. Williams, David A.G. Gervasio, Claire E. Turkal, Mehrshad Hairani, Michael X. Wang, Anna E. Stuhlfire, Ariel H. West, Brandon E. Mauch, Michelle H. Paw, Cooper P. Lathrop, Sophie H. Harris, Jennifer L. Page, Matthew J. Hangauer. Apoptotic DNase DFFB mediates stress-induced mutagenesis [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR003.