Abstract Acute myeloid leukemia (AML) presents significant therapeutic challenges despite advances in targeted therapies, primarily due to acquired resistance. In AML, relapse is frequently driven by drug-tolerant persisters (DTPs), a small subpopulation of cells that survive therapy through non-genetic mechanisms. These DTPs evade initial treatment and re-initiate disease, highlighting the urgent need to define their vulnerabilities and develop strategies to eradicate them. We employed the Watermelon reporter system, integrating H2B-mCherry dilution to track proliferative dynamics, and combined it with single-cell RNA sequencing (scRNA-seq), lineage barcoding, and flow cytometry to characterize persister populations in AML cell lines (MV4-11, AML2, MOLM-13) treated with Cytarabine (AraC) and targeted agents (Venetoclax, Navitoclax, Gilteritinib, S63845). A genome-wide CRISPR knockout screen was performed in AraC-treated AML2 persisters to identify genes essential for DTP survival. We observed heterogeneous emergence of cycling and slow-cycling persister cells depending on drug and cell genotype. scRNA-seq revealed distinct transcriptional programs, with Gilteritinib-derived persisters showing leukemic stem/progenitor cell–like signatures. CRISPR screening identified critical dependencies in persisters, including DNA damage regulators (SAMHD1) and epigenetic factors (JARID2, PCGF1, MBTD1). Functional validation demonstrated that inhibition of these epigenetic regulators using a selective histone deacetylase inhibitor (Mocetinostat) disrupted chromatin compaction and sensitized DTPs to AraC. Sequential Mocetinostat pretreatment followed by AraC completely eradicated persister populations in vitro. In conclusion, our study demonstrates that AML persisters rely on epigenetic plasticity to survive chemotherapy. Genome-wide CRISPR screening revealed epigenetic regulators as actionable vulnerabilities. Pharmacologic targeting of these factors with Mocetinostat reprograms chromatin and restores drug sensitivity, leading to complete elimination of DTPs. These findings provide a strong rationale for incorporating epigenetic modulators into combination therapies to prevent AML relapse. Citation Format: Ankit Jana, Karanpreet Bhatia, Xuejing Lyu, Xiaoyang Qi, Xiao Xian Lin, Fang Qi Lim, Chuqi Wang, Ahmed Mamdouh, Emanuelle Grody, Yogesh Goyal, Yaara Oren, Greg Tucker-Kellogg, Shruti Bhatt. EPIGENETIC VULNERABILITIES DRIVE SURVIVAL OF DRUG-TOLERANT PERSISTERS AND REVEAL THERAPEUTIC OPPORTUNITIES IN AML abstract. In: Proceedings of Frontiers in Cancer Science 2025; 2025 Nov 5-7; Singapore. Philadelphia (PA): AACR; Cancer Res 2026;86 (13Suppl): Abstract nr LT07.
Jana et al. (Thu,) studied this question.