PROJECT SUMMARY Most patients diagnosed with acute myeloid leukemia (AML) die of the disease due to development of resistance to existing chemotherapy or targeted therapy. This problem underscores the compelling need for understanding of relapse emergence mechanisms and identification of effective targeted strategies to overcome leukemia relapse. Recently, by using dynamic BH3 profiling that measures rapid change in drug-induced apoptotic signaling, we identified drugs that can overcome resistance to venetoclax, a selective BCL-2 inhibitor and active drug in leukemia (Bhatt et al, Cancer Cell 2020). Through mechanistic studies using patient derived xenograft (PDX) models, we next found venetoclax resistance emergence is accompanied by reduced sensitivity of mitochondria to apoptotic signaling mechanisms. Surprisingly, we found that resistance to disparate, narrowly-targeted agents in distinct PDX models was consistently accompanied by broad resistance to a wide variety of drugs, indicative of multi-resistance phenotype at relapse (Olesinski et al, Blood Cancer Discovery, 2024). We then reported that selection for reduced mitochondrial priming drives multi-drug resistance phenotype at relapse. By building upon these findings, we hypothesize that stable acquired resistance emerges from drug-tolerant leukemic persister cells and that the persister cells can be targeted by exploiting mitochondrial apoptotic signaling mechanisms. To address this, we propose to utilize Watermelon 2.0 library an innovative lentiviral construct that contains both an expressed barcode, and an inducible histone-2B (H2B-mCherry) florescent dilution system, to trace in vivo clonal evolution. In Aim 1, we will look for an evidence of both genetic and non- genetic modes of clonal evolution of resistance using high-complexity DNA barcoding, proliferative index, and whole genome studies. In Aim 2, we will identify cellular states that contribute to persister development by single-cell transcriptomi