ABSTRACT Acute leukemia is a leading cause of death in the young. The goal of my research program is to identify the genomic drivers of leukemogenesis and treatment failure, and through experimental modeling gain mechanistic insight to enable the development of new diagnostic and therapeutic approaches, and ultimately improve cure rates for this disease. In the last project period, I identified multiple subtypes of leukemia that led to a revision of the molecular taxonomy of acute lymphoblastic and lineage ambiguous leukemia; I defined the relationship of genomic variegation and clonal evolution in leukemia; I demonstrated the role of liquid-liquid phase separation in leukemogenesis; and I developed new therapeutic approaches using targeted protein degradation directed at intractable drivers of high-risk leukemia. In this proposal, the original overarching goal remains unchanged, but my research program will address several conceptually new areas of research that have been stimulated by my prior discoveries. These include (1) defining the mechanism by which enhancer deregulation drives leukemogenesis in T-lineage ALL; (2) investigating how concomitant LMO2-activating and STAG2-inactivating alterations perturb chromatin state to drive leukemogenesis, and how this may be exploited for therapeutic benefit; (3) elucidating how BCL11B deregulation primes primitive hematopoietic cells for lineage ambiguous leukemia; (4) determining how fusion oncoprotein-driven liquid-liquid phase separation (LLPS) drives chromatin and transcriptional deregulation in leukemogenesis, and how this may be targeted for therapeutic benefit; and (5) developing molecular glue-based targeted protein degradation strategies to improve cure rates for childhood leukemia. These research areas explore central questions in childhood and adult leukemia, including the mechanisms by which enhancer hijacking/deregulation events drive leukemogenesis, the basis of cell lineage ambiguity in lineage ambiguous leukemia, the mechanistic role of LLPS in leukemogenesis, and the efficacy of innovative therapeutic approaches to degrade intractable drivers of acute leukemia. To achieve these goals I will use innovative experimental approaches including novel engineered mouse models, single cell barcoding and lineage tracing, multiomic gene expression-chromatin profiling, LLPS-condensate profiling, and exploitation of a unique small molecule library of molecular glues. This research program will be facilitated by collaborations with colleagues with expertise in these approaches. Collectively, these approaches will yield fundamental mechanistic insight and therapeutic advances that will improve cures for currently intractable high risk leukemias.