PROJECT SUMMARY Patients with KMT2A-rearranged (KMT2A-r) B-ALL could potentially benefit from the efficacy of CD19 CAR-T therapy, however they are particularly susceptible to lineage switching (to acute myelogenous leukemia, AML) upon CD19-directed therapies. However, defining the precise origins and mechanistic basis of “lineage- switched” leukemia has proven difficult. Clinical studies have previously relied on bulk tumor analysis of biospecimens obtained pre- and post-CAR-T therapy to infer lineage switching has occurred based on the presence of naturally occurring genetic footprints (e.g., specific immunoglobulin rearrangements or translocation breakpoints) in relapsed myeloid CD19neg blasts as evidence of a B-lineage CD19+ origin. These approaches fail to uncover the clone-specific mechanisms that favor lineage switching throughout CAR-T pressure directed at the CD19 surface protein. With our newly developed system to transform B cell progenitors to B-ALL with KMT2A-fusion oncoproteins, we have developed a system that captures the inherent plasticity of KMT2A-r leukemia. This supplement proposal will rigorously identify leukemia-intrinsic lineage reprogramming mechanisms though a cellular barcoding and tracking approach that will enable prospective identification of cell populations that are prone to become the lineage-switched AML in vivo. These studies will lead to a better understanding of lineage plasticity and the extent to which epigenetic heterogeneity contributes to the relapsed AML, which can directly inform the design of curative therapies.