PROJECT SUMMARY Acute leukemias of ambiguous lineage (ALAL) are high-risk leukemia subtypes and include mixed phenotype acute leukemia (MPAL) and early T cell precursor acute lymphoblastic leukemia (ETP-ALL). These leukemias commonly express markers associated with both the myeloid and T lymphoid lineages which complicates choice of therapy. Moreover, the genomic, molecular, and cellular basis of ALAL remains obscure and hinders our ability to identify more relevant and tailored therapeutic strategies. I recently discovered a new genomic alteration that is specific to a subset of T/myeloid MPAL and ETP-ALL cases, namely noncoding structural variations that aberrantly activate the T cell transcription factor gene BCL11B in a non-T lineage cell of origin. Most of these cases (81%) harbored activating mutations (e.g. internal tandem duplication, ITD) in the FLT3 tyrosine kinase receptor gene, suggesting functional cooperation between these alterations. This discovery enables faithful experimental modeling of the earliest stages of ALAL development. My preliminary data demonstrated that ectopic BCL11B expression is sufficient to drive formation of phenotypic T cells from a pool of extra-thymic human CD34+ hematopoietic stem and progenitor cells (HSPCs). However, HSPCs are a highly heterogeneous population of cells with different stemness capacities and degrees of lineage commitment, and it remains unknown whether a certain subpopulation is most permissive to BCL11B-induced lineage skewing or how BCL11B transcriptional activity disrupts different HSPC gene regulatory programs to drive the lineage ambiguous phenotype. The goal of this proposal is to define the mechanisms by which ectopic BCL11B expression corrupts hematopoietic differentiation to drive development of ALAL. To accomplish this, Aim 1 will use a single cell in vitro differentiation assay to determine how the developmental state of the cell of origin impacts the ability of BCL11B/FLT3-ITD to drive lineage skewing. Aim 2 will complement this cell phenotype-based assay with single cell RNA-seq to identify the spectrum of gene expression changes that accompany changes in differentiation potential and lineage skewing of different cells of origin. I will also use acute protein degradation techniques to identify direct BCL11B target genes which will inform on BCL11B-controlled transcription networks that I will investigate in my future independent research. Collectively, these experiments will clarify the role of the cell of origin in dictating oncogenic BCL11B activity and identify BCL11B-controlled transcription networks. In the independent phase (Aim 3), I will investigate the molecular mechanism of BCL11B oncogenic activity to nominate new therapeutic targets. I will first investigate how BCL11B alters chromatin regulation by identifying changes in chromatin state and transcription factor occupancy. I will then use a new mouse model to screen for epigenetic regulators critical for oncogenic ...