PROJECT SUMMARY This proposal aims to investigate dysregulation of alternative polyadenylation (APA) in acute myeloid leukemia (AML). Dysregulation of APA has been identified in hematopoietic stem and progenitor cells in AML, but previous studies have compared bulk populations of HSPCs from AML patients to healthy donors. Therefore, the extent of dysregulation in malignant hematopoietic stem cells (HSCs) and the molecular regulators driving this phenomenon remain poorly understood. To address these questions, this proposal outlines two specific aims that leverage a previously developed computational platform for identifying shifts in polyA site usage at single-cell resolution. The first aim will use single-cell RNA sequencing data from AML patients to characterize changes in polyA site usage between malignant and healthy HSPCs at matched differentiation stages within the same donor. This will identify if changes in APA originate in leukemic stem cells or are restricted to more committed progenitors. PolyA site usage is heterogenous between patients and even healthy donors, so comparisons within the same donor will improve accuracy on identifying true changes in polyA site usage. The second aim extends a sequence-based deep learning framework to model polyA site usage in human HSPCs with over/under expression of different CPA regulators. This model will elucidate the extent to which CPSF6, a subunit of cleavage-factor I complex that is downregulated at the transcriptional level in AML patients, drives aberrant polyA site usage in AML. If CPSF6 downregulation is not sufficient to explain dysregulated polyA site usage, additional factors that contribute will be identified. Finally, CRISPR perturbation studies will be used to assess if modulating the expression of CPSF6 expression (and other implicated regulators) affects differentiation state in AML cell lines, providing functional evidence that APA is involved in maintaining a leukemic state. The completion of these aims will provide a new understanding of APA in myeloid malignancies and may lead to the identification of novel therapeutic targets.