ROLE OF RNA SEQUESTRATION IN ACUTE MYELOID LEUKEMIA

PROJECT SUMMARY Post-transcriptional mechanisms play crucial roles in maintenance of progenitor cell identity and have been implicated in cancer development, including leukemia. However, our understanding of the role of post- transcriptional regulatory processes in leukemogenesis remains limited. To address this gap, we conducted parallel CRISPR dropout screens in both normal and transformed hematopoietic progenitor cells, identifying the RNA helicase DDX6 as a selective vulnerability of leukemia cells. Our findings reveal that DDX6 is overexpressed in leukemia patients and plays a crucial role in initiating and sustaining acute myeloid leukemia (AML). Importantly, depletion of DDX6 in vivo significantly reduced leukemic burden and extended lifespan in patient-derived xenografts and mouse models of AML. Mechanistically, DDX6 coordinates the sequestration and translational suppression of mRNAs encoding chromatin and transcription factors in cytoplasmic condensates termed P-bodies. Overall, our data unveil DDX6-mediated RNA processing as a central pathway dysregulated in leukemia cells, relative to hematopoietic stem and progenitor cells, and required for AML maintenance. While these insights are significant, the roles of DDX6 in promoting malignant transformation remain unclear. Furthermore, the direct gene-regulatory networks of DDX6 in leukemia are yet to be fully understood. To address these questions, this application proposes three complementary aims. In SPECIFIC AIM 1, we will utilize two novel genetic mouse models for deletion and overexpression of DDX6 in vivo in a cell- and time-specific manner to rigorously dissect the roles of DDX6-mediated RNA sequestration during normal and malignant hematopoiesis. Additionally, we will assess the impact of perturbing DDX6's helicase activity on leukemia cells, both in vitro and in vivo. In SPECIFIC AIM 2, we will explore the relationship between DDX6 and RNA sequestration in cytoplasmic condensates of normal and leukemia cells. Specifically, we will characterize the molecular landscape of P-bodies in human AML as well as normal hematopoietic progenitors and investigate how DDX6 cooperates with other RNA-binding proteins to regulate the storage of critical RNAs during leukemogenesis. We will also identify DDX6's direct protein interactors and assess how these interactors facilitate RNA sequestration and contribute to leukemogenesis. In SPECIFIC AIM 3, we will elucidate the direct role of DDX6 in suppressing the translation of target mRNAs during leukemogenesis. By analyzing the translatome and proteome of leukemia cells following acute degradation of DDX6 using our novel FKBP-degron AML lines, we will identify factors regulated by DDX6 and examine their functional and cellular roles in AML cell self-renewal and leukemogenesis. Collectively, our study will advance the understanding of post- transcriptional regulation in AML and pave the way for innovative therapeutic strategies targeting DDX6 and downstream effectors. Furthermore, our work introduces the concept of RNA condensates as potential biomarkers in myeloid leukemia diagnosis and treatment.