The critical role of PUS1-mediated mRNA Ψ modification in leukemogenesis

About This Grant

Abstract Acute myeloid leukemia (AML) is one of the most common acute leukemias stemming from genetic alterations in hematopoietic stem/progenitor cells (HSPCs). Currently, effective targeted therapies for AML are limited. A comprehensive understanding of the molecular mechanisms driving AML is crucial for developing innovative treatments. RNA modifications, including pseudouridine (Ψ), are crucial for the regulation of gene expression and cellular function. While aberrant Ψ levels have been linked to certain cancers, its role and underlying mechanisms in AML remain elusive. Pseudouridine modification is catalyzed by pseudouridine synthases (PUSs), including PUS1, a major regulator of Ψ modification across RNA species. Although Ψ has been extensively characterized in tRNA and rRNA, its distribution and function in mRNA remain incompletely understood because of limited sensitive genomic approaches. Using Bisulfite-lnduced Deletion sequencing (BlD-seq) to quantify mRNA Ψ at base resolution, we identified abundant Ψ sites in human leukemia cells and found that PUS1 regulates hundreds of transcripts through PUS1-mediated pseudouridylation. PUS1 is significantly upregulated in subsets of AML patients, and PUS1/Pus1 knockdown markedly inhibits growth and induces apoptosis in human leukemia cells and primary human and mouse leukemia cells, with minimal effects on normal human or mouse HSPCs. These data support a previously unrecognized and leukemia-selective role for PUS1 in AML. Our central hypothesis is that PUS1-mediated mRNA Ψ modification post-transcriptionally regulates genes required for leukemogenesis and AML maintenance. We will test this hypothesis by: 1) defining the oncogenic role of PUS1/Pus1 in AML pathogenesis; and 2) elucidating how PUS1-mediated mRNA Ψ promotes leukemogenesis through post-transcriptional regulation of gene expression. Using animal models and integrated multi-omics approaches, we will determine the in vivo function of PUS1 in normal hematopoiesis and leukemia and define how PUS1 regulates downstream targets from transcription to translation. Successful completion of these studies will establish the role and mechanisms of PUS1/Pus1 in AML development and maintenance, particularly in LSC/LICs, and advance understanding of RNA epitranscriptomic regulation in AML pathogenesis. Animal studies are essential to evaluate leukemogenesis and leukemia stem cell function in vivo within the physiologic bone marrow microenvironment, which cannot be adequately modeled in cell culture alone.