Fusion Gene Depletion Eliminates Stemness and Induces Bidirectional Differentiation of Acute Myeloid Leukemia Open Access

• A targeted LNP formulation efficiently delivers fusion transcript-specific siRNAs to primary AML cells leading to fusion gene knockdown.

• Loss of RUNX1::RUNX1T1 promotes granulocytic and eosinophilic differentiation to more normal states while depleting stem cell populations.

Chromosomal rearrangements that generate novel fusion genes are a hallmark of acute myeloid leukemia (AML). Depletion experiments in cell line models have suggested that their continued expression is required for maintaining their leukemic phenotype and that fusion genes therefore represent ideal cancer-specific therapeutic targets. However, to which extent this result holds true for the different stages of hematopoietic development in primary cells and whether therapeutic agents can be efficiently delivered to those cells is still unclear. In this study, we demonstrate that primary AML cells harboring the chromosomal translocation t(8;21) are critically dependent on the corresponding fusion gene, RUNX1::RUNX1T1, to suppress differentiation and maintain stemness. Silencing RUNX1::RUNX1T1 expression using siRNA-loaded lipid nanoparticles induces substantial changes in chromatin accessibility, thereby redirecting the leukemia-associated transcriptional network towards a myeloid differentiation program. Single-cell analyses reveal that this transcriptional reprogramming is associated with the depletion of immature stem and progenitor-like cell populations, accompanied by an expansion of granulocytic and eosinophilic/mast cell-like populations with impaired self-renewal capacity. These findings underscore the essential role of RUNX1::RUNX1T1 in sustaining AML and highlight the therapeutic potential of targeting fusion gene expression in primary AML cells.