EIF4A3-dependent nonsense-mediated decay buffers AML1-ETO9a dosage and modulates outcome in t(8;21) acute myeloid leukemia.

t(8;21) acute myeloid leukemia (AML) is driven by AML1-ETO, which undergoes alternative splicing to generate AML1-ETO9a (AE9a), a truncated isoform with enhanced leukemogenic activity. Although t(8;21) AML is considered favorable-risk, clinical outcomes are heterogeneous, and AE9a expression varies markedly among patients. How cells restrain this oncogenic isoform remains unclear. Here, we identify nonsense-mediated mRNA decay (NMD) as an isoform-specific buffer of AE9a dosage. Inclusion of the ETO9a cassette exon introduces premature termination codons and generates an NMD-sensitive transcript. In primary t(8;21) AML CD34⁺ hematopoietic stem and progenitor cells, AE9a inclusion inversely correlated with NMD-factor expression, and high EIF4A3 expression was associated with improved overall survival specifically in t(8;21) AML, but not in other AML subtypes. Pharmacological inhibition of SMG1 or EIF4A3 and genetic depletion of NMD factors increased AE9a abundance in t(8;21) AML cell lines and primary patient cells, with cytoplasmic transcript accumulation and increased AE9a protein. Conversely, EIF4A3 overexpression reduced AE9a RNA and protein, restrained t(8;21) AML cell growth, spared healthy CD34⁺ progenitor expansion, and enhanced idarubicin sensitivity. These findings define EIF4A3-dependent NMD as a checkpoint linking RNA surveillance to oncogenic fusion-isoform dosage, leukemic fitness, and chemosensitivity in t(8;21) AML, providing a mechanistic explanation for clinical heterogeneity in t(8;21) AML. EIF4A3-dependent NMD buffers AE9a dosage and modulates t(8;21) AML cell fitness and chemosensitivity: Schematic model summarizing the proposed AE9a-NMD axis in t(8;21) AML. Alternative splicing of AML1-ETO generates the ETO9a cassette exon, producing a PTC-containing AE9a transcript. After nuclear export, ribosome engagement with the PTC-containing AE9a mRNA recruits the NMD machinery, including UPF factors, SMG factors, DHX34, and the exon-junction complex component EIF4A3. Efficient NMD promotes AE9a mRNA decay and limits AE9a protein accumulation. High EIF4A3/NMD activity therefore lowers AE9a dosage, restrains t(8;21) AML cell proliferation, enhances chemosensitivity to idarubicin, and is associated with improved patient survival. Conversely, impaired NMD activity permits AE9a accumulation and may increase leukemic fitness. This model defines an isoform-specific, NMD-buffered oncogenic dosage checkpoint in t(8;21) AML.