AbstractBackgroundGenetic mutations ofIKZF1have been frequently delineated in B‐lineage acute leukaemia (B‐ALL) but rarely elucidated in acute myeloid leukaemia (AML).IKZF1mutations confer a poor prognosis in AML, and hotspot mutations ofIKZF1, N159Y and N159S tend to occur in B‐ALL and AML respectively. However, the pathogenesis ofIKZF1N159S in AML andIKZF1lineage susceptibility are largely unknown.MethodsThe genetic and clinical characteristics ofIKZF1‐mutated AML patients were evaluated. Multi‐omics analysis and functional assays were performed in vitro usingIKZF1mutations knock‐in AML cell lines.Results23 (4.84%) small sequence variants ofIKZF1were identified in 475 newly diagnosed AML (non‐M3) patients. Based on RNA sequencing, three classes ofIKZF1‐related AML were defined, including 9 patients (39.13%) withIKZF1N159S mutations, 10 (43.47%) withCEBPAmutations and 4 others (17.39%).IKZF1N159S may define a unique subgroup with higherHOXA/Bexpression and native B‐cell immune fractions. Gene expression data of multiple knock‐in cell lines indicate that the lymphocyte differentiation‐relatedMMEandCD44kept high expression inIKZF1N159Y but were downregulated in N159S. CUT&TAG sequencing showed thatIKZF1N159S reshaped the binding profiles of IKZF1. Integration analysis suggested that the pathogenesis ofIKZF1N159S may depend on the deregulation of several cofactors, such as oncogenicMYCandCPNE7targets.ConclusionsCollectively, we dissected the molecular spectrum and clinical features ofIKZF1‐related AML, which may promote an in‐depth understanding of the pathogenesis, lineage susceptibility and clinical research of AML.