AbstractThe chromosomal translocation t(4;11)(q21;q23), a hallmark of an aggressive form of acute lymphoblastic leukemia (ALL), encodes mixed‐lineage leukemia (MLL)‐AF4 oncogenic chimera that triggers aberrant transcription of genes involved in lymphocyte differentiation, including HOXA9 and MEIS1. The scaffold protein 14‐3‐3θ, which promotes the binding of MLL‐AF4 to the HOXA9 promoter, is a target of MiR‐27a, a tumor suppressor in different human leukemia cell types. We herein study the role of MiR‐27a in the pathogenesis of t(4;11) ALL. Reverse transcription quantitative PCR (qPCR) reveals that MiR‐27a and 14‐3‐3θ expression is inversely correlated in t(4;11) ALL cell lines; interestingly, MiR‐27a relative expression is significantly lower in patients affected by t(4;11) ALL than in patients affected by the less severe t(12;21) leukemia. In t(4;11) leukemia cells, ectopic expression of MiR‐27a decreases protein level of 14‐3‐3θ and of the key transcription factor RUNX1. We show for the first time that MiR‐27a also targets AF4 and MLL‐AF4; in agreement, MiR‐27a overexpression strongly reduces AF4 and MLL‐AF4 protein levels in RS4;11 cells. Consequent to AF4 and MLL‐AF4 downregulation, MiR‐27a overexpression negatively affects transcription of HOXA9 and MEIS1 in different t(4;11) leukemia cell lines. In agreement, we show through chromatin immunoprecipitation experiments that MiR‐27a overexpression impairs the binding of MLL‐AF4 to the HOXA9 promoter. Lastly, we found that MiR‐27a overexpression decreases viability, proliferation, and clonogenicity of t(4;11) cells, whereas it enhances their apoptotic rate. Overall, our study identifies the first microRNAthat strikes in one hit four crucial drivers of blast transformation in t(4;11) leukemia. Therefore, MiR‐27a emerges as a new promising therapeutic target for this aggressive and poorly curable form of leukemia.