Effects of HOXA9 suppression in human t(9;11) MOLM-14 and t(4;11) SEMK2 cells. (A) Fluorescence-activated cell sorter analysis of cell-cycle status after transduction of MOLM-14 cells with 1F3-HOXA9shRNA after PI staining. A progressive increase of cells in G₁ phase and a decrease of cells in G₂ and S phases were noted compared with control GFP-shRNA–transduced cells with onset 24 to 36 hours before induction of apoptosis. (B) Analysis of mRNA expression of myeloid differentiation markers M-CSF1R, PU.1, and ITGAM (CD11b) and the granule molecules lysozyme (LYZ), myeloperoxidase (MPO), and elastaseA2 (ELA2) associated with terminal myeloid differentiation by quantitative real-time PCR 72 hours after HOXA9shRNA transduction. Compared with control GFP-shRNA transduced cells, a significant induction of mRNA expression was observed. (C) Gene expression analysis 44 hours after HOXA9 suppression in t(9;11) MOLM-14 cells with 2 different HOXA9shRNAs (1F3-HOXA9shRNA; 2A5-HOXA9shRNA) in triplicates compared with GFP-shRNA–transduced controls. Analysis of concomitant expression changes in other Homeobox A-D cluster genes demonstrated co–down-regulation of 5′-HOXA cluster gens in the HOXA9 suppression signature. (D) GSEA of the top 500 genes down-regulated after HOXA9 suppression in a gene expression dataset of 56 human AML patients.⁶⁰  The HOXA9 suppression gene set is significantly enriched in a signature previously identified as more highly expressed in MLL-AML compared with other genetically defined AML subtypes (ES = 0.48; P < .001). (E) Confirmation of co–down-regulation of 5′-HOXA cluster genes as well as the HOXA9 cofactors MEIS1 and PBX3 and the transcription factor MEF2C in t(4;11) SEMK2 cells (quantitative real-time PCR analysis).