The Myocyte Enhancer Factor 2C (MEF2C) Is a Direct Transcriptional Target of HOXA9 and Mediates Leukemia Survival in Human Mixed-Lineage Leukemias.

Leukemias that harbor rearrangements of the mixed-lineage leukemia gene (MLL) possess unique clinical and biological characteristics. Aberrant high-level expression of select Homeobox (HOX) genes including HOXA9 is a consistent feature of MLL-rearranged leukemias, implicating an important role of dysregulated HOX gene expression in this entity. Applying HOXA9-directed RNAi, we have previously shown that aberrant HOXA9 expression is a prerequisite for leukemia survival in human MLL-rearranged leukemias and murine Mll-leukemia stem cells as HOXA9 suppression leads to a progressive induction of apoptosis in vitro and in vivo. In this study we aimed to identify potential direct HOXA9 target genes in human MLL-rearranged leukemias utilizing gene expression profiling after shRNA mediated HOXA9 knockdown and subsequent chromatin immunoprecipitation (ChIP) analysis. To establish efficient HOXA9 knockdown, 2 previously validated shRNA constructs targeting human HOXA9 with high efficiency (>80% mRNA suppression) were transduced into t(9;11) MOLM-14 AML cells. 72h after transduction, RNA was hybridized to Affymetrix HU 133A2.0 expression arrays. Supervised analysis identified a large group of genes concomitantly downregulated after HOXA9 suppression. Gene set enrichment analysis demonstrated significant enrichment of the top 300 genes in the HOXA9 suppression signature and genes more highly expressed in human MLL-AML as compared to other genetically defined AML subtypes (p=0.02). Interestingly, genes previously implicated in the pathogenesis of MLL-rearranged leukemias were among the most highly enriched (HOXA5, MEIS1, PBX3, MEF2C). To analyze if any of this genes might be a direct physiological HOXA9 target, we performed ChIP assays from FLAG-tagged HOXA9 expressing MOLM-14 cells. Whereas the promotor regions of HOXA5, MEIS1 and PBX3 did not show HOXA9 binding, the MEF2C promotor region DNA was highly enriched by ChIP. Furthermore HOXA9 binding was confined to the MEF2C promotor region as the regions -5kb 5’-upstream, +5kb and +10kb 3’-downstream of the MEF2C promotor region did not show any enrichment. Motive search analysis of the MEF2C promoter further revealed a perfect consensus binding site (TGATTTAT) for HOXA9 and its DNA binding partner PBX located at −721 to −714 from the transcriptional start site. We then analyzed if direct shRNA mediated targeting of MEF2C also abrogates leukemia survival in human MLL-rearranged leukemias similar to upstream HOXA9 suppression. Analysis of viability after MEF2C knockdown with two different shRNA constructs (>80% mRNA knockdown) in a panel of human 8 AML/ALL cell lines (4 MLL-rearranged; 4 MLL-wildtype) and primary AML patient cells (5 MLL-rearranged; 5 MLL-wildtype) revealed progressive induction of apoptosis which was significantly correlated with the presence of the MLL-fusion oncogene in the cell lines (mean viability at day 7: MLL-rearranged: 14.5% MLL-wildtype: 86.75%: p<0.001) and in the primary AML cells (p=0.006). Our data implicates MEF2C as a direct downstream target of HOXA9 and aberrant MEF2C expression is necessary for survival of MLL-rearranged leukemias. Targeting the HOXA9-MEF2C program may be a novel therapeutic approach.