Recurrent chromosomal translocations are common initiation events and have provided important insights into the pathogenesis of AML, paving the way for the introduction of novel targeted therapies. However, clin. outcomes, in particular for patients with adverse cytogenetic features remain suboptimal. The chromosomal translocation t(7;11)(p15, p15), encoding the fusion protein NUP98-HOXA9 (NHA9), is a rare poor risk cytogenetic event in AML associated with a particularly poor prognosis. NHA9 brings the FG repeat-rich portion of the nucleoporin NUP98 upstream of the homeodomain and PBX heterodimerization domains of HOXA9, and acts as oncogenic transcription factor.1. The pathogenic events underlying NHA9 remain poorly understood and herein, we aim to characterize the downstream mediators of this oncoprotein by determining the effects of the fusion using human cellular models. We set out initially to compare the DNA binding sites of NHA9, HOXA9 and NUP98, by forced expression of these genes alone or the corresponding fusion gene by retroviral transduction of HEK93FT cell line and cord blood-isolated human hematopoietic progenitors (hHP). ChIP-seq anal. in the HEK293FT cellular model identified 4471 significant genomic regions (false discovery rate (FDR) <0.05) as target sites of the fusion protein, all located within -5/+ kb from the annotated transcription start site (TSS) (Supplementary Figure S1A). They correspond to 1368 genes and 17 miRNAs (Supplementary Table S1) of which 399 genes were also shown to be common targets of HOXA9 and 4 of NUP98 (Figure 1a, Supplementary Table S2, Supplementary Figures S1C-D) (Supplementary methods) (Data deposited in GEO http://www.ncbi.nlm.nih.gov/geo/, accession number: GSE62587). Ingenuity pathway anal. of the NHA9 target series demonstrated a significant enrichment of pathways associated with tumorigenesis and leukemic differentiation (Supplementary Figure S1B). We next performed a detailed sequence anal. of the NHA9 binding sites using the MEME-ChIP algorithm and detected a significant overlap with binding of several HOX genes, including HOXA9, supporting a role for this homeodomain in the DNA binding of NHA9. Strikingly, NHA9 sites were enriched for a novel binding motif, CA/gTTT, that was present in one-third (n=1421) of all NHA9 ChIP-seq regions (Supplementary Table S3). This motif had not been previously associated with any known transcription factor and was not observed in wild type HOXA9 or NUP98 binding site experiments, suggesting that it is specific to NHA9 DNA binding. MEME-ChIP (SpaMO) was used to identify significant co-occurrences of other known DNA binding motifs with this novel NHA9 DNA binding motif. Binding motifs corresponding to 12 transcription factors, including other HOX family proteins such as HOXB7 or HOXD11, were found to be overrepresented within the region adjacent to CA/gTTT (Supplementary Table S4), suggesting a possible functional cooperation with the fusion oncoprotein. As the NHA9 target motifs are preferentially located more than 1 kb upstream/downstream of the TSS (Supplementary Figure S1A), we reasoned that NHA9 binding may coincide with particular enhancer elements. A similar distribution was also found for the identified HOXA9 target regions whereas NUP98 binding sites were mostly located within promoters, both in agreement with previous studies.2, 3 We selected eight leukemia-related genes (MEIS1, HOXA9, PBX3, MET, BRAF, AF9, PTEN and NF1) identified as part of our NHA9 ChIP-seq experiments, for locus specific qChIP studies. A significant enrichment of H3K4me1, a chromatin mark that predicts poised and active enhancers, and RNA Polymerase II (PolII), which is consistent with the presence of the active form of the enhancers,4, 5 was shown within the NHA9 binding sites upstream of the eight genes (Figure 1b and Supplementary Figure S1E). NHA9 expression levels were demonstrated to be comparable in our two cellular models (HEK293FT and hHP) (Supplementary Figure S1G). Accordingly, we validated the ChIP-seq results in the HEK293FT model (Supplementary Figure S1F) using the same set of eight NHA9 target genes and also demonstrated binding of NHA9 to the eight enhancers in our second model system of NHA9-expressing hHP cells (Figure 1c), allowing us to confirm these findings in primary human hematopoiesis. We next focused attention on the transcription factors MEIS1, HOXA9 and PBX3, as their overexpression is significantly related to adverse prognosis in AML (The Cancer Genome Atlas;6Supplementary Figure S1H) and were previously reported to drive leukemogenesis through the formation of a transcriptional activator complex.7. To test the importance of these three transcription factors in NHA9 pathogenesis, we completed reporter assays in HEK293FT cells by cloning the identified enhancers of MEIS1, HOXA9 or PBX3 into a luciferase reporter vector. A significant 1.6-2.8 fold induction in luciferase activity was observed when NHA9 was co-expressed for all three enhancers, indicating a direct induction of MEIS1, HOXA9 and PBX3 expression through the NHA9 interaction with their corresponding regulatory regions (Figure 1d) (Supplementary Methods). This observation was accompanied by upregulation of all three transcription factors and of three of their known target genes (MYB, MEF2C and FLT3)7 in NHA9-expressing hHP cells (Figure 1e and Supplementary Figure S1I). Gene Expression Profiling performed in three independent NHA9-expressing hHP clones and AMLs from five patients with t(7;11)(p15,p15), confirmed MEIS1-HOXA9-PBX3 overexpression and it was further validated by RT-qPCR anal. in three addnl. NHA9 primary samples (Supplementary Figure S2A). These observations suggested that the NHA9-expressing hHP cells can be sensitive to HXR9, a specific peptide inhibitor of HOXA9 and PBX3 interaction that leads to disruption of the MEIS1-HOXA9-PBX3 complex.8. We tested this hypothesis by treating these cells with HXR9 that resulted in a selective decrease in their viability (Figure 1f and Supplementary Figure S2B-D) (Supplementary Methods) without affecting cell differentiation (data not shown), therefore confirming the relevance of these downstream mediators in driving the oncogenic activity of NHA9. In order to explore other mechanisms driving NHA9 pathogenesis and to better understand its role in transcriptional regulation, we interrogated our ChIP-seq and gene expression profiling data, which revealed both activation and repression of gene expression induced by this fusion oncoprotein (Figure 2a). The cooperation of MLL1 and CRM1 with NHA9 in the upregulation of some target genes has been shown recently by Xu et al.,9, 10 which was also supported by comparing NHA9 target genes identified in our ChIP-seq experiments with MLL1 and CRM1 targets. We found that 25% and 35% of NHA9 target genes were also in common with MLL1 and CRM1 target genes, resp. (Supplementary Figure S2E). Notably, 151 target genes, including MEIS1 and HOXA9, were shared by all three proteins (NHA9, MLL1 and CRM1), suggesting a possible cooperation among these transcription factors in NHA9-driven leukemias. It has also been reported that NUP98, through its FG repeat domain, may interact with transcriptional activator p300 and repressor HDACs,11 allowing us to postulate that transcriptional effects of NHA9 in enhancers could be mediated by these regulators. We first demonstrated NHA9 binding to both p300 and HDAC1 by co-immunoprecipitation experiments (Figure 2b) (Supplementary Methods) and went on to examine their binding potential in a panel of eight regulatory regions of NHA9 target genes (four upregulated and four downregulated target genes) in the presence of the fusion protein by qChIP. These experiments demonstrated selective binding of p300 to the regulatory regions of the upregulated genes MEIS1, HOXA9, PBX3 and AFF3 (Figure 2c), and of HDAC1 to the downregulated genes BIRC3, SMAD1, FILIP1L and PTEN (Figure 2d). Altogether this data suggests that p300 and HDAC1 are selectively recruited by NHA9 at enhancer regions to modulate the expression of genes involved in leukemogenesis. As the interaction of NHA9 with HDAC1/2 was validated by mass spectrometry anal. using the NHA9-expressing HEK293FT model (Proteomics data have been deposited on the ProteomeXchange Consortium via the PRIDE partner repository, data set identifier PXD001828) (Supplementary Methods), we had a mol. rationale for testing HDAC inhibitors (HDACi) in NHA9 AML. It has to be noted that LBH589 did not induce differentiation in NHA9-expressing cells as no significant changes in the number of CD11b pos. cells were observed by flow cytometry anal. post treatment (data not shown). These observations are in accordance with a recent report suggesting the combination of COX or DNMT inhibitors with HDACi for treatment of NHA9 AML patients,16 however in this study we identified the mol. rationale for HDACi therapy as well as a panel of target genes downstream of NHA9 that can be used as biomarkers for response to this treatment. However, the biol. consequences of this therapy, as well as the best dosage-time relation for the translation into clinics need to be further investigated. In summary, NHA9 deregulates the expression of key leukemic genes, including MEIS1-HOXA9-PBX3 complex, through the enhancer binding and the direct interaction of the fusion protein with HDAC and p300 transcriptional regulators. The oncogenic effects of NHA9 can be overcome by HDACi treatment, demonstrating a significant inhibitory effects against NHA9-driven leukemic cells and suggesting a novel approach to treatment of this high-risk group of patients.