Characterization of the Molecular Interplay Between HOXA9 and Ogt in Leukemia

HOXA9 helps maintain the balance between hematopoietic stem/progenitor cell self-renewal and myeloid/lymphoid cell differentiation in the bone marrow (BM). HOXA9 encodes a homeodomain-containing transcription factor, which mediates its function in collaboration with other co-factors, e.g., MEIS1. Several genetic alterations observed in acute myeloid leukemia (AML) patients, e.g., chromosomal translocations involving the MLL gene, are associated with aberrant upregulation of HOXA9 , thus disrupting the hematopoietic balance towards leukemogenesis. In a proteomic screen combining the use of affinity purification coupled to mass spectrometry analysis (AP-MS) and the yeast two-hybrid system (Y2H), we discovered that HOXA9 interacts physically with OGT, an O-linked N-acetyl glucosamine transferase. We have obtained multiple lines of evidence supporting the HOXA9-OGT interaction: i) Y2H; ii) identification of endogenous OGT by affinity purification using HOXA9 as a bait in both murine leukemic blasts and human THP1 AML cells; and iii) reciprocal protein pulldowns in HEK293T cells (Fig. 1). A domain mapping analysis revealed that the HOXA9 domain D62-135 is required for the OGT-HOXA9 interaction. We also demonstrated that the MEIS interaction motif of HOXA9 is GlcNAcylated by OGT. We used a colony forming assay to measure the clonogenic potential of the OGT-interaction defective allele of HOXA9 , i.e., HOXA9 (Δ62-135). We observed a higher number of colonies for HOXA9 (Δ62-135) in comparison to WT HOXA9 (Fig. 2). In light of these results, we hypothesize that the HOXA9-OGT interaction has an inhibitory effect on HOXA9's ability to promote colony formation. This model is also corroborated by an experiment in which co-transduction of OGT with HOXA9 inhibits clonogenesis, further supporting the hypothesis that the OGT has a suppressive effect on HOXA9 . By quantitative RT-PCR, we show that the level of expression of FLT3, a well-characterized target of HOXA9 , is higher in HOXA9 (Δ62-135)-transduced BM cells than in HOXA9 -transduced ones . To identify genes modulated by HOXA9 in an OGT -dependent manner, we analyzed the transcriptome of HOXA9 (Δ62-135)-transduced BM cells by expression profiling studies using next-generation sequencing (RNA-seq). We identified 1,083 and 551 genes that are further up- or further down-regulated with HOXA9 (Δ62-135) in comparison to HOXA9 . Increased chromatin binding of HOXA9(Δ62-135) at the loci of some of these genes was also confirmed by chromatin immunoprecipitation followed by qPCR (ChIP-qPCR). We note that pro-oncogenic genes such as FLT3, LCK and ERG, which have been previously characterized as being HOXA9 -upregulated, demonstrate even higher levels of expression in cells transformed by HOXA9 (Δ62-135) . The most striking observation, though, arose from the integrative analysis of two RNA-seq datasets [ HOXA9 and HOXA9 (Δ62-135)-transformed BM cells] and two ChIP-seq datasets (HOXA9 and OGT ChIP-seq in BM cells). Remarkably, we observed that HOXA9 -downregulated genes that are further downregulated in presence of HOXA9 (Δ62-135) ( N = 82) are specifically enriched for OGT-bound genes ( N = 41 out of 82; P < 4 x 10-10). In contrast, such enrichment is not observed for HOXA9 -upregulated genes ( N = 22 OGT-bound genes out of 108 HOXA9/HOXA9 (Δ62-135)-upregulated genes; no statistical enrichment). Thus, our transcriptome-wide analysis of the OGT -dependent regulation of HOXA9 gene targets highlights the critical importance of HOXA9 as a negative regulator of transcription, rather than a positive regulator. This observation is further validated by the analysis of gene expression profiles in human leukemia samples. Indeed, a larger than expected number of the human orthologs of the murine HOXA9 -downregulated genes have a low level of expression in MLL translocation-driven AML patient samples compared normal samples. In comparison, such trend is not as prevalent for the human orthologs of the murine HOXA9 -upregulated genes. In conclusion, our data support a model in which OGT inhibits HOXA9's ability to transform primary bone marrow cells, thus defining OGT as a potential tumor suppressor of HOXA9 -driven AML. The biomedical relevance of the OGT-HOXA9 interaction to HOXA9 -driven leukemogenesis is being investigated in vivo using a HOXA9 -induced mouse model of AML. This project is supported by the WES Foundation and the American Society of Hematology. ![][1] Disclosures No relevant conflicts of interest to declare. [1]: pending:yes