NKL homeobox gene activity in normal and malignant myeloid cells. NKL homeobox gene activity in normal and malignant myeloid cells

Recently, we have reported a hematopoietic NKL-code which describes normal expression patterns of NKL homeobox genes in early hematopoiesis and lymphopoiesis including T-cell, B-cell and NK-cell development. This code allows the identification of deregulated NKL homeobox genes in lymphoid malignancies. Here, we report normal activities of NKL homeobox genes in myelopoiesis, thus, extending the NKL-code for the hematopoietic system. Analysis of public expression profiling data for developing and mature granulocytes, mast cells, monocytes, macrophages, megakaryocytes and erythrocytes revealed seven active myeloid NKL homeobox genes including DLX2, HHEX, HLX, HMX1, NANOG, NKX3-1 and VENTX. Furthermore, we analyzed public expression profiling data of 251 acute myeloid leukemia (AML) patients identifying 18 deregulated genes. These results indicated that NKL homeobox genes are frequently deregulated in this myeloid malignancy. For detailed analysis we focused on NKL homeobox gene NANOG which acts as stem cell factor and was accordingly expressed just in hematopoietic stem/progenitor cells. We detected aberrant NANOG expression in a small subset of AML patients and in AML cell line NOMO-1 which served as model to investigate upstream and downstream factors of this gene. Karyotyping and genomic profiling excluded rearrangements of the NANOG locus at 12p13. Analysis of NOMO-1 cells treated for NANOG knockdown, expression profiling analyses of HL-60 cells lentivirally transfected for NANOG overexpression, and of public AML patient data revealed regulators and target genes of NANOG. NKL homeobox genes HHEX and MSX1 and the NOTCH-pathway were located upstream of NANOG and HHEX, HLX, VENTX, MYB, CDK6, MAML2 and MIR17HG represented target genes of NANOG in the myeloid context. In conclusion, we described a myeloid NKL-code and several deregulated NKL homeobox genes in AML. We identified for NKL homeobox gene NANOG deregulating factors and downstream activities in AML. These data indicate a common oncogenic role of NKL homeobox genes in myeloid malignancies. Overall design: Expression profiling analyses of HL-60 cells lentivirally transfected for NANOG overexpression.

近日，我们此前已报道了一种造血NKL编码（hematopoietic NKL-code），该编码描述了NKL同源框基因（NKL homeobox gene）在早期造血与淋巴系造血过程中的正常表达模式，涵盖T细胞、B细胞与NK细胞的发育进程。该编码可用于鉴定淋巴恶性肿瘤中失调的NKL同源框基因。
本文中，我们报道了NKL同源框基因在髓系造血中的正常活性，由此扩展了造血系统的NKL编码。通过分析发育阶段与成熟阶段的粒细胞、肥大细胞、单核细胞、巨噬细胞、巨核细胞及红细胞的公共表达谱数据，我们共鉴定出7个具有活性的髓系NKL同源框基因，包括DLX2、HHEX、HLX、HMX1、NANOG、NKX3-1与VENTX。
此外，我们分析了包含251例急性髓系白血病（acute myeloid leukemia, AML）患者的公共表达谱数据，鉴定出18个失调基因。上述结果表明，NKL同源框基因在该髓系恶性肿瘤中频繁发生表达失调。
为开展详细机制分析，我们聚焦于兼具干细胞因子功能的NKL同源框基因NANOG，该基因仅在造血干/祖细胞中正常表达。我们在一小部分AML患者与AML细胞系NOMO-1中检测到了NANOG的异常表达，并以NOMO-1作为模型，探究该基因的上下游调控因子。
核型分析与基因组谱分析排除了12p13位点上NANOG基因座的重排现象。
通过对经NANOG敲低处理的NOMO-1细胞进行表达谱分析、对经慢病毒转染实现NANOG过表达的HL-60细胞进行表达谱分析，结合公共AML患者数据集，我们鉴定出了NANOG的调控因子与靶基因。
NKL同源框基因HHEX、MSX1以及NOTCH通路位于NANOG的上游调控通路中，而HHEX、HLX、VENTX、MYB、CDK6、MAML2与MIR17HG则为髓系背景下NANOG的靶基因。
综上，我们报道了髓系NKL编码，并鉴定出AML中多个失调的NKL同源框基因。我们明确了NKL同源框基因NANOG在AML中的失调调控因子与下游活性通路。上述数据表明，NKL同源框基因在髓系恶性肿瘤中普遍发挥致癌作用。
整体实验设计：对经慢病毒转染实现NANOG过表达的HL-60细胞进行表达谱分析。