Transcription Regulation of HOX Genes in Normal Hematopoiesis and Leukemogenesis in Children

The homeodomain genes (HOX genes) encode a family of highly conserved transcription factors that play fundamental roles during embryogenesis. HOX genes are also important regulators in hematopoiesis. In leukemogenesis, dysregulated expression of HOX genes has been found. Despite many correlative studies, the mechanism of establishment of leukemia specific HOX gene expression patterns in hematopoietic cells remains to be elucidated.

Histone methylases and demethylases (Trithorax (TrxG), JMJD3 and Polycomb-group (PcG) genes) are chromatin modifiers regulating global gene expression through chromatin remodeling in many biological processes. PcG genes can also interact with DNA methyltransferases and alter their activity. Our previously published data showed that HOX gene expression correlated with the level of DNA methylation. These data together with the stabilizing function of PcG genes on HOX expression in embryogenesis suggest the involvement of histone modifiers in the regulation of hematopoietic HOX gene expression.

To investigate the regulation of HOX expression in leukemogenesis, we determined mRNA levels of the representative groups of HOX genes (HOXA, HOXB, CDX1/2), PcG genes (EZH2, BMI1), MLL and demethylases (JMJD3, UTX) in samples of childhood AML (N=41) and healthy controls (N=5). We also studied the dynamics of HOX genes and chromatin modifiers in preleukemic and diagnostic samples of a patient who underwent secondary leukemia.

Quantification of gene expression was performed using qPCR assays as previously described.

Expression patterns for the majority of HOX genes differed significantly among morphologically defined subgroups of AML with AML M3 having the lowest expression of all HOX genes. Children with AML M5 expressed HOXA cluster at the highest level, while HOXB genes were highly expressed in M5 and M4 subtype. Subgroups defined according to molecular genetics showed similar results. The presence of PML/RARa fusion gene was associated with very low expression of all HOX genes whereas MLL+ and CBFb/MYH11+ patients expressed higher levels of HOXA genes. We also assessed the prognostic significance of particular HOX genes and found that the HOXA cluster was expressed at very low levels in standard risk cases compared to the high risk group (P<0.0001 for most HOXA genes), which is in concordance with previously published results in adult AML (Andreeff et al. 2008).

Determination of mRNA levels of histone modifiers showed an overall level of high expression across various AML subgroups. Nevertheless, some were uniformly expressed in AML patients (EZH2, MLL), while others were differentially expressed with the lowest level in the M3 subtype (BMI1, JMJD3). Interestingly, we found a correlation between HOX gene expression and levels of JMJD3, which was mainly evident in CBFb-MYH11+, PML-RARa+ and AML1-ETO+ patients. JMJD3 levels were also correlated with another demethylase, UTX. A positive trend between HOX gene expression and JMJD3 was identified in healthy controls as well.

Analysis of the sample from preleukemic period of the patient with secondary leukemia (secALL with MLL translocation) allowed us to study the dynamics of HOX gene expression during leukemogenesis. The diagnostic secALL sample showed an expression pattern of HOX genes typical for MLL+ leukemia. However, the profile of HOX genes in preleukemic sample (16 months before secALL) resembled the pattern found in healthy controls. Nonetheless, 90% of these seemingly normal hematopoietic cells were confirmed by FISH analysis to carry MLL/FOXO3A. Thus, even though MLL is a well known regulator of HOX genes, there must be an additional mechanism, that establishes the expression pattern of HOX genes typical in MLL+ patients.

In summary, we identified different expression patterns of HOX genes in particular subtypes of childhood AML that significantly correlated with prognosis. Our results indicate that histone modifiers JMJD3 and UTX might be involved in the regulation of HOX gene expression. Moreover, these data also suggest that histone demethylases could cooperate with specific genetic aberrations implicated in chromatin remodeling on regulation of HOX genes.

The analysis of secondary leukemia suggests that additional alterations are required to deregulate HOX expression in at least some MLL+ patients.

No relevant conflicts of interest to declare.