Abstract

Studies in mice and humans suggests that HOX genes integrate the myelodysplastic and leukemogenic activities of different oncogenes. HOXA9 has been identified as a major downstream target in patients with MLL translocations and amplifications. Since upregulation of HOXA9 and other specific HOX genes is more frequent than MLL deregulation, additional upstream pathways have to be postulated. Given the fact that HOXA9 is targeted by several yet to be identified mechanisms, we asked whether other members of the HOX9 paralog group are also highly expressed in myeloid malignancies. Microarray gene expression analysis of 449 AML patients revealed that besides HOXA9 (61% of all cases), HOXB9 is upregulated in 15% of the cases, whereas the overexpression of HOXC9 and HOXD9 expression is a very rare event (>1% of all cases). High levels of HOXB9 expression did not correlate with low levels of HOXA9 expression ruling out the possibility that HOXB9 compensates for a lack of HOXA9 activity in the leukemogenesis of a HOXA9 negative AML cases. Moreover, within the cytogenetically defined subgroups inv(16), t(15;17), t(8;21) that have no HOXA9 expression at all, HOXB9 was also low or absent. The analysis of 27 AML cell lines revealed a gene expression pattern of HOXA9 and HOXB9 that reflected the frequency of overexpression found in patient samples (14/27 showed HOXA9 expression; 6/27 showed HOXB9 expression). Interestingly, the absolute expression level of HOXB9 mRNA in cell lines is about 10fold higher than HOXA9 as measured by real-time RT-PCR. Western-blotting with an antibody recognizing both HOXA9 and HOXB9 confirmed this result on the protein level. However, the analysis of the absolute levels in normal CD34+ bone marrow cells revealed an inverse ratio, with very low levels of HOXB9. Taken together these results indicate that HOXB9 expression is aberrant in the leukemic clone.

To identify downstream pathways driven by HOXB9 overexpression we designed two highly efficient siRNAs targeting HOXB9 and knocked down its expression by retroviral transduction. In comparison to cells expressing a control siRNA, the resulting cell lines showed <5% of the control HOXB9 protein levels and a reduction in the cellular growth rate. Consequently, HOXB9 expression is required for the rapid growth of these AML cells. To gain insight into the molecular mechanisms underlying this phenotype we are performing global gene expression analysis. Gene set enrichment analysis (GSEA) and related approaches will be used to extract relevant candidate pathways for further analysis.

Our results indicate that aberrant expression of HOXB9 inhibits the differentiation of leukemic myeloid progenitor cells and maintains the cells of the leukemic clone in an undifferentiated and rapidly proliferative state.

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