Pathogenesis of Acute Myeloid Leukemia: In Vitro Studies Using Primary Human Cells.

Abstract

Many cases of acute myeloid leukemia (AML) are associated with non-random chromosomal rearrangements and most of these result in fusions involving retinoic acid receptor α (RARα), CBF transcription factors, MLL, and nucleoporins. Here, we report the effects of key members of these four major groups of AML-associated chimeric fusion proteins on differentiation, proliferation, self-renewal and gene expression in primary human CD34+ hematopoietic cells. We expressed the PML-RARα, AML1-ETO, MLL-AF9 and NUP98-HOXA9 fusion genes in human peripheral blood CD34+ cells by retroviral transduction and compared them to cells transduced with empty vector. By colony forming assays, morphological examination and flow cytometric immunophenotyping we found that PML-RARα causes some degree of myeloid differentiation block. AML1-ETO had no obvious effect on differentiation, while MLL-AF9 and NUP98-HOXA9 caused a block in both myeloid and erythroid differentiation. All fusion oncoproteins, except PML-RARα, induced dramatic long-term proliferation in liquid culture and a marked increase in the numbers of primitive long-term culture-initiating cells (LTC-ICs). In order to understand the molecular basis of these effects, gene expression profiling was performed for each fusion gene by oligonucleotide microarray analysis at 3 different time points (6 h, 3 days, and 8 days post transduction). At the 6 h time point both AML1-ETO and PML-RARα caused extensive changes in gene expression with a predominance of downregulated genes. The number of genes dysregulated by AML1-ETO and PML-RARα decreased dramatically at the 3-day time point. These data are consistent with the fact that these two oncoproteins are DNA-binding transcription factors that are known to repress transcription. In contrast, MLL-AF9 and NUP98-HOXA9 altered the expression of fewer genes at the 6 h time point with a preponderance of upregulated genes; at the 3 day time point, the number of genes dysregulated by NUP98-HOXA9 and MLL-AF9 increased. These findings suggest that NUP98-HOXA9 and MLL-AF9 may have delayed effects that are not due to direct transcriptional regulation. Homeobox transcription factors were upregulated by both MLL-AF9 and NUP98-HOXA9, but not by either AML1-ETO or PML-RARα. The results of the biological assays and gene profiling show marked similarities between NUP98-HOXA9 and MLL-AF9, and suggest that they transform cells by similar pathways that are different from those used by AML1-ETO and PML-RARα.