In vitro transformation of primary human CD34+ cells by AML fusion oncogenes: early gene expression profiling reveals possible drug target in AML

Abstract

Different fusion oncogenes in acute myeloid leukemia (AML) have distinct clinical and laboratory features suggesting different modes of malignant transformation. Here we compare the in vitro effects of representatives of 4 major groups of AML fusion oncogenes on primary human CD34+ cells. As expected from their clinical similarities, MLL-AF9 and NUP98-HOXA9 had very similar effects in vitro. They both caused erythroid hyperplasia and a clear block in erythroid and myeloid maturation. On the other hand, AML1-ETO and PML-RARA had only modest effects on myeloid and erythroid differentiation. All oncogenes except PML-RARA caused a dramatic increase in long-term proliferation and self-renewal. Gene expression profiling revealed two distinct temporal patterns of gene deregulation. Gene deregulation by MLL-AF9 and NUP98-HOXA9 peaked 3 days after transduction. In contrast, the vast majority of gene deregulation by AML1-ETO and PML-RARA occurred within 6 hours, followed by a dramatic drop in the numbers of deregulated genes. Interestingly, the p53 inhibitor MDM2 was upregulated by AML1-ETO at 6 hours. Nutlin-3, an inhibitor of the interaction between MDM2 and p53, specifically inhibited the proliferation and self-renewal of primary human CD34+ cells transduced with AML1-ETO, suggesting that MDM2 upregulation plays a role in cell transformation by AML1-ETO. These data show that differences among AML fusion oncogenes can be recapitulated in vitro using primary human CD34+ cells and that early gene expression profiling in these cells can reveal potential drug targets in AML.

Figure 1. Effects of AML fusion oncogenes on the differentiation of primary human CD34+ cells.

(A) Immunoblotting shows expression of the indicated AML oncogenes in primary human CD34+ cells. (B) CFC assays of primary human CD34+ cells transduced with the indicated oncogenes. Unmagnified plates are shown in the upper panels and representative fields of Giemsa-stained Cytospin preparations from the CFC plates are shown in the lower panels. Differential counts are shown in Table 2. (C) Flow cytometric analysis of myeloid differentiation of cells harvested from the CFC plates. Myeloid (CD33+) cells were gated and the level of CD11b expression was plotted on histograms in comparison to control. (D) Flow cytometric analysis of erythroid differentiation of cells harvested from the CFC plates. Erythroid (CD235+) cells were gated and their level of CD71 expression was plotted on histograms in comparison to control.

Figure 2. Effects of AML fusion oncogenes on the proliferation and self-renewal of primary human CD34+ cells.

(A) Long-term liquid culture of primary human CD34+ cells transduced with the indicated oncogenes. Data are representative of 3 independent experiments. (B) Representative plates from LTC-IC assays of primary human CD34+ cells transduced with the indicated oncogenes. * Due to the much larger number of LTC-ICs in the MLL-AF9 sample, the MLL-AF9 plate was seeded with 1/10 of the sample in order to allow the visualization of discrete colonies. Data are representative of 3 independent experiments. (C) Average numbers of LTC-ICs per 10,000 input cells from 3 independent experiments.

Figure 3. Microarray analysis of the effects of AML fusion oncogenes on primary human CD34+ cells.

Temporal patterns of gene deregulation by the indicated oncogenes. Primary human CD34+ cells were transduced with the indicated oncogenes by nucleofection for the 6-hour time point and retrovirally for the 3- and 8-day time points. Cells were sorted for GFP expression and compared to empty vector controls. The numbers shown are of genes that were up- or down-regulated by 2-fold or more in two separate experiments.

Figure 4. MDM2 overexpression in primary human CD34+ cells transduced with AML1-ETO.

(A) Quantitative RT-PCR confirms early overexpression of MDM2 RNA by CD34+ cells transduced with AML1-ETO. Fold change shown is the average of 3 independent experiments. (B) Immunoblotting shows increased MDM2 protein levels in cells transduced with AML1-ETO. (C) MDM2 protein was quantified by using the ChemiDoc XRS imaging system (Bio-Rad) with Quantity One 1-D software and fold change over control was calculated. Fold change shown is the average of 3 independent experiments.

Figure 5. Nutlin-3 specifically inhibits the proliferation and self-renewal of primary human CD34+ cells expressing AML1-ETO.

(A) Liquid culture of primary human CD34+ cells transduced with empty vector or vector expressing AML1-ETO or MLL-AF9, in the presence or absence of nutlin-3. Data are representative of 3 independent experiments. (B) LTC-IC assays of primary human CD34+ cells transduced with empty vector or vector expressing AML1-ETO or MLL-AF9, in the presence or absence of nutlin-3. Shown are representative LTC-IC plates from 3 independent experiments. * Due to the much larger number of LTC-ICs in the MLL-AF9 sample, the MLL-AF9 plates were seeded with only 1/10 of the sample in order to allow the visualization of discrete colonies. (C) Average numbers of LTC-ICs as percentage of vehicle sample from 3 separate experiments are shown.