t(4;11) leukemias display addiction to MLL-AF4 but not to AF4-MLL

Abstract

The most frequent MLL-gene rearrangement found in leukemia is a reciprocal translocation with AF4 on chromosome 4 resulting in the formation of the MLL-AF4 and the AF4-MLL fusion genes. The oncogenic role of MLL-AF4 is documented but the significance of the reciprocal product - AF4-MLL in leukemia is less clear. In the human leukemia cell lines - RS4;11 and SEMK2-M1, both of which express MLL-AF4 and AF4-MLL, we knocked down the expression of AF4-MLL using siRNA. Loss of AF4-MLL had no effect on the growth of either RS4;11 or SEMK2-M1 cells. Furthermore, in SEMK2-M1 cells there were no changes in cell cycle or apoptosis with loss of AF4-MLL. In contrast, knockdown of MLL-AF4 significantly inhibited growth of both RS4;11 and SEMK2-M1. Additionally, in SEMK2-M1 cells, loss of MLL-AF4 led to G2/M cell cycle arrest and increased apoptosis. Overall, these results demonstrate that in t(4;11) leukemia, the MLL-AF4 fusion protein is critical for leukemia cell proliferation and survival while the AF4-MLL fusion product is dispensable.

Figure 1. siRNAs againt MLL-AF4 and AF4-MLL specifically knockdown their respective targets

Human leukemia cell lines were electroporated with various siRNA duplexes as described and gene expression measured by real time quantitative RT-PCR at 24 hours. Bar graphs show relative expression of AF4-MLL (der4a and der4b), MLL-AF4, AF4 and MLL in RS4;11 (A, C) and SEMK2-M1 (B) cells while HPB-NULL (D) served as a control cell line since it lacks the MLL-translocation. Relative expression levels were derived using beta-actin as internal control and normalized to levels in the scrambled control condition. Panel A shows data from one representative experiment in RS4;11 while for the remaining graphs data represent mean ± std. err of 3 separate experiments. Panel E shows Western blot results for MLL-AF4 knockdown in SEMK2-M1 probed with anti-AF4 (upper panel) or anti-MLL^N (lower panel) antibodies. Lane labels on top represent the siRNA treatments – control (C) or MA6 (S) - the SEMK2-specific MLL-AF4 siRNA. Protein bands are labeled on the left and the antibodies used in each blot are on the right.

Figure 2. Knockdown of AF4-MLL has no effect on cell growth

Leukemia cell lines SEMK2-M1, RS4;11 and HPB-NULL were electroporated with various siRNAs as described and viable cells were counted by Trypan blue exclusion. Daily cell counts were normalized to those on day 4 which were considered 100%. Graphs below depict growth curves for each cell line with time since electroporation indicated on the x-axis. Data represent mean ± std. err. of 3 separate experiments. In the SEMK2-M1 repeat electroporation experiment, SEMK2-M1 cells were electroporated sequentially on days 0, 2, 4 and 7 (indicated by arrows on the x-axis) observed for a total of 9 days. Viable cells were counted on the days of electroporation and normalized to counts on day 9 (100%).

Figure 3. Knockdown of AF4-MLL has no effect on cell cycling or cell survival

SEMK2-M1 cells were electroporated with various siRNA duplexes as described. Cell aliquots were harvested every 24 hours for a total of 4 days and DNA content analyzed by flow cytometry. Line graphs represent the proportions of cells in the subdiploid fraction [<2N, (A)] and various phases of the cell cycle [G0/G1 (B), S (C) and G2/M (D)] with the x-axis representing time in days. Depicted are mean ± std. err. of three separate experiments.