Depletion of the Leukemic Fusion Protein MLL-AF4 with Short Interfering RNAs (siRNAs) Affects Post-Translational Modification of Aldolase A.

The chromosomal translocation t(4;11) marks a therapy-resistant infant leukemia with very poor prognosis. It results in the expression of two fusion-proteins, MLL-AF4 and AF4-MLL. We addressed the role of MLL-AF4 in t(4;11) positive SEM cells by siRNA-mediated suppression. Depletion of MLL-AF4 results in induction of apoptosis, inhibition of proliferation, decrease in colony formation and diminished leukemic engraftment in vivo. Currently, we are analyzing global changes in protein expression. For that, we compare the proteome of MLL-AF4 depleted SEM cells with those of control cells. The analysis is performed by 2D-gelelectrophoresis followed by mass spectrometry identification and immunoblot validation of differentially expressed spots. One of these spots was identified as Aldolase A. Comparison of MLL-AF4 depleted SEM cells with control cells showed neither change in mRNA levels nor in absolute protein levels of Aldolase A. Two-dimensional western blotting, however, revealed differences in the protein pattern, suggesting changes in Aldolase A modifications upon MLL-AF4 depletion. These analyses will provide us with a better insight into the effects of siRNA-mediated MLL-AF4 knockdown on the proteome, and may enable us to identify new targets for molecular therapeutic approaches.