MDM2 Promotes Ubiquitin-Independent Degradation of HoxA9: Implications for MLL-Fusion Protein Mediated Transformation.

Chromosome translocations involving the mixed lineage leukemia gene (MLL) are associated with aggressive lymphoid and myeloid leukemias. Inappropriate activation of crucial developmental genes has been proposed as one important mechanism for triggering leukemia by MLL-fusions. For example, it is known that the HoxA9 homeodomain protein, a key regulator of hematopoiesis and embryonic development, can be induced by expression of MLL-fusion proteins, and that this event is essential for MLL-dependent leukemogenesis. Recently, it has been suggested that functional inactivation of p53 by MLL-fusions may also contribute to this process. In this study, we explored the effect of p53 suppression by MLL-fusions on HoxA9 function. Using a variety of experimental strategies including co-immunoprecipitation, protein turnover assay and in vivo ubiquitylation assay, we found that the well-known p53 target MDM2 plays an important role in HoxA9 stability. To examine whether MDM2 is associated with HoxA9, Flag tagged MDM2 and HA tagged HoxA9 expression vectors were co-transfected into human HEK293T cells. MDM2 was then immunoprecipitated with anti-Flag beads. HoxA9 was readily detected in the MDM2 immnunocomplex, indicating that HoxA9 indeed interacts with MDM2. To study the functional role of the interaction between MDM2 and HoxA9, the effect of MDM2 on HoxA9 stability was next investigated. Increasing amounts of an MDM2 expression vector was co-transfected into HEK293T cells with an equal amount of a HoxA9 expression plasmid. Following the transfections, HoxA9 protein expression in the lysates from the different transfections was compared. MDM2 was found to promote the degradation HoxA9 in a dose-dependent manner. This effect was specific, since MDM2 did not interfere with Nup98-HoxA9 fusion protein expression in the control experiment. To further demonstrate the effect of MDM2 on HoxA9 stability, HoxA9 half-life was examined in wild type mouse embryonic fibroblast cells and MDM2 negative mouse embryonic fibroblast cells by the measuring HoxA9 protein turnover rate upon cycloheximide treatment. In wild type mouse embryonic fibroblast cells, the HoxA9 protein half-life is less then 30 minutes while HoxA9 protein half-life is about 3 hours in MDM2 knockout mouse embryonic fibroblast cells. Finally, to elucidate the molecular mechanism by which MDM2 promotes HoxA9 degradation, an in vivo ubiquitylation assay was used to test whether MDM2 is a ubiquitin E3 ligase for HoxA9. MDM2 was found to be incompetent in promoting the ubiquitylation of HoxA9. However, the proteasome inhibitor MG-132 could stabilize HoxA9 protein in HoxA9 transfected HEK293T cells. Taken together, these data suggest that MDM2 regulates HoxA9 stability by a ubiquitin-independent proteasome-dependent pathway. Furthermore, our findings indicate that indirect inhibition of MDM2 through functional inactivation of p53 by MLL-fusions might stabilize HoxA9 protein at post-translation level and this might be an important mechanism for the full functional activation of HoxA9 by MLL-associated fusion proteins.