Role of the MLL-AF4 chimeric protein in the molecular pathogenesis of t(4;11) acute lymphoblastic leukemia
Imperato, Maria Rosaria (2012) Role of the MLL-AF4 chimeric protein in the molecular pathogenesis of t(4;11) acute lymphoblastic leukemia. [Tesi di dottorato] (Inedito)
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Chromosomal rearrangements involving the Mixed Lineage Leukemia (MLL) gene are associated with very aggressive forms of acute lymphoblastic leukemia (ALL), often refractory to conventional therapies. In particular, patients carrying the translocation t(4;11)(q21;q23) have the worst prognosis among patients with other MLL-associated malignancies. Although it has been largely shown that the MLL-AF4 fusion protein has the capability to up-regulate genes involved in the self-renewal/differentiation balance of the hematopoietic stem cell, the mechanism induced by this oncoprotein is still poorly understood. Previous functional proteomic studies performed in our group identified the molecular partners of the native AF4 protein, the most common MLL translocation partner in infant ALL, and confirmed that this protein is deeply involved in a complex protein network, important for the regulation of the Pol II-dependent transcription. In this study, we cloned in an eukaryotic expression vector the complete cDNA encoding MLL-AF4 and transiently expressed the recombinant protein in Hek293 cells. In order to understand which molecules take part in the aberrant pathway induced by the MLL-AF4 oncoprotein, we aimed to identify some of its molecular interactors, starting from the proteins that are already known to interact with AF4. We showed that MLL-AF4 binds to CdK9 that by interacting with cyclin T1 forms the positive elongation factor (P-TEFb), which is involved in the activation of the Pol II elongation machinery. We also found that MLL-AF4 interacts with CRSP130 and CRSP33, two members of the so-called “Mediator Complex”, thus suggesting that MLL-AF4 is involved in the regulation of the Pol II-dependent transcription. Interestingly, we found that MLL-AF4 also interacts with the tyrosine-kinase receptor FGFR2, and with a protein belonging to the family of 14-3-3s (the isoform θ), involved in diverse intracellular pathways. Moreover, we observed down-regulation in the expression of HoxA9, one of the MLL target genes, in the cells co-expressing both recombinant MLL-AF4 and 14-3-3 θ, thus suggesting that this interaction could modulate transcriptional processes induced by MLL-AF4. Elucidating the role of fusion protein interactors such as FGFR2 and 14-3-3 θ is very important for identifying new molecular targets for the therapy of the MLL-AF4-dependent B-cell ALL.
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