NON GENOMIC LOSS OF FUNCTION OF TUMOR SUPPRESSORS IN CML: BCR-ABL PROMOTES P53 NUCLEAR EXCLUSION THROUGH THE INTERACTION WITH IKB-ALPHA

Introduction. The function of tumor suppressors can be tightly controlled by various non-genomic mechanisms, such as epigenetic silencing, regulation by non-coding RNAs and post translational modifications. The identification of mechanisms that cause non genomic loss of function of tumor suppressors could have tremendous consequences from the therapeutic standpoint. Targeting pathways that inactivates tumors suppressors could indeed promote the re-activation of a specific tumor suppressor with dramatic biological consequences. In this work, we show that BCR-ABL interacts with IkBα which in turn promotes p53 nuclear exclusion in Chronic Myeloid Leukemia. Methods. HEK293T and HeLa cells transfected with IkBα expression vector alone or in combination with Bcr-Abl expression vector and primary cells collected from Chronic Myeloid Leukemia patients at the diagnosis were analysed by immunofluorescence, immunoprecipitation and western blot in order to evaluate IkBα and p53 protein levels, interactions and cellular compartmentalization. Furthermore, kinase assays have been performed with purified proteins to examine the phosphorylation status of IkBα. Results. While assessing the cellular compartmentalization of IkBα in BCR-ABL transfected HeLa cells, we observed that IkBα is expressed mostly in the cytosol, while in parental HeLa cells IkBα showed both cytosolic and nuclear localization. Similarly, primary CD34 positive CML cells are characterized by IkB-alpha expression exclusively in the cytosol. Using BCR-ABL transfected cells, we demonstrate that BCR-ABL physically interacts with IkBα through the BCR portion of the chimeric protein BCR-ABL. In vitro, BCR-ABL does not appear to directly phosphorylate IkBα on tyrosine residues. IkBα is known to negatively regulate the NF-kB pathway through the interaction with p65 subunit. Importantly, it was also reported that IkB-alpha can interact with the tumor suppressor p53, with consequent inhibition of some of its functions. Interestingly, here we observed that in CML primary cells p53 is also delocalized into the cytosol. Strikingly, we observed that IkBα is physically bound to both p53 and BCR-ABL in the cytosol of CML cells. Discussion. In this work, we demonstrate that BCR-ABL promotes p53 nuclear exclusion through the interaction with IkBα. Our data suggest that expression of BCR-ABL could promote non genomic loss of function of the tumor suppressor p53.