P218
NON GENOMIC LOSS OF FUNCTION OF TUMOR SUPPRESSORS IN CML: BCR-ABL PROMOTES P53 NUCLEAR EXCLUSION THROUGH THE INTERACTION WITH IKB-ALPHA
S. Crivellaro1,* C. Panuzzo1, G. Carra1, A. Volpengo1, F. Crasto1, E. Gottardi1, A. Guerrasio1, G. Saglio1, A. Morotti1
1Clinical and Biological Sciences, University of Turin, Orbassano, Italy
Background: The function of tumor suppressors (TS) 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 TS could have tremendous consequences from the therapeutic standpoint. Targeting pathways that
inactivates TS could indeed promote the re-activation of a specific tumor suppressor with strong biological consequences. IκBα is commonly known as the
inhibitor of the transcription factor (TF) NFκB, which in turn regulates the transcription of many genes involved in immune and inflammatory responses, as well
as genes regulating cell proliferation and survival, including IκBα itself. IκBα is also able to physically interact with the tumor suppressor protein p53. In the 20% of blast crisis Chronic Myeloid Leukemia (CML) patients, p53 is mutated, while it was never found mutated/deleted during the chronic phase of the disease. Aims: The aim of this study was to demonstrate that BCR-ABL promotes the formation of a ternary complex with IκBα and p53 in the cytoplasm causing loss of p53 tumor suppressive nuclear pool.
Methods: HEK293T and HeLa cells transfected with IκBα expression vector alone or in combination with Bcr-Abl expression vector and primary cells collected from CML patients at the diagnosis were analysed by immunofluorescence, immunoprecipitation and western blot in order to evaluate IκBα and p53 protein levels, interactions and cellular compartmentalization. Furthermore,
kinase assays have been performed with purified proteins to examine the phosphorylation status of IκBα. CML/healthy subjects and transfected cell line mRNA
were analysed for NFKBIA (IκBα) and p21 expression by Real-TimePCR Results: While assessing the cellular compartmentalization of IkBα in BCRABL transfected HeLa cells, we observed that IκBα is expressed mostly in the cytosol, while in parental HeLa cells IκBα showed a cyto/nuclear localization. Similarly, primary CML cells are characterized by IκBα expression exclusively in the cytosol. Using BCR-ABL transfected cells, we demonstrate that BCRABL physically interacts with IκBα through the BCR portion of the chimeric protein BCR-ABL. in vitro, BCR-ABL does not appear to directly phosphorylate IκBα on tyrosine residues. Next, we investigated whether BCR-ABL regulates IκBα expression and stability. RealTimePCR analysis does not show IκBα mRNA expression differences between normal and CML BM, and between empty and BCR-ABL transfected cell. Incubation of BCR-ABL positive cells with proteasome inhibitor MG-132 shows that BCR-ABL regulates IκBα protein stability at a post-translational level. IκBα is known to negatively regulate the NFkB pathway through the interaction with p65 subunit. Importantly, it was also reported that IκBα can interact with the 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 and that IκBα is physically bound to both p53 and BCR-ABL in the cytosol of CML cells. The delocalization of p53 is associated with impairment of its function. The p21 mRNA levels, a downstream gene of p53, are indeed markedly reduced in CML primary cells, compared to normal bone marrow.
Summary and Conclusions: In this work, we demonstrate that BCR-ABL promotes p53 nuclear exclusion through the interaction with IκBα. Our data suggest
that expression of BCR-ABL could promote non genomic loss of function of the tumor suppressor p53.
