Myc oncoprotein-cofactor interactions : Function, regulation and targeting

The members of the Myc-family of proto-oncoproteins c-Myc, N-Myc and L-Myc are transcription factors exerting their effect via protein-protein interactions. The Myc proteins are basic-helix-loop-helix-leucine-zipper (bHLHzip) transcription factors, which after heterodimerization with their obligatory partner Max, bind to E-boxes in target gene promoters and regulate the expression of 10-15% of all genes by the recruitment of different cofactors. By this mechanism Myc affects cellular functions like growth, proliferation and apoptosis. Deregulation of Myc occurs in many types of tumors and is often associated with an aggressive disease and poor prognosis. In mouse models inactivation of Myc leads to tumor regression with reversible and tolerable side effects. This renders Myc an attractive target for anti-cancer therapy. The work in this thesis can be divided into two parts, one focusing on elucidating the function of Myc in the nucleolus, which is the ribosome production site in the nucleus. The other part focuses on protein-protein interactions and how to inhibit these specifically. Firstly, we investigated the role of c-Myc in the nucleolus. We show that c-Myc associates to rDNA and directly activates polymerase I transcription in response to mitogenic stimuli. Further, we show that the molecular mechanism involves recruitment of histone acetylases and parts of the ubiquitin-proteasome system, similar to the mechanism described for c-Myc activation of polymerase II target genes. Hence, c-Myc is a master regulator of translation, coordinating the activity of all three polymerases. Secondly, we have developed a technique to detect endogenous protein-protein interactions in situ. This method called P-LISA (recently renamed to in situ PLA), is based on proximity ligation and rolling circle amplification and is sensitive enough to detect individual complexes in both cell lines and clinical material. Finally, we used a protein fragment complementation assay in a cell based screen to identify small molecular inhibitors of the Myc-Max interaction. Two of the identified compounds are described in this thesis. The first is a compound that selectively targets the N-Myc-Max interaction, hence named Inhibitor of the NMyc:Max Interaction (INMI). It reduces cellular proliferation rate in an N-Myc dependent manner and inhibits N-Myc dependent transformation. This is the first report of an N-Myc:Max inhibitor and a proof of principle that it is possible to achieve selectivity between c-Myc and N-Myc. The second compound, Terminator of Myc (ToM), has an indirect mechanism of action, increasing the turnover rate of Myc and inducing apoptosis in a Myc-dependent manner. Both these compounds have a potential use as anti-cancer therapy. LIST OF PUBLICATIONS I. Arabi ,A, Wu S, Ridderstråle K, Bierhoff H, Shiue C, Fatyol K, Fahlén S, Hydbring P, Söderberg O, Grummt I, Larsson LG, Wright A. c-Myc associates with ribosomal DNA and activates RNA polymerase I transcription. Nature Cell Biology, 2005, 7, 303-310 II. Söderberg O, Gullberg M, Jarvius M, Ridderstråle K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG, Landegren U. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nature Methods, 2006, 3, 995-1000 III. Ridderstråle K, Hydbring P, Larsson LG. Identification of a low molecular weight compound that targets the NMyc-Max interaction and inhibits N-Myc-dependent transformation. Manuscript IV. Ridderstråle K, Hydbring P, Mannich-Uggla C, Yan Q, Larsson LG. Identification of a small molecule that increases Myc turnover and induces apoptosis in Mycand transformation-dependent manner. Manuscript