DISSECTING THE P53 PATHWAY BY MEANS OF SMALL MOLECULE- MEDIATED REACTIVATION AND COMPUTATIONAL BIOLOGY

The p53 tumor suppressor is a highly connected signaling molecule with paramount importance to tumor suppression. It is activated by diverse stress signals and in response activates the transcription of affector genes that can induce several cellular processes, most importantly cell-cycle arrest and apoptosis. Consequently, the p53 tumor suppressor pathway is inactivated in a large proportion of cancers, across almost all tumor types. In up to 50% of tumors, inactivation is achieved by point mutations in the p53 gene. In tumors that retain wild-type p53, mechanisms of inactivation mainly converge on de-regulation of MDM2, the E3 ligase and the main destructor of p53. This thesis presents the identification and further investigations of the precise mechanism of action of a novel p53-reactivating compound dubbed RITA (Reactivation of p53 and Induction of Tumor cell Apoptosis). RITA was identified in a cell-based screening assay, using a pair of isogenic colon cancer cell-lines that differ only in the status of p53. Thus, RITA was selected based upon its ability to selectively inhibit the growth of the p53-carrying cell line without toxicity on the p53 null derivative. RITA was shown to bind directly to p53 and disrupt its complex with MDM2, thereby stabilizing the protein. Consequently, p53 levels increased in cells leading to massive programmed cell death. Furthermore, RITA inhibited tumor growth in a mouse xenograft model without apparent toxicity to the animals. In order to further characterize the p53 response induced by RITA, we investigated the global transcriptional response upon RITA treatment by oligonucleotide microarrays. Pathway analysis revealed an unusually selective induction of the apoptotic branch of the p53 pathway. Further investigation revealed that hnRNP-K, a transcriptional co-activator of p53, was down-regulated after RITA treatment causing deficient cell-cycle arrest and thereby guiding the p53 response towards induction of apoptosis. p21, the major cell-cycle arrest target gene of p53 was shown not only to be weakly induced due to hnRNP-K reduction, but also degraded on protein level. We present evidence for a model where the cause of both hnRNP-K and p21 degradation is traced back to MDM2, which, after being displaced from p53 by RITA and thereby unable to degrade its primary target, instead induced degradation of these two proteins. In the context of pharmacological reactivation of p53 by RITA, it therefore seems that MDM2 goes out of character and actually co-operates with p53 to boost the apoptotic response. In the third paper we introduce a method, called revarray (from Reverse-engineering Microarray), for analyzing the transcriptional program that underlies the changes observed in an oligonucleotide microarray experiment. The underlying transcriptional model is based on combinations of modules of either single or clusters of Transcripion Factors (TFs), and properly takes into account whether the module is an activator, a repressor or an unspecific enhancer. We apply the method on the p53 response upon treatment with three p53-activating compounds – 5-Fluorouracil, RITA and Nutlin. The results suggest that the FOX family of transcription factors could be important co-factors of p53 and that repression of genes by p53 seems to play an important role in apoptosis induction. LIST OF PUBLICATIONS I. Issaeva N, Bozko P, Enge M, Protopopova M, Verhoef LG, Masucci M, Pramanik A, Selivanova G, . Small-molecule RITA binds to p53, blocks p53HDM2 interaction and activates p53 function in tumors. Nat Med. 2004 Dec;10(12):1321-8. Epub 2004 Nov 21. II. Enge M, Bao W, Hedstrom E, Jackson SP, Moumen A, Selivanova G. , MDM2-Dependent Downregulation of p21 and hnRNP-K Provides a Switch between Apoptosis and Growth Arrest Induced by Pharmacologically Activated p53. Cancer Cell. 2009 Mar 3;15(3):171-83. III. Enge M, Nikulenkov F, Turunen M, Taipale J, Kel A and Selivanova G. , Reverse-engineering microarray data applied to the p53 transcriptional response. Manuscript. ASSOCIATED PUBLICATIONS IV. Grinkevich V, Nikulenkov F, Shi Y, Enge M, Bao W, Maljukova A, Sangfelt O, Gluch A, Kel A, Selivanova G. Ablation of key oncogenic pathways by RITA-reativated p53 is required for efficient apoptosis. Cancer Cell May 2009. V. Hedstrom E, Issaeva N, Enge M, Selivanova G . Tumor-specific induction of apoptosis by a p53-reactivating compound. Exp Cell Res. 2009 Feb 1;315(3):451-61. Epub 2008 Dec 3.