The most essential human tumor suppressor protein p53 is mutated in more than 50% of the human cancers. The tumor suppressor function of p53 is tightly controlled by high density post-translational modifications (PTMs), which are localized on N- and C-terminal tails of the p53 protein. Stress-induced C-terminal lysine acetylation of p53 plays a central role in the p53 transcriptional activities that regulates cell cycle arrest, senescence or apoptosis. Our study revealed that p53 recruitment of the co-activator CBP (CREB binding protein) requires association of the conserved bromodomain of CBP (CBP BRD) with p53 at acetylated Iysine 382 (p53K382ac): a specific molecular interaction that is essential for p53-induced transcriptional activation of the cyclin dependent kinase inhibitor p21, involved in G1 cell cycle arrest. The long-term goal of this research plan is to identify small molecule inhibitors (SMIs) of CBP BRD to modulate the p53 function in cancer cells. While multiple acetylation sites in the C-terminal tail of p53 have been reported, specific effects of individual or combined acetylation of these lysine residues on p53 activity remain elusive. Further, despite p53 being a sequence-specific DNA binding transcription factor, the role of PTMs on chromatin as well as p53 in regulating downstream genes is also unknown. The presented preliminary data evaluates acetylation-induced p53 activation in response to DNA damage is dependent on co-activator recruitment, and further, potential of the p21 promoter-directed luciferase reporter system to be developed as a robust cell based assay for automated high throughput screening assay (HTS). We hypothesize that highly specific and selective SMIs for CBP BRD could alter the fate of the cancer cells from growth arrest to apoptosis. A multifaceted approach is proposed to address mechanistic underpinnings of p53 transcriptional activation with emphasis on the role of C-terminal post-translational modifications in p53 activation.
Three specific aims are proposed to achieve this goal: (1) to develop and validate p21 luciferase reporter cell based assay that could be used for HTS to identify SMI binding to CBP BRD;(2) to evaluate CBP BRD binding SMIs by secondary assays to rule out artifacts by in vitro and in vivo approaches;and (3) to modulate p53 activity between cell growth arrest and apoptosis using the newly developed CBP Bromodomain-binding SMIs. The emerging results from the proposed studies are expected to enhance our understanding of the molecular basis of p53 target gene regulation by its C-terminal modifications. Given the central role of p53 in cancer, these studies will have important implications for the prognosis and treatment of human tumors.

Public Health Relevance

The tumor suppressor protein p53 is mutated in atleast 50% of human cancers. The developed cell based assay will identify small molecule inhibitors (SMIs) to modulate p53 function by changing the fate of cancer cells to cell death. The newly developed CBP BRD SMIs will be also used as innovative tools to dissect molecular interactions of p53, which could open new avenues for therapeutic development.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA143662-03
Application #
8196764
Study Section
Drug Discovery and Molecular Pharmacology Study Section (DMP)
Program Officer
Arya, Suresh
Project Start
2009-12-11
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2013-11-30
Support Year
3
Fiscal Year
2012
Total Cost
$307,045
Indirect Cost
$125,897
Name
Icahn School of Medicine at Mount Sinai
Department
Pharmacology
Type
Schools of Medicine
DUNS #
078861598
City
New York
State
NY
Country
United States
Zip Code
10029
Mujtaba, Shiraz; Winer, Benjamin Y; Jaganathan, Anbalagan et al. (2013) Anthrax SET protein: a potential virulence determinant that epigenetically represses NF-*B activation in infected macrophages. J Biol Chem 288:23458-72