The setup of the Bernal Laboratory was completed late in the fall of 2010. The setup of the laboratory included the installation of a liquid chromatography mass spectrometer for the characterization and purification of biologically active molecules and the setup of an asynchronous, high throughput peptide synthesizer. These instruments form the core infrastructure of the Bernal laboratory, and they are essential components in our synthesis work. Within a span of three months, we were able to synthesize multi-milligram quantities of a wide variety compounds covering not only the p53 project, but other newer research endeavors. At this point, we have the capability to produce materials in high purity with chemical yields exceeding 95% per synthetic step. This has allowed us to maximize the efficiency of our synthetic efforts. We have also secured the assistance of the members of the Protein Chemistry Core in the AIDS and Cancer Virus Program at NCI-Frederick for the complete characterization and quantification of all of the compounds we have synthesized. Previously, the availability of compounds for biological studies was the biggest bottleneck in our work. We have fully surmounted that obstacle, and the synthesis of compounds is now part of the daily routine in our group. The p53 project in the Bernal Lab encompasses a broad spectrum of research endeavors. The inspiration for this work arose from the use of the hydrocarbon-stapled peptide SAH-p53-8. The sequence of this peptide is based on the transactivation domain of the p53 protein. The sequence has been optimized to include two hydrocarbon cross-linking amino acids, the three essential residues required for binding to the oncoproteins HDM2 and HDMX, and other modifications that enable it to traverse cell membranes. We have shown that this compound is capable of reactivating the p53 pathway in cells that overexpress either HDM2 or HDMX. While this finding is significant in the context that SAH-p53-8 is the only compound disclosed to date that is capable of disrupting p53-HDM2 and p53-HDMX protein complexes, this capability enabled us to elucidate a mechanistic framework for determining which cancer cells will be susceptible to single agent HDM2 or HDMX inhibition and overcoming p53 suppression in a resistant cell through synergistic targeting of HDM2 and HDMX. These results were published in November of 2010 (see: Bernal, et al. Cancer Cell 2010, 18, 411). As a segway to the research published in Cancer Cell, we embarked on a research collaboration with the group of Dr. Jean-Christophe Marine from the Laboratory for Molecular Cancer Biology at the Catholic University of Leuven in Belgium. Dr. Marines research entails the determination of the causative agents responsible for the transformation of melanocytes into melanoma. Dr. Marines research has determined melanocyte specific overexpression of HDMX cooperates with mutations in Ras to promote the manifestation of melanoma. Given the utility of SAH-p53-8 in studying the p53-HDM2-HDMX axis, we set forth a plan to identify the key mechanisms that disrupt the function of p53 in melanoma. Per our collaborative plan, we synthesized a large batch of SAH-p53-8 for use in the Marine laboratory for qPCR and animal studies. At the same time, we evaluated the efficacy of SAH-p53-8 alone and in combination with Nutlin-3 in a wide variety of melanoma cells derived from patients. Gratifyingly, we have found that the molecular blueprint we uncovered in our previous work indeed holds for melanoma. The results translate directly to the p53 transcription studies and the melanoma animal models studied in the Marine lab. The final experiments in this project are currently in progress, and we expect to have a manuscript submitted for publication before the end of 2011. While we are encouraged by the prospect of translating SAH-p53-8 to the clinic, we have found that despite its solid pharmacodynamic effects, its pharmacokinetic parameters preclude it from functioning effectively as a therapeutic. This was evidenced by its failure to pass the first layer of screening in the NCI-60. In order to circumvent this problem, we have developed a proposal to develop a small molecule screen using the techniques that were published in the Cancer Cell manuscript. The plan focuses on the use of high throughput fluorescence polarization assays to find selective inhibitors of the p53-HDMX interaction. We are currently in process of validating the assay for high throughput, and we are involved in discussions with members of NCGC to execute this project. The aforementioned study is directly linked to the study of the transcriptional modulation of p53;however, p53 is known to have many other functions that are independent of its ability to function as a transcription factor. These non-transcriptional functions comprise a wide variety cellular processes ranging from mitochondrial apoptosis and stress responses to cell motility and the maintenance of the structural integrity of the cell. Given that the majority of all cancers contain mutations in the sequence of the p53 protein, exploiting its cytoplasmic functions provides a novel avenue to rescue the tumor suppressor functions of mutant p53 despite its inability to activate the transcriptional machinery. Our first observation came about from an experiment which showed reactivation of the apoptosis machinery in a cell line engineered to express the dominant-negative p53-DD mini-protein. Osteosarcoma cells expressing p53-DD (SJSA-DD) treated with SAH-p53-8 displayed a marked increase in caspase-3/7 activation, a hallmark of apoptosis. This result was also replicated in a lymphoma cell line (OCI-Ly3) whose endogenous p53 protein is truncated and lacks a nuclear localization sequence. In order to study the protein-protein interactions that govern the induction of apoptosis in cells with defective p53, we have performed experiments in isolated mitochondria treated with several variants of the SAH-p53 peptides. The data show that, indeed, SAH-p53s induce cytochrome c release while the known inactive stapled p53 peptide SAH-p53-8F19A is completely inert, demonstrating the p53 dependence on this process. In addition to our findings on the reactivation of apoptosis, we have also noticed significant changes in the structural integrity of cells treated with SAH-p53 which also possess defects in endogenous p53. We have conducted cell migration studies in a series of breast cancer cell lines with different degrees of severity in the impairment of p53 function. These experiments have shown that while reactivation of p53 with SAH-p53-8 can induce apoptotic cell death in the breast cancer lines with wild type p53 (such as MCF-7), the same stimulus impairs cell motility in invasive cell lines with mutations in p53 (i.e. MDA-MB-231). Moreover, immunohistochemical evidence shows vestiges of mitotic catastrophe with remnants of actin filaments, suggesting that these effects are mediated by the collapse in cellular structural integrity. This project comprises the majority of the work currently being done in the Bernal Laboratory. We believe that using the tools of chemical synthesis and chemical biology will allow us to investigate protein-protein interactions and functions that, to this date have been elusive.
|Gembarska, Agnieszka; Luciani, Flavie; Fedele, Clare et al. (2012) MDM4 is a key therapeutic target in cutaneous melanoma. Nat Med 18:1239-47|
|de Lange, Job; Teunisse, Amina Fas; Vries, Matty Verlaan-de et al. (2012) High levels of Hdmx promote cell growth in a subset of uveal melanomas. Am J Cancer Res 2:492-507|
|Bernal, Federico; Wade, Mark; Godes, Marina et al. (2010) A stapled p53 helix overcomes HDMX-mediated suppression of p53. Cancer Cell 18:411-22|