Ramanuj Dasgupta - US grants

[unreadable] DESCRIPTION (provided by applicant): The Wnt/wingless (wg) pathway is one of a core set of evolutionarily conserved signaling pathways that regulates many aspects of metazoan development. Misregulation of the Wnt pathway can be detrimental since mutations in several components are associated with tumorigenesis of the liver, colon, breast and skin. It is therefore crucial to develop and implement new technologies in order to generate molecular tools that may be used to modulate the activity of the Wnt/wg signaling pathway. One of the most important effectors of the Wnt pathway is encoded by the transcription factor, ?-catenin (?-cat)/armadillo (arm). Since Catenin Responsive Transcription (CRT) has been implicated in the genesis of many cancers, it makes a good target for developing therapeutics that could modulate the nuclear activity of ?-cat. Recently, we employed a novel methodology of integrating a "sensitized" chemical genetic high-throughput screen (HTS) with RNA-interference (RNAi) screening technology in order to identify specific small molecule inhibitors of the Wnt pathway in Drosophila cells. Objective/hypothesis: We hypothesize that our primary chemical genetic screen will identify small molecule inhibitors that specifically target the activity of the stabilized pool of ?-cat. In this proposal, we outline experiments to investigate the molecular mechanism(s) by which the candidate small molecules impact the activity of stabilized ?-cat and also identify their protein targets. Moreover, since target identification of small molecules has been traditionally difficult, we also propose to identify the comprehensive protein "interactome" network of ?-cat using mass-spectrometry. Finally, we propose that comparative phenotypic analysis of dsRNA-mediated knockdown of the known and newly identified ?-cat-interacting proteins with that of the candidate small molecules will provide us with a novel method for target identification of the inhibitors isolated in the chemical genetic screen. Specific aims: 1) Investigate the molecular mechanisms by which the small molecules identified in the primary screen impact CRT and Wnt-responsive phenotypes in cultured cells. 2) Identify novel protein interaction partners of ?-cat/arm that may regulate the activity/stability of ?-cat. 3) Perform comparative phenotypic analysis of candidate small molecules and dsRNA-mediated knockdown of ?-cat-interacting proteins and assess whether the candidate small molecules alter the binding of the ?-cat to its known and newly identified cognate protein partners. Study design: For the chemical genetic screen, we will activate the Wnt pathway using the dsRNA-mediated knockdown of the negative regulator, Axin which results in the stabilization and activation of the cytosolic pool of ?-cat. These "activated" cells will be treated with large small molecule libraries to test if any of the individual compounds could inhibit ?-cat mediated activation of transcription (CRT) as judged by activity of the Wnt- responsive luciferase reporter gene (dTF12). The candidate small molecules will be tested for their ability to alter ?-cat's interaction with its known protein partners, such as Tcf, Bcl9/legless (lgs), pygopus (pygo), APC and Axin using FRET and co-immunoprecipitation assays. We will also perform epistasis analysis (in cells) using RNAi of known regulators of the pathway in conjunction with candidate small molecules. This will enable us to determine the site/stage at which the candidate small molecules affect the Wnt pathway. Additionally, we will identify novel protein interaction partners of ?-cat using the TAP-tag (Tandem Affinity Purification) technology with the purpose of finding additional partner proteins that might regulate the activity of the stabilized pool of??-cat. Moreover, we will test if the candidate small molecules could abrogate the interaction between ?-cat and the novel interacting proteins identified in the TAP screen. Finally, we will employ comparative phenotypic analysis to test for similarities between phenotypes obtained from small molecules or dsRNA-mediated knockdown of??-cat protein partners. We will employ cell-based and in vivo reporter assays, immuno- cytochemical and morphology-based assays in order to conduct the phenotypic analysis. Project narrative The Wnt/wingless (wg) signaling pathway is an evolutionarily conserved pathway, which is involved in the regulation of many aspects of cell biology and animal development. Misregulation of the Wnt pathway has also been implicated in a variety of human diseases including cancer of the liver, colon, breast and the skin. The primary goal of this project is to develop and implement new technologies in order to generate molecular tools that may be used to modulate the activity of the Wnt signaling pathway. [unreadable] [unreadable] [unreadable]

Since the previous review, there have been 24 recruits to the Cancer Institute: seven translational, eight basic scientists, and nine clinical. Table 7.5.1 shows recruitments to date. While NYUCI institutional funds were used to support the expense of these recruits CCSG funds were used to fund pilot projects in the laboratories of many of these recruits, and to fund new cores without which these recruitments would not have been possible (e.g. Genomics Facility and siRNA core). As mentioned in Section 6.0 NYUCI recruitment took place in three phases during the last funding period. The first phase focused on key recruits with significant cancer focus that would complement the existing community. Thus the recruitment of Drs. Bhardwaj and O'Neill extended existing excellence in immunology to include immunotherapy as well as adding melanoma to our CCSG disease based programs that already included Genitourinary and Breast Cancer. The Chair of the Department of Pathology, David Roth, MD, PhD, and Co-Program Leader of the Immunology Program loaned the Cancer Institute space for the early recruitments of Drs. Bhardwaj, Dynlacht and O'Neill. This year, Drs. Bhardwaj and Dynlacht have since moved into the Cancer Research Center and Dr. O'Neill to the Vaccine Facility. The second phase focused on increasing depth in the clinical program with specific investments in recruits that would expand clinical services and research capabilities. The Clinical Cancer Center opened July 2004, and recruitment for disease based, programmatically aligned clinicians (e.g. Novik, Axelrod, Tiersten, Chandra, Skinner and others) prior to its opening provided for an environment of multi-disciplinary clinical research and care from its inception. Currently the NYUCI has 12 Disease Management Groups. Finally, with the opening of the Smilow Research Center May 2006, the three research floors of the Cancer Research Center opened and the newest recruits arrived on campus (Dasgupta, Yamasaki, Cho, Krogsgaard, Trombetta). Developmental Funds were earmarked to recruit a Deputy Director for Clinical Research to build the clinical research enterprise and foster collaborations across research entities to translate laboratory findings in the clinic, to recruit a molecular epidemiologist to bridge research within various programs, and build a collaborative and interactive Epidemiology and Prevention Program. These priorities were based upon the initiatives of the Strategic Plan and the recommendations of the EAB. In 2005, William Carroll, MD was named the Deputy Director for the Cancer Institute, responsible for the clinical research enterprise. We were, however, unsuccessful in recruiting a Molecular Epidemiologist, and in 2005, Dr. Roy Shore, the Associate Director for Epidemiology and Prevention took a position in Japan, and recruitment efforts to fill this position became our highest priority. In early 2006, a candidate was in final negotiations, yet decided to stay at his institution. The CI is actively recruiting for both positions and is in preliminary negotiations with one of the final candidates for the Associate Director position;our plan is to have this position filled by the Site Visit.

DESCRIPTION (provided by applicant): The Wnt/wingless (wg) pathway is an evolutionarily conserved cell-signaling pathway that regulates many aspects of metazoan development. Dysregulation of the Wnt pathway has been associated with tumorigenesis of the liver, colon, breast and skin. One of the most important effectors of the Wnt pathway is encoded by the transcription factor, ?-catenin (?-cat). Since Catenin Responsive Transcription (CRT) has been implicated in the genesis of many cancers, it makes a good target for developing therapeutics that could modulate the nuclear activity of ?-cat. Recently, we employed an innovative RNAi-based targeted chemical genetic high-throughput-screen (HTS) to identify novel and specific compound modulators of CRT in Drosophila and human cell lines. Objective/Hypothesis: We hypothesize that our primary screening strategy specifically targets CRT and that the novel compound modulators of the Wnt pathway could serve as effective therapeutic reagents in Wnt- relevant disease and developmental models. Specific Aims: 1) Determine (and improve) the specificity as well as efficacy of candidate small molecules in blocking CRT-induced/dependent phenotypes in cell-based assays; 2) Determine the molecular mechanisms by which candidate small molecules identified in the primary screen impact CRT and identify their protein targets; 3) Test the ability/efficacy of lead inhibitory compounds in blocking Wnt/CRT-dependent phenotypes in xenograft models, as well as in mouse models of Wnt-relevant cancers. Study design: The goal of the primary screen was to identify novel inhibitors of nuclear ?-cat activity that act downstream of the Axin-mediated degradation complex. In order to determine the mechanism of candidate small molecules, we will test their ability to alter ?-cat's interaction with its known protein interaction partners, or their ability to alter the DNA binding properties of ?-cat/TCF-transcriptional complex using co- immunoprecipitation, immunolocalization, and EMSA assays. We will utilize ELISA, and pull-down assays (with purified proteins), together with Surface Plasmon Resonance assays to determine direct binding of candidate compounds to purified ?-cat or other target proteins. We will validate the inhibitory effect of candidate compounds from our pilot screen in a variety of Wnt-responsive mammalian and cancer cell lines, including, HEK293 cells, C57mg mouse mammary epithelial cells, MCF7 human breast adenocarcinoma cell line, and the HCT116 & HT29 colon cancer cell lines. We will assess the effect of these compounds in blocking Wnt/CRT-induced tumor establishment and metastasis models in mouse xenografts, in vivo. We will also perform SAR studies coupled with in silico docking models to improve the efficacy/potency of the novel class of compounds identified in the primary screen. The improved candidate compounds will subsequently be validated empirically using the already optimized cell-based and in vivo assays for Wnt/CRT activity. PUBLIC HEALTH RELEVANCE: The overall goal of this proposal is to develop and implement innovative screening technologies for the identification of novel compound modulators of the Wnt/Wingless signaling pathway. Results from this study will have a broad impact in the development of targeted therapeutics for the treatment of Wnt-induced carcinogenesis.