JHU/CCR Fellowship in Molecular Targets and Drug Discovery Technologies Project Details

Project Sponsor/Mentor:	Phillip A. Dennis, M.D., Ph.D.
Title:	Principal Investigator
Address:	NCI/Navy Medical Oncology 8901 Wisconsin Ave., Bldg. 8, Rm. 5101 Bethesda, MD 20889
Telephone:	301-496-0929
Fax:	301-496-0047
Email:	pdennis@nih.gov
Sponsoring Laboratory/Branch:	Medical Oncology Branch

Project Title:	Identifying inducers of the tumor suppressor RhoB
Target(s) of Interest:	RhoB

Project Synopsis:
RhoB is an understudied, bona fide human tumor suppressor gene. It inhibits many important functions in cancer cells, including proliferation and migration. RhoB expression decreases with the development and progression of human cancers, and its ectopic re-expression inhibits cellular transformation and tumorigenesis. Despite the emergence of this compelling data, the pharmaceutical industry has mostly ignored RhoB as a therapeutic target. We became interested in RhoB during the course of microarray studies with Akt inhibitors called phosphatidylinositol ether lipid analogues (PIAs). PIAs rapidly inhibit Akt activation and selectively kill cancer cells with high levels of Akt activation (Cancer Research 64: 2782, 2004). PIAs are widely active in the NCI 60 cell line screen, and their biologic activity correlates with levels of phosphorylated but not total Akt (Molec. Canc. Ther., 5: 713, 2006). Because other molecular targets were identified that correlated more closely than phosphorylated Akt, gene expression profiling of PIAs was performed using microarrays. RhoB was identified as a transcriptional target of all active PIAs as well as another inhibitor of the PI3K/Akt pathway, LY294002. RhoB was rapidly induced, and increased expression was validated using RT-PCR and immunoblotting in a panel of four lung cancer cell lines (manuscript submitted). Mapping of the RhoB promoter revealed a minimum sequence containing CEBP-β and inverted NF-Y sites that were necessary for increased RhoB transcription (manuscript in preparation). This sequence was subsequently sub-cloned into a dual luciferase reporter cassette, and two cancer cell lines (H157 and Rh30) have been stably transfected with this cassette (NCI employee invention report filed). Because PIAs increase luciferase activity 2-10 fold in each cancer cell line in vitro and in vivo, and reporter activity is readily detectable when cells are plated in a 96 well format, these cell lines will be utilized to identify novel inducers of RhoB. Three publicly available compound libraries from the NCIs Developmental Therapeutics Program (DTP) will be tested in 96 well assays (http://www.dtp.nci.nih.gov/branches/dscb/repo_open.html). These include the Diversity Set II (1364 compounds), the Mechanistic Set (879 compounds) and the Natural Product Set (235 compounds). Each compound library will be tested in series and the experimental approach will be similar for all libraries. Briefly, each compound will be dissolved and plated in sextuplet at 10 M on 96 well plates containing H157 or Rh30 cells stably transfected with the luciferase-based reporter systems. An active PIA will serve as a positive control for induction of RhoB on each plate. Plates will be read for firefly and renilla luciferase activity at 2 hr and 6 hr. Two independent experiments will be performed for all compounds. Compounds that induce RhoB with mean values ≥2 fold will be annotated for further studies. These will include experiments to establish dose-dependent induction of luciferase activity, which will identify compounds within a library with the greatest potency. Induction of RhoB will be confirmed at the protein level using immunoblotting. If less than 20 hits at 10 M are obtained from the Diversity Set, compounds from the Mechanistic and Natural Products set will be tested in order. If more than 20 hits are obtained from the Diversity Set, analysis of other compound libraries will be deferred. After these studies identify compounds from one or more library that rapidly induce RhoB transcription and translation, winnowing of compounds will be based on potency and Lipinskis Rule of 5 (Adv. Drug Del. Rev., 2001, 46: 3). Compounds that remain after this step will be tested for inhibition of proliferation and/or migration of parental H157 or Rh30 cells. Proliferation will be assessed using SRB assays and migration will be assessed using Boyden chambers. Compounds that inhibit these processes will be tested in isogenic H157 or Rh30 cells that have been transfected with siRNA against RhoB or scrambled siRNA. (We have previously used commercially available constructs to successful knock down RhoB expression in H157 cells.) A diminished effect in cells that have decreased RhoB expression will help attribute changes in cellular function to RhoB induction. Dependence on RhoB could be further demonstrated by comparing the effects of lead compounds on the proliferation or migration of RhoB-/- or wt mouse embryo fibroblasts that are available in our laboratory. Compounds that induce RhoB and inhibit cellular proliferation and/or migration in a RhoB-dependent manner will move forward in two ways. First, these compounds will be presented to the NCI Joint Development Committee (JDC) for testing in the NCI 60 cell line screen. Second, these compounds will be considered for in vivo experiments. (Although in vivo experiments will depend on supply of these compounds, it is anticipated that some compounds will be commercially available and those that are not could be presented to DTP for assistance with compound synthesis.) H157 and Rh30 cells stably transfected with the luciferase reporters will be grown as subcutaneous xenografts in SCID mice. The endpoints for in vivo experiments will include induction of RhoB as assessed by a small animal IVIS Lumina Imaging System and inhibition of tumor growth. Because PIAs can induce RhoB in vivo and decrease xenograft growth in this system, an active PIA will serve as a positive control. For select lead compounds identified from the compound libraries, a short-term maximum tolerated dose will be established for intraperitoneal injection and toxicities will be correlated with RhoB induction. If a non-toxic dose that induces RhoB can be identified, the compound(s) will be injected daily for 5 days and RhoB induction and tumor inhibition will be assessed. Successful induction of RhoB in vivo and inhibition of tumor growth will prompt presentation of this data to the JDC for further development that could include chemical optimization of lead compounds, pharmacokinetic analysis, and further assessment of in vivo efficacy. To summarize, this project will utilize a novel high throughput assay system to identify inducers of RhoB. Induction of RhoB will be validated in vitro and in vivo and correlated with inhibition of cellular proliferation and/or migration. The training of a JHU/CCR fellow and the successful development of inducers of RhoB will be aided by the experience we have gained through the development and characterization of inhibitors of the Akt/mTOR pathway (e.g. PIAs) and long standing relationships with investigators within DTP. Most scientists in our laboratory have experience with preclinical drug development and will be readily available to assist and train the fellow. Taken together, this project identifying inducers of the tumor suppressor RhoB will provide invaluable experience for a young scientist interested in preclinical drug development.
Fellow Research Plan:
The fellow will be an integral part of the Signal Transduction Section, a team devoted to the preclinical development of novel anti-cancer therapies. The fellow will work closely with Dr. Chunyu Zhang, the research fellow who single-handedly identified RhoB as a target of PIAs and who developed the luciferase reporter system. The fellow will learn to culture and maintain the cells used in the reporter system, and will develop the ability to adeptly use the plate reader and luminometer. The fellow will learn to perform assays to measure cellular proliferation, cell death, and cell migration. Because animal studies are a planned step in the evaluation of inducers of RhoB, the fellow will participate in studies that will develop a dosing schedule for administration of compounds to mice and assess RhoB induction and tumor inhibition. The fellow will present data in our laboratory meetings and will participate in our journal club. With time, it is anticipated that the fellow will become an expert in RhoB and assume a leadership role in the project. Given that the length of the fellowship is two years, it is anticipated that the fellow will identify lead compounds through cell-based assays, and will progress to testing inhibitors in animal models. This data from the cellular and animal assays will form the basis for publications that the fellow will write. Successful development of compounds that induce RhoB will likely lead to filing of employee invention reports and/or patents, where the fellow will be credited as co-inventor.