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

Project Sponsor/Mentor:	Nancy Colburn
Title:	Lab/Branch Chief
Address:	Building 675, Rm 100 NCI-Frederick Frederick, MD 21702
Telephone:	301-846-1342
Fax:	301 846 6903
Email:	colburna@mail.nih.gov
Sponsoring Laboratory/Branch:	LCP/CCR

Project Title:	Small molecule inhibitors for cancer prevention
Target(s) of Interest:	AP-1, NFkappaB

Project Synopsis:
Activation of AP-1 and NFkB is necessary to drive tumor promotion and tumor progression. Genetically engineered mice that express the AP-1 blocker TAM67 are resistant to chemical, UV, viral, and oncogene induced tumorigenesis (1, 2). A relatively small number of genes are targeted by TAM67 when it inhibits skin tumorigenesis without inhibiting cell proliferation or survival. These are genes that drive carcinogenesis including COX-2, osteopontin, urokinase plasminogen activator receptor, MMP-10, and most recently sulfiredoxin (3, 4). Efficacy of TAM67 targeting has been shown in skin, mammary and lung carcinogenesis. In order to translate these findings for clinical application, we have identified several small molecules that inhibit AP-1 without inhibiting cell proliferation in a high throughput screen (5). The aim of this project is to characterize the mechanism of inhibition of AP-1 or NFkB by small molecules recently identified in the AP-1 hit screen. This analysis will define the steps leading to inhibition of transcription factor activity and may identify the binding partners of the active compounds. This characterization will be useful for determining if these compounds are suitable for contributing to CCRs pipeline of promising compounds that might eventually be considered for Phase Zero or Phase One Trials. Should a compound show submicromolar activity in suitable cell lines for hitting the target and suppressing transformation or invasion, show lack of toxicity in a suitable short term mouse assay, and be available in sufficient quantities for a larger scale mouse study, we shall submit a proposal to the Molecular Targets Faculty Steering Committee Joint Development Committee (JDC) that the compound be considered for development. We have used a high-through-put screen (5) to identify a several small molecules that inhibit AP-1 or NFkB activity with little or no toxicity or effects on proliferation. We have shown that NSC676914 targets IKKbeta and hence NFkB activation at submicromolar concentrations (6) and that Quassinoids target AP-1 (7). We are now studying a new set of interesting compounds that show potency and specificity for inhibiting AP-1 and/or NFkB. These compounds present the advantage that structural analogs are available as well as substantial supplies of the compounds. Furthermore, two of the analogs are FDA-approved drugs. Initial analysis indicates that subtle differences in structure confer differential potency for inhibiting AP-1 or NFkB. The set of analogues will be analyzed to define the steps leading to inhibition of transcription factor activity and with suitable collaboration we may identify the binding partner of the compound. We have begun analyzing the next 4 compounds. Data from 2 of these compounds show that they target NFkB with an IC50 that is less than 0.1 uM. Interestingly, the structure of these 2 compounds has not been previously reported. Analysis of the last 2 compounds is pending. The MTDD fellow will continue the analysis of the next 4 compounds. The fellow will begin by characterizing the NFkB targeted compounds using methods similar to those used to characterize NSC676914 (6). This analysis will include determining where in the NFkB signaling pathway the compounds are targeting. Phospho-Western blot analysis will be used to determine which kinases are not activated in the presence of the compounds. Once the target has been identified further analysis is needed to determine the mechanism of inhibition. If the binding target of the compounds can also be identified, binding kinetics and structure analysis will be pursued. In addition to determining the targets that these compounds act on, the fellow will determine the effects of these compounds on cell migration, invasion and metastasis. Using human cancer cell lines, the effects of the compounds on constitutive activation of AP-1 and NFkB will be determined using Luciferase reporter assays. In addition, transcriptional regulation of known endogenous AP-1 or NFB regulated genes will be measured by quantitative RT-PCR. The fellow will also assist in completing the analysis of the first 2 set of compounds. Once the molecular analysis of all the candidate compounds isolated from the screen has been completed, the compounds will be evaluated on the potential for further development for clinical phase 0 or phase 1 trials. Compounds whose functional targets can be identified, which hit the target at sub-micromolar concentrations without toxicity and where there is sufficient amount of compound will be pursued further. The fellow will help set up and perform in vivo toxicity assays. Non-toxic compounds will be assayed to determine if the compounds hit their target in vivo. If sufficient compound is available, in vivo tumorigenesis studies will be conducted. These studies will be done in collaboration with Center for Advanced Preclinical Research (CAPR). Genetically engineered mice designed to develop target specific carcinogenesis and that show activation of the compounds target, AP-1 and or NFkB will be used in preclinical trials to determine efficacy of hitting the target and preventing or treating carcinogenesis in vivo. 1) Young MR, Yang HS, Colburn NH, Promising molecular targets for cancer prevention: AP-1, NF-kappa B and Pdcd4. Trends Mol Med. 2003 2) Shen Q, Uray IP, Li Y, Zhang Y, Hill J, Xu XC, Young MR, Gunther EJ, Hilsenbeck SG, Colburn NH, Chodosh LA, Brown PH. Targeting the activator protein 1 transcription factor for the prevention of estrogen receptor-negative mammary tumors. Cancer Prev Res (Phila Pa). 2008. 3) Matthews CP, Birkholz AM, Baker AR, Perella CM, Beck GR Jr, Young MR, Colburn NH., Dominant-negative activator protein 1 (TAM67) targets cyclooxygenase-2 and osteopontin under conditions in which it specifically inhibits tumorigenesis. Cancer Res. 2007 4) Wei Q, Jiang H, Matthews CP, Colburn NH., Sulfiredoxin is an AP-1 target gene that is required for transformation and shows elevated expression in human skin malignancies. Proc Natl Acad Sci U S A. 2008 5) Ruocco KM, Goncharova EI, Young MR, Colburn NH, McMahon JB, Henrich CJ., A high-throughput cell-based assay to identify specific inhibitors of transcription factor AP-1. J Biomol Screen. 2007 6) Kang MI, Henrich CJ, Bokesch HR, Gustafson KR, McMahon JB, Baker AR, Young MR, Colburn NH. A selective small-molecule nuclear factor-kappaB inhibitor from a high-throughput cell-based assay for "activator protein-1 hits". Mol Cancer Ther. 2009. 7) Beutler JA, Kang MI, Robert F, Clement JA, Pelletier J, Colburn NH, McKee TC, Goncharova E, McMahon JB, Henrich CJ. Quassinoid Inhibition of AP-1 Function Does Not Correlate with Cytotoxicity or Protein Synthesis Inhibition (dagger). J Nat Prod. 2009.
Fellow Research Plan:
Specific aims. 1. Characterize the effects of 4 small molecules derived from our high-through-put screen for inhibition of AP-1 and NFkB. The candidate will use Luciferase reporters to determine the IC50 of the remaining compounds. The inhibitor effects of these compounds on AP-1, NFkB and SRE will be measured. Compounds that show inhibition of AP-1 or NFkB at sub-micromolar concentrations without inhibition of SRE, an indicator of proliferation, will be characterized further. 2. Determine the molecular mechanism of inhibition by the compounds. The candidate will determine if the compounds inhibit the signal transduction leading to activation of AP-1 or NFkB or if they inhibit DNA binding of these transcription factors. The candidate will query the signaling pathway of AP-1 and NFkB to determine where signaling is inhibited. These pathways can be assayed by Phospho-Western blot analysis. DNA binding will be measured by Electrophoresis mobility shift assays (EMSA) and chromosomal immuneprecipitation assays (ChIP). If the compounds are affecting one of the signaling kinases in vitro kinase assays will be used to determine the IC50 and ATP dependence. 3. Determination of binding target. If the compound can be modified to incorporate a chemical tag such as biotin, the binding target of the compound can be identified. Cells treated with the biotin-labeled compound will be lysed under non-denaturing conditions and precipitated with streptavidin. Precipitated proteins will be determined by Mass-spec analysis. 4. Characterization of the effects of the compounds on cell migration and invasion in human cancer cell lines. Human cancer cell lines such as MBA-MB-231, an invasive breast cancer cell line, will be used to determine the effects of the compounds on invasion using Matrigel-coated chambers. 5. Determination of in vivo toxicity. In collaboration with Douglas Figg, Medical Oncology Branch and Affiliates, Molecular Pharmacology Section, CCR, the fellow will determine if promising candidates are toxic in mice.