This project is based on the working hypothesis of the program project that the p70S6 kinase and the JARID1B histone demethylase enzymes play key roles in both melanoma cell growth and survival. The studies proposed here will delineate the biochemical and structural basis for the activities of these enzymes and use this information to develop small molecule inhibitors to study the function of these proteins in vivo using melanoma models through the other projects of the program. These studies will also provide lead compounds for development for therapy of melanoma.
Aim 1 will focus on p70S6K and Aim 2 will focus on JARID1B.
For Aim 1, we have prepared active recombinant full-length p70S6 kinase as well as several truncated protein constructs, prepared a lead p70S6K organometallic inhibitor scaffold (compound EMS) with promising selectivity profile and an IC{50} value in the mid-nanomolar range, and determined the crystal structure of the p70S6 kinase domain bound to the EMS inhibitor. We propose to: (a) biochemically characterize the catalytic activity of the p70S6 kinase;(b) prepare a focused library around the EMS organometallic p70S6 kinase inhibitor for screening in vitro and in vivo (together with Project 2) and determine the X-ray crystal structure of p70S6K/inhibitor complexes;(c) carry out a high-throughput solution screen for organic p70S6 kinase inhibitors, establish the mode of inhibition, and determine the X-ray crystal structure of the p70S6 kinase domain bound to lead organic inhibitors;and (d) use structure-based design, medicinal chemistry, kinase profiling and cell-based studies (together with Project 2) to develop potent and selective second-generation organometallic and organic p70S6 kinase inhibitors.
For Aim 2, we have prepared recombinant and enzymatically active JARID1B and the yeast Jhd2p ortholog for biochemical and crystallographic analysis and have prepared preliminary JARID1B and Jhd2p crystals. We now propose to (a) biochemically characterize the catalytic activity of JARID1B and Jhd2p;(b) determine the X-ray crystal structures of JARID1B and Jhd2p and carry out structure-based mutagenesis to facilitate a structure-function analysis;(c) use the recombinant JARID1 proteins (JARID1B and Jhd2p) and structures to facilitate high throughput solution and in silico small molecule inhibitor screening, respectively, and JARID1 inhibitors will be tested in cells together with Project 1 of the program;and (d) crystallize the most potent JARID1 inhibitors with the JARID1 proteins for structure-based inhibitor optimization. Together, the studies proposed here will provide novel molecular insights and small molecule reagents to delineate the roles of two novel protein targets, p70S6K and JARID1B, in melanoma and will provide lead small molecule compounds to develop therapeutic agents to treat melanoma.

Public Health Relevance

Members of our program project have generated exciting new data showing that the p70S6K and JARID1B proteins play key roles in both melanoma cell growth and survival. The overall goal of this project is to determine the biochemical and structural basis for the activities of these proteins and to use this information to develop small molecule inhibitors to study their function in melanoma cells through the other projects of the program. These studies will also provide novel lead compounds to develop therapeutic agents to treat melanoma.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
5P01CA025874-32
Application #
8450872
Study Section
Special Emphasis Panel (ZCA1-RPRB-O)
Project Start
Project End
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
32
Fiscal Year
2013
Total Cost
$348,384
Indirect Cost
$22,489
Name
Wistar Institute
Department
Type
DUNS #
075524595
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Talevich, Eric; Shain, A Hunter; Botton, Thomas et al. (2016) CNVkit: Genome-Wide Copy Number Detection and Visualization from Targeted DNA Sequencing. PLoS Comput Biol 12:e1004873
Lu, Hezhe; Liu, Shujing; Zhang, Gao et al. (2016) Oncogenic BRAF-Mediated Melanoma Cell Invasion. Cell Rep 15:2012-24
Krepler, Clemens; Xiao, Min; Samanta, Minu et al. (2016) Targeting Notch enhances the efficacy of ERK inhibitors in BRAF-V600E melanoma. Oncotarget :
Shannan, Batool; Chen, Quan; Watters, Andrea et al. (2016) Enhancing the evaluation of PI3K inhibitors through 3D melanoma models. Pigment Cell Melanoma Res 29:317-28
Krepler, Clemens; Xiao, Min; Sproesser, Katrin et al. (2016) Personalized Preclinical Trials in BRAF Inhibitor-Resistant Patient-Derived Xenograft Models Identify Second-Line Combination Therapies. Clin Cancer Res 22:1592-602
Yeh, Iwei; Tee, Meng Kian; Botton, Thomas et al. (2016) NTRK3 kinase fusions in Spitz tumours. J Pathol 240:282-290
Shannan, Batool; Perego, Michela; Somasundaram, Rajasekharan et al. (2016) Heterogeneity in Melanoma. Cancer Treat Res 167:1-15
Cierlitza, Monika; Chauvistré, Heike; Bogeski, Ivan et al. (2015) Mitochondrial oxidative stress as a novel therapeutic target to overcome intrinsic drug resistance in melanoma cell subpopulations. Exp Dermatol 24:155-7
Qin, Jie; Rajaratnam, Rajathees; Feng, Li et al. (2015) Development of organometallic S6K1 inhibitors. J Med Chem 58:305-14
Carvajal, Richard D; Lawrence, Donald P; Weber, Jeffrey S et al. (2015) Phase II Study of Nilotinib in Melanoma Harboring KIT Alterations Following Progression to Prior KIT Inhibition. Clin Cancer Res 21:2289-96

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