The Hedgehog (Hh) signaling pathway is integral to tissue patterning during fetal development and oncogenesis in children and adults. Genetic screens have revealed several of the signaling proteins that regulate Hh target gene expression, which in mammals include the Sonic, Indian, and Desert Hh ligands (Shh, Ihh, and Dhh), the Hh receptor Patched1 (Ptch1), the transmembrane protein Smoothened (Smo), the Gli family of transcription factors (Gli1, Gli2, and Gli3), and the Gli antagonist Suppressor of Fused (Sufu). These genes are potential targets for next-generation chemotherapies, and several compounds that inhibit Smo have demonstrated efficacy in mouse models of Hh pathway-dependent tumors, such as basal cell carcinoma, medulloblastoma, pancreatic adenocarcinoma, and prostate cancer. The Smo antagonist GDC-0449 has even caused the regression of metastatic basal cell carcinoma and medulloblastoma in human clinical trials. It has become increasingly apparent, however, that Hh pathway-dependent cancers can readily gain resistance to Smo antagonists and that certain tumors are initiated or maintained by Smo-independent Hh target gene expression. Since Smo is the most "druggable" target within the Hh pathway and nearly all known pathway inhibitors target this transmembrane protein, there is a clear and urgent need for small molecules that act downstream, preferably at the level of the Gli transcription factors. This application describes a high-throughput screen for small-molecule antagonists of Gli function, using a Sufu null cell line that has been stably transfected with a Gli-dependent firefly luciferase reporter (Sufu-KO- LIGHT cells). Since Sufu directly inhibits Gli function, Sufu-KO-LIGHT cells exhibit constitutive firefly luciferase expression that mediated by endogenous Gli proteins. In contrast to cell-based assays used previously to discover Hh pathway inhibitors, the Sufu-KO-LIGHT cells are unresponsive to the large number of Smo- targeting compounds typically found in chemical libraries and they do not require the overexpression of Gli1 or Gli2 to achieve Smo-independent Hh target gene expression. This novel assay will therefore rapidly identify compounds that block the function of endogenous Gli factors, which are subject to regulatory processes that can be circumvented by Gli overexpression. In addition to this new screening campaign, a comprehensive hit advancement plan is outlined, including several secondary assays for assessing the Hh pathway selectivity of lead compounds, cellular and biochemical experiments for evaluating compound action on Gli function, and studies for determining compound efficacy against Hh pathway-dependent cancer cells. Collectively these investigations will provide valuable mechanistic probes of downstream signaling events within the Hh pathway and structural leads for the development of new anti-cancer therapies.

Public Health Relevance

Uncontrolled activation of the Hedgehog (Hh) pathway contributes to the onset and progression of several cancers and its pharmacological inhibition can induce tumor regression in mouse models and human patients. The emergence of drug-resistant tumors in these studies underscores the need for new Hh pathway antagonists, particularly compounds that inactivate the Gli family of transcription factors. The proposed research will identify novel inhibitors of Gli function through a cell-based high-throughput screen, characterize their mechanisms of action, and assess their ability to block the proliferation of Hh pathway-dependent cancers.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Small Research Grants (R03)
Project #
5R03MH094195-02
Application #
8257909
Study Section
Special Emphasis Panel (ZRG1-BST-F (50))
Program Officer
Yao, Yong
Project Start
2011-04-15
Project End
2013-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
2
Fiscal Year
2012
Total Cost
$39,500
Indirect Cost
$14,500
Name
Stanford University
Department
Biology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305