Fragile X syndrome (FXS) is the most common form of inherited mental retardation, and it is caused in most of cases by epigenetic silencing of the Fmr1 gene. Today, no specific therapy exists for FXS, and current treatments are only directed to improve behavioral symptoms. Neuronal progenitors derived from FXS patient induced pluripotent stem cells (iPSCs) represent a unique model to study the disease and develop assays for large-scale drug discovery screens since they conserve the Fmr1 gene silenced within the disease context. We have established a high-content imaging assay to run a large-scale phenotypic screen aimed to identify compounds that reactivate the silenced Fmr1 gene. A set of 50,000 compounds was tested, including modulators of several epigenetic targets. We describe an integrated drug discovery model comprising iPSC generation, culture scale-up, and quality control and screening with a very sensitive high-content imaging assay assisted by single-cell image analysis and multiparametric data analysis based on machine learning algorithms. The screening identified several compounds that induced a weak expression of fragile X mental retardation protein (FMRP) and thus sets the basis for further large-scale screens to find candidate drugs or targets tackling the underlying mechanism of FXS with potential for therapeutic intervention.

No abstract

Macrophages are key cell types of the innate immune system regulating host defense, inflammation, tissue homeostasis and cancer. Within this functional spectrum diverse and often opposing phenotypes are displayed which are dictated by environmental clues and depend on highly plastic transcriptional programs. Among these the ‘classical’ (M1) and ‘alternative’ (M2) macrophage polarization phenotypes are the best characterized. Understanding macrophage polarization in humans may reveal novel therapeutic intervention possibilities for chronic inflammation, wound healing and cancer. Systematic loss of function screening in human primary macrophages is limited due to lack of robust gene delivery methods and limited sample availability. To overcome these hurdles we developed cell-autonomous assays using the THP-1 cell line allowing genetic screens for human macrophage phenotypes. We screened 648 chromatin and signaling regulators with a pooled shRNA library for M1 and M2 polarization modulators. Validation experiments confirmed the primary screening results and identified OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) as a novel mediator of M2 polarization in human macrophages. Our approach offers a possible avenue to utilize comprehensive genetic tools to identify novel candidate genes regulating macrophage polarization in humans.

SummaryIn fragile X syndrome (FXS), CGG repeat expansion greater than 200 triplets is believed to trigger FMR1 gene silencing and disease etiology. However, FXS siblings have been identified with more than 200 CGGs, termed unmethylated full mutation (UFM) carriers, without gene silencing and disease symptoms. Here, we show that hypomethylation of the FMR1 promoter is maintained in induced pluripotent stem cells (iPSCs) derived from two UFM individuals. However, a subset of iPSC clones with large CGG expansions carries silenced FMR1. Furthermore, we demonstrate de novo silencing upon expansion of the CGG repeat size. FMR1 does not undergo silencing during neuronal differentiation of UFM iPSCs, and expression of large unmethylated CGG repeats has phenotypic consequences resulting in neurodegenerative features. Our data suggest that UFM individuals do not lack the cell-intrinsic ability to silence FMR1 and that inter-individual variability in the CGG repeat size required for silencing exists in the FXS population.

The mTOR pathway is a critical integrator of nutrient and growth factor signaling. Once activated, mTOR promotes cell growth and proliferation. Several components of the mTOR pathway are frequently deregulated in tumors, leading to constitutive activation of the pathway and thus contribute to uncontrolled cell growth. We performed a high-throughput screen with an isogenic cell line system to identify compounds specifically inhibiting proliferation of PTEN/mTOR-pathway addicted cells. We show here the characterization and mode of action of two such compound classes. One compound class inhibits components of the PTEN/mTOR signaling pathway, such as S6 ribosomal protein phosphorylation, and leads to cyclin D3 downregulation. These compounds are not adenosine triphosphate competitive inhibitors for kinases in the pathway, nor do they require FKBP12 for activity like rapamycin. The other compound class turned out to be a farnesylation inhibitor, blocking the activity of GTPases, as well as an inducer of oxidative stress. Our results demonstrate that an isogenic cell system with few specific mutations in oncogenes and tumor suppressor genes can identify different classes of compounds selectively inhibiting proliferation of PTEN/mTOR pathway-addicted isogenic clones. The identified mechanisms are in line with the known cellular signaling networks activated by the altered oncogenes and suppressor genes in the isogenic system.

Proteins encoded by oncogenes represent suitable drug targets and several have been validated clinically. However, certain non-oncogenes, which are downstream effectors of oncogenes, may be similarly useful as potential targets of therapy. We have developed a high-throughput strategy using a shRNA genomic library and tumor cells addicted to the mTOR pathway to identify genes (oncogenes or non-oncogenes) which control the oncogenic phenotype. mTOR addicted cells were generated from IL-3 dependent cells following frame-shift mutagenesis and selection for IL-3 independence. mTOR addicted cells obtained from this selection carried frame-shift mutations in the PTEN gene. mTOR addiction was revealed by sensitivity to rapamycin and confirmed biochemically by examining phosphorylation of the mTOR targets S6K and PKB. Interestingly (and of importance to our shRNA-based screening strategy), addition of IL-3 rescued cells from mTOR addiction, as this growth factor antagonized the apoptotic effect of rapamycin. Using robotics (BioMek-NX) and a genomic retroviral shRNA library we evaluated 14’000 genes for a potential role in maintaining mTOR addicted growth. Our strategy was to screen cells in the presence and absence of IL-3 to identify shRNAs that antagonize growth of cells in the absence of IL-3. We identified around 300 genes required for mTOR addicted growth. This set of genes was enriched in highly significant manner for genes known to be involved in cancer, providing proof of concept for the strategy. Examples of identified genes include ras, raf, PKB and mTOR. Also, genes with no cancer link and genes with unknown function were identified. Use of Ingenuity software allowed to place many of the identified genes into functionally distinct groups or pathways. In addition numerous hits affecting metabolic functions, redox functions, and ROS generating systems were identified including the cytoplasmic NADPH complex and complex I-IV of the respiratory chain. Combining the genes et with an Affymetrix gene overexpression analysis in the same cells and analysing by Oncomine software, we identified a glioblastoma gene signature consisting of 13 genes (p-value 2.4E-5) containing 3 oncogenes and several signaling elements. Downregulation of several of these genes in human cancer lines induced apoptosis. Some of he genes identified represent novel potential drug targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2197.