Additional file 1: Figure S1. of Osteoglycin promotes meningioma development through downregulation of NF2 and activation of mTOR signaling

OGN mRNA expression in tonsil, as a negative control of OGN RNAscope. (PDF 1195 kb

Mutation profiling of pleomorphic xanthoastrocytoma and giant cell glioblastoma arising in a PXA reveals BRAF V600E mutations.

<p>The sections on the left illustrate SNaPshot genotyping and the sections on the right depict Sanger sequencing of <i>BRAF</i> exon 15 for the same samples. The top panel shows genotyping data obtained with normal male genomic DNA (Promega, Madison, WI). The lower panels illustrate BRAF V600E (c.1799T>A) mutation detection (arrows) in tumor DNA derived from formalin-fixed paraffin-embedded specimens of representative examples of: PXA (BT21), anaplastic PXA (BT22) and gcGBM arising in a PXA (BT49). Assays: (1) <i>EGFR</i> 2235_49del R; (2) <i>NRAS</i> 38; (3) <i>BRAF</i> 1799; (4) <i>NRAS</i> 182; (5) <i>PIK3CA</i> 263; (6) <i>TP53</i> 742; (7) <i>CTNNB1</i> 95 and (8) <i>CTNNB1</i> 122.</p

Representative photomicrographs of tumors.

<p>A. H&E-stained section of a pleomorphic xanthoastrocytoma (PXA, BT06) demonstrating fascicular growth pattern and prominent intercellular reticulin deposition (B) corresponding to PXA (m). C. H&E-stained section of PXA (BT21) that demonstrates neither a fascicular growth pattern nor prominent intercellular reticulin deposition (D). E. H&E-stained section of a giant cell glioblastoma (gcGBM) arising from a PXA (BT49) with marked pleomorphism and giant cells and multifocal reticulin deposition (F).</p

Clinical Data and Summary of Results.

<p>PXA – pleomorphic xanthoastrocytoma; PXA (m) – pleomorphic xanthoastrocytoma with mesenchymal-like growth pattern; aPXA – anaplastic pleomorphic xanthoastrocytoma; NA – Not assessed.</p

Mutation analysis of GBM.

<p>SNaPshot clinical genotyping of 62 glioblastoma cases from MGH identified cancer gene mutations that confer a favorable prognosis to the patients (IDH1) or that activate pathways targeted by therapeutic agents under clinical development (BRAF, KRAS and PIK3CA).</p

Using the Heat-Shock Response To Discover Anticancer Compounds that Target Protein Homeostasis

Unlike normal tissues, cancers experience profound alterations in protein homeostasis. Powerful innate adaptive mechanisms, especially the transcriptional response regulated by Heat Shock Factor 1 (HSF1), are activated in cancers to enable survival under these stressful conditions. Natural products that further tax these stress responses can overwhelm the ability to cope and could provide leads for the development of new, broadly effective anticancer drugs. To identify compounds that drive the HSF1-dependent stress response, we evaluated over 80,000 natural and synthetic compounds as well as partially purified natural product extracts using a reporter cell line optimized for high-throughput screening. Surprisingly, many of the strongly active compounds identified were natural products representing five diverse chemical classes (limonoids, curvularins, withanolides, celastraloids, and colletofragarones). All of these compounds share the same chemical motif, an α,β-unsaturated carbonyl functionality, with strong potential for thiol-reactivity. Despite the lack of <i>a priori</i> mechanistic requirements in our primary phenotypic screen, this motif was found to be necessary albeit not sufficient, for both heat-shock activation and inhibition of glioma tumor cell growth. Within the withanolide class, a promising therapeutic index for the compound withaferin A was demonstrated <i>in vivo</i> using a stringent orthotopic human glioma xenograft model in mice. Our findings reveal that diverse organisms elaborate structurally complex thiol-reactive metabolites that act on the stress responses of heterologous organisms including humans. From a chemical biology perspective, they define a robust approach for discovering candidate compounds that target the malignant phenotype by disrupting protein homeostasis

Structure–Activity Relationships for Withanolides as Inducers of the Cellular Heat-Shock Response

To understand the relationship between the structure and the remarkably diverse bioactivities reported for withanolides, we obtained withaferin A (WA; <b>1</b>) and 36 analogues (<b>2</b>–<b>37</b>) and compared their cytotoxicity to cytoprotective heat-shock-inducing activity (HSA). By analyzing structure–activity relationships for the series, we found that the ring A enone is essential for both bioactivities. Acetylation of 27-OH of 4-<i>epi</i>-WA (<b>28</b>) to <b>33</b> enhanced both activities, whereas introduction of β-OH to WA at C-12 (<b>29</b>) and C-15 (<b>30</b>) decreased both activities. Introduction of β-OAc to 4,27-diacetyl-WA (<b>16</b>) at C-15 (<b>37</b>) decreased HSA without affecting cytotoxicity, but at C-12 (<b>36</b>), it had minimal effect. Importantly, acetylation of 27-OH, yielding <b>15</b> from <b>1</b>, <b>16</b> from <b>14</b>, and <b>35</b> from <b>34</b>, enhanced HSA without increasing cytotoxicity. Our findings demonstrate that the withanolide scaffold can be modified to enhance HSA selectively, thereby assisting development of natural product-inspired drugs to combat protein aggregation-associated diseases by stimulating cellular defense mechanisms

HSF1 null MEF cells support significantly less VACV infection.

<p>(A) HSF1 null MEF cells infected with VACV-TrpV show significantly less early (Venus), intermediate (mCherry) and late (TagBFP) gene expression compared to wild type MEFs. Three independent experiments were completed in triplicate; this is a representative plot showing normalized relative fluorescent units (RFU) with standard error. (B) Brightfield images show HSF1 null MEFs exhibit less cytopathic effects than wild type MEFs when infected with VACV at an MOI of 0.1 at 18 hpi. Mock infected HSF1 null and wild type MEFs are included for comparison.</p

Pooled-cell shRNA screen revealed host factors necessary for orthopoxvirus infection.

<p>(A) Schematic of the primary pooled shRNA screen. (B) Table of the significant host functional and signaling pathways necessary for vaccinia infection revealed by the candidate hits. Analysis completed in IPA. (C) Pie chart of overrepresented Biological Process GO terms, annotated using Panther.</p

High confidence hits identified in secondary screen.

<p>(A) Schematic of arrayed shRNA lentivirus secondary screen. (B) High confidence host factor gene hits identified with the secondary screen.</p