Deguelins, Natural Product Modulators of NF1-Defective Astrocytoma Cell Growth Identified by High-Throughput Screening of Partially Purified Natural Product Extracts.
ABSTRACT: A high-throughput screening assay for modulators of Trp53/NF1 mutant astrocytoma cell growth was adapted for use with natural product extracts and applied to a novel collection of prefractionated/partially purified extracts. Screening 68?427 samples identified active fractions from 95 unique extracts, including the terrestrial plant Millettia ichthyotona. Only three of these extracts showed activity in the crude extract form, thus demonstrating the utility of a partial purification approach for natural product screening. The NF1 screening assay was used to guide purification of active compounds from the M. ichthyotona extract, which yielded the two rotenones deguelin (1) and dehydrodeguelin (2). The deguelins have been reported to affect growth of a number of cancer cell lines. They potently inhibited growth of only one of a panel of NF1/Trp53 mutant murine astrocytoma cell lines, possibly related to epigenetic factors, but had no effect on the growth of normal astrocytes. These results suggest the potential utility of deguelins as tools for further investigating NF1 astrocytoma cell growth. These bioprobes were identified only as a result of screening partially purified natural product extracts.
Project description:Neurofibromatosis type 1 (NF1) is the most common genetic disease affecting the nervous system. Patients typically develop many tumors over their lifetime, leading to increased morbidity and mortality. The NF1 gene, mutated in NF1, is also commonly mutated in sporadic glioblastoma multiforme (GBM). Because both NF1 and GBM are currently incurable, new therapeutic approaches are clearly needed. Natural products represent an opportunity to develop new therapies, as they have been evolutionarily selected to play targeted roles in organisms. Schweinfurthin A is a prenylated stilbene natural product that has previously shown specific inhibitory activity against brain and hematopoietic tumor lines. We show that patient-derived GBM and NF1 malignant peripheral nerve sheath tumor (MPNST) lines, as well as tumor lines derived from the Nf1-/+;Trp53-/+ (NPcis) mouse model of astrocytoma and MPNST are highly sensitive to inhibition by schweinfurthin A and its synthetic analogs. In contrast, primary mouse astrocytes are resistant to the growth inhibitory effects of schweinfurthin A, suggesting that schweinfurthin A may act specifically on tumor cells. Stable transfection of the GTPase-activating protein related domain of Nf1 into Nf1-/-;Trp53-/- astrocytoma cells confers resistance to schweinfurthin A. In addition, the profound effect of schweinfurthin A on dynamic reorganization of the actin cytoskeleton led us to discover that schweinfurthin A inhibits growth factor-stimulated Rho signaling. In summary, we have identified a class of small molecules that specifically inhibit growth of cells from both central and peripheral nervous system tumors and seem to act on NF1-deficient cells through cytoskeletal reorganization correlating to changes in Rho signaling.
Project description:A growing body of work suggests that astrocytomas and glioblastoma multiforme will require carefully tailored, molecularly targeted therapy for successful treatment. Recent efforts to comprehensively identify mutations and gene expression changes in glioblastoma have shown that mutation of NF1 is a common alteration in human glioblastoma. We have developed and characterized a panel of 14 tumor lines from grades II through IV astrocytomas developed from our Nf1-/+;Trp53-/+cis mouse model and have used this panel to characterize signal transduction pathways and inhibitors that are candidate therapeutic targets for astrocytoma and glioblastoma. We show that these tumors express platelet-derived growth factor receptor-?, epidermal growth factor receptor, and their respective ligands to varying degrees. We find that both the MEK and PI3K signaling pathways downstream of epidermal growth factor receptor and platelet-derived growth factor receptor-? are necessary for full proliferation of astrocytoma cells; however, inhibition of the PI3K pathway is more effective than inhibition of MEK at blocking cell growth. We have examined inhibitors of the PI3K/Akt/mTOR signaling pathway and find that PI-103 and TCN show particular promise for inhibiting growth in Nf1 and Trp53 mutant astrocytoma cells.
Project description:Astrocytoma is the most common malignant brain tumor in humans. Loss of the p53 signaling pathway and up-regulation of the ras signaling pathway are common during tumor progression. We have shown previously that mice mutant for Trp53 and Nf1 develop astrocytoma, progressing to glioblastoma, on a C57BL/6J strain background. In contrast, here we present data that mice mutant for Trp53 and Nf1 on a 129S4/SvJae background are highly resistant to developing astrocytoma. Through analysis of F1 progeny, we demonstrate that susceptibility to astrocytoma is linked to chromosome 11, and that the modifier gene(s) responsible for differences in susceptibility is closely linked to Nf1 and Trp53. Furthermore, this modifier of astrocytoma susceptibility is itself epigenetically modified. These data demonstrate that epigenetic effects can have a strong effect on whether cancer develops in the context of mutant ras signaling and mutant p53, and that this mouse model of astrocytoma can be used to identify modifier phenotypes with complex inheritance patterns that would be unidentifiable in humans. Because analysis of gene function in the mouse is often performed on a mixed C57BL/6,129 strain background, these data also provide a powerful example of the potential of these strains to mask interesting gene functions.
Project description:Neurofibromatosis type 1 (NF1) is the most common cancer predisposition syndrome affecting the nervous system, with elevated risk for both astrocytoma and peripheral nerve sheath tumors. NF1 is caused by a germline mutation in the NF1 gene, with tumors showing loss of the wild type copy of NF1. In addition, NF1 heterozygosity in surrounding stroma is important for tumor formation, suggesting an additional role of haploinsufficiency for NF1. Studies in mouse models and NF1 families have implicated modifier genes unlinked to NF1 in the severity of the disease and in susceptibility to astrocytoma and peripheral nerve sheath tumors. To determine if differences in Nf1 expression may contribute to the strain-specific effects on tumor predisposition, we examined the levels of Nf1 gene expression in mouse strains with differences in tumor susceptibility using quantitative polymerase chain reaction. The data presented in this paper demonstrate that strain background has as much effect on Nf1 expression levels as mutation of one Nf1 allele, indicating that studies of haploinsufficiency must be carefully interpreted with respect to strain background. Because expression levels do not correlate entirely with the susceptibility or resistance to tumors observed in the strain, these data suggest that either variation in Nf1 levels is not responsible for the differences in astrocytoma and peripheral nerve sheath tumor susceptibility in Nf1-/+;Trp53-/+cis mice, or that certain mouse strains have evolved compensatory mechanisms for differences in Nf1 expression.
Project description:Neurofibromatosis type 1 (NF1) is one of the most common human genetic diseases affecting the nervous system and predisposes individuals to cancer, including peripheral nerve sheath tumors (PNSTs) and astrocytomas. Modifiers in the genetic background affect the severity of the disease and we have previously mapped two modifier loci, Nstr1 and Nstr2, that influence resistance to PNSTs in the Nf1-/+;Trp53-/+cis mouse model of NF1. We report here the analysis of Nstr1 in isolation from other epistatic loci using a chromosome substitution strain, and further show that a modifier locus (or loci) on chromosome 19 influences resistance to both PNSTs and astrocytomas. This modifier locus interacts with sex, resulting in sex-specific modification of tumors. Allele variability on chromosome 19 affects both the timing and the penetrance of the growth of different tumor types associated with NF1, specifically PNSTs and astrocytoma. These results indicate that modifiers of cancer susceptibility interact and affect tumorigenesis under different genetic conditions and demonstrate the power of chromosome substitution strains to study genetic modifiers.
Project description:Although most clinically used antibiotics are derived from natural products, identifying new antibacterial molecules from natural product extracts is difficult due to the complexity of these extracts and the limited tools to correlate biological activity with specific molecules. Here, we show that bacterial cytological profiling (BCP) provides a rapid method for mechanism of action determination on plates and in complex natural product extracts and for activity-guided purification. We prepared an extract from Bacillus subtilis 3610 that killed the Escherichia coli lptD mutant and used BCP to observe two types of bioactivities in the unfractionated extract: inhibition of translation and permeablization of the cytoplasmic membrane. We used BCP to guide purification of the molecules responsible for each activity, identifying the translation inhibitors bacillaene and bacillaene B (glycosylated bacillaene) and demonstrating that two molecules contribute to cell permeabilitization, the bacteriocin subtilosin and the cyclic peptide sporulation killing factor. Our results suggest that bacillaene mediates translational arrest, and show that bacillaene B has a minimum inhibitory concentration 10 × higher than unmodified bacillaene. Finally, we show that BCP can be used to screen strains on an agar plate without the need for extract preparation, greatly saving time and improving throughput. Thus, BCP simplifies the isolation of novel natural products, by identifying strains, crude extracts and fractions with interesting bioactivities even when multiple activities are present, allowing investigators to focus labor-intensive steps on those with desired activities.
Project description:While many cancers show a sex bias, the genetic basis and molecular mechanisms underlying sex bias are not always clear. Astrocytoma and glioblastoma show male predominance in humans. We have shown previously that glial tumors forming in the Nf1-/+; Trp53-/+cis (NPcis) mouse model also show a sex bias in some genetic contexts. Using cross-species comparisons we have identified candidate male-specific modifiers of astrocytoma/glioblastoma. Linkage analysis of B6X(B6X129)-NPcis mice identifies a modifier of astrocytoma resistance specific to males, named Arlm1, on distal mouse Chr 12. Arlm1 is syntenic to human Chr 7p15, 7p21, 7q36, and 14q32 regions that are altered in human glioblastoma. A subset of these genes shows male-specific correlations to glioblastoma patient survival time and represents strong candidates for the Arlm1 modifier gene. Identification of male-specific modifier genes will lead to a better understanding of the molecular basis of male predominance in astrocytoma and glioblastoma.
Project description:Natural products remain an important source of drug candidates, but the difficulties inherent to traditional isolation, coupled with unacceptably high rates of compound rediscovery, limit the pace of natural product detection. Here we describe a reactivity-based screening method to rapidly identify exported bacterial metabolites that contain dehydrated amino acids (i.e., carbonyl- or imine-activated alkenes), a common motif in several classes of natural products. Our strategy entails the use of a commercially available thiol, dithiothreitol, for the covalent labeling of activated alkenes by nucleophilic 1,4-addition. Modification is easily discerned by comparing mass spectra of reacted and unreacted cell surface extracts. When combined with bioinformatic analysis of putative natural product gene clusters, targeted screening and isolation can be performed on a prioritized list of strains. Moreover, known compounds are easily dereplicated, effectively eliminating superfluous isolation and characterization. As a proof of principle, this labeling method was used to identify known natural products belonging to the thiopeptide, lanthipeptide, and linaridin classes. Further, upon screening a panel of only 23 actinomycetes, we discovered and characterized a novel thiopeptide antibiotic, cyclothiazomycin C.
Project description:Malignant peripheral nerve sheath tumors (MPNSTs) are Schwann cell-derived malignancies that arise from plexiform neurofibromas in patients with mutation of the neurofibromin 1 (NF1) gene. We have shown that the growth factor neuregulin-1 (NRG1) also contributes to human neurofibroma and MPNST pathogenesis and that outbred C57BL/6J × SJL/J transgenic mice overexpressing NRG1 in Schwann cells (P0-GGF?3 mice) recapitulate the process of neurofibroma-MPNST progression. However, it is unclear whether NRG1 acts predominantly within NF1-regulated signaling cascades or instead activates other essential cascades that cooperate with NF1 loss to promote tumorigenesis. We now report that tumorigenesis is suppressed in inbred P0-GGF?3 mice on a C57BL/6J background. To determine whether NRG1 overexpression interacts with reduced Nf1 or Trp53 gene dosage to "unmask" tumorigenesis in these animals, we followed cohorts of inbred P0-GGF?3;Nf1+/?, P0-GGF?3;Trp53+/? and control (P0-GGF?3, Nf1+/? and Trp53+/?) mice for 1 year. We found no reduction in survival or tumors in control and P0-GGF?3;Nf1+/? mice. In contrast, P0-GGF?3;Trp53+/? mice died on average at 226 days, with MPNSTs present in 95 % of these mice. MPNSTs in inbred P0-GGF?3;Trp53+/? mice arose de novo from micro-MPNSTs that uniformly develop intraganglionically. These micro-MPNSTs are of lower grade (WHO grade II-III) than the major MPNSTs (WHO grade III-IV); array comparative genomic hybridization showed that lower grade MPNSTs also had fewer genomic abnormalities. Thus, P0-GGF?3;Trp53+/? mice represent a novel model of low- to high-grade MPNST progression. We further conclude that NRG1 promotes peripheral nervous system neoplasia predominantly via its effects on the signaling cascades affected by Nf1 loss.
Project description:Natural product discovery arises through a unique interplay between chromatographic purification and biological assays. Currently, most techniques used for natural product purification deliver leads without a defined biological action. We now describe a technique, referred to herein as functional chromatography, that deploys biological affinity as the matrix for compound isolation.