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.
Clear cell renal cell carcinoma (CCRCC) evolves due to mutations in the Von Hippel-Lindau (VHL) tumor suppressor gene. Although the loss of VHL enables survival and proliferation of CCRCC cells, it ...is also expected to introduce vulnerabilities that may be exploited for therapeutics discovery. To this end, we developed a high-throughput screen to identify small molecules derived from plants, microorganisms, and marine organisms to which CCRCC cells are sensitive. Screening over 8,000 compounds using this approach, we report here the identification of the microbially derived compound carminomycin I (CA) as an effective inhibitor of VHL-defective (VHL(-/-)) CCRCC cell proliferation. CA also induced apoptosis in CCRCC cells by a mechanism independent of p53 or hypoxia-inducible factor 2. We found that P-glycoprotein (P-gp) sequestered CA within the Golgi complex. Interestingly, Golgi sequestration was critical for the antiproliferative effects of CA and P-gp inhibitors abrogated this activity. Furthermore, CA induced cleavage of the Golgi protein p115 and the translocation of its C-terminal fragment to the nucleus. Finally, examination of the activity of the VHL-interacting Golgi protein, endoplasmic reticulum-Golgi intermediate compartment, ERGIC-53 showed that VHL could mediate protection from CA in CCRCC cells. Our natural product-based screening approach has revealed the P-gp-mediated localization of anticancer compounds within the Golgi in CCRCC cells as a potential strategy of targeting VHL-deficient CCRCC cells.
Actin fibers (F‐actin) control the shape and internal organization of cells, and generate force. It has been long appreciated that these functions are tightly coupled, and in some cases drive cell ...behavior and cell fate. The distribution and dynamics of F‐actin is different in cancer versus normal cells and in response to small molecules, including actin‐targeting natural products and anticancer drugs. Therefore, quantifying actin structural changes from high resolution fluorescence micrographs is necessary for further understanding actin cytoskeleton dynamics and phenotypic consequences of drug interactions on cells. We applied an artificial neural network algorithm, which used image intensity and anisotropy measurements, to quantitatively classify F‐actin subcellular features into actin along the edges of cells, actin at the protrusions of cells, internal fibers and punctate signals. The algorithm measured significant increase in F‐actin at cell edges with concomitant decrease in internal punctate actin in astrocytoma cells lacking functional neurofibromin and p53 when treated with three structurally‐distinct anticancer small molecules: OSW1, Schweinfurthin A (SA) and a synthetic marine compound 23'‐dehydroxycephalostatin 1. Distinctly different changes were measured in cells treated with the actin inhibitor cytochalasin B. These measurements support published reports that SA acts on F‐actin in NF1−/− neurofibromin deficient cancer cells through changes in Rho signaling. Quantitative pattern analysis of cells has wide applications for understanding mechanisms of small molecules, because many anti‐cancer drugs directly or indirectly target cytoskeletal proteins. Furthermore, quantitative information about the actin cytoskeleton may make it possible to further understand cell fate decisions using mathematically testable models. Published 2014 Wiley Periodicals Inc.
A novel cyclopentenedione, asterredione (1), two new terrecyclic acid A derivatives, (+)-5(6)-dihydro-6-methoxyterrecyclic acid A (2) and (+)-5(6)-dihydro-6-hydroxyterrecyclic acid A (3), and five ...known compounds, (+)-terrecyclic acid A (4), (-)-quadrone (5), betulinan A (6), asterriquinone D (7), and asterriquinone C-1 (8), were isolated from Aspergillus terreus occurring in the rhizosphere of Opuntia versicolor, using bioassay-guided fractionation. Acid-catalyzed reaction of 2 under mild conditions afforded 4, whereas under harsh conditions 2 yielded 5 and (-)-isoquadrone (9). Catalytic hydrogenation and methylation of 4 afforded 5(6)-dihydro-terrecyclic acid A (10) and (+)-terrecyclic acid A methyl ester (11), respectively. The structures of 1-11 were elucidated by spectroscopic methods. All compounds were evaluated for cytotoxicity in a panel of three sentinel cancer cell lines, NCI-H460 (non-small cell lung cancer), MCF-7 (breast cancer), and SF-268 (CNS glioma), and were found to be moderately active. Cell cycle analysis of 2, 4, and 5 using the NCI-H460 cell line indicated that 4 is capable of disrupting the cell cycle through an apparent arrest to progression at the G(1) and G(2)/M phases in this p53 competent cell line. A pathway for the biosynthetic origin of asterredione (1) from asterriquinone D (7) is proposed.
Abstract
Mutations in the small GTPases HRAS, NRAS, and splice variants KRAS4a and KRAS4b occur in roughly 20% of all cancers1. Each of these four RAS isoforms share a high degree of homology, yet ...play distinct biological roles; mouse models devoid of KRAS die mid-gestation, whereas HRAS and NRAS null mice survive2. The occurrence of RAS isoform mutations and their specific oncogenic codon substitutions are non-uniform across cancer types, with KRAS mutations in the G12 position frequently driving colorectal cancer while NRAS Q61 mutations are commonly present in skin cancer1. Several explanations have been proposed for this, including differential regulation at the level of transcription, mRNA stability, translation, and protein stability, along with distinctions in post-translational modification, localization, and tissue-specific availability of effectors3. Subtle differences in the biochemical properties of amino acid substitutions may also influence the dominant mutational forms across cancer types. However, the question of whether RAS isoforms and their specific codon substitutions preferentially engage with certain effectors within a cellular context remains unclear, despite considerable efforts. Here, we utilize bioluminescence resonance energy transfer (BRET) between mVenus- and NanoLuc-tagged recombinant proteins to derive quantitative measurements of effector affinity across RAS isoforms and common mutants. By transiently expressing a fixed concentration of NanoLuc-tagged effectors while titrating increasing amounts of mVenus-tagged RAS in a cellular system, we generated saturation curves that reflect the nature of binding between the protein pair in vivo to help untangle this longstanding question. We further validated interactions by immunoprecipitation, and determined construct expression and colocalization via western blot and confocal microscopy.
1Prior IA, Hood FE, Hartley JL. The frequency of Ras mutations in cancer. Cancer Research 20202Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nature Reviews Cancer 2003;3:459-653Li S, Balmain A, Counter CM. A model for RAS mutation patterns in cancers: finding the sweet spot. Nature Reviews Cancer 2018;18:767-77
Citation Format: Megan Rigby, John Columbus, Vanessa Wall, Dominic Esposito, Thomas Turbyville. Interrogating RAS isoform and mutational specificity for effectors with bioluminescence resonance energy transfer (BRET) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2272.
Tumors are dependent on cellular stress responses, in particular the heat shock response, for survival in their hypoxic, acidotic,
and nutrient-deprived microenvironments. Using cell-based reporter ...assays, we have identified terrecyclic acid A (TCA) from
Aspergillus terreus , a fungus inhabiting the rhizosphere of Opuntia versicolor of the Sonoran desert, as a small-molecule inducer of the heat shock response that shows anticancer activity. Further characterization
suggested that TCA also affects oxidative and inflammatory cellular stress response pathways. The presence of an α-methylene
ketone moiety suggested that TCA may form adducts with sulfhydryl groups of proteins. Reaction with labile intracellular cysteines
was supported by our finding that the glutathione precursor N -acetyl-cysteine protected tumor cells from the cytotoxic effects of TCA whereas the glutathione-depleting agent buthionine
sulfoximine enhanced its activity. Related sesquiterpenes have been shown to increase levels of reactive oxygen species (ROS)
and to inhibit nuclear factor κB (NF-κB) transcriptional activity. To assess whether TCA could have similar activities, we
used a ROS-sensitive dye and flow cytometry to show that TCA does indeed increase ROS levels in 3LL cells. When tested in
cells carrying NF-κB reporter constructs, TCA also exhibited concentration-dependent inhibition of cytokine-induced NF-κB
transcriptional activity. These findings suggest that TCA modulates multiple stress pathways—the oxidative, heat shock, and
inflammatory responses—in tumor cells that promote their survival. Small-molecule natural products such as TCA may serve as
useful probes for understanding the relationships between these pathways, potentially providing leads for the design of novel
and effective anticancer drugs.
Abstract
Rat sarcoma (RAS) family of proteins function as GTP/GDP-dependent control switch to regulate Raf-MAPK effector pathway for cell proliferation and are frequently mutated in human cancer. RAS ...is anchored to the inner leaflet of the plasma membrane (PM) via C-terminal lipid-tether(s) and hypothesized to concentrate in distinct locations in membrane nano-domains to control the activation step. However, very little is known about the molecular dynamics of RAS on PM and the precise mechanism of activation. Here we characterize the molecular mobility of RAS variants by single-molecule tracking (SMT) method and estimate underlying mobility states. KRAS4b exhibit confined mobility with three diffusive states, a unique characteristic compared to all the other RAS isoforms (KRAS4a, NRAS, and HRAS). Although the major difference across RAS isoforms lies within the C-terminus HVR (hypervariable region) and its post-translational lipid modifications, the additional confinement for KRAS4b is contributed by the G-domain (globular domain) of the protein. Simulation of KRAS4b on membrane revealed a detailed atomistic mechanism that corroborates with the experimental results. Importantly, the oncogenic mutant of KRAS4b shows an altered mode of diffusion, implicating a plausible attribution to/from enhanced effector interaction. This study uncovers a novel underlying principle of KRAS4b functionality on living cell membrane with a potential for innovation in therapeutic strategies against its oncogenic variant.
Citation Format: Debanjan Goswami, De Chen, John Columbus, Thomas Turbyville. Cooperative membrane interaction between G-domain and HVR defines unique diffusion behavior of KRAS4b abstract. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A09.
Abstract
KRAS4b is a major driver of cancers of the pancreas, lung, and colon, and until recently was considered undruggable. However, recent discoveries have revealed that molecules can target KRAS ...directly, and that our understanding of KRAS is incomplete. To exploit this new appreciation of KRAS druggability and biology, we have developed several protein-protein interaction (PPI) assays to target KRAS activity in live cells. Here we report on a campaign where we screened a 20,000-compound library against full length KRAS4b-G12D and RAF1 interactions in live HEK293 cells using the NanoBRET platform. From the list of screening hits, we identified several structurally related active compounds with dose-dependent, albeit weak, inhibition of the PPI signal. We expanded these active compounds to a more complete list of structural analogs which we tested in a full dose response. From this expanded group of compounds, we identified several with low µM IC50s. Follow up with these compounds in secondary assays revealed promising biological readouts, including inhibition of downstream pERK levels in G12D mutant PANC1 cells. In summary, by using well-designed live cell assays, we have identified promising leads, which we are pursuing with mechanism of action studies.
Citation Format: John Columbus, Thomas J. Turbyville, Vanessa Wall, Dominic Esposito, David A. Barda, Sheng-Bin Peng. A live-cell, protein-protein interaction assay identified inhibitors of KRAS4b-G12D interaction with full-length RAF1 abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-062.
Abstract
RAS proteins are GTP-dependent switches that control and regulate signaling pathways involved in cell fate and are frequently mutated in cancer. RAS association with the plasma membrane, or ...with certain endomembrane compartments, is a required step for its activity. Why precisely this is so remains an open question. One possibility is that RAS is merely required for recruitment of RAF and other effectors to the membrane where it can associate with key regulatory molecules that lead to signaling activation. However, four isoforms exist in humans (HRAS, NRAS, and two splice variants, KRAS4b and KRAS4a) and their differences lie within 22 amino acids in the C-terminal hypervariable region (HVR, aa 167-189). These differences in the domain responsible for membrane association result in the recruitment and organization of RAS into distinct membrane nanodomains, and it is thought that this results in differential signaling behavior from RAS isoforms. Isoform-specific RAS nanodomains are thought to both be highly dynamic, differentially composed of a variety of lipids and proteins, and supported by interactions with cytoskeletal structures. How these domains are regulated, and how they influence isoform-specific RAS signaling, remains unclear. Here, we report the molecular mobility of RAS variants in the plasma membrane of living cancer cells using single-molecule tracking methods. Detailed analysis of tracks revealed that KRAS4b molecules exhibit confined mobility with three diffusive states in the active plasma membrane of living cells. This diffusion characteristic was unique to KRAS4b and influenced by both the hypervariable region and globular domain of the protein, compared to all the other Ras isoforms. Importantly, the occupancy of each diffusive states was altered for the oncogenic mutant of KRAS4b, suggesting that the diffusive states we observe are related to signaling events. Our working hypothesis is that the HVR of KRAS4b may be directly involved in assembling the membrane lipid and protein nanodomain necessary for KRAS4b signaling activity. In addition, using two-color single-molecule tracking studies, we are beginning to characterize the interactions of KRAS4b with its major effector RAF. From these studies, we are learning about the kinetics of RAS/RAF interactions in the membrane of living cells. Understanding the underlying principle of KRAS4b functionality on cell membranes is useful for developing novel therapeutic strategies for targeting oncogenic KRAS4b.
Citation Format: Debanjan Goswami, De Chen, John Columbus, Yue Yang, Felice Lightstone, Thomas Turbyville. KRAS4b’s unique diffusion behavior is defined by plasma membrane and effector interactions abstract. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA07.
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