Carleen Klumpp-Thomas

The phosphatidylinositol-3-phosphate 5-kinase inhibitor apilimod blocks filoviral entry and infection

Phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) is a lipid kinase involved in endosome maturation that emerged from a haploid genetic screen as being required for Ebola virus (EBOV) infection. Here we analyzed the effects of apilimod, a PIKfyve inhibitor that was reported to be well tolerated in humans in phase 2 clinical trials, for its effects on entry and infection of EBOV and Marburg virus (MARV). We first found that apilimod blocks infections by EBOV and MARV in Huh 7, Vero E6 and primary human macrophage cells, with notable potency in the macrophages (IC50, 10 nM). We next observed that similar doses of apilimod block EBOV-glycoprotein-virus like particle (VLP) entry and transcription-replication competent VLP infection, suggesting that the primary mode of action of apilimod is as an entry inhibitor, preventing release of the viral genome into the cytoplasm to initiate replication. After providing evidence that the anti-EBOV action of apilimod is via PIKfyve, we showed that it blocks trafficking of EBOV VLPs to endolysosomes containing Niemann-Pick C1 (NPC1), the intracellular receptor for EBOV. Concurrently apilimod caused VLPs to accumulate in early endosome antigen 1-positive endosomes. We did not detect any effects of apilimod on bulk endosome acidification, on the activity of cathepsins B and L, or on cholesterol export from endolysosomes. Hence by antagonizing PIKfyve, apilimod appears to block EBOV trafficking to its site of fusion and entry into the cytoplasm. Given the drug’s observed anti-filoviral activity, relatively unexplored mechanism of entry inhibition, and reported tolerability in humans, we propose that apilimod be further explored as part of a therapeutic regimen to treat filoviral infections.

Evaluation of the Activity of Lamivudine and Zidovudine against Ebola Virus

In the fall of 2014, an international news agency reported that patients suffering from Ebola virus disease (EVD) in Liberia were treated successfully with lamivudine, an antiviral drug used to treat human immunodeficiency virus-1 and hepatitis B virus infections. According to the report, 13 out of 15 patients treated with lamivudine survived and were declared free from Ebola virus disease. In this study, the anti-Ebola virus (EBOV) activity of lamivudine and another antiretroviral, zidovudine, were evaluated in a diverse set of cell lines against two variants of wild-type EBOV. Variable assay parameters were assessed to include different multiplicities of infection, lengths of inoculation times, and durations of dosing. At a multiplicity of infection of 1, lamivudine and zidovudine had no effect on EBOV propagation in Vero E6, Hep G2, or HeLa cells, or in primary human monocyte-derived macrophages. At a multiplicity of infection of 0.1, zidovudine demonstrated limited anti-EBOV activity in Huh 7 cells. Under certain conditions, lamivudine had low anti-EBOV activity at the maximum concentration tested (320 μM). However, lamivudine never achieved greater than 30% viral inhibition, and the activity was not consistently reproducible. Combination of lamivudine and zidovudine showed no synergistic antiviral activity. Independently, a set of in vitro experiments testing lamivudine and zidovudine for antiviral activity against an Ebola-enhanced green fluorescent protein reporter virus was performed at the Centers for Disease Control and Prevention. No antiviral activity was observed for either compound. A study evaluating the efficacy of lamivudine in a guinea pig model of EVD found no survival benefit. This lack of benefit was observed despite plasma lamivudine concentrations in guinea pig of about 4 μg/ml obtained in a separately conducted pharmacokinetics study. These studies found no evidence to support the therapeutic use of lamivudine for the treatment of EVD.

Identification of HDAC Inhibitors Using a Cell-Based HDAC I/II Assay.

Histone deacetylases (HDACs) are a class of epigenetic enzymes that regulate gene expression by histone deacetylation. Altered HDAC function has been linked to cancer and neurodegenerative diseases, making HDACs popular therapeutic targets. In this study, we describe a screening approach for identification of compounds that inhibit endogenous class I and II HDACs. A homogeneous, luminogenic HDAC I/II assay was optimized in a 1536-well plate format in several human cancer cell lines, including HCT116 and human neural stem cells. The assay confirmed 37 known HDAC inhibitors from two libraries of known epigenetics-active compounds. Using the assay, we identified a group of potential HDAC inhibitors by screening the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection of 2527 small-molecule drugs. The selected compounds showed similar HDAC I/II inhibitory potency and efficacy values in both HCT116 and neural stem cells. Several previously unidentified HDAC inhibitors were further evaluated and profiled for their selectivity against a panel of 10 HDAC I/II isoforms using fluorogenic HDAC biochemical assays. In summary, our results show that several novel HDAC inhibitors, including nafamostat and piceatannol, have been identified using the HDAC I/II cell-based assay, and multiple cell types have been validated for high-throughput screening of large chemical libraries.

Functional Genomic Screening Reveals Splicing of the EWS-FLI1 Fusion Transcript as a Vulnerability in Ewing Sarcoma

Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells.

Identification of Combinations of Approved Drugs With Synergistic Activity Against Ebola Virus in Cell Cultures

Background A need to develop therapeutics to treat Ebola virus disease patients in remote and resource-challenged settings remains in the wake of the 2013-2016 epidemic in West Africa. Toward this goal, we screened drugs under consideration as treatment options and other drugs of interest, most being small molecules approved by the Food and Drug Administration. Drugs demonstrating in vitro antiviral activity were advanced for evaluation in combinations because of advantages often provided by drug cocktails. Methods Drugs were screened for blockade of Ebola virus infection in cultured cells. Twelve drugs were tested in all (78 pair-wise) combinations, and 3 were tested in a subset of combinations. Results Multiple synergistic drug pairs emerged, with the majority comprising 2 entry inhibitors. For the pairs of entry inhibitors studied, synergy was demonstrated at the level of virus entry into host cells. Highly synergistic pairs included aripiprazole/piperacetazine, sertraline/toremifene, sertraline/bepridil, and amodiaquine/clomiphene. Conclusions Our study shows the feasibility of identifying pairs of approved drugs that synergistically block Ebola virus infection in cell cultures. We discuss our findings in terms of the theoretic ability of these or alternate combinations to reach therapeutic levels. Future research will assess selected combinations in small-animal models of Ebola virus disease.

Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening

Cell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaign are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for a more in-depth downstream evaluation influence the future direction of projects, so cytotoxicity profiling of the screening libraries at early stage is essential for increasing the likelihood of candidate success. In this study, we analyzed results from a cell-based cytotoxicity screening campaign, comprising nearly 10,000 compounds in NCATS annotated libraries, and over 100,000 compounds in a diversity library, evaluated in four “normal” cell lines (HEK 293, NIH 3T3, CRL-7250 and HaCat) and a cancer cell line (KB 3-1, a HeLa subline) with overall screening outcomes, hit rates, pan-activity and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile large libraries for cytotoxicity in both normal and cancer cell lines. In summary, the results generated here constitutes a valuable resource for the scientific community and provides insight on identifying cytotoxic compounds with particular mechanisms of action, and prioritizing compounds with suitable cytotoxicity profile for further evaluation.

Mutation Profiles in Glioblastoma 3D Oncospheres Modulate Drug Efficacy

Glioblastoma (GBM) is a lethal brain cancer with a median survival time of approximately 15 months following treatment. Common in vitro GBM models for drug screening are adherent and do not recapitulate the features of human GBM in vivo. Here we report the genomic characterization of nine patient-derived, spheroid GBM cell lines that recapitulate human GBM characteristics in orthotopic xenograft models. Genomic sequencing revealed that the spheroid lines contain alterations in GBM driver genes such as PTEN, CDKN2A, and NF1. Two spheroid cell lines, JHH-136 and JHH-520, were utilized in a high-throughput drug screen for cell viability using a 1912-member compound library. Drug mechanisms that were cytotoxic in both cell lines were Hsp90 and proteasome inhibitors. JHH-136 was uniquely sensitive to topoisomerase 1 inhibitors, while JHH-520 was uniquely sensitive to Mek inhibitors. Drug combination screening revealed that PI3 kinase inhibitors combined with Mek or proteasome inhibitors were synergistic. However, animal studies to test these drug combinations in vivo revealed that Mek inhibition alone was superior to the combination treatments. These data show that these GBM spheroid lines are amenable to high-throughput drug screening and that this dataset may deliver promising therapeutic leads for future GBM preclinical studies.

A single nucleotide polymorphism in the Plasmodium falciparum atg18 gene associates with artemisinin resistance and confers enhanced parasite survival under nutrient deprivation

BackgroundArtemisinin-resistant Plasmodium falciparum has been reported throughout the Greater Mekong subregion and threatens to disrupt current malaria control efforts worldwide. Polymorphisms in kelch13 have been associated with clinical and in vitro resistance phenotypes; however, several studies suggest that the genetic determinants of resistance may involve multiple genes. Current proposed mechanisms of resistance conferred by polymorphisms in kelch13 hint at a connection to an autophagy-like pathway in P. falciparum.ResultsA SNP in autophagy-related gene 18 (atg18) was associated with long parasite clearance half-life in patients following artemisinin-based combination therapy. This gene encodes PfAtg18, which is shown to be similar to the mammalian/yeast homologue WIPI/Atg18 in terms of structure, binding abilities, and ability to form puncta in response to stress. To investigate the contribution of this polymorphism, the atg18 gene was edited using CRISPR/Cas9 to introduce a T38I mutation into a k13-edited Dd2 parasite. The presence of this SNP confers a fitness advantage by enabling parasites to grow faster in nutrient-limited settings. The mutant and parent parasites were screened against drug libraries of 6349 unique compounds. While the SNP did not modulate the parasite’s susceptibility to any of the anti-malarial compounds using a 72-h drug pulse, it did alter the parasite’s susceptibility to 227 other compounds.ConclusionsThese results suggest that the atg18 T38I polymorphism may provide additional resistance against artemisinin derivatives, but not partner drugs, even in the absence of kelch13 mutations, and may also be important in parasite survival during nutrient deprivation.

mQC: A Heuristic Quality-Control Metric for High-Throughput Drug Combination Screening.

Quality control (QC) metrics are critical in high throughput screening (HTS) platforms to ensure reliability and confidence in assay data and downstream analyses. Most reported HTS QC metrics are designed for plate level or single well level analysis. With the advent of high throughput combination screening there is a need for QC metrics that quantify the quality of combination response matrices. We introduce a predictive, interpretable, matrix-level QC metric, mQC, based on a mix of data-derived and heuristic features. mQC accurately reproduces the expert assessment of combination response quality and correctly identifies unreliable response matrices that can lead to erroneous or misleading characterization of synergy. When combined with the plate-level QC metric, Z’, mQC provides a more appropriate determination of the quality of a drug combination screen. Retrospective analysis on a number of completed combination screens further shows that mQC is able to identify problematic screens whereas plate-level QC was not able to. In conclusion, our data indicates that mQC is a reliable QC filter that can be used to identify problematic drug combinations matrices and prevent further analysis on erroneously active combinations as well as for troubleshooting failed screens. The R source code of mQC is available at http://matrix.ncats.nih.gov/mQC.

Abstract 479: Inhibition of the splicing of the EWS-FLI1 fusion transcript reverses EWS-FLI1 driven oncogenic expression in Ewing sarcoma

Ewing sarcoma (ES) is a highly aggressive cancer of the bone and soft tissue. In ∼85% of ES tumors the primary oncogenic event is a t(11:22)(q24:q12) translocation that generates a fusion of the 5′ end of EWSR1 and the 3′ end of FLI1 referred to as EWS-FLI1. The exact genomic breakpoints within the EWSR1 and FLI1 genes vary, but typically occur within introns and require the splicing machinery to generate an in-frame EWS-FLI1 transcript. The most common EWS-FLI1 transcripts fuse either exon 7 of EWSR1 to exon 6 of FLI1 (a type I or a 7/6 fusion), or fuse exon 7 of EWSR1 to exon 5 of FLI1 (a type II or 7/5 fusion). In an estimated 40% of EWS-FLI1 driven tumors the generation of an in-frame EWS-FLI1 fusion transcript requires alternative splicing. In particular, translocations that retain exon 8 of EWSR1 generate an out-of-frame transcript unless this exon is removed. Using an assay of EWS-FLI1 activity and genome-wide siRNA screening we have identified RNA processing as a therapeutic vulnerability in ES. Parallel genome-wide siRNA-mediated RNAi screens were conducted in ES TC32 cell lines expressing a luciferase (luc) reporter protein driven by either the promoter of the EWS-FLI1 target gene NR0B1 (TC32-NR0B1-luc) or the CMV promoter (TC32-CMV-luc). The top gene ontology terms associated with the 28 priority candidate genes that when silenced induced a differential decrease in the TC32-NR0B1-luc signal versus the TC32-CMV-luc signal were mRNA splicing (p-value = 1.42E-08) and mRNA processing (p-value = 2.32E-07). To investigate the mechanistic basis for the identification of specific RNA processing proteins as required for the activity of EWS-FLI1 we focused on two lead candidate genes, the heterogeneous nuclear ribonucleoprotein H1, HNRNPH1, and the core splicing factor, SF3B1. Using PCR analysis we determined that HNRNPH1 is required for the splicing of EWS-FLI1 fusion transcripts expressed in ES cells in which the chromosome 22 breakpoint retains EWSR1 exon 8, specifically in TC32 and SKNMC ES cells. We also show ES cell lines harboring 7/ 6 (TC32, SKNMC, and TC71) or 7/ 5 (RD-ES) EWS-FLI1 fusions are all sensitive to the loss-of-function of SF3B1. Quantitative RT-PCR, immunoblot, and whole transcriptome analysis show that disrupted splicing of the EWS-FLI1 transcript alters its expression and reverses the expression of a significant proportion of genes that are targets of EWS-FLI1. These observations were confirmed in four ES cell lines using the splicing inhibitor Pladienolide B. Our results provide the basis for a novel strategy to target fusion oncogenes by interfering with RNA processing. This study has implications for the treatment of ES through inhibition of proteins required for expression of the EWS-FLI1 transcript and identifies a candidate lead compound for further clinical development. Our findings may also open up strategies for treatment of other cancers driven by fusion oncogenes. Citation Format: Patrick J. Grohar, Suntae Kim, Sara Haddock, Guillermo Rangel Rivera, Matt Harlow, Nichole K. Maloney, Konrad Huppi, Kristen Gehlhaus, Magdalena Grandin, Carleen Klumpp-Thomas, Eugen Buehler, Lee J. Helman, Scott E. Martin, Natasha J. Caplen. Inhibition of the splicing of the EWS-FLI1 fusion transcript reverses EWS-FLI1 driven oncogenic expression in Ewing sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 479. doi:10.1158/1538-7445.AM2015-479