University of Texas Southwestern Medical Center (complete)

Lung Cancer Oncogenotype-selective Drug Target Discovery (Natural Products Focus)

The goal of this project is to use small molecules and RNAi to functionally define subtypes of non-small cell lung cancer (NSCLC) using a panel of cell lines prepared and molecularly annotated by Drs. John Minna and Adi Gazdar.

Experimental Approaches

Lung Cancer Natural Products Screening/Chemical Library Screening

Using biological and molecular characteristics, we divided a panel of 108 tumor samples representative of non-small cell lung cancer (NSCLC) into 7 distinct families, or clades. Based on preliminary data, we anticipated that the tumor membership of each clade will have shared genetic and chemical vulnerabilities, which can be exploited for target identification and drug discovery. To pursue this hypothesis, we selected representative cell lines from each NSCLC clade for chemical library screening. The UT Southwestern chemical library consists of ~230,000 compounds and includes ~25,000 natural products, 1,200 approved drugs, and 1,000 compounds prepared by UT Southwestern chemists. Screens were conducted using the Cell Titer Glo assay platform from Promega, Inc., which measures cell viability in terms of ATP levels. Data from primary screening and confirmation and dose-response studies were analyzed using GeneData’s Screener^TM version 9.

Genome-Wide RNAi Screening

Using biological and molecular characteristics, we divided a panel of 108 tumor samples representative of non-small cell lung cancer (NSCLC) into 7 distinct families, or clades. Based on preliminary data, we anticipated that the tumor membership of each clade will have shared genetic and chemical vulnerabilities which can be exploited for target identification and drug discovery. To address genetic vulnerabilities, we selected representative cell lines from each NSCLC clade for genome-wide RNAi screening. Screens were conducted using the Cell Titer Glo assay platform from Promega, Inc., which measures cell viability in terms of ATP levels. Two genome-wide libraries were screened against each cell line (Dharmacon and Ambion libraries) in order to ensure complete coverage of genes and pathways. Data from primary screening and confirmation studies were analyzed using Gene Data’s Screener^TM version 9.

Functional Signature Ontology Tool: Triplicate Measurements of Reporter Gene Expression in Response to Individual Genetic and Chemical Perturbations in HCT116 Cells (Colon Cancer)

The goal of this project is to use an eight-gene expression profile to define functional signatures for small molecules and natural products with heretofore undefined mechanism of action. Two genes in the eight gene set are used as internal controls and do not vary across gene expression array data collected from the public domain. The remaining six genes are found to vary independently across a large collection of publicly available gene expression array datasets. 

Experimental Approaches

Lung Cancer Natural Products Screening/Chemical Library Screening

Reporter gene expression was profiled via QuantiGene Plex 2.0 assays (Panomics) with a Luminex 200 machine.

U01 Natural Products Screening

The goal of this project was to enlarge the chemical space probed by Project 1 (High-Throughput siRNA Screening of a Non-Small Cell Lung Cancer Cell Line Panel) by screening an expanded natural products library (~40,000) in an effort to further define vulnerabilities and therapeutic targets in non-small cell lung cancer. This new library is derived from a diverse collection of marine bacteria (prepared by Dr. John MacMillan, University of Texas Southwestern). Because of the higher potency of natural products observed in previous screening projects, this collection was screened using a limited dose response approach in a viability format (see Experimental Approaches).

Experimental Approaches

Lung Cancer Natural Products Screening/Chemical Library Screening

Screens were conducted using the Cell Titer Glo assay platform from Promega, Inc. (as described for Project 1; High-Throughput siRNA Screening of a Non-Small Cell Lung Cancer Cell Line Panel) which measures cell viability in terms of ATP levels. Each natural product fraction in the MacMillan natural product library (7,500 fractions) was tested at 4 doses in singleton using the CTG assay. Data from primary screening and confirmation and dose-response studies were analyzed using GeneData’s Screener^TM version 9.

High-throughput siRNA Screening of a Non-small Cell Lung Cancer (NSCLC) Cell Line Panel

The goal of this project is to use siRNA screens to identify NSCLC-selective siRNAs from two genome-wide libraries that will allow us to functionally define genetic dependencies of subtypes of NSCLC. Using bioinformatics tools, the CTD² Center at the University of Texas Southwestern Medical Center are discovering associations between this functional data (siRNAs) and NSCLC mutational status, methylation arrays, gene expression arrays, and copy number variation data that will help us identify new targets and enrollment biomarkers. 

Experimental Approaches

Two commercial genome wide siRNA libraries from Ambion (library 1; 21,585 genes) and Dharmacon (library 2; 18,175 genes) were purchased in the 96-well plate format. siRNAs were dissolved in 13 siRNA buffer (Dharmacon) overnight to a final concentration of 10 mM and stored at 80°C prior to use. Library 1 and 2 are mixes of 3 and 4 individual siRNA oligos/genes, respectively. For the primary and secondary screens, cells were cultured in ACL4 (normal cell lines and HCC4017) with 2% FBS or RPMI medium (all other cancer lines) with 5% FBS. For reverse transfections 10 pmole of each siRNA pool was transferred (3 ml) to serum free media (95 ml/well) in empty 96-well assay plates (Costar). Thirty μl of this siRNA solution was transferred to an empty 96-well optical assay plate (BioMek), incubated for 5 minutes, then mixed with 10 ml transfection reagent solution (0.13 ml RNAiMax [Invitrogen] in 10 ml serum free medium), and incubated for 15 minutes. Cells were harvested and diluted in parallel (10,000 cells/100 ml), then added to the siRNA-lipid mix and incubated for 96 hours. CellTiter-Glo (Promega) assays were performed using 15 ml reagent/well followed by a 10 minute incubation prior to quantitation of luminescence with an Envision plate reader (PerkinElmer). Primary screen data were normalized by top quantile per plate. Standard Deviations of the triplicate activities were calculated for each gene.

Identification of ASCL1 as a Therapeutic Target for High-grade Neuroendocrine Lung Cancers

The goal of this project is to use genetic screening of lung cancer cells to reveal novel therapeutic targets.

SW04428 is a Novel Topoisomerase 1 Inhibitor

This study shows the changes in gene expression in response to SW044248, a compound that displays selective toxicity for some NSCLC cell lines. This data led to the discovery that SW044248 is an inhibitor of topoisomerase 1 (Top1) different from other Top1 inhibitors such as camptothecin¹.

Experimental Approaches

RNA was extracted and analyzed by RNAseq from non-small cell lung cancer (NSCLC) cell line HCC4017 treated with SW04428.

NSCLC Cell Lines with Loss of SMARCA4 Expression are Hypersensitive to Inhibitors of Aurora Kinase A

A genome-wide siRNA screen was employed to identify genes that were selectively toxic for a non-small cell lung cancer (NSCLC) cell line that lacked expression of SMARCA4, but were not toxic in non-cancerous immortalized human bronchial epithelial cells lacking SMARCA4 expression. Among the selectively toxic genes were several mapping to the molecular machinery regulating activity of Aurora kinase A on the mitotic spindle. NSCLC cell lines lacking SMARCA4 expression were found to be hypersensitive to inhibition of Aurora kinase A with five different Aurora kinase inhibitors and this sensitivity was confirmed to occur in xenografts of a sensitive and non-sensitive cell line in mice.

Primary data for this work is in the Supplementary Data of the manuscript or under GEO accession number GSE32036.

References

1. Ramirez RD, et al. (2004) Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer research 64(24):9027-9034.
2. Sif S, et al. (2001) Purification and characterization of mSin3A-containing Brg1 and hBrm chromatin remodeling complexes. Genes & Development 15(5):603-618.
3. Wang L, et al. (2015) Genomic profiling of Sezary syndrome identifies alterations of key T cell signaling and differentiation genes. Nature Genetics 47(12):1426-1434.
4. Dobin A, et al. (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29(1):15-21.
5. Wang L, et al. (2012) RSeQC: quality control of RNA-seq experiments. Bioinformatics 28(16):2184-2185.
6. Trapnell C, et al. (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nature Protocols 7(3):562-578.
7. Byers LA, et al. (2013) An epithelial-mesenchymal transition gene signature predicts resistance to EGFR and PI3K inhibitors and identifies Axl as a therapeutic target for overcoming EGFR inhibitor resistance. Clinical Cancer Research 19(1):279-290.

DRC Study of Natural Products Against Non-small Cell Lung Cancer (NSCLC) Cell Lines

In this study, near 1000 natural products (fractionated from marine originated, laboratory produced bacteria) were screened against 55 cancer cell lines at 12 concentrations by half-log dilutions.

Experimental Approaches

Screens were conducted using the Cell Titer Glo (CTG) assay platform from Promega, Inc., which measures cell viability in terms of ATP levels. Data were analyzed using GeneData’s Screener^TM version 13.

Briefly, cell lines were cultured in 10 cm dishes (Corning, Inc.) in NSCLC culture medium (RPMI/L-glutamine medium (Invitrogen, Inc.), 1000 U/ml penicillin (Invitrogen, Inc.), 1 mg/ml streptomycin (Invitrogen, Inc.), and 5% fetal bovine serum (Atlanta Biologicals, Inc.). Cell lines were maintained in a humidified environment in the presence of 5% CO2 at 37^oC. For cell viability assays, Cells (60 µL) were plated at appropriate density (numbers available upon request) in 384 well microtiter assay plates (Bio-one; Greiner, Inc.).

After incubating the assay plates overnight under the growth conditions described above, natural product fractions or control compounds were added to each plate to 12 final compound concentration after serial half-log dilutions. In all experiments, we used a final DMSO concentration of 0.5% as neutral controls. After an incubation of 96 hours under growth conditions, Cell Titer Glo^TM reagent (Promega, Inc.) was added to each well (10 µL of a 1:2 dilution in NSCLC culture medium or ACL4) and mixed. Plates were incubated for 10 minutes at room temperature and luminescence was determined for each well using an EnVision multi-label plate reader (Perkin-Elmer, Inc.). The Genedata Screener® software (version 11 or later, GeneData, Inc. Basel, Switzerland) was used to process and analyze the screening data. The raw data values for all wells were normalized using the equation: Normalized Values= (Raw Values - Median of DMSO controls)/(Median of Positive controls - Median of DMSO controls)*100. Dose-response curves were created by smart fit algorithm with Genedata software, and fit parameters (AC50 mode, qAC50, S0, Sinf and nHill) were exported to a spreadsheet.

High-throughput Screening of CTD² Network Informer Set (CNIS) Compounds Against Non-Small-Cell Lung Cancer (NSCLC) Cell Lines

Small-molecule probes and drugs selectively target distinct nodes in cell circuitry and collectively modulate a broad array of cell processes. In this study, 303 CNIS compounds were screened against a panel of 70 NSCLC cell lines using Cell Titer Glo^TM assay platform. These primary screening results demonstrate that the CNIS compounds have differential cytotoxic effects on NSCLC cell lines, which is valuable information for further studies, e.g., subtype-selective compounds from the CNIS will be used, along with orthogonal datasets, to generate biomarker hypotheses that will be prioritized and validated.

Experimental Approaches

Screens were conducted using the CTG assay platform from Promega, Inc., which measures cell viability in terms of ATP levels. Data were analyzed using GeneData Screener^TM v13.0.

Briefly, cell lines were cultured in 10-cm dishes (Corning, Inc.) in NSCLC culture medium: RPMI/L-glutamine medium (Invitrogen, Inc.), 1000 U/mL penicillin (Invitrogen, Inc.), 1 mg/mL streptomycin (Invitrogen, Inc.), and 5% fetal bovine serum (Atlanta Biologicals, Inc.). Cell lines were maintained in a humidified environment in the presence of 5% CO² at 37 ^oC. For cell-viability assays, cells (60 µL) were plated at appropriate density (numbers available upon request) in 384-well microtiter assay plates (Bio-one; Greiner, Inc.). After incubating the assay plates overnight under these growth conditions, compounds were added to each plate at a final concentration of 5 µM. In all experiments, the final DMSO concentration was maintained at 0.5%. After an incubation for 96 hours under growth conditions, Cell Titer Glo^TM reagent (Promega, Inc.) was added to each well (10 µL of a 1:2 dilution in NSCLC culture medium or ACL4) and mixed. A positive control (brefeldin A, 10 µM in column 1) was included on each assay plate as was a neutral control (DMSO only, columns 2 and 23) and an untreated control (column 24, no DMSO or compound). Plates were incubated for 10 minutes at room temperature and luminescence was determined for each well using an EnVision multi-label plate reader (Perkin-Elmer, Inc.). The assays typically displayed Z’ factors greater than 0.6. The GeneData Screener® software (v13.0, GeneData, Inc., Basel, Switzerland) was used to process and analyze the screening data. The raw data values for all wells were normalized using the following equation:

normalized value = 100 * (raw value - median of DMSO controls) / (median of positive controls - median of DMSO controls)