Isogenic Cell Line Pairs Research Articles

A kinome siRNA screen identifies HGS as a potential target for liver cancers with oncogenic mutations in CTNNB1.

BackgroundAberrant activation of the Wnt/β-catenin pathway is a major and frequent event in liver cancer, but inhibition of oncogenic β-catenin signaling has proven challenging. The identification of genes that are synthetically lethal in β-catenin-activated cancer cells would provide new targets for therapeutic drug design.MethodsWe transfected the parental HuH6 hepatoblastoma cell line with a doxycycline-inducible shRNA against CTNNB1 (gene coding for β-catenin) to obtain an isogenic cell line pair with or without aberrant β-catenin signaling. Using this hepatoblastoma isogenic cell line pair, we performed a human kinome-wide siRNA screen to identify synthetic lethal interactions with oncogenic CTNNB1. The phenotypic readouts of the screen were cell proliferation, cell cycle arrest and apoptosis, which were assessed by image-based analysis. In addition, apoptosis was assessed by flow cytometric experiments and immunoblotting. The potential synthetic lethal relationship between candidates genes identified in the screen and oncogenic CTNNB1 was also investigated in a different cellular context, a colorectal HCT116 isogenic cell line pair.ResultsWe first determined the experimental conditions that led to the efficient expression of shRNA against CTNNB1 and maximal reduction of β-catenin signaling activity in response to doxycycline treatment. Following high throughput screening in which 687 genes coding for kinases and proteins related to kinases (such as pseudokinases and phosphatases) were targeted, we identified 52 genes required for HuH6 survival. The silencing of five of these genes selectively impaired the viability of HuH6 cells with high β-catenin signaling: HGS, STRADA, FES, BRAF and PKMYT1. Among these candidates, HGS depletion had the strongest inhibitory effect on cell growth and led to apoptosis specifically in HuH6 with high β-catenin activity, while HuH6 with low β-catenin activity were spared. In addition, HGS was identified as a potential synthetic lethal partner of oncogenic CTNNB1 in the HCT116 colorectal isogenic cell line pair.ConclusionsThese results demonstrate the existence of crosstalk between β-catenin signaling and HGS. Importantly, HGS depletion specifically affected cells with uncontrolled β-catenin signaling activity in two different types of cancer (Hepatoblastoma HuH6 and colorectal HCT116), and thus may represent a new potential target for novel therapeutic strategies in liver and colorectal cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-2037-8) contains supplementary material, which is available to authorized users.

Abstract A182: Targeting the topoisomerase I enzyme in cancer cells with acquired resistance to SN-38

Abstract De novo or acquired resistance to anti-cancer drugs represents a major obstacle to successful treatment of cancer patients. In order to improve future cancer drug development we have established the DEN-50R cell line panel which when fully developed will consist of isogenic pairs of drug sensitive and resistant human cancer cell lines representing the 5 most common cancer types. Based on the DEN-50R we have tested a number of novel anti-cancer drugs in this panel of cell lines. One class of drugs that includes the indenoisoquinolines LMP400 and LMP776, which have topoisomerase 1 inhibitory activity and recently passed phase 1 clinical studies, showed very interesting features in the cell line studies. When tested in SN-38 (the active metabolite of irinotecan) -sensitive and -resistant isogenic colorectal cancer cell lines and breast cancer cell lines, LMP400 showed significant dose related cytotoxicity independent of the state of SN-38-resistance (Table). As these resistant cell lines have significant up-regulation of either the BCRP and/or mdr-1 protein, it can be concluded that the indenoisoquinoline LMP400 can target topoisomerase 1 enzyme without being a substrate for these drug efflux pumps. We are now planning to initiate two clinical phase II studies (Simon's two stage design) with LMP400; one in irinotecan-failed metastatic colorectal cancer patients and one in late stage metastatic breast cancer patients. Patients for these studies will be preselected based on an LMP400-responsiveness profile we generated by gene expression data where associations between gene expression profiles and growth inhibition were compared in a panel of cell lines exposed to LMP400. A second step included filtering the identified gene expression profile against mRNA expression from a collection of 3200 human tumors. Only genes being differentially expressed in the clinical tumor material were retained in the model. Sensitivity to LMP776 and LMP400 of human colorectal and breast cancer cell lines with acquired resistance to SN-38 Cell lineCross-resistantSensitiveHCT116-SN-38*LMP400; LMP776HT29-SN-38LMP776LMP400LoVo-SN-38LMP776LMP400MDA-MB-231- SN-38LMP776LMP400MCF-7- SN-38LMP776; LMP400* HCT-116-SN38 resistant cells have gained a mutation in the binding site of SN38 and LMP400 during acquisition of SN-38 resistance. Citation Format: Jan Stenvang, Niels Frank Jensen, Haatisha Jandu, Steen Knudsen, Keli Agama, Thomas Jensen, Anker Hansen, Peter Buhl Jensen, Yves Pommier, Mark Cushman, Nils Brünner. Targeting the topoisomerase I enzyme in cancer cells with acquired resistance to SN-38. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A182.

A Comparison of Real-Time and Endpoint Cell Viability Assays for Improved Synthetic Lethal Drug Validation

Cell viability assays fulfill a central role in drug discovery studies. It is therefore important to understand the advantages and disadvantages of the wide variety of available assay methodologies. In this study, we compared the performance of three endpoint assays (resazurin reduction, CellTiter-Glo, and nuclei enumeration) and two real-time systems (IncuCyte and xCELLigence). Of the endpoint approaches, both the resazurin reduction and CellTiter-Glo assays showed higher cell viabilities when compared directly to stained nuclei counts. The IncuCyte and xCELLigence real-time systems were comparable, and both were particularly effective at tracking the effects of drug treatment on cell proliferation at sub-confluent growth. However, the real-time systems failed to evaluate contrasting cell densities between drug-treated and control-treated cells at full growth confluency. Here, we showed that using real-time systems in combination with endpoint assays alleviates the disadvantages posed by each approach alone, providing a more effective means to evaluate drug toxicity in monolayer cell cultures. Such approaches were shown to be effective in elucidating the toxicity of synthetic lethal drugs in an isogenic pair of MCF10A breast cell lines.

Abstract C161: CRISPR/Cas9 genome-wide gRNA library screening platform

Abstract CRISPR/Cas9 gene knock-out technology can be used as a powerful tool for large-scale functional genomic analysis in mammalian cells. With the goal to establish a cost-effective functional genomics platform for the discovery of therapeutic targets, we developed a highly functional high throughput lentiviral gRNA/CRISPR screening platform, enabling scientists to perform pooled format genome-wide CRISPR/Cas9 genetic screens. Three 55K gRNA libraries were designed to cover the whole protein-encoding human genome with a redundancy of 8 gRNAs per gene. As supporting tools, we developed protocols, reagents, and software tools for hit validation and target prioritization. Here we show the results of parallel genetic screens in a pair of isogenic CML cell lines (CML-R and CML-P), where the performance of CRISPR/Cas9 and RNAi screening platforms was compared for each cell line. Citation Format: Donato Tedesco, Paul Diehl, Mikhail Makhanov, Sylvain Baron, Dmitry Suchkov, Alex Chenchik. CRISPR/Cas9 genome-wide gRNA library screening platform. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C161.

Abstract B2-45: Uncovering coordinated regulation of transcription factors by microRNAs using integrated network models

Abstract MicroRNAs (miRNAs) are small non-coding RNAs that play a significant role in development and cancer but induce only moderate repression of direct messenger RNA (mRNA) targets, suggesting that they coordinate with other modes of cellular regulation to affect cellular phenotype. In this work we exhaustively profile the transcriptional and post-transcriptional regulatory changes between an isogenic pair of murine fibroblast cell lines with and without Dicer, an enzyme required for miRNA processing. Through quantitative analysis of diverse high-throughput datasets collected from both cell lines, we were able to decouple transcriptional from post-transcriptional changes reflected in mRNA expression changes between cells with and without miRNAs. We present three major findings. First, we find that direct miRNA-mRNA interactions have a limited impact on gene expression changes upon Dicer deletion when compared to changes at transcription. We then introduce an integrative graphical network approach to identify specific transcription factors that explain the observed changes in gene expression upon loss of Dicer. Validation of this computational model via transcription factor over-expression reveals a subset of the miRNA-mediated transcriptional program that is activated upon Dicer loss, confirming that transcriptional networks amplify the effects of miRNAs. Lastly, we use this network to examine coordination between transcriptional and post-transcriptional regulation. We identify numerous coherent and in-coherent feed-forward loops, network motifs that have been alluded to but only minimally studied in the context of microRNAs and transcription factors, and find that genes regulated within feed-forward loops by microRNAs and transcription factors exhibit distinct properties in development. In summary, our work illustrates how transcriptional networks amplify the effects of microRNAs. We show coordinated regulation of transcription factors by microRNAs that suggests that most gene expression changes attributed to microRNAs in diseases such as cancer are due to changes in transcription rather than microRNA-mediated degradataion. As such, this work has the potential to reframe future studies of microRNAs in the context of cancer by shifting the focus to understanding the impact of microRNAs on transcription factors. Citation Format: Sara JC Gosline, Allan M. Gurtan, Courtney K. JnBaptiste, Andrew Bosson, Pamela Milani, Simona Dalin, Bryan Matthews, Yoon Sing Yap, Phillip A. Sharp, Ernest Fraenkel. Uncovering coordinated regulation of transcription factors by microRNAs using integrated network models. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B2-45.

Abstract 4473: WP760, A new highly potent and selective agent against melanoma including BRAFi resistant melanoma

Abstract Melanoma is expected to be diagnosed in 76,100 patients in the United States, and 9,710 patients are estimated to die of the disease in 2014. Early-stage melanomas can often be treated effectively with surgery alone, but more advanced cancers often require other treatments, including radiation therapy, immunotherapy, chemotherapy and targeted therapy. Targeted therapy with vemurafenib is used to treat approximately 60 percent of melanoma patients with activating mutation in the BRAF gene. V600E mutation in the BRAF gene is detected in over half of the melanoma patients and targeted inhibition of the oncogenic BRAF has shown a very good initial response in melanoma patients, however, this treatment alone turned out to be unsuccessful due to the rapid development of resistance. Development of therapeutics for use in combination with BRAF inhibitors or strategies overcoming the resistance to vemurafenib is a subject of intensive research. Systematic screening of unique DNA bisintercalating agents of our own design using NCI 60 cell line panel led to identification of highly potent and melanoma selective compound WP760. The main aim of this study was to assess antiproliferative activity of WP760 in a panel of melanoma cell lines including BRAFi resistant cell lines. Here we have shown that WP760 inhibited proliferation of melanoma cell in low nanomolar range and was not toxic to normal fibroblasts. Importantly, WP760 inhibited potently proliferation of cells resistant to BRAF inhibitors and wild type BRAF in low nanomolar range. Moreover, it was equally active in pairs of isogenic cell lines: naïve and those which developed resistance to vemurafenib. The second aim of this work was preliminary assessment of WP760 toxicity in vivo. In our first attempt to perform in vivo characterization of WP760, we analyzed its solubility and developed formulation in 5% dextrose in a presence of 5% DMSO suitable for intravenous administration. Our preliminary toxicity study performed in CD-1 mice, indicated low or no apparent toxicity symptoms when WP760 was dosed up to 10 mg/kg (rate of death 14%). For multiple dosing, when animals received three IV injections of WP760, the dose of 2.5 mg/kg appeared to be safe and animals did not present abnormal behavior when observed over 30 days period. Mice receiving higher doses of the compound developed toxicity symptoms after 3rd dose and the time they appeared was dose dependent. The observed melanoma selectivity of WP760 and its low nanomolar range cytotoxic potency in vitro combined with the lack of toxicity in normal fibroblasts were in agreement with results of the in vivo experiments demonstrating relatively high maximum tolerated dose in animals suggesting on the existence of a broad therapeutic window in vivo. Acknowledgment: This research was supported in part by funds from the University Cancer Foundation via the Sister Institution Network Fund at the UT MD Anderson Cancer Center and Melanoma SPORE (2 P50 CA093459) Citation Format: Aleksandra Rusin, Rafal Zielinski, Izabela Fokt, Van Nguyen, Stanislaw Skora, Arumugam Jayakumar, Waldemar Priebe. WP760, A new highly potent and selective agent against melanoma including BRAFi resistant melanoma. [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 4473. doi:10.1158/1538-7445.AM2015-4473

Abstract 2147: Correlating RAS oncogenic allele dependence with drug sensitivity

Abstract KRAS is a small G-protein that is a highly important member of the EGFR pathway. KRAS is frequently mutated in human cancers and predicts a shorter survival for patients with lung and colorectal cancers. Although any mutation at codon 12 or 13 is potentially oncogenic, Cysteine-12, Aspartate-12, Valine-12, and Aspartate 13 mutations predominate in human cancers, and the various oncogenic alleles correlate with distinct clinical outcomes. There is even some indication that Glycine 12 mutations may benefit from EGFR targeted therapy, whereas codon 12 mutations are contraindicated. We hypothesize that these clinical observations may be explained by subtle differences in effector pathway activation. It has been reported that G12C and G12V mutations preferentially activate the Ral-GDS pathway, while cell lines with G12D mutations elevate the PI3′ kinase and MAPK pathways. However, it is difficult to correlate clinical response to differences in biochemical signaling amidst a myriad of potentially confounding genetic alterations present across a panel of cell lines or patient tumor samples. In order to normalize the contributions of cell line heterogeneity, we tested pairs of isogenic cell lines to isolate KRAS allele specific effects, and to identify compounds that selectively inhibit RAS dependent growth in an allele specific manner. To do this, we used an engineered mouse embryonic fibroblast cell line - H-Ras−/−, N-Ras−/− and K-Raslox/lox, which expresses a transgene cre-recombinase - estrogen receptor fusion. Treatment with 4OHT results in excision of the endogenous K-Ras alleles, loss of KRAS expression, suppression of the MAPK pathway and G1 arrest. Reconstitution with any RAS isoform (K- H- or N-Ras), including any of the KRAS oncogenic alleles using a lentiviral vector system, allows the cells to re-enter the cell cycle and proliferate. The doubling time and basal pERK and pAKT levels are comparable across all isogenic uniclonal cell lines regardless of the RAS allele introduced. This system takes advantage of its dependency on a single RAS mutant to help screen for effector pathway dependence or synthetic lethal interactions unilaterally relevant to one mutant KRAS allele. Majority of compounds tested were unbiased towards the cell lines, but a few classes of drug showed selectivity. Cells expressing wild-type RAS were more sensitive to RTK inhibitors compared with cells expressing a mutant KRAS allele. Farnesyl Transferase Inhibitors (FTIs) such as Tipifarnib (R115777/Zarnestra) and Lonafarnib (SCH66336/Sarasar) were more potent against the HRAS WT dependent cell lines as compared to the KRAS dependent lines, validating the escape mechanism by which K-Ras and N-Ras get geranylgeranylated in the presence of FTIs. This strategy provides us with tools to delineate the subtle allele specific sensitivity differences which may translate to meaningful biology and may be used for tailoring mutant specific drug regimens in the clinic. Citation Format: Kanika Sharma, Katie Beam, Nicole Fer, Matthew Holderfield. Correlating RAS oncogenic allele dependence with drug sensitivity. [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 2147. doi:10.1158/1538-7445.AM2015-2147

Abstract 1112: Loss-of-function genetic screening with shRNA and CRISPR libraries

Abstract Genome-wide loss-of-function screens provide a direct approach to identify the genes regulating biological responses and find new therapeutic targets. While RNAi screens have proven an effective tool for these screens, CRISPR/CAS9 provides an alternative approach to implement these screens. To complement our established shRNA screening platform, we have developed pooled format genome-wide modular sgRNA libraries for cost-effective functional CRISPR knockout screens. Pooled sgRNA and shRNA libraries were then run to identify lethal interactions in isogenic PDX-derived cell line pairs. Citation Format: Donato Tedesco, Mikhail Makhanov, Sylvain Baron, Dmitry Suchkov, Alex Chenchik. Loss-of-function genetic screening with shRNA and CRISPR libraries. [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 1112. doi:10.1158/1538-7445.AM2015-1112

High-throughput simultaneous screen and counterscreen identifies homoharringtonine as synthetic lethal with von Hippel-Lindau loss in renal cell carcinoma.

Renal cell carcinoma (RCC) accounts for 85% of primary renal neoplasms, and is rarely curable when metastatic. Approximately 70% of RCCs are clear-cell type (ccRCC), and in >80% the von Hippel-Lindau (VHL) gene is mutated or silenced. We developed a novel, high-content, screening strategy for the identification of small molecules that are synthetic lethal with genes mutated in cancer. In this strategy, the screen and counterscreen are conducted simultaneously by differentially labeling mutant and reconstituted isogenic tumor cell line pairs with different fluorochromes and using a highly sensitive high-throughput imaging-based platform. This approach minimizes confounding factors from sequential screening, and more accurately replicates the in vivo cancer setting where cancer cells are adjacent to normal cells. A screen of ~12,800 small molecules identified homoharringtonine (HHT), an FDA-approved drug for treating chronic myeloid leukemia, as a VHL-synthetic lethal agent in ccRCC. HHT induced apoptosis in VHL-mutant, but not VHL-reconstituted, ccRCC cells, and inhibited tumor growth in 30% of VHL-mutant patient-derived ccRCC tumorgraft lines tested. Building on a novel screening strategy and utilizing a validated RCC tumorgraft model recapitulating the genetics and drug responsiveness of human RCC, these studies identify HHT as a potential therapeutic agent for a subset of VHL-deficient ccRCCs.

Differentiation of multipotent neural stem cells derived from Rett syndrome patients is biased toward the astrocytic lineage.

BackgroundRett syndrome (RTT) is one of the most prevalent neurodevelopmental disorders in females, caused by de novo mutations in the X-linked methyl CpG-binding protein 2 gene, MECP2. Although abnormal regulation of neuronal genes due to mutant MeCP2 is thought to induce autistic behavior and impaired development in RTT patients, precise cellular mechanisms underlying the aberrant neural progression remain unclear.ResultsTwo sets of isogenic pairs of either wild-type or mutant MECP2-expressing human induced pluripotent stem cell (hiPSC) lines were generated from a single pair of 10-year-old RTT-monozygotic (MZ) female twins. Mutant MeCP2-expressing hiPSC lines did not express detectable MeCP2 protein during any stage of differentiation. The lack of MeCP2 reflected altered gene expression patterns in differentiated neural cells rather than in undifferentiated hiPSCs, as assessed by microarray analysis. Furthermore, MeCP2 deficiency in the neural cell lineage increased astrocyte-specific differentiation from multipotent neural stem cells. Additionally, chromatin immunoprecipitation (ChIP) and bisulfite sequencing assays indicated that anomalous glial fibrillary acidic protein gene (GFAP) expression in the MeCP2-negative, differentiated neural cells resulted from the absence of MeCP2 binding to the GFAP gene.ConclusionsAn isogenic RTT-hiPSC model demonstrated that MeCP2 participates in the differentiation of neural cells. Moreover, MeCP2 deficiency triggers perturbation of astrocytic gene expression, yielding accelerated astrocyte formation from RTT-hiPSC-derived neural stem cells. These findings are likely to shed new light on astrocytic abnormalities in RTT, and suggest that astrocytes, which are required for neuronal homeostasis and function, might be a new target of RTT therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0121-2) contains supplementary material, which is available to authorized users.

Epigenetics and miRNA as predictive markers and targets for lung cancer chemotherapy

Lung cancer cells show inherent and acquired resistance to chemotherapy. The lack of good predictive markers/novel targets and the incomplete understanding of the mechanisms of resistance limit the success of lung cancer response to chemotherapy. In the present study, we used an isogenic pair of lung adenocarcinoma cell lines; A549 (wild-type) and A549DOX11 (doxorubicin resistant) to study the role of epigenetics and miRNA in resistance/response of non-small cell lung cancer (NSCLC) cells to doxorubicin. Our results demonstrate differential expression of epigenetic markers whereby the level of HDACs 1, 2, 3 and4, DNA methyltransferase, acetylated H2B and acetylated H3 were lower in A549DOX11 compared to A549 cells. Fourteen miRNAs were dys-regulated in A549DOX11 cells compared to A549 cells, of these 14 miRNAs, 4 (has-mir-1973, 494, 4286 and 29b-3p) have shown 2.99 – 4.44 fold increase in their expression. This was associated with reduced apoptosis and higher resistance of A549DOX11cells to doxorubicin and etoposide. Sequential treatment with the epigenetic modifiers trichostatin A or 5-aza-2'-deoxycytidine followed by doxorubicin resulted in: (i) enhanced sensitivity of both cell lines to doxorubicin especially at low concentrations, (ii) enhanced doxorubicin-induced DNA damage in both cell lines, (iii) dysregulation of some miRNAs in A549 cells. In conclusion, A549DOX11 cells resistant to DNA damaging drugs have epigenetic profile and miRNA expression different from the sensitive cells. Moreover, epigenetic modifiers may reverse the resistance of certain NSCLC cells to DNA damaging agents by enhancing induction of DNA damage. This may open the door for using epigenetic profile/miRNA expression of some cancer cells as resistance markers/targets to improve response of resistant cells to doxorubicin and for the use of combination doxorubicin/epigenetic modifiers to reduce doxorubicin toxicity.

P6.11 - Epigenetics and miRNA expression as mediators of A549 lung cancer cells resistance to DNA damaging anticancer drugs

ABSTRACT Lung cancer cells show both inherent and acquired resistance to chemotherapy. Epigenetics and miRNA are new mediators of response to anticancer agents. In the present study, we used an isogenic pair of lung adenocarcinoma cell lines; A549 (wild-type) and A549DOX11 (doxorubicin resistant) and two epigenetic modifiers (trichostatin A, TSA and 5-aza-2′-deoxycytidine, 5AZA) to study the role of epigenetics and miRNA in resistance of non-small cell lung cancer (NSCLC) cells to doxorubicin. Our results demonstrate differential expression of epigenetic markers whereby the level of HDACs 1, 2, 3 &4, DNA methyltransferase (DNMT1), acetylated H2B and acetylated H3 were lower in A549DOX11 compared to A549 cells. Fourteen miRNAs were dys-regulated in A549DOX11 cells compared to A549 cells. This was associated with reduced apoptosis and higher resistance of A549DOX11cells to doxorubicin and etoposide than A549 cells. Sequential treatment with TSA/5AZA followed by doxorubicin resulted in : (i) enhanced sensitivity of A549DOX11 cells to doxorubicin more than A549 especially at low concentrations, (ii) enhanced doxorubicin-induced DNA damage in both cell lines, (iii) dysregulation of some miRNAs in A549 cells. In conclusion, A549DOX11 cells resistant to DNA damaging drugs have epigenetic profile and miRNA expression different from the sensitive cells. Moreover, epigenetic modifiers may reverse the resistance of certain NSCLC cells to DNA damaging agents by enhancing their ability to induce DNA damage. This may open the door for using epigenetic profile/miRNA expression of some cancer cells as markers for response to doxorubicin and the use of combination doxorubicin/epigenetic modifiers to reduce doxorubicin toxicity.

Label free quantification of time evolving morphologies using time-lapse video microscopy enables identity control of cell lines and discovery of chemically induced differential activity in iso-genic cell line pairs

Label free time-lapse video microscopy based monitoring of time evolving cell population morphology has potential to offer a simple and cost effective method for identity control of cell lines. Such morphology monitoring also has potential to offer discovery of chemically induced differential changes between pairs of cell lines of interest, for example where one in a pair of cell lines is normal/sensitive and the other malignant/resistant. A new simple algorithm, pixel histogram hierarchy comparison (PHHC), for comparison of time evolving morphologies (TEM) in phase contrast time-lapse microscopy movies was applied to a set of 10 different cell lines and three different iso-genic colon cancer cell line pairs, each pair being genetically identical except for a single mutation. PHHC quantifies differences in morphology by comparing pixel histogram intensities at six different resolutions. Unsupervised clustering and machine learning based classification methods were found to accurately identify cell lines, including their respective iso-genic variants, through time-evolving morphology. Using this experimental setting, drugs with differential activity in iso-genic cell line pairs were likewise identified. Thus, this is a cost effective and expedient alternative to conventional molecular profiling techniques and might be useful as part of the quality control in research incorporating cell line models, e.g. in any cell/tumor biology or toxicology project involving drug/agent differential activity in pairs of cell line models.

Loss of the Histone Demethylase UTX Contributes to Multiple Myeloma and Sensitizes Cells to EZH2 Inhibitors

Genetic alterations of epigenetic regulators have become a recurrent theme in hematological malignancies. In particular, aberrations that alter the levels or distribution of methylation of lysine 27 on histone H3 (H3K27me) have emerged as a common feature of a wide variety of cancers, including multiple myeloma (MM). The histone demethylase UTX/KDM6A activates gene expression by removing the H3K27me3 repressive histone mark, counteracting the activity of EZH2, the enzyme that places this modification. UTX somatic inactivating mutations and deletions are found in up to 10% of MM cases; nevertheless, the epigenetic impact of UTX loss in MM and the mechanisms by which it contributes to this disease remain to be elucidated.To ascertain the biological impact of UTX loss, we used a recently identified isogenic cell line pair: ARP-1 (UTX wild-type) and ARD (UTX null). UTX-null ARD cells were engineered to express UTX in a doxycycline-inducible manner. UTX add-back slowed the proliferation rate of ARD cells, without affecting their viability. Soft agar assays demonstrated that UTX-null ARD cells have increased clonogenicity compared to UTX-wild-type ARP-1 cells. Re-expression of UTX partially reversed this effect, decreasing the number and size of colonies formed. ARD cells also showed increased adhesion to Hs-5 bone marrow stromal cells and to fibronectin than ARP-1 cells, an ability associated with cell survival and drug resistance. UTX add-back decreased the adhesive properties of ARD cells demonstrating this effect is dependent on UTX loss.Mass spectrometry analysis of the add-back system and a panel of UTX wild-type and mutant MM cell lines showed that global levels of H3K27me are not altered after UTX loss or upon its add-back. Therefore, UTX depletion may alter H3K27me at specific loci, and control the expression of a limited number of genes. To identify the genes and pathways that are altered upon UTX loss, we performed RNA-sequencing (RNA-seq) on the paired MM cell lines and the add-back system. This analysis revealed approximately 5,000 genes differentially expressed between ARP-1 and ARD cells. Re-expression of UTX in the UTX-null ARD cells reversed the expression of approximately 1,400 genes, most of them being upregulated upon reintroduction of UTX. Gene ontology analysis of genes responsive to UTX manipulation identified pathways such as JAK-STAT, cadherin, integrin and Wnt pathways. Many of these pathways are related to cell adhesion properties, correlating with the effects observed in vitro. Some examples of the genes which expression was restored upon UTX add-back are E-cadherin, whose loss has been associated with MM progression; and PTPN6, a negative regulator of the JAK-STAT pathway. Chromatin immunoprecipitation (ChIP) experiments at UTX target genes revealed a decrease in H3K27me3 and a concomitant increase in H3K4me3 upon UTX add-back, correlating with the observed changes in gene expression.As loss of UTX leads to a failure in the removal of H3K27me3, we hypothesized that UTX-null cells may be more dependent on EZH2 to maintain high H3K27me3 levels at specific loci. Treatment of the paired cell lines with the EZH2 inhibitor GSK343 for 7 days significantly decreased the viability of UTX-null ARD cells, but had no effect on the UTX wild-type ARP-1 cells. This effect was not exclusive to these cell lines, as treatment of a panel of UTX wild-type and mutant MM cells corroborated the increased sensitivity in UTX-mutant cells. RNA-seq of ARD cells treated with GSK343 for 7 days identified approximately 2,000 genes with altered expression in response to this drug, most of them being upregulated upon EZH2 inhibition. These genes partially overlapped with the genes that were responsive to UTX add-back, including E-cadherin, suggesting that treatment with EZH2 inhibitors is somewhat similar to UTX add-back.Collectively, this work demonstrates that loss of UTX alters the epigenetic landscape of MM cells, leading to altered expression of a specific set of genes, ultimately benefiting cells through increased proliferation, clonogenicity and adhesion. Moreover, inhibition of EZH2 partially reverses aberrations promoted by UTX loss and may represent a rationale therapy for the treatment of this type of MM. DisclosuresNo relevant conflicts of interest to declare.

319 Genomic profiling of uterine leiomyosarcomas reveal frequent alterations in Akt/mammalian target of rapamycin (mTOR) pathway genes and other actionable genomic abnormalities linked to targeted therapies

Based on our current understanding of ARID1A function, it is difficult to predict a therapeutic strategy for these tumours. Therefore we adopted an unbiased approach using integrated functional and molecular profiling of ARID1A mutant and wild-type tumour cell models. Methods: We profiled two parallel systems; a panel of ARID1A mutant and wild-type OCCC cell lines models and an isogenic pair of ARID1A proficient and deficient HCT116 cell lines, using high throughput screening with chemical and siRNA libraries. This has been integrated with molecular profiling data generated from exome and RNA sequencing to aid the discovery of novel targets. Results: Using functional screens we have identified critical genetic dependencies and potential therapeutics in a series of clear cell ovarian cancer models. Specifically, we have established that dasatinib is selective for OCCC models with loss of function ARID1A mutations both in vitro and in vivo. Dasatinib is not only selective for ARID1A mutant OCCC models, but experimental induction of ARID1A deficiency drives dasatinib sensitivity in ARID1A wild-type OCCC models as well as in isogenic cell systems (both mouse and human) where ARID1A has been rendered dysfunctional by gene targeting. Conclusions: These results provide a framework for the discovery of therapeutic targets for clear cell ovarian cancer characterised by ARID1A mutations. Using an unbiased drug and siRNA sensitivity screening approach, we have identified dasatinib as a candidate therapeutic approach for OCCC. This Dasatinib/ARID1A synthetic lethal approach can be observed both in in vitro and in vivo and suggests that dasatinib should be assessed in clinical trials in OCCC patients.

O-089 Micrornas And Chemoresistance In Neuroblastoma

Background Neuroblastoma is a neoplasm of the sympathetic nervous system representing the most frequently diagnosed solid tumour in infants. Despite continued improvements in cancer treatment, the overall survival of patients with high risk neuroblastoma is still only 40–50%. Irrespective of risk factors, neuroblastomas generally respond well to initial therapy. However, the majority of high risk patients relapse with tumours refractory to standard chemotherapeutic agents. Therefore, the understanding of biological and molecular aspects of drug resistance in neuroblastoma may provide new opportunities for therapy of aggressive neuroblastoma. Recent evidence has revealed a substantial role of microRNAs (miRNAs) in multidrug resistance in various cancer types. MicroRNAs are small (18–24 nucleotides) non-coding RNA molecules that regulate the expression of genes at the post-transcriptional level by either direct cleavage of target mRNAs or repression of translation. Several studies indicate that deviant expression of certain miRNAs correlate with poor clinical outcome in neuroblastoma. However, the role of miRNAs in neuroblastoma cell resistance to chemotherapeutic drugs is poorly understood. Methods To explore the role of miRNAs in the resistance of neuroblastoma cells to anticancer drugs, we generated miRNA cDNA libraries from six isogenic human neuroblastoma cell line pairs established from the same patients at the time of initial diagnosis and relapse following therapy. To analyse expression patterns of miRNAs, a deep sequencing analysis (SOLiD sequencing) was performed using the miRNA cDNA libraries. Results Deep sequencing analysis (SOLiD sequencing) revealed differential expression patterns of miRNAs before and after treatment. Systematic analysis of these miRNA expression patterns identified potential alterations in pathways associated with drug resistance suggesting that dysregulation of miRNAs might influence sensitivity to therapy. Conclusion We anticipate that our findings will provide new insights into the molecular mechanisms of drug resistance in neuroblastoma.

Abstract 2126: PIK3CA mutation status is not involved in the response of nonsteroidal anti-inflammatory drugs in colorectal cancer cell lines

Abstract Nonsteroidal anti-inflammatory drugs (NSAIDs) have shown a chemopreventive effect for colorectal cancer (CRC) in many epidemiological and large scale population studies. Moreover, regular use of NSAIDs has been recently associated with a reduction in the recurrence rate of CRC in patients previously diagnosed with colorectal tumors harboring PIK3CA mutations in two different retrospective analyses of large clinical cohorts. However, the mechanism by which PIK3CA mutations interacts with the mechanism of action of NSAIDs remains elusive. Our aim was to investigate the contribution of the most common alterations detected in PIK3CA to the sensitivity of NSAIDs in CRC cell line models. We have studied the cytotoxicity activity of two NSAIDs, Aspirin and Naproxen, in a panel of nine CRC cell lines by performing drug cytotoxicity assays. We have compared the sensitivity to Aspirin and Naproxen in cell lines grouped by the PIK3CA mutational status (wild-type versus mutant and by specific mutation hot-spots). Drug concentrations resulting in 50% growth inhibition (IC50) were determined by a curve-fitting analysis. In addition, two pairs of isogenic cell lines were used to analyze the contribution of PIK3CA mutations to drug sensitivity. One pair of each cell line contain only the mutant allele and the other the wild-type copy. The DLD-1 and HCT-116 cell line models containing only the wild-type allele were named 123 and 127, respectively. The DLD-1 and HCT-116 harboring the p.H1047R (exon 20) and p.E545K (exon 9) mutation were named 125 and 129, respectively. Moreover, modulation of classical biomarkers of the effect of NSAIDs, COX-2 and Prostaglandin E2 (PGE2) levels, was studied by qRT-PCR in treated and untreated cell lines. The ß-actin housekeeping gene was used to normalize samples and relative quantification of COX-2 and PGE2 RNA was done by the ΔΔCt approach. PIK3CA mutant cell lines showed no significant differences in IC50 compared to wild-type cells lines. This lack of contribution of the PIK3CA mutations to the cytotoxic effect of NSAIDs was further validated in the isogenic cell line pairs. Also, in the isogenic cell lines pairs, we found a reduced expression of COX-2 and an increased expression of PGE2 when the cell lines were treated with Aspirin or Naproxen as expected based on the known effects of NSAIDs, and thus corroborating their mechanistic effect. Therefore, we conclude that PIK3CA alterations are not correlated with sensitivity to NSAIDs. Our preclinical results do not support the epidemiological observations that PIK3CA tumors are more sensitive to NSADIS. Further in vitro and in vivo analyses to continue elucidating the contribution of the PI3K pathway to the effect of NSAIDs in CRC models are warranted. Citation Format: Ester Borras-Flores, Kara Calhoun, Gita Bhatia, Hong Wu, Eduardo Vilar-Sanchez. PIK3CA mutation status is not involved in the response of nonsteroidal anti-inflammatory drugs in colorectal cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2126. doi:10.1158/1538-7445.AM2014-2126

Abstract 1541: Co-mutations define major subsets of KRAS-driven lung adenocarcinoma with implications for targeted cancer therapy

Abstract Introduction The molecular underpinnings that beget the considerable biological and clinical heterogeneity of lung adenocarcinoma (AC) driven by oncogenic KRAS are poorly characterized. We hypothesized that significantly co-occurring mutations in components of key cellular tumor suppressor pathways are major determinants of signalling diversification downstream of mutant KRAS. Methods We applied un-supervised hierarchical analysis and classical multi-dimensional scaling to determine patterns of somatic (co)-mutations within a curated set of significantly mutated genes (MutSigCV p value <0.001) among 99 KRAS-mutant lung ACs from the TCGA dataset. Independently, we derived expression clusters by applying un-supervised hierarchical analysis to a subset of 68 KRAS-driven lung ACs with available RNASeq data. Non-negative matrix factorization (NMF) was used to determine cluster stability. Results Clustering of KRAS-driven lung ACs based on patterns of somatic mutations revealed six major mutation clades. Five clades could be defined by combinations of somatic mutations in only four genes : KRAS, TP53, STK11/LKB1 and KEAP1, whereas a sixth clade appeared more heterogenous and was associated with a higher overall rate of somatic mutation. Co-mutations in STK11/LKB1 and TP53 accounted for higher branches of the cluster dendrogram and were largely non-overlapping in the context of early-stage disease. Integration of co-mutation analysis with unsupervised hierarchical analysis of RNA Seq differential expression data identified 3 major expression clusters, that were significantly enriched for the KRAS;TP53 (KP), KRAS;LKB1 (KL) and KRAS only (K) somatic mutation clades, as well as sub-clusters characterized by the presence of mutations in KEAP1. On the contrary, the distribution of the main KRAS amino acid substitutions (G12C, G12V and G12D) did not differ significantly between the expression clusters, indicating that co-mutations rather than distinct KRAS codon 12 base substitutions are the major driver of biological heterogeneity within KRAS-mutant lung ACs. Re-analysis of publicly available large scale drug sensitivity data based on the proposed sub-classification revealed novel genotype-specific therapeutic vulnerabilities for the KL and KP subgroups including selective sensitivity of KL AC cell lines to MK-2206 ( a pan-AKT inhibitor), BX-795 (a PDK1 and TBK1/IKKϵ inhibitor) and the type II topoisomerase inhibitor etoposide. Selected hits were subsequently validated using pairs of isogenic cell lines. Conclusions Co-mutations in a small number of key tumor suppressor genes define biologically distinct and therapeutically relevant subgroups of KRAS-driven lung AC. Efforts to target KRAS signalling in the context of specific co-mutations may provide a more rational and personalized approach to therapy for this challenging group of lung cancer patients. Citation Format: Ferdinandos Skoulidis, Kevin R. Coombes, Lixia Diao, Pan Tong, Maria A. Cortez, Uma Giri, Chao Yang, You Hong Fan, John N. Weinstein, Vassiliki Papadimitrakopoulou, John D. Minna, Jing Wang, Lauren A. Byers, John V. Heymach. Co-mutations define major subsets of KRAS-driven lung adenocarcinoma with implications for targeted cancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1541. doi:10.1158/1538-7445.AM2014-1541

Abstract 5158: The H3K9 demethyltransferase gene JMJD1C has tumor suppressor functions in multiple myeloma

Abstract Increasing evidence points towards the importance of epigenetic changes in the pathogenesis of multiple myeloma (MM). The biochemical modifications that govern epigenetics are DNA methylation, and post-translational modifications of histone proteins. Histone methylation is catalyzed by histone methyltransferases (HMT) and histone demethylases (HDMT). The purpose of our study was to interrogate genomics data to identify HMT or HDMT genes that are altered in MM. We mined copy number, gene expression and whole genome sequencing data generated as part of the Multiple Myeloma Research Consortium Genomics Initiative. Collectively, the datasets converged on numerous alterations involving histone methylation of lysine 9 (H3K9). One of these genes, the HDMT JMJD1C, was downregulated in approximately 25% of MM samples examined and was associated with worst clinical outcome. We validated these data by quantitative RT-PCR and by immunohistochemistry (IHC) on a tissue microarray (TMA) consisting of over 60 MM samples. Next we demonstrated a negative correlation between JMJD1C expression and H3K9 methylation by IHC and western blot in 6/10 HMCL and in over 60% of clinical samples examined on the TMA. To study the direct effects of JMJD1C on H3K9 methylation and determine the functional significance, we generated a knockout isogenic cell line pair in KMS11 MM cells using zinc finger nuclease technology. JMJD1C depletion resulted in an increase in H3K9 mono- and dimethylation as demonstrated by western blot and immunofluorescence, confirming its association to H3K9 methylation. H3K9 trimethylation was unaffected by JMJD1C depletion. Differential gene expression analysis demonstrated defects in cell cycle and G2/M transition. Most notably, NEK2, Cyclin B, CDC20, PLK1 and TTK were among genes upregulated in response to JMJD1C depletion. Accordingly, we assessed cell growth using CellTiter-Glo® and demonstrated increased cell growth in JMJD1C-depleted cells. We also demonstrated a more pronounced G2/M peak by FACS. DNA methylation analysis using the HumanMethylation 450K BeadChip showed global hypomethylation in response to JMJD1C loss, a phenotype indicative of myeloma progression. Results to date validate the H3K9 demethylase activity of JMJD1C, and suggest a tumor suppressive function, which may be lost in MM. Data demonstrate a novel biological and molecular understanding of JMJD1C, pointing us to therapeutic vulnerabilities in MM. Studies are ongoing to further characterize the genomic, epigenomic and functional significance of JMJD1C in MM. Citation Format: Danielle DiPerna, Gerald C. Gooden, Brooke Hjelm, Sara Nasser, Janine LoBello, Aprill Watanabe, Bodour Salhia. The H3K9 demethyltransferase gene JMJD1C has tumor suppressor functions in multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5158. doi:10.1158/1538-7445.AM2014-5158

Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress

The interplay between oxidative stress and autophagy is critical for determining the fate of cancer cells exposed to redox-active and cytotoxic chemotherapeutic agents. Mitoquinone (MitoQ), a mitochondrially-targeted redox-active ubiquinone conjugate, selectively kills breast cancer cells over healthy mammary epithelial cells. We reported previously that MitoQ, although a derivative of the antioxidant ubiquinone, can generate excess ROS and trigger the Keap1-Nrf2 antioxidant response in the MDA-MB-231 cell line. Following MitoQ treatment, a greater number of cells underwent autophagy than apoptosis. However, the relationship between MitoQ-induced oxidative stress and autophagy as a primary cellular response was unclear. In this report, we demonstrate that MitoQ induces autophagy related gene 7 (Atg7)-dependent, yet Beclin-1-independent, autophagy marked by an increase in LC3-II. Both the ATG7-deficient human MDA-MB-231 cells and Atg7-knockout mouse embryonic fibroblasts exhibited lower levels of autophagy following MitoQ treatment than their respective wild-type counterparts. Increased apoptosis was confirmed in these autophagy-deficient isogenic cell line pairs, indicating that autophagy was attempted for survival in wild type cell lines. Furthermore, we observed higher levels of ROS in Atg7-deficient cells, as measured by hydroethidine oxidation. In Atg7-deficient cells, redox-sensitive Keap1 degradation was decreased, suggesting autophagy- and Atg7-dependent degradation of Keap1. Conversely, downregulation of Keap1 decreased autophagy levels, increased Nrf2 activation, upregulated cytoprotective antioxidant gene expression, and caused accumulation of p62, suggesting a feedback loop between ROS-regulated Keap1-Nrf2 and Atg7-regulated autophagy. Our data indicate that excessive ROS causes the upregulation of autophagy, and autophagy acts as an antioxidant feedback response triggered by cytotoxic levels of MitoQ.