Jason Borawski

A Smac Mimetic Rescue Screen Reveals Roles for Inhibitor of Apoptosis Proteins in Tumor Necrosis Factor-α Signaling

Smac mimetic compounds targeting the inhibitor of apoptosis proteins (IAP) baculoviral IAP repeat-3 domain are presumed to reduce the threshold for apoptotic cell death by alleviating caspase-9 repression. We explored this tenet in an unbiased manner by searching for small interfering RNAs that are able to confer resistance to the Smac mimetic compound LBW242. Among the screening hits were multiple components of the tumor necrosis factor alpha (TNFalpha) signaling pathway as well as X-linked inhibitor of apoptosis (XIAP) itself. Here, we show that in a subset of highly sensitive tumor cell lines, activity of LBW242 is dependent on TNFalpha signaling. Mechanistic studies indicate that in this context, XIAP is a positive modulator of TNFalpha induction whereas cellular inhibitor of apoptosis protein 1 negatively regulates TNFalpha-mediated apoptosis.

Class III Phosphatidylinositol 4-Kinase Alpha and Beta Are Novel Host Factor Regulators of Hepatitis C Virus Replication

ABSTRACT Host factor pathways are known to be essential for hepatitis C virus (HCV) infection and replication in human liver cells. To search for novel host factor proteins required for HCV replication, we screened a subgenomic genotype 1b replicon cell line (Luc-1b) with a kinome and druggable collection of 20,779 siRNAs. We identified and validated several enzymes required for HCV replication, including class III phosphatidylinositol 4-kinases (PI4KA and PI4KB), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and mevalonate (diphospho) decarboxylase. Knockdown of PI4KA could inhibit the replication and/or HCV RNA levels of the two subgenomic genotype 1b clones (SG-1b and Luc-1b), two subgenomic genotype 1a clones (SG-1a and Luc-1a), JFH-1 genotype 2a infectious virus (JFH1-2a), and the genomic genotype 1a (FL-1a) replicon. In contrast, PI4KB knockdown inhibited replication and/or HCV RNA levels of Luc-1b, SG-1b, and Luc-1a replicons. The small molecule inhibitor, PIK93, was found to block subgenomic genotype 1b (Luc-1b), subgenomic genotype 1a (Luc-1a), and genomic genotype 2a (JFH1-2a) infectious virus replication in the nanomolar range. PIK93 was characterized by using quantitative chemical proteomics and in vitro biochemical assays to demonstrate PIK93 is a bone fide PI4KA and PI4KB inhibitor. Our data demonstrate that genetic or pharmacological modulation of PI4KA and PI4KB inhibits multiple genotypes of HCV and represents a novel druggable class of therapeutic targets for HCV infection.

FGFR Genetic Alterations Predict for Sensitivity to NVP-BGJ398, a Selective Pan-FGFR Inhibitor

UNLABELLED Patient stratification biomarkers that enable the translation of cancer genetic knowledge into clinical use are essential for the successful and rapid development of emerging targeted anticancer therapeutics. Here, we describe the identification of patient stratification biomarkers for NVP-BGJ398, a novel and selective fibroblast growth factor receptor (FGFR) inhibitor. By intersecting genome-wide gene expression and genomic alteration data with cell line-sensitivity data across an annotated collection of cancer cell lines called the Cancer Cell Line Encyclopedia, we show that genetic alterations for FGFR family members predict for sensitivity to NVP-BGJ398. For the first time, we report oncogenic FGFR1 amplification in osteosarcoma as a potential patient selection biomarker. Furthermore, we show that cancer cell lines harboring FGF19 copy number gain at the 11q13 amplicon are sensitive to NVP-BGJ398 only when concomitant expression of β-klotho occurs. Thus, our findings provide the rationale for the clinical development of FGFR inhibitors in selected patients with cancer harboring tumors with the identified predictors of sensitivity. SIGNIFICANCE The success of a personalized medicine approach using targeted therapies ultimately depends on being able to identify the patients who will benefit the most from any given drug. To this end, we have integrated the molecular profiles for more than 500 cancer cell lines with sensitivity data for the novel anticancer drug NVP-BGJ398 and showed that FGFR genetic alterations are the most significant predictors for sensitivity. This work has ultimately endorsed the incorporation of specific patient selection biomakers in the clinical trials for NVP-BGJ398.

Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling

The calcium-activated chloride channel anoctamin 1 (ANO1) is located within the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human cancer, but its functional role in tumorigenesis has remained unclear. The 11q13 region is amplified in ∼15% of breast cancers. Whether ANO1 is amplified in breast tumors, the extent to which gene amplification contributes to ANO1 overexpression, and whether overexpression of ANO1 is important for tumor maintenance have remained unknown. We have found that ANO1 is amplified and highly expressed in breast cancer cell lines and primary tumors. Amplification of ANO1 correlated with disease grade and poor prognosis. Knockdown of ANO1 in ANO1-amplified breast cancer cell lines and other cancers bearing 11q13 amplification inhibited proliferation, induced apoptosis, and reduced tumor growth in established cancer xenografts. Moreover, ANO1 chloride channel activity was important for cell viability. Mechanistically, ANO1 knockdown or pharmacological inhibition of its chloride-channel activity reduced EGF receptor (EGFR) and calmodulin-dependent protein kinase II (CAMKII) signaling, which subsequently attenuated AKT, v-src sarcoma viral oncogene homolog (SRC), and extracellular signal-regulated kinase (ERK) activation in vitro and in vivo. Our results highlight the involvement of the ANO1 chloride channel in tumor progression and provide insights into oncogenic signaling in human cancers with 11q13 amplification, thereby establishing ANO1 as a promising target for therapy in these highly prevalent tumor types.

Multiple cyclophilins involved in different cellular pathways mediate HCV replication.

Three cyclophilin inhibitors (DEBIO-025, SCY635, and NIM811) are currently in clinical trials for hepatitis C therapy. The mechanism of action of these, however, is not completely understood. There are at least 16 cyclophilins expressed in human cells which are involved in a diverse set of cellular processes. Large-scale siRNA experiments, chemoproteomic assays with cyclophilin binding compounds, and mRNA profiling of HCV replicon containing cells were used to identify the cyclophilins that are instrumental to HCV replication. The previously reported cyclophilin A was confirmed and additional cyclophilin containing pathways were identified. Together, the experiments provide strong evidence that NIM811 reduces viral replication by inhibition of multiple cyclophilins and pathways with protein trafficking as the most strongly and persistently affected pathway.

Down-Regulation of Class II Phosphoinositide 3-Kinase α Expression below a Critical Threshold Induces Apoptotic Cell Death

Members of the phosphoinositide 3-kinase (PI3K) family collectively control multiple cellular responses, including proliferation, growth, chemotaxis, and survival. These diverse effects can partly be attributed to the broad range of downstream effectors being regulated by the products of these lipid kinases, the 3′-phosphoinositides. However, an additional layer of complexity is introduced by the existence of multiple PI3K enzyme isoforms. Much has been learned over the last years on the roles of the classes I and III PI3K members in cellular signaling, but little is known about the isoform-specific tasks done by the class II PI3Ks (C2α, β, and γ). In this study, we used quantitative reverse transcription–PCR and RNA interference in mammalian cells to gain further insight into the function of these lesser studied PI3K enzymes. We find that PI3K-C2α, but not PI3K-C2β, has an important role in controlling cell survival and by using a panel of RNA interference reagents, we were able to determine a critical threshold of PI3K-C2α mRNA levels, below which the apoptotic program is switched on, via the intrinsic cell death pathway. In addition, knockdown of PI3K-C2α to levels that by themselves do not induce apoptosis sensitize cells to the anticancer agent Taxol (paclitaxel). Lastly, we report that lowering the levels of PI3K-C2α in a number of cancer cell lines reduces their proliferation and cell viability, arguing that PI3K inhibitors targeting not only the class Iα isoform but also class IIα may contribute to an effective anticancer strategy. (Mol Cancer Res 2008;6(4):614–23)

Optimization Procedure for Small Interfering RNA Transfection in a 384-Well Format

High-throughput screening of RNAi libraries has become an essential part of functional analysis in academic and industrial settings. The transition of a cell-based RNAi assay into a 384-well format requires several optimization steps to ensure the phenotype being screened is appropriately measured and that the signal-to-background ratio is above a certain quantifiable threshold. Methods currently used to assess small interfering RNA (siRNA) efficacy after transfection, including quantitative PCR or branch DNA analysis, face several technical limitations preventing the accurate measurement of mRNA levels in a 384-well format. To overcome these difficulties, the authors developed an approach using a viral-based transfection system that measures siRNA efficacy in a standardized 384-well assay. This method allows measurement of siRNA activity in a phenotypically neutral manner by quantifying the knockdown of an exogenous luciferase gene delivered by a lentiviral vector. In this assay, the efficacy of a luciferase siRNA is compared to a negative control siRNA across many distinct assay parameters including cell type, cell number, lipid type, lipid volume, time of the assay, and concentration of siRNA. Once the siRNA transfection is optimized as a 384-well luciferase knockdown, the biologically relevant phenotypic analysis can proceed using the best siRNA transfection conditions. This approach provides a key technology for 384-well assay development when direct measurement of mRNA knockdown is not possible. It also allows for direct comparison of siRNA activity across cell lines from almost any mammalian species. Defining optimal conditions for siRNA delivery into mammalian cells will greatly increase the speed and quality of large-scale siRNA screening campaigns. (Journal of Biomolecular Screening 2007:546-559)

Distinctive features of lincRNA gene expression suggest widespread RNA-independent functions

Combining single-cell RNA-seq of mouse ESCs and NPCs, lincRNA gene deletions, conditional RNA depletion, and nuclear exosome profiling distinguishes RNA-dependent and RNA-independent lincRNA gene activities. Eukaryotic genomes produce RNAs lacking protein-coding potential, with enigmatic roles. We integrated three approaches to study large intervening noncoding RNA (lincRNA) gene functions. First, we profiled mouse embryonic stem cells and neural precursor cells at single-cell resolution, revealing lincRNAs expressed in specific cell types, cell subpopulations, or cell cycle stages. Second, we assembled a transcriptome-wide atlas of nuclear lincRNA degradation by identifying targets of the exosome cofactor Mtr4. Third, we developed a reversible depletion system to separate the role of a lincRNA gene from that of its RNA. Our approach distinguished lincRNA loci functioning in trans from those modulating local gene expression. Some genes express stable and/or abundant lincRNAs in single cells, but many prematurely terminate transcription and produce lincRNAs rapidly degraded by the nuclear exosome. This suggests that besides RNA-dependent functions, lincRNA loci act as DNA elements or through transcription. Our integrative approach helps distinguish these mechanisms.

Abstract LB-205: The calcium activated chloride channel ANO1 promotes breast cancer progression by activating EGFR- and CAMK-signaling.

Genomic alterations are the underlining cause of many human cancers: Amplified and overexpressed genes can drive neoplastic transformation and become essential survival factors for cancer cells. Thus, they represent promising targets for anti-cancer therapies and their identification and validation is of paramount importance. In a search for novel survival factors contributing to breast cancer oncogenesis, we performed genomic fine mapping of the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human neoplasia, in a large dataset of breast cancer patients. Remarkably, we found ANO1, a calcium-activated chloride channel, to be located within the summit of the most frequently and highly amplified genomic region. Furthermore, amplification of ANO1 correlates with its overexpression and elevated protein levels and is a negative predictor for overall survival in breast cancer patients. Next, we examined how ANO1 contributes to cancer cell survival and proliferation. We found that inhibition of ANO1 expression or function reduced cancer cell viability and colony formation, and triggered apoptosis in 11q13 amplified breast cancer, HNSCC and ESCC. Furthermore, expression of ANO1 in non-transformed mammary cells increased cell viability and established a novel addiction to ANO1 biochemical activity. We next applied inducible shRNAs against ANO1 in vivo to assess the effect of ANO1 inhibition on the maintenance of established tumors. We found in four different 11q13-amplified tumor models that knockdown of ANO1 reduced tumor growth, indicating an important role for ANO1 not only in oncogenesis, but also in tumor maintenance of 11q13 amplified cancers. To explore the mechanism by which inhibition of ANO1 contributes to cancer cell viability and tumor growth, we performed antibody arrays to measure the activity of major oncogenic signaling pathways after knockdown of ANO1. Upon depletion of ANO1, activation of EGFR and several survival signaling pathways (AKT-, SRC- and ERK1/2 pathways) were reduced in different models of human cancer. Subsequent experiments showed that ANO1 modulates both EGFR- and Ca2+/calmodulin-dependent protein kinase (CAMK) signaling in breast cancer and HNSCC cells. Consistently, activation of EGFR- and CAMK correlated with the expression of ANO1 in human primary breast tumor samples. Lastly, we found that only the simultaneous stimulation of EGFR- and CAMK-signaling pathways rescued the effect of the ANO1-inhibition. In summary, our study establishes ANO1 as a key tumor-promoting factor in 11q13-amplified breast and other malignancies. Our results highlight the importance of chloride channels in cancer and provide the first detailed mechanistic insight into the activity of ANO1 in tumorigenesis. Most importantly, our findings open up new opportunities for therapeutic intervention in several prevalent cancers. Citation Format: Adrian Britschgi, Anke Bill, Heike Brinkhaus, Christopher Rothwell, Ieuan Clay, Stephan Duss, Michael Rebhan, Pichai Raman, Chantale Guy, Kristie Wetzel, Elizabeth George, M. Oana Popa, Sarah Lilley, Hedaythul Choudhury, Martin Gosling, Louis Wang, Stephanie Fitzgerald, Jason Borawski, Jonathan Baffoe, Mark Labow, L. Alex Gaither, Mohamed Bentires-Alj. The calcium activated chloride channel ANO1 promotes breast cancer progression by activating EGFR- and CAMK-signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-205. doi:10.1158/1538-7445.AM2013-LB-205

Abstract 854: Patient stratification strategies for NVP-BGJ398, a selective pan-FGFR inhibitor in phase I clinical trials

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The mammalian fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) family comprises 22 FGF ligands, which exert their action through 4 highly conserved transmembrane tyrosine kinase receptors (FGFR1, FGFR2, FGFR3 and FGFR4). This highly complex signaling network regulates fundamental developmental pathways and physiological processes in the adult organism. A growing body of evidence linking deregulation of the FGF/FGFR system, as a consequence of genetic alterations, to the pathogenesis of cancer makes these receptors attractive targets for therapeutic intervention. To this purpose we have developed NVP-BGJ398, an orally bioavailable, selective pan-FGFR kinase inhibitor which is currently in clinical Phase I dose escalation trial. In order to preclinically identify and validate patient stratification biomarkers that accurately predict for response to NVP-BGJ398 in the clinic, we have utilized the Cancer Cell Line Encyclopedia (CCLE). The CCLE is a collection of almost 1000 cancer cell lines representing multiple tumor types that, in a collaborative effort between The Novartis Institutes for BioMedical Research and the Broad Institute, has been comprehensively annotated in terms of genome-scale mRNA expression, gene copy number alterations and gene mutations (http:/www.broadinstitute.org/ccle). In addition, over half of these cell lines were subjected to high-throughput cell viability assays in the presence NVP-BGJ398, with the aim of generating cell line sensitivity data. Here we show the validation of predictive biomarkers of response in primary human tumor xenografts, revealed through the analysis of the CCLE, as well as the identification of potentially new indications for NVP-BGJ398. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 854. doi:1538-7445.AM2012-854