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PDF file - 180K, Table S1. Genes in the VDV signature, ordered by anti-VEGF response in human IBC trial. Table S2. Gene Ontology terms over-represented in VDV signature. Table S3. Summary of tumors models tested.

PDF file - 233K, Fig S4A. Validation and application of a compacted 22-gene VDV subset in human clinical samples. Fig S4B and C. Stratification of NO16966 patients by VEGF and CD31 expression levels.

Supplementary Fig. S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

PDF file - 154K, Fig S5. Most proxVDV genes are not obviously up-regulated by rVEGF in vitro.

Supplementary Table S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

PDF file - 83K, Fig S6. Effect of VDV gene expression and bevacizumab treatment in OS.

The insulin-like growth factor (IGF) system consists of two ligands (IGF-I and IGF-II), which both signal through IGF-I receptor (IGF-IR) to stimulate proliferation and inhibit apoptosis, with activity contributing to malignant growth of many types of human cancers. We have developed a humanized, affinity-matured anti-human IGF-IR monoclonal antibody (h10H5), which binds with high affinity and specificity to the extracellular domain. h10H5 inhibits IGF-IR-mediated signaling by blocking IGF-I and IGF-II binding and by inducing cell surface receptor down-regulation via internalization and degradation, with the extracellular and intracellular domains of IGF-IR being differentially affected by the proteasomal and lysosomal inhibitors. In vitro, h10H5 exhibits antiproliferative effects on cancer cell lines. In vivo, h10H5 shows single-agent antitumor efficacy in human SK-N-AS neuroblastoma and SW527 breast cancer xenograft models and even greater efficacy in combination with the chemotherapeutic agent docetaxel or an anti–vascular endothelial growth factor antibody. Antitumor activity of h10H5 is associated with decreased AKT activation and glucose uptake and a 316-gene transcription profile with significant changes involving DNA metabolic and cell cycle machineries. These data support the clinical testing of h10H5 as a biotherapeutic for IGF-IR-dependent human tumors and furthermore illustrate a new method of monitoring its activity noninvasively in vivo via 2-fluoro-2-deoxy-d-glucose-positron emission tomography imaging. [Mol Cancer Ther 2008;7(9):2599–608]

PDF file - 113K, Fig S1. Kinetics of anti-VEGF effects on proliferation (ki67) and tumor burden in the RIP-TβAg model.

Purpose: The aim of this study was to identify conserved pharmacodynamic and potential predictive biomarkers of response to anti-VEGF therapy using gene expression profiling in preclinical tumor models and in patients.Experimental Design: Surrogate markers of VEGF inhibition [VEGF-dependent genes or VEGF-dependent vasculature (VDV)] were identified by profiling gene expression changes induced in response to VEGF blockade in preclinical tumor models and in human biopsies from patients treated with anti-VEGF monoclonal antibodies. The potential value of VDV genes as candidate predictive biomarkers was tested by correlating high or low VDV gene expression levels in pretreatment clinical samples with the subsequent clinical efficacy of bevacizumab (anti-VEGF)-containing therapy.Results: We show that VDV genes, including direct and more distal VEGF downstream endothelial targets, enable detection of VEGF signaling inhibition in mouse tumor models and human tumor biopsies. Retrospective analyses of clinical trial data indicate that patients with higher VDV expression in pretreatment tumor samples exhibited improved clinical outcome when treated with bevacizumab-containing therapies.Conclusions: In this work, we identified surrogate markers (VDV genes) for in vivo VEGF signaling in tumors and showed clinical data supporting a correlation between pretreatment VEGF bioactivity and the subsequent efficacy of anti-VEGF therapy. We propose that VDV genes are candidate biomarkers with the potential to aid the selection of novel indications as well as patients likely to respond to anti-VEGF therapy. The data presented here define a diagnostic biomarker hypothesis based on translational research that warrants further evaluation in additional retrospective and prospective trials. Clin Cancer Res; 19(13); 3681–92. ©2013 AACR.

PDF file - 63K, Fig S3. Enrichment of VDV markers Tumor-Associated Endothelial Cells.

PDF file - 708K, Fig S2A. Histological evidence for the in vivo activity of VEGF pathway inhibitors in MDA-MB-231 tumors. Fig S2B. In vivo VEGF blockade or VEGFR-2 downstream signaling inhibition induces consistent downregulation of proxVDV genes. Fig S2C. The proxVDV gene ESM1 is an in vivo VEGF target specifically expressed in tumor-associated vasculature

Supplementary Figure 1 from Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection

PDF file - 95K

Antibody-drug conjugates (ADC), potent cytotoxic drugs covalently linked to antibodies via chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Here, we systematically examine the potential targets and linker-drug combinations that could provide an optimal ADC for the treatment for non–Hodgkin's lymphoma. We identified seven antigens (CD19, CD20, CD21, CD22, CD72, CD79b, and CD180) for potential treatment of non–Hodgkin's lymphoma with ADCs. ADCs with cleavable linkers mediated in vivo efficacy via all these targets; ADCs with uncleavable linkers were only effective when targeted to CD22 and CD79b. In target-independent safety studies in rats, the uncleavable linker ADCs showed reduced toxicity, presumably due to the reduced release of free drug or other toxic metabolites into the circulation. Thus, our data suggest that ADCs with cleavable linkers work on a broad range of targets, and for specific targets, ADCs with uncleavable linkers provide a promising opportunity to improve the therapeutic window for ADCs in humans. [Cancer Res 2009;69(6):2358–64]

Supplementary Table 1 from Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection

PDF file - 6626K, Validation of IHC antibody specificity for LDHB, LDHA, MCT1, and MCT4

PDF file - 963K, Additional top targets from RNAi screen that are specifically required for triple negative breast cancer

Supplementary Figure Legend from Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection

XLSX file - 25K, RNAi optimization and screening conditions

XLSX file - 59K, Genes that comprise the glycolysis and oxidative phosphorylation gene signatures

PDF file - 663K, Glycolysis signature genes that correlate to LDHB expression in basal-like tumors

PDF file - 257K, Animal weight change and tumor volume of individual xenograft tumors

PDF file - 791K, mRNA expression profiles of lactate dehydrogenase enzymes in breast cancer subtypes

XLSX file - 31K, Results from the RNAi screen and identification of hits

In mammals, MT1 and MT2 melatonin receptors are high affinity G protein-coupled receptors and are thought to be involved in the integration of the melatonin signaling throughout the brain and periphery. In the present study, we describe a new melatonin binding site, named MTx, with a peculiar pharmacological profile. This site had a low affinity for 2-[125I]-melatonin in saturation assays in hypothalamus and retina (pKD = 9.13 {plus minus} 0.05, Bmax = 1.12 {plus minus} 0.11 fmol/mg protein and pKD = 8.81 {plus minus} 0.50, Bmax = 7.65 {plus minus} 2.64 fmol/mg protein, respectively) and a very high affinity, in competition assays, for melatonin (pKi = 13.08 {plus minus} 0.18), and other endogenous compounds. Using autoradiography, we showed a preferential localization of the MTx in periventricular areas of the sheep brain, with a density 3 to 8 times higher than those observed for ovine MT1 In addition, using a set of well-characterized ligands, we showed that this site did not correspond to any of the following receptors: MT1, MT2, MT3 , D1, D2, noradrenergic, nor 5-HT2 Based on its affinity for melatonin, MTx did not seem to be implicated in the integration of cerebral melatonin concentration variations since they were saturating for MTx. Nevertheless, it remained of prime importance because of its periventricular distribution, in close contact with the CSF, and its peculiar pharmacological profile responding to both melatoninergic and serotoninergic compounds. Significance Statement Herein a putative new melatonin binding site is described in sheep brain parts in close contact with the 3rd ventricle. The characteristics of the pharmacological profile of this site is different from anything previously reported in the literature. The present work forms the basis of future full pharmacological characterization.

Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease defined by ER-, PR- and HER2-negative phenotype and in most cases, a relatively aggressive clinical behaviour. The lack of specific targeted therapies and low efficiency of currently available chemotherapies spurred several clinical trials in the last few years. Despite encouraging results, TNBC still remains a major unmet medical need that prompted us to explore the role of 863 epigenetic modulators in TNBC cell survival.Methods: A comprehensive siRNA library was screened to explore the role of known epigenetic modulators in TNBC cell viability and growth. The knock-down effect was evaluated for 863 epigenetic genes using 4 siRNAs/gene in two TNBC and a non-TNBC cell lines using ATP-based luminescence and nuclei count image-based assays. Considering siRNA off-target effects, four analysis methods including a classical threshold-based analysis and three ranking methods were applied to determine on-target hits for each screen readout. Hit genes common to both phenotypic readouts highlighted strong epigenetic players involved in TNBC cell survival.Results: Overall, knock-down of many epigenetic modulator genes mitigates cell survival in TNBC and a non-TNBC cell lines depicted from both phenotypic readouts. Interestingly, ranking-based analysis confirmed hit genes identified in threshold-based analysis and also revealed additional hits enabling us to confirm CDK1 and KMT5A as important regulators in TNBC cell viability and growth. Surprisingly, CHAF1A appeared as a new candidate gene involved in TNBC cell survival.Conclusions: Taken together, siRNA epigenetic screening results identified CHAF1A as a novel regulator of TNBC cell survival.

Predicting fluorescently labelled cellular structures from brightfield images is a recent application of convolutional neural networks and has already proven a valuable tool in biological imaging studies. These methods reduce the need for time-consuming manual annotations for supervised datasets, potentially reduce the costs in high-throughput imaging screens, and open up a number of potentially novel analyses. However as with any prediction there can be sources of error and uncertainty. Here we present BFNet, a method to visualise the uncertainty in predicted images by applying Monte Carlo dropout during inference and calculating per-pixel variance as an uncertainty map. Our method demonstrates the ability to highlight regions of an image where prediction is difficult or impossible due to imaging artefacts such as occlusions or out-of-focus images, as well as more general uncertainty when a trained model is applied to new data from different imaging of experimental settings. We have provided a python implementation of the method which is available at github.com/swarchal/bfnet.

In recent years, questions about the sustainability of the current drug discovery process have triggered a revival of interest in phenotypic drug discovery approaches. This trend has clearly been amplified by the emergence of multiple cell-based assay technologies enabling a higher degree of translatability between in vitro conditions and physio-pathological situations, including induced pluripotent stem cells, three-dimensional models, co-culture and organ-on-a-chip systems, complemented by advances in gene editing technologies. Progress in High-Content Screening technology has also contributed to the recent excitement for phenotypic drug discovery approaches, bringing image-capture and processing, and data-analysis, to a level of content and throughput fully compatible with large scale drug discovery efforts. Nevertheless, implementation of HCS in discovery projects must be carefully considered, to ensure optimal performance and the generation of relevant data to enable the discovery of first-in-class medicines.

Acquired resistance to cancer drug therapies almost always occurs in advanced-stage patients even following a significant response to treatment. In addition to mutational mechanisms, various non-mutational resistance mechanisms have now been recognized. We previously described a chromatin-mediated subpopulation of reversibly drug-tolerant persisters that is dynamically maintained within a wide variety of tumour cell populations. Here we explore a potential role for microRNAs in such transient drug tolerance. Functional screening of 879 human microRNAs reveals miR-371-3p as a potent suppressor of drug tolerance. We identify PRDX6 (peroxiredoxin 6) as a key target of miR-371-3p in establishing drug tolerance by regulating PLA2/PKCα activity and reactive oxygen species. PRDX6 expression is associated with poor prognosis in cancers of multiple tissue origins. These findings implicate miR-371-3p as a suppressor of PRDX6 and suggest that co-targeting of peroxiredoxin 6 or modulating miR-371-3p expression together with targeted cancer therapies may delay or prevent acquired drug resistance., Acquired resistance significantly limits the efficacy of cancer drug therapies. Here, the authors identify miR-371-3p as a suppressor of drug tolerance in cancer cell lines by its target gene PRDX6, which in turn regulates PLA2/PKCα signalling and ROS levels.

Cardiomyocytes derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) are increasingly used for in vitro assays and represent an interesting opportunity to increase the data throughput for drug development. In this work, we describe a 96-well recording of synchronous electrical activities from spontaneously beating hiPSC-derived cardiomyocyte monolayers. The signal was obtained with a fast-imaging plate reader using a submillisecond-responding membrane potential recording assay, FluoVolt, based on a newly derived voltage-sensitive fluorescent dye. In our conditions, the toxicity of the dye was moderate and compatible with episodic recordings for >3 h. We show that the waveforms recorded from a whole well or from a single cell-sized zone are equivalent and make available critical functional parameters that are usually accessible only with gold standard techniques like intracellular microelectrode recording. This approach allows accurate identification of the electrophysiological effects of reference drugs on the different phases of the cardiac action potential as follows: fast depolarization (lidocaine), early repolarization (nifedipine, Bay K8644, and veratridine), late repolarization (dofetilide), and diastolic slow depolarization (ivabradine). Furthermore, the data generated with the FluoVolt dye can be pertinently complemented with a calcium-sensitive dye for deeper characterization of the pharmacological responses. In a semiautomated plate reader, the two probes used simultaneously in 96-well plates provide an easy and powerful multiparametric assay to rapidly and precisely evaluate the cardiotropic profile of compounds for drug discovery or cardiac safety.

Hibernation is an exceptional physiological response to a hostile environment, characterized by a seasonal period of torpor cycles involving dramatic reductions of body temperature and metabolism, and arousal back to normothermia. As the mechanisms regulating hibernation are still poorly understood, here we analysed the expression of genes involved in energy homeostasis, torpor regulation, and daily or seasonal timing using digital droplet PCR in various central and peripheral tissues sampled at different stages of torpor/arousal cycles in the European hamster. During torpor, the hypothalamus exhibited strongly down-regulated gene expression, suggesting that hypothalamic functions were reduced during this period of low metabolic activity. During both torpor and arousal, many structures (notably the brown adipose tissue) exhibited altered expression of deiodinases, potentially leading to reduced tissular triiodothyronine availability. During the arousal phase, all analysed tissues showed increased expression of the core clock genes Per1 and Per2. Overall, our data indicated that the hypothalamus and brown adipose tissue were the tissues most affected during the torpor/arousal cycle, and that clock genes may play critical roles in resetting the body’s clocks at the beginning of the active period.

The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterized by marked disruption of both metabolism and growth factor signaling.

// Catherine Wilson 1 , Katrina Nicholes 2 , Daisy Bustos 2 , Eva Lin 1 , Qinghua Song 3 , Jean-Philippe Stephan 2 , Donald S. Kirkpatrick 2 and Jeff Settleman 1 1 Department of Discovery Oncology, Genentech, 1 DNA Way, South San Francisco, CA 2 Department of Discovery Protein Chemistry, Genentech, 1 DNA Way, South San Francisco, CA 3 Department of Discovery Nonclinical Biostatistics, Genentech, 1 DNA Way, South San Francisco, CA Correspondence: Jeff Settleman, email: // Keywords : EMT, Src, drug resistance, dasatinib, cancer Received : July 19, 2014 Accepted : August 26, 2014 Published : August 27, 2014 Abstract Epithelial to mesenchymal transition (EMT) is a key process in embryonic development and has been associated with cancer metastasis and drug resistance. For example, in EGFR mutated non-small cell lung cancers (NSCLC), EMT has been associated with acquired resistance to the EGFR inhibitor erlotinib. Moreover, “EGFR-addicted” cancer cell lines induced to undergo EMT become erlotinib-resistant in vitro . To identify potential therapeutic vulnerabilities specifically within these mesenchymal, erlotinib-resistant cells, we performed a small molecule screen of ~200 established anti-cancer agents using the EGFR mutant NSCLC HCC827 cell line and a corresponding mesenchymal derivative line. The mesenchymal cells were more resistant to most tested agents; however, a small number of agents showed selective growth inhibitory activity against the mesenchymal cells, with the most potent being the Abl/Src inhibitor, dasatinib. Analysis of the tyrosine phospho-proteome revealed several Src/FAK pathway kinases that were differentially phosphorylated in the mesenchymal cells, and RNAi depletion of the core Src/FAK pathway components in these mesenchymal cells caused apoptosis. These findings reveal a novel role for Src/FAK pathway kinases in drug resistance and identify dasatinib as a potential therapeutic for treatment of erlotinib resistance associated with EMT.

Purpose: The aim of this study was to identify conserved pharmacodynamic and potential predictive biomarkers of response to anti-VEGF therapy using gene expression profiling in preclinical tumor models and in patients. Experimental Design: Surrogate markers of VEGF inhibition [VEGF-dependent genes or VEGF-dependent vasculature (VDV)] were identified by profiling gene expression changes induced in response to VEGF blockade in preclinical tumor models and in human biopsies from patients treated with anti-VEGF monoclonal antibodies. The potential value of VDV genes as candidate predictive biomarkers was tested by correlating high or low VDV gene expression levels in pretreatment clinical samples with the subsequent clinical efficacy of bevacizumab (anti-VEGF)-containing therapy. Results: We show that VDV genes, including direct and more distal VEGF downstream endothelial targets, enable detection of VEGF signaling inhibition in mouse tumor models and human tumor biopsies. Retrospective analyses of clinical trial data indicate that patients with higher VDV expression in pretreatment tumor samples exhibited improved clinical outcome when treated with bevacizumab-containing therapies. Conclusions: In this work, we identified surrogate markers (VDV genes) for in vivo VEGF signaling in tumors and showed clinical data supporting a correlation between pretreatment VEGF bioactivity and the subsequent efficacy of anti-VEGF therapy. We propose that VDV genes are candidate biomarkers with the potential to aid the selection of novel indications as well as patients likely to respond to anti-VEGF therapy. The data presented here define a diagnostic biomarker hypothesis based on translational research that warrants further evaluation in additional retrospective and prospective trials. Clin Cancer Res; 19(13); 3681–92. ©2013 AACR.

Maintenance of phenotypic heterogeneity within cell populations is an evolutionarily conserved mechanism that underlies population survival upon stressful exposures. We show that the genomes of a cancer cell subpopulation that survives treatment with otherwise lethal drugs, the drug-tolerant persisters (DTPs), exhibit a repressed chromatin state characterized by increased methylation of histone H3 lysines 9 and 27 (H3K9 and H3K27). We also show that survival of DTPs is, in part, maintained by regulators of H3K9me3-mediated heterochromatin formation and that the observed increase in H3K9me3 in DTPs is most prominent over long interspersed repeat element 1 (LINE-1). Disruption of the repressive chromatin over LINE-1 elements in DTPs results in DTP ablation, which is partially rescued by reducing LINE-1 expression or function.

Apoptotic caspase activation mechanisms are well defined, yet inactivation modes remain unclear. The death receptors (DRs), DR4, DR5, and Fas, transduce cell-extrinsic apoptotic signals by recruiting caspase-8 into a death-inducing signaling complex (DISC). At the DISC, Cullin3-dependent polyubiquitination on the small catalytic subunit of caspase-8 augments stimulation. Here we report that tumor necrosis factor receptor-associated factor 2 (TRAF2) interacts with caspase-8 at the DISC, downstream of Cullin3. TRAF2 directly mediates RING-dependent, K48-linked polyubiquitination on the large catalytic domain of caspase-8. This modification destines activated caspase-8 molecules to rapid proteasomal degradation upon autoprocessing and cytoplasmic translocation. TRAF2 depletion lowers the signal threshold for DR-mediated apoptosis, altering cell life versus death decisions in vitro and in vivo. Thus, TRAF2 sets a critical barrier for cell-extrinsic apoptosis commitment by tagging activated caspase-8 with a K48-ubiquitin shutoff timer. These results may have important implications for caspase regulation mechanisms.

The effector functions of therapeutic antibodies are strongly affected by the specific glycans added to the Fc domain during post-translational processing. Antibodies bearing high levels of N-linked mannose-5 glycan (Man5) have been reported to exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared with antibodies with fucosylated complex or hybrid glycans. To better understand the relationship between antibodies with high levels of Man5 and their biological activity in vivo, we developed an approach to generate substantially homogeneous antibodies bearing the Man5 glycoform. A mannosidase inhibitor, kifunensine, was first incorporated in the cell culture process to generate antibodies with a distribution of high mannose glycoforms. Antibodies were then purified and treated with a mannosidase for trimming to Man5 in vitro. This 2-step approach can consistently generate antibodies with > 99% Man5 glycan. Antibodies bearing varying levels of Man5 were studied to compare ADCC and Fcγ receptor binding, and they showed enhanced ADCC activity and increased binding affinity to the FcγRIIIA. In addition, the clearance rate of antibodies bearing Man8/9 and Man5 glycans was determined in a pharmacokinetics study in mice. When compared with historical data, the antibodies bearing the high mannose glycoform exhibited faster clearance rate compared with antibodies bearing the fucosylated complex glycoform, while the pharmacokinetic properties of antibodies with Man8/9 and Man5 glycoforms appeared similar. In addition, we identified the presence of a mannosidase in mouse serum that converted most Man8/9 to Man6 after 24 h.

Sensitive detection of protein interactions is a critical step toward understanding complex cellular processes. As an alternative to fluorescence-based detection, Renilla reniformis luciferase conjugated to quantum dots results in self-illuminating bioluminescence resonance energy transfer quantum dot (BRET-Qdot) nanoprobes that emit red to near-infrared bioluminescence light. Here, we report the development of an ultrasensitive technology based on BRET-Qdot conjugates modified with streptavidin ([BRET-Qdot]-SA) to detect cell-surface protein interactions. Transfected COS7 cells expressing human cell-surface proteins were interrogated with a human Fc tagged protein of interest. Specific protein interactions were detected using a biotinylated anti-human Fc region specific antibody followed by incubation with [BRET-Qdot]-SA. The luciferase substrate coelenterazine activated bioluminescence light emission was detected with an ultra-fast and -sensitive imager. Protein interactions barely detectable by the fluorescence-based approach were readily quantified using this technology. The results demonstrate the successful application and the flexibility of the BRET-Qdot-based imaging technology to the ultrasensitive investigation of cell-surface proteins and protein-protein interactions.

With more than 34 targets being investigated and nearly 20 clinical trials at various phases of development, antibody–drug conjugates (ADCs) hold a lot of promise for improving oncological malignancy therapy. This therapeutic strategy designed to specifically or preferentially deliver a cytotoxic agent to tumor cells through conjugation to a monoclonal antibody is not new. Although this approach is relatively simple conceptually, the history of ADCs clearly attests to the high degree of complexity in their development. Each component of an ADC is important to achieve efficacy with minimal toxicity, and the ability to monitor this multicomponent therapeutic entity is deemed to be critical for their successful optimization. In this article we review the different bioanalytical strategies that have been implemented to characterize various ADCs and discuss the challenges and issues associated with these approaches.

Among Breast Cancer (BC) classification, Triple-Negative (TN) form is the most aggressive, lacking efficient and specific treatment. Our project aims at investigating the possibility to convert TNBC tumor cell lines in Estrogen Receptor (ER), Progesterone Receptor (PR) and/or Human Epidermal growth factor Receptor 2 (HER2)-positive cells using various modalities (siRNA, CRISPR-Cas9, small molecules). Our approach consists in optimizing a multi-parametric High-Content Screening (HCS) relying on phenotypic read-outs such as expression of TNBC positive and negative markers, cell behavior and viability. This poster will present our initial results, validation and future plans. This thesis project fits within a context of drug discovery, innovation, comparison of technologies and understanding of key biological processes and pathways, carried out in partnership between Servier Research Institute and Institut Curie. Citation Format: Albane Gaudeau, Véronique Lamamy, Anne-Laure Jaskowiak, Xavier Scerri, Benjamin Chanrion, Thierry Dorval, Elaine Del Nery, Franck Perez, Jacques Camonis, Jean-Philippe Stéphan. Investigation of triple-negative breast cancer tumor conversion through high-content screening approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1487.

Targeting cytotoxic drugs to cancer cells using antibody–drug conjugates (ADCs), particularly those with stable linkers between the drug and the antibody, could be an effective cancer treatment with low toxicity. However, for stable-linker ADCs to be effective, they must be internalized and degraded, limiting potential targets to surface antigens that are trafficked to lysosomes. CD79a and CD79b comprise the hetrodimeric signaling component of the B-cell receptor, and are attractive targets for the use of ADCs because they are B-cell–specific, expressed in non-Hodgkin lymphomas (NHL), and are trafficked to a lysosomal-like compartment as part of antigen presentation. We show here that the stable-linker ADCs anti-CD79b-MCC-DM1 and anti-CD79b-MC-MMAF are capable of target-dependent killing of nonHodgkin lymphoma cell lines in vitro. Further, these 2 ADCs are equally effective as low doses in xenograft models of follicular, mantle cell, and Burkitt lymphomas, even though several of these cell lines express relatively low levels of CD79b in vivo. In addition, we demonstrate that anti-CD79b ADCs were more effective than anti-CD79a ADCs and that, as hypothesized, anti-CD79b antibodies downregulated surface B-cell receptor and were trafficked to the lysosomal-like major histocompatibility complex class II–positive compartment MIIC. These results suggest that anti-CD79b-MCC-DM1 and anti-CD79b-MC-MMAF are promising therapeutics for the treatment of NHL.

Most mouse models of hepatocellular carcinoma have expressed growth factors and oncogenes under the control of a liver-specific promoter. In contrast, we describe here the formation of liver tumors in transgenic mice overexpressing human fibroblast growth factor 19 (FGF19) in skeletal muscle. FGF19 transgenic mice had elevated hepatic α-fetoprotein mRNA as early as 2 months of age, and hepatocellular carcinomas were evident by 10 months of age. Increased proliferation of pericentral hepatocytes was demonstrated by 5-bromo-2′-deoxyuridine incorporation in the FGF19 transgenic mice before tumor formation and in nontransgenic mice injected with recombinant FGF19 protein. Areas of small cell dysplasia were initially evident pericentrally, and dysplastic/neoplastic foci throughout the hepatic lobule were glutamine synthetase-positive, suggestive of a pericentral origin. Consistent with chronic activation of the Wingless/Wnt pathway, 44% of the hepatocellular tumors from FGF19 transgenic mice had nuclear staining for β-catenin. Sequencing of the tumor DNA encoding β-catenin revealed point mutations that resulted in amino acid substitutions. These findings suggest a previously unknown role for FGF19 in hepatocellular carcinomas.

Despite the knowledge accumulated during the last two decades about programmed cell death, further investigations of the complex regulatory network of apoptosis, including the extrinsic pathways, are still needed to gain an exhaustive and comprehensive understanding of this critical biological process. In addition, the identification of novel modulators of apoptosis may represent a good opportunity for making new paths into an otherwise heavily investigated area, therefore providing a molecular basis for new therapeutic strategies. In the last decade, RNA interference has become the technology of choice for discovering genes that encode molecules with previously unknown functions in biological pathways of interest. Various RNAi reagents and library formats have been developed and harnessed for high-throughput screening technologies to enable almost limitless investigation to uncover gene functions and networks in the context of basic biology and biomedical research including cancer biology. Although RNAi screening has been demonstrated to be a very powerful tool, various caveats and pitfalls have been progressively uncovered, including, but not limited to the enduring off-target effects. As the novelty of its bells and whistles have begun to diminish, functional genomic screens have morphed into a specialized field within the high-throughput screening community, where expert investigators progressively establish rigorous strategies to mitigate most of its possible flaws. Using various examples of RNAi screens conducted to further understand the extrinsic apoptosis pathway, this chapter describes the different RNAi tools and screening formats available and reviews the parameters one has to critically consider in order to be successful in implementing this technology.

The spliceosome machinery is composed of multimeric protein complexes that generate a diverse repertoire of mRNA through coordinated splicing of heteronuclear RNAs. While somatic mutations in spliceosome components have been discovered in several cancer types, the molecular bases and consequences of spliceosome aberrations in cancer are poorly understood. Here we report for the first time that PRPF6, a member of the tri-snRNP (small ribonucleoprotein) spliceosome complex, drives cancer proliferation by preferential splicing of genes associated with growth regulation. Inhibition of PRPF6 and other tri-snRNP complex proteins, but not other snRNP spliceosome complexes, selectively abrogated growth in cancer cells with high tri-snRNP levels. High-resolution transcriptome analyses revealed that reduced PRPF6 alters the constitutive and alternative splicing of a discrete number of genes, including an oncogenic isoform of the ZAK kinase. These findings implicate an essential role for PRPF6 in cancer via splicing of distinct growth-related gene products.

It is well established that the notochord influences the development of adjacent neural and mesodermal tissue. Involvement of the notochord in the differentiation of the dorsal pancreas has been demonstrated. However, our knowledge of the signals involved in pancreatic development is still incomplete. In order to identify proteins potentially implicated during pancreatic differentiation, we raised and characterized monoclonal antibodies against previously established embryonic pancreatic ductal epithelial cell lines (BUD and RED). Using the MAb 2117, the cell surface antigen 2117 (Ag 2117) was cloned. The predicted sequence for Ag 2117 is the rat homologue of BEN. Initially reported as a protein expressed on epithelial cells of the chicken bursa of Fabricius, BEN is expressed in a variety of tissues during development and described as a marker for the developing central and peripheral chicken nervous systems. A role has been suggested for BEN in the adhesion of stem cells and progenitor cells to the blood-forming tissue microenvironment. In this study, we demonstrate that BEN, initially expressed exclusively in the notochord during the early development of rat, is implicated in pancreatic development. We show that Ag 2117 regulates the pancreatic epithelial cell growth through the ras and Jun kinase pathways. In addition, we demonstrate that Ag 2117 is able to regulate the expression of the transcription factor PDX1, required for insulin gene expression, in embryonic pancreas organ cultures.

Despite the knowledge accumulated during the last two decades about programmed cell death, further investigations of the complex regulatory network of apoptosis, including the extrinsic pathways, are still needed to gain an exhaustive and comprehensive understanding of this critical biological process. In addition, the identification of novel modulators of apoptosis may represent a good opportunity for making new paths into an otherwise heavily investigated area, therefore providing a molecular basis for new therapeutic strategies. In the last decade, RNA interference has become the technology of choice for discovering genes that encode molecules with previously unknown functions in biological pathways of interest. Various RNAi reagents and library formats have been developed and harnessed for high-throughput screening technologies to enable almost limitless investigation to uncover gene functions and networks in the context of basic biology and biomedical research including cancer biology. Although RNAi screening has been demonstrated to be a very powerful tool, various caveats and pitfalls have been progressively uncovered, including, but not limited to the enduring off-target effects. As the novelty of its bells and whistles have begun to diminish, functional genomic screens have morphed into a specialized field within the high-throughput screening community, where expert investigators progressively establish rigorous strategies to mitigate most of its possible flaws. Using various examples of RNAi screens conducted to further understand the extrinsic apoptosis pathway, this chapter describes the different RNAi tools and screening formats available and reviews the parameters one has to critically consider in order to be successful in implementing this technology.