David Bond

A Quantitative RNAi Screen for JNK Modifiers Identifies Pvr as a Novel Regulator of Drosophila Immune Signaling

Drosophila melanogaster responds to gram-negative bacterial challenges through the IMD pathway, a signal transduction cassette that is driven by the coordinated activities of JNK, NF-κB and caspase modules. While many modifiers of NF-κB activity were identified in cell culture and in vivo assays, the regulatory apparatus that determines JNK inputs into the IMD pathway is relatively unexplored. In this manuscript, we present the first quantitative screen of the entire genome of Drosophila for novel regulators of JNK activity in the IMD pathway. We identified a large number of gene products that negatively or positively impact on JNK activation in the IMD pathway. In particular, we identified the Pvr receptor tyrosine kinase as a potent inhibitor of JNK activation. In a series of in vivo and cell culture assays, we demonstrated that activation of the IMD pathway drives JNK-dependent expression of the Pvr ligands, Pvf2 and Pvf3, which in turn act through the Pvr/ERK MAP kinase pathway to attenuate the JNK and NF-κB arms of the IMD pathway. Our data illuminate a poorly understood arm of a critical and evolutionarily conserved innate immune response. Furthermore, given the pleiotropic involvement of JNK in eukaryotic cell biology, we believe that many of the novel regulators identified in this screen are of interest beyond immune signaling.

Autocrine Platelet-derived Growth Factor-Vascular Endothelial Growth Factor Receptor-related (Pvr) Pathway Activity Controls Intestinal Stem Cell Proliferation in the Adult Drosophila Midgut

Background: Drosophila midgut intestinal stem cells (ISCs) proliferate and differentiate to replace mature cells types and maintain tissue integrity. Results: The Pvr signal transduction pathway provides an autocrine control of the differentiation of ISCs into mature cells. Conclusion: The Pvr pathway is an intrinsic regulator of ISC differentiation. Significance: Pvr is the first strictly intrinsic regulator of ISC differentiation characterized. A dynamic pool of undifferentiated somatic stem cells proliferate and differentiate to replace dead or dying mature cell types and maintain the integrity and function of adult tissues. Intestinal stem cells (ISCs) in the Drosophila posterior midgut are a well established model to study the complex genetic circuitry that governs stem cell homeostasis. Exposure of the intestinal epithelium to environmental toxins results in the expression of cytokines and growth factors that drive the rapid proliferation and differentiation of ISCs. In the absence of stress signals, ISC homeostasis is maintained through intrinsic pathways. In this study, we uncovered the PDGF- and VEGF-receptor related (Pvr) pathway as an essential regulator of ISC homeostasis under unstressed conditions in the posterior midgut. We found that Pvr is coexpressed with its ligand Pvf2 in ISCs and that hyperactivation of the Pvr pathway distorts the ISC developmental program and drives intestinal dysplasia. In contrast, we show that mutant ISCs in the Pvf/Pvr pathway are defective in homeostatic proliferation and differentiation, resulting in a failure to generate mature cell types. Additionally, we determined that extrinsic stress signals generated by enteropathogenic infection are epistatic to the hypoplasia generated in Pvf/Pvr mutants, making the Pvr pathway unique among all previously studied intrinsic pathways. Our findings illuminate an evolutionarily conserved signal transduction pathway with essential roles in metazoan embryonic development and direct involvement in numerous disease states.

Quantitative evaluation of signaling events in Drosophila S2 cells

Drosophila activates a robust defense response to gram-negative bacteria through the Immune deficiency (Imd) pathway. Imd signaling proceeds through c-Jun N-terminal Kinase (JNK), NF-kB and caspase modules. The individual signaling modules act in a highly coordinated manner to yield a stereotypical response to infection. While considerable attention has focused on NF-kB-mediated antimicrobial activities, more recent studies have highlighted the involvement of JNK signaling in the Imd pathway response. JNK signaling occurs in a transitory burst and drives the expression of a number of gene products through the AP-1 transcription factor. In this report, we describe a simple method for the quantification of JNK activation by Western blot analysis or directly in tissue culture plates.

Therapeutic efficacy of human hepatocyte transplantation in a SCID/uPA mouse model with inducible liver disease.

Background Severe Combined Immune Deficient (SCID)/Urokinase-type Plasminogen Activator (uPA) mice undergo liver failure and are useful hosts for the propagation of transplanted human hepatocytes (HH) which must compete with recipient-derived hepatocytes for replacement of the diseased liver parenchyma. While partial replacement by HH has proven useful for studies with Hepatitis C virus, complete replacement of SCID/uPA mouse liver by HH has never been achieved and limits the broader application of these mice for other areas of biomedical research. The herpes simplex virus type-1 thymidine kinase (HSVtk)/ganciclovir (GCV) system is a powerful tool for cell-specific ablation in transgenic animals. The aim of this study was to selectively eliminate murine-derived parenchymal liver cells from humanized SCID/uPA mouse liver in order to achieve mice with completely humanized liver parenchyma. Thus, we reproduced the HSVtk (vTK)/GCV system of hepatic failure in SCID/uPA mice. Methodology/Principal Findings In vitro experiments demonstrated efficient killing of vTK expressing hepatoma cells after GCV treatment. For in vivo experiments, expression of vTK was targeted to the livers of FVB/N and SCID/uPA mice. Hepatic sensitivity to GCV was first established in FVB/N mice since these mice do not undergo liver failure inherent to SCID/uPA mice. Hepatic vTK expression was found to be an integral component of GCV-induced pathologic and biochemical alterations and caused death due to liver dysfunction in vTK transgenic FVB/N and non-transplanted SCID/uPA mice. In SCID/uPA mice with humanized liver, vTK/GCV caused death despite extensive replacement of the mouse liver parenchyma with HH (ranging from 32–87%). Surprisingly, vTK/GCV-dependent apoptosis and mitochondrial aberrations were also localized to bystander vTK-negative HH. Conclusions/Significance Extensive replacement of mouse liver parenchyma by HH does not provide a secure therapeutic advantage against vTK/GCV-induced cytotoxicity targeted to residual mouse hepatocytes. Functional support by engrafted HH may be secured by strategies aimed at limiting this bystander effect.

Quantitative in vivo whole genome motility screen reveals novel therapeutic targets to block cancer metastasis

Metastasis is the most lethal aspect of cancer, yet current therapeutic strategies do not target its key rate-limiting steps. We have previously shown that the entry of cancer cells into the blood stream, or intravasation, is highly dependent upon in vivo cancer cell motility, making it an attractive therapeutic target. To systemically identify genes required for tumor cell motility in an in vivo tumor microenvironment, we established a novel quantitative in vivo screening platform based on intravital imaging of human cancer metastasis in ex ovo avian embryos. Utilizing this platform to screen a genome-wide shRNA library, we identified a panel of novel genes whose function is required for productive cancer cell motility in vivo, and whose expression is closely associated with metastatic risk in human cancers. The RNAi-mediated inhibition of these gene targets resulted in a nearly total (>99.5%) block of spontaneous cancer metastasis in vivo.Tumour metastasis is dependent on tumour cell motility. Here, the authors investigate genes required for tumour cell motility by establishing a quantitative in vivo screening platform based on intravital imaging of human cancer metastasis in ex ovo avian embryos.

Quantitative Analysis of Human Cancer Cell Extravasation Using Intravital Imaging.

Metastasis, or the spread of cancer cells from a primary tumor to distant sites, is the leading cause of cancer-associated death. Metastasis is a complex multi-step process comprised of invasion, intravasation, survival in circulation, extravasation, and formation of metastatic colonies. Currently, in vitro assays are limited in their ability to investigate these intricate processes and do not faithfully reflect metastasis as it occurs in vivo. Traditional in vivo models of metastasis are limited by their ability to visualize the seemingly sporadic behavior of where and when cancer cells spread (Reymond et al., Nat Rev Cancer 13:858-870, 2013). The avian embryo model of metastasis is a powerful platform to study many of the critical steps in the metastatic cascade including the migration, extravasation, and invasion of human cancer cells in vivo (Sung et al., Nat Commun 6:7164, 2015; Leong et al., Cell Rep 8, 1558-1570, 2014; Kain et al., Dev Dyn 243:216-28, 2014; Leong et al., Nat Protoc 5:1406-17, 2010; Zijlstra et al., Cancer Cell 13:221-234, 2008; Palmer et al., J Vis Exp 51:2815, 2011). The chicken chorioallantoic membrane (CAM) is a readily accessible and well-vascularized tissue that surrounds the developing embryo. When the chicken embryo is grown in a shell-less, ex ovo environment, the nearly transparent CAM provides an ideal environment for high-resolution fluorescent microcopy approaches. In this model, the embryonic chicken vasculature and labeled cancer cells can be visualized simultaneously to investigate specific steps in the metastatic cascade including extravasation. When combined with the proper image analysis tools, the ex ovo chicken embryo model offers a cost-effective and high-throughput platform for the quantitative analysis of tumor cell metastasis in a physiologically relevant in vivo setting. Here we discuss detailed procedures to quantify cancer cell extravasation in the shell-less chicken embryo model with advanced fluorescence microscopy techniques.

Abstract 4972: In vivo whole genome shRNA screen reveals novel targets to block cancer metastasis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The formation of invasive, rapidly growing metastatic lesions is a critical step in cancer metastasis, the cause of more than 90% of cancer deaths. Development of novel therapeutic approaches that block the invasion step of metastasis is one of the highest priorities for clinical cancer research. For this reason we completed the first genome-wide in vivo shRNA screen for genes that directly contribute to invasive metastatic lesion formation. Using state of the art intravital imaging, we identified over fifty novel regulators of invasive metastatic colony formation in vivo. Interactome analysis links these genes to key cellular processes including: transcriptional regulation of gene expression, mRNA processing and cytoskeletal remodeling. The target list was then prioritized based on clinical gene expression profiles that negatively correlated with key cancer endpoints including metastasis, cancer-specific and overall survival. Pharmacological and shRNA-mediated knockdown of the high priority targets in human cancer cell lines such as prostate cancer and melanoma specifically blocked cancer cell migration and invasion in vitro and in vivo. Moreover, shRNA-mediated knockdown of these genes blocked human cancer cell metastasis in the avian embryo and mouse preclinical models of metastasis. Finally, immunohistochemical analysis on clinical prostate cancer and melanoma tissue samples showed strong correlation with disease progression and metastasis. In summary, we have identified numerous novel genes that that are functionally involved in cancer invasion and metastasis that may serve as predictive markers for disease aggressiveness and represent exciting new pharmacological targets to block cancer invasion and metastasis. Citation Format: Konstantin Stoletov, David Bond, Hon Sing Leong, Emma Woolner, Srijan Raha, Amy Robertson, Francis Wong, Andries Zijlstra, John D. Lewis. In vivo whole genome shRNA screen reveals novel targets to block cancer metastasis. [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 4972. doi:10.1158/1538-7445.AM2014-4972

Abstract 4971: Splicing factor kinase regulates metastatic dissemination of human prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Metastasis causes the vast majority of cancer-associated deaths. Despite its clinical significance, the molecular mechanisms that regulate this multi-step process are poorly understood. To successfully disseminate a cancer cell must escape the primary tumor, travel to a distant site, arrest, enter the stroma and grow in a new microenvironment. The presence of circulating tumor cells in patients with metastatic disease and experimental animals with metastatic cancers suggests that tumor invasion is a critical, rate-limiting step in the metastatic process. To identify the key molecular determinants that regulate invasive metastatic lesion formation we performed the first whole-genome, in vivo RNAi screen in an avian embryo model using advanced intravital imaging techniques. We identified numerous novel regulators of cancer cell invasion, including JL1, a splicing factor kinase. In secondary assays, we demonstrated that JL1-depletion inhibits migration of several human cancer cell lines with no effect on proliferation or survival. Furthermore, JL1-deficient human cancer cells display diminished metastatic potential in chicken embryo and mouse models of metastasis. Cancer microarray database mining shows that JL1 gene expression is strongly associated with indicators of poor clinical outcome in prostate cancer, including pathological Gleason score and metastasis. To experimentally determine JL1 association with advanced disease we performed histological examinations of prostate cancer tissue and determined that JL1 staining strongly correlates with prostate cancer progression. JL1 kinase regulates alternative splicing of numerous cellular transcripts in response to stress and growth signals. To examine the molecular mechanism behind JL1 regulation of cancer cell metastasis, we performed exon level microarray analysis on JL1-depleted cancer cell lines. Subsequent interactome analysis identified an enrichment of key regulatory proteins in pro-metastatic networks. Therefore, we hypothesize that abrogated JL1 activity directly promotes prostate cancer cell migration and invasion. In summary, JL1 is a novel regulator of cancer cell metastasis that represents a potential pathological marker for prostate cancer progression, and novel drug target to block invasion and metastasis. Citation Format: David J. Bond, Konstantin Stoletov, Hon Sing Leong, Emma Woolner, Shuhong Liu, Tarek Bismar, Andries Zijlstra, John D. Lewis. Splicing factor kinase regulates metastatic dissemination of human prostate cancer. [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 4971. doi:10.1158/1538-7445.AM2014-4971