Insertional Mutagenesis Screen Research Articles

The mitochondrial thiamine pyrophosphate transporter TptA promotes adaptation to low iron conditions and virulence in fungal pathogen Aspergillus fumigatus

ABSTRACTAspergillus fumigatus is the most prevalent airborne fungal pathogen that causes invasive fungal infections in immunosuppressed individuals. Adaptation to iron limited conditions is crucial for A. fumigatus virulence. To identify novel genes that play roles in adaptation to low iron conditions we performed an insertional mutagenesis screen in A. fumigatus. Using this approach, we identified the tptA gene in A. fumigatus, which shares homology with the Saccharomyces cerevisiae thiamine pyrophosphate (ThPP) transporter encoding gene tpc1. Heterologous expression of tpc1 in the tptA deletion mutant completely restored the ThPP auxotrophy phenotype, suggesting that Tpc1 and TptA are functional orthologues. Importantly, TptA was required for adaptation to low iron conditions in A. fumigatus. The ΔtptA mutant had decreased resistance to the iron chelator bathophenanthroline disulfonate (BPS) with severe growth defects. Moreover, loss of tptA decreased the expression of hapX, which is a major transcription factor indispensable for adaptation to iron starvation in A. fumigatus. Overexpression of hapX in the ΔtptA strain greatly rescued the growth defect and siderophore production by A. fumigatus in iron-depleted conditions. Mutagenesis experiments demonstrated that the conserved residues related to ThPP uptake in TptA were also required for low iron adaptation. Furthermore, TptA-mediated adaptation to low iron conditions was found to be dependent on carbon sources. Finally, loss of tptA resulted in the attenuation of virulence in a murine model of aspergillosis. Taken together, this study demonstrated that the mitochondrial ThPP transporter TptA promotes low iron adaptation and virulence in A. fumigatus.

Identification of Cancer Genes Based on De Novo Transposon Insertion Site Analysis Using RNA and DNA Sequencing.

Forward genetic insertional mutagenesis screens are used by many labs to identify candidate cancer genes. We and others have used the Sleeping Beauty DNA transposon to generate random mutations within the murine genome that cause cancer. Identification of the insertion sites, either via RNA sequencing or DNA sequencing, is required for cancer gene discovery. Multiple sequencing-based approaches have been utilized to identify locations of transposon insertions within a genome including linker-mediated PCR, RNA-Seq, and Seq capture. Here, a bioinformatics pipeline is presented applicable to both the identification of transposon-generated fusions in RNA-Seq data and the direct identification of transposon insertion sites in DNA sequencing data. We are currently utilizing this method to identify transposon insertions generated by Sleeping Beauty transposase-mediated mobilization of the T2/Onc transposon within the murine genome. With slight modification, this approach is amenable to the identification of any mobile genetic element within any genome.

PiggyBac Transposon-Based Insertional Mutagenesis in Mice.

While sequencing and array-based studies are creating catalogues of genetic alterations in cancer, discriminating cancer drivers among the large sets of epigenetically, transcriptionally or posttranslationally dysregulated genes remains a challenge. Transposon-based genetic screening in mice has proven to be a powerful approach to address this challenge. Insertional mutagenesis directly flags biologically relevant genes and, combined with the transposon's unique molecular fingerprint, facilitates the recovery of insertion sites. We have generated transgenic mouse lines harboring different versions of PiggyBac-based oncogenic transposons, which in conjunction with PiggyBac transposase mice can be used for whole-body or tissue-specific insertional mutagenesis screens. We have also developed QiSeq, a method for (semi-)quantitative transposon insertion site sequencing, which overcomes biasing limitations of previous library preparation methods. QiSeq can be used in multiplexed high-throughput formats for candidate cancer gene discovery and gives insights into the clonal distribution of insertions for the study of genetic tumor evolution.

Transposon mutagenesis screen in mice identifies TM9SF2 as a novel colorectal cancer oncogene

New therapeutic targets for advanced colorectal cancer (CRC) are critically needed. Our laboratory recently performed an insertional mutagenesis screen in mice to identify novel CRC driver genes and, thus, potential drug targets. Here, we define Transmembrane 9 Superfamily 2 (TM9SF2) as a novel CRC oncogene. TM9SF2 is an understudied protein, belonging to a well conserved protein family characterized by their nine putative transmembrane domains. Based on our transposon screen we found that TM9SF2 is a candidate progression driver in digestive tract tumors. Analysis of The Cancer Genome Atlas (TCGA) data revealed that approximately 35% of CRC patients have elevated levels of TM9SF2 mRNA, data we validated using an independent set of CRC samples. RNAi silencing of TM9SF2 reduced CRC cell growth in an anchorage-independent manner, a hallmark of cancer. Furthermore, CRISPR/Cas9 knockout of TM9SF2 substantially diminished CRC tumor fitness in vitro and in vivo. Transcriptome analysis of TM9SF2 knockout cells revealed a potential role for TM9SF2 in cell cycle progression, oxidative phosphorylation, and ceramide signaling. Lastly, we report that increased TM9SF2 expression correlates with disease stage and low TM9SF2 expression correlate with a more favorable relapse-free survival. Taken together, this study provides evidence that TM9SF2 is a novel CRC oncogene.

Transcriptomics and Transposon Mutagenesis Identify Multiple Mechanisms of Resistance to the FGFR Inhibitor AZD4547.

In human cancers, FGFR signaling is frequently hyperactivated by deregulation of FGF ligands or by activating mutations in the FGFR receptors such as gene amplifications, point mutations, and gene fusions. As such, FGFR inhibitors are considered an attractive therapeutic strategy for patients with mutations in FGFR family members. We previously identified Fgfr2 as a key driver of invasive lobular carcinoma (ILC) in an in vivo insertional mutagenesis screen using the Sleeping Beauty transposon system. Here we explore whether these FGFR-driven ILCs are sensitive to the FGFR inhibitor AZD4547 and use transposon mutagenesis in these tumors to identify potential mechanisms of resistance to therapy. Combined with RNA sequencing-based analyses of AZD4547-resistant tumors, our in vivo approach identified several known and novel potential resistance mechanisms to FGFR inhibition, most of which converged on reactivation of the canonical MAPK-ERK signaling cascade. Observed resistance mechanisms included mutations in the tyrosine kinase domain of FGFR2, overexpression of MET, inactivation of RASA1, and activation of the drug-efflux transporter ABCG2. ABCG2 and RASA1 were identified only from de novo transposon insertions acquired during AZD4547 treatment, demonstrating that insertional mutagenesis in mice is an effective tool for identifying potential mechanisms of resistance to targeted cancer therapies.Significance: These findings demonstrate that a combined approach of transcriptomics and insertional mutagenesis in vivo is an effective method for identifying potential targets to overcome resistance to therapy in the clinic. Cancer Res; 78(19); 5668-79. ©2018 AACR.

Identifying a novel connection between the fungal plasma membrane and pH-sensing.

The mechanisms by which micro-organisms sense and internalize extracellular pH signals are not completely understood. One example of a known external pH-sensing process is the fungal-specific Rim/Pal signal transduction pathway. Fungi, such as the opportunistic pathogen Cryptococcus neoformans, use Rim signaling to sense and respond to changes in environmental pH. Mutations in this pathway result in strains that are attenuated for survival at alkaline pH, and often for survival within the host. Here, we used an insertional mutagenesis screen to identify novel genes required for C. neoformans growth at host pH. We discovered altered alkaline pH growth in several strains with specific defects in plasma membrane composition and maintenance of phospholipid assembly. Among these, loss of function of the Cdc50 lipid flippase regulatory subunit affected the temporal dynamics of Rim pathway activation. We defined distinct and overlapping cellular processes regulated by Rim101 and Cdc50 through analysis of the transcriptome in these mutant strains. We further explored how pH-induced membrane changes affect membrane-bound pH-sensing proteins, specifically the C-terminal domain of the Rra1 protein, an upstream Rim pathway activator and pH sensor. These results suggest both broadly applicable and phylum-specific molecular interactions that drive microbial environmental sensing.

Abstract 1821: Genome-wide genetic screens define the drug resistance landscape of BRAF mutant colon cancer

Abstract Background: Almost 10% of colon cancers harbor the canonical activating V600 BRAF mutation that in melanoma cancers has been shown to dramatically sensitize cells to targeted therapy and prolong survival. In the colon, such tumors are associated with worse survival. Recent clinical trials of combination therapies targeting the EGFR and MAPK pathways in these tumors have demonstrated impressive response rates. However, the clinical benefit has been limited by the rapid emergence of drug resistance. Identifying and targeting the genes complicit in this process and thus re-sensitize resistant cancer cells may be of clinical benefit. The landscape of drug resistance in cancer patients is composed of (a) loss-of-function (LoF), (b) gain-of-function (GoF) and (c) point mutation-mediated perturbations of genes, with often no single mechanism being predominant. Therefore, capturing the full breadth of resistance genes for any drug would require multiple genetic screens to be executed in parallel. Methods: We performed 3 genome-wide genetic screens in parallel in BRAF mutant colon cancer cells treated with a BRAF/MEK/EGFR inhibitor combination, to capture all of the resistance mechanisms described above. To screen for LoF events we used a genome-wide CRISPR/Cas9 sgRNA library. For GoF resistance events, a lentiviral-based insertional mutagenesis vector was used to randomly integrate the SFFV enhancer/promoter sequence throughout the genome. Finally, to saturate the genome of each cell line with point mutations we used N-Ethyl-N-nitrosourea (ENU) to randomly mutagenize every base in the genome. Each genetic screen was analysed separately to identify the relevant genes and pathways that confer drug resistance before aggregating the outputs of all three screens for a more comprehensive view of the drug resistance landscape. Results: The most recurrent and strongest hit from the CRISPR knockout screen was the inhibitor of Src family kinases, CSK. A Gaussian Kernal Convolution analysis for recurrent integrations detected in resistant clones following the insertional mutagenesis screen identified five genes as likely resistance candidates - BRAF, SOS1, MET, FRS2 and KRAS. Finally, Illumina exome sequencing of resistant clones following ENU mutagenesis revealed statistical enrichment for non-synonymous point mutations in three genes in the MAPK pathway, namely MAP2K1, NRAS and KRAS. Of note, loss of CSK was also shown to confer resistance to targeted therapies in other cancer types. Conclusion: Genome-wide genetic screens identified genes in the MAPK pathway likely to cause resistance clinically, and many of which are amenable to therapeutic targeting. Of note, the resistance as a result of loss of CSK instead activates Src signalling. It can be overcome through the addition of a Src inhibitor, and may be relevant across a range of cancer types as a resistance mechanism. Citation Format: Jonathan Brammeld, Marco Ranzani, Elizabeth A. Coker, Stacey Price, Theodoros I. Roumeliotis, Barbara Lupo, Mi Petljak, Steven P. Williams, Francesco Iorio, Francesco Sassi, Nanne Aben, Kosuke Yusa, Livio Trusolino, Lodewyk Wessels, David J. Adams, Mathew Garnett, Bissan Al-Lazikani, Jyoti S. Choudhary, Andrea Bertotti, Ultan McDermott. Genome-wide genetic screens define the drug resistance landscape of BRAF mutant colon cancer [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 1821.

Abstract A47: Reducing actin contraction in E-cadherin inactivated mammary epithelial cells leads to the development of invasive lobular carcinoma

Abstract Invasive lobular carcinoma (ILC) accounts for 10-15% of breast cancer cases in women. One of the characteristics of ILC is the loss of intercellular adhesion, which occurs as a consequence of inactivation of E-cadherin (CDH1). Inactivation of E-cadherin often occurs through truncating mutations followed by loss of heterozygosity. E-cadherin is a master regulator of cell-cell interactions and thereby functions as an important tumor suppressor. Nevertheless, loss of E-cadherin by itself does not induce mammary tumor formation in mice. Moreover, somatic inactivation of E-cadherin in mouse mammary epithelium resulted in outgrowth mammary ducts with only E-cadherin proficient mammary epithelial cells, suggesting that loss of E-cadherin by itself is not tolerated. Why the inactivation of E-cadherin is not tolerated in mammary epithelial cells remains unclear. To uncover the fate of mammary epithelial cells upon inactivation of E-cadherin, a WapCre;Cdh1F/F;mTmg mouse model was generated. The introduction of the mTmG reporter allows for the identification and tracking of Cre-driven E-cadherin inactivated mammary epithelial cells. Analysis of the mammary ducts of these mice revealed that at least a portion of E-cadherin inactivated mammary epithelial cells survive. At 12 weeks of age clusters of E-cadherin inactivated mammary epithelial cells were found nested between the myoepithelial cells and the basal membrane. Despite the fact that they survive, these cells appear dormant since these clusters do not increase in size over time. Using in vivo imaging of WapCre;Cdh1F/F;mTmg mice, it becomes clear that the E-cadherin inactivated cells are motile and show a high degree of membrane blebbing. The E-cadherin inactivated cells have increased cortical accumulation of MYH9 and myosin light chain phosphorylation, which indicate increased actin contraction. Recently we identified PPP1R12A/B, TP53BP2, and MYH9 as drivers of ILC in an in vivo insertional mutagenesis screen. The identified drivers did not have any effect on the PI3 kinase and MAP kinase pathways nor did they alter P53 activation. They did, however, allow survival of E-cadherin inactivated mammary cells in vitro and in vivo. PPP1R12A/B and MYH9 are primarily known for their critical role in actin contractility. Interestingly, overexpression of the TRP53BP2 truncation variant identified in the screen reduced myosin light chain phosphorylation in a similar manner as the PPP1R12A truncation variant. To provide more evidence that the reduction of actin contraction allows rescue of E-cadherin inactivated cells, we inhibited actin contraction pharmacologically. Both exposure to a Rho kinase inhibitor as well as blebbistatin (inhibits non-muscle myosin ATPase activity) allowed for the growth of E-cadherin inactivated primary epithelial cells. Overall our data suggest that the inactivation of E-cadherin in mammary epithelial cells leads to aberrant actin contraction preventing oncogenic transformation. By reducing actin contraction, it allows for the many benefits of losing E-cadherin to drive the development of ILC. We are currently investigating through which signaling routes the increase in actin contraction prevents growth. Citation Format: Koen Schipper, Micha Nethe, Sjors M. Kas, Julian R. de Ruiter, Anne Paulien Drenth, Eline van der Burg, Ute Boon, Eva Schut, Jos Jonkers. Reducing actin contraction in E-cadherin inactivated mammary epithelial cells leads to the development of invasive lobular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A47.

Haploid genetic screens identify genetic vulnerabilities to microtubule-targeting agents.

The absence of biomarkers to accurately predict anticancer therapy response remains a major obstacle in clinical oncology. We applied a genome‐wide loss‐of‐function screening approach in human haploid cells to characterize genetic vulnerabilities to classical microtubule‐targeting agents. Using docetaxel and vinorelbine, two well‐established chemotherapeutic agents, we sought to identify genetic alterations sensitizing human HAP1 cells to these drugs. Despite the fact that both drugs act on microtubules, a set of distinct genes were identified whose disruption affects drug sensitivity. For docetaxel, this included a number of genes with a function in mitosis, while for vinorelbine we identified inactivation of FBXW7,RB1, and NF2, three frequently mutated tumor suppressor genes, as sensitizing factors. We validated these genes using independent knockout clones and confirmed FBXW7 as an important regulator of the mitotic spindle assembly. Upon FBXW7 depletion, vinorelbine treatment led to decreased survival of cells due to defective mitotic progression and subsequent mitotic catastrophe. We show that haploid insertional mutagenesis screens are a useful tool to study genetic vulnerabilities to classical chemotherapeutic drugs by identifying thus far unknown sensitivity factors. These results provide a rationale for investigating patient response to vinca alkaloid‐based anticancer treatment in relation to the mutational status of these three tumor suppressor genes, and could in the future lead to the establishment of novel predictive biomarkers or suggest new drug combinations based on molecular mechanisms of drug sensitivity.

In vivo insertion pool sequencing identifies virulence factors in a complex fungal-host interaction.

Large-scale insertional mutagenesis screens can be powerful genome-wide tools if they are streamlined with efficient downstream analysis, which is a serious bottleneck in complex biological systems. A major impediment to the success of next-generation sequencing (NGS)-based screens for virulence factors is that the genetic material of pathogens is often underrepresented within the eukaryotic host, making detection extremely challenging. We therefore established insertion Pool-Sequencing (iPool-Seq) on maize infected with the biotrophic fungus U. maydis. iPool-Seq features tagmentation, unique molecular barcodes, and affinity purification of pathogen insertion mutant DNA from in vivo-infected tissues. In a proof of concept using iPool-Seq, we identified 28 virulence factors, including 23 that were previously uncharacterized, from an initial pool of 195 candidate effector mutants. Because of its sensitivity and quantitative nature, iPool-Seq can be applied to any insertional mutagenesis library and is especially suitable for genetically complex setups like pooled infections of eukaryotic hosts.

Replication-incompetent gammaretroviral and lentiviral vector-based insertional mutagenesis screens identify prostate cancer progression genes.

Replication-incompetent gammaretroviral (γRV) and lentiviral (LV) vectors have both been used in insertional mutagenesis screens to identify cancer drivers. In this approach the vectors stably integrate in the host cell genome and induce cancers by dysregulating nearby genes. The cells that contain a retroviral vector provirus in or near a proto-oncogene or tumor suppressor are preferentially enriched in a tumor. γRV and LV vectors have different integration profiles and genotoxic potential, making them potentially complementary tools for insertional mutagenesis screens. We performed screens using both γRV and LV vectors to identify driver genes that mediate progression of androgen-independent prostate cancer (AIPC) using a xenotransplant mouse model. Vector transduced LNCaP cells were injected orthotopically into the prostate gland of immunodeficient mice. Mice that developed tumors were castrated to create an androgen-deficient environment and metastatic tumors that developed were analyzed. A high-throughput modified genomic sequencing PCR (MGS-PCR) approach identified the positions of vector integrations in these metastatic tumors. OR2A14, FER1L6, TAOK3, MAN1A2, MBNL2, SERBP1, PLEKHA2, SPTAN1, ADAMTS1, SLC30A5, ABCC1, SLC7A1 and SLC25A24 were identified as candidate prostate cancer (PC) progression genes. TAOK3 and ABCC1 expression in PC patients predicted the risk of recurrence after androgen deprivation therapy. Our data shows that γRV and LV vectors are complementary approaches to identify cancer driver genes which may be promising potential biomarkers and therapeutic targets.

BRE/BRCC45 regulates CDC25A stability by recruiting USP7 in response to DNA damage

BRCA2 is essential for maintaining genomic integrity. BRCA2-deficient primary cells are either not viable or exhibit severe proliferation defects. Yet, BRCA2 deficiency contributes to tumorigenesis. It is believed that mutations in genes such as TRP53 allow BRCA2 heterozygous cells to overcome growth arrest when they undergo loss of heterozygosity. Here, we report the use of an insertional mutagenesis screen to identify a role for BRE (Brain and Reproductive organ Expressed, also known as BRCC45), known to be a part of the BRCA1-DNA damage sensing complex, in the survival of BRCA2-deficient mouse ES cells. Cell viability by BRE overexpression is mediated by deregulation of CDC25A phosphatase, a key cell cycle regulator and an oncogene. We show that BRE facilitates deubiquitylation of CDC25A by recruiting ubiquitin-specific-processing protease 7 (USP7) in the presence of DNA damage. Additionally, we uncovered the role of CDC25A in BRCA-mediated tumorigenesis, which can have implications in cancer treatment.

Insertional mutagenesis in a HER2-positive breast cancer model reveals ERAS as a driver of cancer and therapy resistance

Personalized medicine for cancer patients requires a deep understanding of the underlying genetics that drive cancer and the subsequent identification of predictive biomarkers. To discover new genes and pathways contributing to oncogenesis and therapy resistance in HER2+ breast cancer, we performed Mouse Mammary Tumor Virus (MMTV)-induced insertional mutagenesis screens in ErbB2/cNeu-transgenic mouse models. The screens revealed 34 common integration sites (CIS) in mammary tumors of MMTV-infected mice, highlighting loci with multiple independent MMTV integrations in which potential oncogenes are activated, most of which had never been reported as MMTV CIS. The CIS most strongly associated with the ErbB2-transgenic genotype was the locus containing Eras (ES cell-expressed Ras), a constitutively active RAS-family GTPase. We show that upon expression, Eras acts as a potent oncogenic driver through hyperactivation of the PI3K/AKT pathway, in contrast to other RAS proteins that signal primarily via the MAPK/ERK pathway and require upstream activation or activating mutations to induce signaling. We additionally show that ERAS synergistically enhances HER2-induced tumorigenesis and, in this role, can functionally replace ERBB3/HER3 by acting as a more powerful activator of PI3K/AKT signaling. Although previously reported as pseudogene in humans, we observed ERAS RNA and protein expression in a substantial subset of human primary breast carcinomas. Importantly, we show that ERAS induces primary resistance to the widely used HER2-targeting drugs Trastuzumab (Herceptin) and Lapatinib (Tykerb/Tyverb) in vivo, and is involved in acquired resistance via selective upregulation during treatment in vitro, indicating that ERAS may serve as a novel clinical biomarker for PI3K/AKT pathway hyperactivation and HER2-targeted therapy resistance.

Phenotypic Lentivirus Screens to Identify Antiviral Single Domain Antibodies.

Our understanding of infection biology is based on experiments in which pathogen or host proteins are perturbed by small compound inhibitors, mutation, or depletion. This approach has been remarkably successful, as, for example, demonstrated by the independent identification of the endosomal membrane protein Niemann-Pick C1 as an essential factor for Ebola virus infection in both small compound and insertional mutagenesis screens (Côté, Nature 477:344-348, 2011; Carette et al., Nature 477:340-343, 2011). However, many aspects of host-pathogen interactions are poorly understood because we cannot target all of the involved molecules with small molecules, or because we cannot deplete essential proteins. Single domain antibody fragments expressed in the cytosol or other organelles constitute a versatile alternative to perturb the function of any given protein by masking protein-protein interaction interfaces, by stabilizing distinct conformations, or by directly interfering with enzymatic activities. The variable domains of heavy chain-only antibodies (VHHs) from camelid species can be cloned from blood samples of animals immunized with the desired target molecules. We can thus exploit the ability of the camelid immune system to generate affinity-matured single domain antibody fragments to obtain highly specific tools. Interesting VHH candidates are typically identified based on their affinity toward immobilized antigens using techniques such as phage display.The phenotypical screening approach described here allows the direct identification of VHHs that prevent infection of cells with influenza A virus (IAV) or other pathogens. The VHH repertoire is cloned into a lentiviral vector, which is used to generate pseudo-typed lentivirus particles. Target cells are transduced with the lentivirus, so that every cell inducibly expresses a different VHH. This cell collection is then challenged with a lethal dose of virus. Only the cells which express a VHH that prevents infection by targeting virus proteins or host cell components essential for infection will survive. We can thus identify critical target molecules including vulnerable epitopes and conformations, render target molecules accessible to informative perturbation studies, and stabilize intermediates of virus entry for detailed analysis.

The Wnt Signaling Landscape of Mammary Stem Cells and Breast Tumors.

Attention has been focused on Wnt signaling in the mouse mammary gland for several decades, firstly by the discovery of several Wnt loci among the oncogenes revealed by MMTV-based insertional mutagenesis screening of mouse mammary gland, and then by the remarkable visualization of Wnt-dependent specification of mammary placodes in embryonic skin. This review aims to summarize the impact of recent data for our understanding of the roles of Wnt signaling in these roles. The amount and identity of both familiar and novel Wnt signaling components is examined for mouse mammary epithelial cells. The hierarchical arrangement of mammary epithelial cell progenitors and stem cells inferred from the study of isolated cells is reinterpreted in an era that has demonstrated almost limitless cellular plasticity. Functional definitions of stem and progenitor activities are reevaluated with the discovery of novel stem cell activities and regulators, and we draw parallels with the arrangement of replication-competent cells in other tissues. Although Wnt signaling is highly oncogenic for mouse mammary epithelia, the data supporting Wnt signaling as a tumor driver for human breast cancer are still flimsy, and there is little support for the recruitment of normal Wnt-dependent breast stem cells as tumor precursor cells for either mouse or human. We discuss possible explanations for this paradox and questions still unanswered, including the potential impact of recent discoveries of Wnt-secreting microenvironments, oncogenic changes in the Rspo/Lgr/Ubiquitin ligase amplifier complex, as they could apply to breast tissues, and the feedback suppression of Wnt signaling that characterizes its developmental activity and may hide Wnt signatures in tumors.

RUNX1 is required for oncogenic Myb and Myc enhancer activity in T-cell acute lymphoblastic leukemia

AbstractThe gene encoding the RUNX1 transcription factor is mutated in a subset of T-cell acute lymphoblastic leukemia (T-ALL) patients, and RUNX1 mutations are associated with a poor prognosis. These mutations cluster in the DNA-binding Runt domain and are thought to represent loss-of-function mutations, indicating that RUNX1 suppresses T-cell transformation. RUNX1 has been proposed to have tumor suppressor roles in T-cell leukemia homeobox 1/3–transformed human T-ALL cell lines and NOTCH1 T-ALL mouse models. Yet, retroviral insertional mutagenesis screens identify RUNX genes as collaborating oncogenes in MYC-driven leukemia mouse models. To elucidate RUNX1 function(s) in leukemogenesis, we generated Tal1/Lmo2/Rosa26-CreERT2Runx1f/f mice and examined leukemia progression in the presence of vehicle or tamoxifen. We found that Runx1 deletion inhibits mouse leukemic growth in vivo and that RUNX silencing in human T-ALL cells triggers apoptosis. We demonstrate that a small molecule inhibitor, designed to interfere with CBFβ binding to RUNX proteins, impairs the growth of human T-ALL cell lines and primary patient samples. We demonstrate that a RUNX1 deficiency alters the expression of a crucial subset of TAL1- and NOTCH1-regulated genes, including the MYB and MYC oncogenes, respectively. These studies provide genetic and pharmacologic evidence that RUNX1 has oncogenic roles and reveal RUNX1 as a novel therapeutic target in T-ALL.

Survival of BRCA2-Deficient Cells Is Promoted by GIPC3, a Novel Genetic Interactor of BRCA2.

BRCA2 loss-of-heterozygosity (LOH) is frequently observed in BRCA2-mutated tumors, but its biallelic loss causes embryonic lethality in mice and inhibits proliferation of normal somatic cells. Therefore, it remains unclear how loss of BRCA2 contributes to tumorigenesis. One possibility is that mutation in potential genetic interactors of BRCA2, such as TRP53, is required for cell survival/proliferation in the absence of BRCA2. In this study, using an insertional mutagenesis screen in mouse embryonic stem cells (mESC), we have identified GIPC3 (GAIP-interacting protein C-terminus 3) as a BRCA2 genetic interactor that contributes to survival of Brca2-null mESC. GIPC3 does not compensate for BRCA2 loss in the repair of double-strand breaks. Mass-spectrometric analysis resulted in the identification of G-protein signaling transducers, APPL1 and APPL2, as potential GIPC3-binding proteins. A mutant GIPC3 (His155Ala) that does not bind to APPL1/2 failed to rescue the lethality of Brca2-null mESC, suggesting that the cell viability by GIPC3 is mediated via APPL1/2. Finally, the physiological significance of GIPC3 as a genetic interactor of BRCA2 is supported by the observation that Brca2-null embryos with Gipc3 overexpression are developmentally more advanced than their control littermates. Taken together, we have uncovered a novel role for GIPC3 as a BRCA2 genetic interactor.

Insertional mutagenesis identifies drivers of a novel oncogenic pathway in invasive lobular breast carcinoma.

Invasive lobular carcinoma (ILC) is the second most common breast cancer subtype and accounts for 8-14% of all cases. Although the majority of human ILCs are characterized by the functional loss of E-cadherin (encoded by CDH1), inactivation of Cdh1 does not predispose mice to develop mammary tumors, implying that mutations in additional genes are required for ILC formation in mice. To identify these genes, we performed an insertional mutagenesis screen using the Sleeping Beauty transposon system in mice with mammary-specific inactivation of Cdh1. These mice developed multiple independent mammary tumors of which the majority resembled human ILC in terms of morphology and gene expression. Recurrent and mutually exclusive transposon insertions were identified in Myh9, Ppp1r12a, Ppp1r12b and Trp53bp2, whose products have been implicated in the regulation of the actin cytoskeleton. Notably, MYH9, PPP1R12B and TP53BP2 were also frequently aberrated in human ILC, highlighting these genes as drivers of a novel oncogenic pathway underlying ILC development.

Identification and characterization of a solute carrier, CIA8, involved in inorganic carbon acclimation in Chlamydomonas reinhardtii.

The supply of inorganic carbon (Ci) at the site of fixation by Rubisco is a key parameter for efficient CO2 fixation in aquatic organisms including the green alga, Chlamydomonas reinhardtii. Chlamydomonas reinhardtii cells, when grown on limiting CO2, have a CO2-concentrating mechanism (CCM) that functions to concentrate CO2 at the site of Rubisco. Proteins thought to be involved in inorganic carbon uptake have been identified and localized to the plasma membrane or chloroplast envelope. However, current CCM models suggest that additional molecular components are involved in Ci uptake. In this study, the gene Cia8 was identified in an insertional mutagenesis screen and characterized. The protein encoded by Cia8 belongs to the sodium bile acid symporter subfamily. Transcript levels for this gene were significantly up-regulated when the cells were grown on low CO2. The cia8 mutant exhibited reduced growth and reduced affinity for Ci when grown in limiting CO2 conditions. Prediction programs localize this protein to the chloroplast. Ci uptake and the photosynthetic rate, particularly at high external pH, were reduced in the mutant. The results are consistent with the model that CIA8 is involved in Ci uptake in C. reinhardtii.

Monkeypox Virus Host Factor Screen Using Haploid Cells Identifies Essential Role of GARP Complex in Extracellular Virus Formation.

Monkeypox virus (MPXV) is a human pathogen that is a member of the Orthopoxvirus genus, which includes Vaccinia virus and Variola virus (the causative agent of smallpox). Human monkeypox is considered an emerging zoonotic infectious disease. To identify host factors required for MPXV infection, we performed a genome-wide insertional mutagenesis screen in human haploid cells. The screen revealed several candidate genes, including those involved in Golgi trafficking, glycosaminoglycan biosynthesis, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. We validated the role of a set of vacuolar protein sorting (VPS) genes during infection, VPS51 to VPS54 (VPS51-54), which comprise the Golgi-associated retrograde protein (GARP) complex. The GARP complex is a tethering complex involved in retrograde transport of endosomes to the trans-Golgi apparatus. Our data demonstrate that VPS52 and VPS54 were dispensable for mature virion (MV) production but were required for extracellular virus (EV) formation. For comparison, a known antiviral compound, ST-246, was used in our experiments, demonstrating that EV titers in VPS52 and VPS54 knockout (KO) cells were comparable to levels exhibited by ST-246-treated wild-type cells. Confocal microscopy was used to examine actin tail formation, one of the viral egress mechanisms for cell-to-cell dissemination, and revealed an absence of actin tails in VPS52KO- or VPS54KO-infected cells. Further evaluation of these cells by electron microscopy demonstrated a decrease in levels of wrapped viruses (WVs) compared to those seen with the wild-type control. Collectively, our data demonstrate the role of GARP complex genes in double-membrane wrapping of MVs necessary for EV formation, implicating the host endosomal trafficking pathway in orthopoxvirus infection.IMPORTANCE Human monkeypox is an emerging zoonotic infectious disease caused by Monkeypox virus (MPXV). Of the two MPXV clades, the Congo Basin strain is associated with severe disease, increased mortality, and increased human-to-human transmission relative to the West African strain. Monkeypox is endemic in regions of western and central Africa but was introduced into the United States in 2003 from the importation of infected animals. The threat of MPXV and other orthopoxviruses is increasing due to the absence of routine smallpox vaccination leading to a higher proportion of naive populations. In this study, we have identified and validated candidate genes that are required for MPXV infection, specifically, those associated with the Golgi-associated retrograde protein (GARP) complex. Identifying host targets required for infection that prevents extracellular virus formation such as the GARP complex or the retrograde pathway can provide a potential target for antiviral therapy.