Publications by authors named "Neal G Copeland"

162 Publications

Identification of cancer driver genes using Sleeping Beauty transposon mutagenesis.

Cancer Sci 2021 Mar 30. Epub 2021 Mar 30.

Genetics Department, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Cancer genome sequencing studies have identified driver genes for a variety of different cancers and helped to understand the genetic landscape of human cancer. It is still challenging, however, to identify cancer driver genes with confidence simply from genetic data alone. In vivo forward genetic screens using Sleeping Beauty (SB) transposon mutagenesis provides another powerful genetic tool for identifying candidate cancer driver genes in wild-type and sensitized mouse tumors. By comparing cancer driver genes identified in human and mouse tumors, cancer driver genes can be identified with additional confidence based upon comparative oncogenomics. This review describes how SB mutagenesis works in mice and focuses on studies that have identified cancer driver genes in the mouse gastrointestinal tract.
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http://dx.doi.org/10.1111/cas.14901DOI Listing
March 2021

Ubiquitin specific peptidase 32 acts as an oncogene in epithelial ovarian cancer by deubiquitylating farnesyl-diphosphate farnesyltransferase 1.

Biochem Biophys Res Commun 2021 May 18;552:120-127. Epub 2021 Mar 18.

Department of Obstetrics and Gynecology, Osaka University, Graduate School of Medicine, Japan.

Epithelial ovarian cancer (EOC) is the seventh most common cancer worldwide and the deadliest gynecological malignancy because of its aggressiveness and high recurrence rate. To discover new therapeutic targets for EOC, we combined public EOC microarray datasets with our previous in vivo shRNA screening dataset. The top-ranked gene ubiquitin specific peptidase 32 (USP32), coding a deubiquitinating enzyme, is a component of the ubiquitin proteasome system. Clinically, USP32 is expressed in primary ovarian cancer, especially in metastatic peritoneal tumors, and negatively impacts the survival outcome. USP32 regulates proliferative and epithelial mesenchymal transition capacities that are associated with EOC progression. Proteomic analysis identified farnesyl-diphosphate farnesyltransferase 1 (FDFT1) as a novel substrate of USP32 that is an enzyme in the mevalonate pathway, essentially associated with cell proliferation and stemness. USP32 and FDFT1 expression was higher in tumor spheres than in adherent cells. Inhibition of USP32, FDFT1, or mevalonate pathway considerably suppressed tumor sphere formation, which was restored by adding squalene, a downstream product of FDFT1. These findings suggested that USP32-FDFT1 axis contributes to EOC progression, and could be novel therapeutic targets for EOC treatment.
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http://dx.doi.org/10.1016/j.bbrc.2021.03.049DOI Listing
May 2021

Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation.

Cancers (Basel) 2021 Jan 9;13(2). Epub 2021 Jan 9.

Department of Molecular Oncology, Moffitt Cancer Center & Research Institute, Tampa, FL 33612, USA.

A central challenge in cancer genomics is the systematic identification of single and cooperating tumor suppressor gene mutations driving cellular transformation and tumor progression in the absence of oncogenic driver mutation(s). Multiple in vitro and in vivo gene inactivation screens have enhanced our understanding of the tumor suppressor gene landscape in various cancers. However, these studies are limited to single or combination gene effects, specific organs, or require sensitizing mutations. In this study, we developed and utilized a transposon mutagenesis system that functions only as a gene trap to exclusively inactivate tumor suppressor genes. Using whole body transposon mobilization in wild type mice, we observed that cumulative gene inactivation can drive tumorigenesis of solid cancers. We provide a quantitative landscape of the tumor suppressor genes inactivated in these cancers and show that, despite the absence of oncogenic drivers, these genes converge on key biological pathways and processes associated with cancer hallmarks.
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http://dx.doi.org/10.3390/cancers13020225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827284PMC
January 2021

is a critical regulator of adult long-term hematopoietic stem cell quiescence.

Proc Natl Acad Sci U S A 2020 12 2;117(50):31945-31953. Epub 2020 Dec 2.

Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814;

Regulation of quiescence is critical for the maintenance of adult hematopoietic stem cells (HSCs). Disruption of transcription factor gene during mouse embryonic development has been shown to cause a severe loss of fetal liver HSCs; however, the underlying mechanisms and the function of in adult HSCs remain unclear. To investigate the role of in adult HSCs, we generated a novel conditional knockout mouse model and deleted in adult mouse hematopoietic system using the IFN-inducible Our results show that deletion in the adult mouse hematopoietic system has a less severe effect on HSCs, causing a gradual decline of adult HSC numbers and a concomitant increase in the multipotent progenitor (MPP) compartment. deletion in the hematopoietic system following transplantation produced the same phenotype, indicating that the defect is intrinsic to adult HSCs. This HSC loss was also exacerbated by stress induced by 5-fluorouracil injections. Annexin V staining showed no difference in apoptosis between wild-type and knockout adult HSCs. In contrast, Bromodeoxyuridine analysis revealed that loss of significantly increased cycling of long-term HSCs (LT-HSCs) with the majority of the cells found in the S to G2/M phase. Consistently, RNA sequencing analysis of mouse LT-HSCs with and without deletion showed that loss induced a significant decrease in the expression of several known cell cycle regulators of HSCs, among which and were identified as direct targets of Our results suggest that preserves the function of adult LT-HSCs by promoting their quiescence.
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http://dx.doi.org/10.1073/pnas.2017626117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749346PMC
December 2020

MRTFB suppresses colorectal cancer development through regulating SPDL1 and MCAM.

Proc Natl Acad Sci U S A 2019 11 5;116(47):23625-23635. Epub 2019 Nov 5.

Houston Methodist Research Institute, Houston Methodist Hospital, Houston, TX 77030;

Myocardin-related transcription factor B (MRTFB) is a candidate tumor-suppressor gene identified in transposon mutagenesis screens of the intestine, liver, and pancreas. Using a combination of cell-based assays, in vivo tumor xenograft assays, and Mrtfb knockout mice, we demonstrate here that MRTFB is a human and mouse colorectal cancer (CRC) tumor suppressor that functions in part by inhibiting cell invasion and migration. To identify possible MRTFB transcriptional targets, we performed whole transcriptome RNA sequencing in MRTFB siRNA knockdown primary human colon cells and identified 15 differentially expressed genes. Among the top candidate tumor-suppressor targets were melanoma cell adhesion molecule (MCAM), a known tumor suppressor, and spindle apparatus coiled-coil protein 1 (SPDL1), which has no confirmed role in cancer. To determine whether these genes play a role in CRC, we knocked down the expression of MCAM and SPDL1 in human CRC cells and showed significantly increased invasion and migration of tumor cells. We also showed that Spdl1 expression is significantly down-regulated in Mrtfb knockout mouse intestine, while lower SPDL1 expression levels are significantly associated with reduced survival in CRC patients. Finally, we show that depletion of MCAM and SPDL1 in human CRC cells significantly increases tumor development in xenograft assays, further confirming their tumor-suppressive roles in CRC. Collectively, our findings demonstrate the tumor-suppressive role of MRTFB in CRC and identify several genes, including 2 tumor suppressors, that act downstream of MRTFB to regulate tumor growth and survival in CRC patients.
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http://dx.doi.org/10.1073/pnas.1910413116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876235PMC
November 2019

CRISPR-Cas9-mediated gene knockout in intestinal tumor organoids provides functional validation for colorectal cancer driver genes.

Proc Natl Acad Sci U S A 2019 07 12;116(31):15635-15644. Epub 2019 Jul 12.

Division of Genetics, Cancer Research Institute, Kanazawa University, 920-1192 Kanazawa, Ishikawa, Japan.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Several genome sequencing studies have provided comprehensive CRC genomic datasets. Likewise, in our previous study, we performed genome-wide transposon-based mutagenesis screening in mice and provided comprehensive datasets of candidate CRC driver genes. However, functional validation for most candidate CRC driver genes, which were commonly identified from both human and mice, has not been performed. Here, we describe a platform for functionally validating CRC driver genes that utilizes CRISPR-Cas9 in mouse intestinal tumor organoids and human CRC-derived organoids in xenograft mouse models. We used genetically defined benign tumor-derived organoids carrying 2 frequent gene mutations ( and mutations), which act in the early stage of CRC development, so that we could clearly evaluate the tumorigenic ability of the mutation in a single gene. These studies showed that , , and could function as tumor suppressor genes (TSGs) in CRC and uncovered a role for in tumor metastasis. We also showed that co-occurrent mutations in receptors for activin and transforming growth factor-β (TGF-β) synergistically promote tumorigenesis, and shed light on the role of activin receptors in CRC. This experimental system can also be applied to mouse intestinal organoids carrying other sensitizing mutations as well as organoids derived from other organs, which could further contribute to identification of novel cancer driver genes and new drug targets.
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http://dx.doi.org/10.1073/pnas.1904714116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681705PMC
July 2019

Author Correction: A recellularized human colon model identifies cancer driver genes.

Nat Biotechnol 2019 Jul;37(7):820

Department of Biomedical Engineering, Cornell University, Ithaca, New York, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41587-019-0163-6DOI Listing
July 2019

The Roles of Initiating Truncal Mutations in Human Cancers: The Order of Mutations and Tumor Cell Type Matters.

Cancer Cell 2019 01;35(1):10-15

Genetics Department, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

We propose that initiating truncal mutations plays a special role in tumor formation by both enhancing the survival of the initiating cancer cell and by selecting for secondary mutations that contribute to tumor progression, and that these mutations often act in a tissue-preferred fashion. Here, we explain why inherited mutations often have different tissue specificities compared with spontaneous mutations in the same gene. Initiating truncal mutations make excellent neo-antigens for immunotherapy, and understanding why one mutation selects for a second mutation in a particular tissue type could one day aid in the design of gene-targeted combination therapies.
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http://dx.doi.org/10.1016/j.ccell.2018.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6376970PMC
January 2019

Molecular profiling of nonalcoholic fatty liver disease-associated hepatocellular carcinoma using SB transposon mutagenesis.

Proc Natl Acad Sci U S A 2018 10 16;115(44):E10417-E10426. Epub 2018 Oct 16.

Genetics Department, The University of Texas MD Anderson Cancer Center, Houston, TX 77030

Nonalcoholic fatty liver disease (NAFLD) is the fastest rising cause of hepatocellular carcinoma (HCC) in Western countries; however, the molecular mechanisms that cause NAFLD-HCC remain elusive. To identify molecular drivers of NAFLD-HCC, we performed Sleeping Beauty (SB) transposon mutagenesis screens in liver-specific Pten knockout and in high-fat diet-fed mice, which are murine models of NAFLD-HCC. SB mutagenesis accelerated liver tumor formation in both models and identified 588 and 376 candidate cancer genes (CCGs), respectively; 257 CCGs were common to both screens and were enriched in signaling pathways known to be important for human HCC. Comparison of these CCGs with those identified in a previous SB screen of hepatitis B virus-induced HCC identified a core set of 141 CCGs that were mutated in all screens. Forty-one CCGs appeared specific for NAFLD-HCC, including Sav1, a component of the Hippo signaling pathway and the most frequently mutated gene identified in both NAFLD-HCC screens. Liver-specific deletion of Sav1 was found to promote hepatic lipid accumulation, apoptosis, and fibrogenesis, leading to the acceleration of hepatocarcinogenesis in liver-specific Pten mutant mice. Sav1/Pten double-mutant livers also showed a striking up-regulation of markers of liver progenitor cells (LPCs), along with synergistic activation of Yap, which is a major downstream effector of Hippo signaling. Lastly, Yap activation, in combination with Pten inactivation, was found to accelerate cell growth and sphere formation of LPCs in vitro and induce their malignant transformation in allografts. Our forward genetic screens in mice have thus identified pathways and genes driving the development of NAFLD-HCC.
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http://dx.doi.org/10.1073/pnas.1808968115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217425PMC
October 2018

Creation of a myosin Va-TAP-tagged mouse and identification of potential myosin Va-interacting proteins in the cerebellum.

Cytoskeleton (Hoboken) 2018 09;75(9):395-409

Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland.

The actin-based motor myosin Va transports numerous cargos, including the smooth endoplasmic reticulum (SER) in cerebellar Purkinje neurons (PNs) and melanosomes in melanocytes. Identifying proteins that interact with this myosin is key to understanding its cellular functions. Toward that end, we used recombineering to insert via homologous recombination a tandem affinity purification (TAP) tag composed of the immunoglobulin G-binding domain of protein A, a tobacco etch virus cleavage site, and a FLAG tag into the mouse MYO5A locus immediately after the initiation codon. Importantly, we provide evidence that the TAP-tagged version of myosin Va (TAP-MyoVa) functions normally in terms of SER transport in PNs and melanosome positioning in melanocytes. Given this and other evidence that TAP-MyoVa is fully functional, we purified it together with associated proteins directly from juvenile mouse cerebella and subjected the samples to mass spectroscopic analyses. As expected, known myosin Va-binding partners like dynein light chain were identified. Importantly, numerous novel interacting proteins were also tentatively identified, including guanine nucleotide-binding protein G(o) subunit alpha (Gnao1), a biomarker for schizophrenia. Consistently, an antibody to Gnao1 immunoprecipitates myosin Va, and Gnao1's localization to PN dendritic spines depends on myosin Va. The mouse model created here should facilitate the identification of novel myosin Va-binding partners, which in turn should advance our understanding of the roles played by this important myosin in vivo.
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http://dx.doi.org/10.1002/cm.21474DOI Listing
September 2018

SB Driver Analysis: a Sleeping Beauty cancer driver analysis framework for identifying and prioritizing experimentally actionable oncogenes and tumor suppressors.

Nucleic Acids Res 2018 09;46(16):e94

Department of Molecular Oncology, Moffitt Cancer Center, Tampa, FL, USA.

Cancer driver prioritization for functional analysis of potential actionable therapeutic targets is a significant challenge. Meta-analyses of mutated genes across different human cancer types for driver prioritization has reaffirmed the role of major players in cancer, including KRAS, TP53 and EGFR, but has had limited success in prioritizing genes with non-recurrent mutations in specific cancer types. Sleeping Beauty (SB) insertional mutagenesis is a powerful experimental gene discovery framework to define driver genes in mouse models of human cancers. Meta-analyses of SB datasets across multiple tumor types is a potentially informative approach to prioritize drivers, and complements efforts in human cancers. Here, we report the development of SB Driver Analysis, an in-silico method for defining cancer driver genes that positively contribute to tumor initiation and progression from population-level SB insertion data sets. We demonstrate that SB Driver Analysis computationally prioritizes drivers and defines distinct driver classes from end-stage tumors that predict their putative functions during tumorigenesis. SB Driver Analysis greatly enhances our ability to analyze, interpret and prioritize drivers from SB cancer datasets and will continue to substantially increase our understanding of the genetic basis of cancer.
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http://dx.doi.org/10.1093/nar/gky450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6144815PMC
September 2018

Stability and Function of Hippocampal Mossy Fiber Synapses Depend on .

Front Mol Neurosci 2018 5;11:103. Epub 2018 Apr 5.

Institute of Molecular and Cellular Anatomy, Ulm University, Ulm, Germany.

Structural and functional plasticity of synapses are critical neuronal mechanisms underlying learning and memory. While activity-dependent regulation of synaptic strength has been extensively studied, much less is known about the transcriptional control of synapse maintenance and plasticity. Hippocampal mossy fiber (MF) synapses connect dentate granule cells to CA3 pyramidal neurons and are important for spatial memory formation and consolidation. The transcription factor is expressed in dentate granule cells and required for postnatal hippocampal development. Ablation of in the adult hippocampus results in impaired adult neurogenesis and spatial memory. The molecular mechanisms underlying the behavioral impairment remained unclear. Here we show that selective deletion of in the adult mouse hippocampus leads to a rapid loss of excitatory synapses in CA3 as well as reduced ultrastructural complexity of remaining mossy fiber boutons (MFBs). Moreover, a dramatic decline of long-term potentiation (LTP) of the dentate gyrus-CA3 (DG-CA3) projection is caused by adult loss of . Differential transcriptomics revealed the deregulation of genes associated with synaptic transmission in mutants. Together, our data suggest to regulate maintenance and function of MF synapses in the adult hippocampus.
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http://dx.doi.org/10.3389/fnmol.2018.00103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5895709PMC
April 2018

p120-Catenin is an obligate haploinsufficient tumor suppressor in intestinal neoplasia.

J Clin Invest 2017 12 13;127(12):4462-4476. Epub 2017 Nov 13.

Department of Cancer Biology, and.

p120-Catenin (p120) functions as a tumor suppressor in intestinal cancer, but the mechanism is unclear. Here, using conditional p120 knockout in Apc-sensitized mouse models of intestinal cancer, we have identified p120 as an "obligatory" haploinsufficient tumor suppressor. Whereas monoallelic loss of p120 was associated with a significant increase in tumor multiplicity, loss of both alleles was never observed in tumors from these mice. Moreover, forced ablation of the second allele did not further enhance tumorigenesis, but instead induced synthetic lethality in combination with Apc loss of heterozygosity. In tumor-derived organoid cultures, elimination of both p120 alleles resulted in caspase-3-dependent apoptosis that was blocked by inhibition of Rho kinase (ROCK). With ROCK inhibition, however, p120-ablated organoids exhibited a branching phenotype and a substantial increase in cell proliferation. Access to data from Sleeping Beauty mutagenesis screens afforded an opportunity to directly assess the tumorigenic impact of p120 haploinsufficiency relative to other candidate drivers. Remarkably, p120 ranked third among the 919 drivers identified. Cofactors α-catenin and epithelial cadherin (E-cadherin) were also among the highest scoring candidates, indicating a mechanism at the level of the intact complex that may play an important role at very early stages of of intestinal tumorigenesis while simultaneously restricting outright loss via synthetic lethality.
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http://dx.doi.org/10.1172/JCI77217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5707165PMC
December 2017

SBCDDB: Sleeping Beauty Cancer Driver Database for gene discovery in mouse models of human cancers.

Nucleic Acids Res 2018 01;46(D1):D1011-D1017

Cancer Research Program, Houston Methodist Research Institute, Houston, Texas, USA.

Large-scale oncogenomic studies have identified few frequently mutated cancer drivers and hundreds of infrequently mutated drivers. Defining the biological context for rare driving events is fundamentally important to increasing our understanding of the druggable pathways in cancer. Sleeping Beauty (SB) insertional mutagenesis is a powerful gene discovery tool used to model human cancers in mice. Our lab and others have published a number of studies that identify cancer drivers from these models using various statistical and computational approaches. Here, we have integrated SB data from primary tumor models into an analysis and reporting framework, the Sleeping Beauty Cancer Driver DataBase (SBCDDB, http://sbcddb.moffitt.org), which identifies drivers in individual tumors or tumor populations. Unique to this effort, the SBCDDB utilizes a single, scalable, statistical analysis method that enables data to be grouped by different biological properties. This allows for SB drivers to be evaluated (and re-evaluated) under different contexts. The SBCDDB provides visual representations highlighting the spatial attributes of transposon mutagenesis and couples this functionality with analysis of gene sets, enabling users to interrogate relationships between drivers. The SBCDDB is a powerful resource for comparative oncogenomic analyses with human cancer genomics datasets for driver prioritization.
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http://dx.doi.org/10.1093/nar/gkx956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753260PMC
January 2018

In vivo loss-of-function screens identify KPNB1 as a new druggable oncogene in epithelial ovarian cancer.

Proc Natl Acad Sci U S A 2017 08 15;114(35):E7301-E7310. Epub 2017 Aug 15.

Cancer Research Program, Houston Methodist Research Institute, Houston, TX 77030;

Epithelial ovarian cancer (EOC) is a deadly cancer, and its prognosis has not been changed significantly during several decades. To seek new therapeutic targets for EOC, we performed an in vivo dropout screen in human tumor xenografts using a pooled shRNA library targeting thousands of druggable genes. Then, in follow-up studies, we performed a second screen using a genome-wide CRISPR/Cas9 library. These screens identified 10 high-confidence drug targets that included well-known oncogenes such as ERBB2 and RAF1, and novel oncogenes, notably KPNB1, which we investigated further. Genetic and pharmacological inhibition showed that KPNB1 exerts its antitumor effects through multiphase cell cycle arrest and apoptosis induction. Mechanistically, proteomic studies revealed that KPNB1 acts as a master regulator of cell cycle-related proteins, including p21, p27, and APC/C. Clinically, EOC patients with higher expression levels of KPNB1 showed earlier recurrence and worse prognosis than those with lower expression levels of KPNB1. Interestingly, ivermectin, a Food and Drug Administration-approved antiparasitic drug, showed KPNB1-dependent antitumor effects on EOC, serving as an alternative therapeutic toward EOC patients through drug repositioning. Last, we found that the combination of ivermectin and paclitaxel produces a stronger antitumor effect on EOC both in vitro and in vivo than either drug alone. Our studies have thus identified a combinatorial therapy for EOC, in addition to a plethora of potential drug targets.
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http://dx.doi.org/10.1073/pnas.1705441114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584430PMC
August 2017

Identification of new tumor suppressor genes in triple-negative breast cancer.

Cancer Res 2017 Jul 19. Epub 2017 Jul 19.

University of Texas MD Anderson Cancer Center

Although genomic sequencing has provided a better understating of the genetic landmarks in triple-negative breast cancer (TNBC), functional validation of candidate cancer genes (CCG) remains unsolved. In this study, we used a transposon mutagenesis strategy based on a two-step Sleeping Beauty (SB) forward genetic screen to identify and validate new tumor suppressors (TS) in this disease. We generated 120 siRNAs targeting 40 SB-identified candidate breast cancer TS genes and used them to downregulate expression of these genes in four human TNBC cell lines. Among CCG whose SB-mediated genetic mutation resulted in increased cellular proliferation in all cell lines tested, the genes ADNP, AP2B1, TOMM70A and ZNF326 showed tumor suppressor (TS) activity in tumor xenograft studies. Subsequent studies showed that ZNF326 regulated expression of multiple EMT and cancer stem cell (CSC) pathway genes. It also modulated expression of TS genes involved in the regulation of migration and cellular invasion and was a direct transcriptional activator of genes that regulate CSC self-renewal. ZNF326 expression associated with TNBC patient survival, with ZNF326 protein levels showing a marked reduction in TNBC. Our validation of several new tumor suppressor genes in TNBC demonstrate the utility of two-step forward genetic screens in mice, and offer an invaluable tool to identify novel candidate therapeutic pathways and targets.
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http://dx.doi.org/10.1158/0008-5472.CAN-17-0785DOI Listing
July 2017

Transposon insertional mutagenesis in mice identifies human breast cancer susceptibility genes and signatures for stratification.

Proc Natl Acad Sci U S A 2017 03 1;114(11):E2215-E2224. Epub 2017 Mar 1.

Institute of Molecular and Cell Biology, Singapore 138673;

Robust prognostic gene signatures and therapeutic targets are difficult to derive from expression profiling because of the significant heterogeneity within breast cancer (BC) subtypes. Here, we performed forward genetic screening in mice using Sleeping Beauty transposon mutagenesis to identify candidate BC driver genes in an unbiased manner, using a stabilized N-terminal truncated β-catenin gene as a sensitizer. We identified 134 mouse susceptibility genes from 129 common insertion sites within 34 mammary tumors. Of these, 126 genes were orthologous to protein-coding genes in the human genome (hereafter, human BC susceptibility genes, hBCSGs), 70% of which are previously reported cancer-associated genes, and ∼16% are known BC suppressor genes. Network analysis revealed a gene hub consisting of E1A binding protein P300 (), CD44 molecule (), neurofibromin () and phosphatase and tensin homolog (), which are linked to a significant number of mutated hBCSGs. From our survival prediction analysis of the expression of human BC genes in 2,333 BC cases, we isolated a six-gene-pair classifier that stratifies BC patients with high confidence into prognostically distinct low-, moderate-, and high-risk subgroups. Furthermore, we proposed prognostic classifiers identifying three basal and three claudin-low tumor subgroups. Intriguingly, our hBCSGs are mostly unrelated to cell cycle/mitosis genes and are distinct from the prognostic signatures currently used for stratifying BC patients. Our findings illustrate the strength and validity of integrating functional mutagenesis screens in mice with human cancer transcriptomic data to identify highly prognostic BC subtyping biomarkers.
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http://dx.doi.org/10.1073/pnas.1701512114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358385PMC
March 2017

Transposon mutagenesis identifies genes that cooperate with mutant Pten in breast cancer progression.

Proc Natl Acad Sci U S A 2016 11 14;113(48):E7749-E7758. Epub 2016 Nov 14.

Cancer Research Program, Houston Methodist Research Institute, Houston, TX 77030;

Triple-negative breast cancer (TNBC) has the worst prognosis of any breast cancer subtype. To better understand the genetic forces driving TNBC, we performed a transposon mutagenesis screen in a phosphatase and tensin homolog (Pten) mutant mice and identified 12 candidate trunk drivers and a much larger number of progression genes. Validation studies identified eight TNBC tumor suppressor genes, including the GATA-like transcriptional repressor TRPS1 Down-regulation of TRPS1 in TNBC cells promoted epithelial-to-mesenchymal transition (EMT) by deregulating multiple EMT pathway genes, in addition to increasing the expression of SERPINE1 and SERPINB2 and the subsequent migration, invasion, and metastasis of tumor cells. Transposon mutagenesis has thus provided a better understanding of the genetic forces driving TNBC and discovered genes with potential clinical importance in TNBC.
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http://dx.doi.org/10.1073/pnas.1613859113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137755PMC
November 2016

KRAS-related proteins in pancreatic cancer.

Pharmacol Ther 2016 12 3;168:29-42. Epub 2016 Sep 3.

Cancer Research Program, Houston Methodist Research Institute, Houston, TX 77030, USA.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease with a high mortality rate. Genetic and biochemical studies have shown that RAS signaling mediated by KRAS plays a pivotal role in disease initiation, progression and drug resistance. RAS signaling affects several cellular processes in PDAC, including cellular proliferation, migration, cellular metabolism and autophagy. 90% of pancreatic cancer patients harbor somatic oncogenic point mutations in KRAS, which lead to constitutive activation of the molecule. Pancreatic cancers lacking KRAS mutations show activation of RAS via upstream signaling through receptor mediated tyrosine kinases, like EGFR, and in a small fraction of patients, oncogenic activation of the downstream B-RAF molecule is detected. RAS-stimulated signaling of RAF/MEK/ERK, PI3K/AKT/mTOR and RalA/B is active in human pancreatic cancers, cancer cell lines and mouse models of PDAC, although activation levels of each signaling arm appear to be variable across different tumors and perhaps within different subclones of single tumors. Recently, several targeted therapies directed towards MEK, ERK, PI3K and mTOR have been assayed in pancreatic cancer cell lines and in mouse models of the disease with promising results for their ability to impede cellular growth or delay tumor formation, and several inhibitors are currently in clinical trials. However, therapy-induced cross activation of RAS effector molecules has elucidated the complexities of targeting RAS signaling. Combinatorial therapies are now being explored as an approach to overcome RAS-induced therapeutic resistance in pancreatic cancer.
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http://dx.doi.org/10.1016/j.pharmthera.2016.09.003DOI Listing
December 2016

Analyzing tumor heterogeneity and driver genes in single myeloid leukemia cells with SBCapSeq.

Nat Biotechnol 2016 09 1;34(9):962-72. Epub 2016 Aug 1.

Cancer Research Program, Houston Methodist Research Institute, Houston, Texas, USA.

A central challenge in oncology is how to kill tumors containing heterogeneous cell populations defined by different combinations of mutated genes. Identifying these mutated genes and understanding how they cooperate requires single-cell analysis, but current single-cell analytic methods, such as PCR-based strategies or whole-exome sequencing, are biased, lack sequencing depth or are cost prohibitive. Transposon-based mutagenesis allows the identification of early cancer drivers, but current sequencing methods have limitations that prevent single-cell analysis. We report a liquid-phase, capture-based sequencing and bioinformatics pipeline, Sleeping Beauty (SB) capture hybridization sequencing (SBCapSeq), that facilitates sequencing of transposon insertion sites from single tumor cells in a SB mouse model of myeloid leukemia (ML). SBCapSeq analysis of just 26 cells from one tumor revealed the tumor's major clonal subpopulations, enabled detection of clonal insertion events not detected by other sequencing methods and led to the identification of dominant subclones, each containing a unique pair of interacting gene drivers along with three to six cooperating cancer genes with SB-driven expression changes.
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http://dx.doi.org/10.1038/nbt.3637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124494PMC
September 2016

A recellularized human colon model identifies cancer driver genes.

Nat Biotechnol 2016 08 11;34(8):845-51. Epub 2016 Jul 11.

Department of Biomedical Engineering, Cornell University, Ithaca, New York, USA.

Refined cancer models are needed to bridge the gaps between cell line, animal and clinical research. Here we describe the engineering of an organotypic colon cancer model by recellularization of a native human matrix that contains cell-populated mucosa and an intact muscularis mucosa layer. This ex vivo system recapitulates the pathophysiological progression from APC-mutant neoplasia to submucosal invasive tumor. We used it to perform a Sleeping Beauty transposon mutagenesis screen to identify genes that cooperate with mutant APC in driving invasive neoplasia. We identified 38 candidate invasion-driver genes, 17 of which, including TCF7L2, TWIST2, MSH2, DCC, EPHB1 and EPHB2 have been previously implicated in colorectal cancer progression. Six invasion-driver genes that have not, to our knowledge, been previously described were validated in vitro using cell proliferation, migration and invasion assays and ex vivo using recellularized human colon. These results demonstrate the utility of our organoid model for studying cancer biology.
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http://dx.doi.org/10.1038/nbt.3586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4980997PMC
August 2016

Transposon mutagenesis identifies genes and cellular processes driving epithelial-mesenchymal transition in hepatocellular carcinoma.

Proc Natl Acad Sci U S A 2016 06 31;113(24):E3384-93. Epub 2016 May 31.

Cancer Research Program, Houston Methodist Research Institute, Houston, TX 77030;

Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), leading to their poor prognosis. The genes driving EMT in HCC are not yet fully understood, however. Here, we show that mobilization of Sleeping Beauty (SB) transposons in immortalized mouse hepatoblasts induces mesenchymal liver tumors on transplantation to nude mice. These tumors show significant down-regulation of epithelial markers, along with up-regulation of mesenchymal markers and EMT-related transcription factors (EMT-TFs). Sequencing of transposon insertion sites from tumors identified 233 candidate cancer genes (CCGs) that were enriched for genes and cellular processes driving EMT. Subsequent trunk driver analysis identified 23 CCGs that are predicted to function early in tumorigenesis and whose mutation or alteration in patients with HCC is correlated with poor patient survival. Validation of the top trunk drivers identified in the screen, including MET (MET proto-oncogene, receptor tyrosine kinase), GRB2-associated binding protein 1 (GAB1), HECT, UBA, and WWE domain containing 1 (HUWE1), lysine-specific demethylase 6A (KDM6A), and protein-tyrosine phosphatase, nonreceptor-type 12 (PTPN12), showed that deregulation of these genes activates an EMT program in human HCC cells that enhances tumor cell migration. Finally, deregulation of these genes in human HCC was found to confer sorafenib resistance through apoptotic tolerance and reduced proliferation, consistent with recent studies showing that EMT contributes to the chemoresistance of tumor cells. Our unique cell-based transposon mutagenesis screen appears to be an excellent resource for discovering genes involved in EMT in human HCC and potentially for identifying new drug targets.
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http://dx.doi.org/10.1073/pnas.1606876113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914200PMC
June 2016

Two-Step Forward Genetic Screen in Mice Identifies Ral GTPase-Activating Proteins as Suppressors of Hepatocellular Carcinoma.

Gastroenterology 2016 08 10;151(2):324-337.e12. Epub 2016 May 10.

Cancer Research Program, Houston Methodist Research Institute, Houston, Texas. Electronic address:

Background & Aims: High-throughput sequencing technologies have identified thousands of infrequently mutated genes in hepatocellular carcinomas (HCCs). However, high intratumor and intertumor heterogeneity, combined with large numbers of passenger mutations, have made it difficult to identify driver mutations that contribute to the development of HCC. We combined transposon mutagenesis with a high-throughput screen of a small-hairpin RNA (shRNA) library to identify genes and pathways that contribute to HCC development.

Methods: Sleeping beauty transposons were mobilized in livers of transgenic mice predisposed to develop hepatocellular adenoma and HCC owing to expression of the hepatitis B virus surface antigen. This whole-genome mutagenesis technique was used to generate an unbiased catalogue of candidate cancer genes (CCGs). Pooled shRNA libraries targeting 250 selected CCGs then were introduced into immortalized mouse liver cells and the cells were monitored for their tumor-forming ability after injection into nude mice.

Results: Transposon-mediated mutagenesis identified 1917 high-confident CCGs and highlighted the importance of Ras signaling in the development of HCC. Subsequent pooled shRNA library screening of 250 selected CCGs validated 27 HCC tumor-suppressor genes. Individual shRNA knockdown of 4 of these genes (Acaa2, Hbs1l, Ralgapa2, and Ubr2) increased the proliferation of multiple human HCC cell lines in culture and accelerated the formation of xenograft tumors in nude mice. The ability of Ralgapa2 to promote HCC cell proliferation and tumor formation required its inhibition of Rala and Ralb. Dual inhibition of Ras signaling via Ral and Raf, using a combination of small-molecule inhibitor RBC8 and sorafenib, reduced the proliferation of HCC cells in culture and completely inhibited their growth as xenograft tumors in nude mice.

Conclusions: In a 2-step forward genetic screen in mice, we identified members of the Ral guanosine triphosphatase-activating protein pathway and other proteins as suppressors of HCC cell proliferation and tumor growth. These proteins might serve as therapeutic targets for liver cancer.
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http://dx.doi.org/10.1053/j.gastro.2016.04.040DOI Listing
August 2016

Sleeping Beauty transposon mutagenesis identifies genes that cooperate with mutant Smad4 in gastric cancer development.

Proc Natl Acad Sci U S A 2016 Apr 22;113(14):E2057-65. Epub 2016 Mar 22.

Division of Genomics and Genetics, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore 138673; Cancer Research Program, Houston Methodist Research Institute, Houston, TX 77030

Mutations in SMAD4 predispose to the development of gastrointestinal cancer, which is the third leading cause of cancer-related deaths. To identify genes driving gastric cancer (GC) development, we performed a Sleeping Beauty (SB) transposon mutagenesis screen in the stomach of Smad4(+/-) mutant mice. This screen identified 59 candidate GC trunk drivers and a much larger number of candidate GC progression genes. Strikingly, 22 SB-identified trunk drivers are known or candidate cancer genes, whereas four SB-identified trunk drivers, including PTEN, SMAD4, RNF43, and NF1, are known human GC trunk drivers. Similar to human GC, pathway analyses identified WNT, TGF-β, and PI3K-PTEN signaling, ubiquitin-mediated proteolysis, adherens junctions, and RNA degradation in addition to genes involved in chromatin modification and organization as highly deregulated pathways in GC. Comparative oncogenomic filtering of the complete list of SB-identified genes showed that they are highly enriched for genes mutated in human GC and identified many candidate human GC genes. Finally, by comparing our complete list of SB-identified genes against the list of mutated genes identified in five large-scale human GC sequencing studies, we identified LDL receptor-related protein 1B (LRP1B) as a previously unidentified human candidate GC tumor suppressor gene. In LRP1B, 129 mutations were found in 462 human GC samples sequenced, and LRP1B is one of the top 10 most deleted genes identified in a panel of 3,312 human cancers. SB mutagenesis has, thus, helped to catalog the cooperative molecular mechanisms driving SMAD4-induced GC growth and discover genes with potential clinical importance in human GC.
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http://dx.doi.org/10.1073/pnas.1603223113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833248PMC
April 2016

Loss of the transcription factor Meis1 prevents sympathetic neurons target-field innervation and increases susceptibility to sudden cardiac death.

Elife 2016 Feb 8;5. Epub 2016 Feb 8.

Institute for Neurosciences of Montpellier, Institut national de la santé et de la recherche médicale, Montpellier, France.

Although cardio-vascular incidents and sudden cardiac death (SCD) are among the leading causes of premature death in the general population, the origins remain unidentified in many cases. Genome-wide association studies have identified Meis1 as a risk factor for SCD. We report that Meis1 inactivation in the mouse neural crest leads to an altered sympatho-vagal regulation of cardiac rhythmicity in adults characterized by a chronotropic incompetence and cardiac conduction defects, thus increasing the susceptibility to SCD. We demonstrated that Meis1 is a major regulator of sympathetic target-field innervation and that Meis1 deficient sympathetic neurons die by apoptosis from early embryonic stages to perinatal stages. In addition, we showed that Meis1 regulates the transcription of key molecules necessary for the endosomal machinery. Accordingly, the traffic of Rab5(+) endosomes is severely altered in Meis1-inactivated sympathetic neurons. These results suggest that Meis1 interacts with various trophic factors signaling pathways during postmitotic neurons differentiation.
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http://dx.doi.org/10.7554/eLife.11627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760953PMC
February 2016

Cardiomyocyte-specific overexpression of the ubiquitin ligase Wwp1 contributes to reduction in Connexin 43 and arrhythmogenesis.

J Mol Cell Cardiol 2015 Nov 16;88:1-13. Epub 2015 Sep 16.

Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA. Electronic address:

Gap junctions (GJ) are intercellular channels composed of connexin subunits that play a critical role in a diverse number of cellular processes in all tissue types. In the heart, GJs mediate electrical coupling between cardiomyocytes and display mislocalization and/or downregulation in cardiac disease (a process known as GJ remodeling), producing an arrhythmogenic substrate. The main constituent of GJs in the ventricular myocardium is Connexin 43 (Cx43), an integral membrane protein that is rapidly turned over and shows decreased expression or function with age. We hypothesized that Wwp1, an ubiquitin ligase whose expression in known to increase in aging-related pathologies, may regulate Cx43 in vivo by targeting it for ubiquitylation and degradation and yield tissue-specific Cx43 loss of function phenotypes. When Wwp1 was globally overexpressed in mice under the control of a β-actin promoter, the highest induction of Wwp1 expression was observed in the heart which was associated with a 90% reduction in cardiac Cx43 protein levels, left ventricular hypertrophy (LVH), and the development of lethal ventricular arrhythmias around 8weeks of age. This phenotype was completely penetrant in two independent founder lines. Cardiomyocyte-specific overexpression of Wwp1 confirmed that this phenotype was cell autonomous and delineated Cx43-dependent and -independent roles for Wwp1 in arrhythmogenesis and LVH, respectively. Using a cell-based system, it was determined that Wwp1 co-immunoprecipitates with and ubiquitylates Cx43, causing a decrease in the steady state levels of Cx43 protein. These findings offer new mechanistic insights into the regulation of Cx43 which may be exploitable in various gap junctionopathies.
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http://dx.doi.org/10.1016/j.yjmcc.2015.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641030PMC
November 2015

Bcl11a (Ctip1) Controls Migration of Cortical Projection Neurons through Regulation of Sema3c.

Neuron 2015 Jul;87(2):311-25

Institute of Molecular and Cellular Anatomy, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany. Electronic address:

During neocortical development, neurons undergo polarization, oriented migration, and layer-type-specific differentiation. The transcriptional programs underlying these processes are not completely understood. Here, we show that the transcription factor Bcl11a regulates polarity and migration of upper layer neurons. Bcl11a-deficient late-born neurons fail to correctly switch from multipolar to bipolar morphology, resulting in impaired radial migration. We show that the expression of Sema3c is increased in migrating Bcl11a-deficient neurons and that Bcl11a is a direct negative regulator of Sema3c transcription. In vivo gain-of-function and rescue experiments demonstrate that Sema3c is a major downstream effector of Bcl11a required for the cell polarity switch and for the migration of upper layer neurons. Our data uncover a novel Bcl11a/Sema3c-dependent regulatory pathway used by migrating cortical neurons.
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http://dx.doi.org/10.1016/j.neuron.2015.06.023DOI Listing
July 2015

The transcription factor Bcl11b is specifically expressed in group 2 innate lymphoid cells and is essential for their development.

J Exp Med 2015 Jun 11;212(6):865-74. Epub 2015 May 11.

Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, England, UK

Group 2 innate lymphoid cells (ILCs), or ILC2s, are a subset of recently identified ILCs, which play important roles in innate immunity by producing type 2 effector cytokines. Several transcription factors have been found to have critical functions in the development of both ILC2s and T cells. We report here that Bcl11b, a transcription factor essential in T cell lineage commitment and maintenance, is specifically expressed in progenitors committed to the ILC2 lineage and is required for ILC2 development. The Bcl11b gene is expressed in ∼28% of ILC progenitors (ILCPs; common helper innate lymphoid progenitors or ILCPs expressing either ID2 or promyelocytic leukemia zinc finger, respectively). Both in vitro and in vivo, these Bcl11b-expressing early ILCPs generate only ILC2s. Inactivation of Bcl11b causes a complete loss of ILC2 development from hematopoietic progenitors, which is confirmed upon immune challenge with either papain administration or influenza virus infection.
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http://dx.doi.org/10.1084/jem.20142318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4451136PMC
June 2015

Transposon mutagenesis identifies genetic drivers of Braf(V600E) melanoma.

Nat Genet 2015 May 13;47(5):486-95. Epub 2015 Apr 13.

1] Cancer Research Program, Houston Methodist Research Institute, Houston, Texas, USA. [2] Institute of Molecular and Cell Biology, Singapore.

Although nearly half of human melanomas harbor oncogenic BRAF(V600E) mutations, the genetic events that cooperate with these mutations to drive melanogenesis are still largely unknown. Here we show that Sleeping Beauty (SB) transposon-mediated mutagenesis drives melanoma progression in Braf(V600E) mutant mice and identify 1,232 recurrently mutated candidate cancer genes (CCGs) from 70 SB-driven melanomas. CCGs are enriched in Wnt, PI3K, MAPK and netrin signaling pathway components and are more highly connected to one another than predicted by chance, indicating that SB targets cooperative genetic networks in melanoma. Human orthologs of >500 CCGs are enriched for mutations in human melanoma or showed statistically significant clinical associations between RNA abundance and survival of patients with metastatic melanoma. We also functionally validate CEP350 as a new tumor-suppressor gene in human melanoma. SB mutagenesis has thus helped to catalog the cooperative molecular mechanisms driving BRAF(V600E) melanoma and discover new genes with potential clinical importance in human melanoma.
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http://dx.doi.org/10.1038/ng.3275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4844184PMC
May 2015

BCL11A is a triple-negative breast cancer gene with critical functions in stem and progenitor cells.

Nat Commun 2015 Jan 9;6:5987. Epub 2015 Jan 9.

Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK.

Triple-negative breast cancer (TNBC) has poor prognostic outcome compared with other types of breast cancer. The molecular and cellular mechanisms underlying TNBC pathology are not fully understood. Here, we report that the transcription factor BCL11A is overexpressed in TNBC including basal-like breast cancer (BLBC) and that its genomic locus is amplified in up to 38% of BLBC tumours. Exogenous BCL11A overexpression promotes tumour formation, whereas its knockdown in TNBC cell lines suppresses their tumourigenic potential in xenograft models. In the DMBA-induced tumour model, Bcl11a deletion substantially decreases tumour formation, even in p53-null cells and inactivation of Bcl11a in established tumours causes their regression. At the cellular level, Bcl11a deletion causes a reduction in the number of mammary epithelial stem and progenitor cells. Thus, BCL11A has an important role in TNBC and normal mammary epithelial cells. This study highlights the importance of further investigation of BCL11A in TNBC-targeted therapies.
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http://dx.doi.org/10.1038/ncomms6987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338552PMC
January 2015