Publications by authors named "Riadh Lobbardi"

14 Publications

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JDP2: An oncogenic bZIP transcription factor in T cell acute lymphoblastic leukemia.

J Exp Med 2018 07 25;215(7):1929-1945. Epub 2018 Jun 25.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA

A substantial subset of patients with T cell acute lymphoblastic leukemia (T-ALL) develops resistance to steroids and succumbs to their disease. encodes a bZIP protein that has been implicated as a T-ALL oncogene from insertional mutagenesis studies in mice, but its role in human T-ALL pathogenesis has remained obscure. Here we show that is aberrantly expressed in a subset of T-ALL patients and is associated with poor survival. JDP2 is required for T-ALL cell survival, as its depletion by short hairpin RNA knockdown leads to apoptosis. Mechanistically, JDP2 regulates prosurvival signaling through direct transcriptional regulation of Furthermore, is one of few oncogenes capable of initiating T-ALL in transgenic zebrafish. Notably, thymocytes from transgenic zebrafish express high levels of and demonstrate resistance to steroids in vivo. These studies establish as a novel oncogene in high-risk T-ALL and implicate overexpression of as a mechanism of steroid resistance in -overexpressing cells.
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http://dx.doi.org/10.1084/jem.20170484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028512PMC
July 2018

Identification and characterization of T reg-like cells in zebrafish.

J Exp Med 2017 Dec 24;214(12):3519-3530. Epub 2017 Oct 24.

Program in Molecular Medicine and Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA

Regulatory T (T reg) cells are a specialized sublineage of T lymphocytes that suppress autoreactive T cells. Functional studies of T reg cells in vitro have defined multiple suppression mechanisms, and studies of T reg-deficient humans and mice have made clear the important role that these cells play in preventing autoimmunity. However, many questions remain about how T reg cells act in vivo. Specifically, it is not clear which suppression mechanisms are most important, where T reg cells act, and how they get there. To begin to address these issues, we sought to identify T reg cells in zebrafish, a model system that provides unparalleled advantages in live-cell imaging and high-throughput genetic analyses. Using a orthologue as a marker, we identified CD4-enriched, mature T lymphocytes with properties of T reg cells. Zebrafish mutant for displayed excess T lymphocytes, splenomegaly, and a profound inflammatory phenotype that was suppressed by genetic ablation of lymphocytes. This study identifies T reg-like cells in zebrafish, providing both a model to study the normal functions of these cells in vivo and mutants to explore the consequences of their loss.
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http://dx.doi.org/10.1084/jem.20162084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716030PMC
December 2017

TOX Regulates Growth, DNA Repair, and Genomic Instability in T-cell Acute Lymphoblastic Leukemia.

Cancer Discov 2017 11 3;7(11):1336-1353. Epub 2017 Oct 3.

Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes. Using a transgenic screen in zebrafish, thymocyte selection-associated high mobility group box protein (TOX) was uncovered as a collaborating oncogenic driver that accelerated T-ALL onset by expanding the initiating pool of transformed clones and elevating genomic instability. TOX is highly expressed in a majority of human T-ALL and is required for proliferation and continued xenograft growth in mice. Using a wide array of functional analyses, we uncovered that TOX binds directly to KU70/80 and suppresses recruitment of this complex to DNA breaks to inhibit nonhomologous end joining (NHEJ) repair. Impaired NHEJ is well known to cause genomic instability, including development of T-cell malignancies in KU70- and KU80-deficient mice. Collectively, our work has uncovered important roles for TOX in regulating NHEJ by elevating genomic instability during leukemia initiation and sustaining leukemic cell proliferation following transformation. TOX is an HMG box-containing protein that has important roles in T-ALL initiation and maintenance. TOX inhibits the recruitment of KU70/KU80 to DNA breaks, thereby inhibiting NHEJ repair. Thus, TOX is likely a dominant oncogenic driver in a large fraction of human T-ALL and enhances genomic instability. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-0267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5683427PMC
November 2017

Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing.

J Exp Med 2017 Oct 6;214(10):2875-2887. Epub 2017 Sep 6.

Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA

Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA-protein kinase catalytic subunit (), interleukin-2 receptor γ a (), and double-homozygous-mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish.
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http://dx.doi.org/10.1084/jem.20170976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626406PMC
October 2017

Quaking RNA-Binding Proteins Control Early Myofibril Formation by Modulating Tropomyosin.

Dev Cell 2017 09 31;42(5):527-541.e4. Epub 2017 Aug 31.

IBENS, Institut de Biologie de l'Ecole Normale Supérieure, 75005 Paris, France; INSERM U1024, 75005 Paris, France; CNRS UMR 8197, 75005 Paris, France. Electronic address:

Skeletal muscle contraction is mediated by myofibrils, complex multi-molecular scaffolds structured into repeated units, the sarcomeres. Myofibril structure and function have been extensively studied, but the molecular processes regulating its formation within the differentiating muscle cell remain largely unknown. Here we show in zebrafish that genetic interference with the Quaking RNA-binding proteins disrupts the initial steps of myofibril assembly without affecting early muscle differentiation. Using RNA sequencing, we demonstrate that Quaking is required for accumulation of the muscle-specific tropomyosin-3 transcript, tpm3.12. Further functional analyses reveal that Tpm3.12 mediates Quaking control of myofibril formation. Moreover, we identified a Quaking-binding site in the 3' UTR of tpm3.12 transcript, which is required in vivo for tpm3.12 accumulation and myofibril formation. Our work uncovers a Quaking/Tpm3 pathway controlling de novo myofibril assembly. This unexpected developmental role for Tpm3 could be at the origin of muscle defects observed in human congenital myopathies associated with tpm3 mutation.
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http://dx.doi.org/10.1016/j.devcel.2017.08.004DOI Listing
September 2017

The NOTCH1/SNAIL1/MEF2C Pathway Regulates Growth and Self-Renewal in Embryonal Rhabdomyosarcoma.

Cell Rep 2017 06;19(11):2304-2318

Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA; Center of Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129, USA; Harvard Stem Cell Institute, Boston, MA 02114, USA. Electronic address:

Tumor-propagating cells (TPCs) share self-renewal properties with normal stem cells and drive continued tumor growth. However, mechanisms regulating TPC self-renewal are largely unknown, especially in embryonal rhabdomyosarcoma (ERMS)-a common pediatric cancer of muscle. Here, we used a zebrafish transgenic model of ERMS to identify a role for intracellular NOTCH1 (ICN1) in increasing TPCs by 23-fold. ICN1 expanded TPCs by enabling the de-differentiation of zebrafish ERMS cells into self-renewing myf5+ TPCs, breaking the rigid differentiation hierarchies reported in normal muscle. ICN1 also had conserved roles in regulating human ERMS self-renewal and growth. Mechanistically, ICN1 upregulated expression of SNAIL1, a transcriptional repressor, to increase TPC number in human ERMS and to block muscle differentiation through suppressing MEF2C, a myogenic differentiation transcription factor. Our data implicate the NOTCH1/SNAI1/MEF2C signaling axis as a major determinant of TPC self-renewal and differentiation in ERMS, raising hope of therapeutically targeting this pathway in the future.
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http://dx.doi.org/10.1016/j.celrep.2017.05.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563075PMC
June 2017

Efficient Transduction of Zebrafish Melanoma Cell Lines and Embryos Using Lentiviral Vectors.

Zebrafish 2017 08 30;14(4):379-382. Epub 2017 May 30.

1 Stem Cell Program, Division of Hematology/Oncology, Boston Children's Hospital , Boston, Massachusetts.

The establishment of in vitro cultures of zebrafish cancer cells has expanded the potential of zebrafish as a disease model. However, the lack of effective methods for gene delivery and genetic manipulation has limited the experimental applications of these cultures. To overcome this barrier, we tested and optimized vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral and retroviral vector transduction protocols. We show that lentivirus successfully and efficiently transduced zebrafish melanoma cell lines in vitro, allowing antibiotic selection, fluorescence-based sorting, and in vivo allotransplantation. In addition, injection of concentrated lentiviral particles into embryos and tumors established the feasibility of in vivo gene delivery.
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http://dx.doi.org/10.1089/zeb.2017.1434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5549793PMC
August 2017

Single-cell imaging of normal and malignant cell engraftment into optically clear prkdc-null SCID zebrafish.

J Exp Med 2016 11 24;213(12):2575-2589. Epub 2016 Oct 24.

Molecular Pathology, Massachusetts General Hospital, Charlestown, MA 02129

Cell transplantation into immunodeficient mice has revolutionized our understanding of regeneration, stem cell self-renewal, and cancer; yet models for direct imaging of engrafted cells has been limited. Here, we characterize zebrafish with mutations in recombination activating gene 2 (rag2), DNA-dependent protein kinase (prkdc), and janus kinase 3 (jak3). Histology, RNA sequencing, and single-cell transcriptional profiling of blood showed that rag2 hypomorphic mutant zebrafish lack T cells, whereas prkdc deficiency results in loss of mature T and B cells and jak3 in T and putative Natural Killer cells. Although all mutant lines engraft fluorescently labeled normal and malignant cells, only the prkdc mutant fish reproduced as homozygotes and also survived injury after cell transplantation. Engraftment into optically clear casper, prkdc-mutant zebrafish facilitated dynamic live cell imaging of muscle regeneration, repopulation of muscle stem cells within their endogenous niche, and muscle fiber fusion at single-cell resolution. Serial imaging approaches also uncovered stochasticity in fluorescently labeled leukemia regrowth after competitive cell transplantation into prkdc mutant fish, providing refined models to assess clonal dominance and progression in the zebrafish. Our experiments provide an optimized and facile transplantation model, the casper, prkdc mutant zebrafish, for efficient engraftment and direct visualization of fluorescently labeled normal and malignant cells at single-cell resolution.
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http://dx.doi.org/10.1084/jem.20160378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5110017PMC
November 2016

T Cell Immune Deficiency in Mutant Zebrafish.

Mol Cell Biol 2016 Dec 14;36(23):2868-2876. Epub 2016 Nov 14.

Molecular Pathology, Massachusetts General Hospital, Charlestown, Massachusetts, USA

[, ], is required for T cell activation. deficiencies in humans and null mutations in mice lead to severe combined immune deficiency. Here, we describe a loss-of-function mutation in zebrafish ( ) that was created using transcription activator-like effector nucleases (TALENs). In contrast to what has been reported for morphant zebrafish, homozygous mutant zebrafish displayed normal development of blood and lymphatic vasculature. Hematopoietic cell development was also largely unaffected in mutant larvae. However, mutant fish had reduced : thymic T cells by 5 days postfertilization that persisted into adult stages. Morphological analysis, RNA sequencing, and single-cell gene expression profiling of whole kidney marrow cells of adult fish revealed complete loss of mature T cells in mutant animals. T cell immune deficiency was confirmed through transplantation of unmatched normal and malignant donor cells into mutant zebrafish, with T cell loss being sufficient for robust allogeneic cell engraftment. mutant zebrafish show remarkable conservation of immune cell dysfunction as found in mice and humans and will serve as a valuable model to study immune deficiency.
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http://dx.doi.org/10.1128/MCB.00281-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5108876PMC
December 2016

Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish.

J Exp Med 2016 05 2;213(6):979-92. Epub 2016 May 2.

Molecular Pathology, Massachusetts General Hospital, Charlestown, MA 02129 Cancer Center, Massachusetts General Hospital, Charlestown, MA 02129 Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114 Harvard Stem Cell Institute, Cambridge, MA 02139

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4(+) cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2(E450fs) mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4(+) cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2(E450fs) mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4(+)/CD8(+) cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia.
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http://dx.doi.org/10.1084/jem.20152013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4886368PMC
May 2016

Optimized cell transplantation using adult rag2 mutant zebrafish.

Nat Methods 2014 Aug 20;11(8):821-4. Epub 2014 Jul 20.

1] Molecular Pathology Unit, Massachusetts General Hospital, Boston, Massachusetts, USA. [2] Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, USA. [3] Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA. [4] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [5].

Cell transplantation into adult zebrafish has lagged behind mouse models owing to the lack of immunocompromised strains. Here we have created rag2(E450fs) mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft muscle, blood stem cells and various cancers. rag2(E450fs) mutant zebrafish are the first immunocompromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer.
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http://dx.doi.org/10.1038/nmeth.3031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4294527PMC
August 2014

Glycogen synthase kinase 3 inhibitors induce the canonical WNT/β-catenin pathway to suppress growth and self-renewal in embryonal rhabdomyosarcoma.

Proc Natl Acad Sci U S A 2014 Apr 24;111(14):5349-54. Epub 2014 Mar 24.

Departments of Pathology and Regenerative Medicine and the Center for Cancer Research, Massachusetts General Hospital, Boston, MA 02114.

Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle, with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress ERMS self-renewal and induce differentiation of TPCs, a large-scale chemical screen was completed. Glycogen synthase kinase 3 (GSK3) inhibitors were identified as potent suppressors of ERMS growth through inhibiting proliferation and inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT/β-catenin pathway, recombinant WNT3A and stabilized β-catenin also enhanced terminal differentiation of human ERMS cells. Treatment of ERMS-bearing zebrafish with GSK3 inhibitors activated the WNT/β-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. Activation of the canonical WNT/β-catenin pathway also significantly reduced self-renewal of human ERMS, indicating a conserved function for this pathway in modulating ERMS self-renewal. In total, we have identified an unconventional tumor suppressive role for the canonical WNT/β-catenin pathway in regulating self-renewal of ERMS and revealed therapeutic strategies to target differentiation of TPCs in ERMS.
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http://dx.doi.org/10.1073/pnas.1317731111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3986146PMC
April 2014

Clonal evolution enhances leukemia-propagating cell frequency in T cell acute lymphoblastic leukemia through Akt/mTORC1 pathway activation.

Cancer Cell 2014 Mar 6;25(3):366-78. Epub 2014 Mar 6.

Department of Pathology, Regenerative Medicine and Center for Cancer Research, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Stem Cell Institute, Boston, MA 02138, USA. Electronic address:

Clonal evolution and intratumoral heterogeneity drive cancer progression through unknown molecular mechanisms. To address this issue, functional differences between single T cell acute lymphoblastic leukemia (T-ALL) clones were assessed using a zebrafish transgenic model. Functional variation was observed within individual clones, with a minority of clones enhancing growth rate and leukemia-propagating potential with time. Akt pathway activation was acquired in a subset of these evolved clones, which increased the number of leukemia-propagating cells through activating mTORC1, elevated growth rate likely by stabilizing the Myc protein, and rendered cells resistant to dexamethasone, which was reversed by combined treatment with an Akt inhibitor. Thus, T-ALL clones spontaneously and continuously evolve to drive leukemia progression even in the absence of therapy-induced selection.
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http://dx.doi.org/10.1016/j.ccr.2014.01.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3992437PMC
March 2014

Fine-tuning of Hh signaling by the RNA-binding protein Quaking to control muscle development.

Development 2011 May 29;138(9):1783-94. Epub 2011 Mar 29.

Ecole Normale Supérieure, Institut de Biologie, 46 rue d'Ulm, 75005 Paris, France.

The development of the different muscles within the somite is a complex process that involves the Hedgehog (Hh) signaling pathway. To specify the proper number of muscle cells and organize them spatially and temporally, the Hh signaling pathway needs to be precisely regulated at different levels, but only a few factors external to the pathway have been described. Here, we report for the first time the role of the STAR family RNA-binding protein Quaking A (QkA) in somite muscle development. We show in zebrafish that the loss of QkA function affects fast muscle fiber maturation as well as Hh-induced muscle derivative specification and/or morphogenesis. Mosaic analysis reveals that fast fiber maturation depends on the activity of QkA in the environment of fast fiber progenitors. We further show that Hh signaling requires QkA activity for muscle development. By an in silico approach, we screened the 3'UTRs of known Hh signaling component mRNAs for the Quaking response element and found the transcription factor Gli2a, a known regulator of muscle fate development. Using destabilized GFP as a reporter, we show that the gli2a mRNA 3'UTR is a functional QkA target. Consistent with this notion, the loss of QkA function rescued slow muscle fibers in yot mutant embryos, which express a dominant-negative Gli2a isoform. Thus, our results reveal a new mechanism to ensure muscle cell fate diversity by fine-tuning of the Hh signaling pathway via RNA-binding proteins.
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http://dx.doi.org/10.1242/dev.059121DOI Listing
May 2011