Riadh Lobbardi

Optimized cell transplantation using adult rag2 mutant zebrafish

Cell transplantation into adult zebrafish has lagged behind mouse models owing to the lack of immunocompromised strains. Here we have created rag2E450fs 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. rag2E450fs mutant zebrafish are the first immunocompromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer.

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

Significance Embryonal rhabdomyosarcoma (ERMS) is a cancer of skeletal muscle and is one of the most common pediatric cancers of soft tissue. There is no effective treatment for patients with relapsed ERMS, with less than 50% surviving the disease. The self-renewing and molecularly defined tumor propagating cells (TPCs) drive continued tumor growth and relapse. Yet to date, drugs targeting ERMS self-renewal and differentiation of TPCs have not been identified. Our study describes a large-scale chemical screen to identify targetable pathways essential for modulating self-renewal and differentiation of ERMS and demonstrates the feasibility of inducing differentiation of TPCs in ERMS by small molecules. 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.

Identification and characterization of T reg–like cells in zebrafish

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 FOXP3 orthologue as a marker, we identified CD4-enriched, mature T lymphocytes with properties of T reg cells. Zebrafish mutant for foxp3a 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.

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

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 (prkdc), interleukin-2 receptor &ggr; a (il2rga), 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.

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

Summary 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 up-regulated 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.

T Cell Immune Deficiency in zap70 Mutant Zebrafish

ABSTRACT ZAP70 [zeta-chain (TCR)-associated protein kinase, 70-kDa], is required for T cell activation. ZAP70 deficiencies in humans and null mutations in mice lead to severe combined immune deficiency. Here, we describe a zap70 loss-of-function mutation in zebrafish (zap70y442) that was created using transcription activator-like effector nucleases (TALENs). In contrast to what has been reported for morphant zebrafish, zap70y442 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 lck:GFP+ 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 zap70y442 mutant animals. T cell immune deficiency was confirmed through transplantation of unmatched normal and malignant donor cells into zap70y442 mutant zebrafish, with T cell loss being sufficient for robust allogeneic cell engraftment. zap70 mutant zebrafish show remarkable conservation of immune cell dysfunction as found in mice and humans and will serve as a valuable model to study zap70 immune deficiency.

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

Moore and colleagues present new strains of optically clear immune-deficient zebrafish that allow for dynamic imaging of regeneration and tumor progression at single-cell resolution in live animals.

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

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.Significance: 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. Cancer Discov; 7(11); 1336-53. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1201.

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

A substantial subset of patients with T cell acute lymphoblastic leukemia (T-ALL) develops resistance to steroids and succumbs to their disease. JDP2 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 JDP2 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 MCL1. Furthermore, JDP2 is one of few oncogenes capable of initiating T-ALL in transgenic zebrafish. Notably, thymocytes from rag2:jdp2 transgenic zebrafish express high levels of mcl1 and demonstrate resistance to steroids in vivo. These studies establish JDP2 as a novel oncogene in high-risk T-ALL and implicate overexpression of MCL1 as a mechanism of steroid resistance in JDP2-overexpressing cells.

Abstract 4177: Dynamic visualization of cancer cell engraftment into immune compromised zebrafish

Cell transplantation into immune compromised mice has transformed our understanding of cancer and is now the gold standard for assessing therapeutic responses in vivo. However, mouse models are expensive and engraftment is often difficult to visualize directly. To overcome these challenges, we have developed immune compromised zebrafish (ICZ) in the transparent casper background using genome editing techniques. We have successfully targeted genes required for immune cell function and are well known to cause immune deficiency in human and mice. To date, we have developed homozygous viable mutants for recombination-activating gene 2 (rag2), DNA-dependent protein kinase (prkdc), janus kinase 3 (jak3), interleukin 2 receptor gamma (Il2rg), zeta-chain (TCR) associated protein kinase 70 (zap70), and forkhead box N1 (foxn1/nude). Gene expression analysis of marrow cells using RNAseq has identified novel transcript changes correlated with loss of specific cell types, and in conjunction with large-scale single cell transcriptional profiling, has identified specific cellular defects associated with T, B, and NK cell loss. For example, homozygous prkdc (SCID) mutant fish lack mature T and B cells, but have intact NK cell signaling. By contrast, il2rg-deficient zebrafish lack T and NK cells. Importantly, these ICZ models accurately recapitulate known human severe combined immune deficiencies and established mouse models that are commonly used for cell transplantation. Thus, it is not unexpected that a subset of zebrafish mutants have reduced immune cell function, permitting engraftment of normal hematopoietic and muscle satellite cells from allogeneic donors. Additionally, we have demonstrated robust and persistent engraftment of fluorescently labeled leukemia, rhabdomyosarcoma, neuroblastoma, and melanoma from a wide range of zebrafish strains. Because mutations have been created in optically-clear, casper-strain zebrafish and cancers are fluorescently labeled, we now have unprecedented access to directly visualize tumor cells at single cell resolution in live animals. To date, we have optimized our models to visualize neovascularization, intratumoral cell heterogeneity, clonal evolution and metastisis. The ability to transplant non-immune matched cell types will likely revolutionize the types and scale of cell transplantation experiments performed in the zebrafish and will likely permit engraftment of mouse and human cells into compound mutant ICZ models in the near future. Citation Format: John C. Moore, Qin Tang, Nora Torres Yordan, Timothy Mulligan, Finola E. Moore, Riadh Lobbardi, Ashwin Ramakrishnan, Anthony Anselmo, Ruslan Sadreyev, Jason Berman, Robert Liwski, Brant Weinstein, John Rawls, David M. Langenau. Dynamic visualization of cancer cell engraftment into immune compromised zebrafish. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4177.