Publications by authors named "Jason N Berman"

78 Publications

High-dose AraC is Essential for the Treatment of ML-DS Independent of Post-Induction MRD: Results of COG AAML1531.

Blood 2021 07 28. Epub 2021 Jul 28.

University of Ottawa/Children’s Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada

Myeloid leukemia in children with Down syndrome (ML-DS) is associated with young age and somatic GATA1 mutations. Due to high event-free survival (EFS) and hypersensitivity of the leukemic blasts to chemotherapy, the prior Children's Oncology Group protocol ML-DS protocol (AAML0431), reduced overall treatment intensity but lacking risk stratification, retained the high-dose cytarabine course (HD-AraC), which was highly associated with infectious morbidity. Despite high EFS of ML-DS, survival for those who relapse is rare. AAML1531 introduced therapeutic risk stratification based on the previously identified prognostic factor, measurable residual disease (MRD) at the end of the first induction course. Standard risk (SR) patients were identified by negative MRD using flow cytometry (<0.05%) and did not receive the historically administered HD-AraC course. Interim analysis of 114 SR patients revealed a 2-year EFS of 85.6% (95% confidence interval (CI), 75.7-95.5%), which was significantly lower than for MRD-negative patients treated with HD-AraC on AAML0431 (p=0.0002). Overall survival at 2 years was 91.0% (95% CI 83.8%-95.0%). Twelve SR patients relapsed, mostly within one year from study entry and had a 1-year OS of 16.7% (95% CI 2.7% - 41.3%). Complex karyotypes were more frequent in SR patients who relapsed compared to those who did not (36% vs. 9%; p=0.0248). MRD by error-corrected sequencing of GATA1 mutations was piloted in 18 SR patients and detectable in 60% who relapsed vs. 23% who did not (p=0.2682). Patients with SR ML-DS had worse outcomes without HD-AraC after risk classification based on flow cytometric MRD. ClinicalTrials.gov NCT02521493.
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http://dx.doi.org/10.1182/blood.2021012206DOI Listing
July 2021

STAG2 loss rewires oncogenic and developmental programs to promote metastasis in Ewing sarcoma.

Cancer Cell 2021 Jun;39(6):827-844.e10

Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA. Electronic address:

The core cohesin subunit STAG2 is recurrently mutated in Ewing sarcoma but its biological role is less clear. Here, we demonstrate that cohesin complexes containing STAG2 occupy enhancer and polycomb repressive complex (PRC2)-marked regulatory regions. Genetic suppression of STAG2 leads to a compensatory increase in cohesin-STAG1 complexes, but not in enhancer-rich regions, and results in reprogramming of cis-chromatin interactions. Strikingly, in STAG2 knockout cells the oncogenic genetic program driven by the fusion transcription factor EWS/FLI1 was highly perturbed, in part due to altered enhancer-promoter contacts. Moreover, loss of STAG2 also disrupted PRC2-mediated regulation of gene expression. Combined, these transcriptional changes converged to modulate EWS/FLI1, migratory, and neurodevelopmental programs. Finally, consistent with clinical observations, functional studies revealed that loss of STAG2 enhances the metastatic potential of Ewing sarcoma xenografts. Our findings demonstrate that STAG2 mutations can alter chromatin architecture and transcriptional programs to promote an aggressive cancer phenotype.
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http://dx.doi.org/10.1016/j.ccell.2021.05.007DOI Listing
June 2021

Zebrafish Cancer Predisposition Models.

Front Cell Dev Biol 2021 27;9:660069. Epub 2021 Apr 27.

Children's Hospital of Eastern Ontario Research Institute, Ottawa, ON, Canada.

Cancer predisposition syndromes are rare, typically monogenic disorders that result from germline mutations that increase the likelihood of developing cancer. Although these disorders are individually rare, resulting cancers collectively represent 5-10% of all malignancies. In addition to a greater incidence of cancer, affected individuals have an earlier tumor onset and are frequently subjected to long-term multi-modal cancer screening protocols for earlier detection and initiation of treatment. models are needed to better understand tumor-driving mechanisms, tailor patient screening approaches and develop targeted therapies to improve patient care and disease prognosis. The zebrafish () has emerged as a robust model for cancer research due to its high fecundity, time- and cost-efficient genetic manipulation and real-time high-resolution imaging. Tumors developing in zebrafish cancer models are histologically and molecularly similar to their human counterparts, confirming the validity of these models. The zebrafish platform supports both large-scale random mutagenesis screens to identify potential candidate/modifier genes and recently optimized genome editing strategies. These techniques have greatly increased our ability to investigate the impact of certain mutations and how these lesions impact tumorigenesis and disease phenotype. These unique characteristics position the zebrafish as a powerful tool to model cancer predisposition syndromes and as such, several have already been created, including those recapitulating Li-Fraumeni syndrome, familial adenomatous polyposis, RASopathies, inherited bone marrow failure syndromes, and several other pathogenic mutations in cancer predisposition genes. In addition, the zebrafish platform supports medium- to high-throughput preclinical drug screening to identify compounds that may represent novel treatment paradigms or even prevent cancer evolution. This review will highlight and synthesize the findings from zebrafish cancer predisposition models created to date. We will discuss emerging trends in how these zebrafish cancer models can improve our understanding of the genetic mechanisms driving cancer predisposition and their potential to discover therapeutic and/or preventative compounds that change the natural history of disease for these vulnerable children, youth and adults.
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http://dx.doi.org/10.3389/fcell.2021.660069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8112447PMC
April 2021

Humanized zebrafish enhance human hematopoietic stem cell survival and promote acute myeloid leukemia clonal diversity.

Haematologica 2020 10 1;105(10):2391-2399. Epub 2020 Oct 1.

University of Ottawa and Dalhousie University, Canada.

Xenograft models are invaluable tools in establishing the current paradigms of hematopoiesis and leukemogenesis. The zebrafish has emerged as a robust alternative xenograft model but, like mice, lack specific cytokines that mimic the microenvironment found in human patients. To address this critical gap, we generated the first humanized zebrafish that express human hematopoietic-specific cytokines (GM-CSF, SCF, and SDF1α). Termed GSS fish, these zebrafish promote survival, self-renewal and multilineage differentiation of human hematopoietic stem and progenitor cells and result in enhanced proliferation and hematopoietic niche-specific homing of primary human leukemia cells. Using error-corrected RNA sequencing, we determined that patient-derived leukemias transplanted into GSS zebrafish exhibit broader clonal representation compared to transplants into control hosts. GSS zebrafish incorporating error-corrected RNA sequencing establish a new standard for zebrafish xenotransplantation that more accurately recapitulates the human context, providing a more representative cost-effective preclinical model system for evaluating personalized response-based treatment in leukemia and therapies to expand human hematopoietic stem and progenitor cells in the transplant setting.
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http://dx.doi.org/10.3324/haematol.2019.223040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556680PMC
October 2020

The identification of dual protective agents against cisplatin-induced oto- and nephrotoxicity using the zebrafish model.

Elife 2020 07 28;9. Epub 2020 Jul 28.

IWK Health Centre, Department of Pediatrics, Halifax, Canada.

Dose-limiting toxicities for cisplatin administration, including ototoxicity and nephrotoxicity, impact the clinical utility of this effective chemotherapy agent and lead to lifelong complications, particularly in pediatric cancer survivors. Using a two-pronged drug screen employing the zebrafish lateral line as an in vivo readout for ototoxicity and kidney cell-based nephrotoxicity assay, we screened 1280 compounds and identified 22 that were both oto- and nephroprotective. Of these, dopamine and L-mimosine, a plant-based amino acid active in the dopamine pathway, were further investigated. Dopamine and L-mimosine protected the hair cells in the zebrafish otic vesicle from cisplatin-induced damage and preserved zebrafish larval glomerular filtration. Importantly, these compounds did not abrogate the cytotoxic effects of cisplatin on human cancer cells. This study provides insights into the mechanisms underlying cisplatin-induced oto- and nephrotoxicity and compelling preclinical evidence for the potential utility of dopamine and L-mimosine in the safer administration of cisplatin.
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http://dx.doi.org/10.7554/eLife.56235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470826PMC
July 2020

The Canadian Rare Diseases Models and Mechanisms (RDMM) Network: Connecting Understudied Genes to Model Organisms.

Am J Hum Genet 2020 02;106(2):143-152

Maternal Infant Child and Youth Research Network (MICYRN), Vancouver, BC V5Z 4H4, Canada; Department of Pediatrics, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.

Advances in genomics have transformed our ability to identify the genetic causes of rare diseases (RDs), yet we have a limited understanding of the mechanistic roles of most genes in health and disease. When a novel RD gene is first discovered, there is minimal insight into its biological function, the pathogenic mechanisms of disease-causing variants, and how therapy might be approached. To address this gap, the Canadian Rare Diseases Models and Mechanisms (RDMM) Network was established to connect clinicians discovering new disease genes with Canadian scientists able to study equivalent genes and pathways in model organisms (MOs). The Network is built around a registry of more than 500 Canadian MO scientists, representing expertise for over 7,500 human genes. RDMM uses a committee process to identify and evaluate clinician-MO scientist collaborations and approve 25,000 Canadian dollars in catalyst funding. To date, we have made 85 clinician-MO scientist connections and funded 105 projects. These collaborations help confirm variant pathogenicity and unravel the molecular mechanisms of RD, and also test novel therapies and lead to long-term collaborations. To expand the impact and reach of this model, we made the RDMM Registry open-source, portable, and customizable, and we freely share our committee structures and processes. We are currently working with emerging networks in Europe, Australia, and Japan to link international RDMM networks and registries and enable matches across borders. We will continue to create meaningful collaborations, generate knowledge, and advance RD research locally and globally for the benefit of patients and families living with RD.
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http://dx.doi.org/10.1016/j.ajhg.2020.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010971PMC
February 2020

Genome Editing in Zebrafish Using High-Fidelity Cas9 Nucleases: Choosing the Right Nuclease for the Task.

Methods Mol Biol 2020 ;2115:385-405

CHEO Research Institute, Ottawa, ON, Canada.

Shortly after the development of the CRISPR/Cas9 system, it was recognized that it is prone to induce off-target mutations at significant frequencies. Therefore, there is a strong motivation to develop Cas9 enzymes with reduced off-target activity. Multiple rational design or selection approaches have been applied to develop several Cas9 versions with reduced off-target activities (high fidelity). To make these high-fidelity Cas9s available for model systems other than human cells and bacterial strains, as, for example, in zebrafish, new specialized expression vectors need to be developed. In this chapter, we focused on the HypaCas9 and HiFi Cas9 high-fidelity enzymes and incorporated the mutations of these Cas9 versions into a codon-optimized zebrafish Cas9 vector. This optimized vector was further improved by introducing an artificial polyadenine insert (A71) since polyadenylation is known to enhance mRNA translational efficiency. The Hypa-nCas9n and HiFi-nCas9n vectors were produced by single-site mutagenesis from pT3TS-nCas9n-A71 vector. We then tested the polyadenylated mRNAs for nCas9n, Hypa-nCas9n, HiFi-nCas9n, and HiFi-Cas9 protein for editing efficiency in five genome editing strategies and found that these high-fidelity Cas9 versions had different performances ranging from activity at 2-4 sites, where the wild-type nCas9n is active, indicating that these Cas9 versions have different sgRNA preferences. In summary, the developed new high-fidelity Cas9 vectors will enable researchers to perform much more accurate genome editing.
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http://dx.doi.org/10.1007/978-1-0716-0290-4_21DOI Listing
February 2021

The Zebrafish Xenograft Platform-A Novel Tool for Modeling KSHV-Associated Diseases.

Viruses 2019 12 20;12(1). Epub 2019 Dec 20.

Department of Microbiology & Immunology, Dalhousie University, 5850 College Street, Halifax, NS B3H 4R2, Canada.

Kaposi's sarcoma associated-herpesvirus (KSHV, also known as human herpesvirus-8) is a gammaherpesvirus that establishes life-long infection in human B lymphocytes. KSHV infection is typically asymptomatic, but immunosuppression can predispose KSHV-infected individuals to primary effusion lymphoma (PEL); a malignancy driven by aberrant proliferation of latently infected B lymphocytes, and supported by pro-inflammatory cytokines and angiogenic factors produced by cells that succumb to lytic viral replication. Here, we report the development of the first model for a virally induced lymphoma in zebrafish, whereby KSHV-infected PEL tumor cells engraft and proliferate in the yolk sac of zebrafish larvae. Using a PEL cell line engineered to produce the viral lytic switch protein RTA in the presence of doxycycline, we demonstrate drug-inducible reactivation from KSHV latency , which enabled real-time observation and evaluation of latent and lytic phases of KSHV infection. In addition, we developed a sensitive droplet digital PCR method to monitor latent and lytic viral gene expression and host cell gene expression in xenografts. The zebrafish yolk sac is not well vascularized, and by using fluorogenic assays, we confirmed that this site provides a hypoxic environment that may mimic the microenvironment of some human tumors. We found that PEL cell proliferation in xenografts was dependent on the host hypoxia-dependent translation initiation factor, eukaryotic initiation factor 4E2 (eIF4E2). This demonstrates that the zebrafish yolk sac is a functionally hypoxic environment, and xenografted cells must switch to dedicated hypoxic gene expression machinery to survive and proliferate. The establishment of the PEL xenograft model enables future studies that exploit the innate advantages of the zebrafish as a model for genetic and pharmacologic screens.
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http://dx.doi.org/10.3390/v12010012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019925PMC
December 2019

Mixed-phenotype acute leukemia: A cohort and consensus research strategy from the Children's Oncology Group Acute Leukemia of Ambiguous Lineage Task Force.

Cancer 2020 02 29;126(3):593-601. Epub 2019 Oct 29.

Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta/Emory University, Atlanta, Georgia.

Background: Optimal chemotherapy for treating mixed-phenotype acute leukemia (MPAL) and the role of hematopoietic stem cell transplantation (HSCT) remain uncertain. Major limitations in interpreting available data are MPAL's rarity and the use of definitions other than the currently widely accepted criteria: the World Health Organization 2016 (WHO2016) classification.

Methods: To assess the relative efficacy of chemotherapy types for treating pediatric MPAL, the Children's Oncology Group (COG) Acute Leukemia of Ambiguous Lineage Task Force assembled a retrospective cohort of centrally reviewed WHO2016 MPAL cases selected from banking studies for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Patients were not treated in COG trials; treatment and outcome data were captured separately. The findings were then integrated with the available, mixed literature to develop a prospective trial in pediatric MPAL.

Results: The central review confirmed that 54 of 70 cases fulfilled WHO2016 criteria for MPAL. ALL induction regimens achieved remission in 72% of the cases (28 of 39), whereas AML regimens achieved remission in 69% (9 of 13). The 5-year event-free survival (EFS) and overall survival (OS) rates for the entire cohort were 72% ± 8% and 77% ± 7%, respectively. EFS and OS were 75% ± 13% and 84% ± 11%, respectively, for those receiving ALL chemotherapy alone without HSCT (n = 21).

Conclusions: The results of the COG MPAL cohort and a literature review suggest that ALL chemotherapy without HSCT may be the preferred initial therapy. A prospective trial within the COG is proposed to investigate this approach; AML chemotherapy and/or HSCT will be reserved for those with treatment failure as assessed by minimal residual disease. Embedded biology studies will provide further insight into MPAL genomics.
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http://dx.doi.org/10.1002/cncr.32552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7489437PMC
February 2020

Frizzled 4 regulates ventral blood vessel remodeling in the zebrafish retina.

Dev Dyn 2019 12 21;248(12):1243-1256. Epub 2019 Oct 21.

Department of Pediatrics, IWK Health Centre/Dalhousie University, Halifax, Nova Scotia, Canada.

Background: Familial exudative vitreoretinopathy (FEVR) is a rare congenital disorder characterized by a lack of blood vessel growth to the periphery of the retina with secondary fibrovascular proliferation at the vascular-avascular junction. These structurally abnormal vessels cause leakage and hemorrhage, while the fibroproliferative scarring results in retinal dragging, detachment and blindness. Mutations in the FZD4 gene represent one of the most common causes of FEVR.

Methods: A loss of function mutation resulting from a 10-nucleotide insertion into exon 1 of the zebrafish fzd4 gene was generated using transcription activator-like effector nucleases (TALENs). Structural and functional integrity of the retinal vasculature was examined by fluorescent microscopy and optokinetic responses.

Results: Zebrafish retinal vasculature is asymmetrically distributed along the dorsoventral axis, with active vascular remodeling on the ventral surface of the retina throughout development. fzd4 mutants exhibit disorganized ventral retinal vasculature with discernable tubular fusion by week 8 of development. Furthermore, fzd4 mutants have impaired optokinetic responses requiring increased illumination.

Conclusion: We have generated a visually impaired zebrafish FEVR model exhibiting abnormal retinal vasculature. These fish provide a tractable system for studying vascular biology in retinovascular disorders, and demonstrate the feasibility of using zebrafish for evaluating future FEVR genes identified in humans.
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http://dx.doi.org/10.1002/dvdy.117DOI Listing
December 2019

Pathologic Features of Down Syndrome Myelodysplastic Syndrome and Acute Myeloid Leukemia: A Report From the Children's Oncology Group Protocol AAML0431.

Arch Pathol Lab Med 2020 04 20;144(4):466-472. Epub 2019 Aug 20.

From the Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee (Drs Mast, Mosse, Jones, and Head); the Division of Hematology/Oncology, Children's Hospital of Michigan, Wayne State University, Detroit (Dr Taub); the Department of Biostatistics, University of Southern California, Monrovia (Dr Alonzo and Mr Wang); the Division of Hematology/Oncology/Bone Marrow Transplantation, Children's Mercy Hospital, Kansas City, Missouri (Dr Gamis); the Pathology and Laboratory Medicine Service, VA Tennessee Valley Healthcare System, Nashville (Dr Mosse); the Department of Pediatrics, University of New Mexico, Albuquerque (Dr Mathew); the Division of Hematology-Oncology, IWK Health Centre, Halifax, Nova Scotia, Canada (Dr Berman); the Departments of Oncology (Dr Campana and Ms Coustan-Smith) and Pathology (Dr Raimondi), St. Jude Children's Research Hospital, Memphis, Tennessee; the Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, The National University Cancer Institute, NUH Medical Centre, Singapore (Dr Campana and Ms Coustan-Smith); the Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis (Dr Hirsch); the Department of Pediatrics, University of Toronto, Toronto, Ontario, Canada (Dr Hitzler); and the Division of Hematology/Oncology, The Hospital for Sick Children Developmental and Stem Cell Biology, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada (Dr Hitzler). Dr Mast now has a joint appointment at the Pathology and Laboratory Medicine Service, VA Tennessee Valley Healthcare System, Nashville. Dr Mathew is currently at the Department of Pediatrics, Presbyterian Health Services, Albuquerque, New Mexico. Dr Berman is currently at the Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada. Dr Jones is currently at Pathgroup Labs, Nashville, Tennessee. Dr Campana is no longer at the Department of Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee. Ms Coustan-Smith is no longer at the Department of Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee.

Context.—: Detailed diagnostic features of acute myeloid leukemia in Down syndrome are lacking, leading to potential misdiagnoses as standard acute myeloid leukemia occurring in patients with Down syndrome.

Objective.—: To evaluate diagnostic features of acute myeloid leukemia and myelodysplastic syndrome in patients with Down syndrome.

Design.—: Diagnostic bone marrow samples from 163 patients enrolled in the Children's Oncology Group study AAML0431 were evaluated by using central morphologic review and institutional immunophenotyping. Results were compared to overall survival, event-free survival, mutation status, cytogenetics, and minimal residual disease results.

Results.—: Sixty myelodysplastic syndrome and 103 acute myeloid leukemia samples were reviewed. Both had distinctive features compared to those of patients without Down syndrome. They showed megakaryocytic and erythroid but little myeloid dysplasia, and marked megakaryocytic hyperplasia with unusual megakaryocyte morphology. In acute myeloid leukemia cases, megakaryoblastic differentiation of blasts was most common (54 of 103, 52%); other cases showed erythroblastic (11 of 103, 11%), mixed erythroid/megakaryoblastic (20 of 103, 19%), or no differentiation (10 of 103, 10%). Myelodysplastic syndrome and acute myeloid leukemia cases had similar event-free survival and overall survival. Leukemic subgroups showed interesting, but not statistically significant, trends for survival and minimal residual disease. Cases with institutional diagnoses of French American British M1-5 morphology showed typical features of Down syndrome disease, with survival approaching that of other cases.

Conclusions.—: Myelodysplastic syndrome and acute myeloid leukemia in Down syndrome display features that allow discrimination from standard cases of disease. These distinctions are important for treatment decisions, and for understanding disease pathogenesis. We propose specific diagnostic criteria for Down syndrome-related subtypes of acute myeloid leukemia and myelodysplastic syndrome.
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http://dx.doi.org/10.5858/arpa.2018-0526-OADOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031019PMC
April 2020

High-throughput Chemical Screening Identifies Focal Adhesion Kinase and Aurora Kinase B Inhibition as a Synergistic Treatment Combination in Ewing Sarcoma.

Clin Cancer Res 2019 07 12;25(14):4552-4566. Epub 2019 Apr 12.

Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Boston, Massachusetts.

Purpose: Ewing sarcoma is an aggressive solid tumor malignancy of childhood. Although current treatment regimens cure approximately 70% of patients with localized disease, they are ineffective for most patients with metastases or relapse. New treatment combinations are necessary for these patients.

Experimental Design: Ewing sarcoma cells are dependent on focal adhesion kinase (FAK) for growth. To identify candidate treatment combinations for Ewing sarcoma, we performed a small-molecule library screen to identify compounds synergistic with FAK inhibitors in impairing Ewing cell growth. The activity of a top-scoring class of compounds was then validated across multiple Ewing cell lines and in multiple xenograft models of Ewing sarcoma.

Results: Numerous Aurora kinase inhibitors scored as synergistic with FAK inhibition in this screen. We found that Aurora kinase B inhibitors were synergistic across a larger range of concentrations than Aurora kinase A inhibitors when combined with FAK inhibitors in multiple Ewing cell lines. The combination of AZD-1152, an Aurora kinase B-selective inhibitor, and PF-562271 or VS-4718, FAK-selective inhibitors, induced apoptosis in Ewing sarcoma cells at concentrations that had minimal effects on survival when cells were treated with either drug alone. We also found that the combination significantly impaired tumor progression in multiple xenograft models of Ewing sarcoma.

Conclusions: FAK and Aurora kinase B inhibitors synergistically impair Ewing sarcoma cell viability and significantly inhibit tumor progression. This study provides preclinical support for the consideration of a clinical trial testing the safety and efficacy of this combination for patients with Ewing sarcoma.
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http://dx.doi.org/10.1158/1078-0432.CCR-17-0375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6634997PMC
July 2019

Fats enhance stem cell emergence.

Science 2019 03;363(6431):1041-1042

Department of Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada.

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http://dx.doi.org/10.1126/science.aaw7059DOI Listing
March 2019

Zebrafish knock-ins swim into the mainstream.

Dis Model Mech 2018 10 24;11(10). Epub 2018 Oct 24.

Department of Pediatrics, Dalhousie University, IWK Health Centre, Halifax, NS B3K 6R8, Canada

The zebrafish is an increasingly popular model organism for human genetic disease research. CRISPR/Cas9-based approaches are currently used for multiple gene-editing purposes in zebrafish, but few studies have developed reliable ways to introduce precise mutations. Point mutation knock-in using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) is currently the most promising technology for this purpose. Despite some progress in applying this technique to zebrafish, there is still a great need for improvements in terms of its efficiency, optimal design of sgRNA and ssODNs and broader applicability. The papers discussed in this Editorial provide excellent case studies on identifying problems inherent in the mutation knock-in technique, quantifying these issues and proposing strategies to overcome them. These reports also illustrate how the procedures for introducing specific mutations can be straightforward, such that ssODNs with only the target mutation are sufficient for generating the intended knock-in animals. Two of the studies also develop interesting point mutant knock-in models for cardiac diseases, validating the translational relevance of generating knock-in mutations and opening the door to many possibilities for their further study.
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http://dx.doi.org/10.1242/dmm.037515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6215421PMC
October 2018

Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9.

Nucleic Acids Res 2018 09;46(17):9252

Departments of Pediatrics, Microbiology & Immunology, and Pathology, Dalhousie University, Halifax, NS, B3H 4R2, Canada.

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http://dx.doi.org/10.1093/nar/gky674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158599PMC
September 2018

Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9.

Nucleic Acids Res 2018 09;46(17):e102

Departments of Pediatrics, Microbiology & Immunology, and Pathology, Dalhousie University, Halifax, NS, B3H 4R2, Canada.

We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.
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http://dx.doi.org/10.1093/nar/gky512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158492PMC
September 2018

Etiology and functional validation of gastrointestinal motility dysfunction in a zebrafish model of CHARGE syndrome.

FEBS J 2018 06 27;285(11):2125-2140. Epub 2018 Apr 27.

Department of Pediatrics, Dalhousie University, Halifax, Canada.

CHARGE syndrome is linked to autosomal-dominant mutations in the CHD7 gene and results in a number of physiological and structural abnormalities, including heart defects, hearing and vision loss, and gastrointestinal (GI) problems. Of these challenges, GI problems have a profound impact throughout an individual's life, resulting in increased morbidity and mortality. A homolog of CHD7 has been identified in the zebrafish, the loss of which recapitulates many of the features of the human disease. Using a morpholino chd7 knockdown model complemented by a chd7 null mutant zebrafish line, we examined GI structure, innervation, and motility in larval zebrafish. Loss of chd7 resulted in physically smaller GI tracts with normal epithelial and muscular histology, but decreased and disorganized vagal projections, particularly in the foregut. chd7 morphant larvae had significantly less ability to empty their GI tract of gavaged fluorescent beads, and this condition was only minimally improved by the prokinetic agents, domperidone and erythromycin, in keeping with mixed responses to these agents in patients with CHARGE syndrome. The conserved genetics and transparency of the zebrafish have provided new insights into the consequences of chd7 gene dysfunction on the GI system and cranial nerve patterning. These findings highlight the opportunity of the zebrafish to serve as a preclinical model for studying compounds that may improve GI motility in individuals with CHARGE syndrome.
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http://dx.doi.org/10.1111/febs.14473DOI Listing
June 2018

How Surrogate and Chemical Genetics in Model Organisms Can Suggest Therapies for Human Genetic Diseases.

Genetics 2018 03;208(3):833-851

Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4R2

Genetic diseases are both inherited and acquired. Many genetic diseases fall under the paradigm of orphan diseases, a disease found in < 1 in 2000 persons. With rapid and cost-effective genome sequencing becoming the norm, many causal mutations for genetic diseases are being rapidly determined. In this regard, model organisms are playing an important role in validating if specific mutations identified in patients drive the observed phenotype. An emerging challenge for model organism researchers is the application of genetic and chemical genetic platforms to discover drug targets and drugs/drug-like molecules for potential treatment options for patients with genetic disease. This review provides an overview of how model organisms have contributed to our understanding of genetic disease, with a focus on the roles of yeast and zebrafish in gene discovery and the identification of compounds that could potentially treat human genetic diseases.
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http://dx.doi.org/10.1534/genetics.117.300124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844338PMC
March 2018

Phloridzin docosahexaenoate, a novel flavonoid derivative, suppresses growth and induces apoptosis in T-cell acute lymphoblastic leukemia cells.

Am J Cancer Res 2017 1;7(12):2452-2464. Epub 2017 Dec 1.

Department of Plant, Food, & Environmental Sciences, Faculty of Agriculture, Dalhousie UniversityTruro, NS B2N 5E3, Canada.

The overall clinical outcome in T-cell acute lymphoblastic leukemia (T-ALL) can be improved by minimizing risk for treatment failure using effective pharmacological adjuvants. Phloridzin (PZ), a flavonoid precursor found in apple peels, was acylated with docosahexaenoic acid (DHA) yielding a novel ester known as phloridzin docosahexaenoate (PZ-DHA). Here, we have studied the cytotoxic effects of PZ-DHA on human leukemia cells using and models. The inhibitory effects of PZ-DHA were tested on human Jurkat T-ALL cells in comparison to K562 chronic myeloid leukemia (CML) cells and non-malignant murine T-cells. PZ-DHA, not PZ or DHA alone, reduced cell viability and ATP levels, increased intracellular LDH release, and caused extensive morphological alterations in both Jurkat and K562 cells. PZ-DHA also inhibited cell proliferation, and selectively induced apoptosis in Jurkat and K562 cells while sparing normal murine T-cells. The cytotoxic effects of PZ-DHA on Jurkat cells were associated with caspase activation, DNA fragmentation, and selective down-regulation of STAT3 phosphorylation. PZ-DHA significantly inhibited Jurkat cell proliferation in zebrafish larvae; however, the proliferation of K562 cells was not affected . We propose that PZ-DHA-induced cytotoxic response is selective towards T-ALL in the presence of a tumor-stromal microenvironment. Prospective studies evaluating the combinatorial effects of PZ-DHA with conventional chemotherapy for T-ALL are underway.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752686PMC
December 2017

New Developments in CRISPR/Cas-based Functional Genomics and their Implications for Research Using Zebrafish.

Curr Gene Ther 2017 ;17(4):286-300

Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada.

Introduction: Genome editing using CRISPR/Cas9 has advanced very rapidly in its scope, versatility and ease of use. Zebrafish (Danio rerio) has been one of the vertebrate model species where CRISPR/Cas9 has been applied very extensively for many different purposes and with great success. In particular, disease modeling in zebrafish is useful for testing specific gene variants for pathogenicity in a preclinical setting. Here we describe multiple advances in diverse species and systems that can improve genome editing in zebrafish.

Objective: To achieve temporal and spatial precision of genome editing, many new technologies can be applied in zebrafish such as artificial transcription factors, drug-inducible or optogenetically-driven expression of Cas9, or chemically-inducible activation of Cas9. Moreover, chemically- or optogenetically- inducible reconstitution of dead Cas9 (catalytically inactive, dCas9) can enable spatiotemporal control of gene regulation. In addition to controlling where and when genome editing occurs, using oligonucleotides allows for the introduction (knock-in) of precise modifications of the genome.

Conclusion: We review recent trends to improve the precision and efficiency of oligo-based point mutation knock-ins and discuss how these improvements can apply to work in zebrafish. Similarly to how chemical mutagenesis enabled the first genetic screens in zebrafish, multiplexed sgRNA libraries and Cas9 can enable the next revolutionary transition in how genetic screens are performed in this species. We discuss the first examples and prospects of approaches using sgRNAs as specific and effective mutagens. Moreover, we have reviewed methods aimed at measuring the phenotypes of single cells after their mutagenic perturbation with vectors encoding individual sgRNAs. These methods can range from different cell-based reporters to single-cell RNA sequencing and can serve as great tools for high-throughput genetic screens.
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http://dx.doi.org/10.2174/1566523217666171121164132DOI Listing
April 2019

Enzalutamide inhibits testosterone-induced growth of human prostate cancer xenografts in zebrafish and can induce bradycardia.

Sci Rep 2017 10 31;7(1):14698. Epub 2017 Oct 31.

Department of Pathology, Dalhousie University, Halifax, Canada.

The zebrafish has become a popular human tumour xenograft model, particularly for solid tumours including prostate cancer (PCa). To date PCa xenotransplantation studies in zebrafish have not been performed in the presence of testosterone, even when employing androgen-dependent cell models, such as the LNCaP cell line. Thus, with the goal of more faithfully modelling the hormonal milieu in which PCa develops in humans, we sought to determine the effects of exogenous testosterone on the growth of LNCaP, or androgen-independent C4-2 cells xenografted into zebrafish embryos. Testosterone significantly increased engrafted LNCaP proliferation compared to control xenografts, which could be inhibited by co-administration of the anti-androgen receptor drug, enzalutamide. By contrast, C4-2 cell growth was not affected by either testosterone or enzalutamide. Enzalutamide also induced bradycardia and death in zebrafish embryos in a dose-dependent manner and strongly synergized with the potassium-channel blocking agent, terfenadine, known to induce long QT syndrome and cardiac arrhythmia. Together, these data not only indicate that testosterone administration should be considered in all PCa xenograft studies in zebrafish but also highlights the unique opportunity of this preclinical platform to simultaneously evaluate efficacy and toxicity of novel therapies and/or protective agents towards developing safer and more effective PCa treatments.
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http://dx.doi.org/10.1038/s41598-017-14413-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5665934PMC
October 2017

hace1 Influences zebrafish cardiac development via ROS-dependent mechanisms.

Dev Dyn 2018 02 27;247(2):289-303. Epub 2017 Oct 27.

Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada.

Background: In this study, we reveal a previously undescribed role of the HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1) tumor suppressor protein in normal vertebrate heart development using the zebrafish (Danio rerio) model. We examined the link between the cardiac phenotypes associated with hace1 loss of function to the expression of the Rho small family GTPase, rac1, which is a known target of HACE1 and promotes ROS production via its interaction with NADPH oxidase holoenzymes.

Results: We demonstrate that loss of hace1 in zebrafish via morpholino knockdown results in cardiac deformities, specifically a looping defect, where the heart is either tubular or "inverted". Whole-mount in situ hybridization of cardiac markers shows distinct abnormalities in ventricular morphology and atrioventricular valve formation in the hearts of these morphants, as well as increased expression of rac1. Importantly, this phenotype appears to be directly related to Nox enzyme-dependent ROS production, as both genetic inhibition by nox1 and nox2 morpholinos or pharmacologic rescue using ROS scavenging agents restores normal cardiac structure.

Conclusions: Our study demonstrates that HACE1 is critical in the normal development and proper function of the vertebrate heart via a ROS-dependent mechanism. Developmental Dynamics 247:289-303, 2018. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/dvdy.24600DOI Listing
February 2018

Validation of PAPSS1 (3'-phosphoadenosine 5'-phosphosulfate synthase 1) as a Cisplatin-sensitizing Therapeutic Target.

Clin Cancer Res 2017 Nov 8;23(21):6555-6566. Epub 2017 Aug 8.

Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.

Our previous screening efforts found that inhibition of PAPSS1 increases the potency of DNA-damaging agents in non-small cell lung cancer (NSCLC) cell lines. Here, we explored the clinical relevance of PAPSS1 and further investigated it as a therapeutic target in preclinical model systems. PAPSS1 expression and cisplatin IC values were assessed in 52 lung adenocarcinoma cell lines. Effects of PAPSS1 inhibition on A549 cisplatin sensitivity under hypoxic and starvation conditions, in 3D spheroids, as well as in zebrafish and mouse xenografts, were evaluated. Finally, the association between PAPSS1 expression levels and survival in patients treated with standard chemotherapy was assessed. Our results show a positive correlation between low PAPSS1 expression and increased cisplatin sensitivity in lung adenocarcinoma. , the potentiation effect was greatest when A549 cells were serum-starved under hypoxic conditions. When treated with low-dose cisplatin, PAPSS1-deficient A549 spheroids showed a 58% reduction in size compared with control cells. , PAPSS1 suppression and low-dose cisplatin treatment inhibited proliferation of lung tumor cells in zebrafish xenografts and significantly delayed development of subcutaneous tumors in mice. Clinical data suggest that NSCLC and ovarian cancer patients with low PAPSS1 expression survive longer following platinum-based chemotherapy. These results suggest that PAPSS1 inhibition enhances cisplatin activity in multiple preclinical model systems and that low PAPSS1 expression may serve as a biomarker for platin sensitivity in cancer patients. Developing strategies to target PAPSS1 activity in conjunction with platinum-based chemotherapy may offer an approach to improving treatment outcomes. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-0700DOI Listing
November 2017

Improved outcomes for myeloid leukemia of Down syndrome: a report from the Children's Oncology Group AAML0431 trial.

Blood 2017 06 7;129(25):3304-3313. Epub 2017 Apr 7.

Division of Hematology/Oncology, Children's Mercy Hospitals and Clinics, Kansas City, MO.

Patients with myeloid leukemia of Down syndrome (ML-DS) have favorable event-free survival (EFS), but experience significant treatment-related morbidity and mortality. ML-DS blast cells ex vivo have increased sensitivity to cytarabine (araC) and daunorubicin, suggesting that optimizing drug dosing may improve outcomes while reducing toxicity. The Children's Oncology Group (COG) AAML0431 trial consisted of 4 cycles of induction and 2 cycles of intensification therapy based on the treatment schema of the previous COG A2971 trial with several modifications. High-dose araC (HD-araC) was used in the second induction cycle instead of the intensification cycle, and 1 of 4 daunorubicin-containing induction cycles was eliminated. For 204 eligible patients, 5-year EFS was 89.9% and overall survival (OS) was 93.0%. The 5-year OS for 17 patients with refractory/relapsed leukemia was 34.3%. We determined the clinical significance of minimal residual disease (MRD) levels as measured by flow cytometry on day 28 of induction I. MRD measurements, available for 146 of the 204 patients, were highly predictive of treatment outcome; 5-year disease-free survival for MRD-negative patients (n = 125) was 92.7% vs 76.2% for MRD-positive patients (n = 21) (log-rank = .011). Our results indicated that earlier use of HD-araC led to better EFS and OS in AAML0431 than in past COG studies. A 25% reduction in the cumulative daunorubicin dose did not impact outcome. MRD, identified as a new prognostic factor for ML-DS patients, can be used for risk stratification in future clinical trials. This trial was registered at www.clinicaltrials.gov as #NCT00369317.
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http://dx.doi.org/10.1182/blood-2017-01-764324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5482102PMC
June 2017

A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish.

Dis Model Mech 2017 06 9;10(6):811-822. Epub 2017 Mar 9.

Department of Pediatrics, Dalhousie University, Halifax, NS, Canada B3K 6R8

Clustered regularly interspaced palindromic repeats (CRISPR)/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish () to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in , and genes. An insertion of seven nucleotides in resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in , suggesting activity of alternative translation sites in the other two mutants even though exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible.
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http://dx.doi.org/10.1242/dmm.026765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5483001PMC
June 2017

FISH identifies a KAT6A/CREBBP fusion caused by a cryptic insertional t(8;16) in a case of spontaneously remitting congenital acute myeloid leukemia with a normal karyotype.

Pediatr Blood Cancer 2017 Aug 18;64(8). Epub 2017 Jan 18.

Department of Pediatrics, IWK Health Centre/Dalhousie University, Halifax, Nova Scotia, Canada.

Cytogenetics can inform risk stratification in pediatric acute myeloid leukemia (AML). We describe the first case of a newborn with leukemia cutis found to have AML harboring a cryptic insertional t(8;16)(p11.2;p13.3) with associated KAT6A/CREBBP fusion identified exclusively by fluorescence in situ hybridization (FISH). Expectant management resulted in spontaneous leukemia resolution. The identification of t(8;16)(p11.2;p13.3) may serve as a biomarker for spontaneous remission in congenital AML. FISH for this translocation is warranted in congenital AML with a normal karyotype, and patients with KAT6A/CREBBP fusion should be conservatively managed. While 50% of spontaneously remitting congenital AML with t(8;16)(p11.2;p13.3) may recur, high salvage rates are attained with standard therapy.
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http://dx.doi.org/10.1002/pbc.26450DOI Listing
August 2017

Roles for APRIN (PDS5B) in homologous recombination and in ovarian cancer prediction.

Nucleic Acids Res 2016 12 24;44(22):10879-10897. Epub 2016 Oct 24.

Genome Stability Laboratory, CHU de Québec Research Center, HDQ Pavilion, Oncology Axis, 9 McMahon, Québec City, QC G1R 2J6, Canada

APRIN (PDS5 cohesin associated factor B) interacts with both the cohesin complex and the BRCA2 tumor suppressor. How APRIN influences cohesion and DNA repair processes is not well understood. Here, we show that APRIN is recruited to DNA damage sites. We find that APRIN interacts directly with RAD51, PALB2 and BRCA2. APRIN stimulates RAD51-mediated DNA strand invasion. APRIN also binds DNA with an affinity for D-loop structures and single-strand (ss) DNA. APRIN is a new homologous recombination (HR) mediator as it counteracts the RPA inhibitory effect on RAD51 loading to ssDNA. We show that APRIN strongly improves the annealing of complementary-strand DNA and that it can stimulate this process in synergy with BRCA2. Unlike cohesin constituents, its depletion has no impact on class switch recombination, supporting a specific role for this protein in HR. Furthermore, we show that low APRIN expression levels correlate with a better survival in ovarian cancer patients and that APRIN depletion sensitizes cells to the PARP inhibitor Olaparib in xenografted zebrafish. Our findings establish APRIN as an important and specific actor of HR, with cohesin-independent functions.
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http://dx.doi.org/10.1093/nar/gkw921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5159559PMC
December 2016

Using zebrafish models of leukemia to streamline drug screening and discovery.

Exp Hematol 2017 Jan 6;45:1-9. Epub 2016 Oct 6.

Department of Pediatrics, IWK Health Centre, Halifax, Nova Scotia, Canada; Departments of Microbiology and Immunology and Pathology, Dalhousie University, Halifax, Nova Scotia, Canada. Electronic address:

Current treatment strategies for acute leukemias largely rely on nonspecific cytotoxic drugs that result in high therapy-related morbidity and mortality. Cost-effective, pertinent animal models are needed to link in vitro studies with the development of new therapeutic agents in clinical trials on a high-throughput scale. However, targeted therapies have had limited success moving from bench to clinic, often due to unexpected off-target effects. The zebrafish has emerged as a reliable in vivo tool for modeling human leukemia. Zebrafish genetic and xenograft models of acute leukemia provide an unprecedented opportunity to conduct rapid, phenotype-based screens. This allows for the identification of relevant therapies while simultaneously evaluating drug toxicity, thus circumventing the limitations of target-centric approaches.
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http://dx.doi.org/10.1016/j.exphem.2016.09.012DOI Listing
January 2017

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

Modeling Leukemogenesis in the Zebrafish Using Genetic and Xenograft Models.

Methods Mol Biol 2016 ;1451:171-89

Department of Microbiology and Immunology, Dalhousie University, Halifax, Canada, B3H 4R2.

The zebrafish is a widely accepted model to study leukemia. The major advantage of studying leukemogenesis in zebrafish is attributed to its short life cycle and superior imaging capacity. This chapter highlights using transgenic- and xenograft-based models in zebrafish to study a specific leukemogenic mutation and analyze therapeutic responses in vivo.
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http://dx.doi.org/10.1007/978-1-4939-3771-4_12DOI Listing
January 2018
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