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Zebrafish ( Danio rerio ) are a good model for cancer research including studies on chemotherapy treatments. We treated wild-type and miR-34a deletion mutant zebrafish embryos at 24 h post-fertilization with 1 µM of the topoisomerase I inhibitor, camptothecin (CPT), for 4 h to catalogue gene expression changes induced by this DNA damage treatment and to understand if these changes are influenced by loss of miR-34a. The 4 sample groups of 3 independent biological samples consisting of 30 embryos each were analyzed by RNA-sequencing using the recently updated zebrafish transcriptome annotation based on GRCz11, which enabled a more complete and sensitive read mapping and gene assignment than standard annotations. Using this gene expression estimates dataset as the primary resource, we performed a differentially expressed gene (DEG) analysis based on treatment as loss of miR-34a had minimal effects on CPT-induced expression changes. The DEGs were analyzed for Gene Ontology and KEGG pathway terms. Enriched terms and pathways among up-regulated genes were mostly related to stress, cell death, cell cycle regulation, transcriptional regulation, cell signalling, developmental processes and synthesis of retinol and steroid hormones. By contrast, down-regulated genes were most strongly associated with genes involved in key developmental processes, adhesion molecules, as well as some transport and metabolic pathways, together suggesting a "developmental shutdown". We also identified interferon-regulated genes and p53 target genes activated or inhibited by DNA damage due to topoisomerase I inhibition, suggesting that they are important components of the response to this type of DNA damage in zebrafish embryos., (© 2024 The Author(s).)

Mutations in the DNAJC21 gene were recently described in Shwachman-Diamond syndrome (SDS), a bone marrow failure syndrome with high predisposition for myeloid malignancies. To study the underlying biology in hematopoiesis regulation and disease, we generated the first in vivo model of Dnajc21 deficiency using the zebrafish. Zebrafish dnajc21 mutants phenocopy key SDS patient phenotypes such as cytopenia, reduced growth, and defective protein synthesis. We show that cytopenia results from impaired hematopoietic differentiation, accumulation of DNA damage, and reduced cell proliferation. The introduction of a biallelic tp53 mutation in the dnajc21 mutants leads to the development of myelodysplastic neoplasia-like features defined by abnormal erythroid morphology and expansion of hematopoietic progenitors. Using transcriptomic and metabolomic analyses, we uncover a novel role for Dnajc21 in nucleotide metabolism. Exogenous nucleoside supplementation restores neutrophil counts, revealing an association between nucleotide imbalance and neutrophil differentiation, suggesting a novel mechanism in dnajc21-mutant SDS biology., (© 2024. The Author(s).)

Li-Fraumeni syndrome is caused by inherited TP53 tumor suppressor gene mutations. MicroRNA miR-34a is a p53 target and modifier gene. Interestingly, miR-34 triple-null mice exhibit normal p53 responses and no overt cancer development, but the lack of miR-34 promotes tumorigenesis in cancer-susceptible backgrounds. miR-34 genes are highly conserved and syntenic between zebrafish and humans. Zebrafish miR-34a and miR-34b/c have similar expression timing in development, but miR-34a is more abundant. DNA damage by camptothecin led to p53-dependent induction of miR-34 genes, while miR-34a mutants were adult-viable and had normal DNA damage-induced apoptosis. Nevertheless, miR-34a-/- compound mutants with a gain-of-function tp53R217H/ R217H or tp53-/- mutants were more cancer-prone than tp53 mutants alone, confirming the tumor-suppressive function of miR-34a. Through transcriptomic comparisons at 28 hours post-fertilization (hpf), we characterized DNA damage-induced transcription, and at 8, 28 and 72 hpf we determined potential miR-34a-regulated genes. At 72 hpf, loss of miR-34a enhanced erythrocyte levels and up-regulated myb-positive hematopoietic stem cells. Overexpression of miR-34a suppressed its reporter mRNA, but not p53 target induction, and sensitized injected embryos to camptothecin but not to γ-irradiation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Prykhozhij et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction or knock-in of precise genomic modifications remains one of the most important applications of CRISPR/Cas9 in all model systems including zebrafish. The most widely used type of donor template containing the desired modification is single-stranded DNA (ssDNA), either in the form of single-stranded oligodeoxynucleotides (ssODN) (<150 nucleotides (nt)) or as long ssDNA (lssDNA) molecules (up to about 2000 nt). Despite the challenges posed by DNA repair after DNA double-strand breaks, knock-in of precise mutations is relatively straightforward in zebrafish. Knock-in efficiency can be enhanced by careful donor template design, using lssDNA as template or tethering the donor template DNA to the Cas9-guide RNA complex. Other point mutation methods such as base editing and prime editing are starting to be applied in zebrafish and many other model systems. However, these methods may not always be sufficiently accessible or may have limited capacity to perform all desired mutation knock-ins which are possible with ssDNA-based knock-in methods. Thus, it is likely that there will be complementarity in the technologies used for generating precise mutants. Here, we review and describe a suite of CRISPR/Cas9 knock-in procedures utilizing ssDNA as the donor template in zebrafish, point out the potential challenges and suggest possible approaches for their solution ultimately leading to successful generation of precise mutant lines., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

KIT, a type III tyrosine kinase receptor, plays a crucial role in haematopoietic development. The KIT receptor forms a dimer after ligand binding; this activates tyrosine kinase activity leading to downstream signal transduction. The D816V KIT mutation is extensively implicated in haematological malignancies, including mastocytosis and leukaemia. KIT D816V is constitutively active, but the molecular nuances that lead to constitutive tyrosine kinase activity are unclear. For the first time, we present experimental evidence that the KIT D816V mutant does not dimerize like KIT wild type. We further show evidence of decreased stabilization of the tyrosine kinase domain in the KIT D816V mutant, a phenomenon that might contribute to its constitutive activity. Since the mechanism of KIT D816V activation varies from that of the wild type, we explored downstream signal transduction events and found that even though KIT D816V targets similar signalling moieties, the signalling is amplified in the mutant compared to stem cell factor-activated wild type receptor. Uniquely, KIT D816V induces infection-related pathways and the spliceosome pathway, providing alternate options for selective as well as combinatorial therapeutic targeting., (© 2022 British Society for Haematology and John Wiley & Sons Ltd.)

Lymphangioleiomyomatosis (LAM) is a rare disease involving cystic lung destruction by invasive LAM cells. These cells harbor loss-of-function mutations in TSC2, conferring hyperactive mTORC1 signaling. Here, tissue engineering tools are employed to model LAM and identify new therapeutic candidates. Biomimetic hydrogel culture of LAM cells is found to recapitulate the molecular and phenotypic characteristics of human disease more faithfully than culture on plastic. A 3D drug screen is conducted, identifying histone deacetylase (HDAC) inhibitors as anti-invasive agents that are also selectively cytotoxic toward TSC2 -/- cells. The anti-invasive effects of HDAC inhibitors are independent of genotype, while selective cell death is mTORC1-dependent and mediated by apoptosis. Genotype-selective cytotoxicity is seen exclusively in hydrogel culture due to potentiated differential mTORC1 signaling, a feature that is abrogated in cell culture on plastic. Importantly, HDAC inhibitors block invasion and selectively eradicate LAM cells in vivo in zebrafish xenografts. These findings demonstrate that tissue-engineered disease modeling exposes a physiologically relevant therapeutic vulnerability that would be otherwise missed by conventional culture on plastic. This work substantiates HDAC inhibitors as possible therapeutic candidates for the treatment of patients with LAM and requires further study., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

CHARGE syndrome typically results from mutations in the gene encoding chromodomain helicase DNA-binding protein 7 ( CHD7 ). CHD7 is involved in regulating neural crest development, which gives rise to tissues of the skull/face and the autonomic nervous system (ANS). Individuals with CHARGE syndrome are frequently born with anomalies requiring multiple surgeries and often experience adverse events post-anesthesia, including oxygen desaturations, decreased respiratory rates, and heart rate abnormalities. Central congenital hypoventilation syndrome (CCHS) affects ANS components that regulate breathing. Its hallmark feature is hypoventilation during sleep, clinically resembling observations in anesthetized CHARGE patients. Loss of PHOX2B (paired-like homeobox 2b) underlies CCHS. Employing a chd7 -null zebrafish model, we investigated physiologic responses to anesthesia and compared these to loss of phox2b . Heart rates were lower in chd7 mutants compared to the wild-type. Exposure to tricaine, a zebrafish anesthetic/muscle relaxant, revealed that chd7 mutants took longer to become anesthetized, with higher respiratory rates during recovery. chd7 mutant larvae demonstrated unique phox2ba expression patterns. The knockdown of phox2ba reduced larval heart rates similar to chd7 mutants. chd7 mutant fish are a valuable preclinical model to investigate anesthesia in CHARGE syndrome and reveal a novel functional link between CHARGE syndrome and CCHS.

While MYCN expression is an important contributing factor to heterogeneity in the natural history of neuroblastoma (NBL), a mechanistic understanding of this often mutationally quiet tumor has remained elusive. In this issue of the JCI, Weichert-Leahey and authors focused on the adrenergic and mesenchymal core regulatory circuitries (CRC) as NBL transcriptional programs. The authors previously showed that overexpression of LIM-domain-only 1 (LMO1), a transcriptional coregulator, synergizes with MYCN to accelerate tumor formation and metastasis in an NBL-zebrafish model. They now demonstrate experimentally, using genome-edited zebrafish, that a polymorphism in the human rs2168101 locus of the LMO1 gene determines which CRC is active in a tumor. In some cases, LMO3 compensated for LMO1 loss and drove the adrenergic CRC in MYCN-positive NBL. This study exemplifies the value of evolutionary relationships and zebrafish models in the investigation of human disease and reveals pathways of NBL development that may affect prevention or intervention strategies.

Hyperactive sphingosine 1-phosphate (S1P) signaling is associated with a poor prognosis of triple-negative breast cancer (TNBC). Despite recent evidence that links the S1P receptor 1 (S1P1) to TNBC cell survival, its role in TNBC invasion and the underlying mechanisms remain elusive. Combining analyses of human TNBC cells with zebrafish xenografts, we found that phosphorylation of S1P receptor 1 (S1P1) at threonine 236 (T236) is critical for TNBC dissemination. Compared to luminal breast cancer cells, TNBC cells exhibit a significant increase of phospho-S1P1 T236 but not the total S1P1 levels. Misexpression of phosphorylation-defective S1P1 T236A (alanine) decreases TNBC cell migration in vitro and disease invasion in zebrafish xenografts. Pharmacologic disruption of S1P1 T236 phosphorylation, using either a pan-AKT inhibitor (MK2206) or an S1P1 functional antagonist (FTY720, an FDA-approved drug for treating multiple sclerosis), suppresses TNBC cell migration in vitro and tumor invasion in vivo. Finally, we show that human TNBC cells with AKT activation and elevated phospho-S1P1 T236 are sensitive to FTY720-induced cytotoxic effects. These findings indicate that the AKT-enhanced phosphorylation of S1P1 T236 mediates much of the TNBC invasiveness, providing a potential biomarker to select TNBC patients for the clinical application of FTY720.

Limb-Girdle Muscular Dystrophy Type R1 (LGMDR1; formerly LGMD2A), characterized by progressive hip and shoulder muscle weakness, is caused by mutations in CAPN3 . In zebrafish, capn3b mediates Def-dependent degradation of p53 in the liver and intestines. We show that capn3b is expressed in the muscle. To model LGMDR1 in zebrafish, we generated three deletion mutants in capn3b and a positive-control dmd mutant (Duchenne muscular dystrophy). Two partial deletion mutants showed transcript-level reduction, whereas the RNA-less mutant lacked capn3b mRNA. All capn3b homozygous mutants were developmentally-normal adult-viable animals. Mutants in dmd were homozygous-lethal. Bathing wild-type and capn3b mutants in 0.8% methylcellulose (MC) for 3 days beginning 2 days post-fertilization resulted in significantly pronounced (20-30%) birefringence-detectable muscle abnormalities in capn3b mutant embryos. Evans Blue staining for sarcolemma integrity loss was strongly positive in dmd homozygotes, negative in wild-type embryos, and negative in MC-treated capn3b mutants, suggesting membrane instability is not a primary muscle pathology determinant. Increased birefringence-detected muscle abnormalities in capn3b mutants compared to wild-type animals were observed following induced hypertonia by exposure to cholinesterase inhibitor, azinphos-methyl, reinforcing the MC results. These mutant fish represent a novel tractable model for studying the mechanisms underlying muscle repair and remodeling, and as a preclinical tool for whole-animal therapeutics and behavioral screening in LGMDR1.

Primary atopic disorders are a group of inborn errors of immunity that skew the immune system toward severe allergic disease. Defining the biology underlying these extreme monogenic phenotypes reveals shared mechanisms underlying common polygenic allergic disease and identifies potential drug targets. Germline gain-of-function (GOF) variants in JAK1 are a cause of severe atopy and eosinophilia. Modeling the JAK1GOF (p.A634D) variant in both zebrafish and human induced pluripotent stem cells (iPSCs) revealed enhanced myelopoiesis. RNA-Seq of JAK1GOF human whole blood, iPSCs, and transgenic zebrafish revealed a shared core set of dysregulated genes involved in IL-4, IL-13, and IFN signaling. Immunophenotypic and transcriptomic analysis of patients carrying a JAK1GOF variant revealed marked Th cell skewing. Moreover, long-term ruxolitinib treatment of 2 children carrying the JAK1GOF (p.A634D) variant remarkably improved their growth, eosinophilia, and clinical features of allergic inflammation. This work highlights the role of JAK1 signaling in atopic immune dysregulation and the clinical impact of JAK1/2 inhibition in treating eosinophilic and allergic disease.

Despite numerous therapeutic options, multidrug resistance (MDR) remains an obstacle to successful breast cancer therapy. Jadomycin B, a natural product derived from Streptomyces venezuelae ISP5230, maintains cytotoxicity in MDR human breast cancer cells. Our objectives were to evaluate the pharmacokinetics, toxicity, anti-tumoral, and anti-metastatic effects of jadomycin B in zebrafish larvae and mice. In a zebrafish larval xenograft model, jadomycin B significantly reduced the proliferation of human MDA-MB-231 cells at or below its maximum tolerated dose (40 µm). In female Balb/C mice, a single intraperitoneal dose (6 mg/kg) was rapidly absorbed with a maximum serum concentration of 3.4 ± 0.27 µm. Jadomycin B concentrations declined biphasically with an elimination half-life of 1.7 ± 0.058 h. In the 4T1 mouse mammary carcinoma model, jadomycin B (12 mg/kg every 12 h from day 6 to 15 after tumor cell injection) decreased primary tumor volume compared to vehicle control. Jadomycin B-treated mice did not exhibit weight loss, nor significant increases in biomarkers of impaired hepatic (alanine aminotransferase) and renal (creatinine) function. In conclusion, jadomycin B demonstrated a good safety profile and provided partial anti-tumoral effects, warranting further dose-escalation safety and efficacy studies in MDR breast cancer models.

Zebrafish offer an excellent tool for studying the vertebrate hematopoietic system thanks to a highly conserved and rapidly developing hematopoietic program, genetic amenability, optical transparency, and experimental accessibility. Zebrafish studies have contributed to our understanding of hematopoiesis, a complex process regulated by signaling cues, inflammation being crucial among them. Hematopoietic stem cells (HSCs) are multipotent cells producing all the functional blood cells, including immune cells. HSCs respond to inflammation during infection and malignancy by proliferating and producing the blood cells in demand for a specific scenario. We first focus on how inflammation plays a crucial part in steady-state HSC development and describe the critical role of the inflammasome complex in regulating HSC expansion and balanced lineage production. Next, we review zebrafish studies of inflammatory innate immune mechanisms focusing on interferon signaling and the downstream JAK-STAT pathway. We also highlight insights gained from zebrafish models harbouring genetic perturbations in the role of inflammation in hematopoietic disorders such as bone marrow failure, myelodysplastic syndrome, and myeloid leukemia. Indeed, inflammation has been recently identified as a potential driver of clonal hematopoiesis and leukemogenesis, where cells acquire somatic mutations that provide a proliferative advantage in the presence of inflammation. Important insights in this area come from mutant zebrafish studies showing that hematopoietic differentiation can be compromised by epigenetic dysregulation and the aberrant induction of signaling pathways., Competing Interests: JB is a member of the Scientific Advisory Board of Oxford Immune Algorithmics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ketharnathan, Rajan, Prykhozhij and Berman.)

Oral metronomic topotecan represents a novel approach to chemotherapy delivery which, in preclinical models, may work synergistically with pazopanib in targeting angiogenesis. A phase I and pharmacokinetic (PK) study of this combination was performed in children with relapsed/refractory solid tumors. Oral topotecan and pazopanib were each administered daily without interruption in 28-day cycles at five dose levels (0.12 to 0.3 mg/m 2 topotecan and 125 to 160 mg/m 2 pazopanib powder for oral suspension (PfOS)), with dose escalation in accordance with the rolling-six design. PK studies were performed on day 1 and at steady state. Thirty patients were enrolled, with 26 evaluable for dose-limiting toxicity (DLT), with median age 12 years (3-20). Toxicities were generally mild; the most common grade 3/4 adverse events related to protocol therapy were neutropenia (18%), thrombocytopenia (11%), lymphopenia (11%), AST elevation (11%), and lipase elevation (11%). Only two cycle 1 DLTs were observed on study, both at the 0.3/160 mg/m 2 dose level comprising persistent grade 3 thrombocytopenia and grade 3 ALT elevation. No AEs experienced beyond cycle 1 required treatment discontinuation. The best response was stable disease in 10/25 patients (40%) for a median duration of 6.4 (1.7-45.1) months. The combination of oral metronomic topotecan and pazopanib is safe and tolerable in pediatric patients with solid tumors, with a recommended phase 2 dose of 0.22 mg/m 2 topotecan and 160 mg/m 2 pazopanib. No objective responses were observed in this heavily pre-treated patient population, although 40% did achieve stable disease for a median of 6 months. While this combination is likely of limited benefit for relapsed disease, it may play a role in the maintenance setting.

TET2 loss-of-function mutations are recurrent events in a wide range of hematological malignancies and a physiologic occurrence in blood cells of healthy older adults. It is currently unknown what determines if a person harboring a somatic TET2 mutation will progress to myelodysplastic syndrome or acute myeloid leukemia. Here we develop a zebrafish tet2 mutant through which we show that tet2 loss leads to restricted hematopoietic differentiation combined with a modest upregulation of p53, which is also characteristic of many inherited bone marrow failure syndromes. Uniquely in the context of emergency hematopoiesis by external stimuli, such as infection or cytokine stimulation, lack of tet2 leads hematopoietic stem cells to undergo excessive proliferation, resulting in an accumulation of immature cells, which are poised to become leukemogenic following additional genetic/epigenetic perturbations. This same phenomenon observed in zebrafish extends to human hematopoietic stem cells, identifying TET2 as a critical relay switch in the context of stress hematopoiesis., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Radiotherapy (RT) is an effective cancer treatment modality, but standard RT often causes collateral damage to nearby healthy tissues. To increase therapeutic ratio, radiosensitization via gold nanoparticles (GNPs) has been shown to be effective. One challenge is that megavoltage beams generated by clinical linear accelerators are poor initiators of the photoelectric effect. Previous computer models predicted that a diamond target beam (DTB) will yield 400% more low-energy photons, increasing the probability of interacting with GNPs to enhance the radiation dose by 7.7-fold in the GNP vicinity. After testing DTB radiation coupled with GNPs in multiple cell types, we demonstrate decreased head-and-neck cancer (HNC) cell viability in vitro and enhanced cell-killing in zebrafish xenografts compared to standard RT. HNC cell lines also displayed increased double-stranded DNA breaks with DTB irradiation in the presence of GNPs. This study presents preclinical responses to GNP-enhanced radiotherapy with the novel DTB, providing the first functional data to support the theoretical evidence for radiosensitization via GNPs in this context, and highlighting the potential of this approach to optimize the efficacy of RT in anatomically difficult-to-treat tumors., (© 2022. The Author(s).)

Myeloid leukemia in children with Down syndrome (ML-DS) is associated with young age and somatic GATA1 mutations. Because of 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- patients treated with HD-AraC on AAML0431 (P = .0002). Overall survival at 2 years was 91.0% (95% CI, 83.8-95.0). Twelve SR patients relapsed, mostly within 1 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 with those who did not (36% vs 9%; P = .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 = .2682). Patients with SR ML-DS had worse outcomes without HD-AraC after risk classification based on flow cytometric MRD., (© 2021 by The American Society of Hematology.)

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., Competing Interests: Declaration of interests None of the authors declare a conflict of interest for this project but report the following relationships. N.V.D. is a current employee of Genentech, Inc., a member of the Roche Group. R.A.Y. is a founder and shareholder of Syros Pharmaceuticals, Camp4 Therapeutics, Omega Therapeutics, and Dewpoint Therapeutics. M.J.A. has financial interests in Monitor Biotechnologies (formerly known as Beacon Genomics). K.S. receives grant funding as part of the DFCI/Novartis Drug Discovery Program, consults for and has stock options in Auron Therapeutics, and consulted for AstraZeneca and Kronos Bio. B.D.C. receives research funding from Gradalis for an unrelated project, and his spouse was previously employed by Shire and Mersana and currently works for Acceleron., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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. In vivo 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 ( Danio rerio ) 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 in vivo 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kobar, Collett, Prykhozhij and Berman.)

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.

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., Competing Interests: JW, NM, MS, OP, SL, BO, BR, JB No competing interests declared, SS Affiliated with Appili Therapeutics Inc. The author has no financial interests to declare, (© 2020, Wertman et al.)

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, GATA1 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.

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., Competing Interests: Declarations of Interest The authors declare no competing interests., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

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., (© 2019 American Cancer Society.)

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.

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 in vivo 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 in vivo , 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.

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., (© 2019 Wiley Periodicals, Inc.)

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 in vitro 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., (©2019 American Association for Cancer Research.)

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., (© 2018. Published by The Company of Biologists Ltd.)

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., (© 2018 Federation of European Biochemical Societies.)

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., (Copyright © 2018 by the Genetics Society of America.)

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., (© 2017 Wiley Periodicals, Inc.)

Anoikis acts as a critical barrier to metastasis by inducing cell death upon cancer cell detachment from the extracellular matrix (ECM), thereby preventing tumor cell dissemination to secondary sites. The induction of anoikis requires the lysosomal-mediated downregulation of epidermal growth factor receptors (EGFRs) leading to termination of pro-survival signaling. In this study, we demonstrate that depletion of pre-mRNA splicing factor 4 kinase (PRP4K; also known as PRPF4B) causes dysregulation of EGFR trafficking and anoikis resistance. We also report a novel cytoplasmic localization of PRP4K at the late endosome, and demonstrate both nuclear and cytoplasmic localization in breast, lung and ovarian cancer tissue. Mechanistically, depletion of PRP4K leads to reduced EGFR degradation following cell detachment from the ECM and correlates with increased TrkB, vimentin and Zeb1 expression. As a result, PRP4K loss promotes sustained growth factor signaling and increased cellular resistance to anoikis in vitro and in a novel zebrafish xenotransplantation model of anoikis sensitivity, as well as increased metastasis in a mouse model of ovarian cancer. Thus, PRP4K may serve as a potential biomarker of anoikis sensitivity in ovarian and other epithelial cancers.

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 in vitro and in vivo 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 in vivo . 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.

Purpose: 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. Experimental Design: PAPSS1 expression and cisplatin IC 50 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. Results: Our results show a positive correlation between low PAPSS1 expression and increased cisplatin sensitivity in lung adenocarcinoma. In vitro , 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. In vivo , 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. Conclusions: 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. Clin Cancer Res; 23(21); 6555-66. ©2017 AACR ., (©2017 American Association for Cancer Research.)

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.

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., (© 2017 Wiley Periodicals, Inc.)

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 P = .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., (© 2017 by The American Society of Hematology.)

Clustered regularly interspaced palindromic repeats (CRISPR)/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish ( Danio rerio ) 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 pycr1a , chd7 and hace1 genes. An insertion of seven nucleotides in pycr1a 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 chd7 , suggesting activity of alternative translation sites in the other two mutants even though pycr1a 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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

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., (Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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 ( zap70 y442 ) that was created using transcription activator-like effector nucleases (TALENs). In contrast to what has been reported for morphant zebrafish, zap70 y442 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 zap70 y442 mutant animals. T cell immune deficiency was confirmed through transplantation of unmatched normal and malignant donor cells into zap70 y442 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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR associated protein 9 (Cas9) system discovered as an adaptive immunity mechanism in prokaryotes has emerged as the most popular tool for the precise alterations of the genomes of diverse species. CRISPR/Cas9 system has taken the world of genome editing by storm in recent years. Its popularity as a tool for altering genomes is due to the ability of Cas9 protein to cause double-stranded breaks in DNA after binding with short guide RNA molecules, which can be produced with dramatically less effort and expense than required for production of transcription-activator like effector nucleases (TALEN) and zinc-finger nucleases (ZFN). This system has been exploited in many species from prokaryotes to higher animals including human cells as evidenced by the literature showing increasing sophistication and ease of CRISPR/Cas9 as well as increasing species variety where it is applicable. This technology is poised to solve several complex molecular biology problems faced in life science research including cancer research. In this review, we highlight the recent advancements in CRISPR/Cas9 system in editing genomes of prokaryotes, fungi, plants and animals and provide details on software tools available for convenient design of CRISPR/Cas9 targeting plasmids. We also discuss the future prospects of this advanced molecular technology., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)