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Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease driven by gain-of-function variants in activin receptor-like kinase 2 (ALK2), the most common variant being ALK2 R206H . In FOP, ALK2 variants display increased and dysregulated signaling through the bone morphogenetic protein (BMP) pathway resulting in progressive and permanent replacement of skeletal muscle and connective tissues with heterotopic bone, ultimately leading to severe debilitation and premature death. Here, we describe the discovery of BLU-782 (IPN60130), a small-molecule ALK2 R206H inhibitor developed for the treatment of FOP. A small-molecule library was screened in a biochemical ALK2 binding assay to identify potent ALK2 binding compounds. Iterative rounds of structure-guided drug design were used to optimize compounds for ALK2 R206H binding, ALK2 selectivity, and other desirable pharmacokinetic properties. BLU-782 preferentially bound to ALK2 R206H with high affinity, inhibiting signaling from ALK2 R206H and other rare FOP variants in cells in vitro without affecting signaling of closely related homologs ALK1, ALK3, and ALK6. In vivo efficacy of BLU-782 was demonstrated using a conditional knock-in ALK2 R206H mouse model, where prophylactic oral dosing reduced edema and prevented cartilage and heterotopic ossification (HO) in both muscle and bone injury models. BLU-782 treatment preserved the normal muscle-healing response in ALK2 R206H mice. Delayed dosing revealed a short 2-day window after injury when BLU-782 treatment prevented HO in ALK2 R206H mice, but dosing delays of 4 days or longer abrogated HO prevention. Together, these data suggest that BLU-782 may be a candidate for prevention of HO in FOP.

Background and Aims: Fibrolamellar carcinoma (FLC) is a rare, difficult-to-treat liver cancer primarily affecting pediatric and adolescent patients, and for which precision medicine approaches have historically not been possible. The DNAJB1-PRKACA gene fusion was identified as a driver of FLC pathogenesis. We aimed to assess whether FLC tumors maintain dependency on this gene fusion and determine if PRKACA is a viable therapeutic target., Methods: FLC patient-derived xenograft (PDX) shRNA cell lines were implanted subcutaneously into female NOD-SCID mice and tumors were allowed to develop prior to randomization to doxycycline (to induce knockdown) or control groups. Tumor development was assessed every 2 days. To assess the effect of treatment with novel selective PRKACA small molecule kinase inhibitors, BLU0588 and BLU2864, FLC PDX tumor cells were implanted subcutaneously into NOD-SCID mice and tumors allowed to develop. Mice were randomized to treatment (BLU0588 and BLU2864, orally, once daily) or control groups and tumor size determined as previously., Results: Knockdown of DNAJB1-PRKACA reversed a FLC-specific gene signature and reduced PDX tumor growth in mice compared to the control group. Furthermore, FLC PDX tumor growth was significantly reduced with BLU0588 and BLU2864 treatment vs control ( P  = .003 and P  = .0005, respectively)., Conclusion: We demonstrated, using an inducible knockdown and small molecule approaches, that FLC PDX tumors were dependent upon DNAJB1-PRKACA fusion activity. In addition, this study serves as a proof-of-concept that PRKACA is a viable therapeutic target for FLC and warrants further investigation., (© 2022 Published by Elsevier, Inc on behalf of the AGA Institute.)

A substantial subset of patients with T cell acute lymphoblastic leukemia (T-ALL) develops resistance to steroids and succumbs to their disease. JDP2 encodes a bZIP protein that has been implicated as a T-ALL oncogene from insertional mutagenesis studies in mice, but its role in human T-ALL pathogenesis has remained obscure. Here we show that JDP2 is aberrantly expressed in a subset of T-ALL patients and is associated with poor survival. JDP2 is required for T-ALL cell survival, as its depletion by short hairpin RNA knockdown leads to apoptosis. Mechanistically, JDP2 regulates prosurvival signaling through direct transcriptional regulation of MCL1. Furthermore, JDP2 is one of few oncogenes capable of initiating T-ALL in transgenic zebrafish. Notably, thymocytes from rag2:jdp2 transgenic zebrafish express high levels of mcl1 and demonstrate resistance to steroids in vivo. These studies establish JDP2 as a novel oncogene in high-risk T-ALL and implicate overexpression of MCL1 as a mechanism of steroid resistance in JDP2 -overexpressing cells., (© 2018 Mansour et al.)

Regulatory T (T reg) cells are a specialized sublineage of T lymphocytes that suppress autoreactive T cells. Functional studies of T reg cells in vitro have defined multiple suppression mechanisms, and studies of T reg-deficient humans and mice have made clear the important role that these cells play in preventing autoimmunity. However, many questions remain about how T reg cells act in vivo. Specifically, it is not clear which suppression mechanisms are most important, where T reg cells act, and how they get there. To begin to address these issues, we sought to identify T reg cells in zebrafish, a model system that provides unparalleled advantages in live-cell imaging and high-throughput genetic analyses. Using a FOXP3 orthologue as a marker, we identified CD4-enriched, mature T lymphocytes with properties of T reg cells. Zebrafish mutant for foxp3a displayed excess T lymphocytes, splenomegaly, and a profound inflammatory phenotype that was suppressed by genetic ablation of lymphocytes. This study identifies T reg-like cells in zebrafish, providing both a model to study the normal functions of these cells in vivo and mutants to explore the consequences of their loss., (© 2017 Kasheta et al.)

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes. Using a transgenic screen in zebrafish, thymocyte selection-associated high mobility group box protein (TOX) was uncovered as a collaborating oncogenic driver that accelerated T-ALL onset by expanding the initiating pool of transformed clones and elevating genomic instability. TOX is highly expressed in a majority of human T-ALL and is required for proliferation and continued xenograft growth in mice. Using a wide array of functional analyses, we uncovered that TOX binds directly to KU70/80 and suppresses recruitment of this complex to DNA breaks to inhibit nonhomologous end joining (NHEJ) repair. Impaired NHEJ is well known to cause genomic instability, including development of T-cell malignancies in KU70- and KU80-deficient mice. Collectively, our work has uncovered important roles for TOX in regulating NHEJ by elevating genomic instability during leukemia initiation and sustaining leukemic cell proliferation following transformation. Significance: TOX is an HMG box-containing protein that has important roles in T-ALL initiation and maintenance. TOX inhibits the recruitment of KU70/KU80 to DNA breaks, thereby inhibiting NHEJ repair. Thus, TOX is likely a dominant oncogenic driver in a large fraction of human T-ALL and enhances genomic instability. Cancer Discov; 7(11); 1336-53. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1201 ., (©2017 American Association for Cancer Research.)

Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA-protein kinase catalytic subunit ( prkdc ), interleukin-2 receptor γ a ( il2rga ), and double-homozygous-mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish., (© 2017 Tang et al.)

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

The establishment of in vitro cultures of zebrafish cancer cells has expanded the potential of zebrafish as a disease model. However, the lack of effective methods for gene delivery and genetic manipulation has limited the experimental applications of these cultures. To overcome this barrier, we tested and optimized vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral and retroviral vector transduction protocols. We show that lentivirus successfully and efficiently transduced zebrafish melanoma cell lines in vitro, allowing antibiotic selection, fluorescence-based sorting, and in vivo allotransplantation. In addition, injection of concentrated lentiviral particles into embryos and tumors established the feasibility of in vivo gene delivery.

Tumor-propagating cells (TPCs) share self-renewal properties with normal stem cells and drive continued tumor growth. However, mechanisms regulating TPC self-renewal are largely unknown, especially in embryonal rhabdomyosarcoma (ERMS)-a common pediatric cancer of muscle. Here, we used a zebrafish transgenic model of ERMS to identify a role for intracellular NOTCH1 (ICN1) in increasing TPCs by 23-fold. ICN1 expanded TPCs by enabling the de-differentiation of zebrafish ERMS cells into self-renewing myf5+ TPCs, breaking the rigid differentiation hierarchies reported in normal muscle. ICN1 also had conserved roles in regulating human ERMS self-renewal and growth. Mechanistically, ICN1 upregulated expression of SNAIL1, a transcriptional repressor, to increase TPC number in human ERMS and to block muscle differentiation through suppressing MEF2C, a myogenic differentiation transcription factor. Our data implicate the NOTCH1/SNAI1/MEF2C signaling axis as a major determinant of TPC self-renewal and differentiation in ERMS, raising hope of therapeutically targeting this pathway in the future., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.)

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

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

Cell transplantation into adult zebrafish has lagged behind mouse models owing to the lack of immunocompromised strains. Here we have created rag2(E450fs) mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft muscle, blood stem cells and various cancers. rag2(E450fs) mutant zebrafish are the first immunocompromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer.

Clonal evolution and intratumoral heterogeneity drive cancer progression through unknown molecular mechanisms. To address this issue, functional differences between single T cell acute lymphoblastic leukemia (T-ALL) clones were assessed using a zebrafish transgenic model. Functional variation was observed within individual clones, with a minority of clones enhancing growth rate and leukemia-propagating potential with time. Akt pathway activation was acquired in a subset of these evolved clones, which increased the number of leukemia-propagating cells through activating mTORC1, elevated growth rate likely by stabilizing the Myc protein, and rendered cells resistant to dexamethasone, which was reversed by combined treatment with an Akt inhibitor. Thus, T-ALL clones spontaneously and continuously evolve to drive leukemia progression even in the absence of therapy-induced selection., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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