This presentation is about providing insights in developing a health monitoring program for zebrafish. The program must aim to detect both infectious and non-infectious diseases, including pathogenic ...and non-pathogenic conditions and quarantine procedures to prevent the spread of disease within the colony and to new colonies. We will examine how different sample types are impacting diagnosis and how prevalence and institutional prevalence are important in determining the sample size. The goal of this presentation is to contribute to the development of effective measures for the prevention and control of diseases in zebrafish.
Abstract
The gene encoding Isocitrate dehydrogenase 1 (IDH1) is frequently mutated in gliomas, chondrosarcomas, acute myeloid leukemia and intrahepatic cholangiocarcinomas. As there are few in-vivo ...model systems for IDH-mutated tumors we have created a transgenic zebrafish (Danio rerio) model expressing mutant IDH1. We have chosen the zebrafish as a model because they are transparent (allowing monitoring of the transgene in-vivo) and drug screening assays are straightforward (they are simply added to the aquarium).
IDH1R132H and IDH1R132C, mutations found in tumors that both produce D-2-hydroxyglutarate (D2HG) instead of alpha ketoglutarate, were cloned into an expression construct that is driven either by the Nestin or GFAP promoter. IDH1G70D (a loss of function mutation), IDH1wildtype and GFP were used as control. All IDH1 constructs were fused to GFP for visualization. These constructs were injected into fertilized zebrafish eggs at the one-cell stage.
All of our transgenic zebrafish lines remain healthy and produce offspring. Transgene expression was detected in the mid/hindbrain of the central nervous system by immunohistochemistry, Western blot and RT-QPCR. A significant increase in the level of D2HG was observed in all transgenic lines expressing IDH1R132C or IDH1R132H, but not in any of the lines expressing control constructs (IDH1wildtype, IDH1G70D or GFP). In contrast to reported, we failed to detect any differences in hydroxymethyl cytosine (the first step in DNA-demethylation) and mature collagen IV levels between wildtype and mutant IDH1 transgenic fish. We also performed microinjections on fertilized eggs to screen for early developmental effects of IDH1R132H and IDH1R132C. Despite of the high expression of the transgene, no developmental effects were found. Our observations therefore suggest that elevated levels of D2HG are insufficient to initiate tumorigenesis or other phenotypic effects in our fish. Treatment of the transgenic zebrafish with an IDH1 mutant inhibitor, AGI-5198, resulted in a reduction in the D2HG level in the mutant zebrafish. The L2HG level was not affected by AGI-5198. As no tumors were formed in our transgenic zebrafish lines, we backcrossed them with Tp53 mutant fish. Analysis of these lines is currently being performed.
In summary, we have generated a transgenic zebrafish model system that expresses mutated IDH1 that can be used to study effects of mutant IDH1 (or elevated levels of D2HG) in vivo and can be used for drug screening.
Citation Format: Ya D. Gao, Maurice de Wit, Eduard A. Struys, Martine L.M. Lamfers, Gajja S. Salomons, Peter A.E. Sillevis Smitt, Pim J. French. A transgenic zebrafish model for gliomas with mutations in isocitrate dehydrogenase 1. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4170.
Abstract
Neuroblastoma is a malignancy of the peripheral sympathetic nervous system (PSNS) and accounts for 10-15% of cancer deaths among children. For the 40% of patients presenting with high-risk ...disease, current therapeutic approaches are insufficient and long-term survival is less than 50%. Along with genomic amplification of the MYCN oncogene, hemizygous loss of the 1p36 chromosomal region is a major risk factor in neuroblastoma. The human CHD5 gene is a neuronal specific chromatin remodeling helicase that maps to 1p36, and is thus frequently lost in high-risk neuroblastoma. Our laboratory has previously generated a faithful model of pediatric neuroblastoma in the zebrafish driven by overexpression of the MYCN oncogene in the PSNS (dbh:MYCN). Additionally, zebrafish chd5 mutant alleles were created using the newly developed gene editing technologies TALEN and CRISPR-Cas9. The resulting chd5 mutant fish exhibit abnormal development of the PSNS in the form of expansion of the superior cervical ganglia and enlargement of the interrenal gland (adrenal medulla). Haploinsufficiency for Chd5 combined with dbh:MYCN expression accelerates the onset and increases the penetrance of neuroblastoma tumorigenesis in zebrafish, indicating a tumor suppressive function. Elevated p-ERK and PCNA+ cells in tumor tissue indicates that loss of Chd5, cooperates with MYCN overexpression to accelerate neuroblast proliferation in vivo. Chd5 (in addition to Chd3 and Chd4) is a core member of the epigenetic regulatory NuRD complex, which also contains HDAC1-2, MTA1-3, MBD2-3, GATAD2A/B and RBBP4/7. The conserved biological function of Chd5 is to silence gene expression through the maintenance of a repressed chromatin state. Tumors deficient for Chd5 expression exhibit reduced levels of the H3K27me3 histone modification, a marker of facultatively repressed genes. Future studies will further explore the mechanism and function of Chd5 so that the pathways mediating tumor suppression can be elucidated and that essential proteins in these pathways can be targeted in ways that exploit the synthetic lethal relationships that are established.
Citation Format: Mark W. Zimmerman, Shuning He, Jimann Shin, Shizhen Zhu, Feng Guo, Marc Mansour, Deepak Reyon, J Keith Joung, Jinhua Quan, Timur Yusufzai, A Thomas Look. Loss of chd5-mediated gene repression synergizes with MYCN to accelerate neuroblastoma tumorigenesis in zebrafish. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2433.
Abstract
Mutation in the tumor suppressor Adenomatous Polyposis Coli (APC) is the primary initiating mutation in a majority of colon tumors. Recent studies have shown that APC positively correlates ...with Mitochondrial Pyruvate Carrier 1 (MPC1), a crucial player in pyruvate metabolism and has been reported to repress the Warburg effect and growth in colon cancer cells. Utilizing the zebrafish to examine the relationship between apc and mpc1, we found that mpc1 expression is reduced in embryos harboring a genetic mutation (apcmcr) or diminished expression (apc mo) of apc. Antisense morpholino knockdown of mpc1 in wild type embryos (mpc1 mo) resulted in an array of developmental defects that recapitulated phenotypes of impaired apc function including failed intestinal differentiation. This was accompanied by mitochondrial dysfunction in mpc1 mo as evidenced by a decrease in metabolic respiration and triglyceride levels. We also observed altered mitochondrial function and dysregulation of enzymes involved in pyruvate metabolism in apcmcr and apc mo. Moreover, hMPC1 RNA rescued intestinal differentiation in both zebrafish models of apc deficiency. Meta-analyses using TCGA human tumor samples corroborated our findings in the zebrafish. Our data demonstrate a novel role for apc in pyruvate metabolism through regulation of mpc1 that drives normal intestinal differentiation and support the broader idea that metabolic changes can dictate cell fate programs.
Citation Format: Imelda T. Sandoval, Richard Glenn C. Delacruz, Braden N. Miller, Christeena Satterfield, Kristophor Olson, Shauna Hill, Holly Van Remmen, Jared Rutter, David A. Jones. A metabolic switch controls cell fate decision-making in intestinal differentiation downstream of the tumor suppressor adenomatous polyposis coli (apc). abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 229.
Abstract
We examined the gene expression signature of zebrafish brain tumors induced by somatic inactivation of the retinoblastoma tumor suppressor rb1 to determine the similarity with human CNS ...primitive neuroectodermal tumors (PNETs). Childhood CNS PNETs are aggressive tumors characterized by undifferentiated cells with features of the embryonic neuroepithelium. Previously our histological analysis showed the majority of zebrafish rb1- lesions were proliferative neuroectodermal-like tumors composed of neoplastic cells with small hyperchromatic nuclei that form rosettes. To obtain the transcription profile of the zebrafish rb1- tumors, RNA-Seq was performed on 10 tumors from fish ranging in age from 3.5 to 10 months of age. Duplicate RNA-Seq libraries were synthesized for each tumor and multiplex sequenced on an Illumina HiSeq NextGen Sequencer at the Genome Sequencing Core, Kansas University. Our initial gene expression profiling of the zebrafish rb1- brain tumors revealed a molecular signature consistent with an undifferentiated tumor phenotype. In each zebrafish tumor sample, standard markers of neuronal differentiation (sypa, sypb, neurod1/2, neurog1/2) and glial differentiation (GFAP, S100β) were not significantly expressed. In contrast, the neural stem/progenitor marker sox2 was highly expressed in all tumors, consistent with the primitive neuroectodermal-like tumor histology. Comparison with human childhood CNS-PNET gene expression analysis revealed a similar profile. Four of the nine genes that define the human oligoneural (group 2) molecular subgroup (olig2, bcan, sox8b, sox10 and ascl1) described by Picard et al, 2012 were highly expressed in the zebrafish tumors. Together, the data indicate the zebrafish brain tumors induced by somatic inactivation of rb1 model human CNS PNETs. Additional gene expression, pathway, and cross-species comparative analyses are in progress. The zebrafish rb1- tumor model will be useful for investigating the molecular pathways driving the aggressive and undifferentiated features of CNS PNETs.
Citation Format: Maura A. McGrail, Staci L. Solin, Jeffrey A. Haltom, Laura E. Schultz, Jeffrey J. Essner, Heather R. Shive. Expression profiling of zebrafish rb1- neuroectodermal-like brain tumors: A new model of CNS PNETs. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4179.
Abstract
The possibility of a radiation disaster from a nuclear detonation or accident has existed for over 50 years and spawned much of the basic research in radiobiology in the 1950-60s. The recent ...Fukushima accident was yet another reminder that there remains a dire need to develop novel therapies against radiation-induced toxicities. Here we report on the development of two novel radiation countermeasure therapies: CJ010 and Yel002. These small, biologically active, drug-like molecules were uncovered in the DEL high throughput assay reducing radiation-induced cyto- and geno-toxicity in yeast. Radiation-modulating activity was further confirmed in yeast plate-based DEL Assay: addition of either CJ010 or Yel002 to irradiated cultures reduced cell death and genomic instability. Further, the compounds increases survival to 75% in vivo following an LD100/30 dose of ionizing radiation (IR) with the first therapeutic injection administered 24 hours post exposure followed by injections at 48,72,96, and 120 hours. Additionally, treatment with Yel001 an analog of CJ010 and Yel002 compounds reduces radiation-induced leukemia from 90% to to 50% and 40% respectively. Of note, treatment with either Yel001 or Yel002 reduced spontaneous leukemia rate from 10% to 0%. Treatment with Yel002 following IR accelerates the recovery of the hematopoietic cells after sub-lethal exposures. In addition, treatment with Yel002 reduces EMS, MMS, UV, radioactive iodine, cigarette smoke extract as well as nitrogen mustard induced toxicity as well as genotoxicity showing a broad application spectrum. It also prolongs live of cells in a senescence assay. In addition Atm deficient mice live 16 weeks longer with weekly injection of Yel002 which is about 12 years in human life expectancy. In addition, Yel002 complements a zebrafish model of Diamond Blackfan Anemia. It works in yeast, CHO cells, different human cells, mice and zebrafish. Toxicity has not been observed in neither in vitro or in vivo administrations. Overall, Yel compounds have much potential as stockpile therapies for radiation-induced lethality and cancer: they are highly effective when administered up to 24hours post exposure, they reduce radiation-induce sequelae such as leukemia, and appear to have an acceptable toxicity profile.
Citation Format: Robert H. Schiestl, Yelena Rivina, Michael Davoren. Novel radiation mitigators and anticancer drugs. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3729.
Abstract
Cancer cells rely on extracellular glutamine for growth and survival. Previously, we showed that asparagine, a nonessential amino acid that can be synthesized de novo from glutamine, is both ...necessary and sufficient for glutamine-dependent cell survival. In addition, asparagine synthetase (ASNS) expression positively correlates with the progression of the disease in human glioma patients, suggesting a critical role of asparagine during tumor progression. Interestingly, mammalian cells cannot catabolize asparagine for use as either a nitrogen source or a bioenergetic substrate, potentially due to the loss of asparaginase activity during evolution. Ectopic expression of yeast or zebrafish asparaginase restores the capacity of mammalian cells to utilize asparagine as both bioenergetic and biosynthetic substrates to support cell growth. Furthermore, asparagine restores translation of fast-turn-over proteins under glutamine starvation. Together, the data suggests that asparagine has been evolved to be a metabolic reserve to regulate cellular adaptation to nonessential amino acid limitation in mammalian cells and is therefore a potential target for cancer therapy.
Citation Format: Ji Zhang, Natalya Pavlova, Richard White, Craig Thompson. Non-metabolic function of asparagine in regulating global protein translation. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2670.
Abstract
MYC and NOTCH are major oncogenic drivers in T-cell Acute Lymphoblastic Leukemia (T-ALL), yet additional collaborating genetic lesions collaborate to induce frank malignancy. To identify ...these factors, a large-scale transgenic screen was completed where 28 amplified and over-expressed genes found in human T-ALL were assessed for accelerating leukemia onset in the zebrafish transgenic model. From this analysis, Thymocyte selection-associated HMG protein (TOX) synergized with both MYC and NOTCH to induce T-ALL. Here, we show that TOX is highly expressed in 95% of human primary and relapse T-ALL when compared with both normal T cells and B-ALL. TOX is highly and specifically expressed in human T-ALL due to both genomic amplification and transcriptional regulation by the master transcription factors MYB/LMO2. Characterization of zebrafish T-ALLs revealed that TOX promoted genomic instability as assessed by changes in DNA content and Whole Genome Sequencing. Effects on genomic instability were confirmed by metaphase spread following TOX expression in MEF cells, confirming roles for TOX in regulating genomic instability and elevated DNA translocation potential in a wider range of cell types. To identify TOX binding partners, Tandem Mass Spectrometry was performed in human T-ALL cells. TOX was found to interact with KU70/KU80 but not other DNA repair enzymes, a result verified by co-immunoprecipitation studies. Given that TOX elevated genomic instability in the zebrafish model, that Ku70 or Ku80 loss lead to genomic instability and T cell lymphoma in mice, and that TOX bound specifically to KU70/KU80 - the initiating factors required for Non-Homologous End Joining (NHEJ) repair - we hypothesized that TOX is a negative regulator of double-strand break repair. Fluorescent repair assays were completed in 3T3 fibroblasts and confirmed that TOX inhibits NHEJ. Dynamic real-time imaging studies showed that TOX suppresses recruitment of fluorescent-tagged KU80 to DNA breaks. Importantly, TOX loss of function increased NHEJ in human T-ALL cells and reduced time to DNA repair as assessed by fluorescent Traffic Light Reporter assays and quantitative assessment of 53BP1 and γH2A.X foci resolution following irradiation. Our results show that TOX is aberrantly re-activated in 95% of human T-ALL, thereby suppressing KU70/KU80 function to promote genomic instability and ultimately elevating rates at which acquired mutations and rearrangements are amassed in developing pre-malignant T cells. Our work shows that TOX is the major oncogenic driver of genomic instability human T-ALL and locks cells in a constant state of dampened repair.
Citation Format: Riadh Lobbardi, Jordan Pinder, Barbara Martinez-Pastor, Jessica Blackburn, Brian J. Abraham, Marc Mansour, Nouran S. Abdelfattah, Aleksey Molodtsov, Gabriela Alexe, Debra Toiber, Manon de Waard, Esha Jain, Deepak Bhere, Khalid Shah, Alejandro Gutierrez, Kimberly Stegmaier, Lewis B. Silverman, Ruslan Sadreyev, John Asara, A Thomas Look, Richard A. Young, Raul Mostoslavsky, Graham Dellaire, David M. Langenau. Thymocyte selection-associated HMG box protein (TOX) induces genomic instability in T-cell acute lymphoblastic leukemia. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3583.
Abstract
Our lab recently performed a DNA transposon forward genetic screen in mice that was designed to identify low-frequency mutations that contribute to colorectal cancer (CRC) initiation and ...progression. Results from this screen identified the WW domain-containing adaptor with coiled-coil (WAC) gene as a top DNA transposon insertion site. WAC has previously been implicated in several cellular processes including amino acid starvation-induced autophagy, golgi biogenesis, and transcription associated histone modification but has never before been linked to tumorigenesis. Transposon mutagenesis screens performed by others (Takeda et al. Nature Genetics 2015) have also identified Wac as a common insertion site, a result that further implicates WAC as a candidate CRC driver gene. Analyses of transposon insertion patterns within Wac predict loss of gene function and a role as a tumor suppressor. Soft agar colony formation assays reveal that shRNA mediated silencing of Wac cooperates with Apc mutations in mouse colorectal cells to promote cellular transformation. Additional colony formation assays using immortalized human colonic epithelial cells and the adenoma derived AAC1 cell line also shows that silencing WAC is protumorigenic. Using a zebrafish model we demonstrated that overexpression of wild type but not cancer-associated mutant forms of WAC induce expression of the cell cycle inhibitor p21, which suggests that loss of WAC may lead to uncontrolled cellular proliferation. Finally, using publicly available mutation data we determined that WAC is somatically mutated in both breast and lung cancers; a finding that indicates WAC may serve a critical tumor suppressive role in several tissues. Currently we are developing a conditional knockout mouse to further investigate the role of WAC in CRC tumor formation.
Citation Format: Christopher R. Clark, Caitlin Conboy, Makayla Maile, Callie Janik, Julia Hatler, Robert Cormier, David Largaespada, Timothy K. Starr. WAC: A candidate tumor suppressor gene in colorectal cancer. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3665.
Abstract
Earlier reports indicated that the role of Nf1 tumor suppressor gene in limiting sympathoadrenal cell growth during embryologic development is independent of its ability to down-modulate ...RAS-MAPK signaling. This finding raised the question of whether neuroblastoma pathogenesis was also accelerated by loss of a similar non-canonical function of NF1. To elucidate how loss of the NF1 tumor suppressor gene contributes to the development of high-risk neuroblastoma, we relied on a transgenic zebrafish model that overexpresses MYCN and harbors loss-of-function nf1 mutations. We show here that loss of nf1 leads to aberrant activation of RAS signaling in MYCN-induced neuroblastoma, promoting both increased tumor cell survival and rapid tumor cell proliferation. We demonstrate further that the GTPase-activating protein (GAP) activity of the (GAP)-related domain (GRD) is sufficient to suppress accelerated initiation of neuroblastoma in nf1-deficient zebrafish, even though this transgene is unable to restrict abnormal sympathoadrenal cell growth during embryologic development. Hence NF1 exhibits different activities in vivo in the normal development and tumorigenesis of the peripheral sympathetic nervous system. Our findings establish nf1-deficient zebrafish that overexpress MYCN as an ideal animal model system for investigating new strategies to improve treatment of very high risk neuroblastomas with aberrant RAS-MAPK activation. We are currently performing high-throughput in vivo drug analysis using these zebrafish with primary tumors.
Citation Format: Shuning He, Marc R. Mansour, Mark W. Zimmerman, Dong Hyuk Ki, Hillary M. Layden, Koshi Akahane, Eric D. de Groh, Antonio R. Perez-Atayde, Shizhen Zhu, Jonathan A. Epstein, A Thomas Look. Synergy between loss of NF1 and overexpression of MYCN in neuroblastoma is mediated by the GAP-related domain. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2456.
