RESUMO
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.
Assuntos
Dano ao DNA , Hematopoese , MicroRNAs , Proteína Supressora de Tumor p53 , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Humanos , Camundongos , Apoptose/genética , Camptotecina/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Supressores de Tumor , Hematopoese/genética , Síndrome de Li-Fraumeni/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
PURPOSE: The functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system. METHODS: Genetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences. RESULTS: Metabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models. CONCLUSION: A general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients.
Assuntos
Proteínas Ferro-Enxofre , Peixe-Zebra , Animais , Humanos , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Masculino , Feminino , Fenótipo , Fibroblastos/metabolismo , Fibroblastos/patologia , Citosol/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Microcefalia/genética , Microcefalia/patologia , Lactente , MetalochaperonasRESUMO
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.
Assuntos
Modelos Animais de Doenças , Marcadores Genéticos , Doenças Raras/genética , Doenças Raras/terapia , Sistema de Registros/normas , Animais , Bases de Dados Factuais , Genômica , Humanos , Doenças Raras/epidemiologiaRESUMO
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.
Assuntos
Mastocitose , Humanos , Dimerização , Mastocitose/genética , Mastocitose/metabolismo , Transdução de Sinais/genética , Fosforilação , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
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.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Citarabina/uso terapêutico , Síndrome de Down/complicações , Leucemia Mieloide/complicações , Leucemia Mieloide/tratamento farmacológico , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/efeitos adversos , Pré-Escolar , Citarabina/administração & dosagem , Citarabina/efeitos adversos , Relação Dose-Resposta a Droga , Síndrome de Down/genética , Feminino , Humanos , Lactente , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/genética , Masculino , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Prognóstico , Resultado do TratamentoRESUMO
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.
Assuntos
Neoplasias da Mama , Peixe-Zebra , Humanos , Feminino , Animais , Camundongos , Projetos Piloto , XenoenxertosRESUMO
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.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Leucemia Aguda Bifenotípica/epidemiologia , Leucemia Aguda Bifenotípica/terapia , Prognóstico , Adolescente , Adulto , Criança , Pré-Escolar , Ensaios Clínicos como Assunto , Intervalo Livre de Doença , Feminino , Humanos , Imunofenotipagem/métodos , Lactente , Leucemia Aguda Bifenotípica/patologia , Masculino , Pediatria/tendências , Organização Mundial da Saúde , Adulto JovemRESUMO
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.
Assuntos
Leucemia Mieloide Aguda , Peixe-Zebra , Animais , Diferenciação Celular , Hematopoese , Células-Tronco Hematopoéticas , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Camundongos , Microambiente TumoralRESUMO
We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Mutação Puntual/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Embrião não Mamífero , Microinjeções , Mutagênese Sítio-Dirigida/métodos , Peixe-Zebra/embriologiaRESUMO
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.
Assuntos
Receptores Frizzled/fisiologia , Retina/patologia , Vasos Retinianos/patologia , Remodelação Vascular/genética , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Modelos Animais de Doenças , Embrião não Mamífero , Vitreorretinopatias Exsudativas Familiares/diagnóstico , Vitreorretinopatias Exsudativas Familiares/genética , Vitreorretinopatias Exsudativas Familiares/patologia , Estudos de Viabilidade , Receptores Frizzled/genética , Humanos , Neovascularização Patológica/embriologia , Neovascularização Patológica/genética , Neovascularização Patológica/fisiopatologia , Retina/diagnóstico por imagem , Retina/embriologia , Retina/metabolismo , Doenças Retinianas/genética , Doenças Retinianas/patologia , Vasos Retinianos/embriologia , Vasos Retinianos/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
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.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Antimetabólitos Antineoplásicos/uso terapêutico , Citarabina/uso terapêutico , Daunorrubicina/uso terapêutico , Síndrome de Down/complicações , Leucemia Mieloide Aguda/tratamento farmacológico , Síndromes Mielodisplásicas/tratamento farmacológico , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/efeitos adversos , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Pré-Escolar , Citarabina/administração & dosagem , Citarabina/efeitos adversos , Análise Citogenética , Daunorrubicina/administração & dosagem , Daunorrubicina/efeitos adversos , Intervalo Livre de Doença , Síndrome de Down/genética , Feminino , Humanos , Lactente , Leucemia Mieloide Aguda/complicações , Leucemia Mieloide Aguda/genética , Masculino , Síndromes Mielodisplásicas/complicações , Síndromes Mielodisplásicas/genética , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Resultado do TratamentoRESUMO
Sideroblastic anemias are acquired or inherited anemias that result in a decreased ability to synthesize hemoglobin in red blood cells and result in the presence of iron deposits in the mitochondria of red blood cell precursors. A common subtype of congenital sideroblastic anemia is due to autosomal recessive mutations in the SLC25A38 gene. The current treatment for SLC25A38 congenital sideroblastic anemia is chronic blood transfusion coupled with iron chelation. The function of SLC25A38 is not known. Here we report that the SLC25A38 protein, and its yeast homolog Hem25, are mitochondrial glycine transporters required for the initiation of heme synthesis. To do so, we took advantage of the fact that mitochondrial glycine has several roles beyond the synthesis of heme, including the synthesis of folate derivatives through the glycine cleavage system. The data were consistent with Hem25 not being the sole mitochondrial glycine importer, and we identify a second SLC25 family member Ymc1, as a potential secondary mitochondrial glycine importer. Based on these findings, we observed that high levels of exogenous glycine, or 5-aminolevulinic acid (5-Ala) a metabolite downstream of Hem25 in heme biosynthetic pathway, were able to restore heme levels to normal in yeast cells lacking Hem25 function. While neither glycine nor 5-Ala could ameliorate SLC25A38 congenital sideroblastic anemia in a zebrafish model, we determined that the addition of folate with glycine was able to restore hemoglobin levels. This difference is likely due to the fact that yeast can synthesize folate, whereas in zebrafish folate is an essential vitamin that must be obtained exogenously. Given the tolerability of glycine and folate in humans, this study points to a potential novel treatment for SLC25A38 congenital sideroblastic anemia.
Assuntos
Anemia Sideroblástica/genética , Ácido Fólico/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Glicina/metabolismo , Hemoglobinas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Anemia Sideroblástica/metabolismo , Anemia Sideroblástica/patologia , Animais , Ácido Fólico/administração & dosagem , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Glicina/administração & dosagem , Heme/biossíntese , Hemoglobinas/efeitos dos fármacos , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mutação , Saccharomyces cerevisiae , Peixe-ZebraRESUMO
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.
Assuntos
Coração/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero , Cardiopatias Congênitas/etiologia , NADPH Oxidases , Proteínas Supressoras de Tumor , Proteínas rac1 de Ligação ao GTPRESUMO
Jason graduated from the University of Toronto Medical School in 1997 followed by a pediatric residency at the Hospital for Sick Children, a fellowship in pediatric hematology/oncology at Boston Children's Hospital and post-doctoral training at the Dana-Farber Cancer Institute. He is a professor at Dalhousie University in the Departments of Pediatrics, Microbiology and Immunology and Pathology, was appointed as the director of the Clinician Investigator Program (CIP) and the Graduate Program in Medical Research at Dalhousie (2014), elected president of the Canadian Society for Clinical Investigation (CSCI) (2017) and currently serves as Associate Chair Research in the Department of Pediatrics and Interim VP Research at the IWK, and as an assistant editor of the Journal of Clinical and Investigative Medicine (CIM).
Assuntos
Pesquisa Biomédica , Canadá , Humanos , PesquisadoresRESUMO
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.
Assuntos
Biomarcadores Tumorais/fisiologia , Proteínas de Ligação a DNA/fisiologia , Neoplasias Ovarianas/metabolismo , Lesões Intraepiteliais Escamosas Cervicais/metabolismo , Fatores de Transcrição/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/farmacologia , Proteína BRCA2/metabolismo , Benzimidazóis/farmacologia , Biomarcadores Tumorais/química , Linhagem Celular Tumoral , Dano ao DNA , Proteínas de Ligação a DNA/química , Resistencia a Medicamentos Antineoplásicos , Proteína do Grupo de Complementação N da Anemia de Fanconi , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/mortalidade , Ftalazinas/farmacologia , Piperazinas/farmacologia , Ligação Proteica , Transporte Proteico , Curva ROC , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Lesões Intraepiteliais Escamosas Cervicais/diagnóstico , Lesões Intraepiteliais Escamosas Cervicais/tratamento farmacológico , Lesões Intraepiteliais Escamosas Cervicais/mortalidade , Fatores de Transcrição/química , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
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.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Genoma , Genômica/métodos , Animais , RNA Guia de Cinetoplastídeos/metabolismo , SoftwareRESUMO
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.
Assuntos
Transplante de Células , Proteínas de Ligação a DNA/genética , Mutação , Peixe-Zebra/genética , Idoso , Animais , HumanosRESUMO
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.
Assuntos
Proteína de Ligação a CREB/genética , Histona Acetiltransferases/genética , Leucemia Mieloide Aguda/congênito , Leucemia Mieloide Aguda/genética , Regressão Neoplásica Espontânea/genética , Translocação Genética/genética , Cromossomos Humanos Par 8/genética , Feminino , Humanos , Hibridização in Situ Fluorescente , Recém-Nascido , Cariótipo , Proteínas de Fusão Oncogênica/genéticaRESUMO
Congenital sideroblastic anemia (CSA) is a hematological disorder characterized by the presence of ringed sideroblasts in bone marrow erythroid precursors. Mutations in the erythroid-specific glycine mitochondrial transporter gene SLC25A38 have been found in a subset of patients with transfusion-dependent congenital CSA. Further studies in a zebrafish model identified a promising ameliorative strategy with combined supplementation with glycine and folate. We tested this combination in three individuals with SLC25A38 CSA, with a primary objective to decrease red blood cell transfusion requirements. No significant impact was observed on transfusion requirements or any hematologic parameters.