JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Distinct effects of concomitant Jak2V617F expression and Tet2 loss in mice combine to promote disease progression in myeloproliferative neoplasms.
Blood
PUBLISHED: 10-05-2014
Show Abstract
Hide Abstract
Signaling mutations (e.g. JAK2V617F) and mutations in genes involved in epigenetic regulation (e.g. TET2) are the most common co-occurring classes of mutations in myeloproliferative neoplasms (MPN). Clinical correlative studies have demonstrated that TET2 mutations are enriched in more advanced phases of MPN such as myelofibrosis and leukemic transformation, suggesting that they may co-operate with JAK2V617F to promote disease progression. To dissect the effects of concomitant Jak2V617F expression and Tet2 loss within distinct hematopoietic compartments in vivo, we generated Jak2V617F/Tet2 compound mutant genetic mice. We found that the combination of Jak2V617F expression and Tet2 loss resulted in a more florid MPN phenotype than that seen with either allele alone. Concordant with this, we found that Tet2 deletion conferred a strong functional competitive advantage to Jak2V617F-mutant hematopoietic stem cells (HSC). Transcriptional profiling revealed that both Jak2V617F expression and Tet2 loss were associated with distinct and non-overlapping gene expression signatures within the HSC compartment. In aggregate, our findings indicate that Tet2 loss drives clonal dominance in HSC and Jak2V617F expression causes expansion of downstream precursor cell populations, resulting in disease progression through combinatorial effects. This works provides insights into the functional consequences of JAK2V617F-TET2 co-mutation in MPN, particularly as it pertains to HSC.
Related JoVE Video
TET2 mutations predict response to hypomethylating agents in myelodysplastic syndrome patients.
Blood
PUBLISHED: 09-15-2014
Show Abstract
Hide Abstract
Only a minority of myelodysplastic syndrome (MDS) patients respond to hypomethylating agents (HMAs), but strong predictors of response are unknown. We sequenced 40 recurrently mutated myeloid malignancy genes in tumor DNA from 213 MDS patients collected before treatment with azacitidine (AZA) or decitabine (DEC). Mutations were examined for association with response and overall survival. The overall response rate of 47% was not different between agents. Clonal TET2 mutations predicted response (odds ratio [OR] 1.99, P = .036) when subclones unlikely to be detected by Sanger sequencing (allele fraction <10%) were treated as wild-type (WT). Response rates were highest in the subset of TET2 mutant patients without clonal ASXL1 mutations (OR 3.65, P = .009). Mutations of TP53 (hazard ratio [HR] 2.01, P = .002) and PTPN11 (HR 3.26, P = .006) were associated with shorter overall survival but not drug response. Murine-competitive bone marrow transplantation followed by treatment with AZA demonstrated that Tet2-null cells have an engraftment advantage over Tet2-WT cells. AZA significantly decreased this advantage for Tet2-null cells (P = .002) but not Tet2-WT cells (P = .212). Overall, Tet2 loss appears to sensitize cells to treatment with AZA in vivo, and TET2 mutations can identify patients more likely to respond to HMAs.
Related JoVE Video
Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation.
Nat. Biotechnol.
PUBLISHED: 09-03-2014
Show Abstract
Hide Abstract
Components of the prokaryotic clustered, regularly interspaced, short palindromic repeats (CRISPR) loci have recently been repurposed for use in mammalian cells. The CRISPR-associated (Cas)9 can be programmed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but there are few known rules governing on-target efficacy of this system. We created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. We discovered sequence features that improved activity, including a further optimization of the protospacer-adjacent motif (PAM) of Streptococcus pyogenes Cas9. The results from 1,841 sgRNAs were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens. We provide an online tool for the design of highly active sgRNAs for any gene of interest.
Related JoVE Video
Somatic mutations predict poor outcome in patients with myelodysplastic syndrome after hematopoietic stem-cell transplantation.
J. Clin. Oncol.
PUBLISHED: 08-04-2014
Show Abstract
Hide Abstract
Recurrently mutated genes in myelodysplastic syndrome (MDS) are pathogenic drivers and powerfully associated with clinical phenotype and prognosis. Whether these types of mutations predict outcome after allogeneic hematopoietic stem-cell transplantation (HSCT) in patients with MDS is not known.
Related JoVE Video
MDS is a stem cell disorder after all.
Cancer Cell
PUBLISHED: 06-18-2014
Show Abstract
Hide Abstract
Myelodysplastic syndrome (MDS) has long been presumed to be a stem cell disorder, but rigorous formal proof has been lacking. In this issue of Cancer Cell, Woll and colleagues demonstrate that driver mutations occurring in MDS definitively occur in cells with a stem cell phenotype.
Related JoVE Video
National trends in hospitalizations for sickle cell disease in the United States following the FDA approval of hydroxyurea, 1998-2008.
Med Care
PUBLISHED: 06-14-2014
Show Abstract
Hide Abstract
Patients with sickle cell disease (SCD) can suffer frequent hospital admissions for painful vasoocclusive crises. Hydroxyurea was approved by the FDA in 1998 to decrease the morbidity of SCD, but nationwide hospitalizations for SCD in the United States since 1998 have not been evaluated. We hypothesized that the availability of hydroxyurea for SCD would be associated with a decrease in hospitalizations for SCD over time.
Related JoVE Video
Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing.
Nat. Biotechnol.
PUBLISHED: 04-29-2014
Show Abstract
Hide Abstract
Genome sequencing studies have shown that human malignancies often bear mutations in four or more driver genes, but it is difficult to recapitulate this degree of genetic complexity in mouse models using conventional breeding. Here we use the CRISPR-Cas9 system of genome editing to overcome this limitation. By delivering combinations of small guide RNAs (sgRNAs) and Cas9 with a lentiviral vector, we modified up to five genes in a single mouse hematopoietic stem cell (HSC), leading to clonal outgrowth and myeloid malignancy. We thereby generated models of acute myeloid leukemia (AML) with cooperating mutations in genes encoding epigenetic modifiers, transcription factors and mediators of cytokine signaling, recapitulating the combinations of mutations observed in patients. Our results suggest that lentivirus-delivered sgRNA:Cas9 genome editing should be useful to engineer a broad array of in vivo cancer models that better reflect the complexity of human disease.
Related JoVE Video
Integrated genomic analysis illustrates the central role of JAK-STAT pathway activation in myeloproliferative neoplasm pathogenesis.
Blood
PUBLISHED: 04-16-2014
Show Abstract
Hide Abstract
Genomic studies have identified somatic alterations in the majority of myeloproliferative neoplasms (MPN) patients, including JAK2 mutations in the majority of MPN patients and CALR mutations in JAK2-negative MPN patients. However, the role of JAK-STAT pathway activation in different MPNs, and in patients without JAK2 mutations, has not been definitively delineated. We used expression profiling, single nucleotide polymorphism arrays, and mutational profiling to investigate a well-characterized cohort of MPN patients. MPN patients with homozygous JAK2V617F mutations were characterized by a distinctive transcriptional profile. Notably, a transcriptional signature consistent with activated JAK2 signaling is seen in all MPN patients regardless of clinical phenotype or mutational status. In addition, the activated JAK2 signature was present in patients with somatic CALR mutations. Conversely, we identified a gene expression signature of CALR mutations; this signature was significantly enriched in JAK2-mutant MPN patients consistent with a shared mechanism of transformation by JAK2 and CALR mutations. We also identified a transcriptional signature of TET2 mutations in MPN patent samples. Our data indicate that MPN patients, regardless of diagnosis or JAK2 mutational status, are characterized by a distinct gene expression signature with upregulation of JAK-STAT target genes, demonstrating the central importance of the JAK-STAT pathway in MPN pathogenesis.
Related JoVE Video
L-Leucine improves the anaemia in models of Diamond Blackfan anaemia and the 5q- syndrome in a TP53-independent way.
Br. J. Haematol.
PUBLISHED: 04-07-2014
Show Abstract
Hide Abstract
Haploinsufficiency of ribosomal proteins (RPs) and upregulation of the tumour suppressor TP53 have been shown to be the common basis for the anaemia observed in Diamond Blackfan anaemia and 5q- myelodysplastic syndrome. We previously demonstrated that treatment with L-Leucine resulted in a marked improvement in anaemia in disease models. To determine if the L-Leucine effect was Tp53-dependent, we used antisense MOs to rps19 and rps14 in zebrafish; expression of tp53 and its downstream target cdkn1a remained elevated following L-leucine treatment. We confirmed this observation in human CD34+ cells. L-Leucine thus alleviates anaemia in RP-deficient cells in a TP53-independent manner.
Related JoVE Video
Role of Casein Kinase 1A1 in the Biology and Targeted Therapy of del(5q) MDS.
Cancer Cell
PUBLISHED: 04-01-2014
Show Abstract
Hide Abstract
The casein kinase 1A1 gene (CSNK1A1) is a putative tumor suppressor gene located in the common deleted region for del(5q) myelodysplastic syndrome (MDS). We generated a murine model with conditional inactivation of Csnk1a1 and found that Csnk1a1 haploinsufficiency induces hematopoietic stem cell expansion and a competitive repopulation advantage, whereas homozygous deletion induces hematopoietic stem cell failure. Based on this finding, we found that heterozygous inactivation of Csnk1a1 sensitizes cells to a CSNK1 inhibitor relative to cells with two intact alleles. In addition, we identified recurrent somatic mutations in CSNK1A1 on the nondeleted allele of patients with del(5q) MDS. These studies demonstrate that CSNK1A1 plays a central role in the biology of del(5q) MDS and is a promising therapeutic target.
Related JoVE Video
Csnk1a1 inhibition has p53-dependent therapeutic efficacy in acute myeloid leukemia.
J. Exp. Med.
PUBLISHED: 03-10-2014
Show Abstract
Hide Abstract
Despite extensive insights into the underlying genetics and biology of acute myeloid leukemia (AML), overall survival remains poor and new therapies are needed. We found that casein kinase 1 ? (Csnk1a1), a serine-threonine kinase, is essential for AML cell survival in vivo. Normal hematopoietic stem and progenitor cells (HSPCs) were relatively less affected by shRNA-mediated knockdown of Csnk1a1. To identify downstream mediators of Csnk1a1 critical for leukemia cells, we performed an in vivo pooled shRNA screen and gene expression profiling. We found that Csnk1a1 knockdown results in decreased Rps6 phosphorylation, increased p53 activity, and myeloid differentiation. Consistent with these observations, p53-null leukemias were insensitive to Csnk1a1 knockdown. We further evaluated whether D4476, a casein kinase 1 inhibitor, would exhibit selective antileukemic effects. Treatment of leukemia stem cells (LSCs) with D4476 showed highly selective killing of LSCs over normal HSPCs. In summary, these findings demonstrate that Csnk1a1 inhibition causes reduced Rps6 phosphorylation and activation of p53, resulting in selective elimination of leukemia cells, revealing Csnk1a1 as a potential therapeutic target for the treatment of AML.
Related JoVE Video
Mutations in epigenetic regulators including SETD2 are gained during relapse in paediatric acute lymphoblastic leukaemia.
Nat Commun
PUBLISHED: 02-18-2014
Show Abstract
Hide Abstract
Relapsed paediatric acute lymphoblastic leukaemia (ALL) has high rates of treatment failure. Epigenetic regulators have been proposed as modulators of chemoresistance, here, we sequence genes encoding epigenetic regulators in matched diagnosis-remission-relapse ALL samples. We find significant enrichment of mutations in epigenetic regulators at relapse with recurrent somatic mutations in SETD2, CREBBP, MSH6, KDM6A and MLL2, mutations in signalling factors are not enriched. Somatic alterations in SETD2, including frameshift and nonsense mutations, are present at 12% in a large de novo ALL patient cohort. We conclude that the enrichment of mutations in epigenetic regulators at relapse is consistent with a role in mediating therapy resistance.
Related JoVE Video
Musashi-2 controls cell fate, lineage bias, and TGF-? signaling in HSCs.
J. Exp. Med.
PUBLISHED: 01-06-2014
Show Abstract
Hide Abstract
Hematopoietic stem cells (HSCs) are maintained through the regulation of symmetric and asymmetric cell division. We report that conditional ablation of the RNA-binding protein Msi2 results in a failure of HSC maintenance and engraftment caused by a loss of quiescence and increased commitment divisions. Contrary to previous studies, we found that these phenotypes were independent of Numb. Global transcriptome profiling and RNA target analysis uncovered Msi2 interactions at multiple nodes within pathways that govern RNA translation, stem cell function, and TGF-? signaling. Msi2-null HSCs are insensitive to TGF-?-mediated expansion and have decreased signaling output, resulting in a loss of myeloid-restricted HSCs and myeloid reconstitution. Thus, Msi2 is an important regulator of the HSC translatome and balances HSC homeostasis and lineage bias.
Related JoVE Video
Loss of function of TET2 cooperates with constitutively active KIT in murine and human models of mastocytosis.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
Systemic Mastocytosis (SM) is a clonal disease characterized by abnormal accumulation of mast cells in multiple organs. Clinical presentations of the disease vary widely from indolent to aggressive forms, and to the exceedingly rare mast cell leukemia. Current treatment of aggressive SM and mast cell leukemia is unsatisfactory. An imatinib-resistant activating mutation of the receptor tyrosine kinase KIT (KIT D816V) is most frequently present in transformed mast cells and is associated with all clinical forms of the disease. Thus the etiology of the variable clinical aggressiveness of abnormal mast cells in SM is unclear. TET2 appears to be mutated in primary human samples in aggressive types of SM, suggesting a possible role in disease modification. In this report, we demonstrate the cooperation between KIT D816V and loss of function of TET2 in mast cell transformation and demonstrate a more aggressive phenotype in a murine model of SM when both mutations are present in progenitor cells. We exploit these findings to validate a combination treatment strategy targeting the epigenetic deregulation caused by loss of TET2 and the constitutively active KIT receptor for the treatment of patients with aggressive SM.
Related JoVE Video
Genome-scale CRISPR-Cas9 knockout screening in human cells.
Science
PUBLISHED: 12-12-2013
Show Abstract
Hide Abstract
The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.
Related JoVE Video
Lenalidomide Causes Selective Degradation of IKZF1 and IKZF3 in Multiple Myeloma Cells.
Science
PUBLISHED: 11-29-2013
Show Abstract
Hide Abstract
Lenalidomide is a drug with clinical efficacy in multiple myeloma and other B cell neoplasms, but its mechanism of action is unknown. Using quantitative proteomics, we found that lenalidomide causes selective ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IKZF3, by the CRBN-CRL4 ubiquitin ligase. IKZF1 and IKZF3 are essential transcription factors in multiple myeloma. A single amino acid substitution of IKZF3 conferred resistance to lenalidomide-induced degradation and rescued lenalidomide-induced inhibition of cell growth. Similarly, we found that lenalidomide-induced IL2 production in T cells is due to depletion of IKZF1 and IKZF3. These findings reveal a novel mechanism of action for a therapeutic agent, alteration of the activity of an E3 ubiquitin ligase leading to selective degradation of specific targets.
Related JoVE Video
Sinister symbiosis: pathological hematopoietic-stromal interactions in CML.
Cell Stem Cell
PUBLISHED: 09-10-2013
Show Abstract
Hide Abstract
The impact of myeloid malignancies on the nonhematopoietic components of the bone marrow remains poorly understood. In this issue of Cell Stem Cell, Schepers et al. (2013) describe how malignant myeloid cells alter the endosteal hematopoietic stem cell (HSC) niche, resulting in the expansion of osteoblastic lineage cells that preferentially support malignant HSCs.
Related JoVE Video
The emerging importance of ribosomal dysfunction in the pathogenesis of hematologic disorders.
Leuk. Lymphoma
PUBLISHED: 08-28-2013
Show Abstract
Hide Abstract
More than a decade has passed since the initial identification of ribosomal protein gene mutations in patients with Diamond-Blackfan anemia (DBA), a hematologic disorder that became the founding member of a class of diseases known as ribosomopathies. In these diseases, genetic abnormalities that result in defective ribosome biogenesis cause strikingly tissue-specific phenotypes in patients, specifically bone marrow failure, craniofacial abnormalities and skeletal defects. Several animal models and numerous in vitro studies have demonstrated that the p53 pathway is central to the ribosomopathy phenotype. Additionally, there is mounting evidence of a link between the dysregulation of components of the translational machinery and the pathology of various malignancies. The importance of the role of ribosomal dysfunction in the pathogenesis of hematologic disorders is becoming clearer, and elucidation of the underlying mechanisms could have broad implications for both basic cellular biology and clinical intervention strategies.
Related JoVE Video
The biology and clinical impact of genetic lesions in myeloid malignancies.
Blood
PUBLISHED: 08-16-2013
Show Abstract
Hide Abstract
A longstanding endeavor to define the genetic lesions that drive myeloid malignances has stimulated a period of remarkable discovery. Enabled by technological advances that have sharply decreased the cost of DNA sequencing, the full compendium of common, recurrent somatic mutations in the coding genome of myeloid malignancies is nearly complete. As the focus of genetic discovery shifts to the noncoding genome, renewed attention is being applied to the clinical and biological implications of recent genomic advances. Although the potential for this newfound knowledge to influence the care of patients has not yet been realized, broad genetic surveys of patient samples are now being used to improve the accuracy of disease diagnosis, define a molecular taxonomy of myeloid malignancies, refine prognostic and predictive models, and identify novel therapeutic strategies. Here, we will review recent advances in the genetics of myeloid malignancies and discuss their potential impact on clinical practice.
Related JoVE Video
Diminutive somatic deletions in the 5q region lead to a phenotype atypical of classical 5q- syndrome.
Blood
PUBLISHED: 08-13-2013
Show Abstract
Hide Abstract
Classical 5q- syndrome is an acquired macrocytic anemia of the elderly. Similar to Diamond Blackfan anemia (DBA), an inherited red cell aplasia, the bone marrow is characterized by a paucity of erythroid precursors. RPS14 deletions in combination with other deletions in the region have been implicated as causative of the 5q- syndrome phenotype. We asked whether smaller, less easily detectable deletions could account for a syndrome with a modified phenotype. We employed single-nucleotide polymorphism array genotyping to identify small deletions in patients diagnosed with DBA and other anemias lacking molecular diagnoses. Diminutive mosaic deletions involving RPS14 were identified in a 5-year-old patient with nonclassical DBA and in a 17-year-old patient with myelodysplastic syndrome. Patients with nonclassical DBA and other hypoproliferative anemias may have somatically acquired 5q deletions with RPS14 haploinsufficiency not identified by fluorescence in situ hybridization or cytogenetic testing, thus refining the spectrum of disorders with 5q- deletions.
Related JoVE Video
Niche-based screening identifies small-molecule inhibitors of leukemia stem cells.
Nat. Chem. Biol.
PUBLISHED: 03-13-2013
Show Abstract
Hide Abstract
Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those compounds that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on target, via inhibition of HMG-CoA reductase. These results illustrate the power of merging physiologically relevant models with high-throughput screening.
Related JoVE Video
Depletion of Jak2V617F myeloproliferative neoplasm-propagating stem cells by interferon-? in a murine model of polycythemia vera.
Blood
PUBLISHED: 03-13-2013
Show Abstract
Hide Abstract
Interferon-? (IFN?) is an effective treatment of patients with myeloproliferative neoplasms (MPNs). In addition to inducing hematological responses in most MPN patients, IFN? reduces the JAK2V617F allelic burden and can render the JAK2V617F mutant clone undetectable in some patients. The precise mechanism underlying these responses is incompletely understood and whether the molecular responses that are seen occur due to the effects of IFN? on JAK2V617F mutant stem cells is debated. Using a murine model of Jak2V617F MPN, we investigated the effects of IFN? on Jak2V617F MPN-propagating stem cells in vivo. We report that IFN? treatment induces hematological responses in the model and causes depletion of Jak2V617F MPN-propagating cells over time, impairing disease transplantation. We demonstrate that IFN? treatment induces cell cycle activation of Jak2V617F mutant long-term hematopoietic stem cells and promotes a predetermined erythroid-lineage differentiation program. These findings provide insights into the differential effects of IFN? on Jak2V617F mutant and normal hematopoiesis and suggest that IFN? achieves molecular remissions in MPN patients through its effects on MPN stem cells. Furthermore, these results support combinatorial therapeutic approaches in MPN by concurrently depleting dormant JAK2V617F MPN-propagating stem cells with IFN? and targeting the proliferating downstream progeny with JAK2 inhibitors or cytotoxic chemotherapy.
Related JoVE Video
In Vivo RNAi screening identifies a leukemia-specific dependence on integrin beta 3 signaling.
Cancer Cell
PUBLISHED: 02-19-2013
Show Abstract
Hide Abstract
We used an in vivo small hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase Syk. In contrast, loss of Itgb3 in normal hematopoietic stem and progenitor cells did not affect engraftment, reconstitution, or differentiation. Finally, using an Itgb3 knockout mouse model, we confirmed that Itgb3 is dispensable for normal hematopoiesis but is required for leukemogenesis. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML.
Related JoVE Video
Macrophages support pathological erythropoiesis in polycythemia vera and ?-thalassemia.
Nat. Med.
PUBLISHED: 02-12-2013
Show Abstract
Hide Abstract
Regulation of erythropoiesis is achieved by the integration of distinct signals. Among them, macrophages are emerging as erythropoietin-complementary regulators of erythroid development, particularly under stress conditions. We investigated the contribution of macrophages to physiological and pathological conditions of enhanced erythropoiesis. We used mouse models of induced anemia, polycythemia vera and ?-thalassemia in which macrophages were chemically depleted. Our data indicate that macrophages contribute decisively to recovery from induced anemia, as well as the pathological progression of polycythemia vera and ?-thalassemia, by modulating erythroid proliferation and differentiation. We validated these observations in primary human cultures, showing a direct impact of macrophages on the proliferation and enucleation of erythroblasts from healthy individuals and patients with polycythemia vera or ?-thalassemia. The contribution of macrophages to stress and pathological erythropoiesis, which we have termed stress erythropoiesis macrophage-supporting activity, may have therapeutic implications.
Related JoVE Video
(R)-2-hydroxyglutarate is sufficient to promote leukemogenesis and its effects are reversible.
Science
PUBLISHED: 02-07-2013
Show Abstract
Hide Abstract
Mutations in IDH1 and IDH2, the genes coding for isocitrate dehydrogenases 1 and 2, are common in several human cancers, including leukemias, and result in overproduction of the (R)-enantiomer of 2-hydroxyglutarate [(R)-2HG]. Elucidation of the role of IDH mutations and (R)-2HG in leukemogenesis has been hampered by a lack of appropriate cell-based models. Here, we show that a canonical IDH1 mutant, IDH1 R132H, promotes cytokine independence and blocks differentiation in hematopoietic cells. These effects can be recapitulated by (R)-2HG, but not (S)-2HG, despite the fact that (S)-2HG more potently inhibits enzymes, such as the 5-methylcytosine hydroxylase TET2, that have previously been linked to the pathogenesis of IDH mutant tumors. We provide evidence that this paradox relates to the ability of (S)-2HG, but not (R)-2HG, to inhibit the EglN prolyl hydroxylases. Additionally, we show that transformation by (R)-2HG is reversible.
Related JoVE Video
New strategies in myelodysplastic syndromes: application of molecular diagnostics to clinical practice.
Clin. Cancer Res.
PUBLISHED: 01-17-2013
Show Abstract
Hide Abstract
An increasingly complete compendium of recurrently mutated genes in myelodysplastic syndromes (MDS) has been defined, and the application of massively parallel sequencing to identify mutations in clinical practice now promises to improve the care of patients with this disease. More than 25 recurrent MDS-associated somatic mutations have been identified, involving biologic pathways as diverse as chromatin remodeling and pre-mRNA splicing. Several of these mutations have been shown to have prognostic implications that are independent of existing risk stratification systems based on clinical and pathologic parameters. Application of these recent discoveries to diagnosis, prognosis, risk stratification, and treatment selection for patients with MDS has the potential to improve patient outcomes. Here, we review recent advances in MDS and discuss potential applications of these discoveries to clinical practice.
Related JoVE Video
Power cut: inhibiting mitochondrial translation to target leukemia.
Cancer Cell
PUBLISHED: 11-19-2011
Show Abstract
Hide Abstract
In this issue of Cancer Cell, Skrtic et al. demonstrate that inhibition of mitochondrial ribosomes with tigecycline, a known antimicrobial, selectively kills leukemia cells. This finding highlights the metabolic susceptibility of leukemia cells to mitochondrial translational inhibition and identifies a compound with significant efficacy in an in vivo preclinical model.
Related JoVE Video
Correction of sickle cell disease in adult mice by interference with fetal hemoglobin silencing.
Science
PUBLISHED: 10-13-2011
Show Abstract
Hide Abstract
Persistence of human fetal hemoglobin (HbF, ?(2)?(2)) in adults lessens the severity of sickle cell disease (SCD) and the ?-thalassemias. Here, we show that the repressor BCL11A is required in vivo for silencing of ?-globin expression in adult animals, yet dispensable for red cell production. BCL11A serves as a barrier to HbF reactivation by known HbF inducing agents. In a proof-of-principle test of BCL11A as a potential therapeutic target, we demonstrate that inactivation of BCL11A in SCD transgenic mice corrects the hematologic and pathologic defects associated with SCD through high-level pancellular HbF induction. Thus, interference with HbF silencing by manipulation of a single target protein is sufficient to reverse SCD.
Related JoVE Video
Molecular dissection of the 5q deletion in myelodysplastic syndrome.
Semin. Oncol.
PUBLISHED: 09-28-2011
Show Abstract
Hide Abstract
The 5q-syndrome is a subtype of myelodysplastic syndrome (MDS) with a defined clinical phenotype associated with heterozygous deletions of chromosome 5q. While no genes have been identified that undergo recurrent homozygous inactivation, functional studies have revealed individual genes that contribute to the clinical phenotype of MDS through haplo-insufficient gene expression. Heterozygous loss of the RPS14 gene on 5q leads to activation of p53 in the erythroid lineage and the macrocytic anemia characteristic of the 5q-syndrome. The megakaryocytic and platelet phenotype of the 5q-syndrome has been attributed to heterozygous deletion of miR145 and miR146a. Murine models have implicated heterozygous loss of APC, EGR1, DIAPH1, and NPM1 in the pathophysiology of del(5q) MDS. These findings indicate that the phenotype of MDS patients with deletions of chromosome 5q is due to haplo-insufficiency of multiple genes.
Related JoVE Video
Coordinate loss of a microRNA and protein-coding gene cooperate in the pathogenesis of 5q- syndrome.
Blood
PUBLISHED: 08-26-2011
Show Abstract
Hide Abstract
Large chromosomal deletions are among the most common molecular abnormalities in cancer, yet the identification of relevant genes has proven difficult. The 5q- syndrome, a subtype of myelodysplastic syndrome (MDS), is a chromosomal deletion syndrome characterized by anemia and thrombocytosis. Although we have previously shown that hemizygous loss of RPS14 recapitulates the failed erythroid differentiation seen in 5q- syndrome, it does not affect thrombocytosis. Here we show that a microRNA located in the common deletion region of 5q- syndrome, miR-145, affects megakaryocyte and erythroid differentiation. We find that miR-145 functions through repression of Fli-1, a megakaryocyte and erythroid regulatory transcription factor. Patients with del(5q) MDS have decreased expression of miR-145 and increased expression of Fli-1. Overexpression of miR-145 or inhibition of Fli-1 decreases the production of megakaryocytic cells relative to erythroid cells, whereas inhibition of miR-145 or overexpression of Fli-1 has a reciprocal effect. Moreover, combined loss of miR-145 and RPS14 cooperates to alter erythroid-megakaryocytic differentiation in a manner similar to the 5q- syndrome. Taken together, these findings demonstrate that coordinate deletion of a miRNA and a protein-coding gene contributes to the phenotype of a human malignancy, the 5q- syndrome.
Related JoVE Video
Clinical effect of point mutations in myelodysplastic syndromes.
N. Engl. J. Med.
PUBLISHED: 07-01-2011
Show Abstract
Hide Abstract
Myelodysplastic syndromes are clinically heterogeneous disorders characterized by clonal hematopoiesis, impaired differentiation, peripheral-blood cytopenias, and a risk of progression to acute myeloid leukemia. Somatic mutations may influence the clinical phenotype but are not included in current prognostic scoring systems.
Related JoVE Video
Stem cell gene expression programs influence clinical outcome in human leukemia.
Nat. Med.
PUBLISHED: 06-09-2011
Show Abstract
Hide Abstract
Xenograft studies indicate that some solid tumors and leukemias are organized as cellular hierarchies sustained by cancer stem cells (CSCs). Despite the promise of the CSC model, its relevance in humans remains uncertain. Here we show that acute myeloid leukemia (AML) follows a CSC model on the basis of sorting multiple populations from each of 16 primary human AML samples and identifying which contain leukemia stem cells (LSCs) using a sensitive xenograft assay. Analysis of gene expression from all functionally validated populations yielded an LSC-specific signature. Similarly, a hematopoietic stem cell (HSC) gene signature was established. Bioinformatic analysis identified a core transcriptional program shared by LSCs and HSCs, revealing the molecular machinery underlying stemness properties. Both stem cell programs were highly significant independent predictors of patient survival and were found in existing prognostic signatures. Thus, determinants of stemness influence the clinical outcome of AML, establishing that LSCs are clinically relevant and not artifacts of xenotransplantation.
Related JoVE Video
Dexamethasone and lenalidomide have distinct functional effects on erythropoiesis.
Blood
PUBLISHED: 04-28-2011
Show Abstract
Hide Abstract
Corticosteroids and lenalidomide decrease red blood cell transfusion dependence in patients with Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome (MDS), respectively. We explored the effects of dexamethasone and lenalidomide, individually and in combination, on the differentiation of primary human bone marrow progenitor cells in vitro. Both agents promote erythropoiesis, increasing the absolute number of erythroid cells produced from normal CD34(+) cells and from CD34(+) cells with the types of ribosome dysfunction found in DBA and del(5q) MDS. However, the drugs had distinct effects on the production of erythroid progenitor colonies; dexamethasone selectively increased the number of burst-forming units-erythroid (BFU-E), whereas lenalidomide specifically increased colony-forming unit-erythroid (CFU-E). Use of the drugs in combination demonstrated that their effects are not redundant. In addition, dexamethasone and lenalidomide induced distinct gene-expression profiles. In coculture experiments, we examined the role of the microenvironment in response to both drugs and found that the presence of macrophages, the central cells in erythroblastic islands, accentuated the effects of both agents. Our findings indicate that dexamethasone and lenalidomide promote different stages of erythropoiesis and support the potential clinical utility of combination therapy for patients with bone marrow failure.
Related JoVE Video
Human tumors instigate granulin-expressing hematopoietic cells that promote malignancy by activating stromal fibroblasts in mice.
J. Clin. Invest.
PUBLISHED: 01-25-2011
Show Abstract
Hide Abstract
Systemic instigation is a process by which endocrine signals sent from certain tumors (instigators) stimulate BM cells (BMCs), which are mobilized into the circulation and subsequently foster the growth of otherwise indolent carcinoma cells (responders) residing at distant anatomical sites. The identity of the BMCs and their specific contribution or contributions to responder tumor growth have been elusive. Here, we have demonstrated that Sca1+ cKit- hematopoietic BMCs of mouse hosts bearing instigating tumors promote the growth of responding tumors that form with a myofibroblast-rich, desmoplastic stroma. Such stroma is almost always observed in malignant human adenocarcinomas and is an indicator of poor prognosis. We then identified granulin (GRN) as the most upregulated gene in instigating Sca1+ cKit- BMCs relative to counterpart control cells. The GRN+ BMCs that were recruited to the responding tumors induced resident tissue fibroblasts to express genes that promoted malignant tumor progression; indeed, treatment with recombinant GRN alone was sufficient to promote desmoplastic responding tumor growth. Further, analysis of tumor tissues from a cohort of breast cancer patients revealed that high GRN expression correlated with the most aggressive triple-negative, basal-like tumor subtype and reduced patient survival. Our data suggest that GRN and the unique hematopoietic BMCs that produce it might serve as novel therapeutic targets.
Related JoVE Video
Ribosome defects in disorders of erythropoiesis.
Int. J. Hematol.
PUBLISHED: 01-18-2011
Show Abstract
Hide Abstract
Over the past decade, genetic lesions that cause ribosome dysfunction have been identified in both congenital and acquired human disorders. These discoveries have established a new category of disorders, known as ribosomopathies, in which the primary pathophysiology is related to impaired ribosome function. The protoptypical disorders are Diamond-Blackfan anemia, a congenital bone marrow failure syndrome, and the 5q- syndrome, a subtype of myelodysplastic syndrome. In both of these disorders, impaired ribosome function causes a severe macrocytic anemia. In this review, we will discuss the evidence that defects in ribosomal biogenesis cause the hematologic phenotype of Diamond-Blackfan anemia and the 5q- syndrome. We will also explore the potential mechanisms by which a ribosomal defect, which would be expected to have widespread consequences, may lead to specific defects in erythropoiesis.
Related JoVE Video
The STAT5 inhibitor pimozide decreases survival of chronic myelogenous leukemia cells resistant to kinase inhibitors.
Blood
PUBLISHED: 01-13-2011
Show Abstract
Hide Abstract
The transcription factor STAT5 is an essential mediator of the pathogenesis of chronic myelogenous leukemia (CML). In CML, the BCR/ABL fusion kinase causes the constitutive activation of STAT5, thereby driving the expression of genes promoting survival. BCR/ABL kinase inhibitors have become the mainstay of therapy for CML, although CML cells can develop resistance through mutations in BCR/ABL. To overcome this problem, we used a cell-based screen to identify drugs that inhibit STAT-dependent gene expression. Using this approach, we identified the psychotropic drug pimozide as a STAT5 inhibitor. Pimozide decreases STAT5 tyrosine phosphorylation, although it does not inhibit BCR/ABL or other tyrosine kinases. Furthermore, pimozide decreases the expression of STAT5 target genes and induces cell cycle arrest and apoptosis in CML cell lines. Pimozide also selectively inhibits colony formation of CD34(+) bone marrow cells from CML patients. Importantly, pimozide induces similar effects in the presence of the T315I BCR/ABL mutation that renders the kinase resistant to presently available inhibitors. Simultaneously inhibiting STAT5 with pimozide and the kinase inhibitors imatinib or nilotinib shows enhanced effects in inhibiting STAT5 phosphorylation and in inducing apoptosis. Thus, targeting STAT5 may be an effective strategy for the treatment of CML and other myeloproliferative diseases.
Related JoVE Video
Unraveling the molecular pathophysiology of myelodysplastic syndromes.
J. Clin. Oncol.
PUBLISHED: 01-10-2011
Show Abstract
Hide Abstract
Somatically acquired genetic abnormalities lead to the salient features that define myelodysplastic syndromes (MDS): clonal hematopoiesis, aberrant differentiation, peripheral cytopenias, and risk of progression to acute myeloid leukemia. Although specific karyotypic abnormalities have been linked to MDS for decades, more recent findings have demonstrated the importance of mutations within individual genes, focal alterations that are not apparent by standard cytogenetics, and aberrant epigenetic regulation of gene expression. The spectrum of genetic abnormalities in MDS implicates a wide range of molecular mechanisms in the pathogenesis of these disorders, including activation of tyrosine kinase signaling, genomic instability, impaired differentiation, altered ribosome function, and changes in the bone marrow microenvironment. Specific alterations present in individual patients with MDS may explain much of the heterogeneity in clinical phenotype associated with this disease and can predict prognosis and response to therapy. Elucidation of the full complement of genetic causes of MDS promises profound insight into the biology of the disease, improved classification and prognostic scoring schemes, and the potential for novel targeted therapies with molecular predictors of response.
Related JoVE Video
Densely interconnected transcriptional circuits control cell states in human hematopoiesis.
Cell
PUBLISHED: 01-04-2011
Show Abstract
Hide Abstract
Though many individual transcription factors are known to regulate hematopoietic differentiation, major aspects of the global architecture of hematopoiesis remain unknown. Here, we profiled gene expression in 38 distinct purified populations of human hematopoietic cells and used probabilistic models of gene expression and analysis of cis-elements in gene promoters to decipher the general organization of their regulatory circuitry. We identified modules of highly coexpressed genes, some of which are restricted to a single lineage but most of which are expressed at variable levels across multiple lineages. We found densely interconnected cis-regulatory circuits and a large number of transcription factors that are differentially expressed across hematopoietic states. These findings suggest a more complex regulatory system for hematopoiesis than previously assumed.
Related JoVE Video
Haploinsufficiency for ribosomal protein genes causes selective activation of p53 in human erythroid progenitor cells.
Blood
PUBLISHED: 11-10-2010
Show Abstract
Hide Abstract
Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q-syndrome, a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction, but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14, the ribosomal protein gene deleted in the 5q-syndrome, or RPS19, the most commonly mutated gene in DBA. Induction of p53 led to lineage-specific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect, whereas nutlin-3, a compound that activates p53 through inhibition of HDM2, selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome, we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53, providing a basis for the failure of erythropoiesis in the 5q-syndrome, DBA, and perhaps other bone marrow failure syndromes.
Related JoVE Video
Genetic deletions in AML and MDS.
Best Pract Res Clin Haematol
PUBLISHED: 11-04-2010
Show Abstract
Hide Abstract
Chromosomal deletions are common molecular events in myeloid malignancies. Heterozygous deletions may contain a tumor suppressor gene that undergoes homozygous inactivation or may contain one or more genes that alter the disease phenotype through haploinsufficiency. The most common karyotypic abnormality in myelodysplastic syndrome (MDS) is deletion of chromosome 5q. A subset of patients with del(5q) as a sole cytogenetic abnormality has a consistent set of clinical features, termed the 5q- syndrome. While no tumor suppressor genes have been identified on 5q that are homozygously inactivated, recent studies have highlighted several genes and micro RNAs (miRNAs) that cause the phenotype of the 5q- syndrome through allelic insufficiency. For example, deletion of one allele of the RPS14 gene causes a severe defect in erythropoiesis, analogous to the congenital syndrome Diamond Blackfan anemia, which is itself caused by mutations that inactivate one allele of a ribosomal gene. Loss of one allele of miR-145 and miR-146a causes an increase in megakaryocyte production and may contribute to the clonal advantage of cells with del(5q). The functional approaches used to dissect the molecular basis of the 5q deletion in MDS have the potential to identify key genes and therapeutic targets within other chromosomal deletions in hematologic malignancies.
Related JoVE Video
Transcriptional analysis of HIV-specific CD8+ T cells shows that PD-1 inhibits T cell function by upregulating BATF.
Nat. Med.
PUBLISHED: 07-13-2010
Show Abstract
Hide Abstract
CD8(+) T cells in chronic viral infections such as HIV develop functional defects including loss of interleukin-2 (IL-2) secretion and decreased proliferative potential that are collectively termed exhaustion. Exhausted T cells express increased amounts of multiple inhibitory receptors, such as programmed death-1 (PD-1), that contribute to impaired virus-specific T cell function. Although reversing PD-1 inhibition is therefore an attractive therapeutic strategy, the cellular mechanisms by which PD-1 ligation results in T cell inhibition are not fully understood. PD-1 is thought to limit T cell activation by attenuating T cell receptor (TCR) signaling. It is not known whether PD-1 also acts by upregulating genes in exhausted T cells that impair their function. Here we analyzed gene expression profiles from HIV-specific CD8(+) T cells in individuals with HIV and show that PD-1 coordinately upregulates a program of genes in exhausted CD8(+) T cells from humans and mice. This program includes upregulation of basic leucine transcription factor, ATF-like (BATF), a transcription factor in the AP-1 family. Enforced expression of BATF was sufficient to impair T cell proliferation and cytokine secretion, whereas BATF knockdown reduced PD-1 inhibition. Silencing BATF in T cells from individuals with chronic viremia rescued HIV-specific T cell function. Thus, inhibitory receptors can cause T cell exhaustion by upregulating genes--such as BATF--that inhibit T cell function. Such genes may provide new therapeutic opportunities to improve T cell immunity to HIV.
Related JoVE Video
NF1 inactivation revs up Ras in adult acute myelogenous leukemia.
Clin. Cancer Res.
PUBLISHED: 06-29-2010
Show Abstract
Hide Abstract
Mutations in the Ras pathway are common in myeloid malignancies. NF1, a tumor suppressor and negative regulator of Ras, is inactivated in a subset of adult acute myelogenous leukemia (AML) cases. Loss of NF1 function sensitizes cells to inhibition of mammalian target of rapamycin (mTOR), a downstream effector of Ras activation, highlighting a potential therapeutic opportunity for some AML patients.
Related JoVE Video
Chemical genetic strategy identifies histone deacetylase 1 (HDAC1) and HDAC2 as therapeutic targets in sickle cell disease.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-28-2010
Show Abstract
Hide Abstract
The worldwide burden of sickle cell disease is enormous, with over 200,000 infants born with the disease each year in Africa alone. Induction of fetal hemoglobin is a validated strategy to improve symptoms and complications of this disease. The development of targeted therapies has been limited by the absence of discrete druggable targets. We developed a unique bead-based strategy for the identification of inducers of fetal hemoglobin transcripts in primary human erythroid cells. A small-molecule screen of bioactive compounds identified remarkable class-associated activity among histone deacetylase (HDAC) inhibitors. Using a chemical genetic strategy combining focused libraries of biased chemical probes and reverse genetics by RNA interference, we have identified HDAC1 and HDAC2 as molecular targets mediating fetal hemoglobin induction. Our findings suggest the potential of isoform-selective inhibitors of HDAC1 and HDAC2 for the treatment of sickle cell disease.
Related JoVE Video
Physiological Jak2V617F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells.
Cancer Cell
PUBLISHED: 04-03-2010
Show Abstract
Hide Abstract
We report a Jak2V617F knockin mouse myeloproliferative neoplasm (MPN) model resembling human polycythemia vera (PV). The MPN is serially transplantable and we demonstrate that the hematopoietic stem cell (HSC) compartment has the unique capacity for disease initiation but does not have a significant selective competitive advantage over wild-type HSCs. In contrast, myeloid progenitor populations are expanded and skewed toward the erythroid lineage, but cannot transplant the disease. Treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. These findings provide insights into the consequences of JAK2 activation on HSC differentiation and function and have the potential to inform therapeutic approaches to JAK2V617F-positive MPN.
Related JoVE Video
The genetic basis of myelodysplastic syndromes.
Hematol. Oncol. Clin. North Am.
PUBLISHED: 04-03-2010
Show Abstract
Hide Abstract
Myelodysplastic syndrome (MDS) disorders are clonal diseases that often carry stereotypic chromosomal abnormalities. A smaller proportion of cases harbor point mutations that activate oncogenes or inactivate tumor suppressor genes. New technologies have accelerated the pace of discovery and are responsible for the identification of novel genetic mutations associated with MDS and other myeloid neoplasms. These discoveries have identified novel mechanisms in the pathogenesis of MDS. This article touches on the better known genetic abnormalities in MDS and explains in greater detail those that have been discovered more recently. Understanding how mutations lead to MDS and how they might cooperate with each other has become more complicated as the number of MDS-associated genetic abnormalities has grown. In some cases, these mutations have prognostic significance that could improve upon the various prognostic scoring systems in common clinical use.
Related JoVE Video
Musashi-2 regulates normal hematopoiesis and promotes aggressive myeloid leukemia.
Nat. Med.
PUBLISHED: 04-02-2010
Show Abstract
Hide Abstract
RNA-binding proteins of the Musashi (Msi) family are expressed in stem cell compartments and in aggressive tumors, but they have not yet been widely explored in the blood. Here we demonstrate that Msi2 is the predominant form expressed in hematopoietic stem cells (HSCs), and its knockdown leads to reduced engraftment and depletion of HSCs in vivo. Overexpression of human MSI2 in a mouse model increases HSC cell cycle progression and cooperates with the chronic myeloid leukemia-associated BCR-ABL1 oncoprotein to induce an aggressive leukemia. MSI2 is overexpressed in human myeloid leukemia cell lines, and its depletion leads to decreased proliferation and increased apoptosis. Expression levels in human myeloid leukemia directly correlate with decreased survival in patients with the disease, thereby defining MSI2 expression as a new prognostic marker and as a new target for therapy in acute myeloid leukemia (AML).
Related JoVE Video
The connectivity map links iron regulatory protein-1-mediated inhibition of hypoxia-inducible factor-2a translation to the anti-inflammatory 15-deoxy-delta12,14-prostaglandin J2.
Cancer Res.
PUBLISHED: 03-30-2010
Show Abstract
Hide Abstract
Hypoxia-inducible factors 1 and 2 (HIF1 and HIF2) are heterodimeric transcription factors consisting of alpha regulatory subunits and a constitutively expressed beta subunit. The expression of alpha regulatory subunits is promoted by hypoxia, cancer-associated mutations, and inflammatory cytokines. Thus, HIF1 and HIF2 provide a molecular link between cancer and inflammation. We have recently identified novel small molecules that selectively inhibit translation of the HIF2a message and thereby powerfully inhibit the expression of HIF2a target genes. We report here that Connectivity Map analysis links three of these compounds to the anti-inflammatory cytokine 15-deoxy-Delta(12,14)-prostaglandin J(2) (PGJ(2)). As with our identified compounds, PGJ(2) inhibits translation of the HIF2a message in a mammalian target of rapamycin-independent manner by promoting the binding of iron regulatory protein-1 (IRP1) to a noncanonical iron responsive element (IRE) embedded within the 5-untranslated region of the HIF2a message. The IRE is necessary and sufficient for mediating the effect. Mutation of the IRE sequence, or downregulation of IRP1 expression, blocks the effect of PGJ(2) on HIF2a translation. This is the first report of an endogenous natural molecule regulating HIF2a translation, and it suggests that part of the anti-inflammatory and putative antineoplastic effects of PGJ(2) may be mediated through inhibition of HIF2a within tumor epithelial cells themselves and/or mesenchymal cells of the tumor microenvironment.
Related JoVE Video
The Apc(min) mouse has altered hematopoietic stem cell function and provides a model for MPD/MDS.
Blood
PUBLISHED: 03-02-2010
Show Abstract
Hide Abstract
Apc, a negative regulator of the canonical Wnt signaling pathway, is a bona-fide tumor suppressor whose loss of function results in intestinal polyposis. APC is located in a commonly deleted region on human chromosome 5q, associated with myelodysplastic syndrome (MDS), suggesting that haploinsufficiency of APC contributes to the MDS phenotype. Analysis of the hematopoietic system of mice with the Apc(min) allele that results in a premature stop codon and loss of function showed no abnormality in steady state hematopoiesis. Bone marrow derived from Apc(min) mice showed enhanced repopulation potential, indicating a cell intrinsic gain of function in the long-term hematopoietic stem cell (HSC) population. However, Apc(min) bone marrow was unable to repopulate secondary recipients because of loss of the quiescent HSC population. Apc(min) mice developed a MDS/myeloproliferative phenotype. Our data indicate that Wnt activation through haploinsufficiency of Apc causes insidious loss of HSC function that is only evident in serial transplantation strategies. These data provide a cautionary note for HSC-expansion strategies through Wnt pathway activation, provide evidence that cell extrinsic factors can contribute to the development of myeloid disease, and indicate that loss of function of APC may contribute to the phenotype observed in patients with MDS and del(5q).
Related JoVE Video
Ribosomopathies: human disorders of ribosome dysfunction.
Blood
PUBLISHED: 03-01-2010
Show Abstract
Hide Abstract
Ribosomopathies compose a collection of disorders in which genetic abnormalities cause impaired ribosome biogenesis and function, resulting in specific clinical phenotypes. Congenital mutations in RPS19 and other genes encoding ribosomal proteins cause Diamond-Blackfan anemia, a disorder characterized by hypoplastic, macrocytic anemia. Mutations in other genes required for normal ribosome biogenesis have been implicated in other rare congenital syndromes, Schwachman-Diamond syndrome, dyskeratosis congenita, cartilage hair hypoplasia, and Treacher Collins syndrome. In addition, the 5q- syndrome, a subtype of myelodysplastic syndrome, is caused by a somatically acquired deletion of chromosome 5q, which leads to haploinsufficiency of the ribosomal protein RPS14 and an erythroid phenotype highly similar to Diamond-Blackfan anemia. Acquired abnormalities in ribosome function have been implicated more broadly in human malignancies. The p53 pathway provides a surveillance mechanism for protein translation as well as genome integrity and is activated by defects in ribosome biogenesis; this pathway appears to be a critical mediator of many of the clinical features of ribosomopathies. Elucidation of the mechanisms whereby selective abnormalities in ribosome biogenesis cause specific clinical syndromes will hopefully lead to novel therapeutic strategies for these diseases.
Related JoVE Video
The landscape of somatic copy-number alteration across human cancers.
Nature
PUBLISHED: 02-19-2010
Show Abstract
Hide Abstract
A powerful way to discover key genes with causal roles in oncogenesis is to identify genomic regions that undergo frequent alteration in human cancers. Here we present high-resolution analyses of somatic copy-number alterations (SCNAs) from 3,131 cancer specimens, belonging largely to 26 histological types. We identify 158 regions of focal SCNA that are altered at significant frequency across several cancer types, of which 122 cannot be explained by the presence of a known cancer target gene located within these regions. Several gene families are enriched among these regions of focal SCNA, including the BCL2 family of apoptosis regulators and the NF-kappaBeta pathway. We show that cancer cells containing amplifications surrounding the MCL1 and BCL2L1 anti-apoptotic genes depend on the expression of these genes for survival. Finally, we demonstrate that a large majority of SCNAs identified in individual cancer types are present in several cancer types.
Related JoVE Video
Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia.
Nature
PUBLISHED: 01-19-2010
Show Abstract
Hide Abstract
Mesenchymal cells contribute to the stroma of most normal and malignant tissues, with specific mesenchymal cells participating in the regulatory niches of stem cells. By examining how mesenchymal osteolineage cells modulate haematopoiesis, here we show that deletion of Dicer1 specifically in mouse osteoprogenitors, but not in mature osteoblasts, disrupts the integrity of haematopoiesis. Myelodysplasia resulted and acute myelogenous leukaemia emerged that had acquired several genetic abnormalities while having intact Dicer1. Examining gene expression altered in osteoprogenitors as a result of Dicer1 deletion showed reduced expression of Sbds, the gene mutated in Schwachman-Bodian-Diamond syndrome-a human bone marrow failure and leukaemia pre-disposition condition. Deletion of Sbds in mouse osteoprogenitors induced bone marrow dysfunction with myelodysplasia. Therefore, perturbation of specific mesenchymal subsets of stromal cells can disorder differentiation, proliferation and apoptosis of heterologous cells, and disrupt tissue homeostasis. Furthermore, primary stromal dysfunction can result in secondary neoplastic disease, supporting the concept of niche-induced oncogenesis.
Related JoVE Video
Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies.
Blood
PUBLISHED: 05-06-2009
Show Abstract
Hide Abstract
Disease alleles that activate signal transduction are common in myeloid malignancies; however, there are additional unidentified mutations that contribute to myeloid transformation. Based on the recent identification of TET2 mutations, we evaluated the mutational status of TET1, TET2, and TET3 in myeloproliferative neoplasms (MPNs), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). Sequencing of TET2 in 408 paired tumor/normal samples distinguished between 68 somatic mutations and 6 novel single nucleotide polymorphisms and identified TET2 mutations in MPN (27 of 354, 7.6%), CMML (29 of 69, 42%), AML (11 of 91, 12%), and M7 AML (1 of 28, 3.6%) samples. We did not identify somatic TET1 or TET3 mutations or TET2 promoter hypermethylation in MPNs. TET2 mutations did not cluster in genetically defined MPN, CMML, or AML subsets but were associated with decreased overall survival in AML (P = .029). These data indicate that TET2 mutations are observed in different myeloid malignancies and may be important in AML prognosis.
Related JoVE Video
Ultrasome: efficient aberration caller for copy number studies of ultra-high resolution.
Bioinformatics
PUBLISHED: 02-19-2009
Show Abstract
Hide Abstract
Multimillion-probe microarrays allow detection of gains and losses of chromosomal material at unprecedented resolution. However, the data generated by these arrays are several-fold larger than data from earlier platforms, creating a need for efficient analysis tools that scale robustly with data size.
Related JoVE Video
A germline JAK2 SNP is associated with predisposition to the development of JAK2(V617F)-positive myeloproliferative neoplasms.
Nat. Genet.
PUBLISHED: 01-06-2009
Show Abstract
Hide Abstract
Polycythemia vera, essential thrombocythemia and primary myelofibrosis are myeloproliferative neoplasms (MPN) characterized by multilineage clonal hematopoiesis. Given that the identical somatic activating mutation in the JAK2 tyrosine kinase gene (JAK2(V617F)) is observed in most individuals with polycythemia vera, essential thrombocythemia and primary myelofibrosis, there likely are additional genetic events that contribute to the pathogenesis of these phenotypically distinct disorders. Moreover, family members of individuals with MPN are at higher risk for the development of MPN, consistent with the existence of MPN predisposition loci. We hypothesized that germline variation contributes to MPN predisposition and phenotypic pleiotropy. Genome-wide analysis identified an allele in the JAK2 locus (rs10974944) that predisposes to the development of JAK2(V617F)-positive MPN, as well as three previously unknown MPN modifier loci. We found that JAK2(V617F) is preferentially acquired in cis with the predisposition allele. These data suggest that germline variation is an important contributor to MPN phenotype and predisposition.
Related JoVE Video
Implantable microenvironments to attract hematopoietic stem/cancer cells.
Proc. Natl. Acad. Sci. U.S.A.
Show Abstract
Hide Abstract
The environments that harbor hematopoietic stem and progenitor cells are critical to explore for a better understanding of hematopoiesis during health and disease. These compartments often are inaccessible for controlled and rapid experimentation, thus limiting studies to the evaluation of conventional cell culture and transgenic animal models. Here we describe the manufacture and image-guided monitoring of an engineered microenvironment with user-defined properties that recruits hematopoietic progenitors into the implant. Using intravital imaging and fluorescence molecular tomography, we show in real time that the cell homing and retention process is efficient and durable for short- and long-term engraftment studies. Our results indicate that bone marrow stromal cells, precoated on the implant, accelerate the formation of new sinusoidal blood vessels with vascular integrity at the microcapillary level that enhances the recruitment hematopoietic progenitor cells to the site. This implantable construct can serve as a tool enabling the study of hematopoiesis.
Related JoVE Video
Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts.
Nature
Show Abstract
Hide Abstract
Defects in the availability of haem substrates or the catalytic activity of the terminal enzyme in haem biosynthesis, ferrochelatase (Fech), impair haem synthesis and thus cause human congenital anaemias. The interdependent functions of regulators of mitochondrial homeostasis and enzymes responsible for haem synthesis are largely unknown. To investigate this we used zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anaemia, pinotage (pnt (tq209)). Here we describe a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize haem. The loss of Atpif1 impairs haemoglobin synthesis in zebrafish, mouse and human haematopoietic models as a consequence of diminished Fech activity and elevated mitochondrial pH. To understand the relationship between mitochondrial pH, redox potential, [2Fe-2S] clusters and Fech activity, we used genetic complementation studies of Fech constructs with or without [2Fe-2S] clusters in pnt, as well as pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe-2S] cluster renders vertebrate Fech vulnerable to perturbations in Atpif1-regulated mitochondrial pH and redox potential. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize haem, resulting in anaemia. The identification of mitochondrial Atpif1 as a regulator of haem synthesis advances our understanding of the mechanisms regulating mitochondrial haem homeostasis and red blood cell development. An ATPIF1 deficiency may contribute to important human diseases, such as congenital sideroblastic anaemias and mitochondriopathies.
Related JoVE Video
Myeloproliferative neoplasm animal models.
Hematol. Oncol. Clin. North Am.
Show Abstract
Hide Abstract
Myeloproliferative neoplasm (MPN) animal models accurately re-capitulate human disease in mice and have been an important tool for the study of MPN biology and therapy. Transplantation of BCR-ABL transduced bone marrow into irradiated syngeneic mice established the field of MPN animal modeling. Genetically engineered MPN animal models have enabled detailed characterization of the effects of specific MPN-associated genetic abnormalities on hematopoietic stem and progenitor cells (HSPCs). Xenograft models have allowed the study of primary human MPN-propagating cells in vivo. JAK2V617F, the most common molecular abnormality in BCR-ABL negative MPN, has been extensively studied using retroviral, transgenic, knock-in and xenograft models.
Related JoVE Video
Molecular pathophysiology of myelodysplastic syndromes.
Annu Rev Pathol
Show Abstract
Hide Abstract
The clinicopathologic heterogeneity of myelodysplastic syndromes (MDS) is driven by diverse, somatically acquired genetic abnormalities. Recent technological advances have enabled the identification of many new mutations, which have implicated novel pathways in MDS pathogenesis, including RNA splicing and epigenetic regulation of gene expression. Molecular abnormalities, either somatic point mutations or chromosomal lesions, can be identified in the vast majority of MDS cases and underlie specific disease phenotypes. As the full array of molecular abnormalities is characterized, genetic variables are likely to complement standard morphologic evaluation in future MDS classification schemes and risk models.
Related JoVE Video
Validation of a prognostic model and the impact of mutations in patients with lower-risk myelodysplastic syndromes.
J. Clin. Oncol.
Show Abstract
Hide Abstract
A subset of patients with myelodysplastic syndromes (MDS) who are predicted to have lower-risk disease as defined by the International Prognostic Scoring System (IPSS) demonstrate more aggressive disease and shorter overall survival than expected. The identification of patients with greater-than-predicted prognostic risk could influence the selection of therapy and improve the care of patients with lower-risk MDS.
Related JoVE Video
Gene sets identified with oncogene cooperativity analysis regulate in vivo growth and survival of leukemia stem cells.
Cell Stem Cell
Show Abstract
Hide Abstract
Leukemia stem cells (LSCs) represent a biologically distinct subpopulation of myeloid leukemias, with reduced cell cycle activity and increased resistance to therapeutic challenge. To better characterize key properties of LSCs, we employed a strategy based on identification of genes synergistically dysregulated by cooperating oncogenes. We hypothesized that such genes, termed "cooperation response genes" (CRGs), would represent regulators of LSC growth and survival. Using both a primary mouse model and human leukemia specimens, we show that CRGs comprise genes previously undescribed in leukemia pathogenesis in which multiple pathways modulate the biology of LSCs. In addition, our findings demonstrate that the CRG expression profile can be used as a drug discovery tool for identification of compounds that selectively target the LSC population. We conclude that CRG-based analyses provide a powerful means to characterize the basic biology of LSCs as well as to identify improved methods for therapeutic targeting.
Related JoVE Video
Heterodimeric JAK-STAT activation as a mechanism of persistence to JAK2 inhibitor therapy.
Nature
Show Abstract
Hide Abstract
The identification of somatic activating mutations in JAK2 (refs?1–4) and in the thrombopoietin receptor gene (MPL) in most patients with myeloproliferative neoplasm (MPN) led to the clinical development of JAK2 kinase inhibitors. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms but does not significantly decrease or eliminate the MPN clone in most patients with MPN. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic inhibition of JAK2. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAK–STAT signalling and with heterodimerization between activated JAK2 and JAK1 or TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible: JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, in murine models and in patients treated with JAK2 inhibitors. RNA interference and pharmacological studies show that JAK2-inhibitor-persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors.
Related JoVE Video
Novel approaches to the treatment of sickle cell disease: the potential of histone deacetylase inhibitors.
Expert Rev Hematol
Show Abstract
Hide Abstract
Sickle cell disease (SCD) is a severe genetic disorder of hemoglobin causing vaso-occlusion. Patients suffer severe anemia, strokes, renal failure, pulmonary compromise and shortened life expectancy. Over 90,000 people in the USA have SCD, and the options for therapy are limited and only partially effective. With the available therapies - hydroxyurea, blood transfusion, hydration and pain medicines - patients continue to suffer the long-term complications of the disease. This review focuses on the pathogenesis of SCD and the role of fetal hemoglobin in disrupting the polymerization of sickle hemoglobin. The authors review the compounds that induce fetal hemoglobin: hydroxyurea, which is currently US FDA approved, and the histone deacetylase inhibitors and discuss their role in the treatment of SCD and other ?-hemoglobinopathies.
Related JoVE Video
L-Leucine improves the anemia and developmental defects associated with Diamond-Blackfan anemia and del(5q) MDS by activating the mTOR pathway.
Blood
Show Abstract
Hide Abstract
Haploinsufficiency of ribosomal proteins (RPs) has been proposed to be the common basis for the anemia observed in Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q [del(5q) MDS]. We have modeled DBA and del(5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14, respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. To address the hypothesis that RP loss results in impaired mRNA translation, we treated Rps19 and Rps14-deficient embryos with the amino acid L-leucine, a known activator of mRNA translation. This resulted in a striking improvement of the anemia associated with RP loss. We confirmed our findings in primary human CD34? cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in both Rps19 and Rps14 morphants. This effect could be abrogated by rapamycin suggesting that mTOR signaling may be responsible for the improvement in anemia associated with L-leucine. Our studies support the rationale for ongoing clinical trials of L-leucine as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS.
Related JoVE Video
Distinct roles for long-term hematopoietic stem cells and erythroid precursor cells in a murine model of Jak2V617F-mediated polycythemia vera.
Blood
Show Abstract
Hide Abstract
In the current model of the pathogenesis of polycythemia vera (PV), the JAK2V617F mutation arises in hematopoietic stem cells (HSCs) that maintain the disease, while erythroid precursor populations expand, resulting in excessive red blood cell production. We examined the role of these specific cell populations using a conditional Jak2V617F knockin murine model. We demonstrate that the most immature long-term (LT) HSCs are solely responsible for initiating and maintaining the disease in vivo and that Jak2V617F mutant LT-HSCs dominate hematopoiesis over time. When we induced Jak2V617F expression in erythropoietin receptor expressing precursor cells, the mice developed elevated hematocrit, expanded erythroid precursors, and suppressed erythropoietin levels. However, the disease phenotype was significantly attenuated compared with mice expressing Jak2V617F in LT-HSCs. In addition to developing a PV phenotype, all mice transplanted with Jak2V617F LT-HSCs underwent myelofibrotic transformation over time. These findings recapitulate the development of post-PV myelofibrosis in human myeloproliferative neoplasms. In aggregate, these results demonstrate the distinct roles of LT-HSCs and erythroid precursors in the pathogenesis of PV.
Related JoVE Video
Stress granules contribute to ?-globin homeostasis in differentiating erythroid cells.
Biochem. Biophys. Res. Commun.
Show Abstract
Hide Abstract
Hemoglobin is the major biosynthetic product of developing erythroid cells. Assembly of hemoglobin requires the balanced production of globin proteins and the oxygen-carrying heme moiety. The heme-regulated inhibitor kinase (HRI) participates in this process by phosphorylating eIF2? and inhibiting the translation of globin proteins when levels of free heme are limiting. HRI is also activated in erythroid cells subjected to oxidative stress. Phospho-eIF2?-mediated translational repression induces the assembly of stress granules (SG), cytoplasmic foci that harbor untranslated mRNAs and promote the survival of cells subjected to adverse environmental conditions. We have found that differentiating erythroid, but not myelomonocytic or megakaryocytic, murine and human progenitor cells assemble SGs, in vitro and in vivo. Targeted knockdown of HRI or G3BP, a protein required for SG assembly, inhibits spontaneous and arsenite-induced assembly of SGs in erythroid progenitor cells. This is accompanied by reduced ?-globin production and increased apoptosis suggesting that G3BP+ SGs facilitate the survival of developing erythroid cells.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.