技术资料

TEL-TRKC is a fusion gene generated by chromosomal translocation and encodes an activated tyrosine kinase. Uniquely,it is found in both solid tumors and leukemia. However,a single exon difference (in TEL) in TEL-TRKC fusions is associated with the two sets of cancer phenotypes. We expressed the two TEL-TRKC variants in vivo by using the 3' regulatory element of SCL that is selectively active in a subset of mesodermal cell lineages,including endothelial and hematopoietic stem cells and progenitors. The leukemia form of TEL-TRKC (-exon 5 of TEL) enhanced hematopoietic stem cell renewal and initiated leukemia. In contrast,the TEL-TRKC solid tumor variant (+ TEL exon 5) elicited an embryonic lethal phenotype with impairment of both angiogenesis and hematopoiesis indicative of an effect at the level of the hemangioblasts. The ability of TEL-TRKC to repress expression of Flk1,a critical regulator of early endothelial and hematopoietic cells,depended on TEL exon 5. These data indicate that related oncogenic fusion proteins similarly expressed in a hierarchy of early stem cells can have selective,cell type-specific developmental impacts. View Publication

Erythropoietin (EPO) and its receptor are expressed in a wide variety of tissues,including the central nervous system. Local expression of both EPO and its receptor is upregulated upon injury or stress and plays a role in tissue homeostasis and cytoprotection. High-dose systemic administration or local injection of recombinant human EPO has demonstrated encouraging results in several models of tissue protection and organ injury,while poor tissue availability of the protein limits its efficacy. Here,we describe the discovery and characterization of the nonpeptidyl compound STS-E412 (2-[2-(4-chlorophenoxy)ethoxy]-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine),which selectively activates the tissue-protective EPO receptor,comprising an EPO receptor subunit (EPOR) and the common β-chain (CD131). STS-E412 triggered EPO receptor phosphorylation in human neuronal cells. STS-E412 also increased phosphorylation of EPOR,CD131,and the EPO-associated signaling molecules JAK2 and AKT in HEK293 transfectants expressing EPOR and CD131. At low nanomolar concentrations,STS-E412 provided EPO-like cytoprotective effects in primary neuronal cells and renal proximal tubular epithelial cells. The receptor selectivity of STS-E412 was confirmed by a lack of phosphorylation of the EPOR/EPOR homodimer,lack of activity in off-target selectivity screening,and lack of functional effects in erythroleukemia cell line TF-1 and CD34(+) progenitor cells. Permeability through artificial membranes and Caco-2 cell monolayers in vitro and penetrance across the blood-brain barrier in vivo suggest potential for central nervous system availability of the compound. To our knowledge,STS-E412 is the first nonpeptidyl,selective activator of the tissue-protective EPOR/CD131 receptor. Further evaluation of the potential of STS-E412 in central nervous system diseases and organ protection is warranted. View Publication

The G protein-coupled receptor (GPCR),chemokine CXC-type receptor 4 (CXCR4),and its ligand,CXCL12,mediate the retention of polymorphonuclear neutrophils (PMNs) and hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Agents that disrupt CXCL12-mediated chemoattraction of CXCR4-expressing cells mobilize PMNs and HSPCs into the peripheral circulation and are therapeutically useful for HSPC collection before autologous bone marrow transplantation (ABMT). Our aim was to develop unique CXCR4-targeted therapeutics using lipopeptide GPCR modulators called pepducins. A pepducin is a synthetic molecule composed of a peptide derived from the amino acid sequence of one of the intracellular (IC) loops of a target GPCR coupled to a lipid tether. We prepared and screened a small CXCR4-targeted pepducin library and identified several pepducins with in vitro agonist activity,including ATI-2341,whose peptide sequence derives from the first IC loop. ATI-2341 induced CXCR4- and G protein-dependent signaling,receptor internalization,and chemotaxis in CXCR4-expressing cells. It also induced dose-dependent peritoneal recruitment of PMNs when administered i.p. to mice. However,when administered systemically by i.v. bolus,ATI-2341 acted as a functional antagonist and dose-dependently mediated release of PMNs from the bone marrow of both mice and cynomolgus monkeys. ATI-2341-mediated release of granulocyte/macrophage progenitor cells from the bone marrow was confirmed by colony-forming assays. We conclude that ATI-2341 is a potent and efficacious mobilizer of bone marrow PMNs and HSPCs and could represent a previously undescribed therapeutic approach for the recruitment of HSPCs before ABMT. View Publication

Molecular markers defining self-renewing pluripotent embryonic stem cells (ESCs) have been identified by relative comparisons between undifferentiated and differentiated cells. Most of analysis has been done under a specific differentiation condition that may present significantly different molecular changes over others. Therefore,it is currently unclear if there are true consensus markers defining undifferentiated hESCs. To identify a set of key genes consistently altered during differentiation of hESCs regardless of differentiation conditions we have performed microarray analysis on undifferentiated hESCs (H1 and H9) and differentiated EB's and validated our results using publicly available expression array data sets. We constructed consensus modules by Weighted Gene Correlation Analysis (WGCNA) and discovered novel markers that are consistently present in undifferentiated hESCs under various differentiation conditions. We have validated top markers (downregulated: LCK,KLKB1 and SLC7A3; upregulated: RhoJ,Zeb2 and Adam12) upon differentiation. Functional validation analysis of LCK in self-renewal of hESCs by using LCK inhibitor or gene silencing with siLCK resulted in a loss of undifferentiation characteristics- morphological change,reduced alkaline phosphatase activity and pluripotency gene expression,demonstrating a potential functional role of LCK in self-renewal of hESCs. We have designated hESC markers to interactive networks in the genome,identifying possible interacting partners and showing how new markers relate to each other. Furthermore,comparison of these data sets with available datasets from iPSCs revealed that the level of these newly identified markers were correlated to the establishment of iPSCs,which may imply a potential role of these markers in gaining of cellular potency. Stem Cells 2014. View Publication

Integrins are cell adhesion receptors that mediate cell-to-cell,or cell-to-extracellular matrix adhesion. They represent an attractive target for treatment of multiple diseases. Two classes of small molecule integrin inhibitors have been developed. Competitive antagonists bind directly to the integrin ligand binding pocket and thus disrupt the ligand-receptor interaction. Allosteric antagonists have been developed primarily for α(L)β(2)- integrin (LFA-1,lymphocyte function-associated antigen-1). Here we present the results of screening the Prestwick Chemical Library using a recently developed assay for the detection of α(4)β(1)-integrin allosteric antagonists. Secondary assays confirmed that the compounds identified: 1) do not behave like competitive (direct) antagonists; 2) decrease ligand binding affinity for VLA-4 ∼2 orders of magnitude; 3) exhibit antagonistic properties at low temperature. In a cell based adhesion assay in vitro,the compounds rapidly disrupted cellular aggregates. In accord with reports that VLA-4 antagonists in vivo induce mobilization of hematopoietic progenitors into the peripheral blood,we found that administration of one of the compounds significantly increased the number of colony-forming units in mice. This effect was comparable to AMD3100,a well known progenitor mobilizing agent. Because all the identified compounds are structurally related,previously used,or currently marketed drugs,this result opens a range of therapeutic possibilities for VLA-4-related pathologies. View Publication

Essential thrombocythemia (ET) and polycythemia vera (PV) are clonal myeloproliferative disorders that are often difficult to distinguish from other causes of elevated blood cell counts. Assays that could reliably detect clonal hematopoiesis would therefore be extremely valuable for diagnosis. We previously reported 3 X-chromosome transcription-based clonality assays (TCAs) involving the G6PD,IDS,and MPP1 genes,which together were informative in about 65% of female subjects. To increase our ability to detect clonality,we developed simple TCA for detecting the transcripts of 2 additional X-chromosome genes: Bruton tyrosine kinase (BTK) and 4-and-a-half LIM domain 1 (FHL1). The combination of TCA established the presence or absence of clonal hematopoiesis in about 90% of female subjects. We show that both genes are subject to X-chromosome inactivation and are polymorphic in all major US ethnic groups. The 5 TCAs were used to examine clonality in 46 female patients along with assays for erythropoietin-independent erythroid colonies (EECs) and granulocyte PRV-1 mRNA levels to discriminate polycythemias and thrombocytoses. Of these,all 19 patients with familial polycythemia or thrombocytosis had polyclonal hematopoiesis,whereas 22 of 26 patients with clinical evidence of myeloproliferative disorder and 1 patient with clinically obscure polycythemia were clonal. Interestingly,interferon alpha therapy in 2 patients with PV was associated with reversion of clonal to polyclonal hematopoiesis. EECs were observed in 14 of 14 patients with PV and 4 of 12 with ET,and increased granulocyte PRV-1 mRNA levels were found in 9 of 13 patients with PV and 2 of 12 with ET. Thus,these novel clonality assays are useful in the diagnosis and follow-up of polycythemic conditions and disorders with increased platelet levels. View Publication

Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions,leading to gene network dysregulation and human disease. Human mutations in GATA4,a cardiogenic transcription factor,cause cardiac septal defects and cardiomyopathy. Here,iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility,calcium handling,and metabolic activity. In human cardiomyocytes,GATA4 broadly co-occupied cardiac enhancers with TBX5,another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment,particularly to cardiac super-enhancers,concomitant with dysregulation of genes related to the phenotypic abnormalities,including cardiac septation. Conversely,the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations can disrupt transcriptional cooperativity,leading to aberrant chromatin states and cellular dysfunction,including those related to morphogenetic defects. View Publication

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA),known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as MELAS demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multi-lineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts,the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA,resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. Upon comparative differentiation of iPSC clones,improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared to isogenic clones with high heteroplasmy. Thus,mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny,and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases. View Publication

Oligomerization is an important regulatory mechanism for many proteins,including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity,suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges,resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations,which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl,reduced cell proliferation,and increased caspase-3/7 activity and DNA segmentation. Importantly,the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention. View Publication

Without effective therapy,chronic-phase chronic myeloid leukemia (CP-CML) evolves into an acute leukemia (blast crisis [BC]) that displays either myeloid or B-lymphoid characteristics. This transition is often preceded by a clinically recognized,but biologically poorly characterized,accelerated phase (AP). Here,we report that IKAROS protein is absent or reduced in bone marrow blasts from most CML patients with advanced myeloid disease (AP or BC). This contrasts with primitive CP-CML cells and BCR-ABL1-negative acute myeloid leukemia blasts,which express readily detectable IKAROS. To investigate whether loss of IKAROS contributes to myeloid disease progression in CP-CML,we examined the effects of forced expression of a dominant-negative isoform of IKAROS (IK6) in CP-CML patients' CD34(+) cells. We confirmed that IK6 disrupts IKAROS activity in transduced CP-CML cells and showed that it confers on them features of AP-CML,including a prolonged increased output in vitro and in xenografted mice of primitive cells with an enhanced ability to differentiate into basophils. Expression of IK6 in CD34(+) CP-CML cells also led to activation of signal transducer and activator of transcription 5 and transcriptional repression of its negative regulators. These findings implicate loss of IKAROS as a frequent step and potential diagnostic harbinger of progressive myeloid disease in CML patients. View Publication

Pluripotent stem cells,both human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC),can give rise to multiple cell types and hence have tremendous potential for regenerative therapies. However,the tumorigenic potential of these cells remains a great concern,as reflected in the formation of teratomas by transplanted pluripotent cells. In clinical practice,most pluripotent cells will be differentiated into useful therapeutic cell types such as neuronal,cardiac,or endothelial cells prior to human transplantation,drastically reducing their tumorigenic potential. Our work investigated the extent to which these differentiated stem cell derivatives are truly devoid of oncogenic potential. In this study,we analyzed the gene expression patterns from three sets of hiPSC- and hESC-derivatives and the corresponding primary cells,and compared their transcriptomes with those of five different types of cancer. Our analysis revealed a significant gene expression overlap of the hiPSC- and hESC-derivatives with cancer,whereas the corresponding primary cells showed minimum overlap. Real-time quantitative PCR analysis of a set of cancer-related genes (selected on the basis of rigorous functional and pathway analyses) confirmed our results. Overall,our findings suggested that pluripotent stem cell derivatives may still bear oncogenic properties even after differentiation,and additional stringent functional assays to purify these cells should be done before they can be used for regenerative therapy. View Publication

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