技术资料

Developing novel therapies that suppress self-renewal of leukemia stem cells may reduce the likelihood of relapses and extend long-term survival of patients with acute myelogenous leukemia (AML). AML1-ETO (AE) is an oncogene that plays an important role in inducing self-renewal of hematopoietic stem/progenitor cells (HSPCs),leading to the development of leukemia stem cells. Previously,using a zebrafish model of AE and a whole-organism chemical suppressor screen,we have discovered that AE induces specific hematopoietic phenotypes in embryonic zebrafish through a cyclooxygenase (COX)-2 and β-catenin-dependent pathway. Here,we show that AE also induces expression of the Cox-2 gene and activates β-catenin in mouse bone marrow cells. Inhibition of COX suppresses β-catenin activation and serial replating of AE(+) mouse HSPCs. Genetic knockdown of β-catenin also abrogates the clonogenic growth of AE(+) mouse HSPCs and human leukemia cells. In addition,treatment with nimesulide,a COX-2 selective inhibitor,dramatically suppresses xenograft tumor formation and inhibits in vivo progression of human leukemia cells. In summary,our data indicate an important role of a COX/β-catenin-dependent signaling pathway in tumor initiation,growth,and self-renewal,and in providing the rationale for testing potential benefits from common COX inhibitors as a part of AML treatments. View Publication

Growth factor signaling pathways are tightly regulated by phosphorylation and include many important kinase targets of interest for drug discovery. Small molecule inhibitors of the bone morphogenetic protein (BMP) receptor kinase ALK2 (ACVR1) are needed urgently to treat the progressively debilitating musculoskeletal disease fibrodysplasia ossificans progressiva (FOP). Dorsomorphin analogues,first identified in zebrafish,remain the only BMP inhibitor chemotype reported to date. By screening an assay panel of 250 recombinant human kinases we identified a highly selective 2-aminopyridine-based inhibitor K02288 with in vitro activity against ALK2 at low nanomolar concentrations similar to the current lead compound LDN-193189. K02288 specifically inhibited the BMP-induced Smad pathway without affecting TGF-β signaling and induced dorsalization of zebrafish embryos. Comparison of the crystal structures of ALK2 with K02288 and LDN-193189 revealed additional contacts in the K02288 complex affording improved shape complementarity and identified the exposed phenol group for further optimization of pharmacokinetics. The discovery of a new chemical series provides an independent pharmacological tool to investigate BMP signaling and offers multiple opportunities for pre-clinical development. View Publication

Human embryonic stem cells and mouse epiblast stem cells represent a primed pluripotent stem cell state that requires TGF-β/activin signaling. TGF-β and/or activin are commonly thought to regulate transcription through both Smad2 and Smad3. However,the different contributions of these two Smads to primed pluripotency and the downstream events that they may regulate remain poorly understood. We addressed the individual roles of Smad2 and Smad3 in the maintenance of primed pluripotency. We found that Smad2,but not Smad3,is required to maintain the undifferentiated pluripotent state. We defined a Smad2 regulatory circuit in human embryonic stem cells and mouse epiblast stem cells,in which Smad2 acts through binding to regulatory promoter sequences to activate Nanog expression while in parallel repressing autocrine bone morphogenetic protein signaling. Increased autocrine bone morphogenetic protein signaling caused by Smad2 down-regulation leads to cell differentiation toward the trophectoderm,mesoderm,and germ cell lineages. Additionally,induction of Cdx2 expression,as a result of decreased Smad2 expression,leads to repression of Oct4 expression,which,together with the decreased Nanog expression,accelerates the loss of pluripotency. These findings reveal that Smad2 is a unique integrator of transcription and signaling events and is essential for the maintenance of the mouse and human primed pluripotent stem cell state. View Publication

Tumor heterogeneity of high-grade glioma (HGG) is recognized by four clinically relevant subtypes based on core gene signatures. However,molecular signaling in glioma stem cells (GSCs) in individual HGG subtypes is poorly characterized. Here we identified and characterized two mutually exclusive GSC subtypes with distinct dysregulated signaling pathways. Analysis of mRNA profiles distinguished proneural (PN) from mesenchymal (Mes) GSCs and revealed a pronounced correlation with the corresponding PN or Mes HGGs. Mes GSCs displayed more aggressive phenotypes in vitro and as intracranial xenografts in mice. Further,Mes GSCs were markedly resistant to radiation compared with PN GSCs. The glycolytic pathway,comprising aldehyde dehydrogenase (ALDH) family genes and in particular ALDH1A3,were enriched in Mes GSCs. Glycolytic activity and ALDH activity were significantly elevated in Mes GSCs but not in PN GSCs. Expression of ALDH1A3 was also increased in clinical HGG compared with low-grade glioma or normal brain tissue. Moreover,inhibition of ALDH1A3 attenuated the growth of Mes but not PN GSCs. Last,radiation treatment of PN GSCs up-regulated Mes-associated markers and down-regulated PN-associated markers,whereas inhibition of ALDH1A3 attenuated an irradiation-induced gain of Mes identity in PN GSCs. Taken together,our data suggest that two subtypes of GSCs,harboring distinct metabolic signaling pathways,represent intertumoral glioma heterogeneity and highlight previously unidentified roles of ALDH1A3-associated signaling that promotes aberrant proliferation of Mes HGGs and GSCs. Inhibition of ALDH1A3-mediated pathways therefore might provide a promising therapeutic approach for a subset of HGGs with the Mes signature. View Publication

Lung cancer is the leading cause of cancer-related mortality worldwide. Recent evidence indicates that tumors contain a subpopulation of cancer stem cells (CSCs) that are responsible for tumor maintenance and spread. CSCs have recently been linked to the occurrence of epithelial-to-mesenchymal transition (EMT). Neurotrophins (NTs) are growth factors that regulate the biology of embryonic stem cells and cancer cells,but still little is known about the role NTs in the progression of lung cancer. In this work,we investigated the role of the NTs and their receptors using as a study system primary cell cultures derived from malignant pleural effusions (MPEs) of patients with adenocarcinoma of the lung. We assessed the expression of NTs and their receptors in MPE-derived adherent cultures vs. spheroids enriched in CSC markers. We observed in spheroids a selectively enhanced expression of TrkB,both at the mRNA and protein levels. Both K252a,a known inhibitor of Trk activity,and a siRNA against TrkB strongly affected spheroid morphology,induced anoikis and decreased spheroid forming efficiency. Treatment with neurotrophins reversed the inhibitory effect of K252a. Importantly,TrkB inhibition caused loss of vimentin expression as well as that of a set of transcription factors known to be linked to EMT. These ex vivo results nicely correlated with an inverse relationship between TrkB and E-cadherin expression measured by immunohistochemistry in a panel of lung adenocarcinoma samples. We conclude that TrkB is involved in full acquisition of EMT in lung cancer,and that its inhibition results in a less aggressive phenotype. View Publication

Everolimus is a potent,oral inhibitor of the mammalian target of rapamycin (mTOR) that has been investigated in multiple clinical development programs since 1996. A unique collaboration between academic and pharmaceutical experts fostered research that progressed rapidly,with simultaneous indication findings across numerous tumor types. Initially developed for the prophylaxis of organ transplant rejection,everolimus has demonstrated efficacy and safety for the treatment of patients with various types of cancer (renal cell carcinoma,neuroendocrine tumors of pancreatic origin,and breast cancer) and for adult and pediatric patients with tuberous sclerosis complex. The FDA approval of everolimus for these diseases has addressed several unmet medical needs and is widely accepted by the medical community where treatment options may be limited. An extensive clinical development program is ongoing to establish the role of everolimus as monotherapy,or in combination with other agents,in the treatment of a broad spectrum of malignancies. View Publication

The generation of human induced pluripotent stem cells (hiPSCs) requires the collection of donor tissue,but clinical circumstances in which the interests of patients have highest priority may compromise the quality and availability of cells that are eventually used for reprogramming. Here we compared (i) skin biopsies stored in standard physiological salt solution for up to two weeks (ii) blood outgrowth endothelial cells (BOECs) isolated from fresh peripheral blood and (iii) children's milk teeth lost during normal replacement for their ability to form somatic cell cultures suitable for reprogramming to hiPSCs. We derived all hiPSC lines using the same reprogramming method (a conditional (FLPe) polycistronic lentivirus) and under similar conditions (same batch of virus,fetal calf serum and feeder cells). Skin fibroblasts could be reprogrammed robustly even after long-term biopsy storage. Generation of hiPSCs from juvenile dental pulp cells gave similar high efficiencies,but that of BOECs was lower. In terms of invasiveness of biopsy sampling,biopsy storage and reprogramming efficiencies skin fibroblasts appeared best for the generation of hiPSCs,but where non-invasive procedures are required (e.g. for children and minors) dental pulp cells from milk teeth represent a valuable alternative. View Publication

A satisfactory in vitro model of vocal fold mucosa does not exist,thus precluding a systematic,controlled study of vocal fold biology and biomechanics. We sought to create a valid,reproducible three-dimensional (3D) in vitro model of human origin of vocal fold mucosa of human origin. We hypothesized that coculture of human embryonic stem cell (hESC)-derived simple epithelial cells with primary vocal fold fibroblasts under appropriate conditions would elicit morphogenesis of progenitor cells into vocal fold epithelial-like cells and creation of a basement membrane. Using an in vitro prospective study design,hESCs were differentiated into cells that coexpressed the simple epithelial cell marker,keratin 18 (K18),and the transcription factor,p63. These simple epithelial cells were cocultured with primary vocal fold fibroblasts seeded in a collagen gel scaffold. The cells were cultured for 3 weeks in a keratinocyte medium at an air–liquid interface. After that time,the engineered mucosa demonstrated a stratified,squamous epithelium and a continuous basement membrane recapitulating the key morphologic and phenotypic characteristics of native vocal fold mucosa. hESC-derived epithelial cells exhibited positive staining for vocal fold stratified,squamous epithelial markers,keratin 13 (K13) and 14 (K14),as well as tight junctions,adherens junctions,gap junctions,and desmosomes. Despite the presence of components critical for epithelial structural integrity,the epithelium demonstrated greater permeability than native tissue indicating compromised functional integrity. While further work is warranted to improve functional barrier integrity,this study demonstrates that hESC-derived epithelial progenitor cells can be engineered to create a replicable 3D in vitro model of vocal fold mucosa featuring a multilayered,terminally differentiated epithelium. View Publication

Mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors. Somatic cell reprogramming involves epigenomic reconfiguration,conferring iPSCs with characteristics similar to embryonic stem cells (ESCs). Human ESCs (hESCs) contain 5-hydroxymethylcytosine (5hmC),which is generated through the oxidation of 5-methylcytosine by the TET enzyme family. Here we show that 5hmC levels increase significantly during reprogramming to human iPSCs mainly owing to TET1 activation,and this hydroxymethylation change is critical for optimal epigenetic reprogramming,but does not compromise primed pluripotency. Compared with hESCs,we find that iPSCs tend to form large-scale (100 kb–1.3 Mb) aberrant reprogramming hotspots in subtelomeric regions,most of which exhibit incomplete hydroxymethylation on CG sites. Strikingly,these 5hmC aberrant hotspots largely coincide (∼ 80%) with aberrant iPSC–ESC non-CG methylation regions. Our results suggest that TET1-mediated 5hmC modification could contribute to the epigenetic variation of iPSCs and iPSC–hESC differences. View Publication

RAF kinases have a prominent role in cancer. Their mode of activation is complex but critically requires dimerization of their kinase domains. Unexpectedly,several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and,as a result,undesirably stimulate RAS/ERK pathway-mediated cell growth. The mechanism by which these inhibitors induce RAF kinase domain dimerization remains unclear. Here we describe bioluminescence resonance energy transfer-based biosensors for the extended RAF family that enable the detection of RAF dimerization in living cells. Notably,we demonstrate the utility of these tools for profiling kinase inhibitors that selectively modulate RAF dimerization and for probing structural determinants of RAF dimerization in vivo. Our findings,which seem generalizable to other kinase families allosterically regulated by kinase domain dimerization,suggest a model whereby ATP-competitive inhibitors mediate RAF dimerization by stabilizing a rigid closed conformation of the kinase domain. View Publication

Retinoic acid (RA),a vitamin A metabolite,modulates mucosal T helper cell responses. Here we examined the role of RA in regulating IL-22 production by γδ T cells and innate lymphoid cells in intestinal inflammation. RA significantly enhanced IL-22 production by γδ T cells stimulated in vitro with IL-1β or IL-18 and IL-23. In vivo RA attenuated colon inflammation induced by dextran sodium sulfate treatment or Citrobacter rodentium infection. This was associated with a significant increase in IL-22 secretion by γδ T cells and innate lymphoid cells. In addition,RA treatment enhanced production of the IL-22-responsive antimicrobial peptides Reg3β and Reg3γ in the colon. The attenuating effects of RA on colitis were reversed by treatment with an anti-IL-22 neutralizing antibody,demonstrating that RA mediates protection by enhancing IL-22 production. To define the molecular events involved,we used chromatin immunoprecipitation assays and found that RA promoted binding of RA receptor to the IL-22 promoter in γδ T cells. Our findings provide novel insights into the molecular events controlling IL-22 transcription and suggest that one key outcome of RA signaling may be to shape early intestinal immune responses by promoting IL-22 synthesis by γδ T cells and innate lymphoid cells. View Publication

Ion channels are involved in a large variety of cellular processes including stem cell differentiation. Numerous families of ion channels are present in the organism which can be distinguished by means of,for example,ion selectivity,gating mechanism,composition,or cell biological function. To characterize the distinct expression of this group of ion channels we have compared the mRNA expression levels of ion channel genes between human keratinocyte-derived induced pluripotent stem cells (hiPSCs) and their somatic cell source,keratinocytes from plucked human hair. This comparison revealed that 26&x25; of the analyzed probes showed an upregulation of ion channels in hiPSCs while just 6&x25; were downregulated. Additionally,iPSCs express a much higher number of ion channels compared to keratinocytes. Further,to narrow down specificity of ion channel expression in iPS cells we compared their expression patterns with differentiated progeny,namely,neurons and cardiomyocytes derived from iPS cells. To conclude,hiPSCs exhibit a very considerable and diverse ion channel expression pattern. Their detailed analysis could give an insight into their contribution to many cellular processes and even disease mechanisms. View Publication