技术资料

Extracellular signal-regulated kinases (ERKs) mediate downstream signaling of RAS-RAF-MEK as key regulators of the mitogen-activated protein kinase (MAPK) pathway. Activation of ERK signaling is a hallmark of cancer and upstream MAPK proteins have been extensively pursued as drug targets for cancer therapies. However,the rapid rise of resistance to clinical RAF and MEK inhibitors has prompted interest in targeting ERK (ERK1 and ERK2 isoforms) directly for cancer therapy. Current methods for evaluating activity of inhibitors against ERK isoforms are based primarily on analysis of recombinant proteins. Strategies to directly and independently profile native ERK1 and ERK2 activity would greatly complement current cell biological tools used to probe and target ERK function. Here,we present a quantitative chemoproteomic strategy that utilizes active-site directed probes to directly quantify native ERK activity in an isoform-specific fashion. We exploit a single isoleucine/leucine difference in ERK substrate binding sites to enable activity-based profiling of ERK1 versus ERK2 across a variety of cell types,tissues,and species. We used our chemoproteomic strategy to determine potency and selectivity of academic (VX-11e) and clinical (Ulixertinib) ERK inhibitors. Correlation of potency estimates by chemoproteomics with anti-proliferative activity of VX-11e and Ulixertinib revealed that {\textgreater}90{\%} inactivation of both native ERK1 and ERK2 is needed to mediate cellular activity of inhibitors. Our findings introduce one of the first assays capable of independent evaluation of native ERK1 and ERK2 activity to advance drug discovery of oncogenic MAPK pathways. View Publication

ONC201 is a first-in-class,orally active antitumor agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. ONC201 has demonstrated safety and preliminary efficacy in a first-in-human trial in which patients were dosed every 3 weeks. We hypothesized that dose intensification of ONC201 may impact antitumor efficacy. We discovered that ONC201 exerts dose- and schedule-dependent effects on tumor progression and cell death signaling in vivo. With dose intensification,we note a potent anti-metastasis effect and inhibition of cancer cell migration and invasion. Our preclinical results prompted a change in ONC201 dosing in all open clinical trials. We observed accumulation of activated NK+ and CD3+ cells within ONC201-treated tumors and that NK cell depletion inhibits ONC201 efficacy in vivo,including against TRAIL/ONC201-resistant Bax-/- tumors. Immunocompetent NCR1-GFP mice,in which NK cells express GFP,demonstrated GFP+ NK cell infiltration of syngeneic MC38 colorectal tumors. Activation of primary human NK cells and increased degranulation occurred in response to ONC201. Coculture experiments identified a role for TRAIL in human NK-mediated antitumor cytotoxicity. Preclinical results indicate the potential utility for ONC201 plus anti-PD-1 therapy. We observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients after ONC201 treatment. The results offer what we believe to be a unique pathway of immune stimulation for cancer therapy. View Publication

T cell development requires a period of postthymic maturation. Why this is the case has remained a mystery,particularly given the rigors of intrathymic developmental checkpoints,successfully traversed by only ∼5% of thymocytes. We now show that the first few weeks of T cell residence in the lymphoid periphery define a period of heightened susceptibility to tolerance induction to tissue-restricted antigens (TRAs),the outcome of which depends on the context in which recent thymic emigrants (RTEs) encounter antigen. After encounter with TRAs in the absence of inflammation,RTEs exhibited defects in proliferation,diminished cytokine production,elevated expression of anergy-associated genes,and diminished diabetogenicity. These properties were mirrored in vitro by enhanced RTE susceptibility to regulatory T cell-mediated suppression. In the presence of inflammation,RTEs and mature T cells were,in contrast,equally capable of inducing diabetes,proliferating,and producing cytokines. Thus,recirculating RTEs encounter TRAs during a transitional developmental stage that facilitates tolerance induction,but inflammation converts antigen-exposed,tolerance-prone RTEs into competent effector cells. View Publication

Doxorubicin is a highly efficacious anti-cancer drug but causes cardiotoxicity in many patients. The mechanisms of doxorubicin-induced cardiotoxicity (DIC) remain incompletely understood. We investigated the characteristics and molecular mechanisms of DIC in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). We found that doxorubicin causes dose-dependent increases in apoptotic and necrotic cell death,reactive oxygen species production,mitochondrial dysfunction and increased intracellular calcium concentration. We characterized genome-wide changes in gene expression caused by doxorubicin using RNA-seq,as well as electrophysiological abnormalities caused by doxorubicin with multi-electrode array technology. Finally,we show that CRISPR-Cas9-mediated disruption of TOP2B,a gene implicated in DIC in mouse studies,significantly reduces the sensitivity of hPSC-CMs to doxorubicin-induced double stranded DNA breaks and cell death. These data establish a human cellular model of DIC that recapitulates many of the cardinal features of this adverse drug reaction and could enable screening for protective agents against DIC as well as assessment of genetic variants involved in doxorubicin response. View Publication

Many pathogens evade cytotoxic T lymphocytes (CTLs) by downregulating HLA molecules on infected cells,but the loss of HLA can trigger NK cell-mediated lysis. HIV-1 is thought to subvert CTLs while preserving NK cell inhibition by Nef-mediated downregulation of HLA-A and -B but not HLA-C molecules. We find that HLA-C is downregulated by most primary HIV-1 clones,including transmitted founder viruses,in contrast to the laboratory-adapted NL4-3 virus. HLA-C reduction is mediated by viral Vpu and reduces the ability of HLA-C restricted CTLs to suppress viral replication in CD4+ cells in vitro. HLA-A/B are unaffected by Vpu,and primary HIV-1 clones vary in their ability to downregulate HLA-C,possibly in response to whether CTLs or NK cells dominate immune pressure through HLA-C. HIV-2 also suppresses HLA-C expression through distinct mechanisms,underscoring the immune pressure HLA-C exerts on HIV. This viral immune evasion casts new light on the roles of CTLs and NK cells in immune responses against HIV. View Publication

Induced pluripotent stem cells (iPSCs) have enormous potential in regenerative medicine and disease modeling. It is now felt that clinical trials should be performed with iPSCs derived with non-integrative constructs. Numerous studies,however,including those describing disease models,are still being published using cells derived from iPSCs generated with integrative constructs. Our experimental work presents the first evidence of spontaneous transgene reactivation in vitro in several cellular types. Our results show that the transgenes were predominantly silent in parent iPSCs,but in mesenchymal and endothelial iPSC derivatives,the transgenes experienced random up-regulation of Nanog and c-Myc. Additionally,we provide evidence of spontaneous secondary reprogramming and reversion to pluripotency in mesenchymal stem cells derived from iPSCs. These findings strongly suggest that the studies,which utilize cellular products derived from iPSCs generated with retro- or lentiviruses,should be evaluated with consideration of the possibility of transgene reactivation. The in vitro model described here provides insight into the earliest events of culture transformation and suggests the hypothesis that reversion to pluripotency may be responsible for the development of tumors in cell replacement experiments. The main goal of this work,however,is to communicate the possibility of transgene reactivation in retro- or lenti- iPSC derivatives and the associated loss of cellular fidelity in vitro,which may impact the outcomes of disease modeling and related experimentation. View Publication

Predictive toxicology using stem cells or their derived tissues has gained increasing importance in biomedical and pharmaceutical research. Here,we show that toxicity category prediction by support vector machines (SVMs),which uses qRT-PCR data from 20 categorized chemicals based on a human embryonic stem cell (hESC) system,is improved by the adoption of gene networks,in which network edge weights are added as feature vectors when noisy qRT-PCR data fail to make accurate predictions. The accuracies of our system were 97.5-100% for three toxicity categories: neurotoxins (NTs),genotoxic carcinogens (GCs) and non-genotoxic carcinogens (NGCs). For two uncategorized chemicals,bisphenol-A and permethrin,our system yielded reasonable results: bisphenol-A was categorized as an NGC,and permethrin was categorized as an NT; both predictions were supported by recently published papers. Our study has two important features: (i) as the first study to employ gene networks without using conventional quantitative structure-activity relationships (QSARs) as input data for SVMs to analyze toxicogenomics data in an hESC validation system,it uses additional information of gene-to-gene interactions to significantly increase prediction accuracies for noisy gene expression data; and (ii) using only undifferentiated hESCs,our study has considerable potential to predict late-onset chemical toxicities,including abnormalities that occur during embryonic development. View Publication

Three different label-free,minimally invasive,live single cell analysis techniques were used to characterize embryonic stem cells,and the hepatocytes into which they were differentiated. Atomic Force Microscopy measures the cell's mechanical properties,Raman spectroscopy measures its chemical properties,and dielectrophoresis measures the membrane's capacitance. We were able to assign cell type of individual cells with accuracies of 96.5% (Atomic Force Microscopy),92.5 % (Raman spectroscopy),and *** % (Dielectrophoresis). These techniques,used either independently or in combination,offer label-free methods to study individual living cells. Although they can be applied to any phenotypical or environmental change,these techniques have most potential in human cell therapies where the use of biomarkers is best avoided. If all three properties are independent,then a combined accuracy of *** % can be achieved in cell characterization. We suggest how these methods could be combined into one microfluidic chip for cell sorting,and how they can be applied to cell culture. View Publication

INTRODUCTION The Comprehensive in vitro Proarrhythmia Assay (CiPA) is a nonclinical Safety Pharmacology paradigm for discovering electrophysiological mechanisms that are likely to confer proarrhythmic liability to drug candidates intended for human use. TOPICS COVERED Key talks delivered at the 'CiPA on my mind' session,held during the 2015 Annual Meeting of the Safety Pharmacology Society (SPS),are summarized. Issues and potential solutions relating to crucial constituents [e.g.,biological materials (ion channels and pluripotent stem cell-derived cardiomyocytes),study platforms,drug solutions,and data analysis] of CiPA core assays are critically examined. DISCUSSION In order to advance the CiPA paradigm from the current testing and validation stages to a research and regulatory drug development strategy,systematic guidance by CiPA stakeholders is necessary to expedite solutions to pending and newly arising issues. Once a study protocol is proved to yield robust and reproducible results within and across laboratories,it can be implemented as qualified regulatory procedure. View Publication

NK cells possess inhibitory receptors that are responsible for self-MHC class I recognition; beyond their inhibitory function,accumulating evidence indicates that such receptors confer NK cell functional competence through an unclear process termed licensing." Ly49C is the main self-specific inhibitory Ly49 receptor in H-2(b) C57BL/6 (B6) mice. We used B6 Ly49C-transgenic and B6 β2 microglobulin (β2m)-knockout Ly49C-transgenic mice to investigate the impact of licensing through this inhibitory receptor in precursor and mature NK cells. We found that self-specific inhibitory receptors affected NK cell precursor survival and proliferation at particular developmental stages in an MHC class I-dependent manner. The presence of Ly49C impacted the NK cell repertoire in a β2m-dependent manner� View Publication

$$-Thalassemia ($$-Thal) is one of the most common genetic diseases in the world. The generation of patient-specific $$-Thal-induced pluripotent stem cells (iPSCs),correction of the disease-causing mutations in those cells,and then differentiation into hematopoietic stem cells offers a new therapeutic strategy for this disease. Here,we designed a CRISPR/Cas9 to specifically target the Homo sapiens hemoglobin $$ (HBB) gene CD41/42(-CTTT) mutation. We demonstrated that the combination of single strand oligodeoxynucleotides with CRISPR/Cas9 was capable of correcting the HBB gene CD41/42 mutation in $$-Thal iPSCs. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm mutation correction,we verified that the purified clones retained full pluripotency and exhibited normal karyotyping. Additionally,whole-exome sequencing showed that the mutation load to the exomes was minimal after CRISPR/Cas9 targeting. Furthermore,the corrected iPSCs were selected for erythroblast differentiation and restored the expression of HBB protein compared with the parental iPSCs. This method provides an efficient and safe strategy to correct the HBB gene mutation in $$-Thal iPSCs. View Publication

Generating human podocytes in vitro could offer a unique opportunity to study human diseases. Here,we describe a simple and efficient protocol for obtaining functional podocytes in vitro from human induced pluripotent stem cells. Cells were exposed to a three-step protocol,which induced their differentiation into intermediate mesoderm,then into nephron progenitors and,finally,into mature podocytes. After differentiation,cells expressed the main podocyte markers,such as synaptopodin,WT1,α-Actinin-4,P-cadherin and nephrin at the protein and mRNA level,and showed the low proliferation rate typical of mature podocytes. Exposure to Angiotensin II significantly decreased the expression of podocyte genes and cells underwent cytoskeleton rearrangement. Cells were able to internalize albumin and self-assembled into chimeric 3D structures in combination with dissociated embryonic mouse kidney cells. Overall,these findings demonstrate the establishment of a robust protocol that,mimicking developmental stages,makes it possible to derive functional podocytes in vitro. View Publication