技术资料

The generation of insulin-producing pancreatic $\beta$ cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. However,insulin-producing cells previously generated from human pluripotent stem cells (hPSC) lack many functional characteristics of bona fide $\beta$ cells. Here,we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive $\beta$ cells from hPSC in vitro. These stem-cell-derived $\beta$ cells (SC-$\beta$) express markers found in mature $\beta$ cells,flux Ca(2+) in response to glucose,package insulin into secretory granules,and secrete quantities of insulin comparable to adult $\beta$ cells in response to multiple sequential glucose challenges in vitro. Furthermore,these cells secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner,and transplantation of these cells ameliorates hyperglycemia in diabetic mice. View Publication

Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here,we demonstrate that the NRF2 antioxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further,we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular antiviral program that potently inhibits replication of SARS-CoV2 across cell lines. The inhibitory effect of 4-OI and DMF extends to the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2,Vaccinia virus,and Zika virus through a type I interferon (IFN)-independent mechanism. In addition,4-OI and DMF limit host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion,NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and in suppressing the pro-inflammatory responses of human pathogenic viruses,including SARS-CoV2. View Publication

The protease responsible for the cleavage of poly(ADP-ribose) polymerase and necessary for apoptosis has been purified and characterized. This enzyme,named apopain,is composed of two subunits of relative molecular mass (M(r)) 17K and 12K that are derived from a common proenzyme identified as CPP32. This proenzyme is related to interleukin-1 beta-converting enzyme (ICE) and CED-3,the product of a gene required for programmed cell death in Caenorhabditis elegans. A potent peptide aldehyde inhibitor has been developed and shown to prevent apoptotic events in vitro,suggesting that apopain/CPP32 is important for the initiation of apoptotic cell death. View Publication

In situ molecular architecture analysis of organelles and protein assemblies is essential to understanding the role of individual components and their cellular function,and to engineering new molecular functionalities. Through a super-resolution-driven approach,here we characterize the organization of the ciliary basal foot,an appendage of basal bodies whose main role is to provide a point of anchoring to the microtubule cytoskeleton. Quantitative image analysis shows that the basal foot is organized into three main regions linked by elongated coiled-coil proteins,revealing a conserved modular architecture in primary and motile cilia,but showing distinct features reflecting its specialized functions. Using domain-specific BioID proximity labeling and super-resolution imaging,we identify CEP112 as a basal foot protein and other candidate components of this assembly,aiding future investigations on the role of basal foot across different cilia systems. View Publication

Mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) cause Blau syndrome,an inflammatory disorder characterized by uveitis. The antimicrobial functions of Nod2 are well-established,yet the cellular mechanisms by which dysregulated Nod2 causes uveitis remain unknown. Here,we report a non-conventional,T cell-intrinsic function for Nod2 in suppression of Th17 immunity and experimental uveitis. Reconstitution of lymphopenic hosts with Nod2-/- CD4+ T cells or retina-specific autoreactive CD4+ T cells lacking Nod2 reveals a T cell-autonomous,Rip2-independent mechanism for Nod2 in uveitis. In naive animals,Nod2 operates downstream of TCR ligation to suppress activation of memory CD4+ T cells that associate with an autoreactive-like profile involving IL-17 and Ccr7. Interestingly,CD4+ T cells from two Blau syndrome patients show elevated IL-17 and increased CCR7. Our data define Nod2 as a T cell-intrinsic rheostat of Th17 immunity,and open new avenues for T cell-based therapies for Nod2-associated disorders such as Blau syndrome. View Publication

Protein arginine deiminase 2 (PAD2) plays a key role in the onset and progression of multiple sclerosis,rheumatoid arthritis,and breast cancer. To date,no PAD2-selective inhibitor has been developed. Such a compound will be critical for elucidating the biological roles of this isozyme and may ultimately be useful for treating specific diseases in which PAD2 activity is dysregulated. To achieve this goal,we synthesized a series of benzimidazole-based derivatives of Cl-amidine,hypothesizing that this scaffold would allow access to a series of PAD2-selective inhibitors with enhanced cellular efficacy. Herein,we demonstrate that substitutions at both the N-terminus and C-terminus of Cl-amidine result in {\textgreater}100-fold increases in PAD2 potency and selectivity (30a,41a,and 49a) as well as cellular efficacy (30a). Notably,these compounds use the far less reactive fluoroacetamidine warhead. In total,we predict that 30a will be a critical tool for understanding cellular PAD2 function and sets the stage for treating diseases in which PAD2 activity is dysregulated. View Publication

Imaging strategies to monitor chimeric antigen receptor (CAR) T-cell biodistribution and proliferation harbor the potential to facilitate clinical translation for the treatment of both liquid and solid tumors. In addition,the potential adverse effects of CAR T cells highlight the need for mechanisms to modulate CAR T-cell activity. The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene has previously been translated as a PET reporter gene for imaging of T-cell trafficking in patients with brain tumor. The HSV1-TK enzyme can act as a suicide gene of transduced cells through treatment with the prodrug ganciclovir. Here we report the molecular engineering,imaging,and ganciclovir-mediated destruction of B7H3 CAR T cells incorporating a mutated version of the HSV1-tk gene (sr39tk) with improved enzymatic activity for ganciclovir. The sr39tk gene did not affect B7H3 CAR T-cell functionality and in vitro and in vivo studies in osteosarcoma models showed no significant effect on B7H3 CAR T-cell antitumor activity. PET/CT imaging with 9-(4-[18F]-fluoro-3-[hydroxymethyl]butyl)guanine ([18F]FHBG) of B7H3-sr39tk CAR T cells in an orthotopic model of osteosarcoma revealed tumor homing and systemic immune expansion. Bioluminescence and PET imaging of B7H3-sr39tk CAR T cells confirmed complete tumor ablation with intraperitoneal ganciclovir administration. This imaging and suicide ablation system can provide insight into CAR T-cell migration and proliferation during clinical trials while serving as a suicide switch to limit potential toxicities. SIGNIFICANCE: This study showcases the only genetically engineered system capable of serving the dual role both as an effective PET imaging reporter and as a suicide switch for CAR T cells. View Publication

Evasion of programmed cell death represents a critical form of oncogene addiction in cancer cells. Understanding the molecular mechanisms underpinning cancer cell survival despite the oncogenic stress could provide a molecular basis for potential therapeutic interventions. Here we explore the role of pro-survival genes in cancer cell integrity during clonal evolution in non-small cell lung cancer (NSCLC). We identify gains of MCL-1 at high frequency in multiple independent NSCLC cohorts,occurring both clonally and subclonally. Clonal loss of functional TP53 is significantly associated with subclonal gains of MCL-1. In mice,tumour progression is delayed upon pharmacologic or genetic inhibition of MCL-1. These findings reveal that MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically. View Publication

Ligand-activated glucocorticoid receptor (GR) elicits variable glucocorticoid-modulated transcriptomes in different cell types. However,some genes,including Kr{\{u}}ppel-like factor 9 (KLF9) a putative transcriptional repressor demonstrate conserved responses. We show that glucocorticoids induce KLF9 expression in the human airways in vivo and in differentiated human bronchial epithelial (HBE) cells grown at air-liquid interface (ALI). In A549 and BEAS-2B pulmonary epithelial cells glucocorticoids induce KLF9 expression with similar kinetics to primary HBE cells in submersion culture. A549 and BEAS-2B ChIP-seq data reveal four common glucocorticoid-induced GR binding sites (GBSs). Two GBSs mapped to the 5'-proximal region relative to KLF9 transcription start site (TSS) and two occurred at distal sites. These were all confirmed in primary HBE cells. Global run-on (GRO)-sequencing indicated robust enhancer RNA (eRNA) production from three of these GBSs in BEAS-2B cells. This was confirmed in A549 cells plus submersion and ALI culture of HBE cells. Cloning each GBS into luciferase reporters revealed glucocorticoid-induced activity requiring a glucocorticoid response element (GRE) within each distal GBS. While the proximal GBSs drove modest reporter induction by glucocorticoids this region exhibited basal eRNA production RNA polymerase II enrichment and looping to the TSS plausibly underlying constitutive KLF9 expression. Post-glucocorticoid treatment interactions between distal and proximal GBSs and the TSS correlated with KLF9 induction. CBP/P300 silencing reduced proximal GBS activity but negligibly effected KLF9 expression. Overall a model for glucocorticoid-mediated regulation of KLF9 involving multiple GBSs is depicted. This work unequivocally demonstrates that mechanistic insights gained from cell-lines can translate to physiologically relevant systems." View Publication

Chronic lymphocytic leukaemia (CLL) is characterised by a heterogeneous clinical course. Such heterogeneity is associated with a number of markers,including TP53 gene inactivation. While TP53 gene alterations determine resistance to chemotherapy,it is not clear whether they can influence early disease progression. To clarify this issue,TP53 mutations and deletions of the corresponding locus [del(17p)] were evaluated in 469 cases from the O-CLL1 observational study that recruited a cohort of clinically and molecularly characterised Binet stage A patients. Twenty-four cases harboured somatic TP53 mutations [accompanied by del(17p) in 9 cases],2 patients had del(17p) only,and 5 patients had TP53 germ-line variants. While del(17p) with or without TP53 mutations was capable of significantly predicting the time to first treatment,a reliable measure of disease progression,TP53 mutations were not. This was true for cases with high or low variant allele frequency. The lack of predictive ability was independent of the functional features of the mutant P53 protein in terms of transactivation and dominant negative potential. TP53 mutations alone were more frequent in patients with mutated IGHV genes,whereas del(17p) was associated with the presence of adverse prognostic factors,including CD38 positivity,unmutated-IGHV gene status,and NOTCH1 mutations. View Publication

The thyroid hormone,3,5,3'-Triiodo-L-thyronine (T3),is essential for growth,differentiation,and regulation of metabolic functions in multicellular organisms,although the specific mechanisms of this control are still unknown. In this study,treatment of a human pancreatic duct cell line (hPANC-1) with T3 blocks cell growth by an increase of cells in G(0)/G(1) cell cycle phase and enhances morphological and functional changes as indicated by the marked increase in the synthesis of insulin and the parallel decrease of the ductal differentiation marker cytokeratin19. Expression analysis of some of the genes regulating pancreatic beta-cell differentiation revealed a time-dependent increase in insulin and glut2 mRNA levels in response to T3. As last step of the acquisition of a beta-cell-like phenotype,we present evidence that thyroid hormones are able to increase the release of insulin into the culture medium. In conclusion,our results suggest,for the first time,that thyroid hormones induce cell cycle perturbations and play an important role in the process of transdifferentiation of a human pancreatic duct line (hPANC-1) into pancreatic-beta-cell-like cells. These findings have important implications in cell-therapy based treatment of diabetes and may provide important insights in the designing of novel therapeutic agents to restore normal glycemia in subjects with diabetes. View Publication

This study quantifies uptake of a fluorescent glucose analog,(2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) (2-NBDG),in a large panel of breast cancer cells and demonstrates potential to monitor changes in glycolysis caused by anticancer and endocrine therapies. Expressions of glucose transporter (GLUT 1) and hexokinase (HK I),which phosphorylates 2-NBDG,were measured via western blot in two normal mammary epithelial and eight breast cancer cell lines of varying biological subtype. Fluorescence intensity of each cell line labeled with 100 lM 2-NBDG for 20 min or unlabeled control was quantified. A subset of cancer cells was treated with anticancer and endocrine therapies,and 2-NBDG fluorescence changes were measured. Expression of GLUT 1 was necessary for uptake of 2-NBDG,as demonstrated by lack of 2-NBDG uptake in normal human mammary epithelial cells (HMECs). GLUT 1 expression and 2-NBDG uptake was ubiquitous among all breast cancer lines. Reduction and stimulation of 2-NBDG uptake was demonstrated by perturbation with anticancer agents,lonidamine (LND),and a-cyano-hydroxycinnamate (a-Cinn),respectively. LND directly inhibits HK and significantly reduced 2-NBDG fluorescence in a subset of two breast cancer cell lines. Conversely,when cells were treated with a-Cinn,a drug used to increase glycolysis,2-NBDG uptake was increased. Furthermore,tamoxifen (tam),a common endocrine therapy,was administered to estrogen receptor positive and negative (ER?/-) breast cells and demonstrated a decreased 2-NBDG uptake in ER? cells,reflecting a decrease in glycolysis. Results indicate that 2-NBDG uptake can be used to measure changes in glycolysis and has potential for use in early drug development. View Publication