技术资料

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

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

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

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

Autophagy is a cell-protective and degradative process that recycles damaged and long-lived cellular components. Cancer cells are thought to take advantage of autophagy to help them to cope with the stress of tumorigenesis; thus targeting autophagy is an attractive therapeutic approach. However,there are currently no specific inhibitors of autophagy. ULK1,a serine/threonine protein kinase,is essential for the initial stages of autophagy,and here we report that two compounds,MRT67307 and MRT68921,potently inhibit ULK1 and ULK2 in vitro and block autophagy in cells. Using a drug-resistant ULK1 mutant,we show that the autophagy-inhibiting capacity of the compounds is specifically through ULK1. ULK1 inhibition results in accumulation of stalled early autophagosomal structures,indicating a role for ULK1 in the maturation of autophagosomes as well as initiation. View Publication

The Smoothened receptor (Smo) mediates hedgehog (Hh) signaling critical for development,cell growth,and migration,as well as stem cell maintenance. Aberrant Hh signaling pathway activation has been implicated in a variety of cancers,and small-molecule antagonists of Smo have entered human clinical trials for the treatment of cancer. Here,we report the biochemical characterization of allosteric interactions of agonists and antagonists for Smo. Binding of two radioligands,[(3)H]3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{\{}[3-(4-pyridinyl)-phenyl]methyl{\}}-1-benzothiophene-2-carboxamide (SAG-1.3) (agonist) and [(3)H]cyclopamine (antagonist),was characterized using human Smo expressed in human embryonic kidney 293F membranes. We observed full displacement of [(3)H]cyclopamine by all Smo agonist and antagonist ligands examined. N-[(1E)-(3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene]-4-(phenylmethyl)-1-piperazinamine (SANT-1),an antagonist,did not fully inhibit the binding of [(3)H]SAG-1.3. In a functional cell-based beta-lactamase reporter gene assay,SANT-1 and N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-tris(ethyloxy)-benzamide (SANT-2) fully inhibited 3-chloro-4,7-difluoro-N-[trans-4-(methylamino)cyclohexyl]-N-{\{}[3-(4-pyridinyl)phenyl]methyl{\}}-1-benzothiophene-2-carboxamide (SAG-1.5)-induced Hh pathway activation. Detailed Schild-type" radioligand binding analysis with [(3)H]SAG-1.3 revealed that two structurally distinct Smoothened receptor antagonists SANT-1 and SANT-2 bound in a manner consistent with that of allosteric modulation. Our mechanism of action characterization of radioligand binding to Smo combined with functional data provides a better understanding of small-molecule interactions with Smo and their influence on the Hh pathway." View Publication

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2,an emerging virus that utilizes host proteins ACE2 and TMPRSS2 as entry factors. Understanding the factors affecting the pattern and levels of expression of these genes is important for deeper understanding of SARS-CoV-2 tropism and pathogenesis. Here we explore the role of genetics and co-expression networks in regulating these genes in the airway,through the analysis of nasal airway transcriptome data from 695 children. We identify expression quantitative trait loci for both ACE2 and TMPRSS2,that vary in frequency across world populations. We find TMPRSS2 is part of a mucus secretory network,highly upregulated by type 2 (T2) inflammation through the action of interleukin-13,and that the interferon response to respiratory viruses highly upregulates ACE2 expression. IL-13 and virus infection mediated effects on ACE2 expression were also observed at the protein level in the airway epithelium. Finally,we define airway responses to common coronavirus infections in children,finding that these infections generate host responses similar to other viral species,including upregulation of IL6 and ACE2. Our results reveal possible mechanisms influencing SARS-CoV-2 infectivity and COVID-19 clinical outcomes. View Publication

Pygopus 2 (Pygo2) is a coactivator of Wnt/$\beta$-catenin signaling that can bind bi- or trimethylated lysine 4 of histone-3 (H3K4me2/3) and participate in chromatin reading and writing. It remains unknown whether the Pygo2-H3K4me2/3 association has a functional relevance in breast cancer progression in vivo. To investigate the functional relevance of histone-binding activity of Pygo2 in malignant progression of breast cancer,we generated a knock-in mouse model where binding of Pygo2 to H3K4me2/3 was rendered ineffective. Loss of Pygo2-histone interaction resulted in smaller,differentiated,and less metastatic tumors,due,in part,to decreased canonical Wnt/$\beta$-catenin signaling. RNA- and ATAC-sequencing analyses of tumor-derived cell lines revealed downregulation of TGF$\beta$ signaling and upregulation of differentiation pathways such as PDGFR signaling. Increased differentiation correlated with a luminal cell fate that could be reversed by inhibition of PDGFR activity. Mechanistically,the Pygo2-histone interaction potentiated Wnt/$\beta$-catenin signaling,in part,by repressing the expression of Wnt signaling antagonists. Furthermore,Pygo2 and $\beta$-catenin regulated the expression of miR-29 family members,which,in turn,repressed PDGFR expression to promote dedifferentiation of wild-type Pygo2 mammary epithelial tumor cells. Collectively,these results demonstrate that the histone binding function of Pygo2 is important for driving dedifferentiation and malignancy of breast tumors,and loss of this binding activates various differentiation pathways that attenuate primary tumor growth and metastasis formation. Interfering with the Pygo2-H3K4me2/3 interaction may therefore serve as an attractive therapeutic target for metastatic breast cancer. SIGNIFICANCE: Pygo2 represents a potential therapeutic target in metastatic breast cancer,as its histone-binding capability promotes $\beta$-catenin-mediated Wnt signaling and transcriptional control in breast cancer cell dedifferentiation,EMT,and metastasis. View Publication

Small molecules that promote the metabolic activity of the pyruvate kinase isoform PKM2,such as TEPP-46 and DASA-58,limit tumorigenesis and inflammation. To understand how these compounds alter T cell function,we assessed their therapeutic activity in a mouse model of T cell-mediated autoimmunity that mimics multiple sclerosis (MS). TH17 cells are believed to orchestrate MS pathology,in part,through the production of two proinflammatory cytokines: interleukin-17 (IL-17) and GM-CSF. We found that both TEPP-46 and DASA-58 suppressed the development of IL-17-producing TH17 cells but increased the generation of those producing GM-CSF. This switch redirected disease pathology from the spinal cord to the brain. In addition,we found that activation of PKM2 interfered with TGF-$\beta$1 signaling,which is necessary for the development of TH17 and regulatory T cells. Collectively,our data clarify the therapeutic potential of PKM2 activators in MS-like disease and how these agents alter T cell function. View Publication

Enterotoxin-mediated secretory diarrheas such as cholera involve chloride secretion by enterocytes into the intestinal lumen by the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. We previously identified glycine hydrazide CFTR blockers that by electrophysiological studies appeared to block the CFTR anion pore at its lumen-facing surface. Here,we synthesize highly water-soluble,nonabsorbable malondihydrazides by coupling 2,4-disulfobenzaldehyde,4-sulfophenylisothiocyante,and polyethylene glycol (PEG) moieties to 2-naphthalenylamino-[(3,5-dibromo-2,4-dihydroxyphenyl) methylene] propanedioic acid dihydrazide,and aminoacethydrazides by coupling PEG to [(N-2-naphthalenyl)-2-(2-hydroxyethyl)]-glycine-2-[(3,5-dibromo-2,4-dihydroxyphenyl) methylene] hydrazide. Compounds rapidly,fully and reversibly blocked CFTR-mediated chloride current with Ki of 2-8 microM when added to the apical surface of epithelial cell monolayers. Compounds did not pass across Caco-2 monolayers,and were absorbed by {\textless}2{\%}/hr in mouse intestine. Luminally added compounds blocked by {\textgreater}90{\%} cholera toxin-induced fluid secretion in mouse intestinal loops,without inhibiting intestinal fluid absorption. These orally administered,nonabsorbable,nontoxic CFTR inhibitors may reduce intestinal fluid losses in cholera. View Publication

Mifepristone is a potent antagonist of steroid hormone receptors such as glucocorticoid and progesterone receptors. We investigated the potential for mifepristone to act as an antiandrogen and compared it with partial androgen receptor (AR) agonists and antagonists,in particular bicalutamide. Mifepristone was an effective antiandrogen in vitro that inhibited transcription from three androgen-responsive promoters and blocked the agonist R1881 in a dose-dependent manner. Like bicalutamide,mifepristone also antagonized the action of androgen receptor with a (T877A) mutation. Mifepristone competed effectively with R1881 with a relative binding affinity comparable to that of cyproterone acetate,and much higher than that of hydroxyflutamide and bicalutamide in a binding assay. Mifepristone could effectively induce the binding of the herpes simplex viral protein 16/AR fusion protein to the hormone response elements in the murine mammary tumor virus-luciferase reporter. With either wild-type or T877A mutant AR,mifepristone alone was unable to induce any detectable interaction with coactivators transcriptional intermediary factor-2 or beta-catenin but could inhibit the R1881-induced binding of AR to transcriptional intermediary factor-2 and beta-catenin. Similarly,mifepristone could inhibit the R1881-induced N/C-terminal interaction in a dose-dependent manner even though mifepristone alone has no effect on the N/C-terminal interaction of AR. We found that mifepristone could induce a strong interaction between AR and corepressors nuclear receptor corepressor and silencing mediator for retinoid and thyroid hormone receptors in both transactivation and two-hybrid assays to a greater degree than hydroxyflutamide,cyproterone acetate,and bicalutamide. The AR-corepressor interaction was also seen in coimmunoprecipitation assays. Finally,mifepristone at high concentrations induced a low level of prostate-specific antigen expression in LNCaP and antagonized prostate-specific antigen expression induced by R1881. Mifepristone also antagonized R1881 action on the growth of LNCaP prostate cancer cells. View Publication

2-Hydroxyglutarate (2HG) exists as two enantiomers,(R)-2HG and (S)-2HG,and both are implicated in tumor progression via their inhibitory effects on $\alpha$-ketoglutarate ($\alpha$KG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations,whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme and conversely it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH rather than attempting to block 2HG production by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore our results uncover an unexpected link between oncometabolites altered DNA repair and genetic instability." View Publication