技术资料

Synthetic lethality triggered by PARP inhibitor (PARPi) yields promising therapeutic results. Unfortunately,tumor cells acquire PARPi resistance,which is usually associated with the restoration of homologous recombination,loss of PARP1 expression,and/or loss of DNA double-strand break (DSB) end resection regulation. Here,we identify a constitutive mechanism of resistance to PARPi. We report that the bone marrow microenvironment (BMM) facilitates DSB repair activity in leukemia cells to protect them against PARPi-mediated synthetic lethality. This effect depends on the hypoxia-induced overexpression of transforming growth factor beta receptor (TGF$\beta$R) kinase on malignant cells,which is activated by bone marrow stromal cells-derived transforming growth factor beta 1 (TGF-$\beta$1). Genetic and/or pharmacological targeting of the TGF-$\beta$1-TGF$\beta$R kinase axis results in the restoration of the sensitivity of malignant cells to PARPi in BMM and prolongs the survival of leukemia-bearing mice. Our finding may lead to the therapeutic application of the TGF$\beta$R inhibitor in patients receiving PARPis. View Publication

BACKGROUND The nonsense mutation,c.3846G{\textgreater}A (aka: W1282X-CFTR) leads to a truncated transcript that is susceptible to nonsense-mediated decay (NMD) and produces a shorter protein that is unstable and lacks normal channel activity in patient-derived tissues. However,if overexpressed in a heterologous expression system,the truncated mutant protein has been shown to mediate CFTR channel function following the addition of potentiators. In this study,we asked if a quadruple combination of small molecules that together inhibit nonsense mediated decay,stabilize both halves of the mutant protein and potentiate CFTR channel activity could rescue the functional expression of W1282X-CFTR in patient derived nasal cultures. METHODS We identified the CFTR domains stabilized by corrector compounds supplied from AbbVie using a fragment based,biochemical approach. Rescue of the channel function of W1282X.-CFTR protein by NMD inhibition and small molecule protein modulators was studied using a bronchial cell line engineered to express W1282X and in primary nasal epithelial cultures derived from four patients homozygous for this mutation. RESULTS We confirmed previous studies showing that inhibition of NMD using the inhibitor: SMG1i,led to an increased abundance of the shorter transcript in a bronchial cell line. Interestingly,on top of SMG1i,treatment with a combination of two new correctors developed by Galapagos/AbbVie (AC1 and AC2-2,separately targeting either the first or second half of CFTR and promoting assembly,significantly increased the potentiated channel activity by the mutant in the bronchial epithelial cell line and in patient-derived nasal epithelial cultures. The average rescue effect in primary cultures was approximately 50{\%} of the regulated chloride conductance measured in non-CF cultures. CONCLUSIONS These studies provide the first in-vitro evidence in patient derived airway cultures that the functional defects incurred by W1282X,has the potential to be effectively repaired pharmacologically. View Publication

Introducing a reactive carbonyl to a scaffold that does not otherwise have an electrophilic functionality to create a reversible covalent inhibitor is a potentially useful strategy for enhancing compound potency. However,aldehydes are metabolically unstable,which precludes the use of this strategy for compounds to be tested in animal models or in human clinical studies. To overcome this limitation,we designed ketone-based functionalities capable of forming reversible covalent adducts,while displaying high metabolic stability,and imparting improved water solubility to their pendant scaffold. We tested this strategy on the ferroptosis inducer and experimental therapeutic erastin,and observed substantial increases in compound potency. In particular,a new carbonyl erastin analog,termed IKE,displayed improved potency,solubility and metabolic stability,thus representing an ideal candidate for future in vivo cancer therapeutic applications. View Publication

INTRODUCTION Bronchial asthma is a chronic respiratory disease characterized by airway inflammation,allergen-induced hypersensitivity and dyspnea. Most asthmatic patients demonstrate oscillations of disease symptoms within 24 hours regulated by circadian clock genes. We hypothesized that these genes may be regulators of childhood asthma risk. OBJECTIVES The aim was to investigate whether single-nucleotide polymorphisms (SNPs) in the circadian clock genes are associated with childhood asthma risk. We also aimed to analyze the mRNA level of clock genes in the blood of asthmatic children and NHBE cells stimulated with IL-13. MATERIALS AND METHODS Peripheral blood was collected from 165 asthmatic and 138 healthy Polish children. NHBE cells were culture at the air-liquid interface (ALI) with IL-13 as an in vitro model of allergic inflammation. Using TaqMan probes,we genotyped 32 SNPs in: CLOCK,BMAL1,PER3 and TIMELESS. Expression analysis for TIMELESS was performed using real-time PCR with SYBR Green. For haplotype and genotype statistical analysis we used Haploview 4.2 and STATISTICA version 12,respectively. Gene expression analysis was performed in DataAssist v3.01. RESULTS We found that three polymorphisms in TIMELESS (rs2291739,rs10876890,rs11171856) and two haplotypes (TTTT and CTAC) were associated with asthma risk. We also found significantly decreased expression of TIMELESS in the blood of asthmatic children as compared to the healthy children (P = 0.0289) and in NHBE cells stimulated with IL-13 (P = 0.0302). CONCLUSIONS In our study,we showed for the first time that TIMELESS variants and expression may be associated with childhood asthma. View Publication

Existing antibiotics are inadequate to defeat tuberculosis (TB),a leading cause of death worldwide. We sought potential targets for host-directed therapies (HDTs) by investigating the host immune response to mycobacterial infection. We used high-throughput CRISPR knockout and CRISPR interference (CRISPRi) screens to identify perturbations that improve the survival of human phagocytic cells infected with Mycobacterium bovis BCG (Bacillus Calmette-Gu{\'{e}}rin),as a proxy for Mycobacterium tuberculosis (Mtb). Many of these perturbations constrained the growth of intracellular mycobacteria. We identified over 100 genes associated with diverse biological pathways as potential HDT targets. We validated key components of the type I interferon and aryl hydrocarbon receptor signaling pathways that respond to the small-molecule inhibitors cerdulatinib and CH223191,respectively; these inhibitors enhanced human macrophage survival and limited the intracellular growth of Mtb. Thus,high-throughput functional genomic screens,by elucidating highly complex host-pathogen interactions,can serve to identify HDTs to potentially improve TB treatment. View Publication

Two groups recently reported the in vitro differentiation of human embryonic stem cells into insulin-secreting cells,achieving an elusive goal for regenerative medicine. Herein we provide a perspective regarding these developments,compare phenotypes of the insulin-containing cells to human $\beta$ cells,and discuss implications for type 1 diabetes research and clinical care. View Publication

Pulmonary Fibrosis (PF) is a devastating progressive disease in which normal lung structure and function is compromised by scarring. Lung fibrosis can be caused by thoracic radiation,injury from chemotherapy and systemic diseases such as rheumatoid arthritis that involve inflammatory responses. CDDO-Me (Methyl 2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate,Bardoxolone methyl) is a novel triterpenoid with anti-fibrotic and anti-inflammatory properties as shown by our in vitro studies. Based on this evidence,we hypothesized that CDDO-Me would reduce lung inflammation,fibrosis and lung function impairment in a bleomycin model of lung injury and fibrosis. To test this hypothesis,mice received bleomycin via oropharyngeal aspiration (OA) on day zero and CDDO-Me during the inflammatory phase from days -1 to 9 every other day. Bronchoalveolar lavage fluid (BALF) and lung tissue were harvested on day 7 to evaluate inflammation,while fibrosis and lung function were evaluated on day 21. On day 7,CDDO-Me reduced total BALF protein by 50{\%},alveolar macrophage infiltration by 40{\%},neutrophil infiltration by 90{\%} (p≤0.01),inhibited production of the inflammatory cytokines KC and IL-6 by over 90{\%} (p≤0.001),and excess production of the pro-fibrotic cytokine TGF$\beta$ by 50{\%}. CDDO-Me also inhibited $\alpha$-smooth muscle actin and fibronectin mRNA by 50{\%} (p≤0.05). On day 21,CDDO-Me treatment reduced histological fibrosis,collagen deposition and $\alpha$SMA production. Lung function was significantly improved at day 21 by treatment with CDDO-Me,as demonstrated by respiratory rate and dynamic compliance. These new findings reveal that CDDO-Me exhibits potent anti-fibrotic and anti-inflammatory properties in vivo. CDDO-Me is a potential new class of drugs to arrest inflammation and ameliorate fibrosis in patients who are predisposed to lung injury and fibrosis incited by cancer treatments (e.g. chemotherapy and radiation) and by systemic autoimmune diseases. View Publication

CDC7 is a serine/threonine kinase that has been shown to be required for the initiation and maintenance of DNA replication. Up-regulation of CDC7 is detected in multiple tumor cell lines,with inhibition of CDC7 resulting in cell cycle arrest. In this paper,we disclose the discovery of a potent and selective CDC7 inhibitor,XL413 (14),which was advanced into Phase 1 clinical trials. Starting from advanced lead 3,described in a preceding communication,we optimized the CDC7 potency and selectivity to demonstrate in vitro CDC7 dependent cell cycle arrest and in vivo tumor growth inhibition in a Colo-205 xenograft model. View Publication

Protein citrullination has been shown to regulate numerous physiological pathways (e.g.,the innate immune response and gene transcription) and is,when dysregulated,known to be associated with numerous human diseases,including cancer,rheumatoid arthritis,and multiple sclerosis. This modification,also termed deimination,is catalyzed by a group of enzymes called the protein arginine deiminases (PADs). In mammals,there are five PAD family members (i.e.,PADs 1,2,3,4,and 6) that exhibit tissue-specific expression patterns and vary in their subcellular localization. The kinetic characterization of PAD4 was recently reported,and these efforts guided the development of the two most potent PAD4 inhibitors (i.e.,F- and Cl-amidine) known to date. In addition to being potent PAD4 inhibitors,we show here that Cl-amidine also exhibits a strong inhibitory effect against PADs 1 and 3,thus indicating its utility as a pan PAD inhibitor. Given the increasing number of diseases in which dysregulated PAD activity has been implicated,the development of PAD-selective inhibitors is of paramount importance. To aid that goal,we characterized the catalytic mechanism and substrate specificity of PADs 1 and 3. Herein,we report the results of these studies,which suggest that,like PAD4,PADs 1 and 3 employ a reverse protonation mechanism. Additionally,the substrate specificity studies provided critical information that aided the identification of PAD3-selective inhibitors. These compounds,denoted F4- and Cl4-amidine,are the most potent PAD3 inhibitors ever described. View Publication

OBJECTIVES An imbalance between neutrophil extracellular trap (NET) formation and degradation has been described in systemic lupus erythematosus (SLE),potentially contributing to autoantigen externalisation,type I interferon synthesis and endothelial damage. We have demonstrated that peptidylarginine deiminase (PAD) inhibition reduces NET formation and protects against lupus-related vascular damage in the New Zealand Mixed model of lupus. However,another strategy for inhibiting NETs--knockout of NOX2--accelerates lupus in a different murine model,MRL/lpr. Here,we test the effects of PAD inhibition on MRL/lpr mice in order to clarify whether some NET inhibitory pathways may be consistently therapeutic across models of SLE. METHODS NET formation and autoantibodies to NETs were characterised in lupus-prone MRL/lpr mice. MRL/lpr mice were also treated with two different PAD inhibitors,Cl-amidine and the newly described BB-Cl-amidine. NET formation,endothelial function,interferon signature,nephritis and skin disease were examined in treated mice. RESULTS Neutrophils from MRL/lpr mice demonstrate accelerated NET formation compared with controls. MRL/lpr mice also form autoantibodies to NETs and have evidence of endothelial dysfunction. PAD inhibition markedly improves endothelial function,while downregulating the expression of type I interferon-regulated genes. PAD inhibition also reduces proteinuria and immune complex deposition in the kidneys,while protecting against skin disease. CONCLUSIONS PAD inhibition reduces NET formation,while protecting against lupus-related damage to the vasculature,kidneys and skin in various lupus models. The strategy by which NETs are inhibited will have to be carefully considered if human studies are to be undertaken. View Publication

Recent evidence suggests that enhanced neutrophil extracellular trap (NET) formation activates plasmacytoid dendritic cells and serves as a source of autoantigens in SLE. We propose that aberrant NET formation is also linked to organ damage and to the premature vascular disease characteristic of human SLE. Here,we demonstrate enhanced NET formation in the New Zealand mixed 2328 (NZM) model of murine lupus. NZM mice also developed autoantibodies to NETs as well as the ortholog of human cathelicidin/LL37 (CRAMP),a molecule externalized in the NETs. NZM mice were treated with Cl-amidine,an inhibitor of peptidylarginine deiminases (PAD),to block NET formation and were evaluated for lupus-like disease activity,endothelial function,and prothrombotic phenotype. Cl-amidine treatment inhibited NZM NET formation in vivo and significantly altered circulating autoantibody profiles and complement levels while reducing glomerular IgG deposition. Further,Cl-amidine increased the differentiation capacity of bone marrow endothelial progenitor cells,improved endothelium-dependent vasorelaxation,and markedly delayed time to arterial thrombosis induced by photochemical injury. Overall,these findings suggest that PAD inhibition can modulate phenotypes crucial for lupus pathogenesis and disease activity and may represent an important strategy for mitigating cardiovascular risk in lupus patients. View Publication

CD8+ effector T (TE) cell proliferation and cytokine production depends on enhanced glucose metabolism. However,circulating T cells continuously adapt to glucose fluctuations caused by diet and inter-organ metabolite exchange. Here we show that transient glucose restriction (TGR) in activated CD8+ TE cells metabolically primes effector functions and enhances tumour clearance in mice. Tumour-specific TGR CD8+ TE cells co-cultured with tumour spheroids in replete conditions display enhanced effector molecule expression,and adoptive transfer of these cells in a murine lymphoma model leads to greater numbers of immunologically functional circulating donor cells and complete tumour clearance. Mechanistically,TE cells treated with TGR undergo metabolic remodelling that,after glucose re-exposure,supports enhanced glucose uptake,increased carbon allocation to the pentose phosphate pathway (PPP) and a cellular redox shift towards a more reduced state-all indicators of a more anabolic programme to support their enhanced functionality. Thus,metabolic conditioning could be used to promote efficiency of T-cell products for adoptive cellular therapy. View Publication