技术资料

Interpreting how multicellular interactions in the tumor affect resistance pathways to BRAF and MEK1/2 MAPK inhibitors (MAPKi) remains a challenge. To investigate this,we profiled global ligand-receptor interactions among tumor and stromal/immune cells from biopsies of MAPK-driven disease. MAPKi increased tumor-associated macrophages (TAMs) in some patients,which correlated with poor clinical response,and MAPKi coamplified bidirectional tumor-TAM signaling via receptor tyrosine kinases (RTKs) including AXL,MERTK,and their ligand GAS6. In xenograft tumors,intravital microscopy simultaneously monitored in situ single-cell activities of multiple kinases downstream of RTKs,revealing MAPKi increased TAMs and enhanced bypass signaling in TAM-proximal tumor cells. As a proof-of-principle strategy to block this signaling,we developed a multi-RTK kinase inhibitor nanoformulation that accumulated in TAMs and delayed disease progression. Thus,bypass signaling can reciprocally amplify across nearby cell types,offering new opportunities for therapeutic design. View Publication

Glioblastoma (GBM) is a lethal brain cancer with a median survival time of approximately 15 months following treatment. Common in vitro GBM models for drug screening are adherent and do not recapitulate the features of human GBM in vivo. Here we report the genomic characterization of nine patient-derived,spheroid GBM cell lines that recapitulate human GBM characteristics in orthotopic xenograft models. Genomic sequencing revealed that the spheroid lines contain alterations in GBM driver genes such as PTEN,CDKN2A,and NF1. Two spheroid cell lines,JHH-136 and JHH-520,were utilized in a high-throughput drug screen for cell viability using a 1912-member compound library. Drug mechanisms that were cytotoxic in both cell lines were Hsp90 and proteasome inhibitors. JHH-136 was uniquely sensitive to topoisomerase 1 inhibitors,while JHH-520 was uniquely sensitive to Mek inhibitors. Drug combination screening revealed that PI3 kinase inhibitors combined with Mek or proteasome inhibitors were synergistic. However,animal studies to test these drug combinations in vivo revealed that Mek inhibition alone was superior to the combination treatments. These data show that these GBM spheroid lines are amenable to high-throughput drug screening and that this dataset may deliver promising therapeutic leads for future GBM preclinical studies. View Publication

Blood oxygen saturation (SaO2) is one of the most important environmental factors in clinical heart protection. This study used human heart samples and human induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) to assess how SaO2 affects human CM cell cycle activities. The results showed that there were significantly more cell cycle markers in the moderate hypoxia group (SaO2: 75{\%} to 85{\%}) than in the other 2 groups (SaO2 {\textless}75{\%} or {\textgreater}85{\%}). In iPSC-CMs 15{\%} and 10{\%} oxygen (O2) treatment increased cell cycle markers,whereas 5{\%} and rapid change of O2 decreased the markers. Moderate hypoxia is beneficial to the cell cycle activities of post-natal human CMs. View Publication

Purpose: Despite high initial response rates with cytoreductive surgery,conventional chemotherapy and the incorporation of biologic agents,ovarian cancer patients often relapse and die from their disease. New approaches are needed to improve patient outcomes. This study was designed to evaluate the antitumor activity of NEO-201 monoclonal antibody (mAb) in preclinical models of ovarian cancer where the NEO-201 target is highly expressed. Experimental Design: Functional analysis of NEO-201 against tumor cell lines was performed by antibody-dependent cellular cytotoxicity (ADCC) assays. Binding of NEO-201 to tumor tissues and cell lines were determined by immunohistochemistry (IHC) and flow cytometry,respectively. Further characterization of the antigen recognized by NEO-201 was performed by mass spectrometry. Ovarian cancer models were used to evaluate the anti-tumor activity of NEO-201 in vivo. NEO-201 at a concentration of 250 g/mouse was injected intraperitoneally (IP) on days 1,4,and 8. Human PBMCs were injected IP simultaneously as effector cells. Results: Both IHC and flow cytometry revealed that NEO-201 binds prominently to the colon,pancreatic,and mucinous ovarian cancer tissues and cell lines. Immunoprecipitation of the antigen recognized by NEO-201 was performed in human ovarian,colon,and pancreatic cancer cell lines. From these screening,carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and CEACAM6 were identified as the most likely targets of NEO-201. Our results confirmed that NEO-201 binds different types of cancers; the binding is highly selective for the tumor cells without cross reactivity with the surrounding healthy tissue. Functional analysis revealed that NEO-201 mediates ADCC killing against human ovarian and colorectal carcinoma cell lines in vitro. In addition,NEO-201 inhibited tumor growth in the presence of activated human PBMCs in orthotopic mouse models of both primary and metastatic ovarian cancer. Importantly,NEO-201 prolonged survival of tumor-bearing mice. Conclusions: These data suggested that NEO-201 has an antitumor activity against tumor cells expressing its antigen. Targeting an antigen expressed in tumors,but not in normal tissues,allows patient selection for optimal treatment. These findings strongly indicate that NEO-201 warrants clinical testing as both a novel therapeutic and diagnostic agent for treatment of ovarian carcinomas. A first in human clinical trial evaluating NEO-201 in adults with chemo-resistant solid tumors is ongoing at the NIH clinical Center. View Publication

The discovery of TMEM173/STING-dependent innate immunity has recently provided guidance for the prevention and management of inflammatory disorders. Here,we show that myeloid TMEM173 occupies an essential role in regulating coagulation in bacterial infections through a mechanism independent of type I interferon response. Mechanistically,TMEM173 binding to ITPR1 controls calcium release from the endoplasmic reticulum in macrophages and monocytes. The TMEM173-dependent increase in cytosolic calcium drives Gasdermin D (GSDMD) cleavage and activation,which triggers the release of F3,the key initiator of blood coagulation. Genetic or pharmacological inhibition of the TMEM173-GSDMD-F3 pathway blocks systemic coagulation and improves animal survival in three models of sepsis (cecal ligation and puncture or bacteremia with Escherichia coli or Streptococcus pneumoniae infection). The upregulation of the TMEM173 pathway correlates with the severity of disseminated intravascular coagulation and mortality in patients with sepsis. Thus,TMEM173 is a key regulator of blood clotting during lethal bacterial infections. View Publication

A continuing quest for specific inhibitors of proinflammatory cytokines brings promise for effective therapies designed for inflammatory and autoimmune disorders. Cefazolin,a safe,first-generation cephalosporin antibiotic,has been recently shown to specifically interact with interleukin 15 (IL-15) receptor subunit $\alpha$ (IL-15R$\alpha$) and to inhibit IL-15-dependent TNF-$\alpha$ and IL-17 synthesis. The aim of this study was to elucidate cefazolin activity against IL-2,IL-4,IL-15 and IL-21,i.e. four cytokines sharing the common cytokine receptor $\gamma$ chain ($\gamma$c). In silico,molecular docking unveiled two potential cefazolin binding sites within the IL-2/IL-15R$\beta$ subunit and two within the $\gamma$c subunit. In vitro,cefazolin decreased proliferation of PBMC (peripheral blood mononuclear cells) following IL-2,IL-4 and IL-15 stimulation,reduced production of IFN-$\gamma$,IL-17 and TNF-$\alpha$ in IL-2- and IL-15-treated PBMC and in IL-15 stimulated natural killer (NK) cells,attenuated IL-4-dependent expression of CD11c in monocyte-derived dendritic cells and suppressed phosphorylation of JAK3 in response to IL-2 and IL-15 in PBMC,to IL-4 in TF-1 (erythroleukemic cell line) and to IL-21 in NK-92 (NK cell line). The results of the study suggest that cefazolin may exert inhibitory activity against all of the $\gamma$c receptor-dependent cytokines,i.e. IL-2,IL-4,IL-7,IL-9,IL-15 and IL-21. View Publication

Aberrant activation of the hedgehog (Hh) signaling pathway has been implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human cancer. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells,while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells,accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression,resulting in gefitinib tolerance (P {\textless} 0.001) in EGFR-TKI-sensitive cells. Blockade of the Hh signaling pathway using the SMO antagonist SANT-1 restored E-cadherin expression and downregulate Snail and ABCG2 in EGFR-TKI-resistant cells. A combination of SANT-1 and gefitinib markedly inhibited tumorigenesis and proliferation in EGFR-TKI-resistant cells (P {\textless} 0.001). These findings indicate that hyperactivity of Hh signaling resulted in EGFR-TKI resistance,by EMT introduction and ABCG2 upregulation,and blockade of Hh signaling synergistically increased sensitivity to EGFR-TKIs in primary and secondary resistant NSCLC cells. E-cadherin expression may be a potential biomarker of the suitability of the combined application of an Hh inhibitor and EGFR-TKIs in EGFR-TKI-resistant NSCLCs. View Publication

BACKGROUND Mammalian target of rapamycin complex 1 (mTORC1) is a protein kinase that relays nutrient availability signals to control numerous cellular functions including autophagy,a process of cellular self-eating activated by nutrient depletion. Addressing the therapeutic potential of modulating mTORC1 signaling and autophagy in human disease requires active chemicals with pharmacologically desirable properties. METHODOLOGY/PRINCIPAL FINDINGS Using an automated cell-based assay,we screened a collection of {\textgreater}3,500 chemicals and identified three approved drugs (perhexiline,niclosamide,amiodarone) and one pharmacological reagent (rottlerin) capable of rapidly increasing autophagosome content. Biochemical assays showed that the four compounds stimulate autophagy and inhibit mTORC1 signaling in cells maintained in nutrient-rich conditions. The compounds did not inhibit mTORC2,which also contains mTOR as a catalytic subunit,suggesting that they do not inhibit mTOR catalytic activity but rather inhibit signaling to mTORC1. mTORC1 inhibition and autophagosome accumulation induced by perhexiline,niclosamide or rottlerin were rapidly reversed upon drug withdrawal whereas amiodarone inhibited mTORC1 essentially irreversibly. TSC2,a negative regulator of mTORC1,was required for inhibition of mTORC1 signaling by rottlerin but not for mTORC1 inhibition by perhexiline,niclosamide and amiodarone. Transient exposure of immortalized mouse embryo fibroblasts to these drugs was not toxic in nutrient-rich conditions but led to rapid cell death by apoptosis in starvation conditions,by a mechanism determined in large part by the tuberous sclerosis complex protein TSC2,an upstream regulator of mTORC1. By contrast,transient exposure to the mTORC1 inhibitor rapamycin caused essentially irreversible mTORC1 inhibition,sustained inhibition of cell growth and no selective cell killing in starvation. CONCLUSION/SIGNIFICANCE The observation that drugs already approved for human use can reversibly inhibit mTORC1 and stimulate autophagy should greatly facilitate the preclinical and clinical testing of mTORC1 inhibition for indications such as tuberous sclerosis,diabetes,cardiovascular disease and cancer. View Publication

NAD is a multifunctional molecule involved in both metabolic processes and signaling pathways. Such signalling pathways consume NAD which is replenished via one of several biosynthesis pathways. We show that influx of NAD across the plasma membrane may be able to contribute to the homeostasis of intracellular NAD levels. Indeed,extracellular application of NAD was able to replete NAD levels that had been lowered pharmacologically using the novel drug FK866 and was also able to rescue cells from FK866-induced cell death. A marked lag between the drop in NAD levels and cell death prompted us to investigate the mechanism of cell death. We were unable to find evidence of apoptosis as assessed by immunoblotting for the Caspase 3 activation fragment and immunostaining for cytochrome C and AIF translocation. We,therefore,investigated whether autophagy was initiated by FK866. Indeed,we were able to observe the formation of LC3-positive vesicles that had fused with lysosomes in FK866-treated but not control cells. Furthermore,this autophagic phenotype could be reverted by the addition of NAD to the extracellular medium. View Publication

INTRODUCTION One method for assessing the in vitro response to CFTR-modulating compounds is by analysis of epithelial monolayers in an Ussing chamber,where the apical and basolateral surfaces are isolated and the potential difference,short-circuit current,and transepithelial resistance can be monitored. The effect of a chloride ion gradient across airway epithelia on transepithelial chloride transport and the magnitude of CFTR modulator efficacy were examined. METHODS CFTR-mediated changes in the potential difference and transepithelial currents of primary human nasal epithelial cell cultures were quantified in Ussing chambers with either symmetrical solutions or reduced chloride solutions in the apical chamber. CFTR activity in homozygous F508del CFTR epithelia was rescued by treatment with VX-661,C4/C18,4-phenylbutyrate (4-PBA) for 24 hr at 37°C or by incubation at 29°C for 48 hr. RESULTS Imposing a chloride gradient increased CFTR-mediated and CaCC-mediated ion transport. Treatment of F508del CFTR homozygous cells with CFTR modulating compounds increased CFTR activity,which was significantly more evident in the presence of a chloride gradient. This observation was recapitulated with temperature-mediated F508del CFTR correction. CONCLUSIONS Imposing a chloride gradient during Ussing chamber measurements resulted in increased CFTR-mediated ion transport in expanded non-CF and F508del CFTR homozygous epithelia. In F508del CFTR homozygous epithelia,the magnitude of response to CFTR modulating compounds or low temperature was greater when assayed with a chloride gradient compared to symmetrical chloride,resulting in an apparent increase in measured efficacy. Future work may direct which methodologies utilized to quantify CFTR modulator response in vitro are most appropriate for the estimation of in vivo efficacy. View Publication

Substoichiometric concentrations of cytochalasin D inhibited the rate of polymerization of actin in 0.5 mM MgCl2,increased its critical concentration and lowered its steady state viscosity. Stoichiometric concentrations of cytochalasin D in 0.5 mM MgCl2 and even substoichiometric concentrations of cytochalasin D in 30 mM KCl,however,accelerated the rate of actin polymerization,although still lowering the final steady state viscosity. Cytochalasin B,at all concentrations in 0.5 mM MgCl2 or in 30 mM KCl,accelerated the rate of polymerization and lowered the final steady state viscosity. In 0.5 mM MgCl2,cytochalasin D uncoupled the actin ATPase activity from actin polymerization,increasing the ATPase rate by at least 20 times while inhibiting polymerization. Cytochalasin B had a very much lower stimulating effect. Neither cytochalasin D nor B affected the actin ATPase activity in 30 mM KCl. The properties of cytochalasin E were intermediate between those of cytochalasin D and B. Cytochalasin D also stimulated the ATPase activity of monomeric actin in the absence of MgCl2 and KCl and,to a much greater extent,stimulated the ATPase activity of monomeric actin below its critical concentration in 0.5 mM MgCl2. Both above and below its critical concentration and in the presence and absence of cytochalasin D,the initial rate of actin ATPase activity,when little or no polymerization had occurred,was directly proportional to the actin concentration and,therefore,apparently was independent of actin-actin interactions. To rationalize all these data,a working model has been proposed in which the first step of actin polymerization is the conversion of monomeric actin-bound ATP,A . ATP,to monomeric actin-bound ADP and Pi,A* . ADP . Pi,which,like the preferred growing end of an actin filament,can bind cytochalasins. View Publication

Malignant cells have a higher nicotinamide adenine dinucleotide (NAD(+)) turnover rate than normal cells,making this biosynthetic pathway an attractive target for cancer treatment. Here we investigated the biologic role of a rate-limiting enzyme involved in NAD(+) synthesis,Nampt,in multiple myeloma (MM). Nampt-specific chemical inhibitor FK866 triggered cytotoxicity in MM cell lines and patient MM cells,but not normal donor as well as MM patients PBMCs. Importantly,FK866 in a dose-dependent fashion triggered cytotoxicity in MM cells resistant to conventional and novel anti-MM therapies and overcomes the protective effects of cytokines (IL-6,IGF-1) and bone marrow stromal cells. Nampt knockdown by RNAi confirmed its pivotal role in maintenance of both MM cell viability and intracellular NAD(+) stores. Interestingly,cytotoxicity of FK866 triggered autophagy,but not apoptosis. A transcriptional-dependent (TFEB) and independent (PI3K/mTORC1) activation of autophagy mediated FK866 MM cytotoxicity. Finally,FK866 demonstrated significant anti-MM activity in a xenograft-murine MM model,associated with down-regulation of ERK1/2 phosphorylation and proteolytic cleavage of LC3 in tumor cells. Our data therefore define a key role of Nampt in MM biology,providing the basis for a novel targeted therapeutic approach. View Publication