Intrinsic Resistance to MEK Inhibition through BET Protein-Mediated Kinome Reprogramming in NF1-Deficient Ovarian Cancer.

Mutation or deletion of Neurofibromin 1 (NF1), an inhibitor of RAS signaling, frequently occurs in epithelial ovarian cancer (EOC), supporting therapies that target downstream RAS effectors, such as the RAF-MEK-ERK pathway. However, no comprehensive studies have been carried out testing the efficacy of MEK inhibition in NF1-deficient EOC. Here, we performed a detailed characterization of MEK inhibition in NF1-deficient EOC cell lines using kinome profiling and RNA sequencing. Our studies showed MEK inhibitors (MEKi) were ineffective at providing durable growth inhibition in NF1-deficient cells due to kinome reprogramming. MEKi-mediated destabilization of FOSL1 resulted in induced expression of receptor tyrosine kinases (RTK) and their downstream RAF and PI3K signaling, thus overcoming MEKi therapy. MEKi synthetic enhancement screens identified BRD2 and BRD4 as integral mediators of the MEKi-induced RTK signatures. Inhibition of bromo and extra terminal (BET) proteins using BET bromodomain inhibitors blocked MEKi-induced RTK reprogramming, indicating that BRD2 and BRD4 represent promising therapeutic targets in combination with MEKi to block resistance due to kinome reprogramming in NF1-deficient EOC. IMPLICATIONS: Our findings suggest MEK inhibitors will likely not be effective as single-agent therapies in NF1-deficient EOC due to kinome reprogramming. Cotargeting BET proteins in combination with MEKis to block reprogramming at the transcriptional level may provide an epigenetic strategy to overcome MEKi resistance in NF1-deficient EOC.

Resistance to BET Bromodomain Inhibitors Is Mediated by Kinome Reprogramming in Ovarian Cancer.

Small-molecule BET bromodomain inhibitors (BETis) are actively being pursued in clinical trials for the treatment of a variety of cancers, but the mechanisms of resistance to BETis remain poorly understood. Using a mass spectrometry approach that globally measures kinase signaling at the proteomic level, we evaluated the response of the kinome to targeted BETi treatment in a panel of BRD4-dependent ovarian carcinoma (OC) cell lines. Despite initial inhibitory effects of BETi, OC cells acquired resistance following sustained treatment with the BETi JQ1. Through application of multiplexed inhibitor beads (MIBs) and mass spectrometry, we demonstrate that BETi resistance is mediated by adaptive kinome reprogramming, where activation of compensatory pro-survival kinase networks overcomes BET protein inhibition. Furthermore, drug combinations blocking these kinases may prevent or delay the development of drug resistance and enhance the efficacy of BETi therapy.

A Novel HSP90 Inhibitor-Drug Conjugate to SN38 Is Highly Effective in Small Cell Lung Cancer.

PURPOSE: Small cell lung cancer (SCLC) is a highly aggressive disease representing 12% to 13% of total lung cancers, with median survival of <2 years. No targeted therapies have proven effective in SCLC. Although most patients respond initially to cytotoxic chemotherapies, resistance rapidly emerges, response to second-line agents is limited, and dose-limiting toxicities (DLT) are a major issue. This study performs preclinical evaluation of a new compound, STA-8666, in SCLC. EXPERIMENTAL DESIGN: To avoid DLT for useful cytotoxic agents, the recently developed drug STA-8666 combines a chemical moiety targeting active HSP90 (concentrated in tumors) fused via cleavable linker to SN38, the active metabolite of irinotecan. We compare potency and mechanism of action of STA-8666 and irinotecan in vitro and in vivo RESULTS: In two SCLC xenograft and patient-derived xenograft models, STA-8666 was tolerated without side effects up to 150 mg/kg. At this dose, STA-8666 controlled or eliminated established tumors whether used in a first-line setting or in tumors that had progressed following treatment on standard first- and second-line agents for SCLC. At 50 mg/kg, STA-8666 strongly enhanced the action of carboplatin. Pharmacokinetic profiling confirmed durable STA-8666 exposure in tumors compared with irinotecan. STA-8666 induced a more rapid, robust, and stable induction of cell-cycle arrest, expression of signaling proteins associated with DNA damage and cell-cycle checkpoints, and apoptosis in vitro and in vivo, in comparison with irinotecan. CONCLUSIONS: Together, these results strongly support clinical development of STA-8666 for use in the first- or second-line setting for SCLC. Clin Cancer Res; 22(20); 5120-9. ©2016 AACR.

Interaction with epithelial cells modifies airway macrophage response to ozone.

The initial innate immune response to ozone (O3) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived signals affect Mac response to O3. Macs from the bronchoalveolar lavage (BAL) of healthy volunteers were cocultured with the human bronchial epithelial (16HBE) or alveolar (A549) epithelial cell lines. Cocultures, Mac monocultures, and epithelial cell monocultures were exposed to O3 or air, and Mac immunophenotype, phagocytosis, and cytotoxicity were assessed. Quantities of hyaluronic acid (HA) and IL-8 were compared across cultures and in BAL fluid from healthy volunteers exposed to O3 or air for in vivo confirmation. We show that Macs in coculture had increased markers of alternative activation, enhanced cytotoxicity, and reduced phagocytosis compared with Macs in monoculture that differed based on coculture with A549 or 16HBE. Production of HA by epithelial cell monocultures was not affected by O3, but quantities of HA in the in vitro coculture and BAL fluid from volunteers exposed in vivo were increased with O3 exposure, indicating that O3 exposure impairs Mac regulation of HA. Together, we show epithelial cell-Mac coculture models that have many similarities to the in vivo responses to O3, and demonstrate that epithelial cell-derived signals are important determinants of Mac immunophenotype and response to O3.

Metalation kinetics of the human α-metallothionein†1a fragment is dependent on the fluxional structure of the apo-protein.

Mammalian metallothioneins are cysteine rich metal-binding proteins comprising, when fully metalated, two metal-binding domains: the α-domain binds with M4(II)S(Cys)11 stoichiometry and the ß domain binds as M3(II)S(Cys)9 stoichiometry. While the fully metalated species have been widely studied, the metalation of the apoprotein is poorly understood. Key to a description of the metalation pathway(s) is the initial conformation of the apoprotein and the arrangement of the metal-coordinating cysteines prior to metalation. We report the effect of the ill-defined, globular structure of apoMT on metalation rates. In order to overcome the experimental limitations inherent in structural determinations of a fluxional protein we used a detailed analysis of the apo-α-metallothionein conformation based on the differential rate of cysteine modification with benzoquinone. The ESI mass spectral data show the presence of two distinct conformational families: one a folded conformational family at neutral pH and a second an unfolded family of conformations at low pH. The Cd(II) metalation properties of these two conformationally distinct families were studied using stopped-flow kinetics. Surprisingly, the unfolded apoprotein metalated significantly slower than the folded apoprotein, a result interpreted as being due to the longer time required to fold into the cluster structure when the fourth Cd(II) binds. These results provide the first evidence for the role of the structure of the apo-αMT in the metalation reaction pathways and show that cysteine modification coupled with ESI-MS can be used to probe structure in cysteine-rich proteins.

Progress in assessing air pollutant risks from in vitro exposures: matching ozone dose and effect in human airway cells.

In vitro exposures to air pollutants could, in theory, facilitate a rapid and detailed assessment of molecular mechanisms of toxicity. However, it is difficult to ensure that the dose of a gaseous pollutant to cells in tissue culture is similar to that of the same cells during in vivo exposure of a living person. The goal of the present study was to compare the dose and effect of O3 in airway cells of humans exposed in vivo to that of human cells exposed in vitro. Ten subjects breathed labeled O3 ((18)O3, 0.3 ppm, 2 h) while exercising intermittently. Bronchial brush biopsies and lung lavage fluids were collected 1 h post exposure for in vivo data whereas in vitro data were obtained from primary cultures of human bronchial epithelial cells exposed to 0.25-1.0 ppm (18)O3 for 2 h. The O3 dose to the cells was defined as the level of (18)O incorporation and the O3 effect as the fold increase in expression of inflammatory marker genes (IL-8 and COX-2). Dose and effect in cells removed from in vivo exposed subjects were lower than in cells exposed to the same (18)O3 concentration in vitro suggesting upper airway O3 scrubbing in vivo. Cells collected by lavage as well as previous studies in monkeys show that cells deeper in the lung receive a higher O3 dose than cells in the bronchus. We conclude that the methods used herein show promise for replicating and comparing the in vivo dose and effect of O3 in an in vitro system.

Iron decreases biological effects of ozone exposure.

CONTEXT: Ozone (O3) exposure is associated with a disruption of iron homeostasis and increased availability of this metal which potentially contributes to an oxidative stress and biological effects. OBJECTIVE: We tested the postulate that increased concentrations of iron in cells, an animal model and human subjects would significantly impact the biological effects of O3 exposure. RESULTS: Exposure to 0.4 ppm O3 for 5 h increased mRNA for both Superoxide Dismutase-1 (SOD1) and Cyclooxygenase-2 (COX2) in normal human bronchial epithelial (NHBE) cells. Pre-treatment of NHBE cells with 200 µM ferric ammonium citrate (FAC) for 4 h diminished changes in both SOD1 and COX2 following O3 exposure. mRNA transcript levels and associated protein release of the pro-inflammatory mediators IL-6 and IL-8 were increased by O3 exposure of NHBE cells; changes in these endpoints after O3 exposure were significantly decreased by FAC pre-treatment of the cells. Exposure of CD-1 mice to 2 ppm O3 for 3 h significantly increased lavage indices of inflammation and airflow limitation. Pre-treatment of the animals with pharyngeal aspiration of FAC diminished the same endpoints. Finally, the mean loss of pulmonary function in 19 healthy volunteers exposed to 0.3 ppm O3 for 2 h demonstrated significant correlations with either their pre-exposure plasma ferritin or iron concentrations. DISCUSSION AND CONCLUSION: We conclude that greater availability of iron after O3 exposure does not augment biological effects. On the contrary, increased available iron decreases the biological effects of O3 exposure in cells, animals and humans.

Ozone induces a proinflammatory response in primary human bronchial epithelial cells through mitogen-activated protein kinase activation without nuclear factor-κB activation.

Ground-level ozone (O3) is a ubiquitous environmental air pollutant that is a potent inducer of airway inflammation and has been linked with respiratory and cardiovascular morbidity and mortality. Some studies using transformed or immortalized cells have attributed O3-mediated expression of inflammatory cytokines with activation of the canonical NF-κB pathway. In this study, we sought to characterize the O3-mediated activation of cellular signaling pathways using primary human bronchial epithelial cells obtained from a panel of donors. We demonstrate that the O3-induced expression of proinflammatory cytokines requires the activation of the epidermal growth factor receptor/MEK/ERK and MKK4/p38 mitogen-activated signaling pathways but does not appear to involve activation of canonical NF-κB signaling. In addition to providing a novel mechanistic model for the O3-mediated induction of proinflammatory cytokines, these findings highlight the importance of using primary cells over cell lines in mechanistic studies.

In vitro determinants of asbestos fiber toxicity: effect on the relative toxicity of Libby amphibole in primary human airway epithelial cells.

BACKGROUND: An abnormally high incidence of lung disease has been observed in the residents of Libby, Montana, which has been attributed to occupational and environmental exposure to fibrous amphiboles originating from a nearby contaminated vermiculite mine. The composition of Libby amphibole (LA) is complex and minimal toxicity data are available. In this study, we conduct a comparative particle toxicity analysis of LA compared with standard reference asbestiform amphibole samples. METHODS: Primary human airway epithelial cells (HAEC) were exposed to two different LA samples as well as standard amphibole reference samples. Analysis of the samples included a complete particle size distribution analysis, calculation of surface area by electron microscopy and by gas adsorption and quantification of surface-conjugated iron and hydroxyl radical production by the fibers. Interleukin-8 mRNA levels were quantified by qRT-PCR to measure relative pro-inflammatory response induced in HAEC in response to amphibole fiber exposure. The relative contribution of key physicochemical determinants on the observed pro-inflammatory response were also evaluated. RESULTS: The RTI amosite reference sample contained the longest fibers and demonstrated the greatest potency at increasing IL-8 transcript levels when evaluated on an equal mass basis. The two LA samples and the UICC amosite reference sample consisted of similar particle numbers per milligram as well as similar particle size distributions and induced comparable levels of IL-8 mRNA. A strong correlation was observed between the elongated particle (aspect ratio ≥3:1) dose metrics of length and external surface area. Expression of the IL-8 data with respect to either of these metrics eliminated the differential response between the RTI amosite sample and the other samples that was observed when HAEC were exposed on an equal mass basis. CONCLUSIONS: On an equal mass basis, LA is as potent as the UICC amosite reference sample at inducing a pro-inflammatory response in HAEC but is less potent than the RTI amosite sample. The results of this study show that the particle length and particle surface area are highly correlated metrics that contribute significantly to the toxicological potential of these amphibole samples with respect to the inflammogenic response induced in airway epithelial cells.

Discovery and characterization of a nonphosphorylated cyclic peptide inhibitor of the peptidylprolyl isomerase, Pin1.

Phage panning led to the discovery of a disulfide-cyclized peptide CRYPEVEIC that inhibits Pin1 activity with a K(I) of 0.5 µM. NMR chemical shift perturbation experiments show that cyclic CRYPEVEIC binds to the active site of Pin1. Pin1 residues K63 and R68, which bind the phosphate of substrate peptides, do not show a significant chemical shift change in response to binding of cyclic CRYPEVEIC, consistent with absence of phosphate on the peptide. Cyclic CRYPEVEIC adopts a stable conformation with the side chains of the Y, P, V, and I residues packed together on one side of the ring. Cyclic CRYPEVEIC in solution exists as a mixture of two species, with a 1:4 cis/trans ratio for the Y-P bond. This mixture is stabilized to a single conformation when bound to Pin1. The constrained structure of cyclic CRYPEVEIC apparently facilitates high affinity binding without the presence of a phosphate group.

Effect of size fractionation on the toxicity of amosite and Libby amphibole asbestos.

Abnormally high incidences of asbestos-related pulmonary disease have been reported in residents of Libby, Montana, because of occupational and environmental exposure to asbestos-contaminated vermiculite. The mechanism by which Libby amphibole (LA) causes pulmonary injury is not known. The purpose of this study is to compare the cellular stress responses induced in primary human airway epithelial cells (HAECs) exposed to a respirable size fraction (≤ 2.5 µm) of Libby amphibole (LA(2.5)) to a similar size fraction of a reference amphibole sample amosite (AM(2.5)). HAEC were exposed to 0, 2.64, 13.2, or 26.4 µg/cm(2) AM(2.5) or LA(2.5) or to equivalent doses of unfractionated amosite (AM) or LA for 2 or 24 h. Comparable messenger RNA transcript levels were observed for interleukin-8, cyclooxygenase-2, and heme oxygenase-1 in HAEC following a 24-h exposure to AM or LA. Conversely, exposure to AM(2.5) resulted in a 4- to 10-fold greater induction in these proinflammatory mediators compared with LA(2.5) after 24 h. Evaluation of the expression of 84 additional genes involved in cellular stress and toxicity responses confirmed a more robust response for AM(2.5) compared with LA(2.5) on an equal mass basis. Differences in total surface area (TSA) by gas adsorption, total particle number, or oxidant generation by the size-fractionated particles did not account for the observed difference in response. In summary, AM(2.5) and LA(2.5) are at least as potent in stimulating production of proinflammatory cytokines as unfractionated AM and LA. Interestingly, AM(2.5) was more potent at inducing a proinflammatory response than LA(2.5). This difference could not be explained by differences in mineral contamination between the two samples, TSA, or oxidant generation by the samples.

Metal exchange in metallothioneins: a novel structurally significant Cd(5) species in the alpha domain of human metallothionein 1a.

Metallothioneins (MTs) are cysteine-rich, metal-binding proteins known to provide protection against cadmium toxicity in mammals. Metal exchange of Zn(2+) ions for Cd(2+) ions in metallothioneins is a critical process for which no mechanistic or structural information is currently available. The recombinant human alpha domain of metallothionein isoform 1a, which encompasses the metal-binding cysteines between Cys33 and Cys60 of the alpha domain of native human metallothionein 1a, was studied. Characteristically this fragment coordinates four Cd(2+) ions to the 11 cysteinyl sulfurs, and is shown to bind an additional Cd(2+) ion to form a novel Cd(5)alpha-MT species. This species is proposed here to represent an intermediate in the metal-exchange mechanism. The ESI mass spectrum shows the appearance of charge state peaks corresponding to a Cd(5)alpha species following addition of 5.0 molar equivalents of Cd(2+) to a solution of Cd(4)alpha-MT. Significantly, the structurally sensitive CD spectrum shows a sharp monophasic peak at 254 nm for the Cd(5)alpha species in contrast to the derivative-shaped spectrum of the Cd(4)alpha-MT species, with peak maxima at 260 nm (+) and 240 nm (-), indicating Cd-induced disruption of the exciton coupling between the original four Cd(2+) ions in the Cd(4)alpha species. The (113)Cd chemical shift of the fifth Cd(2+) is significantly shielded (approximately 400 p.p.m.) when compared with the data for the Cd(2+) ions in Cd(4)alpha-MT by both direct and indirect (113)Cd NMR spectroscopy. Three of the four original NMR peaks move significantly upon binding the fifth cadmium. Evidence from indirect (1)H-(113)Cd HSQC NMR spectra suggests that the coordination environment of the additional Cd(2+) is not tetrahedral to four thiolates, as is the case with the four Cd(2+) ions in the Cd(4)alpha-MT, but has two thiolate ligands as part of its ligand environment, with additional coordination to either water or anions in solution.

Peptide folding, metal-binding mechanisms, and binding site structures in metallothioneins.

This minireview specifically focuses on recent studies carried out on structural aspects of metal-free metallothionein (MT), the mechanism of metal binding for copper and arsenic, structural studies using x-ray absorption spectroscopy and molecular mechanics modeling, and speciation studies of a novel cadmium and arsenic binding algal MT. Molecular mechanics-molecular dynamics calculations of apo-MT show that significant secondary structural features are retained by the polypeptide backbone upon sequential removal of the metal ions, which is stabilized by a possible H-bonding network. In addition, the cysteinyl sulfurs were shown to rotate from within the domain core, where they are found in the metallated state, to the exterior surface of the domain, suggesting an explanation for the rapid metallation reactions that were measured. Mixing Cu6beta-MT with Cd4alpha-MT and Cu6alpha-MT with Cd3beta-MT resulted in redistribution of the metal ions to mixed metal species in each domain; however, the Cu+ ions preferentially coordinated to the beta domain in each case. Reaction of As3+ with the individual metal-free beta and alpha domains of MT resulted in three As3+ ions coordinating to each of the domains, respectively, in a proposed distorted trigonal pyramid structure. Kinetic analysis provides parameters that allow simulation of the binding of each of the As3+ ions. X-ray absorption spectroscopy provides detailed information about the coordination environment of the absorbing element. We have combined measurement of x-ray absorption near edge structure (XANES) and extended x-ray absorption fine structure (EXAFS) data with extensive molecular dynamics calculations to determine accurate metal-thiolate structures. Simulation of the XANES data provides a powerful technique for probing the coordination structures of metals in metalloproteins. The metal binding properties of an algal MT, Fucus vesiculosus, has been investigated by UV absorption and circular dichroism spectroscopy and electrospray ionization-mass spectrometry. The 16 cysteine residues of this algal MT were found to coordinate six Cd2+ ions in two domains with stoichiometries of a novel Cd3S7 cluster and a beta-like Cd3S9 cluster.

Metal-dependent protein folding: metallation of metallothionein.

Metallothionein (MT), a low molecular mass, cysteine-rich protein, is a model system for metal ion-directed folding due to its diverse metal binding properties. In this minireview, the current status of theoretical and experimental studies that have focused primarily on the initial metallation steps involving the metal-free, or apo, MT and divalent metals, Zn(2+) and Cd(2+) is described. Apo-MT has recently been reported to be present in the cell in quantities equal to that of the metallated protein, which might indicate a potential role for the protein in the absence of metals. Molecular mechanics-molecular dynamics (MM3/MD) calculations carried out on the demetallation of cadmium-coordinated MT isoform 1a indicate structural stability of the metal-free protein with significant retention of the backbone conformation imposed by the metal-thiolate clusters present in the metallated holo-protein. Significantly, the cysteinyl sulfurs were found inverted to the outside of a quite compact sphere. In contrast, MM3/MD calculations of apo-MT starting from a linear strand did not possess any structural stability and can be described as a random coil conformation. Evidence for the sequence of metallation is discussed, together with current experimental data to support either a cooperative or sequential binding mechanisms.