Proteins In Cell Lysates Research Articles

Combination of Selective Inhibitor of Nuclear Export (SINE) Compounds, Selinexor and KPT-8602, with Venetoclax (ABT-199) Displays Enhanced Activity in Leukemia and Large Cell Lymphoma

Introduction: Exportin 1 (XPO1) is a well characterized and essential nucleo-cytoplasmic transport protein in the karyopherin family, and is responsible for the nuclear export of over 200 cargo proteins, including the major tumor suppressor proteins (TSPs) p53, p21, FOXO and the translation regulator elF4E. XPO1 is overexpressed in numerous cancer types including solid and hematological malignancies, often correlating with poor prognosis. Recently, a novel class of Selective Inhibitors of Nuclear Export (SINE) compounds, selinexor (KPT-330) and the second generation KPT-8602, have been developed for the treatment of advanced cancers. We have previously shown that selinexor has marked activity in AML and DLBCL pre-clinical models.The BCL-2 family of anti-apoptotic proteins are deregulated and linked to maintenance and survival in AML and DLBCL. For its translation, the mRNA for BCL-2 is transported from the nucleus to the cytoplasm by forming a complex with XPO1 cargo, elF4E. Other important mRNAs exported from the nucleus via this mechanism include BCL6 and MYC. We hypothesize that SINE compounds inhibit XPO1/elF4E-mediated nuclear-cytoplasmic transport by covalently binding to the XPO1 cargo binding site and that in the absence of protein translation, BCL-2, BCL6 and MYC levels rapidly decline. Venetoclax (VEN; ABT-199) is a potent, selective inhibitor of BCL-2. In vitro, AML cells acquire resistance to VEN over time, often due to up-regulation of another BCL-2 family anti-apoptotic protein, MCL-1. MCL-1 is regulated by the anti-apoptotic transcription factor and XPO1 cargo NF-kB. We have previously shown that SINE compounds significantly decreased MCL-1 levels, presumably via inactivation of NF-kB.The goal of this study was to test whether SINE compounds will synergize with VEN via BCL-2 modulation and whether the combination would diminish MCL-1 mediated resistance to BCL-2 inhibition in DLBCL and AML models, respectively.Methods: BH3 profiling was performed in a sample of cell lines using a cytochrome c release assay to identify anti-apoptotic dependencies. The effects of SINE compounds and VEN as single agents or in combination on cell viability were performed in AML (K-562, MOLM-13, MV-4-11, and U-937) and DLBCL cell lines (SU-DHL-6, DoHH-2 and Toledo). Whole cell protein lysates were extracted 24 hours after treatment for immunoblot analysis. The activity of SINE compounds (5 mg/kg) and VEN (25 mg/kg) as single agents, or in combination were measured in AML (MV-4-11) and DLBCL (DoHH-2 and Toledo) xenografts in NSGS and nude mice, respectively. Tumor growth and survival were measured throughout these animal studies. Tumor tissue was collected at the end of treatment for flow cytometric analysis, western blotting and immunohistochemistry (IHC).Results: By employing BH3 profiling, we identified AML cell lines that were dependent (MV-4-11 and MOLM-13) and not dependent (U-937 and K-562) on MCL-1. Dose response analysis demonstrated that each of the AML cell lines was sensitive to the SINE compounds, while VEN only reduced viability in the MV-4-11 and MOLM-13 cells. Additionally, there was enhanced growth inhibition when the SINE compounds were combined with VEN in the MCL-1 dependent cells. SINE compound treatment synergistically decreased c-MYC protein levels in all 4 AML cell lines with the combination treatment (Figure 1), whereas PARP cleavage was only enhanced with the combination in the MV-4-11 and MOLM-13 cells. Likewise, MCL-1 is reduced in the presence of SINE compound or SINE compound-VEN combinations. In DLBCL xenograft studies (DoHH-2 and Toledo), combination of selinexor with VEN was synergistic for tumor reduction and increased animal survival when compared to either single agent alone. By IHC we observed a concomitant reduction in BCL-2 and BCL-6 and an increase in cleaved caspase 3 in DLBCL tumors after combination treatment.Conclusions:SINE compound-VEN combinations show enhanced antitumor effect, with reduction of oncogenic activity. SINE compounds reduce MCL-1 in VEN-resistant cells. As MCL-1 driven anti-apoptotic machinery is responsible for resistance to inhibition of BCL-2 in DLBCL and AML, SINE compound regulation of MCL-1 may lead to rescue of VEN resistance. SINE compounds and VEN are excellent candidate partners for combination therapies in AML and DLBCL. [Display omitted] DisclosuresFriedlander:Karyopharm Therapeutics: Employment. Chang:Karyopharm Therapeutics: Employment, Equity Ownership. Kashyap:Karyopharm Therapeutics: Employment, Equity Ownership. Argueta:Karyopharm Therapeutics: Employment, Equity Ownership. Klebanov:Karyopharm Therapeutics: Employment, Equity Ownership. Senapedis:Karyopharm Therapeutics: Employment, Equity Ownership. Baloglu:Karyopharm Therapeutics: Employment, Equity Ownership. Lee:Karyopharm Therapeutics: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Savona:TG Therapeutics: Research Funding; Amgen Inc.: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Sunesis: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Disposable Amperometric Immunosensor for the Determination of Human P53 Protein in Cell Lysates Using Magnetic Micro-Carriers.

An amperometric magnetoimmunosensor for the determination of human p53 protein is described in this work using a sandwich configuration involving the covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic beads (HOOC-MBs) and incubation of the modified MBs with a mixture of the target protein and horseradish peroxidase-labeled antibody (HRP-anti-p53). The resulting modified MBs are captured by a magnet placed under the surface of a disposable carbon screen-printed electrode (SPCE) and the amperometric responses are measured at −0.20 V (vs. an Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as a redox mediator and H2O2 as the enzyme substrate. The magnetoimmunosensing platform was successfully applied for the detection of p53 protein in different cell lysates without any matrix effect after a simple sample dilution. The results correlated accurately with those provided by a commercial ELISA kit, thus confirming the immunosensor as an attractive alternative for rapid and simple determination of this protein using portable and affordable instrumentation.

Discovery of Age-Related Protein Folding Stability Differences in the Mouse Brain Proteome.

Described here is the application of thermodynamic stability measurements to study age-related differences in the folding and stability of proteins in a rodent model of aging. Thermodynamic stability profiles were generated for 809 proteins in brain cell lysates from mice, aged 6 (n = 7) and 18 months (n = 9) using the Stability of Proteins from Rates of Oxidation (SPROX) technique. The biological variability of the protein stability measurements was low and within the experimental error of SPROX. A total of 83 protein hits were detected with age-related stability differences in the brain samples. Remarkably, the large majority of the brain protein hits were destabilized in the old mice, and the hits were enriched in proteins that have slow turnover rates (p < 0.07). Furthermore, 70% of the hits have been previously linked to aging or age-related diseases. These results help validate the use of thermodynamic stability measurements to capture relevant age-related proteomic changes and establish a new biophysical link between these proteins and aging.

A simple enrichment procedure improves detection of membrane proteins by immunoblotting.

We developed a novel approach to improve detection of membrane-associated proteins in yeast cell lysates by immunoblotting. Our method consists of a simple enrichment procedure using sedimentation to remove soluble proteins and the use of an alternative electrophoresis sample buffer, which allows for protein solubilization without heating. The efficacy of this approach was demonstrated for membrane proteins in Hansenula polymorpha (Pho87, Gas1, and Pmr1) and Saccharomyces cerevisiae (Gas1). Immunoblot analysis of proteins that are not membrane-associated showed that the precipitate fraction was depleted of Sup45, carboxypeptidase Y, and Hog1; however, tubulin and, to some extent, Sup35 and Tpd3 were precipitated together with the membrane proteins. The presence of tubulin in the same fraction as the membrane proteins allows its use as a reference protein.

Targeted Mass Spectrometry-Based Approach for Protein-Ligand Binding Analyses in Complex Biological Mixtures Using a Phenacyl Bromide Modification Strategy.

The characterization of protein folding stability changes on the proteomic scale is useful for protein-target discovery and for the characterization of biological states. The Stability of Proteins from Rates of Oxidation (SPROX) technique is one of several mass spectrometry-based techniques recently established for the making proteome-wide measurements of protein folding and stability. A critical part of proteome-wide applications of SPROX is the identification and quantitation of methionine-containing peptides. Demonstrated here is a targeted mass spectrometry-based proteomics strategy for the detection and quantitation of methionine-containing peptides in SPROX experiments. The strategy involves the use of phenacyl bromide (PAB) for the targeted detection and quantitation of methionine-containing peptides in SPROX using selective reaction monitoring (SRM) on a triple quadrupole mass spectrometer (QQQ-MS). As proof-of-principle, the known binding interaction of Cyclosporine A with cyclophilin A protein in a yeast cell lysate is successfully detected and quantified using a targeted SRM workflow. Advantages of the described workflow over other SPROX protocols include a 20-fold reduction in the amount of total protein needed for analysis and the ability to work with the endogenous proteins in a given sample (e.g., stabile isotope labeling with amino acids in cell culture is not necessary).

An OGA-Resistant Probe Allows Specific Visualization and Accurate Identification of O-GlcNAc-Modified Proteins in Cells.

O-linked β-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chemical probes such as GlcNAc or GalNAc analogues bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modification. However, these chemical probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification. We report here the development of a novel probe, 1,3,6-tri-O-acetyl-2-azidoacetamido-2,4-dideoxy-d-glucopyranose (Ac34dGlcNAz), that can be processed by the GalNAc salvage pathway and transferred by O-GlcNAc transferase (OGT) to O-GlcNAc proteins. Due to the absence of a hydroxyl group at C4, this probe is less incorporated into α/β 4-GlcNAc or GalNAc containing glycoconjugates. Furthermore, the O-4dGlcNAz modification was resistant to the hydrolysis of O-GlcNAcase (OGA), which greatly enhanced the efficiency of incorporation for O-GlcNAcylation. Combined with a click reaction, Ac34dGlcNAz allowed the selective visualization of O-GlcNAc in cells and accurate identification of O-GlcNAc-modified proteins with LC-MS/MS. This probe represents a more potent and selective tool in tracking, capturing, and identifying O-GlcNAc-modified proteins in cells and cell lysates.

Abstract P171: Superoxide via Transcription Factor Sp3 Up-regulates Renal Angiotensin II Receptor Function

Oxidative stress is linked to the up-regulation of angiotensin II type 1 receptor (AT1R) function and hypertension. Here we tested the mechanism of oxidative stress-associated up-regulation of AT1 receptor function in human kidney HK2 cells. Diethyldithiocarbamate (DETC), a superoxide dismutase inhibitor, and hydrogen peroxide (H 2 O 2 ) increased DHE and DCFHDA fluorescence respectively [DHE fluorescence (Control vs DETC vs DETC + tempol vs H 2 O 2 : 0.5397 ± 0.07057, 1.463 ± 0.1671, 0.2661 ± 0.01776, 0.6406 ± 0.04821); DCFH fluorescence (Control vs H 2 O 2 vs 3-AT vs DETC: 555.9 ± 21.22, 673.5 ± 37.05, 736.1 ± 33.79, 427.7 ± 22.17)]. DETC increased nuclear levels of Sp3 protein that were attenuated with tempol (Control vs DETC, vs DETC + tempol: 0.50 ± 0.08, 1.28 ± 0.21, 0.52 ± 0.12 densities). However, H 2 O 2 had no significant effect on nuclear Sp3 (Control vs H 2 O 2, vs H 2 O 2 + tempol: Sp3, 0.50 ± 0.08, 0.68 ± 0.14, 1.04 ± 0.30 densities). In transfection studies, Sp3 plasmid increased (Control vs Sp3: 0.1165 ± 0.01, 0.3810 ± 0.03, densities) while Sp3 siRNA decreased (Control siRNA vs Sp3 siRNA: 1.11 ± 0.25, 0.64 ± 0.06, densities) the levels of AT1R protein in cell lysate. DETC increased AT1R protein expression that was attenuated by tempol, measured by immunoblotting (Control vs DETC vs DETC + tempol: 0.3535 ± 0.99, 0.9975 ± 0.19, 0.3250 ± 0.02 densities). Furthermore, DETC increased cell membrane levels of AT1R protein as measured by biotinylation and immunoblotting. This effect was attenuated by tempol. (Control vs DETC vs DETC + tempol: 100 ± 0.0, 189.8 ± 0.34.60, 102.3 ± 10.77 densities, percent change). Moreover, DETC increased PKC activity in response to angiotensin II, an indicator of AT1R function. This effect was attenuated with candesartan and tempol. (Control vs Ang II vs DETC + AngII vs DETC + Cand + AngII vs DETC + tempol: 100.0 ± 0.0, 162.0 ± 12.97, 224.9 ± 52.97, 126.0 ± 6.133, 73.32 ± 35.01 percent change) These results suggest that superoxide but not H 2 O 2 increase nuclear Sp3 protein that up-regulates renal AT1R expression and function.

Clickable 4-Oxo-β-lactam-Based Selective Probing for Human Neutrophil Elastase Related Proteomes.

Human neutrophil elastase (HNE) is a serine protease associated with several inflammatory processes such as chronic obstructive pulmonary disease (COPD). The precise involvement of HNE in COPD and other inflammatory disease mechanisms has yet to be clarified. Herein we report a copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC, or 'click' chemistry) approach based on the 4-oxo-β-lactam warhead that yielded potent HNE inhibitors containing a triazole moiety. The resulting structure-activity relationships set the basis to develop fluorescent and biotinylated activity-based probes as tools for molecular functional analysis. Attaching the tags to the 4-oxo-β-lactam scaffold did not affect HNE inhibitory activity, as revealed by the IC50 values in the nanomolar range (56-118 nm) displayed by the probes. The nitrobenzoxadiazole (NBD)-based probe presented the best binding properties (ligand efficiency (LE)=0.31) combined with an excellent lipophilic ligand efficiency (LLE=4.7). Moreover, the probes showed adequate fluorescence properties, internalization in human neutrophils, and suitable detection of HNE in the presence of a large excess of cell lysate proteins. This allows the development of activity-based probes with promising applications in target validation and identification, as well as diagnostic tools.

Abstract 3872: Quantification of mutant SPOP proteins in prostate cancer using targeted proteomics

Abstract Speckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor protein that functions as a potential tumor suppressor, and SPOP mutations have been identified in ∼10% of human prostate cancers. However, it remains unclear if mutant SPOP proteins can be utilized as biomarkers for diagnosis, prognosis or targeted therapy of prostate cancer. To address this issue, selected reaction monitoring (SRM) and PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) -SRM mass spectrometry assays have been developed for quantifying mutant SPOP proteins. SRM assays for wild-type SPOP protein and 11 prostate cancer-derived mutations were developed. The presence of multiple lysine residues in the mutation regions precludes the use of conventional tryptic digestion. Arg-C was selected instead due to its superior performance in generating mutation site(s) containing SPOP peptides that are more suitable for SRM analysis comparing to other proteases (e.g., Asp-N). Although the resulting Arg-C peptides are longer and more hydrophobic than typical tryptic peptides, all the SRM assays showed a linear dynamic range of more than two orders of magnitude. The limits of quantification for the mutation site(s) containing peptides range from 10 to 100 fmol/μg of total protein in the cell lysate. Applying these SRM assays to analyze 293T cells with and without expression of the three most frequent SPOP mutations in prostate cancer (Y87N, F102C or F133V) led to confident detection of all three SPOP mutations in corresponding positive cell lines but not in the negative cell lines. Expression of the F133V mutation and wild-type SPOP was at much lower levels compared to that of F102C and Y87N mutations, which agrees with RT-PCR results. It is unknown if this is related to activity of the SPOP protein. PRISM-SRM assays have shown further improvement in sensitivity. SRM enables multiplexed, isoform-specific detection of mutant SPOP proteins in cell lysates, which holds great potential in biomarker development for prostate cancer. Citation Format: Hui Wang, Christopher Barbieri, Jintang He, Yuqian Gao, Chaochao Wu, Athena Schepmoes, Thomas Fillmore, Tujin Shi, Sung-Suk Chae, Dennis Huang, Juan Miguel Mosquera, Wei-Jun Qian, Richard Smith, Sudhir Srivastava, Jacob Kagan, David Camp, Karin Rodland, Mark Rubin, Tao Liu. Quantification of mutant SPOP proteins in prostate cancer using targeted proteomics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3872.

Abstract 4552: Profiling signalling protein expression, modifications and interactions with multi-dimensional antibody microarrays

Abstract Antibody microarrays permit sensitive and semi-quantitative analysis of the expression, covalent modification and interactions of proteins in lysates of cells and tissues. At Kinexus, we have developed high content Kinex KAM microarrays that feature nearly 900 pan- and phosphosite-specific antibodies for monitoring protein kinases, phosphatases and other low abundance cell signalling proteins with combinations of different detection systems. One method involved capture of in vitro dye-labeled proteins (e.g. with Cy3) from lysates from cells subjected to diverse treatments. Another method involved the detection of changes in their total phosphorylation with biotinylated pIMAGO stain and an anti-biotin antibody that is labeled with a different dye (e.g. Cy5). Alteration in protein-tyrosine phosphorylation were monitored with a dye-labelled, generic phosphotyrosine-specific PYK antibody in a sandwich antibody microarray (SAM) format. The SAM technique was also used to explore the interactions of adapter, scaffolding and chaperone proteins with hundreds of potential target signal transduction proteins with dye-labeled reporter antibodies for these highly interactive proteins. We used several human cancer cell lines (e.g. A431, HeLa, Jurkat, MCF7) subjected to diverse treatments (e.g. growth factors) to identify biomarkers for the actions of these agents. Reproducible results were obtained with as little as 25 μg of lysate protein, with a dynamic range of detection exceeding 6000-fold, and a median error range for duplicates measurements of ±12%. Typically 10-15% of the proteins tracked with these arrays demonstrated perturbations exceeding 50%. More than a third of the leads from our antibody microarrays were confirmed by immunoblotting studies. The major limitation associated with validation by Western blotting was the much lower sensitivity with immunoblotting compared with antibody microarrays. We also explored the specific interactions of heat shock proteins, adapter proteins, 14-3-3 and calcium-binding proteins with the antibody microarray captured lysate proteins from cancer cell lines. By combining these detection strategies, it was feasible to obtain over 7000 data points from use of a single antibody microarray slide with two lysate samples and duplicate measurements. The goal of our proteomics and bioinformatics studies is to use the experimental results from the application of these microarrays to map the architecture of signalling networks in a cell- or tissue-specific manner. Such multi-tiered microarray-based analyses permit target-directed identification of diverse regulatory protein changes in different experimental model systems with greater sensitivity, breadth, selectivity and economy when compared to any other competing proteomics methodologies. Citation Format: Steven Pelech, Lambert Yue, Jeffrey White, Ryan Hounjet, Dirk Winkler. Profiling signalling protein expression, modifications and interactions with multi-dimensional antibody microarrays. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4552.

Abstract 1299: Selinexor, a selective inhibitor of nuclear export (SINE) compound shows enhanced anti-tumor activity when combined with either venetoclax or bendamustine in diffuse large B cell lymphoma (DLBCL) mouse models

Abstract Introduction: Selinexor (sel) is a small molecule inhibitor of CRM1/XPO1, the primary nuclear exporter of over 200 proteins. As such, it affects multiple cellular pathways and has been shown to be broadly synergistic with various drugs in multiple indications. We have previously shown that sel has marked activity in double-hit DLBCL in pre-clinical models and in a small cohort of patients with heavily pre-treated relapsed / refractory double or triple-hit DLBCL. The goal of this study was to test whether combination of sel with either venetoclax (ven), a selective BCL2 inhibitor or bendamustine (benda), a DNA damaging agent, can further enhance the anti-tumor effect of sel in DLBCL. Methods: The effects of sel, benda and ven as single agents and the effects of sel in combination with either benda or ven on cell viability were tested on DLBCL cell lines including Toledo, DoHH2 and SUDHL6 using MTT assays. Total RNA and whole protein cell lysates were extracted and analyzed by qPCR and by immunoblots, respectively. Mice were implanted with either Toledo or DoHH2 cells. Toledo inoculated mice were treated with either sel or ven alone or in combination and DoHH2 inoculated mice were treated with sel, ven or benda alone or with sel-benda or sel- ven combinations.%Tumor growth inhibition (%TGI) and overall survival were determined. Xenografts were harvested and analyzed by Immunohistochemestry (IHC). Results: Sel-ven and sel-benda were highly effective both in-vitro and in-vivo. Using MTT assay, we showed that each of the drugs have low IC50 values (nanomolar) and they function synergistically/ additively when combined. In-vivo, in the sel-benda model, treatment with each drug showed%TGI of 37% (sel) and 86% (benda) but in combination%TGI was 107%. Western and IHC analyses showed that sel reduces the expression of key DNA Damage Response (DDR) proteins presumably disabling the cells from over coming the damage induced by benda. In the sel-ven model, in both Toledo and DoHH2 mouse models, individual drugs had little effect on%TGI (Toledo: sel 4%, ABT 28%; DoHH2: sel 37%, ABT 24%) but when combined, treatment showed a synergistic effect (Toledo: 109%; DoHH2: 93%). Moreover, combination treatment of Toledo-derived large xenografts resulted in 30% tumor volume shrinkage that was sustained until the end of the study. Interestingly, BCL2 protein levels were reduced by each drug and to a further extent in the combination-treated group suggesting that the synergistic effect is induced via BCL2. Conclusion: Selinexor is an excellent candidate partner for combination therapies in DLBCL. It shows enhanced antitumor effect with both bendamustine and venetoclax modulating DDR and BCL2 pathway activity, respectively. These data provide rational support for study of sel-ven and sel-benda combination in clinical trials. Citation Format: Sivan Elloul, Hua Chang, Boris Klebanov, Trinayan Kashyap, Maxwell Werman, Margaret Lee, Yosef Landesman, Sharon Shacham, Michael Kauffman, Sharon Y. Friedlander. Selinexor, a selective inhibitor of nuclear export (SINE) compound shows enhanced anti-tumor activity when combined with either venetoclax or bendamustine in diffuse large B cell lymphoma (DLBCL) mouse models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1299.

Abstract 4646: Synergistic antitumor effect of selinexor, a selective inhibitor of nuclear export (SINE) compound and trastuzumab in a mouse model of breast cancer

Abstract Introduction: Selinexor (sel) is an oral, first-in-class SINE compound that binds to the primary nuclear exporter XPO1/CRM1. XPO1 exports include major tumor suppressor proteins (TSPs) leading to their inactivation. Inhibition of XPO1 results in nuclear retention of TSPs and restores their normal functions. We have previously shown that HER2+ breast cancer (BC) cells are highly sensitive to sel (SKBR3 and MCF7 IC50 0.21 and 0.073 μM, respectively). Here we tested the hypothesis that inhibition of HER2 signaling with trastuzumab (tras) synergizes with sel as both mediators (PI3K-AKT and Ras-Raf-MEK pathways) and effectors (FOXO3A, MDM2) of the HER2 pathway are among direct cargos of XPO1. Methods: The effects of sel and tras alone or in combination on cell viability were tested on MCF7 and BT474 HER2+ BC cells using MTT assays. Total RNA and whole protein cell lysates were extracted and analyzed by qPCR and by immunoblots. In-vivo, BT474 cells were used to derive a xenograft mouse model. Mice were treated with sub-therapeutic doses of sel and tras alone or in combination and with the therapeutic dose of sel. Tumor Growth (TG) was monitored for 60 days. Xenografts were harvested and analyzed by immunohistochemestry (IHC). Results: Sel-tras combination was highly effective in-vitro and in-vivo. In MTT assays, both sel and tras demonstrated low IC50 values (nanomolar) and when combined, they synergistically inhibit proliferation. In-vivo, the combination resulted in significant survival benefit and enhanced TG inhibition of 88% compared to the monotherapy groups 57% (sel) and 25% (tras). Interestingly, an increase in pro-survival cytoplasmic FOXO3A protein was observed in the tras-treated group however, in the combination group, FOXO3A, which is an XPO1cargo, was restricted to the nucleus where it can trigger apoptosis. In addition, a synergistic increase in P27 protein, the G1 phase arrest regulator, was observed in the sel-tras-treated group. While in the monotherapy groups p-ERK was not modulated, in the combination group an increase in cytoplasmic p-ERK was observed, suggesting an affect on its downstream regulators of apoptosis. Indeed, apoptosis was evident in the combination samples by increased IHC staining to Cleaved caspase3 and Apoptag. In addition, an increase in PARP protein was observed in the tras-treated group however, in the combination group, PARP was dramatically reduced. We have previously shown that selinxor modulates DNA Damage Response (DDR) proteins suggesting that one mechanism by which the combination enhances cancer cell death is by accumulation of DNA damage that cannot be resolved. Conclusion: Sel-tras combination is a novel candidate therapy to HER2+ BC modulating DDR and apoptosis. It synergizes to induce survival benefit and TG inhibition. These data provide rational support for study of sel-tras combination in clinical trials. Citation Format: Sivan Elloul, Hua Hua Chang, Boris Klebanov, Trinayan Kashyap, Maxwell Werman, Margaret Lee, Yosef Landesman, Sharon Shacham, Michael Kauffman, Sharon Y. Friedlander. Synergistic antitumor effect of selinexor, a selective inhibitor of nuclear export (SINE) compound and trastuzumab in a mouse model of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4646.

Abstract 2481: Targeting XPO1 overexpression with selinexor disrupts the survivin pathway in neuroblastoma

Abstract Background: Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and accounts for a disproportionately high fraction (12%) of childhood cancer deaths. More than half of patients with high-risk NB will not survive their disease despite intensive treatment. Therefore new therapies that rationally target unique vulnerabilities in NB are urgently needed. The nuclear export protein Exportin-1 (XPO1) is more abundant in primary refractory NB (PRN), the most aggressive form of disease, making it an attractive therapeutic target. Overexpression of XPO1 has been implicated in tumor development and progression of multiple cancers. Selinexor (KPT-330, Karyopharm Therapeutics) targets XPO1 and inhibits its normal function of translocating tumor suppressor and growth regulatory proteins out of the nucleus. Inhibition of XPO1 results in forced nuclear retention of such proteins, which decreases cellular proliferation. Selinexor has demonstrated efficacy across a range of adult malignancies and is being developed for childhood cancer clinical studies. In this study we look at the effects of selinexor treatment in NB, specifically looking at proteins involved in apoptosis. Methods: Whole protein cell lysates were extracted from NB cell lines IMR5 and NB-EBC1 after treatment with selinexor. Cells were treated across a range of concentrations (100nM-1000nM) and evaluated at three time points (24, 48, and 72 hours). Cytotoxic effects were measured by MTT proliferation assays.. Expression of XPO1, nuclear exported proteins, and downstream targets of the Survivin pathway were evaluated by immunoblots. Results: Proliferation defects were observed in all NB cell lines treated with selinexor as compared to vehicle (DMSO) control. Expression of XPO1 was decreased in treated cells after 24, 48, and 72 hours of drug exposure. Expression of the anti-apoptotic protein Survivin was also decreased, as were STAT3 and acetylated STAT3. Cleaved Caspase-3 levels were increased and associated with apoptosis and decreased cellular proliferation. Conclusions: This study provides a mechanism for XPO1 overexpression contributing to tumor progression in highly aggressive NB and explores its potential as a prognostic biological marker and therapeutic target in PRN. Treatment of NB cell lines with selinexor leads to apoptosis and associated decreased cellular proliferation. Inhibition of XPO1 function may have implications aside from nuclear export. Survivin, a pro-apoptotic protein, is decreased in the presence of selinexor, and downstream targets in the Survivin pathway are also disrupted. These data provide additional rationale for clinical development of selinexor for pediatric solid tumors, and particularly for diseases such as PRN where XPO1 is known to be overabundant. This work also suggests rational strategies for the optimal combinatorial approaches using selinexor for refractory neuroblastoma. Citation Format: Raquel Castellanos, Basia Galinski, David Tauber, Yosef Landesman, Edward Attiyeh, Daniel Weiser. Targeting XPO1 overexpression with selinexor disrupts the survivin pathway in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2481.

Abstract 2481A: A novel chemically-modified curcumin (CMC 2.24) promotes chemosensitivity in neuroblastoma

Abstract Introduction The chemically modified curcumin, CMC 2.24 (TRB-N0224, Traverse Biosciences Inc.), was designed to overcome the poor solubility, bioavailability, and biological potency of naturally occurring curcumin in an effort to achieve superior safety and efficacy in chronic inflammatory conditions and cancer. It has been demonstrated that CMC 2.24 can sensitize cancer cells that are otherwise refractory to chemotherapy. Patients with neuroblastoma, a highly lethal childhood cancer of the peripheral sympathetic nervous system, often have de novo chemotherapy resistance and therefore succumb rapidly to their disease. We hypothesize that chemoresistance in primary refractory neuroblastoma is mediated by anti-apoptotic mechanisms that can be overcome when conventional therapy is combined with CMC 2.24. Experimental Procedures A heterogeneous panel of neuroblastoma cell lines was cultured under routine conditions and subjected to varying concentrations of CMC 2.24 to establish IC50s. Subsequent studies were focused on NB-EBc1, a MYCN non-amplified cell line, and IMR-5, a MYCN amplified cell line. IC50s were also generated for cisplatin as a single agent, and then when administered in combination with CMC 2.24. Whole protein cell lysates were extracted at 24, 48, and 72 hours after treatment and cell viability was measured by MTT proliferation assays. Results Proliferation defects were observed in all neuroblastoma cell lines treated with CMC 2.24 as compared to vehicle (DMSO) control. Relatively cisplatin-resistant cell lines had diminished cell viability when CMC 2.24 was added in combination with cisplatin, suggesting a synergistic effect. Alterations in response to CMC 2.24 treatment occur in anti-apoptotic proteins survivin and BARD1, found to be upregulated in chemoresistant cell lines. Additional mechanistic evaluation is ongoing. Conclusions There is no known curative therapy for patients with primary refractory neuroblastoma. CMC 2.24 enhances chemosensitivity in a panel of neuroblastoma cell lines and may provide a new therapeutic approach that can overcome de novo chemotherapy resistance. Further mechanistic and in vivo studies are needed to facilitate clinical development of a promising novel anti-cancer agent. Citation Format: David Tauber, Basia Galinski, Raquel Castellanos, Joseph Scaduto, Francis Johnson, Lorne Golub, Daniel A. Weiser. A novel chemically-modified curcumin (CMC 2.24) promotes chemosensitivity in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2481A.

Abstract 4720: Synergistic antitumor effect of selinexor, a selective inhibitor of nuclear export (SINE) compound and panobinostat in a mouse model of multiple myeloma

Abstract Introduction: Selinexor is an oral, first-in-class SINE compound that binds to the primary nuclear exporter XPO1/CRM1. XPO1 exports over 200 cargos, including major tumor suppressor proteins (TSPs) leading to their inactivation. Inhibition of XPO1 results in nuclear retention of TSPs and restores their normal functions. XPO1 also mediates the export of key signaling molecules in multiple myeloma (MM) including c-MYC mRNA and NF-κB. Selinexor is currently being investigated in phase 2 clinical trials in MM in combination with dexamethasone, pomalidamide, lenalidomide, bortezomib and carfilzomib. In preclinical studies selinexor has been shown to synergize broadly with these anti-MM drugs making it an excellent candidate partner for combination therapies in MM. To identify additional synergistic pairings, we investigated the use of selinexor in combination with panobinostat, a pan-histone deacetylase inhibitor (HDAC) recently approved by the FDA in combination with bortezomib for 3rd line treatment of MM, as a potential treatment for MM both in-vitro and in-vivo. Methods: The effects of selinexor and panobinostat alone or in combination on cell viability were tested on MM1.S and NCI-H929 cell lines using MTT assays. Total RNA and whole protein cell lysates were extracted and analyzed by qPCR and by immunoblots. In-vivo, MM.1S cells were used to derive a xenograft mouse model. Mice were treated with sub-therapeutic doses of selinexor and panobinostat alone or in combination and with the therapeutic dose of selinexor. Tumor growth was monitored for 17 days and% Tumor Growth Inhibition (%TGI) was determined. Xenografts were harvested and analyzed microscopically and by immunohistochemestry (IHC). Results: Selinexor-panobinostat combination was highly effective both in-vitro and in-vivo. In MTT assays, both selinexor and panobinostat demonstrated low IC50 values (nanomolar) and when combined, they synergistically/ additively inhibit proliferation. Gene expression and western blot analyses showed that the combination treatment leads to a synergistic reduction in c-MYC mRNA and protein levels. In addition, an overall reduction in expression of anti-apoptotic signaling molecules including NF-κB and p21 and an increase of expression of pro-apoptotic molecules including cleaved caspase 3 and BAX were observed. Importantly, in-vivo, while%TGI of sub-therapeutic doses of selinexor and panobinostat measured 58% and 52% respectively, the combination showed a synergistic effect, measuring 93%. Importantly this high%TGI exceeded the effect of the therapeutic dose of selinexor alone 79%. Conclusion: Selinexor-panobinostat combination synergizes to induce apoptosis in MM cells and amplifies anti-tumor effect in a MM xenograft model. These data provide rational support for study of selinexor/ panobinostat combination in clinical trials. Citation Format: Sivan Elloul, Hua Chang, Boris Klebanov, Trinayan Kashyap, Maxwell Werman, Margaret Lee, Yosef Landesman, Sharon Shacham, Michael Kauffman, Sharon Y. Friedlander. Synergistic antitumor effect of selinexor, a selective inhibitor of nuclear export (SINE) compound and panobinostat in a mouse model of multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4720.

Abstract 3895: Immunoproteomic profiling in African American men with prostate cancer: Evidence for an autoimmune response to glycolytic enzymes

Abstract Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer-related male deaths in the U.S. This cancer is more aggressive in African American (AA) men, who are twice as likely to die from the disease when compared to other racial groups. Despite these racial disparities in PCa mortality, studies on PCa biology and biomarker discovery include mostly patients from European American (EA) backgrounds. There is a critical need to find minimally invasive PCa biomarkers that could aid in diagnosis and prognosis, especially in high risk populations like AA men. Immunoproteomics offers a minimally invasive approach to detect early malignant processes that trigger production of autoantibodies to tumor-associated antigens (TAA). We used serum samples from AA (n = 59) and EA (n = 50) men with PCa to probe one- and two-dimensional Western blots of protein lysates from aggressive PCa cell lines. AA PCa patient sera showed stronger immunoreactivity to proteins in PC3 cell lysates compared to the EA sera, with a 50 kD protein band frequently targeted by autoantibodies in several AA PCa sera. Serological proteomic analysis with mass spectrometry showed that these AA PCa autoantibodies recognized alpha-enolase (ENO1), a protein important in tumor formation, expansion, and glucose metabolism. In addition, several other glycolytic enzymes important for tumor proliferation were also identified as candidate prostate TAA, including GAPDH, bisphosphate aldolase, phosphoglycerate kinase, and lactate dehydrogenase. The ENO1 autoantibody frequency, measured by ELISA in sera from 400 racially diverse men with and without PCa, was four-fold higher in PCa compared to non-PCa patients. Interestingly, although sera from AA men with PCa had stronger WB reactivity to ENO1 present in PC3 lysates compared to EA men, and the ENO1 autoantibodies were initially discovered in eight AA PCa sera but only in two EA sera, the anti-ENO1 autoantibody frequency measured by ELISA, which used yeast-purified recombinant human ENO1 as a substrate, was higher in the EA PCa cohort. In addition, several anti-ENO1 positive sera from AA men with PCa showed increased immunoreactivity to this protein in the PCa cell lines LNCaP, MDA-PC2b, PC3, and DU145, but not against the purified ENO1 used in ELISA. The opposite was true in the EA PCa cohort, where strong reactivity against the purified ENO1 did not correlate with an equally strong immunoreactivity against the cellular ENO1. The EA PCa sera did not recognize the pattern of ENO1 expression observed with the AA sera in the same panel of prostate cell lines. These differences of immunoreactivity between AA and EA sera against PC3-ENO1 vs recombinant human ENO1 suggest that select AA sera may predominantly recognize ENO1 epitopes not displayed in the purified protein. These discrepancies point to racial differences in the immune response to prostate tumors. Citation Format: Tino Wilson Sanchez, Jian-Ying Zhang, Liping Dai, Susanne Montgomery, Colwick Wilson, Guangyu Zhang, Saied Mirshahidi, Nathan Wall, Carlos A. Casiano. Immunoproteomic profiling in African American men with prostate cancer: Evidence for an autoimmune response to glycolytic enzymes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3895.

Abstract 444: Hyaluronan-mediated motility receptor (RHAMM) in ovarian cancer

Abstract This study focuses on Hyaluronan Mediated Motility Receptor (RHAMM) protein expression in ovarian carcinoma (OC). RHAMM protein increases migration, invasion and is associated with poor prognosis when overexpressed. It is a dualistic oncogenic protein located within and on the cell surface acting as a receptor for Hyaluronan (HA). RHAMM is not generally overexpressed in normal tissues, but is upregulated in a number of cancers and is associated with poor clinical outcome. However, the role of RHAMM in OC progression is unknown. Hypothesis: Elevated RHAMM levels promote OC progression and represent a novel urinary biomarker for OC. Procedures: Measuring RHAMM expression in OC cell protein lysate and conditioned media (CM) was done by western blot (WB) and densitometry. With institution approval, patient cohort urine from male and female healthy controls and patients with cancer of the ovary, lung, breast, prostate, cervix, colorectum, skin, endometrium, head and neck, and brain were acquired from the H. Lee Moffit Cancer Center at the University of South Florida. Enzyme-linked immunosorbent assay (ELISA) designed for RHAMM detection, was used to measure patient urine expression levels. Results: Cultured OC cells overexpress and secrete RHAMM. Using OC, SV-40 large T-antigen transfected human ovarian surface epithelial (IOSE) cells (HIO118, IOSE-121) and human dermal fibroblasts (HDF), we found &amp;gt;2x the expression of RHAMM protein in BC (MCF7) and OC (OVCAR5, OV2008, C13) cells by WB. WB of CM indicated &amp;gt;3x RHAMM levels secreted by OC compared to IOSE cells. Urinary levels of RHAMM are elevated in OC patients. Since RHAMM is not an integral membrane protein, but rather loosely tethered to the plasma membrane, we determined whether RHAMM could be detected in bodily fluids of OC patients. ELISA showed detection of urinary RHAMM protein levels averaged 0.140 ng/ml (n = 26) in healthy patient controls whereas OC patients averaged 0.411 ng/ml (n = 132). Conclusions: Prior to this study, overexpression and secretion of RHAMM by OC cells had not been reported. This has expanded our knowledge of OC etiology by delineating a functional role for RHAMM-driven OC progression. Validating elevated urinary levels of RHAMM suggests it is a potential diagnostic marker alone or in combination with other markers for OC detection. Further, the ability to monitor OC through the course of disease using urinary RHAMM levels could indicate recurrent disease or therapeutic efficacy. Understanding RHAMM expression in OC may lead to a possible diagnostic/prognostic indicator or therapeutic target for OC. Citation Format: Stephanie Buttermore, Patricia Kruk. Hyaluronan-mediated motility receptor (RHAMM) in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 444.

Two-dimensional blue native/SDS-PAGE analysis of whole cell lysate protein complexes of rice in response to salt stress

To understand the biology of a plant in response to stress, insight into protein-protein interactions, which almost define cell behavior, is thought to be crucial. Here, we provide a comparative complexomics analysis of leaf whole cell lysate of two rice genotypes with contrasting responses to salt using two-dimensional blue native/SDS-PAGE (2D-BN/SDS-PAGE). We aimed to identify changes in subunit composition and stoichiometry of protein complexes elicited by salt. Using mild detergent for protein complex solubilization, we were able to identify 9 protein assemblies as hetero-oligomeric and 30 as homo-oligomeric complexes. A total of 20 proteins were identified as monomers in the 2D-BN/SDS-PAGE gels. In addition to identifying known protein complexes that confirm the technical validity of our analysis, we were also able to discover novel protein-protein interactions. Interestingly, an interaction was detected for glycolytic enzymes enolase (ENO1) and triosephosphate isomerase (TPI) and also for a chlorophyll a-b binding protein and RuBisCo small subunit. To show changes in subunit composition and stoichiometry of protein assemblies during salt stress, the differential abundance of interacting proteins was compared between salt-treated and control plants. A detailed exploration of some of the protein complexes provided novel insight into the function, composition, stoichiometry and dynamics of known and previously uncharacterized protein complexes in response to salt stress.

Direct Tracking of Amyloid and Tu Dynamics in Neuroblastoma Cells Using Nanoplasmonic Fiber Tip Probes.

Amyloid plaques and neurofibrillary tangles are the pathological hallmarks of Alzheimer's disease. However, there has been a long-standing discussion on the dynamic relations between Aβ and tau proteins, partially due to the lack of a tool to track protein dynamics in individual live neurons at the early stage of Aβ generation and tau phosphorylation. Here, we developed nanoplasmonic fiber tip probe (nFTP) technology to simultaneously monitor Aβ42 generation and tau phosphorylation (at serine 262) in living, single neuroblastoma cells over 12 h. We observed that Aβ42 generation, under clinically relevant anesthetic treatment, preceded tau phosphorylation, which then facilitated Aβ42 generation. This observation is also supported by measuring proteins in cell lysates using the ultrasensitive label-free photonic crystal nanosensors. nFTP therefore provides an advanced method to investigate protein expression and post-translational modification in live cells and determine outcomes of intervention of Alzheimer's disease and other neurodegenerative disorders.

Comparative proteomic analysis of chicken macrophages stimulated with Salmonella Lipopolysaccharide (LPS) and Monosodium Urate (MSU) Crystals

Abstract Immune system cells are armed with recognition receptors by means of which they can sense pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). Interaction of pathogen as well as damage associated ligands with their cognate receptors drives transcriptional and translational changes. To study the proteomic differences between signal transduction pathways involved in microbial induced and sterile injury, we used chicken macrophages HTC as a model system. Cells were labeled and treated with LPS or MSU by using SILAC proteomic approach. After 20 hrs of stimulation, the protein was extracted from secretome and cell lysate, quantified and subjected to in-solution digestion followed by liquid chromatography/tandem mass spectrometry. We quantified 150 proteins using Skyline software, 30 were upregulated and 18 down regulated in LPS treated cells and 40 upregulated and 11 down regulated in MSU treated cells. Interleukin 8, a secretory protein is differentially upregulated in LPS group. Cathepsin K, vimentin, kinases are some of the significantly upregulated cell lysate proteins in LPS group. Lactate dehydrogenase, heat shock, microtubule, annexin are significantly upregulated in MSU stimulated cell lysate. Functional annotation of proteins in both the groups by gene ontology software shows most of the proteins are involved in cytoskeleton remodeling, cell migration and cell signaling, which may be important in immune activation except nucleic acid binding transcription activity factor proteins which are uniquely expressed in LPS group. In conclusion our study gives an insight about the functional differences between pathogen and sterile associated inflammation at cellular level.