Cell Receptor Function Research Articles

Abstract B119: Blocking metabolic stress response with genetic knockdown and selective ligands of sigma-1 receptor in cancer cells

Abstract There is increasing interest in the biological pathways that determine the ability of cancer cells to adapt and survive to metabolic and micro-environmental stress and sustain tumor-initiating and metastatic properties. A better understanding of these pathways may lead to the identification of key nodes and targetable elements for development novel therapeutic strategies for cancer. The sigma-1 receptor (Sig1R) is a ubiquitously expressed membrane-bound protein that acts as ligand-activated molecular chaperone. Sig1R is localized preferentially at the endoplasmic reticulum (ER) and the mitochondria-associated ER membrane (MAM) domains and controls calcium signaling between ER and mitochondria in response to stress signals. Sig1R is often up-regulated in cancer cell lines and human tumors suggesting that it might have a role in tumorigenesis, although detailed functional studies are missing. In this study we examined whether Sig1R sustains proliferation, survival and tumorigenic properties of human cancer cells. Knockdown of Sig1R using small interfering RNA (siRNA) in human prostate and lung cancer cell lines had profound effect on proliferation, clonogenic capability and tumor-sphere formation, indicating reversal of the tumorigenic and stem-like phenotype in absence of Sig1R. Next, in the attempt to discover pharmacological agents that could phenocopy the effects of the genetic knockdown in cancer cells we tested a series of structurally diverse Sig1R ligands selected for high affinity and selectivity for the receptor. We identified various Sig1R ligands that behaved as antagonists of the receptor functions in cancer cells inhibiting clonogenicity and tumor-sphere formation. Effective concentrations of the ligands were in the micromolar range (1-10 μM). At these doses, Sig1R antagonistic ligands almost completely suppressed clonogenic and tumor sphere forming capability of cancer cells. Furthermore, whereas cell proliferation and viability under standard culture conditions were minimally affected by Sig1R ligands, their effects were more pronounced under glucose starvation, a condition that causes metabolic stress in cancer cells. Notably, both Sig1R knockdown and pharmacological antagonists led to impaired mitochondrial function, which was more evident under glucose starvation. Thus, the absence of functional Sig1R reduced mitochondrial activity and adaptability of cancer cells to metabolic stress. These results indicate that the presence of functional Sig1R sustains tumorigenic and stem-like properties by enhancing the metabolic flexibility of cancer cells through the control of ER-mitochondria functions. Highly selective Sig1R antagonists are promising leads for discovering innovative therapeutic strategies and represent effective candidates for pharmacological targeting of stem-like and tumor-initiating cells in human cancers. Citation Format: Gianluca Civenni, Celeste De Monte, Federica Sereni, Sara Allegrini, Roberto Bosotti, Erik Laurini, Bernhard Wunsch, Sabrina Pricl, Giuseppina M. Carbone, Carlo V. Catapano. Blocking metabolic stress response with genetic knockdown and selective ligands of sigma-1 receptor in cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B119.

Loss and Gain of Natural Killer Cell Receptor Function in an African Hunter-Gatherer Population.

Modulating natural killer cell functions in human immunity and reproduction are diverse interactions between the killer cell immunoglobulin-like receptors (KIR) of Natural Killer (NK) cells and HLA class I ligands on the surface of tissue cells. Dominant interactions are between KIR2DL1 and the C2 epitope of HLA-C and between KIR2DL2/3 and the C1 epitope of HLA-C. KhoeSan hunter-gatherers of Southern Africa represent the earliest population divergence known and are the most genetically diverse indigenous people, qualities reflected in their KIR and HLA genes. Of the ten KhoeSan KIR2DL1 alleles, KIR2DL1*022 and KIR2DL1*026 likely originated in the KhoeSan, and later were transmitted at low frequency to the neighboring Zulus through gene flow. These alleles arose by point mutation from other KhoeSan KIR2DL1 alleles that are more widespread globally. Mutation of KIR2DL1*001 gave rise to KIR2DL1*022, causing loss of C2 recognition and gain of C1 recognition. This makes KIR2DL1*022 a more avid and specific C1 receptor than any KIR2DL2/3 allotype. Mutation of KIR2DL1*012 gave rise to KIR2DL1*026, causing premature termination of translation at the end of the transmembrane domain. This makes KIR2DL1*026 a membrane-associated receptor that lacks both a cytoplasmic tail and signaling function. At higher frequencies than their parental allotypes, the combined effect of the KhoeSan-specific KIR2DL1*022 and KIR2DL1*026 is to reduce the frequency of strong inhibitory C2 receptors and increase the frequency of strong inhibitory C1 receptors. Because interaction of KIR2DL1 with C2 is associated with risk of pregnancy disorder, these functional changes are potentially advantageous. Whereas all other KhoeSan KIR2DL1 alleles are present on a wide diversity of centromeric KIR haplotypes, KIR2DL1*026 is present on a single KIR haplotype and KIR2DL1*022 is present on two very similar haplotypes. The high linkage disequilibrium across their haplotypes is consistent with a recent emergence for these KIR2DL1 alleles that have distinctive functions.

Suppressive effect of membrane-permeable peptides derived from autophosphorylation sites of the IGF-1 receptor on breast cancer cells

Insulin-like growth factor-1 (IGF-1) receptors play a crucial role in the biology of human cancer, making them an attractive target for anti-cancer agents. We previously designed oligopeptides containing the amino-acid sequences surrounding the autophosphorylation sites of the insulin receptor and found that two of them, namely, Ac-DIYET-NH2 and Ac-DYYRK-NH2, suppressed phosphorylation of purified insulin receptors in a non-ATP-competitive manner, whereas Ac-NIYQT-NH2 and Ac-NYYRK-NH2 suppressed in an ATP-competitive manner. Because the IGF-1 receptor is closely related to the insulin receptor, the aim of this study was to observe the effects of these peptides, which correspond to the amino-acid sequences of the autophosphorylation sites of the IGF-1 receptor, on the activity of the human breast cancer cell lines MCF-7, T47D, MDA-MB-231, and MDA-MB-453. To facilitate peptide delivery into breast cancer cells, the cell-penetrating peptide, human immunodeficiency virus type 1-transactivator of transcription (Tat), was linked to these peptides. When breast cancer cells were treated with each of these synthetic Tat-conjugated peptides, the conjugated peptides penetrated into the cells and suppressed cell proliferation. An inhibitory effect of Tat-conjugated peptides against IGF-1-stimulated phosphorylation of IGF-1 receptors was observed. In addition, we found that combinations of these peptides suppressed phosphorylation of IGF-1 receptors to a greater extent than the peptides did individually. In conclusion, IGF-1 receptor autophosphorylation site-derived membrane-permeable peptides have the potential to suppress IGF-1 receptor function in breast cancer cells and to be developed into novel and useful agents for cancer therapy.

Abstract 1043: Blocking constitutive aryl hydrocarbon receptor (AhR) signaling attenuates androgen receptor activity

Abstract PURPOSE: The aryl hydrocarbon receptor (AhR) is a member of the basic-helix-loop-helix family of transcription factors. AhR is widely known for regulating the transcription of drug metabolizing enzymes involved in the xenobiotic metabolism of carcinogens and therapeutic agents, such as cytochrome P450-1B1 (CYP1B1). Additionally, AhR has also been reported to interact with multiple signaling pathways during prostate development. Here we investigate the effect of sustained AhR signaling on androgen receptor function in prostate cancer cells. METHODS/RESULTS: Immunoblot analysis shows that AhR expression is increased in androgen independent (C4-2) prostate cancer cells when compared to androgen sensitive (LNCaP) cells. qRT-PCR studies revealed constitutive AhR signaling in C4-2 cells without the ligand induced activation required in LNCaP cells. A reduction of AhR activity by short RNA mediated silencing in C4-2 cells reduced expression of both AhR and androgen responsive genes. The decrease in androgen responsive genes correlates to a decrease in phosphorylated androgen receptor and androgen receptor expression in the nucleus. CONCLUSION: These data indicates that AhR is required to maintain hormone independent signaling by the androgen receptor in C4-2 cells. Collectively, these data provide evidence of a direct role for AhR in androgen independent signaling and provides insight into the molecular mechanisms responsible for sustained androgen receptor signaling in hormone refractory prostate cancer. These studies were supported by the RCMI grant 2G12RR003062-22. Citation Format: Maryam Ghotbaddini, Aaron Hemphill, Joann B. Powell. Blocking constitutive aryl hydrocarbon receptor (AhR) signaling attenuates androgen receptor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1043. doi:10.1158/1538-7445.AM2015-1043

Toll-like receptor 9 dependent interferon-α release is impaired in severe asthma but is not associated with exacerbation frequency

Patients with asthma and chronic obstructive pulmonary disease (COPD) are susceptible to exacerbations, often caused by microbial pathogens. We hypothesised that intracellular Toll-like receptor (TLR) function in blood mononuclear cells (PBMCs) from these subjects would be impaired and that this impairment is related to exacerbation frequency. PBMCs stimulated with a TLR-9 agonist (but not TLR-3 or 7/8) produced significantly less IFN-α in asthma (26 [3–696]pg/ml) compared to control (943 [164–1651]) and COPD (597 [127–1186]) subjects (p=0.0019) but this was not related to the number of exacerbations per year in asthma or COPD. In COPD, IFN-α levels were related to KCO (% predicted) in COPD (r=−0.41, p=0.01). IFN-α was derived from plasmacytoid dendritic cells (pDCs) and their frequency was lower in asthma compared to control subjects (control 0.48% [0.33–0.64] versus asthma 0.29% [0.13–0.34], p=0.019) whereas pDC function per se was not significantly impaired between groups. The mechanism underlying reduced IFN-α production and the clinical consequences in severe asthma remains to be established.

Direct myocardial ischemia imaging: a new cardiovascular nuclear imaging paradigm.

Myocardial perfusion imaging (MPI), using radiotracers, has been in routine clinical use for over 40 years. This modality is used for the detection of coronary artery disease (CAD), risk stratification, optimizing therapy, and follow-up of patients with CAD. Molecular cardiovascular imaging using targeted radiotracers provides a unique opportunity for imaging biochemical and metabolic processes, and cell membrane transporter and receptor functions at a cellular and molecular level in experimental animal models as well as in humans. Cardiac imaging using radiolabeled free fatty acid analogues and glucose analogues enable us to image myocardial ischemia directly as an alternative to stress-rest MPI. Direct ischemia imaging techniques can avoid and overcome some of the limitations of standard stress-rest MPI. This article describes recent studies using (18) F-fluorodeoxyglucose ((18) FDG) for myocardial ischemia imaging.

Identification of seven genes essential for male fertility through a genome-wide association study of non-obstructive azoospermia and RNA interference-mediated large-scale functional screening in Drosophila.

Non-obstructive azoospermia (NOA) is a complex and severe condition whose etiology remains largely unknown. In a genome-wide association study (GWAS) of NOA in Chinese men, few loci reached genome-wide significance, although this might be a result of genetic heterogeneity. Single nucleotide polymorphisms (SNPs) without genome-wide significance may also indicate genes that are essential for fertility, and multiple stage validation can lead to false-negative results. To perform large-scale functional screening of the genes surrounding these SNPs, we used in vivo RNA interference (RNAi) in Drosophila, which has a short maturation cycle and is suitable for high-throughput analysis. The analysis found that 7 (31.8%) of the 22 analyzed orthologous Drosophila genes were essential for male fertility. These genes corresponded to nine loci. Of these genes, leukocyte-antigen-related-like (Lar) is primarily required in germ cells to sustain spermatogenesis, whereas CG12404, doublesex-Mab-related 11E (dmrt11E), CG6769, estrogen-related receptor (ERR) and sulfateless (sfl) function in somatic cells. Interestingly, ERR and sfl are also required for testis morphogenesis. Our study thus demonstrates that SNPs without genome-wide significance in GWAS may also provide clues to disease-related genes and therefore warrant functional analysis.

Quantitative evaluation of the reticuloendothelial system function with dynamic MRI.

PurposeTo evaluate the reticuloendothelial system (RES) function by real-time imaging blood clearance as well as hepatic uptake of superparamagnetic iron oxide nanoparticle (SPIO) using dynamic magnetic resonance imaging (MRI) with two-compartment pharmacokinetic modeling.Materials and MethodsKinetics of blood clearance and hepatic accumulation were recorded in young adult male 01b74 athymic nude mice by dynamic T2* weighted MRI after the injection of different doses of SPIO nanoparticles (0.5, 3 or 10 mg Fe/kg). Association parameter, Kin, dissociation parameter, Kout, and elimination constant, Ke, derived from dynamic data with two-compartment model, were used to describe active binding to Kupffer cells and extrahepatic clearance. The clodrosome and liposome were utilized to deplete macrophages and block the RES function to evaluate the capability of the kinetic parameters for investigation of macrophage function and density.ResultsThe two-compartment model provided a good description for all data and showed a low sum squared residual for all mice (0.27±0.03). A lower Kin, a lower Kout and a lower Ke were found after clodrosome treatment, whereas a lower Kin, a higher Kout and a lower Ke were observed after liposome treatment in comparison to saline treatment (P<0.005).ConclusionDynamic SPIO-enhanced MR imaging with two-compartment modeling can provide information on RES function on both a cell number and receptor function level.

Carbohydrate-mediated modulation of NK cell receptor function: structural and functional influences of heparan sulfate moieties expressed on NK cell surface.

OPINION article Front. Oncol., 16 July 2014Sec. Molecular and Cellular Oncology Volume 4 - 2014 | https://doi.org/10.3389/fonc.2014.00185

DNA-scaffolded multivalent ligands to modulate cell function.

We report a simple, versatile, multivalent ligand system that is capable of specifically and efficiently modulating cell-surface receptor clustering and function. The multivalent ligand is made of a polymeric DNA scaffold decorated with biorecognition ligands (i.e., antibodies) to interrogate and modulate cell receptor signaling and function. Using CD20 clustering-mediated apoptosis in B-cell cancer cells as a model system, we demonstrated that our multivalent ligand is significantly more effective at inducing apoptosis of target cancer cells than its monovalent counterpart. This multivalent DNA material approach represents a new chemical biology tool to interrogate cell receptor signaling and functions and to potentially manipulate such functions for the development of therapeutics.

Three epilepsy-associated GABRG2 missense mutations at the γ +/β − interface disrupt GABAA receptor assembly and trafficking by similar mechanisms but to different extents

We compared the effects of three missense mutations in the GABAA receptor γ2 subunit on GABAA receptor assembly, trafficking and function in HEK293T cells cotransfected with α1, β2, and wildtype or mutant γ2 subunits. The mutations R82Q and P83S were identified in families with genetic epilepsy with febrile seizures plus (GEFS+), and N79S was found in a single patient with generalized tonic–clonic seizures (GTCS). Although all three mutations were located in an N-terminal loop that contributes to the γ+/β− subunit–subunit interface, we found that each mutation impaired GABAA receptor assembly to a different extent. The γ2(R82Q) and γ2(P83S) subunits had reduced α1β2γ2 receptor surface expression due to impaired assembly into pentamers, endoplasmic reticulum (ER) retention and degradation. In contrast, γ2(N79S) subunits were efficiently assembled into GABAA receptors with only minimally altered receptor trafficking, suggesting that N79S was a rare or susceptibility variant rather than an epilepsy mutation. Increased structural variability at assembly motifs was predicted by R82Q and P83S, but not N79S, substitution, suggesting that R82Q and P83S substitutions were less tolerated. Membrane proteins with missense mutations that impair folding and assembly often can be “rescued” by decreased temperatures. We coexpressed wildtype or mutant γ2 subunits with α1 and β2 subunits and found increased surface and total levels of both wildtype and mutant γ2 subunits after decreasing the incubation temperature to 30°C for 24h, suggesting that lower temperatures increased GABAA receptor stability. Thus epilepsy-associated mutations N79S, R82Q and P83S disrupted GABAA receptor assembly to different extents, an effect that could be potentially rescued by facilitating protein folding and assembly.

Protein tyrosine kinase 7 promotes PI3K/AKT signaling and survival in primary and neoplastic CD4+ T-lineage cells. (HEM4P.231)

Abstract Protein tyrosine kinase 7 (PTK7), a catalytically inactive receptor tyrosine kinase (RTK) that is highly expressed in intrathymic development, is a novel marker for human CD4+ recent thymic emigrants (RTEs), and is also highly expressed on some T-lineage thymomas, e.g., Jurkat cells. The function of PTK7 in normal human T-cell development and tumors remains unclear. Here, using RNAi-mediated gene silencing in T-lineage tumor cells, primary human peripheral T-cells and thymocytes, we found that targeting PTK7 consistently decreased cell survival by augmenting caspase-3 activation of apoptosis. The PTK7 knockdown also decreased AKT phosphorylation and PI3 kinase activity, suggesting an essential role for PTK7 in survival of RTEs and developing thymocytes involving the PI3K/AKT pathway. Using mass spectrometry of we identified insulin-like growth factor-1 (IGF-1) receptor as an active kinase partner of PTK7. Knockdown of PTK7 reduced IGF-1 receptor function in primary T-lineage cells, confirming the biologic importance of this physical association. This role for PTK7 in promoting cell survival via its effects on cell survival via the PI3K/AKT pathway may not only be relevant to normal T-cell development, including the RTE compartment, but also to the oncogenesis of T-lineage and epithelial cell tumors.

Investigation of antitumor effects of sorafenib and lapatinib alone and in combination on MCF-7 breast cancer cells.

Breast cancer evolution and tumor progression are controlled by complex interactions between steroid receptors and growth factor receptor signaling. Aberrant growth factor receptor signaling can augment or suppress estrogen receptor function in hormone-dependent breast cancer cells. Thus, we aimed to investigate antitumor effects of sorafenib and lapatinib alone and in combination on MCF-7 breast cancer cells. Cytotoxicity of the sorafenib and lapatinib was tested in MCF-7 cells by XTT assays. 50, 25, 12.5 and 6.25μM concentrations of sorafenib and 200, 100, 50 and 25μM concentrations of lapatinib were administered alone and in combination. Results were evaluated as absorbance at 450nM and IC50 values are calculated according to the absorbance data Both sorafenib and lapatinib showed concentration dependent cytotoxic effects on MCF-7 cells. Sorafenib exerted cytotoxic effects with an IC50 value of 32.0μM; in contrast with lapatinib the IC50 was 136.6μM. When sorafenib and lapatinib combined, lapatinib increased cytotoxic effects of sorafenib at its ineffective concentrations. Also at the concentrations where both drugs had cytotoxic effects, combination show strong anticancer effects and killed approximately 70 percent of breast cancer cells. Combinations of tyrosine kinase inhibitors and cytotoxic agents or molecular targeted therapy has been successful for many types of cancer. The present study shows that both sorafenib and lapatinib alone are effective in the treatment of breast cancer. Also a combination of these two agents may be a promising therapeutic option in treatment of breast cancer.

G protein‐coupled receptor expression and function in pulmonary artery smooth muscle cells: new targets in pulmonary arterial hypertension (1090.3)

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance, in part due to increased proliferation of pulmonary artery smooth muscle cells (PASMC). Since 3’5’‐cyclic adenosine monophosphate (cAMP) decreases proliferation of PASMC, G protein‐coupled receptors (GPCRs) that couple to Gαs or Gαi are attractive targets for the treatment of PAH. We used a real‐time PCR GPCR array to identify and quantify the GPCRs expressed by PASMC isolated from normal subjects and from patients with PAH. We found that human PASMC express &gt;135 GPCRs, at least 60 of which regulate cAMP formation. GPCR expression correlates with function: for Gαs‐coupled GPCRs with formation of cAMP and inhibition of cell proliferation, thus documenting that the GPCR array identifies physiologically relevant GPCRs. PAH‐PASMC (both from idiopathic and secondary PAH patients) have an increase (&gt;2‐fold) in the expression of 41 GPCRs compared to control‐PASMC, including multiple GPCRs that link to Gαs or Gαi. In addition, PAH‐PASMC have a higher expression of GPCRs that preferentially couple to Gαq/11 or to Gα12/13. Taken together these data provide evidence that a GPCRomic approach can identify GPCRs that may contribute to the physiology of PASMC and that may be new druggable targets for PAH, a disease for which no currently optimal therapies exist.Grant Funding Source: NIH

Enhancement of Cell Surface Expression and Receptor Functions of Membrane Progestin Receptor α (mPRα) by Progesterone Receptor Membrane Component 1 (PGRMC1): Evidence for a Role of PGRMC1 as an Adaptor Protein for Steroid Receptors

A variety of functions have been proposed for progesterone receptor membrane component 1 (PGRMC1), including acting as a component of a membrane progestin receptor and as an adaptor protein. Here we show that stable overexpression of human PGRMC1 in nuclear progesterone receptor (PR)-negative breast cancer cell lines causes increased expression of PGRMC1 and membrane progesterone receptor α (mPRα) on cell membranes that is associated with increased specific [(3)H]progesterone binding. The membrane progestin binding affinity and specificity were characteristic of mPRα, with a Kd of 4.7 nM and high affinity for the mPR-specific agonist, Org OD 02-0, and low affinity for corticosteroids. Progestin treatment caused activation of G proteins, further evidence for increased expression of functional mPRs on PGRMC1-transfected cell membranes. Immunocytochemical and coimmunoprecipitation studies showed a close association of PGRMC1 with mPRα in cell membranes. Transfection of PGRMC1 into spontaneously immortalized rat granulosa cells was associated with membrane expression of PGRMC1 and mPRα as well as antiapoptotic effects of progestins that were abolished after cotransfection with small interfering RNA for mPRα. These data demonstrate that PGRMC1 can act as an adaptor protein, transporting mPRα to the cell surface, and that the progestin binding and apoptotic functions previously ascribed to PGRMC1 are dependent on cell surface expression of mPRα. Collectively, the results suggest PGRMC1 and mPRα are components of a membrane progesterone receptor protein complex. Increased expression of estrogen receptor β was also observed in the membranes of PGRMC1-transfected cells, suggesting that PGRMC1 can act as an adaptor protein for multiple classes of steroid receptors.

Self-Engagement Of An NKT Cell Inhibitory Receptor During Development Alters Receptor Expression and Cellular Function

Invariant NKT (iNKT) cells have been shown to be pro-inflammatory or anti-inflammatory in many pathologic conditions including infections, cancer, autoimmune diseases and allergy. Additionally, early expansion of iNKT cells correlates with successful engraftment and low incidence of graft-versus host disease suggesting a role for iNKT cells in tolerance to allogeneic transplantation. iNKT cells recognize glycolipids bound to CD1d via their T cell receptor (TCR), but also express the inhibitory receptors for MHC class I ligands (MHC I.) Little is known regarding the importance of iNKT cell self-recognition of MHC I during development and the occurrence of autoimmunity, allergy or tolerance to in utero hematopoietic cellular transplantation (IUHCT). Given the importance of NK cell self-recognition during development, we hypothesized that, like NK cells, self-engagement of MHC I inhibitory receptors during development alters the phenotype and function of self-responsive iNKT cells. Prenatal allogeneic chimeras were established by in utero transplantation of Balb/c fetal liver light density cells at E14 into age-matched C57BL/6 fetal recipients. A kinetic evaluation of iNKT cell receptor expression and function was performed after birth. iNKT cells were evaluated for expression of activating (Ly49D) and inhibitory (Ly49A, F, G) receptors specific for the donor MHC I (H-2d.) The functional response of iNKT cell subsets was measured following in vivo activation with KRN7000, the ligand for iNKT cells. Intracellular cytokine production (IFN-gamma and IL-4) production was assessed in the alloresponsive Ly49A+ iNKT cell subset and compared to naïve controls and B10.D2 (H2d) mice. We found that a negligible fraction of iNKT cells expressed the activating Ly49D receptor in either chimera or controls (&lt;1%.) In chimeric mice, the frequency of iNKT cells expressing Ly49A, F, or G did not change significantly. However, the levels of Ly49A expression were significantly reduced on iNKT cells from all organs tested suggesting developmental recognition of donor class I ligands had occurred. These reduced receptor levels did not appear to be due to sequestration as acid stripping did not alter the phenotype. Lastly, Ly49A+ but not Ly49A- iNKT cells in chimeric mice exhibited significantly increased production of IL-4 in response to TCR stimulation suggesting a shift toward a regulatory phenotype. Consistent with this finding, IFN-gamma levels in chimeras were minimally changed compared to controls. These findings indicate that inhibitory receptor self-engagement during development leads to: 1) alterations in receptor expression; 2) shift toward a tolerogenic phenotype in self-responsive iNKT cell phenotypes; and 3) no change in the receptor expression or function of non-responsive iNKT cell phenotypes. Further studies will directly evaluate the impact of self-recognition of iNKT cells in the pathogenesis of autoimmunity and allergy or tolerance following IUHCT. Disclosures: No relevant conflicts of interest to declare.

SF3B1 Mutation Alters The Selection Of 3' RNA Splice Sites In Chronic Lymphocytic Leukemia

Large-scale cancer sequencing efforts have enabled the discovery of previously unknown paths to carcinogenesis. In chronic lymphocytic leukemia (CLL), these initiatives have identified highly recurrent mutations in the core spliceosome component SF3B1 at restricted sites (i.e. 50% at K700E), implicating a role of altered RNA splicing in CLL. Mutated SF3B1 has been associated with adverse clinical outcome in CLL and a higher incidence in refractory/relapsed CLL. However, mechanistic insights into downstream paths affected by mutated SF3B1 and its role in the oncogenic phenotype are still lacking.To this end, we first examined if mutated SF3B1 alters known disrupted pathways in CLL. We tested the effect of expressing constructs encoding full-length mutated vs. wild-type SF3B1 into hematopoietic cell lines on established pathway readout assays but observed no discernible changes in cell cycle, Notch or Wnt signaling. On the other hand, given the key role of SF3B1 in pre-mRNA splicing, we hypothesized that features of altered splicing associated with this driver might provide important mechanistic insights. We therefore generated RNA-seq data from 22 CLL cases (9 with mutant and 13 with wild-type SF3B1; with 45-128 million reads mapped per sample) and identified splicing effects associated with SF3B1 mutation using an update of the computational approach, JuncBASE (Junction Based Analysis of Spliced Events).SF3B1 is the key catalytic component of the U2 snRNP, which is critical in the initial formation of the core spliceosome at 3' exon/intron boundaries. Indeed, we identified pervasive changes in 3' splice site selection in which 642 introns showed altered 3' splice site selection associated with SF3B1 mutation (p < 0.05). This effect was relatively specific to 3' splice sites as only 362 introns showed changes in 5' splice site selection associated with SF3B1 mutation (p < 0.05). Alterations in 3' splice site choice resulted in both partial exon skipping and partial intron retention, equivalently. Although splicing changes were significant and consistent amongst SF3B1 mutated CLLs, the majority of these splicing changes (63%) was subtle, such that there was a <10% change in the proportion of transcripts selecting a given alternative 3' splice site. Additionally, we did not observe specific sequences at altered 3' splice sites in association with SF3B1 mutation, in contrast to the 3' splice sites motifs associated with mutation in the splicing factor U2AF1 (common to AML), suggesting different mechanisms of spliceosome alterations between the 2 spliceosomal component mutations.Altered splicing might be expected to generate specific splice variants that could in turn affect cellular circuitry. To identify likely candidates, we detected 192 genes with splicing changes strongly associated with SF3B1 mutation, using a conservative statistical approach (q < 0.1). As expected, these genes were significantly enriched for 3' splice-site variants (160 of 192, p < 0.00001), 25 of 192 altered splicing events involved upregulation of isoforms virtually undetectable in CLL samples without SF3B1 mutation. We validated these candidates using three complementary approaches. First, an analysis of an independent RNA-seq dataset (5 CLLs with SF3B1 mutation, 67 with wildtype SF3B1) confirmed the set of the altered splicing events identified from the discovery cohort. Second, we detected 5 of 5 tested candidate SF3B1 mutation-associated splice variants (in C11orf2, GCC2, MAP3K7, TPP2, ZNF91) by quantitative real-time RT-PCR in 6 of 6 CLLs with SF3B1 mutation, but in none of wild type SF3B1 CLLs (n=3) or normal B cell samples (n=3). Finally, K562 cells transfected with mutated (K700E) SF3B1 revealed a significant increase in the candidate splice variants (by ∼20-fold) compared to K562 cells transfected with the wild type variant.A detailed examination of the candidate list revealed several genes with known roles in B cell biology, e.g., BLNK, a critical adaptor of B cell receptor function involved in lymphoproliferation; USP15, a modulator of NFkB signaling. Genes with significantly altered splicing were enriched with immune system gene sets (q <0.0001) and with genes unregulated in uveal melanoma (q < 10-7), which carries a high rate of SF3B1 mutation. These analyses will be used to guide selection of candidates for downstream functional assessment and to identify variants with highest oncogenic potential. Disclosures:Brown:Pharmacyclics: Consultancy; Genentech: Consultancy; Celgene: Consultancy, Research Funding; Emergent: Consultancy; Onyx: Consultancy; Sanofi Aventis: Consultancy; Vertex: Consultancy; Novartis: Consultancy; Genzyme: Research Funding. Meyerson:Foundation Medicine: Consultancy, Equity Ownership; Novartis: Consultancy.

The distinctive germinal center phase of IgE+ B lymphocytes limits their contribution to the classical memory response

The mechanisms involved in the maintenance of memory IgE responses are poorly understood, and the role played by germinal center (GC) IgE(+) cells in memory responses is particularly unclear. IgE(+) B cell differentiation is characterized by a transient GC phase, a bias toward the plasma cell (PC) fate, and dependence on sequential switching for the production of high-affinity IgE. We show here that IgE(+) GC B cells are unfit to undergo the conventional GC differentiation program due to impaired B cell receptor function and increased apoptosis. IgE(+) GC cells fail to populate the GC light zone and are unable to contribute to the memory and long-lived PC compartments. Furthermore, we demonstrate that direct and sequential switching are linked to distinct B cell differentiation fates: direct switching generates IgE(+) GC cells, whereas sequential switching gives rise to IgE(+) PCs. We propose a comprehensive model for the generation and memory of IgE responses.

Drosophila Adiponectin Receptor in Insulin Producing Cells Regulates Glucose and Lipid Metabolism by Controlling Insulin Secretion

Adipokines secreted from adipose tissue are key regulators of metabolism in animals. Adiponectin, one of the adipokines, modulates pancreatic beta cell function to maintain energy homeostasis. Recently, significant conservation between Drosophila melanogaster and mammalian metabolism has been discovered. Drosophila insulin like peptides (Dilps) regulate energy metabolism similarly to mammalian insulin. However, in Drosophila, the regulatory mechanism of insulin producing cells (IPCs) by adipokine signaling is largely unknown. Here, we describe the discovery of the Drosophila adiponectin receptor and its function in IPCs. Drosophila adiponectin receptor (dAdipoR) has high homology with the human adiponectin receptor 1. The dAdipoR antibody staining revealed that dAdipoR was expressed in IPCs of larval and adult brains. IPC- specific dAdipoR inhibition (Dilp2>dAdipoR-Ri) showed the increased sugar level in the hemolymph and the elevated triglyceride level in whole body. Dilps mRNA levels in the Dilp2>dAdipoR-Ri flies were similar with those of controls. However, in the Dilp2>dAdipoR-Ri flies, Dilp2 protein was accumulated in IPCs, the level of circulating Dilp2 was decreased, and insulin signaling was reduced in the fat body. In ex vivo fly brain culture with the human adiponectin, Dilp2 was secreted from IPCs. These results indicate that adiponectin receptor in insulin producing cells regulates insulin secretion and controls glucose and lipid metabolism in Drosophila melanogaster. This study demonstrates a new adipokine signaling in Drosophila and provides insights for the mammalian adiponectin receptor function in pancreatic beta cells, which could be useful for therapeutic application.

Designing Novel Therapies Against Sarcomas in the Era of Personalized Medicine and Economic Crisis

Drug "repurposing" is the process of finding new therapeutic indications for existing drugs, and can be considered as a more efficient and realistic strategy for the design of therapies against rare diseases than the current efforts to develop targeted-drugs. In this review, we explore the difficulties related to the identification and development of tailored therapies for individual patients with sarcomas, which are relatively rare diseases characterized by an extreme genetic and histologic variability. Overall, sarcomas comprise about 1% of all adult tumors and 10% of pediatric cancers. They are conventionally divided in bone and soft-tissue sarcomas, considering their site of origin. However, each group is highly heterogeneous and recent global characterization of their genetic alterations has clearly identified the existence of peculiarities that render these group of tumors even more "orphan" for pharmaceutical companies to develop and market specific- targeted drugs. The present review highlights key examples of molecular targets identification in bone sarcomas, reexamining the history of insulin-like growth factor receptor (IGF-IR) and its role in physiology and in cancer as well as developments regarding phase I to II clinical trials of agents directed against this receptor. The IGF system is quite complex, with many players in the field. Insulin receptor function in cancer cells has certainly been underestimated, but also little attention was paid to the type of ligands that are mainly involved in each tumor type. Strategies considering the system in its complex are encouraged and, in this context, drugs aimed at reducing circulating insulin levels, such as metformin, should receive attention as potential anti-cancer agents.