WO2008131426A1 - RIBOSOMAL PROTEIN S3: A FUNCTIONAL COMPONENT OF NF-KAPPA B p65 BINDING COMPLEXES - Google Patents

Abstract

The invention provides a method of inhibiting the expression of NF-kB target genes in a mammal comprising administering an effective amount of an inhibitor of ribosomal protein S3 (RPS3) to the mammal. The invention also provides a method of inhibiting the induction of immunoglobulin k light chain gene expression in B cells comprising administering an effective amount of an inhibitor of RPS3 to the B cells. The invention further provides a method of treating an inflammatory disease or disorder in a mammal comprising administering an effective amount of an inhibitor of RPS3 to the mammal.

Description

RIBOSOMAL PROTEIN S3: A FUNCTIONAL COMPONENT OF NF-KAPPA B p65

BINDING COMPLEXES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 60/913,336 filed April 23, 2007, which is incorporated by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

[0002] Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 44,527 Byte ASCII (Text) file named "702802ST25.TXT," created on April 19, 2008.

BACKGROUND OF THE INVENTION

[0003] Because of its extensive regulation of key processes in immune and inflammatory responses and cellular metabolism, the NF-κB signaling pathway has been targeted for pharmacological interventions (see Gilmore et al., Oncogene, 25: 6887-6899 (2006); Verma et al., Ann. Rheum. Dis., 63 Suppl. 2: 1157-U61 (2004)). Over 750 inhibitors of the NF-κB pathway have been identified, including a variety of natural and synthetic molecules (see Gilmore et al., supra). These inhibitors act at all levels of the NF-κB pathway, but typically exert a global inhibition of NF-κB function rather than having specific effects in disease settings. [0004] There exists a desire to provide additional inhibitors of NF-κB function.

BRIEF SUMMARY OF THE INVENTION

[0005] The invention provides a method of inhibiting the expression of NF-κB target genes in a mammal comprising administering an effective amount of an inhibitor of ribosomal protein S3 (RPS3) to the mammal, such that the expression of NF-κB target genes in the mammal is inhibited.

[0006] The invention further provides a method of inhibiting the induction of immunoglobulin K light chain gene expression in B cells comprising administering an effective amount of an inhibitor of RPS3 to the B cells, such that the induction of immunoglobulin K light chain gene expression in B cells is inhibited. [0007] The invention also provides a method of treating an inflammatory disease or disorder in a mammal comprising administering an effective amount of an inhibitor of RPS3 to the mammal, such that the inflammatory disease or disorder is treated.

DETAILED DESCRIPTION OF THE INVENTION

[0008] NF-κB originally was detected as a DNA-binding complex governing immunoglobulin (Ig) light chain gene intronic enhancer (see Lenardo et al., Science, 236: 1573-1577 (1987); Sen et al., Cell, 47: 921-928 (1986)). The constitutive presence of NF-κB in mature B lymphocytes, in concert with Ig K expression, resulted in the name Nuclear Factor (NF)-κB (see Lenardo et al., Cell, 58: 227-229 (1989)). The mammalian NF-κB/Rel gene family comprises five proteins (p65 (also known as ReIA), c-Rel, ReIB, p50, and p52) that mediate function as homodimers or heterodimers to control gene transcription and are conserved from human to simple organisms, such as insects (see Chen et al.., Nat. Rev. MoI. Cell Biol, 5: 392-401 (2004); Ghosh et al., Cell, 109 Suppl.: S81-96 (2002), Hayden et al., Genes Dev.: 18, 2195-2224 (2004), Silverman et al., Genes Dev., 15: 2321-2342 (2001)). These dimers exhibit varying binding affinities for specific recognition sites in DNA, as well as differences in their regulatory function (see Natoli et al., Nat. Immunol, 6: 439-445 (2005)). Each of the proteins in the family harbors an amino-terminal ReI homology region (RHR) that controls dimerization, DNA binding, and cytoplasmic retention by inhibitors of KB (IKBS) (see Chen et al., supra; Rothwarf et al., ScL STKE, REl (1999)). ReI B, c-Rel, and p65 also have transcriptional activating domains, and dimers containing one of these subunits are potent transcriptional activators. In contrast, dimers lacking these subunits, such as p50 homodimers, are believed to repress transcription (see Kang et al., Science, 256: 1452-1456 (1992)).

[0009] The most abundant form of NF-κB is a p65-p50 heterodimer that can inducibly regulate a large number of important immune, inflammatory, and anti-apoptosis genes (see Bottero et al., Cell Death Differ., 13: 785-797 (2006); Lenardo et al., Cell, 58: 227-229 (1989); Rothwarf et al., supra; Sen et al., supra; Siebenlist et al., Nat. Rev. Immunol., 5: 435- 445 (2005); Takayanagi et al., Nature, 408: 600-605 (2000)). The biochemical pathways resulting in NF-κB activation have been studied extensively (see Baldwin, Ann. Rev. Immunol, 14: 649-683 (1996); Chen, Nat. Cell. Biol, 7: 758-765 (2005); Weil et al., Cell Death Differ., 13: 826-833 (2006); Wertz et al., Nature, 430, 694-699 (2004); Zhou et al., Nature, 427: 167-171 (2004)). An activating stimulus, such as TNFα triggering its cell surface receptors, invokes a cytoplasmic switch that removes IKB proteins from latent, cytoplasmic NF-κB dimers, thereby allowing the dimers to translocate to the nucleus and bind control regions of target genes (see Scheidereit, Oncogene, 25: 6685-6705 (2006)). Degradation of IKBS is due to phosphorylation by protein kinase complexes called IKB kinases (IKK), which leads to ubiquitination and dispatch of IKB to the proteasome. Latent NF-κB is present in essentially all nucleated cell types, allowing NF-κB to be utilized for the induction of many different genes in response to a multiplicity of different stimuli and signaling pathways. However, the manner in which NF-κB modulates effects at selected DNA biding sites and whether NF-κB action depends on other inducible factors is not completely understood.

[0010] NF-κB exerts its fundamental role as a DNA-binding transcription factor by binding to variations of the consensus sequence GGRNYYYCC (SEQ ID NO: 17; in which R is a purine, Y is a pyrimidine, and N is any nucleotide), which is known as a KB site (see Chen et al., Nature, 391: 410-413 (1998)). The presence of KB sites in target genes is a minimal requirement for NF-κB regulation; however it is unknown whether the presence of these sites is sufficient for gene induction. Evidence suggests that both recruitment of NF-κB to target genes and transcriptional events after recruitment are actively controlled (see Natoli et al., supra). The direct cross-linking of gel-purified NF-κB proteins to DNA (see Urban et &\., Embo J., 10: 1817-1825 (1991)) and the crystal structure of p65-p50 heterodimer bound to DNA (see Chen et al., supra) demonstrate that, while p50 binds to the relatively invariant 5' region of the KB site, p65 binds to the 3' region of the KB site, which is where most of the KB sequence variation occurs (see Grilli et al., Int. Rev. CytoL, 143: 1-62 (1993)). The complexity inherent in p65 binding to target genes was highlighted by a genomic microarray using chromatin immunoprecipitation (ChIP) that showed binding to 209 regions on chromosome 22 in cells stimulated with TNFα, of which only 60% contained either canonical or variant KB sites (see Martone et al., Proc. Natl. Acad. ScL USA, 100: 12247-12252 (2003)). Furthermore, NF-κB complexes bind to KB sites with high affinity, which cannot be explained solely by the number or character of protein:DNA contacts (see Chen et al., supra). [0011] To further elucidate the NF-κB complexes and the action on target genes, the inventors used NF-κB as a bait protein in a proteomic screen involving tandem affinity purification and mass spectrometry on mammalian cell extracts. The inventors identified ribosomal protein S3 (RPS3) as an essential component of certain NF-κB gene regulatory complexes that contain p65.

[0012] RPS3 is a 26 kD protein that shuttles between the cytoplasm and nucleus with functions in both cellular compartments. The primary sequence of the protein is highly conserved across phylogeny with close relatives even in prokaryotes (see Lyamouri et al., Gene, 294, 147-156 (2002)). The solution structure of rat RPS3 reveals a "K homology" domain (vide infra) followed by large unstructured region of 120 amino acids. Notably, the protein lacks any obvious transcriptional activation domain, ReI homology, or the conserved consensus sequence present in most coactivator proteins (see Heery et al., Nature, 387: 733- 736 (1997)).

[0013] RPS3 contains a nuclear localization sequence (NLS) that apparently is necessary for its nuclear localization and RPS3's role in DNA damage responses. RPS3 also contains a KH domain, which is a structural motif that can bind single-stranded RNA and DNA with some sequence specificity (see Siomi et al., Nucl. Acids Res., 21: 1193-1198 (1993)). The amino acid sequence of RPS3 is set forth in SEQ ID NO: 16, with the NLS corresponding to residues 7-10 of SEQ ID NO: 16 and the KH domain corresponding to residues 42-111 of SEQ ID NO: 16.

[0014] While not wishing to be bound by any particular theory, RPS3 could influence NF-κB by several means. A first possibility is that RPS3 could be a DNA-binding exponent that stabilizes the interaction of NF-κB with cognate sites. RPS3 could provide a selective and inducible mechanism to generate a high affinity binding complex in a similar manner as constitutively nuclear HMG proteins selectively stabilize NF-κB on the β-interferon promoter (see Thanos et al., Cell, 83: 1091-1100 (1995)). RPS3 could serve to stabilize NF-κB association with the separated strands or serve as a bookmark for NF-κB on the active gene. [0015] A second possibility is that RPS3 could be a specifier that selects particular genomic KB sites to be activated in a certain manner under specific conditions. This possibility is supported by the microarray results described in Example 5 that only a subset of p65-dependent genes activated in T cells by TCR stimulation are contingent upon RPS3. Induction of a larger number of genes was impaired by the removal of RPS3, but not by p65. Selectivity could be conferred by the DNA binding specificity of RPS3 or more complex conformations of a DNA:protein holocomplex. [0016] A third possibility is that RPS3 could be a translational facilitator that is inducibly brought to the transcribed gene by NF-κB association. RPS3 then could bind to newly synthesized transcript through the KH domain and guide the gene to pre-ribosomes forming in the nucleus to facilitate translation. Within the ribosome, RPS3 can be chemically cross- linked to translated mRNA, eIF2, and eIF3, implying a key role in translation initiation (see Loar et al., Genetics, 168: 1877-1889 (2004)). Connecting transcription with translation initiation could accelerate critical gene responses, such as during a lymphocyte's response to a pathogen, that characteristically involve inducible NF-κB.

[0017] The inventors determined that RPS3 was essential for normal expression of NF- KB target genes (e.g., NF-κB target genes requiring p65), including key physiological events, such as the induction of immunoglobulin K light chain gene expression in B cells. Additionally, the inventors determined that RPS3 knockdown resulted in enhanced ultraviolet-induced cell apoptosis in cells and dramatically blocked TCR stimulation-induced cell proliferation and interleukin-2 production in cells (e.g., resting primary peripheral blood lymphocytes).

[0018] Accordingly, the invention provides a method of inhibiting the expression of NF- KB target genes (e.g., by disrupting NF-κB signaling) in a mammal comprising administering an effective amount of an inhibitor of ribosomal protein S3 (RPS3) to the mammal, such that the expression of NF-κB target genes in the mammal is inhibited.

[0019] The mammal can be any suitable mammal, such as a mammal selected from the group consisting of a mouse, rat, guinea pig, hamster, cat, dog, pig, cow, horse, and a primate. The mammal preferably is a human, especially a human patient. [0020] NF-κB target genes are known in the art and include, but are not limited to, cytokines, chemokines, modulators of cytokines and chemokines, immunoreceptors, proteins involved in antigen presentation, cell adhesion molecules, acute phase proteins, stress response genes, cell-surface receptors, regulators of apoptosis, growth factors, ligands of growth factors, modulators of growth factors and their ligands, early response genes, transcription factors and regulators, viruses, and enzymes (see, e.g., Pahl, Oncogene, 18(49): 6853-6866 (1999)).

[0021] Examples of cytokines, chemokines, and their modulators targeted by NF-κB include, but are not limited to, BAFF (TNFSF13B), CCL5, CCL15/Leukotactin/SCYA15, CCL17, CCL19, CCL20, CCL22, CCL23/SYA23, CCL28, CINC-I (CXCLl), CXCLIl, exotaxin (CXCLlO), fractalkine (CXCL3), GRO-alpha (CXCLl), GRO-beta, GRO-gamma, GRO-I (CXCLl), ICOS, INF-g, IL-Ia, IL-Ib, ILl receptor antagonist(ILlA), IL-2, IL-6, IL- 8, IL-9, IL-IO, IL-11, IL-12B (p40), IL-12A (p35), IL-13, IL-15, IL-17, IL-23A (pl9), IL-27 (p28), EBI3/IL-27B, beta-interferon (IFNBl), IP-IO (CXCL5), KC, ligpl, GCP-2 (CXCL6), lymphotoxin a (LTA), lymphotoxin b (LTB), MCP-I /JE (CCL2), MIG (CXCL9), Mip-la,b (CCL3, CCL4), LAG-I (CCL4), MIP-2 (CCL3), MIP-3alpha/CCL20, mob-1 (CXCLlO), neutrophil activating peptide-78 (CXCL5), RANTES (CCL5), TCA3 (CCLl), TNFalpha (TNF), TNFbeta (LTA), TRAIL (Apo2 ligand, TNFSFlO), TFF3, and VEGI (TNFSF15). [0022] Examples of immunoreceptors targeted by NF-κB include, but are not limited to, B7.1 (CD80), BLR-I, CCR5, CCR7, CXCR (IL8RA), CXCR2 (IL8RB), CD137 (TNFRSF9), CDl 54 (CD40LG), CD3gamma (CD3G), CD21 (CR2), CD38, CD40, CD48, CD83, CD86, CD98 (SLC3A2), CD 134 (TNFRSF4), Fl 1 -receptor (Fl IR), FcRn (FCGRT), Fc epsilon receptor II (CD23, FCER2), HLA-G, ICOS, IL-2 receptor a-chain (IL2RA), immunoglobulin Cgammal (IGHG2), immunoglobulin gammal (IGHGl), immunoglobulin gamma4 (IGHG4), immunoglobulin e heavy chain (IGHE), immunoglobulin k light chain (IGKC), invariant chain I₁, kinin Bl receptor (BDKRBl), MHC Class I (H-2K_b), MHC Class I HLA- B7 (HLA-B), b2 microglobulin (B2M), NOD2, polymeric Ig receptor (plgR), PGRP-S (PGLYRPl), T-cell receptor b chain (TCRB), T-cell receptor/CD3 gamma (CD3G), TLR2, TLR9, TNF-receptor, p75/80 (CD120B, TNFRSFlB), and TREMl.

[0023] Examples of proteins involved in antigen presentation that are targeted by NF-κB include, but are not limited to, complement B (CFB), complement component 3 (C3), complement receptor 2 (CR2), proteasome subunit LMP2 (PSMB9), peptide transporter TAPl (TAPl), and tapasin (TAPBP).

[0024] Examples of cell adhesion molecules targeted by NF-κB include, but are not limited to, DC-SIGN (CD209), ELAM-I (CD62E, E-selectin, SELE), endoglin (ENG), fibornectin (FNl), ICAM-I (CD54), MadCAM, NCAM, P-selectin (SELP), tenascin (TNC), and VCAM-I.

[0025] Examples of acute phase proteins targeted by NF-κB include, but are not limited to, angiotensinogen (angiotensin II, AGT), beta-defensin-2 (DEFB2), C4b binding proteing (C4BPA), complement factor B (CFB), complement factor C4 (C4A), C-reactive protein (CRP), hepcidin (HAMP), lipopolysaccharide binding protein (LBP), pentraxin (PTX3), serum amyloid A proteins (SAAl, SAA2, SAA3), tissue factor-1 (F3), and urokinase-type plasminogen activator (PLAU).

[0026] Examples of stress response genes targeted by NF-κB include, but are not limited to, CYP2E1, CYP2C11, CYP7b (CYP7B1), COX-2 (PTGS2), ferritin H chain (FTHl), HSP90-alpha (HSP90AA1), 5 -lipoxygenase (ALOX5), 12-lipoxygenase (ALOX12), inducible NO-synthase (NOS2A), MAP4K1, Cu/Zn SOD (SODl), SOD2, MXl, NAD(P)H quinone oxidoreductase (DT-diaphorase, NQOl), phospholipase A2 (PLA2), and SEPSl (SELS).

[0027] Examples of cell-surface receptors targeted by NF-κB include, but are not limited to, ABCAl, ABCC6, Al adenosine receptor (ADORAl), A2A (AD0RA2A), ADAM19, amiloride-sensitive sodium channel (SCNNAl), alpha2B-adrenergic receptor (ADRA2B), bradykinin Bl receptor (BDKRBl), CD23 (FCER2/CD23), CD69 (C69), DOR (OPRDl), epidermal growth factor receptor (EGFR), ERBB2, Gall receptor (KISSl), Lox-1 (OLRl), Ly49 (KLRAl), Mdrl (ABCB4), Mu-opioid receptor (OPRMl), mGlu2 (GRM2), neuropeptide Y-Yl receptor (NPYlR), NMDA receptor subunit 2A (GRIN2A), NMDA receptor subunite NR-I (GRINl), oxytocin receptor (OXTR), PAF receptor 1 (PTAFR), P-gp (ABCBl), and RAGE-receptor for advanced glycation end products (AGER). [0028] Regulators of apoptosis targeted by NF-κB include, but are not limited to, ASC (PYCARD), BAX, Bfll/Al (BCL2A1), Bcl-xL (BCL2L1), Bcl-2, B7-H1 (CD274), BNIP3, caspase-11 (CASP4), Nrl3, c-FLIP (CFLAR), CD95 (FAS), Fas-associated phosphatase- 1 (PTPNl 3), Fas-ligand (FASLG), IAPs, IEX-IL (IER3), TRAFl, TRAF2, TRAF-2 binidng protein (Carp, TIFA), and XIAP.

[0029] Growth factors, ligands, and their modulators that are targeted by NF-κB include, but are not limited to, activin A (INHBA), angiopoeitin (ANGPTl), BCAP (P13KAP1), BDNF, BLyS (TNFSF 13B), BLNK, BMP-2, BMP-4, CGRP (CALCB), FGF8, FLRG (FSTL3), G-CSF (CSF3), GM-CSF (CSF2), HGF/SF (HGF), EPO, IGFBP-I, IGFBP-2, M- CSF (CSFl), midkine (neurite growth promoting factor-2, MDK), NGF (NGFB), NK-IR (TACRl), NK4, NRGl, OPN (SPPl), PDGF B chain (PDGFB), PIGF, proenkephalin (PENK), prolactin (PRL), stem cell factor (KITLG), thrombospondin-1 (THBSl), thrombospondin-2 (THBS2), VEGFC, and WNTlOB.

[0030] Early response genes targeted by NF-κB include, but are not limited to, B94 (TNFAIP2), EGR-I, p22/PRGl (IER3), p62 (DCTN4), and TIEG (KLFlO). [0031] Transcription factors and their modulators that are targeted by NF-κB include, but are not limited to, A20 (TNFAIP3), ABIN-3 (TNIP3), androgen receptor (AR), BCL-3, BMI- 1, CDXl, c-fos (FOS), c-myb (MYB), c-myc (MYC), c-rel (REL), C/EMPdelta (CEBPD), DC-SCRIPT (ZNF366), DMPl, E2F3a (E2F3), ELF3, ELYS (AHCTFl), ETRlOl (IER2), GATA3, glucocorticoid receptor (NR3C1), HIF-lalpha (HIFlA), H0XA9, IRF-I, IRF-2, IRF-4, IRF-7, IkB-a (NFKBlA), IkB-e (NFKBIE), JUNB, JMJD3, LEFl, LZIP (CREB3), Mail (NFKBIZ), nfkb2 (NFKB2), nfkbl (NFKBl), NLRP2, NURRl, Osterix, p53 (TP53), progesterone receptor (PGR), relB, Snail (SNAIl), Sox-9, Stat5A, Tfec, Twist (TWISTl), WTl, and YYl.

[0032] Viruses targeted by NF-κB include, but are not limited to, adenovirus (E3 region), avian leukosis virus, bovine leukosis virus, CMV, EBV (Wp promoter), HBV (pregenomic promoter), HIV-I, HSV (ICP90, ICPO), JC virus, HPV type 16, SIV, and SV-40. [0033] Enzymes targeted by NF-κB include, but are not limited to, ABC transporters (ABCB9), N-acetylglucosaminyltransferase 1 (GCNTl), ADH (ADHlH), AID (AICDA), AMACR, ARF-related protein- 1 (ARFRPl), argininosuccinate synthetase (ASSl), aromatase (promoter II, CYP19A1), ART2.1 (ARTl), alpha IACT (SERPINA3), BACE-I, Btk, cathepsin B (CTSB), cathepsin L (CTSLl), ceramide glycosyltransferase (UGCGLl), chitinase 3-like protein (CHI3L1), cis-retinoid/androgen dehydrogenase type 1 (CRADl, Rdhl), CRAD2 (Rdh7), collagenase 1 (MMPl), dihydrodiol dehydrogenase (AKRlCl), DYPD, DNASIL2 (DNASE1L2), EL (LIPG), ENO2, GAD67 (GADl), GD3-synthase (ST8SIA1), gp91 phox (NOXl), gelatinase B (MMP9), GSTPl-I (GSTPl), glutamate- cysteine ligase (GCLC), glutamate-cysteine ligase modifier (GCLM), gamma glutamylcystein synthetase (GCLC), glucose 6-phosphate dehydrogenase (G6PD), glucose-6- phosphatase (G6PC), GnRH II (GNRH2), granzyme B (GZMB), soluble guanylyl cyclase alpha 1 (GUCY1A2), heparanase (HPSE), HO-I (HMOXl), hyaluronan synthase (HASl), 1 lbHSD2 (HSDl 1B2), 17bHSD (HSD17B8), H⁺-K⁺ATPaSe alpha 2 (ATPl A2), iodothyronine deiodinase (type 2, DIO2), lipcalin-type prostaglandin D synthase (L-PGDS; PTGDS), lysozyme (LYZ), MTHFR, MKP-I (DUSPl), MMP-3, MMP-9, MLCK (MYLK), iNOS (NOS2A), n-NOS (NOSl), PDE7A1 (PDE7A), PIM-I, PLK3, PIK3CA, PP5 (PPP5C), PKCdelta (PRKCD), PLCdelta 1 (PLCDl), PTGIS, PGES (PTGES), PTPlB (PTPNl), PTHrP (PTHLH), RACKl (GNB2L1), REV3 (REV3L), Slfn-2, serpin 2A (SERPINA2), SIATl (ST6GAL1), SNARK (NUAK2), SSAT (SATl), SUV3 (SUPV3L1), TERT, transglutaminase (TGMl), TTG (TGM2), typell-secreted phospholipase A2 (PAFAH2), uridine phosphorylase (UPPl), and xanthine dehydrogenase (XDH). [0034] Other NF-κB target genes include, but are not limited to, ABCG5, ABCG8, AbetaH-J-J (ASPH), alpha- 1 acid glycoprotein (ORMl), alpha-fetoprotein (AFP), AMH, beta-amyloid (A4), APOBEC2, apolipoprotein C III (APOBEC2), apoliprotein D (APOD), APOE, AQP4, biglycan (BGN), BRCA2, calsarcin-1 (MYOZl), caveolin-1 (CAVl), ρ21- CIPl (CDKNlA), claudin-2 (CLDN2), a2(I) collagen (C0L1A2), connexin32 (GJBl), cyclin Dl (CCNDl), cyclin D2 (CCND2), cyclin D3 (CCND3), DIF2 (IER3), DMTl (SLCl 1A2), Elafm (SKALP, PI3), endothelin 1 (EDNl), ephrin-Al (EPHAl), Factor VIII (F8), ferritin heavy chain (FTHl), Gadd45beta (GADD45B), Galpha i2 (GNAI2), GIF (MT3), Galectin 3 (LGALS3), GBP-I, epsilon-globin (HBEl), zeta-globin (HBZ), GS3686 (IFI44L), hair K5 keratin (KRT5), HCCSl (VPS53), HMG14 (HMGNl), IBABP (FABP6), IMP2 (IGFBP2), K3 keratin (KRT3), K6b keratin (KRT6B), Kl 5 keratin (KRTl 5), lactoferrin (LTF), laminin B2 chain (LAMB2), lipcalin-2 (LCN2), Mtsl (S100A4), Mirl25b, Mirl4a,b, Mirl55, MNEl (SERPINBl), Mucin (MUC-2), myelin basic protein (MBP), MCTl (SLC16A1), NaH (TNIPl), neutrophil gelatinase-associated lipocalin (LCN2), NLF (FAM148A), pll (SlOOAlO), PA28 alpha (PSMEl), PA28 beta (PSME2), PAI-I (SERPINEl), PAX8, PCBD (PTS), Perforin (PRFl), PGKl, POMC, pregnancy specific glycoprotein rnCGM3 (CGM3), prodynorphin (PDYN), prostate-specific antigen (KLK3), PTEN, RAG-I, RAG2, RbAp48 (RBBP4), RICK (RIPK2), SERPINE2, S100A6 (calcyclin), SH3BGRL, SK2 channels (KCNN2), SKP2, Spergen-1 (SPATA19), SWSl (OPNlSW), Syncytin-1 (ERVWEl), Syndecan-4 (SDC4), TAUT (SLC6A6), TASK-2 (KCNK5), tissue factor pathway inhibitor-2 (TFPI-2), transferring (TF), TRIF (TICAMl), TRPCl, UBE2M, UCP-2, uroplakin Ib (UPKlB), 25-hydroxyvitamin D3 1 alpha hydroxylase (CYP27B1), vimentin (VIM), al- antitrypsin (SERPINAl), and Gro-1 (CXCLl).

[0035] Particularly preferred NF-κB target genes whose expression can be inhibited by the methods of the invention include those identified during the microarray analysis described in Example 5, which include NFKBIA, CD83, BIRC3, BACH2, SGK, PLKl, FAS, TNFAIP3, GBPl, and SAMSNl.

[0036] The invention also provides a method of inhibiting the induction of immunoglobulin K light chain gene expression in B cells comprising administering an effective amount of an inhibitor of RPS3 to the B cells, such that the induction of immunoglobulin K light chain gene expression in B cells is inhibited. [0037] Inhibition of gene expression (e.g., NF -KB target gene expression or immunoglobulin K light chain gene expression) refers to a reduction in gene expression by at least about 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) relative to a control (e.g., a mammal or cell of a mammal, such as a B cell, that has not been administered the RPS3 inhibitor).

[0038] The invention further provides a method of treating an inflammatory disease or disorder in a mammal comprising administering an effective amount of an inhibitor of RPS3 to the mammal, such that the inflammatory disease or disorder is treated. [0039] The inflammatory disease or disorder can be any suitable inflammatory disease or disorder. Suitable inflammatory diseases or disorders include, but are not limited to, uveitis, allergy, autoimmune disease, allograft rejection, arthritis, colitis, psoriasis, and combinations thereof. Specific examples of inflammatory diseases or disorders include, but are not limited to, rheumatoid arthritis, rheumatoid spondylitis, ostero-arthritis, asthma, adult respiratory distress syndrome, stroke, reperfusion injury, neural trauma, neural ischemia, restenosis, chronic pulmonary inflammatory disease, grafit-versus-host reaction, Crohn's disease, ulcerative colitis, inflammatory bowel disease, inflammatory lung disease, lupus, lupus nephritis, systemic lupus erythematosis, multiple sclerosis, vasculitis, atherosclerosis, chronic inflammatory demyelinating polyradiculoneuritis, Helicobacter pylori-associated gastritis, systemic inflammatory response syndrome, and combinations thereof (see, e.g., Tak et al., J. Clin. Invest, 107(1): 7-11 (2001)).

[0040] Other diseases and disorders in which activation of NF-κB has been implicated include, but are not limited to, Complex Regional Pain Syndrome, Cardiac Hypertrophy, Muscular Dystrophy (type 2A), muscle wasting, catabolic disorders, diabetes (types 1 and 2), obesity, fetal growth retardation, hypercholesterolemia, atherosclerosis, heart disease, chronic heart failure, ischemia/reperfusion, stroke, cerebral aneurysm, angina pectoris, pulmonary disease, cystic fibrosis, acid-induced lung injury, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), hyaline membrane disease, kidney disease, glomerular disease, alcoholic liver disease, leptospiriosis renal disease, gut diseases, peritoneal endometriosis, skin diseases, nasal sinusitis, mesothelioma, anhidrotic ecodermal dysplasia-ID, Behcet's Disease, incontinentia pigmenti, tuberculosis, glaucoma, appendicitis, Paget's Disease, pancreatitis, periodonitis, endometriosis, sepsis, sleep apnea, AIDS (HIV-I), Antiphospholipid Syndrome, Waldenstrom macroglobulinemia, Chronic Disease Syndrome, Familial Mediterranean Fever, Hereditary Periodic Fever Syndrome, psychosocial stress diseases, neuropathological diseases, familial amyloidotic polyneuropathy, traumatic brain injury, spinal cord injury, Parkinson's Disease, Rheumatic Disease, Alzheimer's Disease, amyotropic lateral sclerosis, Huntington's Disease, retinal disease, cataracts, hearing loss, and cancer (such as cancer of the breast, cervix, ovary, vulva, prostate, kidney, bladder, liver, pancreas, stomach, colon, thyroid, parathyroid, and lung, as well as mesothelioma, non small- cell lung cancer, esophygeal/gastric cancer, laryngeal cancer, melanoma, squamous cell carcinoma, head and neck cancer, endometrial (uterus) cancer, cylindromatosis, trichoepithelioma, Hilar Cholangiocarcinoma, oral carcinoma, astrocytoma/glioblastoma, neuroblastoma, glioblastoma, Hodgkin's lymphoma, acute lymphoblastic leukemia, acute myelogenous leukemia, acute T-cell leukemia (+/-HTLV-1), acute non-lymphocytic leukemia, chronic lymphocytic leukemia, Burkitt's Lymphoma (EBV), mantle cell lymphoma, myelodysplastic syndrome, multiple myeloma, diffuse large B-cell lymphoma, and MALT lymphoma). The inhibitors of RPS3 also can be used to treat the above-described diseases and disorders.

[0041] Inhibitors of RPS3 include, but are not limited to, small molecules, short interfering nucleic acid (siNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), antibodies, antisense compounds, aptamers, peptidomimetics, and combinations thereof.

[0042] Methods of disrupting gene expression are known in the art (see, e.g., Marx, Science, 288: 1370-1372 (2000)). For example, small nucleic acid molecules, such as siNA, siRNA, dsRNA, miRNA, and shRNA can be used to mediate RNA interference (RNAi) against RPS3 gene expression.

[0043] shRNA is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNAi. Selection of suitable shRNA, as well as the vectors for the expression of shRNA, are known in the art and described in Example 6. For example, a suitable shRNA sequence for inhibiting RPS3 includes SEQ ID NO: 14. [0044] siRNA are a class of double-stranded RNA molecules that are about 18 to about 25 (e.g., 19, 20, 21, 22, 23, or 24) nucleotides in length. siRNA interferes with the expression of a specific gene (RPS3). When the inhibitor is a siRNA, the inhibitor can be selected from the group consisting of SEQ ID NOs: 1-4, as described in Example 2. [0045] When the inhibitor is an antisense compound, the inhibitor preferably is an antisense oligonucleotide. The oligonucleotide (e.g., DNA or RNA) employed can have a sequence that is complementary to the sequence of the target RNA (i.e., RPS3 RNA). Absolute complementarity is not required, and any oligonucleotide having sufficient complementarity to form a stable duplex with the target RNA so that translation of the RNA is inhibited is suitable. Oligonucleotides of about 8 to about 40 nucleotides in length (e.g., about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, or about 35 nucleotides), preferably of about 8 to about 30 nucleotides in length, and more preferably of about 8 to about 20 nucleotides in length, and having a sufficient complementarity to form a duplex having a melting temperature of greater than about 40° C are particularly suitable. The antisense oligonucleotide is considered effective as long as the translation of the mRNA to which the oligonucleotide is complementary is inhibited.

[0046] Aptamers are nucleic acid macromolecules that can bind to proteins, nucleotides, and complexes. When the inhibitor of RPS3 is an apatamer, the aptamer is about 10 to about 50 nucleotides (about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides) in length and, preferably, about 15 to about 40 nucleotides in length.

[0047] Antibodies to RPS3 are known in the art and include commercially available antibodies. Antibodies to RPS3 can be prepared using well-established methodologies (see, e.g., Harlow and Lane, in Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988, pages 1-725). Such antibodies can comprise both polyclonal and monoclonal antibodies.

[0048] An "effective amount" refers to a dose of the inhibitor that is adequate to inhibit the expression of NF-κB target genes, inhibit the induction of immunoglobulin K light chain gene expression in B cells, and/or inhibit or treat a disease or disorder (e.g., an inflammatory disease or disorder, cancer, etc.) in a mammal. Amounts effective for a therapeutic or prophylactic use will depend on, for example, the stage and severity of the disease or disorder being treated, the age, weight, and general state of health of the patient, and the judgment of the prescribing physician. The size of the dose will also be determined by the compound selected, method of administration, timing and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound and the desired physiological effect. It will be appreciated by one of skill in the art that various diseases or disorders could require prolonged treatment involving multiple administrations, perhaps using a series of different RPS3 inhibitors, anti-inflammatory agents, and/or NF-κB inhibitors in each or various rounds of administration. [0049] The RPS3 inhibitor can be administered alone or in a composition (e.g., pharmaceutical composition) that can comprise at least one carrier (e.g., a pharmaceutically acceptable carrier), as well as other therapeutic agents (e.g., different RPS3 inhibitors, antiinflammatory agents, and/or NF-κB inhibitors). The inhibitor or composition can be administered by any suitable route, including parenteral, topical, oral, or local administration. [0050] The pharmaceutically acceptable carrier (or excipient) is preferably one that is chemically inert to the RSP3 inhibitor and one that has no detrimental side effects or toxicity under the conditions of use. Such pharmaceutically acceptable carriers include, but are not limited to, water, saline, Cremophor EL (Sigma Chemical Co., St. Louis, MO), propylene glycol, polyethylene glycol, alcohol, and combinations thereof. The choice of carrier will be determined in part by the particular RPS3 inhibitor, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the composition.

[0051] The following formulations for oral, aerosol, parenteral (e.g., subcutaneous, intravenous, intraarterial, intramuscular, intradermal, interperitoneal, and intrathecal), rectal, and vaginal administration are merely exemplary and are in no way limiting. [0052] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and cornstarch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.

[0053] The RPS3 inhibitors alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifiuoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. [0054] Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The RPS3 inhibitor can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane- 4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants. [0055] Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0056] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene-polypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (3) mixtures thereof. [0057] Suitable preservatives and buffers can be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0058] The RPS3 inhibitor can be administered as an injectable formulation. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986). [0059] Topical formulations, including those that are useful for transdermal drug release, are well known to those of skill in the art and are suitable in the context of the invention for application to skin. [0060] The RPS3 inhibitor can be administered as a suppository by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

[0061] The concentration of a compound of the present invention in the pharmaceutical formulations can vary, e.g., from less than about 1%, usually at or at least about 10%, to as much as 20% to 50% or more by weight, and can be selected primarily by fluid volumes, and viscosities, in accordance with the particular mode of administration selected. [0062] Methods for preparing administrable (e.g., parenterally administrable) inhibitors of RPS3 are known or apparent to those skilled in the art and are described in more detail in, for example, Remington 's Pharmaceutical Science (17th ed., Mack Publishing Company, Easton, PA, 1985).

[0063] In addition to the aforedescribed pharmaceutical compositions, the inhibitors of RPS3 can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes. Liposomes can serve to target the inhibitor to a particular tissue. Liposomes also can be used to increase the half-life of the inhibitor. Many methods are available for preparing liposomes, as described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9, 467 (1980) and U.S. Patents 4,235,871, 4,501,728, 4,837,028, and 5,019,369. [0064] When the inhibitor of RPS3 is administered with one or more additional therapeutic agents (e.g., different RPS3 inhibitors, anti-inflammatory agents, and/or NF-κB inhibitors), the inhibitor and one or more additional therapeutic agents can be coadministered to the mammal. By "coadministering" is meant administering one or more additional therapeutic agents and the inhibitor of RPS3 sufficiently close in time such that the inhibitor can enhance the effect of one or more additional therapeutic agents. In this regard, the inhibitor of RPS3 can be administered first and the one or more additional therapeutic agents can be administered second, or vice versa. Alternatively, the inhibitor of RPS3 and the one or more additional therapeutic agents can be administered simultaneously. For example, the one or more additional therapeutic agents can be selected from the group selected from nonsteroidal anti-inflammatory drugs (NSAIDS) and steroids. [0065] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. Additional details of the experiments set forth in the Examples are described in Wan et al., Cell, 131(5): 927-939 (2007).

EXAMPLE 1

[0066] This example demonstrates that RPS3 interacts with p65.

[0067] To identify proteins involved in p65 recruitment to DNA, p65 was fused with two high affinity peptides, a streptavidin binding peptide and a calmodulin binding peptide as tandem affinity peptides (TAP), and overexpressed in HEK 293T cells. This allowed the coprecipitation of p65 binding proteins from whole cell lysates through two consecutive affinity purification steps. Mass spectrometry identified RPS3 as a major associated protein. No other ribosomal proteins were identified in this matter, excluding a contamination of the protein isolates with whole ribosomes.

[0068] To exclude the possibility that the association of RPS3 is due to nonspecific binding in the TAP system, the interaction between p65 and RPS3 was analyzed. The binding of endogenous p65, as well as other NF-κB family proteins, to RPS3 was assessed by coimmunoprecipitation of extracts prepared from cells transfected with a vector expressing an epitope-tagged version of RPS3. These studies revealed a strong interaction between RPS3 and p65, but not between RPS3 and either p50, ReI B, or c-Rel. [0069] The interaction between RPS3 and p65 also was analyzed using nuclear extracts from TCR-stimulated Jurkat A3 cells in a glutathione S-transferase (GST)-RPS3 pulldown assay, which demonstrated that RPS3, but not the GST control, bound strongly to p65. [0070] The presence of endogenous RPS3 also was detected in immunoprecipitates of endogenous p65. The specificity of this interaction was verified by the fact that p65 also brought down IκBα, but none of the proteins were precipitated by isotype control antibody. In contrast, RPS 16, another component of the small ribosome 4OS subunit, was not precipitated, thereby indicating there was no specific interaction between p65 and intact ribosomes or another ribosome protein.

[0071] These experiments demonstrate a strong and specific physical interaction between RPS3 and p65. EXAMPLE 2

[0072] This example demonstrates that knockdown of RPS3 specifically impairs NF-κB signaling.

[0073] To determine whether RPS3 plays a role in NF-κB signaling, the effect of RNAi- mediated knockdown of RPS3 on the expression of a NF-κB-driven luciferase reporter construct in Jurkat cells was measured. Jurkat cells were transiently transfected with 200 pmol of siRNA by electroporation using Electrocell Manipulator (600) (BTX) as described in Su et al., Science, 307: 1465-1468 (2005). Four different RPS3 siRNA duplexes (namely, siRNA duplex 1 sense strand corresponding to SEQ ID NO: 1; siRNA duplex 2 sense strand corresponding to SEQ ID NO: 2; siRNA duplex 3 sense strand corresponding to SEQ ID NO: 3; and siRNA duplex 4 sense strand corresponding to SEQ ID NO: 4) decreased RPS3 protein by roughly 50% compared to nonspecific (NS) control siRNA. As expected, T cell receptor (TCR) and tumor necrosis factor alpha (TNFα) stimulation increased NF-κB-driven luciferase activity in NS siRNA treated cells in a time- and dose-dependent manner. In contrast, knockdown of RPS3 using specific siRNA duplexes significantly reduced the induced NF-κB signaling, thereby indicating that RPS3 is necessary for TCR- and TNFα- induced NF-κB transcriptional activity. A similar decrease in luciferase activity also was observed following knockdown of p65 (siRNA duplex sense strand corresponding to SEQ ID NO: 9).

[0074] NF-κB activity could be restored by overexpression of a RPS3 expression construct containing a mutated nucleotide sequence that could not be targeted by RPS3 siRNA, which confirmed that the loss of NF-κB -driven transcription was not due to off-target affects of the siRNA duplexes. Briefly, to generate RPS3 expression constructs that were not recognized by siRNA duplexes, RPS3 was subcloned into a 3FLAG expression vector (Sigma), and the 3FLAG-RPS3 plasmid was mutated using the primers of SEQ ID NO: 5 and SEQ ID NO: 6 for siRNA duplex 1 and the primers of SEQ ID NO: 7 and SEQ ID NO: 8 for siRNA duplex 3.

[0075] Furthermore, RNAi knockdown of a different 4OS ribosomal component, RPS 16 (i.e., siRNA duplex sense strand corresponding to SEQ ID NO: 10), had no effect on NF-κB- dependent reporter gene activity. These data specifically implicate RPS3 in the NF-κB activation of a reporter gene triggered by TCR or TNF simulation in T cells. [0076] To confirm that decreased NF-κB signaling in RPS3-silenced cells did not result from general inhibition of protein synthesis, protein synthesis in cells that received NS versus RPS3 siRNA were compared. Two different reporter constructs driven independently of NF- KB binding, pTKRL and pE-selectin (ΔκB)-Luc, showed comparable reporter gene induction in both NS- and RPS3-silenced cells. In addition, the Coomassie staining pattern and relative amount of protein found in whole cell lysates prepared from equal numbers of NS- or RPS3- silenced cells were essentially the same, thereby indicating that partially decreasing RPS3 expression in these cells did not reduce overall protein level per cell. Furthermore, EGFP expression from a transfected plasmid was nearly equal in both NS- and RPS16-silenced cells and modestly higher in RPS3 silenced cells, which demonstrated de novo protein synthesis was intact in both RPS3- and RPS16-silenced cells. Thus, abrogation of NF-κB signaling in the presence of RPS3 silencing is not due to a general protein translational defect, but instead is indicative of a role for RPS3 in NF-κB signaling beyond the traditional function in the ribosome.

[0077] To determine the effect of RPS3 knockdown on other TCR-induced signaling pathways, SP-I and NF-AT signaling following T cell stimulation was tested. Knockdown of RPS3 did not impair phorbol myristate acetate (PMA)-induced AP-I signaling as assessed by an AP-I reporter gene, which further demonstrated that RPS3 silencing did not alter de novo protein synthesis. Furthermore, TCR-induced NF-AT nuclear translocation as detected by immunoblotting nuclear extracts was normal after RPS3 knockdown. Therefore, the knockdown of RPS3 selectively affected NF-κB signaling and not other branches of TCR- induced signal transduction. [0078] This example demonstrates that NF-κB signaling is affected by RPS3 knockdown.

EXAMPLE 3

[0079] This example demonstrates the extra-ribosomal function of RPS3 in NF-κB signaling.

[0080] Based on the reduction of NF-κB signaling upon RPS3 knockdown and the physical interaction between RPS3 and p65, the method by which RPS3 exerts its effect on p65 liberation (from IκBα or nuclear translocation) was explored. IκBα degradation occurred normally in cells transfected with RPS3 siRNA, indicating that RPS3 does not function at the level of NF-κB liberation. As expected, p65 rapidly accumulated in the nucleus following TCR stimulation when assessed by immunofluorescence staining and immunoblotting. Remarkably, a fraction of RPS3 also translocated to the nucleus after TCR and TNFα stimulation with similar kinetics.

[0081] To exclude the possibility that TCR triggering stimulated nuclear shuttling of whole ribosomes containing RPS3, RPS 16 was probed under the same conditions. The cellular distribution of RPS 16 remained unchanged, indicating that nuclear translocation of RPS3 in response to T cell stimulation did not involve other ribosomal proteins and, therefore, not whole ribosomes.

[0082] To determine whether RPS3 translocates independently of p65 or whether the two proteins cotranslocate into the nucleus via their physical interaction, immunofluorescence confocal microscopy was used to examine the effect of RPS3 knockdown on p65 nuclear accumulation. TCR simulation caused nuclear translocation of p65 in both NS- and RPS3- silenced cells. RPS3-independent p65 transfer from the cytoplasm to the nucleus also was observed in Western blots of p65 in cellular fractions over time in RPS3-silenced cells. Thus, RPS3 does not appear to govern p65 nuclear accumulation. In the reverse experiment, TCR stimulation induced RPS3 nuclear accumulation. Each protein contains an N-terminal nuclear localization signal (NLS) that could permit the independent nuclear translocation after stimulation. Nuclear translocation of RPS3, independently of whole ribosomes, further highlights its extra-ribosomal function in NF-κB signaling, since the translation function of RPS3 is believed to operate in the cytoplasm.

EXAMPLE 4

[0083] This example demonstrates that RPS3 contributes to NF-κB signaling. [0084] A variety of coactivators and corepressors have been shown to influence NF-κB signaling, most of which physically associate with NF-κB family proteins (see, e.g., Benezra et al., J. Biol. Chem., 278: 26333-26341 (2003); Covic et al., Embo. J., 24: 85-96 (2005); Gao et al., J. Biol. Chem., 280: 21091-21098 (2005); Koyanagi et al.,J. Biol. Chem., 280: 12430- 12437 (2005); Lara-Pezzi et al., J. Biol. Chem., 279: 6553-6559 (2004); and Leung et al., Cell, 118: 453-464 (2004)). Since RPS3 knockdown severely impaired NF-κB signaling, RPS3 could operate as a coactivator for p65. However, RPS3 overexpression did not boost NF-κB signaling either before or after TCR or TNFα stimulation. The reporter construct pG5-Luc with GAL4 fused to RPS3, in both full length and truncated variations, triggered almost no luciferase activity compared to two positive controls, GAL4-VP16 and GAL4-E1 A (see Michelotti et al., J. Biol. Chem., 272: 22526-2253 (1997)). This suggests that RPS3 possesses little intrinsic transcriptional activity and, therefore, does not conform to the role of a traditional coactivator (see Gao et al., supra; Rosenfield et al., Genes Dev., 20: 1405-1428 (2006)).

[0085] To determine whether p65 overexpression could bypass the requirement of RPS3 for NF-κB signaling, cells were transfected with NS or RPS3 siRNA. In cells that received NS siRNA, p65 overexpression significantly increased NF-κB signaling. In contrast, NF-κB activity failed to increase proportionately with p65 expression in cells transfected with RPS3 siRNA, thereby indicating that excess p65 does not override the requirement for RPS3. To further confirm that RPS3 is critical for endogenous p65, the inhibitor of KB kinase beta (IKKβ), which functions upstream of p65, was overexpressed to activate endogenous p65. In NS siRNA-treated cells, overexpression of IKKβ boosted NF-κB signaling, whereas RPS3 knockdown substantially blunted the induced signal. These data are consistent with the finding that silencing RPS3 does not impair IκBα degradation, but does reduce a later step in NF-κB signaling induced by p65 overexpression. The failure of excess p65 to bypass the NF- KB defect in the absence of RPS3 indicates that RPS3 functions downstream or in conjunction with p65. Coexpression of RPS3 and p65 further increased NF-κB signaling in an RPS3 dose-dependent manner from 0.5 to 2 fold higher than the elevated signal induced by p65 overexpression alone. These data indicate that RPS3 plays a critical role in p65 mediated NF-κB signaling, most likely in stoichiometric relation to the concentration of p65 present in the cell. A mutual transcriptional function involving RPS3 and p65 also was supported by enhanced transcription of pG5-Luc using coexpression of GAL4-RPS3 and p65 compared with p65 alone, recognizing that GAL4-RPS3 itself possesses little, if any, transcriptional activity. Therefore, RPS3 is important for NF-κB signaling, depending on a mutual transcriptional function involved in cellular concentration of p65.

EXAMPLE 5

[0086] This example further demonstrates that RPS3 interacts with p65. [0087] To determine if RPS3 regulates a nuclear step in NF-κB target gene transcription, as series of experiments were undertaken. First, a FLAG-tagged version of RPS3 for immunoprecipitation (IP) was employed in order to test whether RPS3 could be recruited to promoters in NF-κB target genes in vivo using a chromatin IP (ChIP) assay (see Ainbinder et al., MoI. Cell, Biol, 22: 6354-6362 (2002)). The recruitment of FLAG-RPS3 to the 200-300 base pair region containing KB binding sites in the promoters of two known NF-κB-driven genes, IκBα and IL-8, was detected, whereas IP with an isotype control antibody was negative. This indicated that RPS3 could be a component of the NF-κB transcriptional complex formed at relevant promoters.

[0088] To further define how RPS3 could be incorporated into the transcriptional complex, electrophoretic mobility shift assays (EMSAs) using nuclear extracts prepared from 293T cells transfected with or without FLAG-tagged RPS3 were employed. In nuclear extracts from both untransfected and RPS3-overexpressing cells, TNFα stimulation strongly increased the signal for two presumed NF-κB specific bands due to nuclear translocation of NF-κB proteins. The specificity of these two NF-κB dependent bands was confirmed by cold oligonucleotide competition: wild type KB oligonucleotides (double-stranded oligonucleotide probe corresponding to SEQ ID NO: 11) competed away both NF-κB dependent bands, as well as non-specific bands, whereas mutant KB (double-stranded oligonucleotide probe corresponding to SEQ ID NO: 12) and unrelated OCTl oligonucleotides (double-stranded oligonucleotide probe corresponding to SEQ ID NO: 13) only diminished the nonspecific bands.

[0089] A supershift assay with p65 and p50 antibodies further confirmed this specificity and indicated that the upper and lower bands represented p65 homodimers and p65-p50 heterodimers, respectively, consistent with previous reports (see Sathe et al., Proc. Natl. Acad. ScL USA, 101: 192-197 (2004)). αFLAG antibody supershifted both NF-κB bands, similar to αp65 antibody in cells overexpressing FLAG-tagged RPS3, whereas no shift was observed with the same antibody in untransfected cells. This result suggests that RPS3 was present in both NF-κB bands containing p65, which supports the physical interaction between RPS3 and p65. Since the supershift assay with isotype control antibody had no effect whether RPS3 was overexpressed or not, this result excluded the possibility that the αFLAG- induced supershift was due to a nonspecific antibody effect. These results, combined with the ChIP data, strongly indicate that RPS3 and p65 are bound to each other and jointly recruited to KB sites in promoters of NF-κB targeted genes.

[0090] To determine the effects on the NF-κB transcriptome after knockdown of RPS3 in stimulated T cells, microarray assays comparing RPS3-silenced versus NS-treated samples on the same chip were performed (see Shaffer et al., Immunity, 13: 199-212 (2000); Lam et al., Clin. Cancer Res., 11: 28-40 (2005)). mRNA from Jurkat cells transfected with nonspecific siRNA was labeled with Cy3 dye and mRNA from Jurkat cells transfected with p65 or RPS3 siRNA was labeled with Cy5 dye. A gene was selected as an NF-κB target gene if both p65 and RPS3 siRNA transfected cells decreased expression of the gene by > 1.4 fold at >1 time points in both time courses.

[0091] Although numerous NF-κB target genes and genomic binding sites have been identified, it is unclear how many functional binding sites are direct targets of the transcription factor in the whole human genome (see Natoli et al., Nat. Immunol, 6: 439-445 (2005)). TCR stimulation induces a plethora of NF-κB-dependent, as well as NF-κB- independent, genes evident on the microarray. The transcription of the genes induced by TCR stimulation was diminished in p65-defϊcient cells versus NS controls, evident by 151 spots on the chip, of which 66 spots (44%) also were dramatically downregulated in the absence of RPS3. Among those common downregulated spots, some known NF-κB genes, such as NFKBIA, NFKBl, NFKB2, BCL2A1, CD83, and FAS, were shown to be controlled by both p65 and RPS3, indicating parallel in vivo function of RPS3 and p65 at the transcriptional level of these genes. Conversely, a similar panel of TCR-induced genes, including many that are known to be NF-κB -independent, were unaffected by either p65 or RPS3 knockdown.

[0092] These results demonstrate that there was substantial, but not total, overlap of the RPS3 requirement for the expression of NF-κB target genes driven by p65.

EXAMPLE 6

[0093] This example demonstrates that RPS3 mediates cellular responses involving NF- KB signaling.

[0094] To determine whether endogenous RPS3 is required for NF-κB binding to and activation of target genes, an additional ChIP analysis involving endogenous p65, without RPS3 knockdown, was performed. As expected, p65 was recruited to IκBα and IL-8 promoters after stimulation in NS oligoribonucleotide-treated cells. In contrast, p65 recruitment to these promoters was diminished or almost abolished under RPS3-deficient conditions. These data indicate that RPS3 is required for and facilitates p65 binding to KB sites in gene control sequences. This view was further supported by an in vitro TransAm™ assay with recombinant p65 and RPS3 proteins, in which p65 DNA binding activity was measured by ELISA with the KB site-containing DNA immobilized on a microtiter plate. Recombinant p65 bound to KB DNA, assumed to be the p65 homodimer. Preincubating recombinant p65 with GST-RPS3 protein significantly increased p65 DNA binding activity in a dose-dependent manner, whereas the GST carrier protein did not. GST or GST-RPS3 protein alone was not recognized by p65 ELISA, excluding the possibility that the increased signaling in the presence of GST-RPS3 was due to a cross-reaction between p65 antibody and GST or RPS3 protein. These data suggest that at least in vitro, RPS3 could facilitate p65 binding to the KB site-containing DNA. The DNA binding activity assays also were used with nuclear extracts from the cells transfected with NS or RPS3 siRNAs. As expected, in NS oligoribonucleotide-treated cells, TNFα stimulation enhanced p65 nuclear translocation and, thus, DNA binding activity. In contrast, knockdown of RPS3 significantly decreased p65 DNA binding activity, but did not impair p65 nuclear translocation. These data indicate that RPS3 increases p65 binding to the KB sequence, which is a critical event for p65 transcriptional activity.

[0095] Repeating the assay in which the NF-κB transcriptional factor was first identified described in Example 1, lipopolysaccharide (LPS)-induced Ig K light chain expression was measured in murine pre-B 70Z/3 cells in which the p65-p50 heterodimer is a key regulator (see Miyamoto et al., Proc. Natl. Acad. Sd. USA, 91: 5056-5060 (1994); Sen et al., Cell, 47: 921-928 (1986)). To optimize and stabilize the RPS3 knockdown, a short hairpin RNA (shRNA) including the RPS3 sequence was inserted into a green fluorescent protein (GFP)- expressing lentiviral vector (see Furumoto et al., J. Immunol, 176: 5167-5171 (2006)). Briefly, mouse RPS3 (NM_012052) shRNA (sense and antisense strands corresponding to SEQ ID NOs: 14 and 15, respectively) were ligated into the GFP expressing pNUTS vector via Apa I/Bst Xl sites (see Furumoto, supra).

[0096] The shRNA-containing vector silenced RPS3 in 70Z/3 cells by lentiviral-mediated transduction. Lentiviral transduction efficiency ranged from 90% to 94% (as assessed by GFP expression) in both RPS3 downregulated and control (empty vector) 70Z/3 cells. Silencing RPS3 with shRNA slightly diminished basal K light chain expression, but dramatically inhibited LPS-induced K light chain expression on the cell surface as assessed by both flow cytometry and confocal microscopy. IFNγ stimulation, which is known to induce K light chain upregulation in 70Z/3 cells via an NF-κB independent mechanism (see Briskin et al., Science, 242: 1036-1037 (1988)), was used as a negative control. The level of K light chain expression induced by IFNγ was the same whether or not RPS3 was knocked down. Mean fluorescence intensity quantification confirmed that reduction of RPS3 specifically curtailed the LPS-induced, NF-κB-dependent pathway without affecting the IFNγ-induced pathway. Measurements of the level of K light chain mRNA by quantitative polymerase chain reaction (Q-PCR) showed that decreasing RPS3 also impaired the induction of K light chain message. Taken together, these data indicated that the RPS3 effects were primarily transcriptional, not translational, and the ability of NF-κB to exert its full physiological regulatory function on Ig gene expression depended on RPS3. [0097] The effects of RPS3 knockdown on NF-κB function in other physiological processes, including cell proliferation and anti-apoptosis, also were screened. Knockdown of RPS3 enhanced ultraviolet irradiation-induced apoptosis in Jurkat cells and dramatically blocked TCR stimulation-induced cell proliferation and interleukin-2 production in resting primary peripheral blood lymphocytes.

[0098] These findings support a prominent role for RPS3 in mediating important physiological cellular responses involving NF-κB signaling.

[0099] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[00100] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [00101] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A method of inhibiting the expression of NF-κB target genes in a mammal comprising administering an effective amount of an inhibitor of ribosomal protein S3 (RPS3) to the mammal, such that the expression of NF-κB target genes in the mammal is inhibited.

2. The method of claim 1 , wherein the inhibitor inhibits binding of NF-κB complexes to the target genes.

3. A method of inhibiting the induction of immunoglobulin K light chain gene expression in B cells comprising administering an effective amount of an inhibitor of ribosomal protein S3 (RPS3) to the B cells, such that the induction of immunoglobulin K light chain gene expression in B cells is inhibited.

4. A method of treating an inflammatory disease or disorder in a mammal comprising administering an effective amount of an inhibitor of ribosomal protein S3 (RPS3) to the mammal, such that the inflammatory disease or disorder is treated.

5. The method of claim 4, wherein the inflammatory disease or disorder is selected from the group consisting of uveitis, allergy, autoimmune disease, allograft rejection, arthritis, colitis, psoriasis, and combinations thereof.

6. The method of claim 4, wherein the inflammatory disease or disorder is selected from the group consisting of rheumatoid arthritis, rheumatoid spondylitis, ostero- arthritis, asthma, gouty arthritis, adult respiratory distress syndrome, stroke, reperfusion injury, neural trauma, neural ischemia, restenosis, chronic pulmonary inflammatory disease, graft-versus-host reaction, Crohn's disease, ulcerative colitis, inflammatory bowel disease, systemic lupus erythematosis, multiple sclerosis, vasculitis, and combinations thereof.

7. The method of any of claims 1-6, wherein the inhibitor is selected from the group consisting of a small molecule, short interfering nucleic acid (siNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), antibody, antisense compound, aptamer, peptidomimetic, and combinations thereof.

8. The method of any of claims 1-6, wherein the inhibitor is siRNA.

9. The method of claim 8, wherein the siRNA is selected from the group consisting of SEQ ID NOs: 1-4.

10. The method of any of claims 1 -6, wherein the inhibitor is shRNA.

11. The method of claim 10, wherein the shRNA is SEQ ID NO: 14.

12. The method of any of claims 1-11, wherein the inhibitor is administered in a composition comprising at least one carrier.

13. The method of claim 12, wherein the composition is a pharmaceutically acceptable composition and the at least one carrier is a pharmaceutically acceptable carrier.

14. The method of claim 12 or 13, wherein the composition comprises at least one additional therapeutic agent selected from the group consisting of anti-inflammatory agents andNF-κB inhibitors.