HSP90 INHIBITOR FOR THE TREATMENT OF CANCER AND INFLAMMATORY

DISEASES

[0001] This application claims the benefit of United States Provisional Patent Application No. 61/606,551, filed March 5, 2012, incorporated herein by reference in its entirety.

[0002] This invention was made with government support under grants AI070603 and AI077283 awarded by the National Institute of Health. The government has certain rights in the invention.

INCORPORATION OF SEQUENCE LISTING [0003] The sequence listing that is contained in the file named "MESCP0060WO_ST25.txt", which is 5 KB (as measured in Microsoft Windows®) and was created on February 22, 2013, is filed herewith by electronic submission and is incorporated by reference herein.

BACKGROUND OF THE INVENTION 1. Field of the Invention

[0004] The invention concerns the fields of molecular biology and specifically concerns methods and compositions involving anticancer and anti-inflammatory agents.

2. Description of Related Art

[0005] HSP90 is a housekeeping protein essential in chaperoning a large number of proteins in the cytosol. Its clients include molecules in multiple signaling pathways and tumor-related gene products. It is widely observed that cancer cells rely more heavily on the chaperoning function of HSP90 compared to normal cells. Therefore, targeting HSP90 could potentially kill tumor cells specifically but not normal cells.

[0006] Existing technologies targeting HSP90 are comprised mainly of small-molecule inhibitors, such as 17AAG, PU70 etc. Therefore, there remains a need to develop novel biological peptide inhibitors that could potentially increase the specificity of HSP90 targeting and reduce the side effects caused by small-molecule inhibitors. SUMMARY OF THE INVENTION

[0007] In a first embodiment, the invention provides an isolated peptide of 50 amino acids or less in length that comprises SEQ ID NO: l (LNISREMLQQSKILKVIRKNIVKKCLELFSELAEDKE), or a sequence that is at least 80%, 85%, 86%, 87%, 88%, 89% or 90% identical thereto. This isolated peptide may include a sequence that is about 90, 92, 94, 95, 96, 98, 100% identical to SEQ ID NO: l. In certain cases the isolated peptide may comprise a deletion or truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids from SEQ ID NO: l. In still further aspects, an isolated polypeptide may comprise a sequence of SEQ ID NO: l, but include, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions or insertions. The isolated peptide may be further defined as being 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30 amino acids or less in length.

[0008] Furthermore it will be understood by the skilled artisan that this isolated peptide may comprise amino acid substitutions relative to SEQ ID NO: l . In some very specific aspects the isolated peptide may be identical to the sequence given by SEQ ID NO: 1 (referred herein as PIER3). Further embodiments of the isolated peptide contemplated by the invention are provided in the detailed description of the embodiments.

[0009] In some further aspects the isolated peptide may comprise a cell internalization moiety. In some cases a cell internalization moiety may be conjugated to the isolated peptide. For example, the isolated peptide may be provided in complex with a liposomal vesicle thereby enabling the polypeptide to traverse the cell membrane. Furthermore, in some specific embodiments a cell internalization moiety may be a peptide, a polypeptide, an aptamer or an avimer (see for example U.S. Applns. 20060234299 and 20060223114) sequence. For example, a cell internalization moiety may comprise amino acids from the HIV TAT, HSV-1 tegument protein VP22, or Drosophila antennopedia homeodomain. In certain further aspects, a cell internalization moiety may be an engineered internalization moiety such as a poly-Arginine, a poly-methionine and/or a poly-glycine peptide such as Methionine and Glycine peptides described by Wright et al. (2003) and Rothbard et al. (2000). For example, a cell internalization moiety may be the sequence of RMRRMRRMRR (SEQ ID NO:5) exemplified herein. In certain aspects, the cell internalization moiety may be at the N-terminus of the isolated peptide or, alternatively, at the C-terminus of the isolated peptide. In specific embodiments, the fusion protein may comprise a Tat sequence GRKKRRQRRRPQ (SEQ ID NO: 6) fused to the N-terminus of SEQ ID NO: 1 or its variant. [0010] Thus, in some cases a peptide or polypeptide cell internalization moiety and the isolated peptide may form a fusion protein. The skilled artisan will understand that such fusion proteins may additionally comprises one or more amino acid sequences separating the cell internalizing moiety and the isolated peptide sequence. For example, in some cases a linker sequence may separate these two domains. For example, a linker sequences may comprise a "flexible" amino acids with a large number or degrees of conformational freedom such as a poly glycine linker. In some cases, a linker sequence may comprise a proteinase cleavage site. For instance, a linker sequence may comprise a cleavage site that is recognized and cleaved by an intracellular proteinase, thereby releasing the isolated peptide sequence from the cell internalization sequence once the fusion protein has been internalized.

[0011] In further aspects of the invention a cell internalization moiety may be further defined as a cell targeting moiety, which is a moiety that binds to or is internalized by only a selected population of cells such as cells expressing a particular cellular receptor. Such a cell targeting may, for example, comprise an antibody, a growth factor, a hormone, a cytokine, an aptamer or an avimer that binds to a cell surface protein. As used herein the term antibody may refer to an IgA, IgM, IgE, IgG, a Fab, a F(ab')2, single chain antibody or paratope peptide. In certain cases, a cell targeting moiety of the invention may target a particular type of cells such as a liver, skin, kidney, blood, retinal, endothelial, iris or neuronal cell. In still further aspects a cell targeting moiety of the invention may be defined as cancer cell binding moiety. For example, in some very specific cases a cell targeting moiety of the invention may target a cancer cell associated antigen such a gp240 or Her-2/neu.

[0012] In still further aspects of the invention the isolated peptide may comprise additional amino acid sequences such as a cell trafficking signal (e.g., a cell secretion signal, a nuclear localization signal or a nuclear export signal) or a reporter polypeptide such as an enzyme or a fluorescence protein. In a preferred aspect for example, the isolated peptide comprises a cellular secretion signal. For example, the isolated peptide may comprise a secretion sequence from a human gene such as the IL-2 secretion signal sequence. Thus, in certain cases, the isolated peptide may comprise a cell internalization moiety and cell secretion signal, thereby allowing the polypeptide to be secreted by one cells and internalized into a surrounding a cell.

[0013] In a further embodiment of the invention there is provided an isolated nucleic acid sequence comprising a sequence encoding the isolated peptide as described supra. Thus, a nucleic acid sequence encoding any of the isolated peptides or polypeptide fusion proteins described herein are also included as part of the instant invention. The skilled artisan will understand that a variety of nucleic acid sequences may be used to encode identical polypeptides in view of the degeneracy of genetic code. In certain cases for example the codon encoding any particular amino acid may be altered to improve cellular expression.

[0014] In preferred aspects, a nucleic acid sequence encoding the isolated peptide is comprised in an expression cassette. As used herein the term "expression cassette" means that additional nucleic acids sequences are included that enable expression of the isolated peptide in a cell, or more particularly in a eukaryotic cell. Such additional sequences may, for examples, comprise a promoter, an enhancer, intron sequences (e.g., before after or within the isolated peptide-encoding region) or a polyadenylation signal sequence. The skilled artisan will recognize that sequences included in an expression cassette may be used to alter the expression characteristics of the isolated peptide. For instance, cell type specific, conditional or inducible promoter sequences may be used to restrict expression of the isolated peptide to selected cell types or growth conditions. For example, in certain cases a hypoxia inducible promoter may be used in the expression cassettes of the invention. Furthermore, in some instances promoters with enhanced activity in cancer cells or cells of the eye may be employed. Furthermore, it is contemplated that certain alterations may be made to the isolated peptide-encoding sequence in order to enhance expression from an expression cassette for example, as exemplified herein, the initiation codon of the coding sequence of the isolated peptide may be changed to ATG to facilitate efficient translation.

[0015] In still further aspects of the invention a coding sequence of the isolated peptide may be comprised in an expression vector such as a viral expression vector. Viral expression vectors for use according to the invention include but are not limited to adenovirus, adeno- associated virus, herpes virus, SV-40, retrovirus and vaccinia virus vector systems. In certain preferred aspects, a retroviral vector may be further defined as a lentiviral vector. In some cases such lentiviral vectors may be self-inactivating (SIN) lentiviral vector such as those described in U.S. Applns. 20030008374 and 20030082789 incorporated herein by reference.

[0016] The isolated peptide may bind to HSP90 and inhibit its activity in a cell, specially a cancer cell or an inflammatory cell. There may be provided a pharmaceutical composition comprising the isolated peptide and a pharmaceutically acceptable carrier. In some respects, the invention provides methods for inhibiting or reducing HSP90 activity comprising expressing the isolated peptide in a cell.

[0017] Thus, in a specific embodiment, there is provided a method for treating a patient with cancer or an inflammatory disease comprising administering to the patient an effective amount of a therapeutic composition comprising the isolated peptide or a nucleic acid expression vector encoding the isolated peptide as described supra. In preferred aspects, methods described herein may be used to treat a human patient.

[0018] As described above, in certain aspects, the invention provides methods for treating cancer. In certain cases, the methods herein may be used to inhibit or treat metastatic cancers. A variety of cancer types may be treated with methods of the invention, for example a cancer for treatment may be a bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, eye, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus cancer. Furthermore additional anticancer therapies may be used in combination or in conjunction with methods of the invention. Such additional therapies may be administered before, after or concomitantly with methods of the invention. For example an additional anticancer therapy may be a chemotherapy, surgical therapy, an immunotherapy or a radiation therapy. In other aspects, the invention provides methods for treating inflammatory diseases such as sepsis, graft rejection (e.g., transplant rejection) or an inflammatory autoimmune diseases, such as rheumatoid arthritis, inflammatory bowel disease, type I diabetes, multiple sclerosis, Sjogren's disease, myasthenia gravis, psoriasis, lupus, allergic reactions, and asthma.

[0019] It is contemplated that compositions of the invention may be administered to a patient locally or systemically. For example, methods of the invention may involve administering a composition topically, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, or via a lavage. [0020] Embodiments discussed in the context of a methods and/or composition of the invention may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the invention as well. [0021] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.

[0022] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.

[0023] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. [0024] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The following drawings are part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein.

[0026] FIGS. 1A-1D. PIER1 specifically inhibits responses to LPS but not to Pam ₃ CSK ₄ or CpG. FIG. 1A, PIER1 inhibits LPS responses in THP-1 cells. THP-1 cells were matured with PMA prior to LPS stimulation in the presence or absence of PIER1. The supernatant was harvested at 24 h and examined for TNFa by ELISA. FIG. IB, PIER1 inhibits LPS responses in pre-B cells. Pre-B cells stably expressing NFKB-GFP reporter were stimulated by LPS for 16-18 h. GFP expression was examined by flow cytometry. The mean fluorescence intensity was normalized to percentage of maximum NFKB-GFP expression (after stimulation with PMA and ionomycin). *, p < 0.01. FIG. 1C, Same as FIG. IB except Pam ₃ CSK ₄ was used as the stimulus. FIG. ID, Same as FIGS. 1B-1C except CpG was used as the stimulus. Experiments were repeated more than three times with similar results. Error bars represent standard error of the mean. AU, absorbance units.

[0027] FIG. 2. PIER1 inhibits binding of LPS to cell surface independently of gp96. WT or gp96 mutant (KO) pre-B cells were incubated with biotin-LPS in the presence or absence of PIER1 followed by flow cytometry for LPS-binding cells with streptavidin-allophycocyanin. Data are presented as both dot plots and histograms. Numbers indicate the percentage of LPS-binding cells. Multiple experiments were performed with similar results.

[0028] FIGS. 3A-3B. Dose-dependent inhibition of LPS binding by PIER1. LPS binding was performed as in FIG. 2 for both WT (FIG. 3A) and gp96 mutant pre-B cells (FIG. 3B) in the presence of increasing concentrations of PIER1 or control peptide. The percentage of LPS-binding cells was plotted. Two experiments were performed with similar results.

[0029] FIG. 4. PIER1 inhibits the interaction between LPS and HSP90 in vitro. Purified HSP90 was incubated with biotin-LPS with or without PIER1 peptide (5 μΜ) prior to incubation with streptavidin beads. Beads were then washed and boiled. The eluates were resolved on SDS-PAGE followed by immunoblotting for HSP90. The density of HSP90 bands was determined by ImageJ software and normalized to the second lane.

[0030] FIGS. 5A-5C. Inhibition of LPS responsiveness by HSP90-based inhibitors. Both HSP90 and gp96-derived PIER peptides inhibit LPS responsiveness. FIG. 5A, Sequence alignment of PIER peptides (SEQ ID NOs: 2, 8 and 1). FIG. 5B, WT pre-B cells were stimulated with LPS in the presence of various PIER peptides at 5 μΜ for 16-18 h. The mean fluorescence intensity of NFKB-GFP was normalized as in FIG. IB. Experiments were repeated for at least three times with similar results. *, p < 0.05. FIG. 5C, WT gp96- expressing pre-B cells were pretreated with 20 μΜ NPGA for 1 h and then stimulated with LPS (200 ng/ml) or with both PMA (50 ng/ml) and ionomycin (1 μg/ml) (PI) for 5 h following by flow cytometric analysis for GFP (open histogram). The shaded histograms represent GFP of unstimulated cells. AU, absorbance units. [0031] FIGS. 6A-6B. Cell-permeable PIER1 has anticancer effect and induces degradation of HSP90 client proteins. FIG. 6A, Killing curve of breast cancer cell line SKBR3 after 48-h treatment with TAT-PIERl and control peptide. FIG. 6B, SKBR3 cells were treated with PIER peptides for 12 h, followed by Western blot analysis of HSP90 client proteins. [0032] FIGS. 7A-7B. CD14 ^high RAW264.7 cells are less sensitive to PIER 1 -mediated inhibition of LPS binding and cytokine production. FIG. 7A, TNFa intracellular stain in response to LPS or PMA/ionomycin (PI) in the presence or absence of PIER1. Numbers represents percentages of TNFa-producing cells. FIG. 7B, RAW264.7 cells were incubated with biotin-LPS in the presence or absence of PIER1 followed by flow cytometry for LPS- binding cells with streptavidin-allophycocyanin (SA-APC). Number represents percentage of LPS-binding cells.

[0033] FIGS. 8A-8C. Enforced expression of CD 14 partially abolished the inhibition of PIER1 peptide. FIG. 8A, CD 14 expression (open histogram) on pre-B cells measured by flow cytometry. The filled grey histogram represents staining with isotype control antibody. FIGS. 8B-8C, WT (FIG. 8B) or CD14-transduced pre-B cells (FIG. 8C) were stimulated with LPS in the absence or presence of PIER1 peptide (5 μΜ) for 16-18 h. NFKB-GFP was detected by FACS. The absolute value of the mean fluorescence intensity (MFI) is shown. Data from one representative experiment of multiple experiments are shown. Error bars represent standard error of the mean. *, p < 0.05. DETAILED DESCRIPTION OF THE INVENTION

[0034] A component of bacteria cell wall, LPS can initiate effective immune responses to bacteria. However, excessive inflammation triggered by LPS can cause lethal diseases, such as sepsis. Certain aspects of the present invention disclose isolated peptides that can specifically inhibit the binding of LPS to cell surface HSP90, further inhibiting the binding to TLR4/MD-2 complex in a CD14-independent fashion. These peptides may be named as peptide inhibitors of endotoxin responsiveness or PIERs. Because these peptides may effectively inhibit the LPS responses in multiple cell types, they are promising therapeutic agents for future clinical application.

[0035] In further aspects, these peptides may be conjugated to cell permeable moieties, such as a TAT cell permeable sequence. The conjugated peptides may effectively trespass cell membranes and interact with cytosolic HSP90, therefore they can inhibit the chaperoning function of HSP90 and render multiple clients of HSP90 unstable. Preferably, these peptides can effectively kill multiple cancer cell lines, but not normal cells.

I. Peptides

[0036] Isolated peptides disclosed herein in certain aspects of the invention include a number of PIER peptide variants. For example, the three sequence specifically studied here comprise the following amino acid sequences.

[0037] SEQ ID NO: 1 (PIER3) is a 37-amino acid peptide having the sequence:

LNISREMLQQSKILKVIRKNrVKKCLELFSELAEDKE [0038] SEQ ID NO: 2 is a 37-amino acid peptide having the sequence:

LNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKY

[0039] SEQ ID NO: 3 is a peptide having the sequence of Tat-PIER3 :

GRKKRRQRRRPQ-Linker- LNISREMLQQSKILKVIRKNIVKKCLELFSELAEDKE [0040] SEQ ID NO: 4 is a peptide having the sequence of Tat-PIERl :

GRKKRRQRRRPQ-Linker- LNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKY

[0041] As described supra, in certain aspects of the invention the isolated peptides may comprise one or more internal or external amino acid deletions, additions or substitutions while maintaining their HSP90-inhibitory or HSP90-binding functions. For example, amino acid substitutions can be made at one or more positions wherein the substitution is for an amino acid having a similar hydrophilicity. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Thus such conservative substitution can be made in an PIER peptide and will likely only have minor effects on their activity and ability to repress VEGF promoter activity. As detailed in U.S. Patent 4,554, 101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine ( 0.5); histidine - 0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). These values can be used as a guide and thus substitution of amino acids whose hydrophilicity values are within ±2 are preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. Thus, any of the isolated peptides described herein may be modified by the substitution of an amino acid, for different, but homologous amino acid with a similar hydrophilicity value. Amino acids with hydrophilicities within +/- 1.0, or +/- 0.5 points are considered homologous.

[0042] Proteins or peptides may be made by any technique known to those of skill in the art, including the- expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteins or peptides from natural sources, or the chemical synthesis of proteins or peptides. The nucleotide and protein, polypeptide and peptide sequences corresponding to various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (world wide web at ncbi.nlm.nih.gov). The coding regions for known genes may be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art. [0043] Another embodiment for the preparation of polypeptides is the use of peptide mimetics. Mimetics are peptide-containing molecules that mimic elements of protein secondary structure. The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is expected to permit molecular interactions similar to the natural molecule. These principles may be used to engineer second generation molecules having many of the natural properties of the targeting peptides disclosed herein, but with altered and even improved characteristics. II. Hsp90 inhibition

[0044] Methods and compositions involving peptides that inhibit Hsp90 are provided. Heat-shock protein 90 (HSP90) is a family of molecular chaperones that are highly conserved in bacteria and eukaryotes (Pearl and Prodromou, 2006). The cytoplasmic form of HSP90 includes HSP90AA1 and HSP90AB1, known also as HSPC1 and HSPC3, respectively (Kampinga et al, 2009). There exists several paralogs of HSP90 in other cellular compartments, including the endoplasmic reticulum (ER) gp96 (also known as grp94, HSPC4) and mitochondrion TRAP-1 (HSPC5). Cytosolic HSP90 chaperones a large number of clients, and it is essential for the survival in yeast but not in bacteria. As a member of the GHKL (Gyrase, HSP90, Histidine Kinases and MutL) superfamily, HSP90 adopts a "V"- shaped parallel homodimer structure (Ali et al, 2006) comprising of the N-terminal ATPase domain, the relative flexible charged middle domain, and the C-terminal dimerization domain. The ATPase activity is conserved among all its orthologs and paralogs and is critical for its role as a molecular chaperone. The ATP hydrolysis cycle of HSP90 is coupled with the binding and release of clients and is critical for its chaperon function.

[0045] LPS or endotoxin is one of the most potent microbial products that can stimulate robust host inflammatory responses (Beutler and Rietschel, 2003). Study of a spontaneous LPS-resistant C3H/HeJ mouse strain led to the discovery of TLR4 as the receptor for LPS (Poltorak et al, 1998). The dimer of TLR4 forms a complex with two MD-2 molecules (Kim et al, 2007). The binding of LPS by MD-2 brings the two protomers of TLR4 together, triggering conformational changes in the cytoplasmic toll-interleukin 1 receptor (TIR) domains of TLR4 to activate downstream signaling. TLR4 is the only TLRs that utilize all four known adaptors: MyD88, TRIF, TRAM, and TIRAP (Horng et al, 2002; Kawai and Akira, 201 1). The signaling of TLR4 is regulated spatially and temporarily. LPS binds to TLR4/MD-2 complex on the cell surface, which then engages TIRAP and MyD88 to initiate the proinflammatory pathway. LPS-bound TLR4/MD-2 complex can also be endocytosed and transported into the endolysosome compartment (Kagan et al, 2008). It is in the endolysosome that TIR domains of TLR4 binds to TRAM and TRIF, which initiates the late NFKB signaling. The event in the endolysosome also triggers signaling of interferon regulatory factor (IRF) pathways, leading to the production of type I IFNs (Kagan and Medzhitov, 2006). [0046] Two other molecules, LPS-binding protein (LBP) and CD 14, are known to play important roles in LPS binding. LBP is a soluble protein that helps to extract LPS from Gram-negative bacterial cell walls (Tobias et al, 1988). Mostly expressed as a membrane bound form on cells of myeloid lineages, CD 14 is a glycosylphosphatidylinositol (GPI)- linked membrane protein (Gupta et al, 1996). Without a signaling tail, CD 14 functions as a co-receptor by transferring LPS from LBP to TLR4/MD-2 complex. However, additional molecules other than CD 14 might act as co-receptors for TLR4. The earliest evidence for the involvement of HSP90 came from a LPS-like molecule Taxol, which induces TNFa in mouse macrophages (Byrd et al, 1999). It was found that HSP70 and HSP90 were the two major Taxol-binding proteins. The role of HSP70 and HSP90 was further suggested by their direct binding to LPS and the indirect evidence of interaction between HSP70/HSP90 and LPS through fluorescence resonance energy transfer studies (Triantafilou et al, 2001a; Triantafilou et al, 2001b; Triantafilou et al, 2001c). Later on, it was shown that HSP70 and HSP90 function in a complex that also includes CXCR4 and growth differentiation factor 5 (Triantafilou and Triantafilou, 2004).

[0047] As an ER paralog of HSP90, gp96 is the master chaperone for TLRs (Randow and Seed, 2001 ; Yang et al, 2007; Liu and Li, 2008). With the exception of TLR3, the rest of the TLRs are exclusively dependent on gp96 for folding and functional expression despite the abundance of other HSPs in the ER (Liu et al, 2010). Overexpression of gp96 causes lupus- like diseases in a manner that is dependent on TLR4 (Liu et al, 2003; Liu et al, 2006). On the other hand, deletion of gp96 from the macrophage compartment leads to LPS resistance (Yang et al, 2007). Therefore, gp96 appears to be an attractive drug target for inflammation, sepsis, and autoimmune disease. Utilizing in silico methods, a recent report designed a peptide inhibitor of gp96 by targeting the N-terminal helix-loop-helix sequence, and demonstrated that this peptide could effectively inhibit LPS responses both in vitro and in vivo (Kliger et al, 2009). The proposed mechanism of action was that this peptide mimics the sequence of the helix, therefore disrupting the helix-helix interaction and the chaperoning function of gp96. However, based on the crystal structure of gp96 (Dollins et al, 2007), this N-terminal helix structure is unlikely to be a substrate-binding site. In addition, the inhibitory effect of this peptide has a very rapid kinetics, arguing against its roles in inhibiting gp96- mediated TLR folding as the mechanism of its action. It was demonstrated that N-terminal helix-based peptides from both HSP90 and gp96 are able to inhibit LPS binding to HSP90 and to attenuate LPS-mediated NFKB signaling in a manner that is independent of their activity against gp96.

[0048] Results in the Examples demonstrated that peptide-based inhibitors targeting an N- terminal helix structure of both gp96 and HSP90 can effectively inhibit NFKB responses to LPS but not to other TLR ligands. Further study suggested that these peptides disrupt LPS binding to cell surface in the absence of TLR4 or CD 14.

[0049] These results are novel in several aspects. First, evidence was provided that a peptide-based inhibitor that was previously reported to inhibit gp96 (Kliger et al, 2009), is in fact most likely targeting HSP90. PIER1 inhibits LPS binding to both WT and gp96-null cells. In addition, direct binding of LPS to HSP90 is compromised in the presence of PIER1. More importantly, it was demonstrated that PIER1 only inhibits TLR4 function, but not that of other TLRs, such as TLR2 and TLR9, which is incompatible with the claim that PIER1 suppress the function of gp96. Second, it was demonstrated that a cell-permeable PIER1 is a novel inhibitor of cytosolic HSP90 and has anti-cancer property. Third, since PIER1 does not enter cells readily, these results are consistent with the notion that HSP90, a cytosolic HSP, can indeed be expressed on the cell surface to serve as another important molecule in mediating LPS recognition. PIER1 was designed originally to target residues 100-137 of gp96. Based on the consideration above, most likely, the analogous region in the HSP90, residues 39-77 (FLRELISNASDALDKIRLISLTDENALSGNEELTVKIK (SEQ ID NO:7)), is the target of PIER1. This region is more than 80% identical to the gp96 sequence and appears to bind favorably to PIER1 as well as a corresponding region of PIER1 sequence on HSP90, PIER3. Fourth, these results also reinforced the notion that the cell surface HSP90- mediated LPS recognition is a dominant pathway in cells that do not co-express CD 14. In the case of Raw264.7 or CD14 ⁺ pre-B cells, the inhibitory effect of PIER1 became marginal. In this context, it is noteworthy that PIER1 has been shown to attenuate sepsis in vivo (Triantafilou et al, 2001b). Together with this finding, the inventors contemplate that CD 14 non-expressors, such as B cells, T cells, and non-hematopoietic parenchymal cells, are the most important cellular types to mediate endotoxin shock. CD14 ⁺ cells maybe more important in protecting the host and in generating subsequent adaptive T immune responses in the presence of the subclinical dose of LPS.

[0050] Multiple HSP90 inhibitors have been reported to inhibit inflammation and TLR4 responses. For example, EC 144, a synthetic HSP90 inhibitor, was shown to block LPS- induced TLR4 signaling in macrophages by inhibiting activation of ERK1/2, MEK1/2, J K, and p38 MAPK (Yun et al, 2011). SNX-7081, another small molecule inhibitor of HSP90, can inhibit NFKB in vitro and attenuate a mouse model of rheumatoid arthritis (Rice et al, 2008). Inhibition of HSP90 by 17-allylamino-17-demethoxy-geldanamycin (17-AAG) was successful in the treatment of endotoxin- induced uveitis (Poulaki et al, 2007). However, these studies utilized HSP90 inhibitors that effectively penetrate the cell membrane, resulting in the global inhibition of HSP90 function. Thus, the inhibition of LPS responsiveness by these inhibitors is exerted not at the ligand binding level on the cell surface, but at the level of downstream signaling, given the known roles of HSP90 in chaperoning many critical kinases in the TLR pathway. In this regard, PIER is a novel class of HSP90 inhibitors that could be used specifically to probe the function of cell surface HSP90 in sepsis as well as in oncogenesis. It is well known that HSP90 clients include molecules in multiple signaling pathways that are crucial for cancer (Pearl and Prodromou, 2006; Zhao et al, 2005; Kamal et al, 2003; Trepel et al, 2010; Whitesell and Lindquist, 2005; Usmani et al, 2009). Indeed, a cell-permeable PIER was found to have anti-cancer activity via inhibiting multiple HSP90 clients. Moreover, cell surface HSP90 has also been linked to increased cancer invasiveness (Picard, 2004; Eustace et al, 2004). Thus, the knowledge from the PIER inhibitors could potentially be applied to develop inhibitors to block and uncover other aspects of HSP90 function. Given the increased appreciation of inflammation in oncogenesis (Grivennikov et al, 2010), it will be of interests to determine if more potent PIER inhibitors can be developed to specifically targeting surface-bound HSP90 for attenuating both inflammation and cancer invasion as a new generation of cancer therapeutics.

III. Cell internalization and targeting moieties

[0051] Cell internalization moieties for use herein may be any molecule in complex (covalently or non-covalently) with an isolated peptide described herein that mediates transport of the peptide across a cell membrane. Such internalization moieties may be peptides, polypeptides, hormones, growth factors, cytokines, aptamers or avimers. Furthermore, cell internalization moiety may mediate non-specific cell internalization or be a cell targeting moiety that is internalized in a subpopulation of targeted cells.

[0052] For example, in certain embodiments, cell targeting moieties for use in the current invention are antibodies. In general the term antibody includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, single chain antibodies, humanized antibodies, minibodies, dibodies, tribodies as well as antibody fragments, such as Fab', Fab, F(ab')2, single domain antibodies and any mixture thereof. In some cases it is preferred that the cell targeting moiety is a single chain antibody (scFv). In a related embodiment, the cell targeting domain may be an avimer polypeptide. Therefore, in certain cases the cell targeting constructs of the invention are fusion proteins comprising an isolated peptide described herein and a scFv or an avimer. In some very specific embodiments the cell targeting construct is a fusion protein comprising an isolated peptide described herein fused to a single chain antibody. [0053] In certain aspects of the invention, a cell targeting moieties may be a growth factor. For example, transforming growth factor, epidermal growth factor, insulin-like growth factor, fibroblast growth factor, B lymphocyte stimulator (BLyS), heregulin, platelet-derived growth factor, vascular endothelial growth factor (VEGF), or hypoxia inducible factor may be used as a cell targeting moiety according to the invention. These growth factors enable the targeting of constructs to cells that express the cognate growth factor receptors. For example, VEGF can be used to target cells that express FLK-1 and/or Fit- 1. In still further aspects the cell targeting moiety may be a polypeptide BLyS (see U.S. Appln. 20060171919).

[0054] In further aspects of the invention, a cell targeting moiety may be a hormone. Some examples of hormones for use in the invention include, but are not limited to, human chorionic gonadotropin, gonadotropin releasing hormone, an androgen, an estrogen, thyroid- stimulating hormone, follicle-stimulating hormone, luteinizing hormone, prolactin, growth hormone, adrenocorticotropic hormone, antidiuretic hormone, oxytocin, thyrotropin-releasing hormone, growth hormone releasing hormone, corticotropin-releasing hormone, somatostatin, dopamine, melatonin, thyroxine, calcitonin, parathyroid hormone, glucocorticoids, mineralocorticoids, adrenaline, noradrenaline, progesterone, insulin, glucagon, amylin, erythropoitin, calcitriol, calciferol, atrial-natriuretic peptide, gastrin, secretin, cholecystokinin, neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor- 1, leptin, thrombopoietin, angiotensinogen, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL- 24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, or IL-36. As discussed above targeting constructs that comprise a hormone enable method of targeting cell populations that comprise extracellular receptors for the indicated hormone. [0055] In yet further embodiments of the invention, cell targeting moieties may be cytokines. For example, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL1 1, IL12, IL13, IL14, IL15, IL-16, IL-17, IL-18, granulocyte-colony stimulating factor, macrophage-colony stimulating factor, granulocyte-macrophage colony stimulating factor, leukemia inhibitory factor, erythropoietin, granulocyte macrophage colony stimulating factor, oncostatin M, leukemia inhibitory factor, IFN-γ, IFN-a, IFN-β, LT-β, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, TGF-β, IL la, IL-1 β, IL-1 RA, MIF and IGIF may all be used as targeting moieties according to the invention.

[0056] In certain aspects of the invention a cell targeting moiety of the invention may be a cancer cell targeting moiety. It is well known that certain types of cancer cells aberrantly express surface molecules that are unique as compared to surrounding tissue. Thus, cell targeting moieties that bind to these surface molecules enable the targeted delivery of an isolated peptide described herein specifically to the cancers cells. For example, a cell targeting moiety may bind to and be internalized by a lung, breast, brain, prostate, spleen, pancreatic, cervical, ovarian, head and neck, esophageal, liver, skin, kidney, leukemia, bone, testicular, colon or bladder cancer cell. The skilled artisan will understand that the effectiveness of cancer cell targeted PIER peptide may, in some cases, be contingent upon the expression or expression level of a particular cancer marker on the cancer cell. Thus, in certain aspects there is provided a method for treating a cancer with targeted PIER peptide comprising determining whether (or to what extent) the cancer cell expresses a particular cell surface marker and administering PIER-peptide targeted therapy (or another anticancer therapy) to the cancer cells depending on the expression level of a marker gene or polypeptide.

[0057] As discussed above cell targeting moieties according to the invention may be, for example, an antibody. For instance, a cell targeting moiety according the invention may bind to a skin cancer cell such as a melanoma cell. It has been demonstrated that the gp240 antigen is expressed in variety of melanomas but not in normal tissues. Thus, in certain aspects of the invention, there is provided a cell targeting construct comprising an isolated peptide described herein and a cell targeting moiety that binds to gp240. In some instances, the gp240 binding molecule may be an antibody, such as the ZME-018 (225.28S) antibody or the 9.2.27 antibody. In an even more preferred embodiment, the gp240 binding molecule may be a single chain antibody such as the scFvMEL antibody. [0058] In yet further specific embodiments of the invention, cell targeting constructs may be directed to breast cancer cells. For example cell targeting moieties that bind to Her-2/neu, such as anti-Her-2/neu antibodies, may be conjugated to an isolated peptide described herein. One example of such cell targeting constructs are fusion proteins comprising the single chain anti-Her-2/neu antibody scFv23 and an isolated peptide described herein. Other scFv antibodies such as scFv(FRP5) that bind to Her-2/neu may also be used in the compositions and methods of the current invention (von Minckwitz et al., 2005).

[0059] In certain additional embodiments of the invention, it is envisioned that cancer cell targeting moieties according to invention may have the ability to bind to multiple types of cancer cells. For example, the 8H9 monoclonal antibody and the single chain antibodies derived therefrom bind to a glycoprotein that is expressed on breast cancers, sarcomas and neuroblastomas (Onda et al. , 2004). Another example is the cell targeting agents described in U.S. Appln. 20040005647 and in Winthrop et al, 2003 that bind to MUC-1 an antigen that is expressed on a variety cancer types. Thus, it will be understood that in certain embodiments, cell targeting constructs according the invention may be targeted against a plurality of cancer or tumor types.

IV. Therapeutic compositions

[0060] Therapeutic compositions for use in methods of the invention may be formulated into a pharmacologically acceptable format. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one isolated peptide described herein or nucleic acid active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g. , human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0061] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference). A pharmaceutically acceptable carrier is preferably formulated for administration to a human, although in certain embodiments it may be desirable to use a pharmaceutically acceptable carrier that is formulated for administration to a non-human animal, such as a canine, but which would not be acceptable (e.g., due to governmental regulations) for administration to a human. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated. [0062] The actual dosage amount of a composition of the present invention administered to a subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0063] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an isolate peptide or its variant. In other embodiments, the peptide or its variant may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above. [0064] In particular embodiments, the compositions of the present invention are suitable for application to mammalian eyes. For example, the formulation may be a solution, a suspension, or a gel. In some embodiments, the composition is administered via a bioerodible implant, such as an intravitreal implant or an ocular insert, such as an ocular insert designed for placement against a conjunctival surface. In some embodiments, the therapeutic agent coats a medical device or implantable device.

[0065] Furthermore, the therapeutic compositions of the present invention may be administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. [0066] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well known parameters.

[0067] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. When the route is topical, the form may be a cream, ointment, salve or spray.

[0068] An effective amount of the therapeutic composition is determined based on the intended goal. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses, discussed above, in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection desired. Thus, in some case dosages can be determined by measuring for example changes in serum insulin or glucose levels of a subject. [0069] Precise amounts of the therapeutic composition may also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus attaining a particular serum insulin or glucose concentration) and the potency, stability and toxicity of the particular therapeutic substance. [0070] For example, the composition may be a solution, a suspension, or a gel. In some embodiments, the composition is administered via a bioerodible implant, such as an intravitreal implant or an ocular insert, such as an ocular insert designed for placement against a conjunctival surface. In some embodiments, the therapeutic agent coats a medical device or implantable device. V. Fusion peptides or conjugated peptides

[0071] In certain embodiments, therapeutic peptides or agents described above may be operatively coupled to a targeting peptide or a second therapeutic agent, for example to form fusion or conjugated peptides. Agents or factors suitable for use may include any chemical compound that induces apoptosis, cell death, cell stasis and/or anti-angiogenesis. A second therapeutic agent may be a drug, a chemotherapeutic agent, a radioisotope, a pro-apoptosis agent, an anti-angiogenic agent, a hormone, a cytokine, a cytotoxic agent, a cytocidal agent, a cytostatic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, a hormone antagonist, a nucleic acid or an antigen.

A. Regulators of programmed cell death

[0072] Apoptosis, or programmed cell death, is an essential process for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing carcinogenesis (Kerr et ah, 1972). The Bcl-2 family of proteins and ICE-like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems. The Bcl-2 protein, discovered in association with follicular lymphoma, plays a prominent role in controlling apoptosis and enhancing cell survival in response to diverse apoptotic stimuli (Bakhshi et ah, 1985; Cleary and Sklar, 1985; Cleary et ah, 1986; Tsujimoto et ah, 1985; Tsujimoto and Croce, 1986). The evolutionarily conserved Bcl-2 protein now is recognized to be a member of a family of related proteins, which can be categorized as death agonists or death antagonists.

[0073] Subsequent to its discovery, it was shown that Bcl-2 acts to suppress cell death triggered by a variety of stimuli. Also, it now is apparent that there is a family of Bcl-2 cell death regulatory proteins that share in common structural and sequence homologies. These different family members have been shown to either possess similar functions to Bcl-2 (e.g., BclXL, BclW, BclS, Mcl-1, Al, Bfl-1) or counteract Bcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri). B. Angiogenic inhibitors

[0074] In certain embodiments the present invention may concern administration of identified peptides operatively coupled to anti-angiogenic agents, such as angiotensin, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, platelet factor 4, IP- 10, Gro-β, thrombospondin, 2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron), interferon-alpha, herbimycin A, P U145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline. [0075] Proliferation of tumors cells relies heavily on extensive tumor vascularization, which accompanies cancer progression. Thus, inhibition of new blood vessel formation with anti-angiogenic agents and targeted destruction of existing blood vessels have been introduced as an effective and relatively non-toxic approach to tumor treatment. (Arap et al., 1998; Arap et al., 1998; Ellerby et al., 1999). A variety of anti-angiogenic agents and/or blood vessel inhibitors are known, (e.g., Folkman, 1997; Eliceiri and Cheresh, 2001).

[0076] The anti-angiogenic agent may also be selected from the group consisting of thrombospondin, angiostatin 5, pigment epithelium-derived factor, angiotensin, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, platelet factor 4, IP-10, Gro-β, thrombospondin, 2- methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron), interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, Docetaxel, polyamines, a proteasome inhibitor, a kinase inhibitor, a signaling peptide, accutin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 and minocycline. Whereas, the pro-apoptosis agent is selected from the group consisting of etoposide, ceramide sphingomyelin, Bax, Bid, Bik, Bad, caspase-3, caspase-8, caspase-9, fas, fas ligand, fadd, fap-1, tradd, faf, rip, reaper, apoptin, interleukin-2 converting enzyme or annexin V. Additional apoptotic agents include gramicidin, magainin, mellitin, defensin, cecropin, (KLAKLAK)2 (SEQ ID NO: 10), (KLAKKLA)2 (SEQ ID NO: 11), (KAAKKAA)2 (SEQ ID NO: 12) or (KLGKKLG)3 (SEQ ID NO: 13). Furthermore, a cytokine may be selected from the group consisting of interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-1 1, IL-12, IL-18, interferon-γ (IF-γ), IF-a, IF-β, tumor necrosis factor-a (TNF-a), or GM- CSF (granulocyte macrophage colony stimulating factor).

C. Cytotoxic agents

[0077] Chemotherapeutic (cytotoxic) agents may be used to treat various disease states, including cancer. Chemotherapeutic (cytotoxic) agents of potential use include, but are not limited to, 5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin, estrogen receptor binding agents, etoposide (VP 16), farnesyl-protein transferase inhibitors, gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, navelbine, nitrosurea, plicomycin, procarbazine, raloxifene, tamoxifen, taxol, temazolomide (an aqueous form of DTIC), transplatinum, vinblastine and methotrexate, vincristine, or any analog or derivative variant of the foregoing. Most chemotherapeutic agents fall into the categories of alkylating agents, antimetabolites, antitumor antibiotics, corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormone agents, miscellaneous agents, and any analog or derivative variant thereof.

[0078] Chemotherapeutic agents and methods of administration, dosages, etc. are well known to those of skill in the art (see for example, the "Physicians Desk Reference", Goodman & Gilman's "The Pharmacological Basis of Therapeutics" and in "Remington's Pharmaceutical Sciences" 15th ed., pp 1035-1038 and 1570-1580, incorporated herein by reference in relevant parts), and may be combined with the invention in light of the disclosures herein. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Of course, all dosages and agents described herein are exemplary rather than limiting, and other doses or agents may be used by a skilled artisan for a specific patient or application. Any dosage in-between these points, or range derivable therein is also expected to be of use in the invention. D. Alkylating agents

[0079] Alkylating agents are drugs that directly interact with genomic DNA to prevent cells from proliferating. This category of chemotherapeutic drugs represents agents that affect all phases of the cell cycle, that is, they are not phase-specific. An alkylating agent, may include, but is not limited to, a nitrogen mustard, an ethylenimene, a methylmelamine, an alkyl sulfonate, a nitrosourea or a triazines. They include but are not limited to: busulfan, chlorambucil, cisplatin, cyclophosphamide (Cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan.

E. Antimetabolites

[0080] Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents, they specifically influence the cell cycle during S phase. Antimetabolites can be differentiated into various categories, such as folic acid analogs, pyrimidine analogs and purine analogs and related inhibitory compounds. Antimetabolites include but are not limited to, 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate.

F. Natural products

[0081] Natural products generally refer to compounds originally isolated from a natural source, and identified as having a pharmacological activity. Such compounds, analogs and derivatives thereof may be, isolated from a natural source, chemically synthesized or recombinantly produced by any technique known to those of skill in the art. Natural products include such categories as mitotic inhibitors, antitumor antibiotics, enzymes and biological response modifiers.

[0082] Mitotic inhibitors include plant alkaloids and other natural agents that can inhibit either protein synthesis required for cell division or mitosis. They operate during a specific phase during the cell cycle. Mitotic inhibitors include, for example, docetaxel, etoposide (VP 16), teniposide, paclitaxel, taxol, vinblastine, vincristine, and vinorelbine. [0083] Taxoids are a class of related compounds isolated from the bark of the ash tree, Taxus brevifolia. Taxoids include but are not limited to compounds such as docetaxel and paclitaxel. Paclitaxel binds to tubulin (at a site distinct from that used by the vinca alkaloids) and promotes the assembly of microtubules. [0084] Vinca alkaloids are a type of plant alkaloid identified to have pharmaceutical activity. They include such compounds as vinblastine (VLB) and vincristine.

G. Antibiotics

[0085] Certain antibiotics have both antimicrobial and cytotoxic activity. These drugs also interfere with DNA by chemically inhibiting enzymes and mitosis or altering cellular membranes. These agents are not phase specific so they work in all phases of the cell cycle. Examples of cytotoxic antibiotics include, but are not limited to, bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), plicamycin (mithramycin) and idarubicin.

H. Miscellaneous agents

[0086] Miscellaneous cytotoxic agents that do not fall into the previous categories include, but are not limited to, platinum coordination complexes, anthracenediones, substituted ureas, methyl hydrazine derivatives, amsacrine, L-asparaginase, and tretinoin. Platinum coordination complexes include such compounds as carboplatin and cisplatin (cis-DDP). An exemplary anthracenedione is mitoxantrone. An exemplary substituted urea is hydroxyurea.

An exemplary methyl hydrazine derivative is procarbazine (N-methylhydrazine, MIH). These examples are not limiting and it is contemplated that any known cytotoxic, cytostatic or cytocidal agent may be attached to targeting peptides and administered to a targeted organ, tissue or cell type within the scope of the invention.

VI. Additional therapies

[0087] As discussed supra in certain aspects therapeutic methods of the invention may be used in combination or in conjunction with additional antiangiogenic or anticancer therapies.

A. Chemotherapy

[0088] In certain embodiments of the invention an isolated peptide described herein is administered in conjunction with a chemo therapeutic agent. For example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, paclitaxel, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, Velcade, vinblastin and methotrexate, or any analog or derivative variant of the foregoing may be used in methods according to the invention.

B. Radiotherapy

[0089] In certain further embodiments of the invention the PIER petpide compositions may be used to sensitize cell to radiation therapy. Radio therapy may include, for example, γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. In certain instances microwaves and/or UV-irradiation may also used according to methods of the invention. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6510 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. [0090] The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radio therapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing. C. Immunotherapy

[0091] Immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0092] Immunotherapy, thus, could be used as part of a combined therapy, in conjunction with gene therapy. The general approach for combined therapy is discussed below. Generally, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B, Her-2/neu, gp240 and pi 55.

D. Genes

[0093] In yet another embodiment, gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as a cell targeting construct of the present invention. Delivery of PIER3 -encoding nucleic acids in conjunction with a vector encoding one or more additional gene products may have a combined anti-hyperproliferative effect on target tissues. A variety of genes are encompassed within the invention, for example a gene encoding p53 may be delivered in conjunction with PIER peptide compositions.

E. Surgery

[0094] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies. A PIER peptide therapy of the invention may be employed alone or in combination with a cytotoxic therapy as neoadjuvant surgical therapy, such as to reduce tumor size prior to resection, or it may be employed as postadjuvant surgical therapy, such as to sterilize a surgical bed following removal of part or all of a tumor.

[0095] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue. [0096] Upon excision of part of all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, or 12 months. These treatments may be of varying dosages as well. F. Other agents

[0097] Hormonal therapy may also be used in conjunction with the present invention or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.

EXAMPLES

[0098] The following examples are included to further illustrate various aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques and/or compositions discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 - Experimental methods

Cell lines and plasmids

[0099] THP-1 and SKBR3 cell lines were obtained from ATCC. Wild type or gp96 KO mutant pre-B cells lines were kind gifts from B. Seed and were described previously (Randow and Seed, 2001). All culture conditions have been reported before (Morales et ah, 2009). MigR 1 -mCD 14TLR4HA was cloned into MigRl vector from puno-murine CD14 (InvivoGen). [00100] Peptides

[00101] All peptides were synthesized by NEO group to more than 98% purity as verified by HPLC and mass spectrometry. All peptides were dissolved in sterile PBS. Sequences of peptides are as follows: [00102] PIER1 : NH ₂ -LNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKY-COOH (SEQ ID NO:2);

[00103] PIER2: NH ₂ -LNVSRETLQQHKLLKVIRKKL KTLDMIKKIADDKY-COOH (SEQ ID NO:8);

[00104] PIER3: NH ₂ -LNISREMLQQSKILKVIRKNIVKKCLELFSELAEDKEN-COOH (SEQ ID NO:9); and

[00105] PIER4 (Tat-PIERl): GRKKRRQRRRPQ-PIER1 (SEQ ID NO:6).

[00106] Reagents

[00107] Biotin-LPS was obtained from InvivoGen. Purified human HSP90 was purchased from Enzo Life Sciences. Streptavidin-allophycocyanin (APC) secondary antibody was purchased from eBioscience. The non-permeable geldanamycin ( PGA), also known as dimethylaminoethylamino-17-demethoxygeldanamycin-N-oxide (Tsutsumi et ah, 2008), was synthesized by Zuping Xia (Pharmaceutical Sciences, Medical University of South Carolina) and was described previously (Gopal et ah, 2011).

[00108] TNFa ELISA [00109] THP-1 cells were pretreated with phorbol 12-myristate 13 -acetate (PMA) at 20 ng/ml for 48 h followed by stimulation with LPS for 24 h in the presence or absence of PIER. The supernatant was then collected, and TNFa was measured by an ELISA kit according to the manufacturer's specifications (BD Biosciences).

[00110] NFKB-GFP reporter assay [00111] As described previously (Randow and Seed, 2001), all cells are derived from E4.126 parental cell line, which contains NFKB-driven GFP reporter. Cells were stimulated with Pam ₃ CSK ₄ (10 μ^πιΐ), LPS (10 μ^ηιΐ), CpG ODN1826 (5 μΜ), PMA (100 ng/ml), and ionomycin (2 μg/ml) for 16-18 h before FACS instrumentation.

[00112] Western blot and antibodies

[00113] Antibodies were purchased from Cell Signaling Technology unless otherwise specified. Essentially all procedures were performed as described in Morales et al. (2009) without significant changes.

[00114] Flow cytometry

[00115] All staining protocol, flow cytometry instrumentation, and data analysis were performed essentially as described without significant modifications (Liu and Li, 2008; Morales et al, 2009). For cell surface staining, a single cell suspension of live cells was obtained and washed in FACS buffer twice. Fc receptor blocking with or without serum was performed depending on individual primary antibody used for staining. Primary and secondary antibody staining was performed stepwise with FACS buffer washing in between. Propidium iodide was added right before FACS instrumentation to gate out dead cells. For intracellular cytokine staining, cells were stimulated in the presence of 10 μg/ml brefeldin A before harvesting and washing with FACS buffer. Cell permeabilization was done with 0.25% saponin in FACS buffer. The same buffer was used in subsequent steps including blocking, washing, and antibody staining. The last washing step before FACS instrumentation was done with FACS buffer alone without detergent. Stained cells were acquired on a FACSCalibur (BD Biosciences) and analyzed using FlowJo software (Tree Star).

[00116] HSP90 in vitro binding assay

[00117] Purified HSP90 (1 μg) was incubated on ice in the presence of biotin-LPS (2 μg/ml) and/or PIER1 peptide (5 μΜ) at 4°C for 30 min. The mixture was then incubated with streptavidin-agarose beads at 4°C overnight. Beads were then washed with Tris lysis buffer and subjected to boiling in SDS loading buffer for 5 min prior to resolving on SDS-PAGE. The intensity of Western blot bands was quantified by ImageJ software.

[00118] Analysis of LPS binding to cell surface [00119] Biotin-LPS stock (500 μ^ηιΐ) was diluted in complete RPMI 1640 medium to the desired working concentration. About 0.2 million cells were resuspended in 50 μΐ of medium/well in a 96-well plate. Another 50 μΐ biotin-LPS suspension was added into the same well. The plate was then placed on a gentle shaker and rocked at 37°C for 30 min. After the incubation, cells were harvested from the well into cold PBS, washed with PBS and FACS buffer, and stained with streptavidin-APC antibody at 4°C for 30 min. Samples were then thoroughly washed with FACS buffer and analyzed on a FACSCalibur.

Example 2 - PIER peptide inhibits NFKB response to LPS but not to other

TLR ligands

[00120] A 37-mer peptide corresponding to residues 444-480 of gp96 was synthesized based on the study by Kliger et al (2009). For consistency in this study, this peptide was named PIER1. PIER1 activity was tested using a murine preB cell line that stably expresses NFKB- GFP reporter. PIER1 activity was tested using a murine pre-B cell line that stably expresses the NFKB-GFP reporter. When stimulated with LPS for 16-18 h, untreated pre-B cell line had a dose-dependent induction of NFKB-GFP (FIG. 1A). Consistent with the Kliger study, it was found that concurrent treatment of the cells with 5 μΜ PIER1 without preincubation significantly suppressed NFKB-GFP in response to LPS. gp96 is the master chaperone for TLRs, including TLR4, TLR2, and TLR9 (Yang et al, 2007; Liu et al, 2010). Inhibition of gp96 affects the de novo TLR biogenesis but it is not expected to affect the preexisting mature TLRs, which argues against gp96 being the target of PIER1. To further address this possibility, the effect of PIER1 was examined on TLR2 and TLR9 signaling using PamsCSIQ and CpG, respectively. The folding and assembly of both TLR2 and TLR9 are dependent on gp96 in the endoplasmic reticulum lumen before transporting to cell surface and endolysosome, respectively. If PIER 1 -mediated suppression of LPS responsiveness were via inhibition of the chaperone function of gp96, significant inhibition of TLR2 and TLR9 function would be expected. Contrary to this prediction, it was found that PIER1 had no activity against NFKB activation induced by TLR2 or TLR9 ligands. Therefore, PIER1 inhibits LPS responsiveness in a gp96-independent manner. Example 3 - PIER1 inhibits LPS response by disrupting binding of LPS to

cell surface

[00121] Next, the effect of PIER1 on LPS binding to cell surface was examined. Given the fact that PIER1 inhibition does not require pre-incubation of cells before adding LPS, it was contemplated that the inhibitory effect of PIER1 is at the upstream level of LPS response, i.e. the binding of LPS to the TLR4/MD-2 complex. This consideration is also consistent with the fact that PIER1 is not expected to enter cells readily.

[00122] A standard LPS binding assay was performed with biotinylated LPS followed by streptavidin-conjugated FITC to detect bound LPS on cell surface (FIG. 2). Strikingly, it was found that LPS was able to bind to both WT and gp96-null pre-B cells, and this binding was significantly inhibited by PIER1 (FIGS. 3A-3B). Since gp96 mutant pre-B cells do not express cell surface gp96, TLR4/MD-2, or CD 14 (Randow and Seed, 2001 ; Yang et ah, 2007; Liu and Li, 2008), the surface binding by LPS must be mediated by other LPS-binding proteins, such as HSP90, but not gp96.

Example 4 - PIER peptide binds to HSP90

[00123] Next, the study was focused on the possibility of PIER1 to inhibit LPS binding to HSP90 as previous studies have suggested that cell surface HSP90 is another LPS-binding protein for TLR4 signaling (Triantafilou et ah, 2001a; Triantafilou et ah, 2001b; Triantafilou et ah, 2001c; Triantafilou and Triantafilou, 2004). Additionally, a sequence alignment between HSP90 and gp96 of both human and mouse origin demonstrated that the target sequence of PIER1 peptide is highly conserved, especially in the first half of the HSP90 sequence. A direct binding assay was performed by incubating purified HSP90 with biotin- LPS in the presence or absence of PIER1. After a 30-min incubation period, the HSP90/LPS complex was pulled down with streptavidin beads followed by SDS-PAGE analysis. It was found that HSP90 could directly bind to LPS, and this binding was significantly inhibited by 5 μΜ PIER1 (FIG. 4).

[00124] As proposed by the Kliger study, the design of PIER1 peptide was based on a potential helix-helix interaction between PIER1 and the target sequence in the HSP that requires a conformation-dependent interaction. To examine this possibility, another peptide, PIER2 (this is a negative control), was designed by substituting the middle two residues in the helix of PIERl, Val and Arg, with two Pro residues. It was contemplated that the inflexible backbone of two Pro residues will kink the helix-loop-helix structure of PIERl peptide, therefore abolishing its inhibitory effect. In addition, PIER3 peptide was designed based on the target region of HSP90 (FIG. 5A). [00125] The efficacy of these two peptides in the same murine pre-B cell line was tested as described earlier. PIER3 was equally effective in inhibiting LPS-mediated NFKB-GFP activation (FIG. 5B). Introduction of Pro to PIERl (PIER2) completely abolished the inhibitory effect of PIERl (FIG. 5B). These results suggest that PIERl and PIER3 inhibit LPS responsiveness by blocking LPS binding in a conformation-dependent manner. [00126] To further determine the roles of cell surface HSP90 in LPS signaling, the inventors took advantage of a well characterized cell-impermeable HSP90 inhibitor, dimethylaminoethylamino-17-demethoxygeldanamycin- N-oxide (Tsutsumi et al, 2008), also known as NPGA (Gopal et al, 201 1). Cells were pretreated with NPGA or vehicle control followed by stimulation with either LPS or a combination of PMA and ionomycin. It was found that NPGA specifically inhibits NFKB activity in response to LPS but not to PMA and ionomycin (FIG. 5C).

Example 5 - Cell-permeable PIERl inhibits the chaperone function of

HSP90 [00127] If PIERl indeed inhibits HSP90 function on the cell surface, it would be expected to have a negative impact on the HSP90 clientele inside the cell. To address this possibility, a cell-permeable PIERl was generated by fusing PIERl with a TAT peptide, GRKKRRQRRRPQ (SEQ ID NO:6). It was found that TAT-PIER1 dose-dependently killed a breast cancer cell line, SKBR3. Importantly, by Western blot, it was found that TAT-PIER1 treatment led to degradation of a variety of well known HSP90 clients, including Her2/neu, AKT, CDK2, and p53 (FIGS. 6A-6B). Example 6 - Over expression of CD14 partially abolishes the inhibitory

effect of PIER1

[00128] Previous studies suggest that HSP90/HSP70 complex on the cell surface contributes preferentially to CD14-independent TLR signaling (Triantafilou et ah, 2001b). It was found that cells without CD 14 expression, such as pre-B cells and THP-1 cells, were more sensitive to PIER1 compared to CD 14 ⁺ RAW264.7 cells (FIGS. 7A-7B). The data is thus consistent with the notion that HSP90 plays more important roles for LPS recognition in cells that do not express CD14, such as epithelial cells, B cells, and hepatocytes. If so, ectopic expression of CD 14 should make it more resistant to PIER1 inhibition. To address this hypothesis, murine CD14 was stably expressed in pre-B cells (FIGS. 8A-8C). When stimulated with a range of concentrations of LPS, CD 14-expressing cells were less sensitive than WT cells to PIER 1 -mediated NFKB inhibition, particularly at the higher concentration of LPS. Thus, PIER1 preferentially inhibits CD14-independent LPS recognition on the cell surface.

* * * All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incoiporated herein by reference.

U.S. Patent 4,554, 101

U.S. Patent Appln. 20030008374

U.S. Patent Appln. 20030082789

U.S. Patent Appln. 20040005647

U.S. Patent Appln. 20060171919

U.S. Patent Appln. 200602231 14

U.S. Patent Appln. 20060234299

Ali et al. Nature, 440: 1013-1017, 2006.

Arap ei a/., Curr. Opin. Oncol, 10:560-565, 1998.

Bakhshi et al, Cell, 41(3):899-906, 1985.

Beutler and ietschel, Nat. Rev. Immunol, 3: 169-176, 2003.

Byrd ei fl/., Proc. Natl. Acad. Sci. USA, 96:5645-5650, 1999.

Cleary and Sklar, Proc. Natl. Acad. Sci. USA, 82(21):7439-7443, 1985.

Cleary et al, J. Exp. Med., 164(1):315-320, 1986.

Dollins et al, Mol Cell, 28:41-56, 2007.

Eliceiri and Cheresh, Curr. Opin. Cell Biol, 13(5):563-568, 2001.

Ellerby et al . Nature Med, 5(9): 1032-1038, 1999.

Eustace et al, Nat. Cell Biol, 6:507-514, 2004.

Folkman, In: Cancer: Principles and Practice, eds. DeVita et al, 3075-3085, Lippincott-

Raven, NY, 1997.

Goodman & Gilman's "The Pharmacological Basis of Therapeutics."

Gopal et al, PLoS One, 6:el7649, 201 1.

Grivennikov et al, Cell, 140:883-899, 2010.

Gupta a/., J Biol. Chem., 271 :23310-23316, 1996.

Horng et al, Nature, 420:329-333, 2002.

Kagan and Medzhitov, Cell, 125:943-955, 2006.

Kagan et al, Nature Immunol, 9:361-368, 2008. Kamal et al, Nature, 425:407-410, 2003.

Kampinga et al, Cell Stress Chaperones, 14: 105-11 1, 2009.

Kawai and Akira, Immunity, 34:637-650, 2001.

Kerr et al, Br. J. Cancer, 26(4):239-257, 1972.

Kim et al, Cell, 130:906-917, 2007.

Kliger, et al, Proc. Natl Acad. Sci. USA, 106: 13797- 13801, 2009.

Kyte and Doolittle, J. Mol. Biol, 157(1): 105- 132, 1982.

Liu and Li, Blood, 1 12: 1223-1230, 2008.

Liu et al, J. Immunol, 177:6880-6888, 2006.

iu et al, Nat. Commun., l :doi: 10 1038/ncomms 1070, 2010.

Liu et al, Proc. Natl Acad. Sci. USA, 100: 15824-15829, 2003.

Morales et al , J. Immunol, 183:5121-5128, 2009.

Onda ei a/., Cancer Res., 64: 1419-1424, 2004.

Pearl and Prodromou, Ann. Rev. Biochem., 75:271-294, 2006.

Physicians Desk Reference

Picard, Nat. Cell Biol, 6:479-480, 2004.

YoW x^k et al, Science, 282:2085-2088, 1998.

Poulaki et al , FASEB J., 21 :21 13-2123, 2007.

Randow and Seed, Nat. Cell Biol, 3 :891-896, 2001.

Remington's Pharmaceutical Sciences, 15 ^th Ed., 1035-1038 and 1570-1580, 1990.

Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990

Rice et al, Arthritis Rheum., 58:3765-3775, 2008.

Rothbard et al, Nat. Med., 6(1 1): 1253-1257, 2000.

Tobias et al, J. Biol. Chem., 263: 13479- 13481, 1988.

Trepel et al, Nat. Rev. Cancer, 10:537-549, 2010.

Triantafilou and Triantafilou, Biochem. Soc. Trans., 32:636-639, 2004.

Triantafilou et al, Hum. Immunol, 62:50-63, 2001c.

Triantafilou et al, J. Cell Sci., 1 14:2535-2545, 2001a.

Triantafilou et al, Nature Immunol, 2:338-345, 2001b.

Tsujimoto and Croce, Proc. Natl. Acad. Sci. USA, 83(14):5214-5218, 1986.

Tsujimoto et al, Nature, 315:340-343, 1985.

Tsujimoto et al, Science, 228(4706): 1440-1443, 1985.

Tsutsumi et al, Oncogene, 27:2478-2487, 2008.

Usmani et al, Curr. Mol Med., 9:654-664, 2009. von Minckwitz et al, Breast Cancer Res., 7:R616-626, 2005. Whitesell and Lindquist, Nat. Rev. Cancer, 5:761-772, 2005. Winthrop et al, Clin. Cancer Res., 9:3845s-3853s, 2003. Wright et al, Dev. Biol, 256(l):73-88, 2003.

Yang et al, Immunity, 26:215-226, 2007.

Yun et al, J. Immunol, 186:563-575, 2011.

Zhao et al, Cell, 120:715-727, 2005.