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WO2012112869A2 - Thérapie anticancéreuse ciblée utilisant des inhibiteurs de la transcription d'ets - Google Patents

Thérapie anticancéreuse ciblée utilisant des inhibiteurs de la transcription d'ets Download PDF

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WO2012112869A2
WO2012112869A2 PCT/US2012/025608 US2012025608W WO2012112869A2 WO 2012112869 A2 WO2012112869 A2 WO 2012112869A2 US 2012025608 W US2012025608 W US 2012025608W WO 2012112869 A2 WO2012112869 A2 WO 2012112869A2
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tel
polypeptide
domain
seq
amino acid
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WO2012112869A3 (fr
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Xiaobo CAO
William Roy SMYTHE
Dawit GIZACHEW
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Baylor Scott and White Health
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Scott and White Healthcare
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention generally concerns the fields of cell biology, molecular biology, and medicine, including cancer medicine.
  • the present invention concerns mutants of an E-twenty six (ETS) transcriptional repressor protein for cancer therapy.
  • ETS E-twenty six
  • MM malignant mesothelioma
  • non-small cell lung cancer Dazzi et al, 1990; Herbst and Shin, 2002; Sridhar et al, 2003; Rogers et al, 2009; Mukohara et al, 2005.
  • EGFR, c-Met (HGFR) and IGF1R may cooperate to regulate tumor growth and survival.
  • the inhibition of one receptor can be compensated by activation of the others, because there is a degree of functional redundancy between these receptors (Bachleitner-Hofmann et al, 2008; Guo et al., 2008; Whittaker et al., 2010).
  • the complex crosstalk between these pathways could therefore significantly reduce the efficacy of single receptor targeted therapies. Therefore, elucidating and targeting common downstream effectors is of some importance.
  • the ETS family consists of more than 30 members, which include both transcription factors and repressors (Silverman et al., 2002; Wei et al., 2010).
  • Most ETS proteins are nuclear targets of diverse signaling pathways, such as the mitogen- activated protein (MAP) kinase signaling pathway, and undergo post-translational modifications including phosphorylation, glycosylation, acetylation, ubiquitination, and sumoylation (Cao et al., 2009). These modifications have a profound impact on the activity and subcellular localization of all ETS proteins.
  • ETS-1, 2 and PU are nuclear targets of diverse signaling pathways, such as the mitogen- activated protein (MAP) kinase signaling pathway.
  • l which are known positive transcriptional regulators of Bcl-xl, are imported to the nucleus following MAPK phosphorylation, and can be exported by an active transport pathway (Sevilla et al., 2001; Sevilla et al., 1999; Nelson et al., 2010).
  • Tel is a transcriptional repressor of Bcl-xl, and is also negatively regulated by phosphorylation and sumoylation, by nuclear export, or as a result of localization to Tel-bodies (Roukens et al., 2008; Maki et al., 2004). Tel interactions with relevant corepressors, mSin3A, N-CoR and HDAC3, are believed to mediate the transcriptional repression of multiple target genes, including Bcl-xl (Wang et al., 2001).
  • the present invention concerns methods and/or compositions related to E-twenty six (ETS) protein inhibitors for cancer therapy and/or prevention in a mammal.
  • Tel a transcription repressor member in the ETS family
  • Tel has been identified as a tumor suppressor. Delivery of Tel into tumor cells leads to tumor cell death, growth arrest and inhibition of invasion. Tel can serve as a pan ETS inhibitor, because it competes with other members of ETS family proteins on similar ETS binding sites.
  • there is a mutated ETS transcriptional repressor protein that is capable of binding a particular DNA promoter sequence and that abolishes other ETS transcription factors' activities.
  • ETS proteins are nuclear targets of diverse signaling pathways, such as the mitogen-activated protein (MAP) kinase signaling pathway, and undergo post-translational modifications, including phosphorylation, glycosylation, acetylation, ubiquitination, and sumoylation, for example.
  • Tel is a transcriptional repressor that is negatively regulated by phosphorylation and sumoylation, by nuclear export, and/or as a result of localization to Tel- bodies, for example.
  • Tel interactions with relevant co-repressors including mSin3A, N-CoR and HDAC3, mediate the transcriptional repression of multiple target genes, including Bcl-xl, in certain aspects of the invention.
  • the present invention there are Tel repressors that have altered post-transcriptional modifications compared to wild-type Tel.
  • the modifications can occur at one or more of the sites for ubiquitination, sumoylation, phosphorylation, and/or auto- inhibition, in certain embodiments.
  • the transcriptional repressor activity of the mutant Tel is greater than that for wild-type Tel.
  • the mutants have enhanced Tel repressive function compared to wild-type Tel.
  • Tel is negatively regulated at the post-transcriptional level, such as through ubiquitination, sumoylation, phosphorylation, and/or auto-inhibition.
  • Tel can be modified by sumoylation at Kl l and K99 and can be modified by phosphorylation at S213.
  • the exemplary results provided herein indicate that the phosphorylation of Tel at Ser213 blocks the Tel's repression on Bcl-xl transcription. This negative regulation of Tel function following receptor activation contributes to the overexpression of the Bcl-xl protein in cancer cells.
  • the one or more mutations provide enhanced repressor activity, such as compared to wild type.
  • a Tel mutant having two or more mutations has greater repressor activity compared to the single mutant activity.
  • the exemplary double mutant Tel K11R/S213A is a stronger repressor of Bcl-xl transcription (for example) than either single mutation alone. Whereas both phosphorylation and sumoylation are useful for Tel export, for example, in certain embodiments these two types of modification function cooperatively or independently.
  • the phosphorylation and sumoylation work synergistically, for example to mediate the downregulatory nuclear export of the ETS transcriptional repressors.
  • the mutant Tel polypeptide is hypophosphorylated compared to wild-type Tel.
  • Exemplary phosphorylation sites on Tel reside within the linker inhibitory damper (LID) domain outside of its ETS DNA-binding domain.
  • LID linker inhibitory damper
  • the Tel polypeptide may have mutations of any suitable kind, but in specific embodiments the polypeptide retains transcriptional repressor activity, including activity that is greater than wild-type when measured by identical or similar assays.
  • the mutation may be a point mutation, inversion, or deletion, for example.
  • the mutation may alter the ability to be subject to one or more post-translational modifications.
  • the amino acid that may be mutated such that phosphorylation cannot occur includes serine, tyrosine, or threonine, and one or more of these amino acids may be changed to another amino acid, particularly one that is not capable of being phosphorylated.
  • the mutation may change the respective encoded amino acid, but in some cases there may be mutations that do not change the respective encoded amino acid.
  • Tel mutants encompassed by the invention also include those that lack part of the protein.
  • the mutant may lack the N-terminal and/or C-terminal.
  • the protein lacks part or all of the inhibitory CID domain.
  • the protein is modified such that it has greater protein solubility than wild type.
  • the Tel protein has single, double, or triple or more modifications that render greater solubility for the protein.
  • the Tel V112E mutant has increased protein solubility.
  • a protein transduction domain (such as the TAT peptide sequence or Antennapedia homeodomain, for example), for example, is present on Tel to enable the polypeptide to penetrate into human cells.
  • the protein transduction domain is present on the N-terminus of the Tel mutant, whereas in other cases the protein transduction domain is present on the C-terminus of the Tel mutant.
  • the methods and compositions of the invention are employed for cancer treatment and/or prevention, wherein the cancer is of any kind of cancer.
  • the cancer is mesothelioma, thoracic cancer (including lung cancer, such as non- small cell lung carcinoma, for example), breast, colon, pancreas, prostate, skin, liver, kidney, bone, spleen, cervix, ovary, testes, bladder, gall bladder, or brain cancer.
  • the individual to be treated with the invention is a mammal, including a human, dog, cat, horse, pig, sheep, or goat, for example.
  • TEL polypeptide having one or both of the following: a) an amino acid substitution at one or more sites corresponding to SEQ ID NO:3, said sites selected from the group consisting of a serine corresponding to position 213, a serine corresponding to position 257, a lysine corresponding to position 11, a lysine corresponding to position 99, and a valine corresponding to position 112; and/or b) a deletion in the polypeptide corresponding to SEQ ID NO:3, said deletion occurring in one or more of a protein transduction domain, a TAT domain, a SAM domain, a LID domain, an ETS domain, or a CID domain, wherein the polypeptide has transcriptional repressor activity.
  • the polypeptide has increased transcriptional repressor activity over wild- type TEL polypeptide.
  • the polypeptide is further defined as having an amino acid substitution at the serine corresponding to position 213, at the serine corresponding to position 257, at the lysine corresponding to position 11, at the lysine corresponding to position 99, and/or at the lysine corresponding to position 99.
  • the substitution at the serine corresponding to position 213 is alanine
  • the substitution at the serine corresponding to position 257 is alanine
  • the substitution at the lysine corresponding to position 11 is arginine
  • the substitution at the lysine corresponding to position 99 is arginine.
  • the polypeptide further comprises a protein transduction domain.
  • TEL polypeptide having a mutation at a phosphorylation site, a ubiquitination site, a sumoylation site, a glycosylation site, an acetylation site, or a combination thereof, wherein the polypeptide has transcriptional repressor activity.
  • the polypeptide has increased transcriptional repressor activity over wild-type TEL polypeptide.
  • the mutation occurs within a C-terminal inhibitory domain (CID), a linker inhibitory damper domain (LID), ETS DNA-binding domain (ETS), or a sterile alpha motif (SAM) domain.
  • CID C-terminal inhibitory domain
  • LID linker inhibitory damper domain
  • ETS ETS DNA-binding domain
  • SAM sterile alpha motif
  • the polypeptide may be further defined as comprising (corresponding to positions in SEQ ID NO:3) a mutation at serine 213, comprising a mutation at serine 257, comprising a mutation at valine 112, and/or having a mutation at lysine 11.
  • the polypeptide may further comprise deletion of part or all of the CID domain and/or the polypeptide may further comprise a protein transduction domain, such as from HIV TAT, Antennapedia, penetratin, SynBl, SynB3, PTD-4, PTD-5, FHV Coat- (35-49), BMV Gag-(7-25), HTLV-II Rex-(4-16), D-Tat, R9-Tat, Transportan, MAP, SBP, FBP, MPG, MPG(ANLS), Pep-1, or Pep-2, for example.
  • a protein transduction domain such as from HIV TAT, Antennapedia, penetratin, SynBl, SynB3, PTD-4, PTD-5, FHV Coat- (35-49), BMV Gag-(7-25), HTLV-II Rex-(4-16), D-Tat, R9-Tat, Transportan, MAP, SBP, FBP, MPG, MPG(ANLS), Pep-1,
  • R0 is H or an amino acid sequence comprising 1 to 500 consecutive amino acids
  • Rl is an amino acid sequence comprising 1 to 11 consecutive amino acid residues of a protein transduction domain
  • R2 is a bond or an amino acid sequence comprising 10 or more consecutive amino acid residues of a SAM domain
  • R3 is a bond or an amino acid sequence comprising 10 or more consecutive amino acid residues of a LID domain
  • R4 is a bond or an amino acid sequence comprising 10 or more consecutive amino acid residues of an ETS domain
  • R5 is a bond or an amino acid sequence comprising 10 or more consecutive amino acid residues of a CID domain
  • R6 is H or an amino acid sequence comprising 1 to 500 consecutive amino acids, provided that R2, R3, R4, and R5 are not all bonds, provided that the isolated recombinant amino acid sequence
  • Rl is selected from the group consisting of SEQ ID NO:5-23; R2 comprises SEQ ID NO:24; R3 comprises SEQ ID NO:25; R4 comprises SEQ ID NO:26; and/or R5 comprises SEQ ID NO:27.
  • the amino acid sequence of the polypeptide of the invention comprises SEQ ID NO:28.
  • R2, R3, and R4 are not bonds but R5 is a bond.
  • Rl comprises a sequence selected from the group consisting of SEQ ID NO:5-23;
  • R2 comprises SEQ ID NO:24;
  • R3 comprises SEQ ID NO:25;
  • R4 comprises SEQ ID NO:26.
  • the amino acid sequence of the polypeptide of the invention comprises SEQ ID NO:29.
  • R2 is a bond and R3, R4, and R5 are not bonds.
  • the amino acid sequence of the polypeptide of the invention comprises SEQ ID NO:30.
  • R2 and R5 are bonds and R3 and R4 are not bonds.
  • R2 is a bond and R3, R4 and R5 are not bonds.
  • the amino acid sequence of the polypeptide of the invention comprises SEQ ID NO:32. In some cases,
  • Rl comprises a TAT sequence (SEQ ID NO:6); R2 comprises SEQ ID NO:35; R2 comprises SEQ ID NO:36; R2 comprises SEQ ID NO:37; R3 comprises SEQ ID NO:38; R3 comprises SEQ ID NO:39; and/or R3 comprises SEQ ID NO:40.
  • wild-type Tel without at least part of the CID domain is employed.
  • there is a Tel lacking the CID domain but the remainder of the Tel protein variant is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the corresponding region in the wild-type Tel protein.
  • there is a Tel lacking the CID domain but the remainder of the Tel protein variant has one, two, three, four, five, or more amino acid substitutions or deletions.
  • there is a Tel lacking the CID domain but the remainder of the Tel protein variant has no more than one, two, three, four, five, or more amino acid substitutions or deletions.
  • there is a Tel lacking the CID domain but the remainder of the Tel protein variant has at least one, two, three, four, five, or more amino acid substitutions or deletions.
  • all of the CID domain is absent from the Tel protein, whereas in certain cases part of the CID domain is absent, such as 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s) from the CID domain are absent.
  • a Tel protein comprising TAT, the V112E mutation, and/or lacking part or all of the CID domain is employed.
  • An exemplary TAT-Tel wild-type with V112E but without CID is provided in SEQ ID NO:43 and may be employed in the invention, in certain embodiments.
  • a recombinant TEL polypeptide comprising an ETS DNA-binding domain (ETS) and a) a sterile alpha motif (SAM) domain having a deletion or an amino acid substitution at a lysine or a valine; and/or b) a linker inhibitory damper domain (LID) domain having a deletion or an amino acid substitution at one or more serines; and/or c) an amino acid substitution at a lysine corresponding to position 11 in SEQ ID NO:3.
  • the polypeptide further comprises a protein transduction domain, a nuclear localization signal, and/or a C-terminal inhibitory domain (CID) domain.
  • the polypeptide has increased transcriptional repressor activity over wild-type TEL polypeptide.
  • the polypeptide may be further defined as having an amino acid substitution at the serine corresponding to position 213 in SEQ ID NO:3, as having an amino acid substitution at the serine corresponding to position 257 in SEQ ID NO:3, as having an amino acid substitution at the lysine corresponding to position 99 in SEQ ID NO:3, and/or as having an amino acid substitution at the valine corresponding to position 112 in SEQ ID NO:3.
  • Exemplary protein transduction domains utilized in the invention include, for example, HIV TAT, Antennapedia, penetratin, SynBl, SynB3, PTD-4, PTD-5, FHV Coat- (35-49), BMV Gag-(7-25), HTLV-II Rex-(4-16), D-Tat, R9-Tat, Transportan, MAP, SBP, FBP, MPG, MPG(ANLS), Pep-1, or Pep-2.
  • an isolated recombinant TEL polypeptide comprising: a) an amino acid substitution at one or more sites in the polypeptide corresponding to SEQ ID NO:3, said sites selected from the group consisting of a serine corresponding to position 213, a serine corresponding to position 257, a lysine corresponding to position 11, a lysine corresponding to position 99, and a valine corresponding to position 112; and/or b) a deletion in the polypeptide corresponding to SEQ ID NO:3, said deletion occurring in one or more of a protein transduction domain, a TAT domain, a SAM domain, a LID domain, an ETS domain, or a CID domain, wherein the polypeptide has transcriptional repressor activity.
  • the method comprises the step of administering to the individual a therapeutically effective amount of a polypeptide of the invention.
  • the individual has a thoracic malignancy.
  • the individual has mesothelioma.
  • the method comprises an additional therapy for cancer.
  • Kits that comprise the polypeptide of the invention are encompassed, said polypeptide housed in a suitable container.
  • FIG. 1A provides that stimulation of cells by HGF and EGF causes identical phosphorylation events by multiple kinases as measured using the proteome profile human phosphor-MAPK array.
  • FIG IB shows that activated Map kinase phosphorylates ETS proteins including Tel.
  • the mesothelioma cell line, 145 was grown under conditions of serum starvation or serum starvation plus 20 minutes of HGF (100 ng/ml) stimulation.
  • the endogenous ETS-2, PU.l and Tel proteins were immunoprecipitated from the cell lysates using the respective antibodies. The immunoprecipitates were then analyzed by western blot using phosphor-Ser/Thr antibodies.
  • Tel is phosphorylated on Serine 213 after HGF stimulation.
  • the Tel serine mutant S213A, S257A and wild- type Tel were transfected into 145 cells which were then serum starved or HGF stimulated. Tel was immunoprecipitated from cell lysates and its phosphorylation status was determined using anti phosphor-Ser/Thr antibodies.
  • FIG. 2 shows that CHIP (IP) assay evaluation of the impact of Tel phosphorylation on its binding to the Bcl-xl promoter.
  • 145 and A549 cells transfected with wild- type or Tel S213A mutants were serum-starved for 24 hours and then subjected to treatment with or without HGF or EGF for 20 minutes.
  • Cross-linked Tel-DNA complexes were immunoprecipitated using a Tel antibody and Bcl-xl promoter fragments were PCR amplified.
  • FIG. 3 A demonstrates that the Tel phosphorylation site mutant S213A is not exported from the nucleus after EGF stimulation.
  • 145 cells were transfected with Tel or Tel S213A expression vectors and 24 hours after serum starvation, these transfected cells were exposed to HGF stimulation for 20 minutes. Acetone-fixed cells were then incubated with Tel antibodies followed by a fluorescein isothiocyanate (FITC)-labeled secondary antibody. Fluorescein signals were visualized under a Nikon 80i fluorescent microscope. 4,6-Diamidino-2- phenylindole (DAPI) was used as a counterstain for nuclear DNA.
  • FIG. 3B Tel S213A achieves a stronger repression of the Bcl-xl promoter in the presence of growth factor activation.
  • A549 and 145 cells seeded in 24-well plates were cotransfected with Bcl-xl promoter pXL-luc and either Tel S213A or Tel vectors along with p-CMV-P-galactosidase. Twenty-four hours after transfection, the cells were lysed, and luciferase and ⁇ -galactosidase activities were measured with a luminometer. The luciferase activities were normalized to those of ⁇ -galactosidase, and the data shown are the average of triplicate determinations. This experiment was repeated twice.
  • FIG. 4A shows that growth factor stimulation of cells fails to decrease the repression of Bcl-xl by Tel S213A.
  • 145 mesothelioma cells were transfected with GFP, Tel wild type or Tel S213A and grown under conditions of serum starvation or serum starvation plus HGF stimulation (100 ng/ml) for 48 hours. The Bcl-xl expression levels in these cells were then determined by western blotting.
  • FIG. 4B To determine the gene targets of Tel repression, both A549 and 145 cells were transfected with GFP, Tel wild type or Tel S213A. Forty-eight hours after transfection the Blc-2 target protein levels were detected by western blotting.
  • FIG. 5A demonstrates measurement of the short-term tumor growth suppression of Tel S213A by cell proliferation assay.
  • A549 and 145 cells were seeded in 96 well plates and transfected with 0.2 ⁇ g of Tel or Tel S213A cDNA, and cultured for 48 hours. Cell growth inhibition was measured by the XTT assay. The data shown are the average of four independent determinations.
  • FIG. 5B Demonstration of long-term tumor growth suppression by Tel S213A in a colony formation assay.
  • A549 and 145 cells were transfected with various doses of Tel or Tel S213A cDNA and plated in 10 cm cell culture plates at 10 4 cells per plate.
  • Tel S213A induces a stronger apoptotic response in cancer cells.
  • A549 cells were transfected with pGL-2, Tel wild type and Tel S213A, collected and stained with FITC-labeled Annexin V and PL Annexin V-positive populations were quantified by flow cytometry. The data shown are the results of triplicate determinations.
  • FIG. 6A illustrates the impact of multiple-site mutations of Tel on Bcl-xl promoter repression.
  • A549 cells were seeded in 96-well plates and transfected with a Bcl-xl promoter reporter, pXL-luc, and co-transfected with Tel wild-type, Tel K11R, Tel S213A, Tel Kl lR, Tel K11R/S213A and a p-CMV-P-galactosidase transfection control vector.
  • the cells were lysed and both the luciferase and ⁇ -galactosidase activities were measured using a luminometer.
  • FIG. 6B Comparison of Bcl-xl protein levels following transfection with the indicated Tel mutants. A549 cells were seeded into 6 well plates (10 6 cells per well) and were transfected with Tel, Tel mutants and GFP expression vectors. Forty-eight hours after transfection, the Bcl-xl protein levels were determined by western blot.
  • FIG. 7 shows analysis of EGFR phosphorylation and Bcl-xl using human non-small cell lung carcinoma and mesothelioma tissue arrays. Correlations between the Bcl-xl levels and phosphorylated EGFR levels were demonstrated by Chi-square analysis (P ⁇ 0.01).
  • FIG. 8 provides an exemplary schematic representation of the negative regulation of Tel by phosphorylation.
  • FIG. 9 illustrates exemplary modifications of wild-type TEL.
  • FIG. 10 provides the sequence of Tel, SEQ ID NO:3, which illustrates different exemplary domains in the Tel protein.
  • the ETS domain comprises amino acids 338-422, the figure marks it as 331-422 merely to illustrate a nuclear localization signal.
  • FIG. 11 illustrates one embodiment by which phosphorylations at S213/S257 decrease Tel's repression on Bcl-xl transcription and increase Bcl-xl transcription.
  • FIG. 12 shows an embodiment of another mechanism by which LID can repress CID.
  • FIG. 13 illustrates an embodiment wherein there is synergism of sumoylation and phosphorylation on negative regulation of Tel function.
  • an effective amount or "therapeutically effective amount” as used herein is defined as an amount of the agent that will decrease, reduce, inhibit or otherwise abrogate the growth of a neoplasm, induce apoptosis, inhibit angiogenesis of a neoplasm, inhibit metastasis, or induce cytotoxicity in a neoplasm.
  • an effective amount is an amount sufficient to detectably and repeatedly ameliorate, reduce, minimize or limit the extent of the disease or its symptoms.
  • ETS transcriptional factors are molecular targets for selective cancer therapy, because they play roles in maintaining malignancy of cancer cells. It is difficult to develop small molecules that directly inhibit the function of ETS transcription factors, because these proteins bind to the same DNA sequence in humans, and the loss of function of one transcription factor due to a small molecular inhibitor, for example, can be compensated by other ETS transcription factors. This difficulty can be supported by the ineffectiveness of dominant negative mutant and siRNA/antisense approaches, for example.
  • the present invention provides novel ETS transcriptional inhibitors and encompasses Tel polypeptides that are modified compared to wild type but retain transcriptional repressor activity, including enhanced transcriptional repressor activity compared to wild type, for example.
  • the Tel gene can be negatively regulated at the post-transcriptional level through ubiquitination, glycosylation, acetylation, sumoylation and phosphorylation, for example.
  • the mutant may have a single mutation or more than one mutation.
  • the exemplary double-mutants TEL 11-213 enhance Tel's repressive function in tumor cells by abolishing one or more negative modifications, in certain embodiments.
  • the present invention includes mutants with increased protein solubility, including the Tel V112E mutant having increased protein solubility.
  • the invention also includes mutants with improved binding to promoter DNA compared to wild type Tel including, for example, a mutant with a deleted part or all CID domain that improves its binding to promoter DNA.
  • the Tel ETS domain is a therapeutic protein, in certain embodiments.
  • TAT peptide sequence is attached at its N terminus to enable protein to penetrate into human cells, in particular aspects of the invention.
  • the phosphorylation of Tel increases Bcl-xl transcription through tyrosine kinase receptors' phosphorylation and activation of ETS2, a transcriptional activator.
  • stimulation by HGF or EGF activates an identical downstream signal transduction pathway and causes the activation of ERK, leading to the nuclear export of Tel.
  • the exemplary phosphorylation site mutant TelS213A enhances Tel repression of Bcl-xl transcriptional activity.
  • the nuclear localization of the Tel S213A mutant was detected under HGF/EGF exposure.
  • a luciferase reporter assay revealed that the transient expression of Tel S213A results in further repression of Bcl-xl promoter activity.
  • the present invention encompasses the impact of the activation of tyrosine kinase receptors upon transcriptional regulation, including Bcl-xl transcriptional regulation.
  • Tel Ser213 phosphorylation is responsible for receptor kinase activation via the MEK-ERK pathway in cancer cells.
  • At least the phosphorylation of Tel at Ser213 blocks the repression of Bcl-xl transcription by Tel. This negative regulation of Tel function following receptor activiation contributes to the overexpression of the prosurvival Bcl-xl protein in cancer cells.
  • Tel Protein and Exemplary Modifications Thereof there are mutants of Tel protein utilized in methods and/or compositions for cancer therapy and/or prevention.
  • the Tel protein mutants may be of any kind, but in particular aspects there are one, two, three, four, five, or more mutations compared to the wild-type Tel protein.
  • the mutation(s) result in a functionally detectable difference compared to the wild-type protein, although in some cases the mutations result in a Tel protein having no detectable functional difference compared to the wild-type protein.
  • the function that is characterized is transcriptional repressor activity.
  • the function that is characterized is as an inducer of apoptosis.
  • both the transcriptional repressor activity and the apoptotic inducer functions are characterized.
  • Measurement of transcriptional repressor activity may occur by any suitable means in the art, but in specific embodiments the measurement comprises cell transfection with the candidate mutant in a cell having detectable transcription of a reporter polynucleotide or other polynucleotide, followed by Western or northern. Transcription repression can be measured by luciferase assay using several genes' promoter constructs.
  • electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) can be used to measure the interaction between transcription factor or repressor and DNA promoters.
  • Real-time RT-PCR also can be adopted to measure the mRNAs levels of multiple ETV6/TEL regulated genes.
  • Measurement of apoptosis inducer activity may occur by any suitable means in the art, but in specific embodiments the measurement comprises XTT assay (conversion of the water-soluble XTT assay (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide) reagent to an orange formazan product by actively respiring cells); colony formation assay; Annexin V binding; and/or flow cytometry. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay can also be applied.
  • TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling
  • One or more amino acids of Tel may be mutated, in certain aspects of the invention, and in some cases, the amino acid is within a functional domain, for example related to a particular activity for the protein.
  • the mutation may be in the C-terminal inhibitory domain, CID, (amino acids 423-452 of the human wild-type protein), which interferes with the DNA binding domain.
  • CID C-terminal inhibitory domain
  • An additional example of a domain that might be inhibited includes the linker inhibitory damper domain, LID, (amino acids 124-338 of the human wild- type protein, and S213 and S257 are present therein).
  • SAM domain amino acids 41-123, and K99 is present therein
  • ETS domain amino acids 338-422
  • CTBP1 amino acids 423-427
  • Exemplary sequences for wild- type Tel domains are as follows: SEQ ID NO:24 (SAM); SEQ ID NO:25 (LID); SEQ ID NO:26 (ETS); SEQ ID NO:27 (CID). Sequence for an exemplary construct having TAT, SAM, LID, ETS, and CID is in SEQ ID NO:28. Sequence for an exemplary construct having TAT, SAM, LID, and ETS is in SEQ ID NO:29. Sequence for an exemplary construct having TAT, LID, ETS, and CID is in SEQ ID NO:30. Sequence for an exemplary construct having TAT, LID, and ETS is in SEQ ID NO:31.
  • Sequence for an exemplary construct having TAT, ETS, and CID is in SEQ ID NO:32. Sequence for an exemplary construct having TAT and ETS is in SEQ ID NO:33.
  • Exemplary sequences for mutated Tel domains are as follows: SEQ ID NO:35 (SAM having K99R mutation); SEQ ID NO:36 (SAM having V112E mutation); SEQ ID NO:37 (SAM having K99R and V112E mutations); SEQ ID NO:38 (LID having S213A mutation); SEQ ID NO:39 (LID having S257A mutation); and SEQ ID NO:40 (LID having S213A and S257A mutations).
  • Exemplary nuclear localization signals are provided in SEQ ID NO:34 (from Tel, located in the LID domain); SEQ ID NO:41 (ETS1 NLS signal); and SEQ ID NO:42 (SV40 large antigen NLS signal).
  • an "amino acid” can include any naturally or non-naturally occurring amino acid that is phosphorylated, ubiquinated, sumoylated, glycosylated, acetylated and that any amino acid sequence of the present invention may include one or a combination of such amino acids.
  • Specific naturally- occurring amino acids (or moieties of naturally occurring amino acids) that are capable of particular modifications are as follows: serine, threonine, and tyrosine can be phosphorylated; lysine can be ubiquinated, and lysine can be sumoylated.
  • N-linked glycans attach to a nitrogen of asparagine or arginine side-chains; O- linked glycans attach to the hydroxy oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains, or to oxygens on lipids such as ceramide; phospho-glycans linked through the phosphate of a phospho-serine; and C-linked glycans are a rare form of glycosylation where a sugar is added to a carbon on a tryptophan side-chain.
  • a lysine may be acetylated.
  • FIG. 9 illustrates exemplary modifications of wild-type TEL.
  • the polypeptide comprises a protein transduction domain, for example on the N-terminus or C-terminus.
  • FIG. 10 provides SEQ ID NO:3, which illustrates different exemplary domains in the Tel protein.
  • the wild-type protein amino acid sequence is also at GenBank® Accession No. P41212 (SEQ ID NO:3), which is incorporated by reference herein in its entirety.
  • Other names for Tel include at least transcription factor ETV6, ETS translocation variant 6, and ETV6.
  • Tel2 which is a naturally occurring variant of Tell having a truncation on the N-terminus, may be employed in the invention, such as the modification occurring in a Tel2 polypeptide sequence (NP_057219 in GenBank® is the protein sequence, incorporated by reference herein; SEQ ID NO:4).
  • FIG. 11 illustrates one embodiment by which phosphorylations at S213/S257 decrease Tel's repression on Bcl-xl transcription and increase Bcl-xl transcription.
  • Tel is the bcl-xl transcriptional repressor.
  • CID within the Tel protein, has an inhibitory interaction with Tel's ETS domain. The existence of CID can weaken Tel-Bcl-xl promoter binding and promote the Tel moving away from promoter.
  • LID domain of Tel can negatively regulate CID's negative impact on Tel ETS domain, (as in, a negative of a negative is positive). Therefore Tel can repress Bcl-xl transcription at the promoter level. This mechanism is illustrated in FIG. 11 in the upper left picture.
  • CID can negatively regulate Tel ETS domain and promote Bcl-xl transcription (FIG. 11, bottom-right picture).
  • mutations at S213/S257 are useful to regulate Tel function. Blockage of phosphorylation of S213/S257 enhances Tel function, in particular aspects. Deletion of CID is also beneficial, in specific embodiments. K11/K99 are sumoylation sites, and in specific embodiments Kl l/99 sumoylation can promote Tel nuclear exportation, which abolishes Tel's repression. Therefore, in certain embodiments of the invevntion, sumoylation and phosphorylation of Tel are blocked to achieve enhanced Bcl-xl repression.
  • FIG. 13 illustrates an embodiment wherein there is synergism of sumoylation and phosphorylation on negative regulation of Tel function. It also illustrates that Tel as well as ETS 1/2 have several co-factors to work together at DNA promoter level.
  • the present invention concerns novel mutant Tel compositions comprising at least one proteinaceous molecule.
  • a "proteinaceous molecule,” “proteinaceous composition,” “proteinaceous compound,” “proteinaceous chain” or “proteinaceous material” generally refers, but is not limited to, a protein of greater than about 400 amino acids or the full length endogenous sequence translated from a gene; a polypeptide of greater than about 350 amino acids; a polypeptide of greater than about 300 amino acids; a polypeptide of greater than about 250 amino acids; a polypeptide of greater than about 200 amino acids; a polypeptide of greater than about 150 amino acids; and/or a peptide of from about 3 to about 100 amino acids. All the "proteinaceous” terms described above may be used interchangeably herein.
  • the size of the at least one proteinaceous molecule may comprise, but is not limited to, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about
  • an "amino molecule” refers to any amino acid, amino acid derivitive or amino acid mimic as would be known to one of ordinary skill in the art.
  • the residues of the proteinaceous molecule are sequential, without any non-amino molecule interrupting the sequence of amino molecule residues.
  • the sequence may comprise one or more non-amino molecule moieties.
  • the sequence of residues of the proteinaceous molecule may be interrupted by one or more non- amino molecule moieties.
  • the Tel molecule is modified such that it comprises a protein transduction domain (PTD), which may also be referred to as a cell- penetrating peptide, for example.
  • PTD protein transduction domain
  • exemplary cell penetrating peptides that are hydrophilic include the following:
  • R9-Tat GRRRRRRRRRPPQ (SEQ ID NO: 15)
  • Exemplary amphiphilic cell-penetrating peptides include the following:
  • FBP GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 19)
  • MPG ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya (SEQ ID NO: 19)
  • Pep-2 ac-KETWFETWFTEWSQPKKKRKV-cya (SEQ ID NO:23)
  • Cya brings more positive charge to the peptide, which in certain embodiments helps the peptide penetrate through cell membrane.
  • proteinaceous composition encompasses amino molecule sequences comprising at least one of the 20 common amino acids in naturally synthesized proteins, or at least one modified or unusual amino acid, including but not limited to those shown on Table 1 below.
  • the proteinaceous composition comprises at least one protein, polypeptide or peptide.
  • the proteinaceous composition comprises a biocompatible protein, polypeptide or peptide.
  • biocompatible refers to a substance that produces no significant untoward effects when applied to, or administered to, a given organism according to the methods and amounts described herein. Organisms include, but are not limited to mammals, such as humans, dogs, cats, horses, pigs, goats, and sheep. Such untoward or undesirable effects are those such as significant toxicity or adverse immunological reactions.
  • biocompatible protein, polypeptide or peptide containing compositions will generally be mammalian proteins or peptides or synthetic proteins or peptides each essentially free from toxins, pathogens and harmful immunogens.
  • Proteinaceous compositions 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 proteinaceous compounds from natural sources, or the chemical synthesis of proteinaceous materials.
  • the nucleotide and protein, polypeptide and peptide sequences for 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.
  • the coding regions for these 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.
  • various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art.
  • a proteinaceous compound may be purified.
  • purified will refer to a specific or protein, polypeptide, or peptide composition that has been subjected to fractionation to remove various other proteins, polypeptides, or peptides, and which composition substantially retains its activity, as may be assessed, for example, by the protein assays, as would be known to one of ordinary skill in the art for the specific or desired protein, polypeptide or peptide.
  • any protein, polypeptide or peptide containing component may be used in the compositions and methods disclosed herein.
  • the proteinaceous material is biocompatible.
  • the formation of a more viscous composition will be advantageous in that will allow the composition to be more precisely or easily applied to the tissue and to be maintained in contact with the tissue throughout the procedure.
  • the use of a peptide composition, or more preferably, a polypeptide or protein composition is contemplated.
  • Ranges of viscosity include, but are not limited to, about 40 to about 100 poise. In certain aspects, a viscosity of about 80 to about 100 poise is preferred.
  • Proteins and peptides suitable for use in this invention may be autologous proteins or peptides, although the invention is clearly not limited to the use of such autologous proteins.
  • autologous protein, polypeptide or peptide refers to a protein, polypeptide or peptide which is derived or obtained from an organism.
  • Organisms that may be used include, but are not limited to, a bovine, a reptilian, an amphibian, a piscine, a rodent, an avian, a canine, a feline, a fungal, a plant, or a prokaryotic organism, with a selected animal or human subject being preferred.
  • the "autologous protein, polypeptide or peptide” may then be used as a component of a composition intended for application to the selected animal or human subject.
  • the autologous proteins or peptides are prepared, for example from whole plasma of the selected donor.
  • the plasma is placed in tubes and placed in a freezer at about 80°C for at least about 12 hours and then centrifuged at about 12,000 times g for about 15 minutes to obtain the precipitate.
  • the precipitate, such as fibrinogen may be stored for up to about one year (Oz, 1990).
  • compositions of the present invention are particularly suitable for use in tissue adhesion and wound healing
  • preferred proteins are contemplated.
  • Preferred protein include albumin, fibrinogen or gelatin, with albumin being most preferred.
  • a proteinaceous material that possesses one or more of the following characteristics: it forms a solution with a high percentage of protenaceous material solubilized; it possesses a high viscosity (i.e. about 40 to about 100 poise); it has the correct molecular charge to bind the dye if it is a non-covalent mixture (i.e. anionic protein and cationic dye, or cationic protein and anionic dye); it has the correct amino-acids present to form covalent cross-links (i.e.
  • tyrosines one or more tyrosines, histidines, tryptophans and/or methionines
  • biocompatible i.e. from mammalian origin for mammals, preferably from human origin for humans, from canine origin for canines, etc. ; it is autologous; it is non-allergenic, and/or it is non-immunogenic).
  • the biological functional equivalent may comprise a Tel polynucleotide that has been engineered to contain distinct sequences while at the same time retaining the capacity to encode the respective mutant protein. This can be accomplished to the degeneracy of the genetic code, i.e., the presence of multiple codons, which encode for the same amino acids.
  • one of skill in the art may wish to introduce a restriction enzyme recognition sequence into a polynucleotide while not disturbing the ability of that polynucleotide to encode a mutant Tel protein.
  • a polynucleotide may encode a biologically functionally equivalent polypeptide wherein certain amino acids may be substituted for other amino acids without appreciable loss of repressor activity and, in certain cases, with enhanced repressor activity.
  • these are "conservative" changes that do not disrupt the biological activity of the protein, as the structural change is not one that impinges of the protein's ability to carry out its designed function.
  • the change(s) is not conservative but still allows the polypeptide to retain functional activity. It is thus contemplated by the inventors that various changes may be made in the sequence of genes and proteins disclosed herein, while still fulfilling the goals of the present invention.
  • biologically functional equivalents it is well understood by the skilled artisan that, inherent in the definition of a "biologically functional equivalent" protein and/or polynucleotide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule while retaining a molecule with an acceptable level of equivalent biological activity.
  • Biologically functional equivalents are thus defined herein as those proteins (and polynucleotides) wherein selected amino acids (or codons) may be substituted.
  • Functional activity includes at least the ability to repress transcription of a polynucleotide, such as Bcl-xl, for example.
  • Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and/or the like.
  • An analysis of the size, shape and/or type of the amino acid side-chain substituents reveals that arginine, lysine and/or histidine are all positively charged residues; that alanine, glycine and/or serine are all a similar size; and/or that phenylalanine, tryptophan and/or tyrosine all have a generally similar shape.
  • arginine, lysine and/or histidine; alanine, glycine and/or serine; and/or phenylalanine, tryptophan and/or tyrosine; are defined herein as biologically functional equivalents.
  • hydropathic index of amino acids may be considered.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and/or charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine ( 0.4); threonine ( 0.7); serine ( 0.8); tryptophan ( 0.9); tyrosine ( 1.3); proline ( 1.6); histidine ( 3.2); glutamate ( 3.5); glutamine ( 3.5); aspartate ( 3.5); asparagine ( 3.5); lysine ( 3.9); and/or arginine ( 4.5).
  • the present invention in many aspects, relies on the synthesis of peptides and polypeptides in cyto, via transcription and translation of appropriate polynucleotides. These peptides and polypeptides will include the twenty "natural" amino acids, and post-translational modifications thereof. However, in vitro peptide synthesis permits the use of modified and/or unusual amino acids. A table of exemplary, but not limiting, modified and/or unusual amino acids is provided herein.
  • peptide mimetics that mimic elements of protein secondary and tertiary structure are described in Johnson et al. (1993).
  • 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/or antigen.
  • a peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule.
  • ⁇ -turns within proteins which are known to be highly antigenic. Likely ⁇ turn structure within a polypeptide can be predicted by computer-based algorithms, as discussed herein. Once the component amino acids of the turn are determined, mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains.
  • Other approaches have focused on the use of small, multidisulfide- containing proteins as attractive structural templates for producing biologically active conformations that mimic the binding sites of large proteins. Vita et al. (1998). A structural motif that appears to be evolutionarily conserved in certain toxins is small (30-40 amino acids), stable, and high permissive for mutation. This motif is composed of a beta sheet and an alpha helix bridged in the interior core by three disulfides.
  • Beta II turns have been mimicked successfully using cyclic L-pentapeptides and those with D-amino acids.
  • Johannesson et al. (1999) report on bicyclic tripeptides with reverse turn inducing properties.
  • alpha-helix mimetics are disclosed in U.S. Patents 5,446,128; 5,710,245; 5,840,833; and 5,859,184. These structures render the peptide or protein more thermally stable, also increase resistance to proteolytic degradation. Six, seven, eleven, twelve, thirteen and fourteen membered ring structures are disclosed.
  • Beta- turns permit changed side substituents without having changes in corresponding backbone conformation, and have appropriate termini for incorporation into peptides by standard synthesis procedures.
  • Other types of mimetic turns include reverse and gamma turns. Reverse turn mimetics are disclosed in U.S. Patents 5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S. Patents 5,672,681 and 5,674,976.
  • compositions of the Tel mutant protein of the invention it may be desirable to combine these compositions with other agents effective in the treatment of hyperproliferative disease, such as anti-cancer agents.
  • An "anticancer” agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, and/or increasing the lifespan of a subject with cancer.
  • these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell.
  • This process may involve contacting the cells with the expression construct and the agent(s) or multiple factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the second agent(s).
  • Tumor cell resistance to chemotherapy and radiotherapy agents represents a major problem in clinical oncology.
  • One goal of current cancer research is to find ways to improve the efficacy of cancer therapy by combining it with other cancer therapy.
  • Tel mutant therapy of the present invention could be used similarly in conjunction with chemotherapeutic, radiotherapeutic, immunotherapeutic, hormonal, or gene therapy intervention, for example.
  • the other cancer therapy may include surgery, radiation, immunotherapy, hormonal therapy, or chemotherapy, including carboplatin, cisplatin, docetaxel, erlotinib, etoposide, gemcitabine, irinotecan, paclitaxel, pemetrexed, topotecan, vinorelbine, and/or combinations thereof.
  • the other cancer therapy may include surgery, radiation, immunotherapy, hormonal therapy, or chemotherapy, such as cisplatin, raltitrexed, pemetrexed, gemcitabine, vinorelbine, carboplatin, and/or combinations thereof.
  • the present invention may be used simultaneously or may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
  • the other agent and Tel mutant are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the other agent and Tel mutant would still be able to exert an advantageously combined effect on the cell.
  • Tel mutant therapy is "A” and the secondary agent, such as radiation therapy or chemotherapy, for example, is "B":
  • Cancer therapies also include a variety of combination therapies with both chemical-and radiation-based treatments.
  • Combination chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing.
  • One or more chemotherapy agents may be included in the chemotherapy.
  • ⁇ -rays X-rays
  • X-rays X-rays
  • UV-irradiation UV-irradiation
  • 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 6000 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.
  • contacted and “exposed,” when applied to a cell are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell.
  • both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.
  • 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.
  • 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.
  • Immunotherapy thus, could be used as part of a combined therapy, in conjunction with the present therapy.
  • the general approach for combined therapy is discussed below.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
  • 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 and pi 55.
  • the secondary treatment is a gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the Tel mutant.
  • a variety of proteins are encompassed within the invention, including antisense directed against inducers of cellular proliferation; inhibitors of cellular proliferation, such as tumor suppressors, including p53, pl6, C-CAM, Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zacl, p73, VHL, MMAC1 / PTEN, DBCCR- 1, FCC, rsk-3, p27, p27/pl6 fusions, p21/p27 fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb, fms, trk, ret, gsp,
  • VEGF vascular endothelial growth factor
  • FGF FGF
  • thrombospondin BAI- 1, GDAIF, or their receptors
  • MCC MCC
  • regulators of programmed cell death such as members of the Bcl-2 family, ICE-like proteases, and so forth.
  • 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.
  • 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.
  • treatment by surgery includes laser surgery, cryosurgery, electro surgery, 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.
  • 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, 11, or 12 months.
  • These treatments may be of varying dosages as well.
  • agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment.
  • additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adehesion, or agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers.
  • Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL- 2 and other cytokines; F42K and other cytokine analogs; or MIP-1 , MIP-lbeta, MCP-1, RANTES, and other chemokines.
  • cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL would potentiate the apoptotic inducing abililties of the present invention by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.
  • cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyerproliferative efficacy of the treatments. Inhibitors of cell adehesion are contemplated to improve the efficacy of the present invention.
  • cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.
  • FAKs focal adhesion kinase
  • Lovastatin Lovastatin
  • 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.
  • compositions of the present invention comprise an effective amount of one or more Tel mutant compositions of the invention dissolved or dispersed in a pharmaceutically acceptable carrier.
  • pharmaceutically 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 an pharmaceutical composition that contains at least one composition of the present invention or additional 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.
  • for animal e.g.
  • 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. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
  • composition of the present invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • the present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g. , aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g. , liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
  • composition of the present invention may be formulated into a composition in a free base, neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g. , those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.
  • the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent.
  • the carrier should be assimilable and includes liquid, semisolid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate.
  • carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof.
  • composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • parabens e.g., methylparabens, propylparabens
  • chlorobutanol phenol
  • sorbic acid thimerosal or combinations thereof.
  • the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.
  • the composition is combined or mixed thoroughly with a semi- solid or solid carrier.
  • the mixing can be carried out in any convenient manner such as grinding.
  • Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach.
  • stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
  • the present invention may concern the use of a pharmaceutical lipid vehicle compositions that include one or more compositions of the present invention, one or more lipids, and an aqueous solvent.
  • lipid will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term "lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance.
  • Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof.
  • neutral fats phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof.
  • lipids are also encompassed by the compositions and methods of the present invention.
  • composition of the present invention may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art.
  • the dispersion may or may not result in the formation of liposomes.
  • the actual dosage amount of a composition of the present invention administered to an animal patient 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. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. 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.
  • compositions may comprise, for example, at least about 0.1% of an active compound.
  • the an active compound 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.
  • the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • 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.
  • 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.
  • the Tel mutant compositions of the present invention are formulated to be administered via an alimentary route.
  • Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually.
  • these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et ah, 1997; Hwang et ah, 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety).
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.
  • a binder such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof
  • an excipient such as, for
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001.
  • the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells.
  • a syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained-release preparation and formulations.
  • compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally- administered formulation.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically- effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
  • suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids.
  • traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof.
  • suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
  • the Tel mutant composition of the invention may be administered via a parenteral route.
  • parenteral includes routes that bypass the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally (see U.S. Pat. Nos. 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363, each specifically incorporated herein by reference in its entirety).
  • Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety).
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • a coating such as lecithin
  • surfactants for example
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • aqueous solutions for parenteral administration in an aqueous solution
  • the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • a powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
  • the Tel mutant compound may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.
  • Pharmaceutical compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder. Ointments include all oleaginous, adsorption, emulsion and water-solubly based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only.
  • Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin.
  • Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram.
  • Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base.
  • Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture.
  • Transdermal administration of the present invention may also comprise the use of a "patch".
  • the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.
  • the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
  • Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety).
  • the delivery of drugs using intranasal microparticle resins Takenaga et ah, 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725, 871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts.
  • transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).
  • aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant.
  • the typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent.
  • Suitable propellants include hydrocarbons and hydrocarbon ethers.
  • Suitable containers will vary according to the pressure requirements of the propellant.
  • Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms. VII. Kits of the Invention
  • compositions described herein may be comprised in a kit.
  • one or more Tel mutants are comprised in a kit and are housed in suitable container means.
  • kits may comprise a suitably aliquoted Tel mutant of the present invention.
  • the components of the kits may be packaged either in aqueous media or in lyophilized form, for example.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.
  • the kits of the present invention also will typically include a means for containing the Tel mutant and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the Tel mutants may also be formulated into a syringeable composition, in which case, the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an individual, and/or even applied to and/or mixed with the other components of the kit.
  • the components of the kit may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
  • kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.
  • a means for containing the vials in close confinement for commercial sale such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.
  • kits of the invention may also comprise, and/or be packaged with, an instrument for assisting with the injection/administration and/or placement of the ultimate therapeutic composition within the body of an animal.
  • an instrument may be a syringe, pipette, forceps, and/or any such medically approved delivery vehicle.
  • the human mesothelioma cell line 145 and the human lung cancer cell line HI 299 were maintained in RPMI 1640 medium (Thermo Scientific Waltham, MA) supplemented with 10% Fetal Bovine Serum (Invitrogen, Carlsbad, CA).
  • A549 human lung adenocarcinoma cells were maintained in F-12K medium (ATCC, Manassas, VA).
  • Tel cDNA was purchased from Origene Corp (Rockville, MD). All Tel mutants were generated through standard DNA mutagenesis.
  • Anti-Bcl-xl antibodies and all anti-MAP kinase antibodies were purchased from Cell Signaling Technology (Beverly, MA). All antibodies used to detect ETS proteins were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
  • Anti-Actin monoclonal antibody was purchased from Sigma- Aldrich (St. Louis, MO).
  • Chromatin immunoprecipitation assay 145 and A549 cells were plated at a density of 5 X 10 5 per well in a 6 well plate and transfected with Tel and Tel S213A expressing plasmids using Lipofectamine 2000. The cells were serum- starved with or without a lOOng/ml HGF or EGF simulation for 20 minutes. The chromatin was crosslinked by treating the transfected cells with 1% formaldehyde for 15 minutes, sonicated to an average length of 1000 bp and immunoprecipitated with Tel antibody (Santa Cruz) at 4oC overnight.
  • the immunoprecipitated chromatin was then reverse crosslinked and PCR amplified with primers specific for BCL-XL promoter (forward 5 '-GCCTAAGGCGGATTTGA ATGTAG-3 ' (SEQ ID NO: l); reverse 5 '-GAAGGGAGAGAAAGAGATTC AGGAA-3 '(SEQ ID NO:2)).
  • Luciferase assay A549, H1299 and 145 cells were transfected in triplicate in a 24 well plate using Lipofectamine 2000 with 200 ng of Tel or Tel S213A cDNA and pXL plasmid generated by cloning the human Bcl-xl promoter into the pGL2 vector (Promega, Madison, WI). A plasmid expressing ⁇ -Gal (25 ng) was used as an internal control for transfection efficiency. The transfected cells were cultured in serum containing media for 24 hours and then subjected to serum starvation and stimulation by HGF/EGF for 20 minutes. Luciferase and ⁇ - Galactosidase activities were measured and the luciferase values were normalized to the ⁇ - Galactosidase control.
  • XTT assays A549 and I 45 cells were seeded in 96 well plates at 5xl0 3 cells per well and then transfected with various amounts of Tel and Tel S213A using Lipofectamine 2000. The transfected cells were cultured for a further 48 hours and assayed following the standard protocol provided with an XTT kit (Roche, Indianapolis, IN).
  • Clonogenic assays were performed to determine the cytotoxic effects of Tel and the Tel S213A. I 45 and A549 cells were transfected with Tel and Tel S213A at various concentrations by electroporation using nucleofector solution L (Lonza Group, Basel, Switzerland). lxlO 4 cells were then plated per 10 mm dish in triplicate for each concentration and cultured for 2 weeks. The cells were then fixed using 2.5% formaldehyde and stained with 1% crystal violet.
  • Western blotting was performed using a standard protocol. Briefly, I 45, H1299 and A549 cells were transfected with Tel, Tel S213A and Tel S213A/K11R cDNA and cultured for 48 hours. The cells were then lysed and equal amounts of total protein were resolved on 4-20 % Tris-glycine gels. The resolved proteins were then transferred onto a nitrocellulose membrane and the membranes were incubated with antibodies raised against Bcl2 family proteins. The ECL system was used to visualize the signals.
  • Tissue arrays (US Biomax, Rockville, MD) were deparaffinized in xylene substitute and rehydrated in PBS. Antigen retrieval was performed with citrate buffer (pH 6.5) for 20 minutes at 99°C, followed by the block of endogenous peroxidase activity. Sections were incubated with blocking serum in PBS containing 5% bovine serum albumin, followed by incubation with rabbit anti-human Bcl-xl polyclonal antibody or with rabbit anti-phosphorylated human EGFR polyclonal antibody for 24 hours, followed by incubation with a biotinylated goat secondary anti-rabbit antibody (1:200 dilution, Vector Laboratories, Burlingame, CA).
  • Immunoreactive signals were detected using a streptavidin- biotin-peroxidase complex (Vector Laboratories), according to the manufacturer's recommended procedures. All of the slides were counterstained with hematoxylin (Sigma- Aldrich).
  • MAPKs mitogen- activated protein kinases
  • Akt Akt
  • GSK-3 Akt
  • p70 S6 kinase Proteome ProfilerTM Human Phospho-MAPK Arrays.
  • FIG. 1A The array signals, shown in FIG. 1A, indicated that kinase activities from growth factor stimulated cells were much stronger than those from serum-starved 145 cells. There were no significant differences found between the c-Met and EGFR groups. The activation of MAPKs was further confirmed by western blotting.
  • ETS-2, PU. l and Tel proteins were analyzed in 145 cells under conditions of serum starvation or HGF stimulation by immunoprecipitation and western blot analysis. Whereas the levels of total ETS proteins were observed to be equivalent in the cells, the levels of phosphorylated ETS-2, PU.l and Tel were clearly elevated (FIG. IB). Similar results were observed in lung cancer cells upon EGF stimulation. The inventors then mapped the serine sites that were phosphorylated after growth factor exposure. FIG. 1C demonstrated that only S213A mutation prevented Tel being phosphorylated.
  • TEL PHOSPHORYLATION NEGATIVELY REGULATES THE TEL-BCL-XL PROMOTER INTERACTION UPON THE GROWTH FACTOR STIMULATION OF
  • Bcl-xl promoter construct XLp-Luc, Tel and Tel S213A cDNA expression vectors were co-transfected into A549 and I 45 cells.
  • Bcl-xl promoter activity is significantly decreased in serum-starved Tel transfected 145 cells in comparison with the I 45 cells under growth factor stimulation.
  • Tel S213A expression leads to decreased Bcl-xl expression under both growth factor stimulated and growth factor deprived conditions.
  • TEL S213A IS A MORE POTENT REPRESSOR OF BCL-XL EXPRESSION THAN
  • Bcl-2 family proteins were evaluated by western blotting following the transfection of cancer cells with Tel, Tel S213A and control expression vector. As shown in FIG. 4B, the expression of Bcl-xl was decreased below basal levels after Tel cDNA transfection. Bcl-xl expression was further reduced when Tel S213A was expressed in both A549 and 145 cells, whereas the Bcl-2 levels remained unchanged. Mcl-1 was found to be upregulated in these cells on a western blot suggesting that an apoptosis "defense mechanism" operates in these tumor cells (Cao et ah, 2007; Xu et ah, 2010).
  • TEL S213A IS MORE POTENT INDUCER OF APOPTOSIS THAN WILD TYPE TEL
  • the current data also indicate that Tel Ser213 phosphorylation is the key response to Bcl-xl transcriptional repression. Tel has multiple serine and threonine sites that can potentially be phosphorylated by ERK kinase (Irvin et ah, 2003). Although in certain embodiments Ser213 (not Ser257) is the only Tel site phosphorylated by ERK in the tested cell lines, Ser 257 has been reported to be the target of p38, which is an activator of different stress signals (Hanson et ah, 2008). Hence, the phosphorylation of Tel by both ERK and p38 is a unique property of this ETS protein. Both ERK and stress signaling pathways converge via Tel in the nucleus under certain circumstances and provide further stimulation of Bcl-xl transcription in cancer cells.
  • the identified phosphorylation sites on Tel reside within the linker inhibitory damper (LID) domain, outside its ETS DNA-binding domain.
  • LID linker inhibitory damper
  • CID amino acids 423-452
  • LID domain amino acids 124-3378
  • CID amino acids 423-452
  • LID domain serves as negative regulator of CID (Green et ah, 2010). Decreased Tel binding to DNA due to the phosphorylation of S213 indicates that LID, once phosphorylated, in certain embodiments of the invention loses its inhibitory interaction with the CID, which would negatively regulate Tel- promoter binding.
  • Tel is also modified by sumoylation at Kl l and K99 (Wood et ah, 2003; Chakrabarti et ah, 2000).
  • the present studies indicate that the double mutant Tel K11R/S213A is a stronger repressor of Bcl-xl transcription than either single mutation alone.
  • both phosphorylation and sumoylation are required for Tel export, the order in which Tel is phosphorylated and sumoylated is unclear, as is whether these two types of modification function cooperatively or independently.
  • ETS members such as NET and YAN
  • phosphorylation and sumoylation generally work synergistically to mediate the downregulatory nuclear export of the ETS transcriptional repressors (Tootle and Rebay, 2005).
  • Dazzi H Hasleton PS, Thatcher N, et al. Malignant pleural mesothelioma and epidermal growth factor receptor (EGF-R). Relationship of EGF-R with histology and survival using fixed paraffin embedded tissue and the F4, monoclonal antibody. Br J Cancer 1990; 61: 924-926.
  • Foulds CE Nelson ML, Blaszczak AG, et al. Ras/mitogen- activated protein kinase signaling activates Ets-1 and Ets-2 by CBP/p300 recruitment. Mol Cell Biol 2004; 24: 10954-10964.
  • Wood LD Irvin BJ, Nucifora G, et al. Small ubiquitin-like modifier conjugation regulates nuclear export of TEL, a putative tumor suppressor. Proc Natl Acad Sci U S A 2003; 100: 3257-3262.
  • Xu H Krystal GW. Actinomycin D decreases Mcl-1 expression and acts synergistically with ABT-737 against small cell lung cancer cell lines. Clin Cancer Res; 16: 4392-4400.

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Abstract

La présente invention concerne généralement des procédés et des compositions de traitement d'un individu atteint du cancer en utilisant des polypeptides mutants de TEL. Dans des modes de réalisation particuliers, les peptides ou polypeptides TEL qui sont utilisés présentent des mutations au niveau d'un ou plusieurs sites de phosphorylation, d'acétylation, de glycosylation, d'ubiquitination, ou de sumoylation, par exemple.
PCT/US2012/025608 2011-02-17 2012-02-17 Thérapie anticancéreuse ciblée utilisant des inhibiteurs de la transcription d'ets Ceased WO2012112869A2 (fr)

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Publication number Priority date Publication date Assignee Title
US20180141977A1 (en) * 2015-05-06 2018-05-24 Norwegian University Of Science And Technology (Ntnu) Anti-bacterial agents and their use in therapy

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GREEN ET AL.: 'DNA binding by the ETS protein TEL (ETV6) is regulated by autoinhibition and self-association' THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 285, no. 24, 11 June 2010, pages 18498 - 18504 *
MAKI ET AL.: 'Leukemia-related transcription factor TEL is negatively regulated through extracellular signal-regulated kinase-induced phosphorylation' MOLECULAR AND CELLULAR BIOLOGY vol. 24, no. 8, April 2004, pages 3227 - 3237 *
ROUKENS ET AL.: 'Identification of a new site of sumoylation on Tel (ETV6) uncovers a PIAS-dependent mode of regulating Tel function' MOLECULAR AND CELLULAR BIOLOGY vol. 28, no. 7, April 2008, pages 2342 - 4357 *
WOOD ET AL.: 'Small ubiquitin-like modifier conjugation regulates nuclear export of TEL, a putative tumor suppressor' PNAS vol. 100, no. 6, 18 March 2003, pages 3257 - 3262 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180141977A1 (en) * 2015-05-06 2018-05-24 Norwegian University Of Science And Technology (Ntnu) Anti-bacterial agents and their use in therapy
US10570180B2 (en) * 2015-05-06 2020-02-25 Norwegian University Of Science And Technology (Ntnu) Anti-bacterial agents and their use in therapy

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