EP1432732A2 - Composition pharmaceutique comprenant un agent modulateur de l'etat de polymerisation de l'actine - Google Patents

Composition pharmaceutique comprenant un agent modulateur de l'etat de polymerisation de l'actine

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Publication number
EP1432732A2
EP1432732A2 EP02745538A EP02745538A EP1432732A2 EP 1432732 A2 EP1432732 A2 EP 1432732A2 EP 02745538 A EP02745538 A EP 02745538A EP 02745538 A EP02745538 A EP 02745538A EP 1432732 A2 EP1432732 A2 EP 1432732A2
Authority
EP
European Patent Office
Prior art keywords
zyxin
cell
expression
cells
pharmaceutical composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02745538A
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German (de)
English (en)
French (fr)
Inventor
Christian Auclair
Valérie AMSELLEM
Martial Hervy
Frédéric SUBRA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Institut Gustave Roussy (IGR)
Bioalliance Pharma SA
Ecole Normale Superieure de Cachan
Original Assignee
Centre National de la Recherche Scientifique CNRS
Institut Gustave Roussy (IGR)
Bioalliance Pharma SA
Ecole Normale Superieure de Cachan
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Application filed by Centre National de la Recherche Scientifique CNRS, Institut Gustave Roussy (IGR), Bioalliance Pharma SA, Ecole Normale Superieure de Cachan filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP1432732A2 publication Critical patent/EP1432732A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • 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

Definitions

  • the present invention relates to the field of oncology, it is based on observations of the modification of certain cellular phenotypic characteristics linked to the structure of the cytoskeleton, such as adhesion and motility, of cells when said cells evolve towards a phenotype. tumor.
  • the invention is based on the correlation observed by the applicant between the under-expression of genes involved in the stabilization of the actin network of the cytoskeleton of cells, such as for example the under-expression of the zyxin gene and the phenotypic transformation d 'a normal phenotype versus a tumor phenotype of said cells.
  • the invention is also based on the demonstration, thanks to the experimental work carried out, that the administration of a pharmaceutical composition capable of stabilizing the actin network of the cytoskeleton of the cell, such as for example compounds inducing the overexpression of the discomfort of zyxin in a cell comprising a tumor phenotype causes the phenotypic reversion of said cell to a normal phenotype.
  • the subject of the invention is the identification of compounds capable of modulating the state of polymerization of actin and the use of said compounds for the preparation of medicaments useful for the diagnosis, prevention and / or treatment of pathologies tumor.
  • Such compounds capable of stabilizing the actin network can for example be inhibitors of cofilin which is an enzyme known for its action on the depolymerization mechanism of actin F, in its active form (dephosphorylated cofilin) it induces rupture of propellers and promotes depolymerization of actin F.
  • the lines must have a well-characterized phenotype. They must have an immortal non-tumor phenotype and the malignant transformation of the immortal lines should be induced by a single genetic event. Finally, we must be able to easily obtain phenotypic revertants.
  • tumor phenotypes induced by oncogenic fusion proteins such as BCR-Abl, PML-RAR which lead to leukemic phenotypes as well as EWS-Fli-
  • Ewing's sarcoma is a neuroectodermal tumor which is characterized by a chromosomal translocation involving the ql2 band of chromosome 22 reworked with the q24 band of chromosome 11: t (ll; 22) (q24; ql2) (Turc-Carel et al., 1984) leading to the formation of a chimeric gene associating the protooncogene EWS with a member of the ETS family of genes.
  • the breakpoints associated with the majority translocation t (ll; 22) are located in a region of 7 kb belonging to the EWS gene for chromosome 22 and a region of 50 kb belonging to FLI-1 for chromosome 11 (Zucman et al. ., 1993).
  • the result of this chromosomal translocation generates a derivative of chromosome 22 where the 5 'part of the EWS gene is associated with the 3' part of the FLI-1 gene (Delattre et al., 1992).
  • the fusion gene expresses the chimeric protein
  • EWS-FLI-1 with oncogenic properties.
  • the chimeric protein EWS-Fli-1 is capable of. transform murine fibroblasts of NIH3T3 type in culture (Ohno et al., 1993) and induce tumors in "nude” mice.
  • the association of the N-terminal domains of EWS and C-terminal of FLI-1 is necessary for its transforming power (March et al., 1993).
  • a study model has been developed consisting on the one hand of normal murine NIH3T3 fibroblasts, non-tumor immortals and on the other hand of fibroblasts expressing the EWS-Fli-1 fusion protein in a constitutive way and presenting a tumor phenotype in nude mice.
  • This pair of cells makes it possible to carry out an evaluation of the differential expression characterizing the acquisition of the tumor phenotype.
  • phenotypic revertants were obtained by infecting the transformed cells with retroviral vectors coding for antisense RNAs directed against the oncogenic fusion gene.
  • a second pair of cells tumor cells / non-tumor revertant cells was thus obtained and could be the subject of a differential expression study.
  • the immunostaining of actin filaments on fibroblasts shows that the malignant transformation of said fibroblasts mediated by a chimeric protein EWS-Fli-1 results in a profound modification of the morphology of fibroblasts with in particular a reduction in focal points. This is accompanied by a reshaping of the cytoskeleton and in particular of the polymerized actin networks.
  • the tumor phenotype is influenced by the level of expression of zyxin and that the under-expression of the zyxin gene is a sufficient condition to transform a normal fibroblast into a tumorigenic ibroblast.
  • zyxin is a protein comprising LIM domains present in the focal adhesion plates of fibroblasts and the lamellipods of higher eukaryotic cells. These LIM motifs, in the form of a zinc finger, are involved in protein-protein interactions. (Scheimechel et al. 1994. "The LIM domain a new structural motif found in zinc-printer-like proteins”. Trends Genêt. 10: 315-320.
  • Zyxin is involved in regulating the polymerization of actin filaments and has structural and functional properties in common with ActA from Listeria (Golsteyn et al., 1997). Zyxin is supposed to act as an anchoring intermediary between the plasma membrane via ⁇ -actinin and the integrins and actin filaments. It is clearly involved in the architecture of the cytoskeleton, adhesion and cell motility (Crawford and Beckerle, 1991). Structurally, zyxin comprises an N-terminal region rich in proline, a nuclear export signal peptide (NES), as well as regions rich in amino acids Histidine and Cysteine forming the LIM motifs in the C-terminal part. (Sadler et al. 1992. "Zyxin and cCRP: Two interactive LIM domain proteins associated with the cytoskeleton". J.Cell.Biol. 119: 1573-1587).
  • NES nuclear export signal peptide
  • the mechanism of zyxin-dependent tumorigenesis involves changes in motility, adhesion and signaling related to cell-cell and cell-extracellular matrix interactions.
  • the object of the invention is to provide new pharmaceutical compositions for the treatment and prevention of cancers comprising compounds capable of stabilizing the actin network of the cytoskeleton of a cell for the preparation of a medicament intended for the treatment or cancer prevention.
  • a composition according to the invention is capable of restoring a non-tumor phenotype unlike most of the compositions of the prior art which destroy st cells are therefore capable of inducing side effects.
  • the present invention therefore relates to a pharmaceutical composition for the treatment, prevention or diagnosis of a tumor pathology, characterized in that it comprises an active agent capable of stabilizing the actin network of the cytoskeleton of a selected cell in the group comprising: - the protein zyxin
  • nucleic acid molecule comprising or consisting of the zyxin gene, a fragment thereof or their complementary sequence, or an antisense nucleic acid thereof, a cell or a set of cells overexpressing the gene zyxin or a protein encoded by a fragment thereof.
  • zyxin fragment is understood to mean any polypeptide fragment thereof capable of conserving the biological function of zyxin and in particular its function of stabilizing the actin network of the cytoskeleton.
  • fragment of the zyxin gene is intended to mean any fragment of the nucleic acid of zyxin and in particular the cDNAs of said gene coding for zyxin and / or the various functional domains thereof, such as those coding for:
  • zyxin gene any nucleic acid chemically modified, or by genetic recombination but retaining the function of said gene, in particular its capacity to code for a polypeptide, when it is expressed in an appropriate host, which retains biological functions of the protein zyxin and in particular its function of stabilizing the actin network of the cell cytoskeleton.
  • modifications include, for example, modification, addition or removal of bases, by fusion with other acids nucleic acids, such as, for example, regulatory elements, or chimeric molecules comprising heterologous cDNAs obtained by fusion of the corresponding cDNAs according to techniques known to those skilled in the art.
  • zyxin protein means any polypeptide modified, for example chemically by association with functional chemical groups, said functional chemical groups being chosen from groups capable of coupling said protein or a fragment thereof. to other molecules, such as, for example, markers, of carrier proteins for the production of an immunogen, enzymes, either on solid supports, such as mineral supports, or organic polymers.
  • a first embodiment of the invention relates to a pharmaceutical composition comprising the protein zyxin or a functional fragment thereof.
  • the pharmaceutical composition of the invention comprises the protein zyxin or functional fragments thereof associated with transport vectors chosen from the group comprising:
  • lipid systems such as anionic or neutral liposomes, and in particular liposomes based on phosphatidylcholoine (PC) or dioleylphosphatidylcoline (DPE), cationic liposomes, in particular liposomes based on dioctadecyldimethylam onium bromide (diodylylpropyl bromide) trimethylammonium (DOTMA), DOGS (Transfectam ® ), DDPPES etc., cationic emulsions such as emulsions based on soybean oil and 1,2 diolcoyl-glycero-3-trimethylammonium propane (DOTAP), etc.
  • DOTAP 1,2 diolcoyl-glycero-3-trimethylammonium propane
  • microparticles based on co-glycolide polydactide acid) PLAG, cetylmethylammonium bromide (PLG-CTAB), PLG-PEI, or microparticles based on PLG-poly-L -Lysine etc., i.e. nanoparticles based on chitosan, nanoparticles of PLG, gelatin, etc.
  • - cationic polyene antibiotics such as cationic derivatives of Amphotericin B.
  • zyxin is associated with said intracellular transport vectors by covalent or non-covalent chemical bonds.
  • the pharmaceutical composition comprises a nucleic acid comprising a cDNA of the zyxin gene, a fragment or a derivative thereof associated with a viral recombinant expression vector or with a non-viral transport vector of particulate type.
  • association between the active agent and the transport vector means either the fixing of said active agent to the transport vector, such as for example by non-covalent bonds, for example of hydrophobic type, or by covalent chemical bonds by means of coupling agents or not, according to techniques well known in the art.
  • person skilled in the art that is to say the insertion of said active compound into a viral or bacterial recombinant expression vector.
  • the active compound is brought to its target either by infection with viral particles expressing said active compound, or by transfection with recombinant expression vectors capable of expressing the active compound during their integration into said host cell.
  • the pharmaceutical composition of the invention comprises a recombinant viral expression vector comprising elements necessary for transcriptional control as well as for controlling the translation of the sequence of a cDNA of the zyxin gene when said expression vector is introduced into target cells.
  • the pharmaceutical composition of the invention comprises a recombinant viral expression vector comprising regulatory sequences, such as constitutive or inducible promoters, or even non-coding sequences of the zyxin gene, allowing the expression of zyxin in the cells of the host to which the composition of the invention is administered.
  • regulatory sequences such as constitutive or inducible promoters, or even non-coding sequences of the zyxin gene
  • the pharmaceutical composition of the invention comprises a recombinant viral expression vector comprising regulatory sequences chosen from LTR sequences, such as for example the LTR sequences of the Moloney leukemia virus, under the dependence of a promoter of the LTR in 5 '.
  • LTR sequences such as for example the LTR sequences of the Moloney leukemia virus
  • the pharmaceutical composition of the invention comprises as transport vector intracellular, any recombinant viral expression vector placed under the control of promoters of the host cell allowing the expression of zyxin in the host cell according to genetic recombination techniques well known to those skilled in the art.
  • expression vectors derived from recombinant Adenoviruses from viruses associated with recombinant Adenoviruses (AAV), from baculoviruses or from recombinant retroviruses, and most preferably a vector of the recombinant lentivirus type.
  • AAV recombinant Adenoviruses
  • the pharmaceutical composition of the invention comprises a viral expression vector comprising promoter sequences, chosen for example and without limitation from the CMV promoter, the EF1 alpha promoter or the PGK promoter.
  • the pharmaceutical composition of the invention comprises as active agent a cell from a patient suffering from a tumor pathology genetically modified to express the discomfort of zyxin.
  • the pharmaceutical composition of the invention is useful for the preparation of a medicament intended for the treatment or prevention of tumor pathologies.
  • the pharmaceutical composition of the invention is useful for the preparation of a medicament intended for the treatment of pathologies such as malignant hemopathies associated with chromosomal abnormalities of the localization region of the zyxin 7q34 / q35 gene.
  • composition of the invention is useful for the preparation of a medicament intended for the treatment or prevention of hepatocarcinomas, neuroectodermal cancers, Ewing's sarcoma.
  • the invention also relates to the non-viral and viral intracellular transfer vectors associated with the active agent, capable of being used in the above pharmaceutical composition.
  • Another subject of the invention relates to a viral vector capable of being used in a pharmaceutical composition defined above.
  • the subject of the invention is therefore a viral vector comprising a cDNA coding for the zyxin gene or a functional fragment thereof.
  • the viral vector according to the invention is chosen from a recombinant vector derived from an Adenovirus, a virus associated with Adenoviruses (AAV) or a retrovirus.
  • a third embodiment of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising as active agent a cell characterized in that it is genetically modified to overexpress the gene for zyxin or a functional fragment thereof.
  • the overexpression of the zyxin gene in said cell is obtained either by transfection of a cell with expression vectors comprising a cDNA of the zyxin gene or by infection of a cell with viral particles expressing said gene for the zyxin.
  • the pharmaceutical composition according to the invention comprises, as active principle, a cell chosen from a stem cell, a cell of bone marrow, a hematopoietic cell or a hepatocarcinoma cell genetically engineered to overexpress the zyxin gene or a functional fragment thereof.
  • the pharmaceutical composition of the invention comprises as active agent a CD34 + cell genetically modified to overexpress the discomfort of zyxin or a functional fragment thereof.
  • the pharmaceutical composition of the invention comprises as active agent a cell originating from a patient suffering from a tumor pathology genetically modified to express the zyxin gene or a functional fragment thereof.
  • the invention also relates to a genetically modified cell overexpressing the zyxin gene.
  • the invention also relates to a genetically modified cell under-expressing the zyxin gene.
  • Such a cell can be obtained, for example, using an antisense RNA targeting the AUG of zyxin and introduced into the cells via a synthetic oligonucleotide cloned in the shuttle comprising the transport vector. .
  • the genetically modified cells according to the invention are chosen from a stem cell, a bone marrow cell, a hematopoietic cell or a hepatocarcinoma cell.
  • the genetically modified cells according to the invention are CD34 + cells.
  • the genetically modified cells according to the invention are from a patient with a tumor pathology.
  • Another embodiment of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising as active agent a compound binding the polymerized actin F with an affinity constant greater by at least two logs than the affinity constant with which said active agent binds unpolymerized actin G.
  • the active agents of the composition of the invention have an affinity constant of the order of 10 7 -10 8 M "1 for the polymerized actin F.
  • the active agent binding the polymerized actin is a cyclic peptide.
  • the invention also relates to a non-human transgenic mammal comprising at least one genetically modified cell sub-expressing the zyxin gene or a functional fragment thereof.
  • the subject of the invention is also a non-human transgenic mammal comprising at least one genetically modified cell sub-expressing the gene for zyxin or a functional fragment thereof.
  • the invention also relates to a method for identifying compounds capable of stabilizing the actin network of the cytoskeleton of a cell, comprising detecting a phenotypic reversion of the expression of zyxin induced by said compounds, characterized in that it comprises the following stages: a) bringing the compounds to be tested into contact with said cell, b) quantifying the expression of zyxin in said cell.
  • the quantification of the expression of zyxin is carried out by comparison of the expression of the messenger RNAs of zyxin in said cell in the presence and in the absence of said test compound.
  • the quantification of the expression of zyxin is carried out by comparison of the expression of the protein zyxin in said cell in the presence and in the absence of said compound to be tested
  • the invention also relates to a method for diagnosing a tumor pathology comprising the following steps: a) the removal of cells from a patient, b) the quantification of the expression of zyxin in the cells removed
  • the quantification of the expression of zyxin is carried out by measuring the expression of the messenger RNAs of zyxin.
  • the quantification of the expression of zyxin is carried out by comparison of the expression of the protein zyxin of the cells sampled at different intervals.
  • the subject of the invention is a method of analyzing a tumor phenotype of a patient, characterized in that it comprises the following stages: a) the removal of the cells from the patient at two different time intervals, b) the quantification of the expression of zyxin in the cells sampled at different intervals. c) comparing the two levels of expression in order to constitute a phenotypic differential profile of said patient.
  • the quantification of the expression of zyxin is carried out by comparison of the expression of the messenger RNAs of the cells sampled at different intervals.
  • the quantification of the expression of zyxin is carried out by comparison of the expression of the zyxin protein of the cells sampled at different intervals.
  • Another subject of the invention relates to a method of screening for an active compound in the treatment of cancers, comprising the following steps: a) incubation of tumor cells with said active compound, b) measuring the stabilization of the polymerization of the actin network of said cells.
  • the invention also relates to the use of a substance capable of restoring the actin networks of a cell for the preparation of a non-cytotoxic anti-tumor medicament.
  • Another subject of the invention is the use of such a substance for the preparation of a medicament intended for the treatment and / or prevention of a pathology resulting from a chromosomal abnormality in the long arm of chromosome 7, more particularly at the level of the region 7q34 / q35.
  • the invention also relates to the use of such a substance for the preparation of a medicament intended for the treatment of a malignant hemopathy associated with a chromosomal abnormality in the region of the gene of zyxin 7q34 / q35.
  • Another subject of the invention is the use of a substance for the preparation of a medicament intended for the treatment or prevention of hepatocarcinomas or neuroectodermal cancers.
  • Another subject of the invention is the use of a substance for the preparation of a medicament intended for the treatment or prevention of mesenchymal tumors, in particular sarcomas.
  • Another subject of the invention is the use of such a substance for the preparation of a medicament intended for the treatment or prevention of Ewing's sarcoma.
  • Figure 1 is a diagram of the interactions between zyxin and its cellular partners at the level of the plasma membrane.
  • Figure 2 shows a diagram of the construction of the viral transport vector associated with zyxin.
  • FIG. 3A shows actin filaments revealed by labeling the cells with a phalloidin probe coupled to the FITC.
  • FIG. 2B shows the location of the zyxin revealed by labeling the cells with an anti-zyxin antibody revealed with an anti-mouse IgG antibody coupled to TRITC.
  • FIG. 4A illustrates the results of the
  • FIG. 4B is a schematic representation of the retroviral shuttle containing the zyxin reading frame, mRNAs containing the open reading frame of human zyxin and of the neo / CMV and zyxin probes used for the revelation of the Northern blots.
  • FIG. 5 illustrates the results of the Northern blot with the detection of the mRNA originating from the 5 'LTR, from 10 ⁇ g of total RNA deposited on denaturing agarose gel, by the neo / CMV probe of 1081 bp.
  • FIG. 6 shows the Western blot and immunodetection images of the zyxin protein in the different cell lines from 60 ⁇ g of protein extracts from the different cell lines.
  • FIG. 7 illustrates the Western Blot images obtained after immunoprecipitation of the protein extracts the protein EWS-FLI-1 extracted from the clones E-F zyxin.
  • FIG 8A shows the sequence of
  • FIG. 8B is a diagram of the construction of the retroviral shuttle expressing the antisense against the AUG of zyxin.
  • FIG. 8C shows the detection by RTPCR of the antisense RNA directed against the AUG of zyxin.
  • FIG. 9 shows the Western blot and immunodetection images of the zyxin protein from 40 ⁇ g of protein extract from different cell lines.
  • FIG. 10 is a graphic representation of the comparative study of the variations in gene expression rates, carried out by macro-arrays, between the line NIH3T3 and the lines EWS-FLI, as zyxine 1 and as zyxine 2.
  • FIG. 11 illustrates the in vitro measurement of actin polymerization by fluorescence anisotropy.
  • FIGS. 12A to 12G show the results of the study of the morphological modification of the NIH3T3 and EWS-Fli cells in the presence of Dolastatin (DU: Untreated NIH3T3 cells (FIG. 12A); NIH3T3 cells transfected with EWS-Fli (Figs 12B , 12C); (NIH3T3 cells transfected with EWS-Fli incubated in the presence of 10 ⁇ M ( Figure 12D) or 100 ⁇ M ( Figure 12E) of Jasplakinolide or incubated with 10 ⁇ M ( Figure 12F) or 20 ⁇ M ( Figure 12G) of DU.
  • DU Untreated NIH3T3 cells
  • Figs 12B , 12C NIH3T3 cells transfected with EWS-Fli incubated in the presence of 10 ⁇ M
  • Figure 12E 100 ⁇ M
  • Jasplakinolide or incubated with 10 ⁇ M ( Figure 12F) or 20
  • FIG. 13 shows the results of the study of the toxicity of Dolastatin 11 (DU) on NIH3T3 and EWS / Fli cells.
  • FIG. 14 shows the results of the study of the development of tumors in nude mice in the presence of Dolastatin 11.
  • GEBCO newborn calf serum
  • antibiotics penicillin lOOUI / mL and streptomycin lOO ⁇ g / mL
  • the EWS-FLI line contains a cDNA encoding the EWS-FLI-1 fusion protein in its genome.
  • the expression of this protein is selected using 2.5 ⁇ g / ml of puromycin.
  • the AS-A line produced by M. Hervy et al. , produces a small antisense RNA directed against the mRNA encoding the protein EWS-Fli-1.
  • This line is selected, in addition to puromycin, lmg / ml of geneticin. Geneticin makes it possible to select the cells which produce the small antisense RNA directed against the mRNA coding for the protein EWS-FLI-1.
  • NIH3T3 AS zyxin lines are cells that produce a small antisense RNA directed against the AUG of mRNA encoding zyxin. They are cultivated in a medium supplemented with geneticin at 1 mg / ml in order to select the expression of the antisense.
  • GP + env Am12 cells are transcomplementing cells capable of providing in trans the proteins coded by the gag and pol genes, carried on a plasmid, and env carried by another plasmid.
  • This line is capable of producing amphotropic viral particles.
  • This line is cultivated in a medium containing DMEM and 10% fetal calf serum (GIBCO) supplemented with penicillin and streptomycin.
  • GEBCO fetal calf serum
  • These cells are selected using a mixture of three compounds (200 ⁇ g / ml of hygromycin B, 15 ⁇ g / ml of hypoxanthine, 250 ⁇ g / ml of mycophenolic acid) for two weeks before transfection with the retroviral shuttle.
  • the cells are seeded on glass slides until they adhere (from 24H to 48H).
  • the cells are fixed with a 3% paraformaldehyde solution rinsed with PBS and permeabilized with a PBS / 0.2% triton X100 solution.
  • the permeabilized cells are saturated with a PBS / 2% BSA solution.
  • the cells are incubated with the primary antibody (anti-zyxin from J. Wehland) diluted twice for 40 min, rinsed 3 times 5 min with PBS and then incubated with the secondary anti-mouse IgG antibody coupled to Texas Red (TRITC) for 40 min.
  • the cells are incubated directly with phalloidin coupled to FITC for 40 min.
  • the coverslips are observed under a fluorescence microscope.
  • the retroviral vector pLNCX contains, on the one hand, the LTR sequences and the psi sequence originating from the Moloney murine leukemia virus (MoMLV) and, on the other hand, the resistance gene to neomycin, conferring resistance to geneticin, under the dependence of the promoter of the LTR in 5 ′.
  • This vector also contains a multicloning site (MCS) directly dependent on the early promoter of the cytomegalovirus (pCMV).
  • MCS multicloning site
  • the vector pLNCX is digested with HindIII / ClaI at the level of the MCS in order to insert therein a sequence containing two adapters which are capable of self-associating in a complementary manner. Between these two adapters, this sequence contains other unique restriction sites including Nsil and Sali.
  • the plasmid pzyxine GFP contains the cDNA coding for human zyxin coupled in phase to the gene for the "green fluorescent protein" GFP (given by M. Beckerle). It is digested with Hind III and BamH I then cloned into the vector PLNCX at the level of the MCS (HindH I / BglII); BamHI and BglII are compatible sites. Digestion with Hind II / BglII eliminates one of the two adapters, this preventing self-association of the RNA coding for the zyxin produced by this vector.
  • the result of this construction is a retroviral vector named pLNCX ADA zyxin, coding for human zyxin under the direct influence of pCMV.
  • pLNCX ADA as zyxin
  • pLNCX ADA sa zyxin is a vector which has the capacity to produce a small RNA in rod-loop structure directed against the AUG of the mRNA coding for zyxin, directly dependent on the pCMV promoter.
  • the construction of the vector is carried out by inserting at the level of the Nsil and Sal I sites of the MCS a small sequence directed against the AUG of the mRNA of zyxin.
  • the vector retroviral shuttle is transformed into the corresponding virus by transfecting the retroviral vector into a transcomplementing cell line GP + envAM12 (GPA).
  • the GPA transcomplementing line is transfected by the retroviral shuttle (pLNCX zyxine or PLNCX ADA as zyxine) in the presence of Superfect (Qiagen) according to the supplier's recommendations.
  • the cells expressing the neo r gene are selected in a medium containing l ⁇ g / ml of G418. Resistant cells are collected, amplified and seeded in a 75cm 2 bottle at a rate of 2.10 6 cells. Two days later, the medium is replaced by a non-selective medium. The supernatant is collected every 24 hours for 3 days, pooled, aliquoted and frozen.
  • the retroviral titer is evaluated on NIH3T3 cells after selection of cells with geneticin (1 mg / ml). The viral supernatant of the GPA cells is then used to infect the desired cells with a multiplicity of infection of the order of 0.1. Three days after infection, the cells are selected with l ⁇ g / mL of G418. Only cells that have integrated the retroviral shuttle resist G418 and form clones that are isolated and amplified to produce permanent cell lines.
  • the adherent cells are trypsinized, pelletized and rinsed with PBS.
  • the cells are lysed with cold RIPA (10 mM Tris-HCL pH7.4, 100 mM NaCl, ImM EDTA, 1% Triton X100, 0.5% Na deoxycholate, 0.1% SDS) in the presence of a mixture of protease (Boehringer) .
  • RIPA cold RIPA
  • the samples are centrifuged for 15 min at 14,000 rpm.
  • the supernatants are recovered and the protein concentration is estimated by the Bradford test.
  • An aliquot containing 1.5 mg of total protein extract is incubated for 1 hour 30 minutes at 4 ° C. with 0.2 ⁇ g of antibody directed against terminal C dominor of Fli-1.
  • the analysis of the rate of production of the zyxin protein is carried out by Western blot on a total cell extract using as primary antibody, the anti-zyxin monoclonal mouse antibody given by J. Wehland directed against the region between the NES and the LIM domains. This membrane is revealed by a chemiluminescent reagent (Immunostar: Biorad).
  • RNAplus lysis solution Quantum Biotechnology
  • An aliquot of 10 ⁇ g of total RNA is denatured in a solution containing 0.04 M MOPS pH7, 0.01M of sodium acetate, 2.2 M formaldehyde and 50% formamide.
  • the samples are analyzed on agarose gel denatured with formaldehyde and transferred to a charged nylon membrane (Hybond N +: Amersham Pharmacia).
  • the membrane is hybridized with a cDNA probe radiolabeled with [ 32 P] dCTP by random priming (Prime-a-gene ® labeling System: Promega) in a prehybridization solution containing 5X SSC, 5X Denhart's, 0, 1 mg / mL of salmon sperm DNA, 0.1 mg / mL of yeast t-RNA, 0.1% SDS, 25 M pH7 KH 2 P0 4 and 50% formamide at 42 ° C overnight. The next day the membrane is rinsed three times with SSC2X / 0.1% SDS at room temperature and once or twice at 42 ° C with 0.5 X SSC / 0.1% SDS. The membrane signals are then observed at the phospholmager.
  • Total RNAs are produced by the same technique described above. The quality and cleanliness of the RNA is checked on denaturing gel. The retrotranscription is carried out using the Omniscript TM kit from Qiagen specifically using a 20-sea LTR primer. The conditions used are 10 ng of LTR primer, 2.5 mM dNTP, I ⁇ g of total RNA supplemented with 2OU of RNase inhibitor
  • RNAsin ® RNAsin ®
  • RT buffer 40U reverse transcriptase.
  • the mixture is incubated for one hour at 37 ° C and 5 min at 94 ° C.
  • the RT products of specific cDNAs which are amplified by PCR using two other primers named as 1 and as 2.
  • reaction conditions are 0.25 mM dNTP, lOOng of each of the primers and 1/20 is the product of RT, l.5mM
  • the cycles used are 4 min 94 ° C and 30cycles (30s 94 ° C, 45s 61 ° C, 1 min 72 ° C) and 10 min at 72 ° C.
  • the cells from the different clones to be tested are trypsinized, pelletized and resuspended in sterile PBS at the rate of 5.10 6 cells per ml.
  • An aliquot of 200 ⁇ L of cells is injected subcutaneously into nude mice aged 6 to 8 weeks, irradiated the day before at 5 Gray.
  • the mice are reared in a sterile, air-conditioned atmosphere. Observation of tumor development is carried out every week for 5 to 6 weeks.
  • the "Atlas cDNA expression Arrays" cDNA expression card (Clontech) is produced according to the supplier's recommendations. Two identical nylon membranes (No. 7741-1) on which are deposited 588 mouse cDNA samples, corresponding to 588 genes, makes it possible to hybridize cDNAs from two different cell lines in parallel. Information concerning the 588 genes is available on the Clontech site (http://www.clontech.com/atlas.genelist/search.htlm). The preparation of the radiolabelled cDNA probes is carried out by retrotranscription with [ 32 P] dATP using the Clontech kit. Hybridization is performed according to the instructions from the supplier. The signals are observed at the Phospholmager.
  • EWS-FLI EWS-Fli fusion protein
  • the production of virus is obtained by transfection of this plasmid into an amphotropic murine packaging line called GPA.
  • EWS-FLI cells are infected using the viral supernatant produced by GPA cells and selected in the presence of Geneticin.
  • the clones thus obtained are called E-F / Zyxin.
  • the study by fluorescence microscopy shows that the EWS-FLI cells have lost the bundles of actin microfilament and the ability to spread, characteristics typical of transformed fibroblasts (Pollack, R et al; 1975, Maness, P, E; 1981).
  • the cells of the EF / Zyxin clones partially recovered the structure of the actin microfilaments as well as the spreading capacity of the NIH3T3 cells (FIG. 4).
  • these cells no longer have the growth capacity in multilayers, typical of transformed cells.
  • a relocation of zyxin is observed at the level of the adhesion plates, in the intercellular junctions and along the stress cables (FIG. 3).
  • RNA coding for zyxin is more weakly expressed in the tumorigenic line EWS-FLI than in the line NIH3T3 (FIG. 4). This result confirms the difference in expression observed previously, by microarrays, between the NIH3T3 and EWS-FLI cells.
  • the expected size of the human zyxin mRNA from the retroviral shuttle and expressed from the CMV promoter (2.2 kb) (FIG. 4B) is identical to that of the endogenous zyxin mRNA. It is therefore very likely that the increase in the intensity of the band that is observed in the three E-F / zyxin clones is due to the expression of the zyxin RNA expressed from the retroviral shuttle.
  • RNA larger than 4.7 kb is represented only in the RNAs from the E-F / zyxin clones.
  • This RNA has a size between 5.5 and 6 kb compatible with an RNA which would be expressed from the promoter located in the U3 region of the 5 'LTR (5.7 kb) of the retroviral shuttle (FIG. 4B).
  • Northern blot was carried out using a 108lbp (neo / CMV) probe, corresponding to a restriction fragment derived from the plasmid pLNCX ADA zyxin, capable of detecting only the RNA derived from the 5 'LTR (FIG. 5).
  • the result presented in FIG. 5 reveals a hybridization only in the EF zyxin clones.
  • the detected band is positioned at the same level as the indeterminate band present in the Northern blot using the zyxin probe. It can therefore be concluded that the retroviral shuttle produces two RNAs containing the zyxin sequence, one from the CMV promoter and the other from the promoter present in the 5 'LTR.
  • the difference in intensity detected in the presence of two different amounts of protein extracts shows that the amount of antibody used is not limiting.
  • the amount of EWS-FLI-1 immunodetected by the amount of anti-Fli-1 antibody detected has been corrected.
  • the results presented in the form of a histogram (FIG. 7B) indicate the absence of the EWS-FLI-1 protein in the NIH3T3 line and an under expression in the AS-A line (expressing an antisense directed against the EWS-FLI junction sequence ) compared to the EWS-FLI line.
  • the amount of EWS-FLI-1 protein is clearly greater than the amount of protein detected in the non-tumorigenic AS-A line and remains comparable to the amount present in tumorigenic EWS-FLI cells.
  • NIH3T3 cells do not cause tumor development.
  • This line is used as a negative control since it is known to be non-tumorigenic.
  • EWS-FLI line known to be tumorigenic
  • all the mice developed tumors between the 2 nd and 3 rd week.
  • the tumorigenicity of the EF zyxin clone two cases may arise, either there is no tumor development (three out of five mice) or there is a delay in tumor development of about two to three weeks (two out of five mice). Analysis of these tumors shows that the DNA of the retroviral shuttle is still present, however, the exogenous RNA coding for zyxin could not be detected. It therefore appears that the development of delayed tumors in mice is due to a loss of expression of the exogenous protein zyxin.
  • the tumor development tests in nude mice corroborate the morphological changes and the growth modifications observed between the cells expressing the antisense directed against zyxin and the parental NIH3T3 cells (Table 2).
  • the rate of tumor development after the injection of EWS-FLI cells is also faster than that observed following the injection of the AS-Zyxin clones.
  • Table 2 Tumorigenicity test: study of the number of tumors developed after subcutaneous injection in nude mice of 10 6 cells from different cell lines for six weeks.
  • EGR1 and p53 tumor suppressors
  • ERCC-1 proteins involved in repair
  • ADAP proteins playing a role in cell differentiation and growth
  • ICE proteins playing a role in cell differentiation and growth
  • TIMP2 proteins involved in the cell matrix
  • PN-1 proteins involved in the cell matrix
  • urokinase plasminogen activator proteins involved in the cell matrix
  • Cell extracts of NIH3T3 cells or EWS-Fli are placed in the presence of actin Alexa 488 in a buffer G (4.3 ⁇ M Tris pH 8.1; 170 ⁇ M CaCl 2 ; 170 ⁇ M DTT; 170 ⁇ M ATP).
  • the polymerization reaction is triggered by the addition of a P buffer (51 mM KC1, 1 mM MgCl 2 and 0.5 mM ATP).
  • Dolastatin ( ⁇ M) is added to the cellular extracts of EWS-Fli cells at the same time as the buffer P.
  • the actin polymerization is followed by anisotopy of fluorecence on a Beacon 2000. The results obtained are illustrated in FIG. 11.
  • the cells are seeded (20% of confluence) on glass slides 20 mm in diameter. Three days after sowing, the glass slides are placed in airtight chambers, regulated in temperature (37 ° C) and in C0 2 (5%).
  • the motility measurement of the cells is carried out by taking a photograph by optical phase contrast microscopy every 4 minutes for 24 hours.
  • the motility analyzes, carried out using the “metamorph” program, are carried out for each cell type, on ten cells. The results are illustrated in Figure 15.
  • EWS-Fli cells with 10 or 20 nM of Dolastatin 11 not only makes it possible to restore the stress fibers of the cells, with a morphology which approximates that of NIH3T3 cells, but it also makes it possible to restore the contact structures between cells (Figure 12).
  • compositions of the invention require, for the phenotypic reversal of the tumor phenotype, doses much lower than those known from one prior art.
  • One of the families of cancers capable of being treated with the pharmaceutical compositions of the invention is the family of melanomas, in which the applicant has characterized the under-expression of the zyxin gene.
  • the Applicant has characterized the expression of several genes in a murine melanoma line, compared to the expression of these same genes in a non-tumor line.
  • B16 / F10 cells are a very aggressive murine melanoma line. They are compared to the non-tumorigenic NIH3T3 line. The total RNA of the two cell lines are amplified and radiolabeled by RT PCR. RT PCR products (cDNA) are incubated with Atlas ® Mouse cDNA Expression Array Clontech membranes (ref 7741-1). The membranes are then exposed and the image is analyzed with MicroArray ® software.
  • B16 / F10 cells are a melanoma line
  • zyxin is a very conserved protein (97% homology between man and mouse) the sequence difference between human and murine zyxin is very probably not at the origin of this phenomenon.

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