WO1999067380A1 - c-Cbl SUPPRESSION OF NEOPLASTIC TRANSFORMATION - Google Patents

Abstract

Introduction of a vector achieving overexpression of the proto-oncogene c-Cbl in transformed cells, particularly cells transformed by an oncogenic protein tyrosine kinase, reverses the transformed phenotype. A method for suppression of neoplastic transformation is provided.

Description

C-CBL SUPPRESSION OF NEOPLASTIC TRANSFORMATION

Cross-Referenee to Related Application

This application claims priority from United States provisional application Ser. No.60/090,195, filed June 22, 1998., the entire disclosure of which is incorporated herein by reference.

Reference to Government Grant

The invention described herein was supported in part by National Institutes of Health grant CA78499. The U.S. government has certain rights in the invention.

Field of the Invention

This invention relates to use of the c-Cbl gene for the suppression of neoplastic transformation. The invention further relates to methods for the diagnosis or prognosis of a malignancy comprising determining the expression or activity level of c-Cbl.

Background of the Invention

Neoplastic transformation of cells is the cause of cancer diseases. This transformation can be caused by oncogenic proteins, including hyperactivated protein tyrosine kinases.

Several presently known proteins, for example p53 , Rb and Rapl , are capable of suppressing growth of transformed cells. Also, dominant- inhibitory mutant forms of proteins that are crucial for cellular activation may suppress cellular transformation. Although the use of tumor suppressor genes and dominant-inhibitory mutants for gene therapy of cancer is feasible, none of the available therapies appears to be absolutely effective in spite of extensive experimentation in the field. The protein c-Cbl, the cellular homolog of the transforming gag-v-

Cbl protein encoded by the murine Cas NS-1 retrovirus (Blake et al. , Oncogene 1991, 6:653-657) is expressed in a wide variety of cell types and becomes tyrosine phosphorylated in response to the stimulation of various multichain immune recognition receptors, cytokine and growth factor receptors, and T-cell accessory molecules. c-Cbl can interact with other proteins in both SH2- dependent and SH2-independent ways. SH2-dependent interactions of c-Cbl are mediated by its phosphotyrosine residues mapped to tyrosines 700, 731 and 774 (Feshchenko et al. , J. Biol. Chem. 273:8323-8331, 1998).

It was initially suggested that c-Cbl is a transcriptional regulator (Blake et al. , Oncogene 1991 , 6:653-657). However, it was later determined that c-Cbl and its transforming mutant forms, other than v-Cbl, were cytoplasmic proteins, and that stimulation did not cause their translocation to the nucleus (Blake et al , J. EMBO 1993, 12:2017-2026). The presence of various interactive sites, such as phosphotyrosine residues (Feshchenko et al. , supra), proline-rich motifs (Blake et al. , 1991. supra), and a non-SH2 phosphotyrosine - binding domain (Lupher et al. , J. Biol. Chem. 1996, 271 :24063-24068), in the c-Cbl molecule suggests that c-Cbl acts as a multivalent adaptor protein. Several reports have been published implicating c-Cbl in the negative regulation of protein tyrosine kinase-mediated signaling (Bonita et al. , Mol. Cell. Biol.1997 ^', 17:4597-4610; Bowtell and Langdon, Oncogene 1995, 11 : 1561-1567; Levkowitz et al , Genes & Development 1998, 12:3663-3674; Miyake et al , Proc. Natl Acad. Sci. USA 1998, 95:7927-7932; Ota and Samelson, Science 1997, 276:418- 420; Thien and Langdon, Oncogene 1997, 14:2239-2249; Yoon et al , Science 1995, 269: 1102-1105). Furthermore, c-Cbl has recently been implicated in the regulation of adhesion in T cells (Zell et al. , Current Biol. 1998, 8:814-822) and macrophages (Meng and Lowell. EMBO J. 1998, 17:4391-4403). Therefore, biological functions of c-Cbl remain to be elucidated. What is needed is an additional strategy for suppressing cell transformation, to augment or supplant existing strategies.

Summary of the Invention

It is an object of the invention to provide a method for the suppression of neoplastic transformation in cells and tissues, as a treatment for neoplastic proliferative disorders.

It is an object of the invention to provide genetically engineered constructs and methods for cancer therapy .

These and other objects will be apparent from the following disclosure.

A method for suppressing neoplastic transformation of a cell is provided comprising administering to the cell a vector expressing c-Cbl so that neoplastic transformation is suppressed in the cell. According to one embodiment of the invention, the neoplastic transformation which is suppressed is transformation by an oncogenic protein tyrosine kinase, such as an oncogenic Abl protein tyrosine kinase.

A method for suppressing neoplastic transformation of cells in an animal is provided comprising administering to the animal a vector expressing c- Cbl so that neoplastic transformation of cells of the animal is suppressed. The following definitions are intended as an aid to understanding the scope and practice of the present invention.

By "animal" it is meant to include, but is not limited to, mammals including humans. The animal treated according to the present invention is preferably a human being. "C-Cbl gene'^' is used to describe the gene of the aforementioned name, and all allelic forms, both naturally occurring and created in the laboratory, without regard to the species of origin. The human c-Cbl proto-oncogene has been isolated, sequenced and cloned. See Blake et al, Oncogene 1991, 6:653-657 and GenBank accession no. X571 10, the entire disclosures of which are incorporated herein by reference. "C-Cbl protein" or "c-Cbl gene product" means the protein encoded by the c-Cbl gene. The terms include the wild-type c-Cbl protein, as well as other forms, both naturally occurring and created in the laboratory.

A "vector" is any means for the transfer of a nucleic acid into a host cell. The term vector includes both viral and nonviral means for introducing the nucleic acid into a cell in vitro, ex vivo, or in vivo. Non-viral vectors include but are not limited to plasmids, liposomes. electrically charged lipids (such as cytofectins), DNA-protein complexes, and biopolymers. Viral vectors include but are not limited to vectors derived from retrovirus, adeno-associated virus, pox viruses, baculovirus, vaccinia virus, herpes simplex virus, Epstein-Barr virus, adenovirus and hybrids of two or more viral vector types. In addition to an antiviral construct according to the invention, a vector may contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).

By "neoplastic transformation" is meant the process by which a cell has acquired certain properties of a tumor cell, such as the capacity for unlimited proliferation and the capacity for anchorage-independent growth in culture. Essentially, neoplastic transformation means the conversion of a normal cell into a tumor cell.

By "protein tyrosine kinase'^" is meant a an enzyme which catalyzes protein phosphorylation by transferring a terminal phosphate from ATP to the side chain of a tyrosine amino acid of the protein. By "oncogenic protein tyrosine kinase" is meant a protein tyrosine kinase which can be identified as oncogenic by its capacity for neoplastic transformation of NIH 3T3 fibroblasts. By "oncogenic Abl protein tyrosine kinase" is meant the product of an oncogenic abl gene, such as but not limited to v-abl and bcr-abl.

By "suppression" of neoplastic transformation as applied to a cell is meant the prevention of the cell from initiating the process of neoplastic transformation, the interruption of an ongoing neoplastic transformation process in the cell, or the reversal of a neoplastic transformation already substantially completed. "Pharmaceutically acceptable carrier" includes diluents and fillers which are pharmaceutically acceptable for method of administration, are sterile, and may be aqueous or oleaginous suspensions formulated using suitable dispersing or wetting agents and suspending agents. The particular pharmaceutically acceptable carrier and the ratio of active compound to carrier are determined by the solubility and chemical properties of the composition, the particular mode of administration, and standard pharmaceutical practice.

Generally, the nomenclature used hereafter and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, cell culture, and transgene incorporation. Generally enzymatic reactions, oligonucleotide syntheses, and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references which are known to the skilled artisan, including Maniatis (Molecular Cloning, Cold Spring Harbor Laboratories. 1982), and Ausubel (Current Protocols in Molecular Biology. Wiley and sons. 1987), which are incorporated herein by reference.

Description of the Figures

Figs. 1A-1C are phase-contrast microscopic views showing the effects of c-Cbl overexpression on morphology of NIH3T3 cells transformed by Bcr-AblX (Fig. IB) or v-Abl (Fig. 1C). The two panels of each cell type in Fig. 1 C represent duplicate experiments. The transformed cells were transduced with a vector expressing wild-type c-Cbl, the 5 Y->F c-Cbl mutant, or with vector alone. Fig. 1A shows untransformed NIH3T3 cells.

Figs.2A-2G are confocal microscopic views of immunofluorescent staining of v-Abl-transformed NIH3T3 cells transduced by v-Abl. v-Abl- transformed NIH3T3 cells transduced to express either 5 Y->F c-Cbl (Figs 2A and 2E) or wild-type c-Cbl (Figs 2B, 2C, 2D, 2F and 2G) were fixed, permeabilized and stained with anti-Cbl (Figs 2A-2G), phalloidin-Texas Red (Figs 2A-2D), and anti- paxillin (Figs 2E, 2F and 2G). Anti-Cbl and anti-paxillin were visualized using secondary antibodies labeled with FITC and Texas Red, respectively. No staining was observed when samples were stained with irrelevant antibodies (data not shown).

Detailed Description of the Invention

The inventors have made the unexpected discovery that overexpression of the c-Cbl protein suppresses transformation of cells, and transformation by transforming oncogenes in particular, most particularly transformation by oncogenic protein tyrosine kinases. Transforming oncogenes are oncogenes that can be identified by their capacity for neoplastic transformation of NIH3T3 fibroblasts, a cell line of immortal but nonmalignant mouse cells. Transforming oncogenes generally encode an abnormal form of some protein involved in pathways of cell growth or differentiation.

The transformation-suppressing effect of c-Cbl is dependent on its phosphorylation. The 5Y->F mutant form of c-Cbl in which tyrosines were mutated to phenylalanine residues, is ineffective in suppressing transformation.

Phosphorylation of the mutant protein is prevented by the lack of tyrosine residues.

The suppression of cell neoplastic transformation by overexpression of c-Cbl was demonstrated with v-Abl transformed NIH3T3 fibroblasts. The v- Abl protein is an oncogenic protein tyrosine kinase. v-Abl transformed cells transfected with a retroviral vector which induced 20- to 30-fold increased c-Cbl expression. The increase in c-Cbl expression correlated with at least a 3-fold decrease in colony formation in soft agarose compared to untransfected v-Abl transformed cells. The c-Cbl-transfected cells also became adherent and retained a flat morphology typical for untransformed cells, while the v-Abl transformed cells exhibited anchorage-independent growth and the round or spindle-like morphology typical for transformed cells.

Suppression of cell transformation is caused by stable overexpression of recombinant wild-type c-Cbl in transformed cells. To achieve this overexpression, the c-Cbl cDNA is transferred into transformed cells using a mammalian expression vector (preferably, a retroviral vector) with the c-Cbl cDN A insert. The insertion of c-Cbl cDNA into a vector is achieved using standard subcloning procedures, as hereinafter exemplified, namely (1 ) enzymatic digestion of the plasmid pMSCV and the plasmid containing the c-Cbl cDNA, (2) purification of the obtained fragments from an agarose gel, (3) ligation of the obtained fragments using T4 DNA ligase, (4) transformation of E. coli with the resultant ligation mixture and selection of positive transformants, and (5) purification of the resultant vector from E. coli.

The promoter is selected and the vector is constructed such that c- Cbl becomes over-expressed in the target cell. By "over-expressed" is meant expression at a level substantially above (at least two-fold higher) that the level in the target cell before the manipulation. The level of c-Cbl expression obtained in the target cell is preferably at least 5- fold, more preferably at least 10 fold, and most preferably at least 20-fold, in comparison to the level of c-Cbl in the cell prior to transfer of the c-Cbl vector. The relative level of c-Cbl expression in cells may be determined by methods well-known to those skilled in the art. Such methods principally include a determination of the level of mRNA transcripts (e.g., Northern hybridization), or the level of protein production (i.e., Western blot).

The c-Cbl gene can be used as a gene therapeutic to suppress neoplastic transformation of cells and thereby provide a favorable therapeutic outcome in patients afflicted with neoplastic proliferative disorders. In a preferred embodiment, the c-Cbl gene becomes stably integrated into the cellular genome.

Expression vectors encoding c-Cbl can be generated in several ways using standard techniques of molecular biology. Such a vector should be suitable for stable, high-level expression of c-Cbl in mammalian cells. Retroviral vectors containing strong viral promoters, such as the immediate-early human cytomegalovirus promoter/enhancer, to drive the expression of inserted c-Cbl, are the preferred vehicle for the c-Cbl DNA transfer. Transformation of NIH3T3 cells by both v-Abl and Bcr-AblX is suppressed by the overexpression of c-Cbl.

The invention provides a method for the suppression of neoplastic transformation of a cell comprising inducing overexpression of the c-Cbl gene in the cell. In one preferred embodiment the c-Cbl gene is delivered into transformed cells using a viral vector in which transcription of c-Cbl is controlled by a highly active promoter, for instance the immediate early promoter of human CMV virus. In a more preferred embodiment the viral vector is an adenoviral vector or a retroviral vector. In a most preferred embodiment the vector is a retroviral vector. The c-Cbl gene is preferably delivered to the patient in the form of a viral vector. Viral vectors that may be used in the present invention include adenoviral vectors and retroviral vectors. Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver nucleic acid to a variety of cell types. Various serotypes of adenovirus exist, including type 2 and type 5 human adenoviruses and adenoviruses of animal origin. Preferably, the replication defective adenoviral vectors according to the invention comprise the LTRs, an encapsidation sequence, and the nucleic acid of interest. Still more preferably, at least the El region of the adenoviral vector is nonfunctional. Other regions may also be modified, including the E3 region (see WO95/02697), the E2 region (see W094/28938). the E4 region (see

W094/28152. W094/12649, and WO 95/02697), or in any of the late genes L 1 -L5

Replication defective recombinant adenoviruses according to the invention can be prepared by techniques known to a person skilled in the art. In particular they can be prepared by homologous recombination between an adenovirus and a plasmid which carries the DNA sequence of interest. Homologous recombination is effected following cotransfection of the adenovirus and plasmid into an appropriate cell line. The cell line employed should be transformable by said components and contain sequences which are able to complement the defective regions in the replication defective adenovirus. Examples of cell lines which may be used are the human embryonic cell line 293 (Graham et al., J. Gen. Virol. 36, 59 (1977)) which contains the left-hand portion of the genome of an Ad5 adenovirus (12%) integrated into its genome, and cell lines which are able to complement the El and E4 functions, as described in W094/26914 and WO95/02697. Recombinant adenoviruses are recovered and purified using standard biological techniques which are well known to those having ordinary skill in the art.

Adenoviral vectors can be produced at high titers (e.g. 10¹⁰-10'² infectious units per ml), and can be used to transiently express the c-Cbl gene in a non-tissue-specific manner.

Retroviruses are integrating viruses which generally infect dividing cells. The retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag,pol and env). The construction of recombinant retroviral vectors is known to those of skill in the art.

In recombinant retroviral vectors, the gag, pol, and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest. These vectors can be constructed from different types of retrovirus, such as M-MuLV, MSV (murine Moloney sarcoma virus), HaSV (Harvey sarcoma virus), SNV (spleen necrosis virus), RSV (Rous sarcoma virus) and Friend virus.

In general, in order to construct recombinant retroviruses containing a sequence according to the invention, a plasmid is constructed which contains the LTRs, the encapsidation sequence and the coding sequence. This construct is used to transfect a packaging cell line, which cell line is able to supply in trans the retroviral functions which are deficient in the plasmid. In general, the packaging cell lines are thus able to express the gag, pol and env genes. Such packaging cell lines have been described in the prior art. In particular the cell line PA317 (US 4,861,719). the PsiCRIP cell line (WO90/02806). and the GP+envAm-12 cell line (WO89/07150) may be mentioned. Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.

Retroviral vectors derived from lentiviruses such as HIV-1. HIV-2, and SIV can be used for delivery to nondividing cells. These viruses can be pseudotyped with the surface glycoproteins of other viruses, such as M-MuLV or vesicular stomatitis virus (VSV). The production of high titer HIV-1 pseudotyped with VSV glycoprotein has been disclosed by Bartz and Vodicka (Methods 12(4):337-42 (August 1997)), and multiply attenuated lentiviral vectors have been disclosed by Zufferey et al. (Nature Biotechnology 15:871-75 (September 1997)). Such lentiviral vectors can infect nondividing cells, have a broad host range, and can be concentrated to high titers by ultracentrifugation.

Chimeric adenoviral/retroviral vector systems can also be used to achieve efficient gene delivery and long term gene expression. A chimeric viral system in which adenoviral vectors are used to produce transient retroviral producer cells in vivo, such that progeny retroviral particles infect neighboring cells has been described by Feng et al. (Nature Biotechnology 15:866-70 (September 1997)). Retroviral vectors are generally preferred for the practice of the present invention. Recombinant viruses are administered to a patient in an amount sufficient to treat or prevent the occurrence of cancer. Effective amounts vary depending on the characteristics of the patient, the type and severity of the condition being treated, the desired duration of treatment, the method of administration, and other parameters. Effective amounts may be determined by the physician or by another qualified medical professional. Recombinant viruses according to the invention are generally formulated and administered in the form of doses of between about 10⁴ and 10¹⁴ pfu. The retrovirus may be administered systemically or locally at the site of a tumor.

As an alternative to in vivo vector delivery to a tumor or tumor site, cells may be excised from the patient and transfected ex vivo to enhance c-Cbl expression. The C-Cbl expressing cells may then be reimplanted in the body of the patient. The vector may be combined with a pharmaceutically acceptable carrier. Such carriers are known to those skilled in the art.

C-Cbl gene therapy may be utilized to treat any neoplastic disease. The treatment is carried out on individuals suffering from a hematologic neoplastic disease, or neoplastic disease characterized by the occurrence of one or more solid tumor masses. Tumors treatable include, for example, osteosarcoma; fibrosarcoma; colon carcinoma; melanoma; epidermal carcinoma; breast carcinoma; various forms of brain tumor, such as glioma, glioblastoma. astrocytoma, meningioma and ependymoma, for example; mesothelioma; lung carcinoma; pancreatic carcinoma; prostate carcinoma; testicular carcinoma; various gynecological carcinomas such as ovarian carcinoma and endometrial carcinoma; head and neck carcinoma; carcinoma of the oral cavity, throat or stomach. Moreover, the treatment may be undertaken for solid tumors which may arise from leukemias and lymphomas, as well as the treatment of leukemias and lymphomas per se.

Most preferably, the present invention is utilized in the treatment of neoplastic disease characterized by neoplastic transformation induced by an oncogenic protein tyrosine kinase.

The protein tyrosine kinases encompass a large diverse group of oncogenes and proto-oncogenes which encode proteins which catalyze the transfer of a phosphate residue from a nucleoside triphosphate to the side chain of a tyrosine residue in a protein. The transforming potential of protein tyrosine kinases is primarily activated by N-terminal or C-terminal rearrangements. These alterations may remove down-regulating domains of the protein and result in the constitutive activation of what is normally a conditionally regulated enzyme activity. Thus, when suitably mutated (or, in some instances, anomalously expressed), protein tyrosine kinases themselves become transforming proteins, acting through unwanted phosphorylation of their diverse substrates. Further, protein tyrosine kinases can be vehicles for transformation by disturbances elsewhere in signaling pathways., e.g. , constitutive production of growth factors that act through protein tyrosine kinase receptors (Aaronson and Pierce, Cancer Cells 1990, 2:212-214) and the effects of phosphatases, which play crucial roles in governing the activity of protein tyrosine kinases (Hunter, Cell 1989, 58:1013-1016).

One type of tyrosine protein kinase comprises the transmembrane protein kinases which span the plasma membrane . They contain large extracellular and cytoplasmic domains. One such category comprises the EGF family of growth factor receptors. The receptor has intrinsic tyrosine kinase activity that is activated by the binding of its ligand. EGF-1 is expressed in breast cancers and glioblasto- mas. EGF₂ is found expressed in neuroblastomas. Further examples of the tyrosine kinase growth factor receptor family include erbB,fms,fos, kit, met. trk and neu oncogenes.

Another type of tyrosine kinases includes a large number of nonintegral membrane-associated protein tyrosine kinases. The protein product of v-src, the prototype of this family, is associated with the plasma membrane but does not traverse the membrane. Oncogenic p60^{v src} encoded in Rous sarcoma virus and its cellular homolog p60^c"src, are membrane-localized phosphoproteins that possess protein tyrosine kinase activity. The cDNA sequence of the normal cellular homologue, the proto-oncogene c-src, has been reported (Braeuninger et al. , Proc. Natl. Acad. Sci. USA 1991, 88:10411-10415). Normal p60^c"src is tightly regulated in its kinase activity relative to p60^v"src and generally is not oncogenic. Mutations in p60^c"src that elevate its kinase activity also activate its oncogenic potential.

Other members of the tyrosine kinase family include/es, abl gr and yes. All of these proto-oncogene products are homologous in their tyrosine kinase domains. The tyrosine kinase domain, as in the growth factor receptor tyrosine kinase family, is responsible for catalyzing the transfer of phosphate groups from ATP to tyrosine residues during autophosphorylation or transphosphorylation of target molecules. The aberrant expression of a nonintegral membrane associated tyrosine kinase is best illustrated by the c-abl proto-oncogene (Shtivelman et al, Cell 1986, 47:277-284). Aberrant expression of c-abl results from the gene's translocation from the long arm of chromosome 9 to the breakpoint cluster region (bcr) on chromosome 22, resulting in the formation of bcr-abl hybrid genes. The chimeric message is in turn translated into a larger chimeric Abl protein (210 kDa) that has increased tyrosine kinase activity (Konopka et al, Cell 1984. 37: 1035. The 210 kDa protein is considerably larger than the normal human Abl protein of 145 kDa, and has a very high tyrosine kinase activity.

The present invention may be utilized in the treatment of neoplastic disorders driven by abnormal or aberrant expression of the aforementioned protein tyrosine kinases.

According to one preferred embodiment of the invention, the present invention comprises a therapy for disorders wherein the oncogenic protein tyrosine kinase inducing or triggering the disorder is an Abl protein tyrosine kinase. In particular, the invention is believed useful in suppressing the neoplastic transformation which occurs with expression of the bcr-abl oncogene. The latter is manifested by the Philadelphia¹ -positive (Ph¹ -positive) chromosome characteristic of most chronic myelogenous leukemias and a significant portion of acute lymphocytic leukemias. Throughout the course of therapy according to the invention, the patient is preferably monitored to assess the treatment effectiveness. The primary indicia of treatment success is reduction in tumor dimensions. The size of the tumor treated may be monitored over the course of the therapy by established tumor imaging techniques such as x-ray. CAT scan, magnetic resonance imaging, direct visual or microscopic examination where feasible, and the like.

For non-solid tumors, i.e. the leukemias, treatment is monitored primarily by histological examination of the bone marrow for surviving leukemic cells. However, a significant number of leukemic cells may still exist when marrow examination provides normal results. For this reason, more recent methods for detecting leukemic cells have focused on detecting the presence of the gene for the relevant oncogene, or its corresponding mRNA, in cells of the bone marrow as a more sensitive test. See, for example, the following U.S. Patents: 4,681,840, 4,857,466 and 4,874,853. The presence of even a few copies of the target oncogene can be effectively detected by amplification using reverse transcriptase polymerase chain reaction technology. For a detailed discussion of such methods, see for example, Cancer Principles & Practice of Oncology, edited by V. T. DeVita, S. Hellman and S.A. Rosenberg, J.B. Lippincott Company, Philadelphia, PA (3rd ed., 1989). Methods for diagnosing and monitoring the progress of neoplastic disorders vary depending upon the nature of the particular disease.

The suppression of cell transformation by overexpression of c-Cbl was demonstrated as follows. The hemagglutinin (HA)-tagged c-Cbl was inserted into the MSCVpac retroviral vector. The MSCV/?ac/c-Cbl construct was co- transfected into 293T cells with pHIT60 and pHIT456 plasmids, containing the gag-pol or env cDNA of MLV, respectively. Co-expression of these plasmids resulted in a production of recombinant retroviral particles capable of integrating c-Cbl cDNA. as well s pac, the puromycin resistance gene, into genomic DNA of fibroblasts. v-Abl transformed NIH3T3 fibroblasts were incubated with the obtained supernatants of 293T cells and then cultured in the presence of puromycin to select positive transformants. The selected puromycin-resistant fibroblasts were cloned, and the obtained clones were tested for the expression of recombinant HA- tagged c-Cbl. This expression exceeded the expression of endogenous c-Cbl in fibroblasts by 20 to 30 fold. Growth and morphology of v- Abl-transformed NIH3T3 overexpressing HA-tagged wild-type c-Cbl were compared to those of v- Abl-transformed NIH3T3 transduced with the MSCVpac vector alone (the latter cells did not overexpress c-Cbl). Colony-forming activity in soft agarose was approximately 3-fold lower for v- Abl-transformed NIH3T3 clones overexpressing wild-type c-Cbl than for v-Abl-transformed NIH3T3 cells expressing endogenous c-Cbl alone or overexpressing functionally deficient c-Cbl. The colony-forming activity in soft agarose is a measure of anchorage-independent growth, which is a hallmark of cellular transformation. Furthermore, v-Abl-transformed NIH3T3 transduced with the vector alone or with the functionally deficient form of c-Cbl are typically detached from the surface of cell culture dishes, whereas v-Abl- transformed NIH3T3 transduced with wild-type c-Cbl become adherent, like untransformedNIH3T3 cells. Finally, v-Abl-transformed NIH3T3 transduced with the vector alone or with the functionally deficient form of c-Cbl have round or spindle-like morphology typical for transformed cells, whereas many of v-Abl- transformed NIH3T3 transduced with the c-Cbl cDNA returned to flat morphology typical for untransformed cells.

Examples The following examples illustrate the invention. These examples are illustrative only, and do not limit the scope of the invention.

EXAMPLE 1 Materials and Methods

A. Cell lines and clones.

Human renal embryocarcinoma 293T cells (Pear et al. , Proc. Natl. Acad. Sci. USA 1993; 90:8392-8396) were obtained from ATCC upon authorization from the Rockefeller University. Mouse fibroblast NIH3T3 cells expressing v-Abl or Bcr-AblX were obtained form Dr. S. Shore (Temple University). v-Abl and Bcr-AblX are two different oncogenic forms of Abl . v- Abl-transformed NIH3T3 cells have been previously described (Shore and Reddy, Oncogene 1989; 4: 1411-1413). Cells were grown in DMEM medium supplemented with 10% bovine calf serum, L-glutamine, antibiotics and HEPES (Gibco/BRL, Grand Island, NY).

B. Plasmids and cDNAs.

The MSCVpac retroviral vector was provided by Dr. R. Hawley

(University of Toronto, Toronto, Canada) (Hawley et al. , Gene Therapy 1994;

1 : 136-138). Plasmids pHIT60 and pHIT456 were provided by Dr. A. Kingsman

(University of Oxford, Oxford, U.K.). Packaging plasmids pHIT60 and pHIT456 are expression vectors containing, respectively, the murine leukemia virus (MLV) gag-pol and env genes under control of the strong, human cytomegalovirus (CMV) immediate-early promoter.

The wild-type c-Cbl cDNA was obtained from Dr. W. Langdon (University of Western Australia, Nedlands, Australia). The 5Y->F c-Cbl mutant, a mutated form of c-Cbl in which tyrosines at positions 674, 700, 731, 735 and 774 are mutated to phenylalanines, was generated using oligonucleotide- directed mutagenesis as previously described (Feshchenko et al. , J. Biol. Chem. 1998; 273:8323-8331). The 5Y- > F c-Cbl mutant cannot be tyrosine phosphorylated. c-Cbl cDNAs were subcloned into pMSCVpac using standard ligation techniques. Briefly, pMSCVpac and the c-Cbl cDNA insert were linearized using restriction endonucleases, the linearized fragments were purified using electrophoresis in an agarose gel, and the obtained fragments were then ligated using T4 DNA ligase. Orientation of the inserts was determined by restriction endonuclease mapping.

C. Production of retroviral stocks and transduction of NIH3T3 cells.

To generate fibroblast cell lines stably expressing wild-type and mutant forms of c-Cbl, a retroviral vector system based on transient co- expression of packaging plasmids and a transfer vector was employed (Soneoka et al. , Nucleic Acids Research 1995; 23:628-633). Retroviral stocks were produced by the method of Merlo et al. , Anticancer Res. 1998: 18:2389-2396.

Accordingly, the expression plasmids pHIT60 and pHIT456, containing the gag-pol or env cDNA of MLV, respectively (Soneoka et al, supra), together with the MSCV transfer vector (Hawley et al , Gene Therapy 1994; 1 : 136-138) containing cDNA inserts under transcriptional control of the MLV LTR promoter, were transfected into 293T cells (Pear et al, Proc. Natl. Acad. Sci. USA 1993; 90:8392-8396). To produce retroviral stocks, 293T cells (Pear et al , Proc. Natl. Acad. Sci. USA 1993; 90:8392-8396) were transiently co-transfected with the MSCV-based plasmids along with pHIT60 and pHIT456 expression vectors encoding the MLV packaging components. Co-transfection was achieved by calcium phosphate precipitation, using The Calcium Phosphate Transfection Kit (Promega, Madison, WI) according to the manufacturer's recommendations. Expression of these plasmids resulted in a production of recombinant retroviral particles capable of integrating c-Cbl cDNA, as well as pac, the puromycin resistance gene, into genomic DNA of untransformed, v-Abl- and Bcr-AblX-transformed NIH3T3 fibroblasts. Supernatants of 293T cells were collected after transfection, filtered through 0.45 μm sterile filters and added to the target cells for 14-16 hours. The cells were incubated with retroviral supernatant overnight. They were then washed and kept in fresh medium for 24 h prior to selection. Positive transformants were selected using 3 μg/ml puromycin (Sigma, St. Louis, MO), because the MSCV vector used in this study carries the puromycin-resistance gene (pac). Cells were then cloned by limiting dilution. Both clonal and polyclonal lines were generated from each parental line using the empty MSCV vector and the MSCV vectors containing HA-tagged cDNA for either wild-type c-Cbl or 5Y- > F c-Cbl. All the obtained cell lines and clones were analyzed for the expression of c-Cbl.

D. Antibodies.

Antisera specific to hemagglutinin (HA) were raised in New Zealand white rabbits in accordance with institutional guidelines using the HA epitope fused to the C-terminus of glutathione S-transferase (GST) as the immunogen. Anti-HA reacts with the hemagglutinin (HA) epitope tagging the N-terminus of recombinant c-Cbl. GST-fusion proteins were produced in Escherichia coli and purified on glutathione-Sepharose according to the manufacturer's recommendations. Monoclonal antibody to Abl was purchased from Pharmingen. Polyclonal antibodies to c-Cbl was purchased from Santa Cruz Biotechnology. Monoclonal antibody to paxillin was from Transduction Laboratories.

E. Microscopic analysis.

For phase-contrast or laser-scanning (confocal) microscopy cells were grown in DMEM supplemented with calf serum as described above to 50- 90% confluence. For phase-contrast microscopy, cell cultures were analyzed without any additional treatment. For confocal microscopy, cells were fixed with 100% methanol, washed, blocked with blocking buffer containing 1% BSA and 0.05%) Tween-20 in PBS. Then cells were stained with phalloidin- Texas Red (Molecular Probes), anti-paxillin mAb followed by anti-mouse IgG-Texas Red, and anti-Cbl followed by anti-rabbit Ig-FITC. The laser-scanning microscope LSM 410 (Karl Zeiss) was used in these experiments.

F. Cell growth in soft agar. To assess colony-forming efficiency of cells in the absence of surface attachment (i.e. anchorage-independent growth), cells were seeded in a layer of soft agarose. This method has been described elsewhere (Shore and Reddy, Oncogene 1989; 4:1411-1413). Briefly, 3xl0³ cells were resuspended in warm 0.3 % agarose and then plated on the surface of a layer of more concentrated, 0.9% , agarose in a 60-mm Petri dish. Both solutions of agarose were prepared using DMEM supplemented with serum. L-glutamine and antibiotics as described above. Cells were allowed to grow for 3 weeks, and then macroscopic colonies (more than 20 cells) were counted.

EXAMPLE 2 Assessment of c-Cbl expression in v-Abl-transformed NIH3T3 cells

The clones generated by retroviral transfection were screened for the expression of c-Cbl by immunoblotting with anti-Cbl and anti-HA antibodies. The immunoblotting methods were described previously (Feshchenko et al. , J. Biol. Chem. 1998; 273:8323-8331). Briefly, whole cell lysates or immunoprecipitates were treated with SDS-PAGE sample buffer. Then proteins of these lysates or immunoprecipitates were separated by SDS-PAGE and transferred to nitrocellulose (Bio-Rad Laboratories, Richmond, CA). Nitrocellulose was blocked in Tris-buffered saline containing 1 % BSA and 0.1 % Tween-20 and incubated with an appropriate antibody diluted in blocking buffer. Protein bands were visualized by autoradiography.

Anti-HA reacts with the hemagglutinin (HA) epitope tagging the N- terminus of recombinant c-Cbl Neither endogenous c-Cbl, nor other cellular proteins, possesses this epitope. Therefore, anti-HA blotting allows the specific detection of MSCV-encoded recombinant c-Cbl. In turn, anti-Cbl immunoblotting can indicate a substantial increase in the total expression of c-Cbl.

The expression of retrovirally transduced c-Cbl appeared to be 20- 30-fold higher than that of endogenous c-Cbl, as determined by this immunoblotting procedure.

EXAMPLE 3

Evaluation of biological effects of wild-type and tyrosine phosphorylation- deficient c-Cbl in fibroblasts transformed by oncogenic forms of Abl

To evaluate the biological effects of overexpression of wild-type and tyrosine-phosphorylation-deficient c-Cbl on transformed and untransformed fibroblasts, the following parameters of these cells were determined: (a) proliferation rate in culture; (b) fraction of adherent cells in culture; (c) overall morphology based on phase-contrast microscopy; (d) colony-forming efficiency in soft agar and (e) formation of focal adhesion complexes and actin fibers based on confocal microscopy of cells stained for paxillin and F-actin. The studies were conducted on Bcr-AblX- or v-Abl-transformed fibroblasts transduced with (i) empty vector, (ii) vector expressing wild-type c-Cbl or (iii) vector expressing 5Y- > F c-Cbl mutant.

A. Proliferation rate in culture Untransformed NIH3T3 cell continued to proliferate at the same rate. Proliferation of Bcr-AblX-transformed cells in culture was unaffected by overexpression of either wild-type or 5Y->F c-Cbl. The results of one experiment are shown in Table I. The experiment was repeated three times with similar results.

B. Fraction of adherent cells in culture Cell adhesion was evaluated in standard 6-well tissue culture plates by washing each well three times with phosphate-buffered saline. Cells remaining attached after washing were then removed using a cell lifter. Both adherent and detached cells were counted. The percentage of adherent cells was determined. The results are shown in Table 1 (Bcr-AblX-transformed cells) and Table II (Abl- transformed cells). Unlike untransformed NIH3T3 cells, the v-Abl- and Bcr-AblX- transformed cells exhibited poor adherence. Overexpression of wild-type c-Cbl in the transformed cells however increased their adhesion, while overexpression of 5Y->F c-Cbl had no effect (Tables 1 and II). For Abl-transformed cells, adhesion was assessed with and without daily addition of 0.5 μM wortmannin, an inhibitor of phosphatidylinositol-3 ' kinase (PI-3 ')• Wortmannin reduced the effect of c-Cbl on adhesion, indicating that the biological effect of c-Cbl was dependent on the enzymatic activity of PI-3 ' (Table II). Without wishing to be bound by any theory, this result suggests a role for PI-3 ' in c-Cbl-mediated signaling.

C. Overall morphology based on phase-contrast microscopy Cells were plated in 60-mm Petri dishes at 10⁵ cells/dish. In 3 days, cell cultures reached 60-90% confluence and were photographed using phase contrast. The results are shown in Figs. IB (Bcr-AblX-transformed), 1C (v-Abl- transformed) and 1A (untransformed NIH3T3 cells). The untransformed NIH3T3 cells retained untransformed flat morphology. In contrast, v-Abl- and Bcr-AblX- transformed cells demonstrated spindle-like or rounded morphology. Overexpression of wild-type c-Cbl dramatically altered the transformed cell morphology, however. Wild-type c-Cbl transduced cells reverted in substantial portion to a flat morphology. Overexpression of 5Y->F c-Cbl in the transformed cells had no effect on morphology. These cells retained the transformed morphology.

D. Colony-forming efficiency in soft agar

Cells were plated in 60-mm Petri dishes at 1 x 10³ (v-Abl-transformed fibroblasts) or 3 x 10³ (Bcr-AblX-transformed fibroblasts) cells/dish with soft agarose. The number of colonies was scored visually after 3 weeks. The results are listed in Tables III and IV. Untransformed NIH3T3 cells did not form colonies in soft agar. Consistent with the effects of c-Cbl on fibroblast morphology, colony- forming efficiency of Bcr-AblX- and v-Abl-transformed fibroblasts was inhibited by overexpression of wild-type c-Cbl, but unchanged by expression of 5Y->F (Tables III, IV). E. Formation of focal adhesion complexes and actin fibers based on confocal microscopy of cells stained for paxillin and F-actin

In these experiments, actin fibers and focal adhesion complexes were stained with phalloidin-Texas Red and anti-paxillin mAb followed by anti- mouse IgG-Texas Red, respectively. Accordingly, v-Abl-transformed NIH3T3 cells transduced to express either 5 Y->F c-Cbl (Figs 2A and 2E) or wild-type c-Cbl (Figs 2B, 2C, 2D, 2F and 2G) were fixed, permeabilized and stained with anti-Cbl (Figs 2A-2G), phalloidin-Texas Red (Figs 2A-2D), and anti-paxillin (Figs 2E, 2F and 2G). Anti-Cbl and anti-paxillin were visualized using secondary antibodies labeled with FITC and Texas Red, respectively. No staining was observed when samples were stained with irrelevant antibodies (data not shown).

Untransformed NIH3T3 cells demonstrated unchanged paxillin and

F-actin staining. There was a dramatic increase in formation of focal adhesion complexes and actin fibers in v-Abl-transformed cells overexpressing wild-type c- Cbl relative to v-Abl-transformed cells overexpressing 5 Y->F c-Cbl, and relative to transformed cells transduced with vector alone.

Table I Effects of wild-type and tyrosine phosphorylation-defective (5Y->F) c- Cbl on proliferation rate and adhesion of Bcr-AblX-transformed fibroblasts in cell culture

Days Total cell number (x10⁵) Fraction of adherent c ells^* (%) in culture Cells transduced with Cells transduced with vector wild-type 5Y->F vector wild-type 5Y->F

0 0 10# 0 10 0 10 ND ND ND 3 0 16 0 11 0 18 ND ND ND 4 0 39 0 41 0 44 18 89 38 5 0 68 0 63 0 69 55 95 36

^•Defined as adherent after triple washing with PBS #Cell number per one well of a 6-well plate

ND=not determined Table II Effects of c-Cbl and wortmannin on adhesion of v Abl-transformed fibroblasts*

Clone Cell transduced with Adherent cells (%) wortmannin (-) wortmannin (+)

C6v vector 0 3 0 0

D1v vector 1 8 0 0

A4wt wild-type c-Cbl 82 18

A5wt wild-type c-Cbl 85 13

C6wt wild-type c-Cbl 50 15

A5mut 5Y->F c-Cbl 13 0 0

B5mut 5Y->F c-Cbl 17 0.3

'Cells were grown in liquid culture to 60-70% confluence, then washed three times with PBS Cells remaining attached to the surface were scored as adherent Wortmannin was added daily at a concentration of 0 5 μM The results of one representative experiment of three are shown

Table III Effects of wild-type and 5Y->F c-Cbl on colony-forming efficiency of v-Abl-transformed fibroblasts*

CClloonnee CCeellll ttrraannssdduucceedd wwiitthh Colony-forming efficiency (%)

C6v vector 13 5

D1 v vector 11 2

^•Cells of each clone were seeded on two 60-mm Petri dishes with soft agarose at 1x10 cells/dish Number of colonies was scored visually after 3 weeks of incubation Mean values are shown The experiment was performed three times with similar results

Table IV Effects of wild-type and tyrosine phosphorylation-defective (5Y->F) c- Cbl on colony-forming efficiency of Bcr-AblX-transformed fibroblasts in soft agarose* Cell transduced with Colony-forming efficiency (%) Large (>1 mm) colonies* vector 2 2

^*3x10 cells were plated per one 60-mm Petri dish Number of colonies was scored visually after 3 weeks of incubation Results of one experiment are shown The experiment was performed twice and yielded similar results "Fewer than 10% of colonies are large (-), 10-25% are large (+), 25-50% are large (++), more than 50% (+++)

The foregoing studies demonstrate that overexpression of wild-type c-Cbl enhances adhesion and spreading of Abl-transformed fibroblasts. Overexpression of either wild-type or 5 Y->F c-Cbl exceeding its endogenous level by 20-30-fold failed to induce phenotypic changes in untransformed NIH3T3 cells, which remained flat and attached to the substratum, and retained anchorage dependence and sensitivity to contact inhibition (data not shown). In contrast, overexpression of c-Cbl to a similar extent significantly affected v-Abl- and Bcr- AblX-transformed cells. Although the clones analyzed demonstrated similar growth rates in culture, a substantial portion of v-Abl-transformed cells overexpressing wild-type c-Cbl regained ability to attach to the substratum, whereas overexpression of 5Y->F c-Cbl induced only a minor increase in attachment compared to the level corresponding the empty vector controls (Table II).

The effect of c-Cbl on adhesion of Abl-transformed NIH3T3 cells was consistent with its effect on their moφhology. Whereas vector controls were indistinguishable from parental Bcr-AblX-transformed and v-Abl-transformed cells (data not shown), overexpression of wild-type c-Cbl reversed morphology of a large fraction of Bcr-AblX-transformed and v-Abl-transformed NIH3T3 fibroblasts from round to the flat morphology characteristic of untransformed cells (Figure

1 ).

The effect of c-Cbl on both adhesion and moφhology of v-Abl- transformed NIH3T3 cells correlates well with its effect on formation of focal adhesion complexes and actin fibers, as determined by confocal microscopy for F- actin and paxillin, a prominent component of focal adhesion complexes. Cells overexpressing wild-type c-Cbl demonstrated a dramatic increase in formation of focal adhesion complexes and actin fibers typical of untransformed NIH3T3 cells, whereas neither focal adhesions nor actin fibers were detectable in v-Abl- transformed NIH3T3 cells overexpressing 5Y->F c-Cbl, or cells transduced with a vector alone (Figure 2). Consistent with the results of phase-contrast microscopy, confocal images indicated that adherent v-Abl-transformed NIH3T3 cells overexpressing wild-type c-Cbl were mostly flat, whereas those overexpressing 5 Y- >F c-Cbl were round or spindle-like and displayed long processes (Figure 1) . In conclusion, the overexpression of c-Cbl in transformed cells, particularly transformation caused by oncogenic forms of Abl, significantly suppresses the transformed phenotype.

Without wishing to be bound by any theory, it is believed that both PI-3 ' kinase and one or members of the Crk-family of adaptor proteins are involved in mediating the observed effects of c-Cbl. Disruption of c-Cbl binding to either Crk or PI-3 ' kinase using site-directed mutagenesis completely abrogated the ability of c-Cbl to facilitate adhesion and spreading of v-Abl-transformed fibroblasts and to reduce colony-forming efficiency of theses cells in soft agar (data not shown). Likewise, inhibition of enzymatic activity of PI-3 ' kinase using several unrelated inhibitors blocked the observed effects of c-Cbl on adhesion, spreading and colony-forming efficiency of v-Abl-transformed fibroblasts (data not shown).

All references discussed herein are incoφorated by reference. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof.

Claims

1. A method for suppressing neoplastic transformation of a cell comprising administering to the cell a vector expressing c-Cbl so that neoplastic transformation of the cell is suppressed.

2. The method of claim 2 wherein the vector is a viral vector.

3. The method of claim 3 wherein the viral vector is a retroviral vector.

4. The method of claim 3 wherein the viral vector is an adenoviral vector.

5. The method of claim 2 wherein the neoplastic transformation which is suppressed is transformation by an oncogenic protein tyrosine kinase.

6. The method of claim 5 wherein the neoplastic transformation which is suppressed is transformation by an oncogenic Abl protein tyrosine kinase.

7. A method for suppressing neoplastic transformation of cells in an animal comprising administering to said animal a vector expressing c-Cbl so that neoplastic transformation of said cells is suppressed.

8. The method of claim 7 wherein the vector is a viral vector.

9. The method of claim 8 wherein the viral vector is a retroviral vector.

10. The method of claim 8 wherein the viral vector is an adenoviral vector.

11. The method of claim 8 wherein the vector is administered to cells of the patient ex vivo to obtain over-expression of the c-Cbl gene product by such cells, which cells are then returned to the body of the patient.

12. The method of claim 8 wherein the vector is administered systemically.

13. The method according of claim 8 wherein the vector is administered locally to a body site containing the cells for which suppression of neoplastic transformation is desired.

14. The method of claim 8 wherein the neoplastic transformation which is suppressed is transformation induced by an oncogenic protein tyrosine kinase.

15. The method of claim 14 wherein the neoplastic transformation which is suppressed is transformation by an oncogenic Abl protein tyrosine kinase.