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Title:  Lymphoma-susceptible transgenic mice and methods for studying drug sensitivity of lymphomas

United States Patent:  6,583,333

Issued:  June 24, 2003

Inventors:  Lowe; Scott W. (Cold Spring Harbor, NY); Wallace-Brodeur; Rachel R. (Huntington Station, NY)

Assignee:  Cold Spring Harbor Laboratory (Cold Spring Harbor, NY)

Appl. No.:  076776

Filed:  May 12, 1998

Abstract

A mouse expressing myc in B cells, because of defective function of one or more tumor suppressor genes, is useful for the testing of anti-lymphoma agents and for the testing of genes which may have an effect on the apoptotic pathway. Preferred embodiments include mice of genotypes E.mu.-myc/p53+/-, E.mu.-myc/Rb+/- and E.mu.-myc/p16+/-, and cells derived from lymphomas arising in these mice, wherein the cells may have undergone further genetic alteration. Mouse strains, lymphoma cells and cell lines of the invention can be used in methods to discover new anti-lymphoma agents, methods to characterize tumors, and to characterize genes which may affect the development of resistance to anti-tumor agents. Such methods are also part of the invention.

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the invention to provide a mouse which expresses myc in B cells, wherein myc expression is manifested by lymphoma. Such mice are characterized by having B cells that harbor mutations affecting the function of one or more so-called tumor suppressor genes, and that express myc, with the result that the mice develop lymphomas. Such mice can be of genotype E.mu.-myc/mutant tumor suppressor gene, for example E.mu.-myc/p53+/-, E.mu.-myc/Rb+/- or E.mu.-myc/p16+/-. Such mice can also be of a genotype which produces a phenotype mimicking that resulting from an E.mu.-myc/mutant tumor suppressor gene genotype.

The invention comprises strains of mice that may be syngeneic, harboring a combination of genes: (1) a myc gene and (2) a mutant tumor suppressor gene (one or more mutant alleles) which results in lack of a function which in the wild type causes suppression of myc-induced tumor formation. The myc gene can be under the control of a promoter/enhancer region specific to B cells, such that the myc gene is specifically expressed in B cells, for example E.mu.-myc (Adams, J. M. et al., Nature 318:533-538, 1985).

E.mu.-myc/tumor suppressor gene mutation mice are mice having the genotype of the myc oncogene, under the control of the EA IgH enhancer, in combination with a tumor suppressor gene mutation whose presence results in an increase in the probability of the development of tumors in an animal or human (relative to the probability of tumor development in animals in which wild type alleles of the suppressor gene are present). The myc gene can be one as described in Harris, A. W., J. Exp. Med. 167:353-371 (1988) or the allelle described by Langdon, W. Y. et al., Cell 47:11-18 (1986), for example. The myc gene can also be a naturally-occurring gene, either cellular or viral, a natural variant or an artificially altered variant of myc.

The p53 gene can be a mutant allele described by Donehower, L. A. et al., 356:215-221 (1992) or as described by Jacks, T. et al., Curr. Biol. 4:1-7 (1994) or Purdie, C. A. et al. Oncogene 9:603-609 (1994). The p53 allele can also be one made by various methods of mutagenesis. Loss of p53 (or of another tumor suppressor gene) function can occur, for instance, through point mutation, allelic loss, rearrangements, and intragenic deletions.

The Rb gene can be one described in Jacks, T. et al., Nature 359:295-300 (1992), in Clarke, A. R. et al., Nature 359:328-330, 1992, or in Lee, E. Y. et al, Nature 359:288-294, 1992. The Rb gene can also be one that is isolated from a source in nature or produced in vitro by methods of genetic manipulation.

The p16 gene can be the allele described by Serrano, M. et al., Cell 85:27-37, 1996, or can be other alleles found in nature or altered by in vitro or in vivo manipulations.

Other mutated tumor suppressor genes can be used in combination with E.mu.-myc in genetically engineered mice. For example, mutated caspase genes or mutated genes of the bcl-2 family (e.g., bax, bak, bik, bad) can be used.

Preferred embodiments of mice which express myc and are defective in tumor suppressor function are those mice having the genotype E.mu.-myc/p53+/-, E.mu.-myc/Rb+/- or E.mu.-myc/p16+/-.

E.mu.-myc/tumor suppressor gene mutation mice can be made by matings to take advantage of existing mutations, as illustrated in the Examples, or they can be made in a number of other ways. For example, it is possible to start with a mouse strain that harbors a mutation in a tumor suppressor gene, and by known methods of producing transgenic animals, introduce the E.mu.-myc transgene into the genome, either by introducing the transgene into a fertilized ovum, using a c-myc vector construct such as the one described by Adams (Adams, J. M. et al., Nature 318:533-538, 1985) by the method of Wagner et al., U.S. Pat. No. 4,873,191 (1989), or by introducing the transgene into embryonic stem (ES) cells (see, for example, Capecchi, M. R., Science 244:1288-1292, 1989).

Where embryonic stem cells are used, the tumor suppressor gene can be specifically inactivated in cultured embryonic stem cells. For instance, a normal gene can be replaced with a copy of itself that contains a bacterial antibiotic resistance gene, such as neo. The neo gene not only inactivates the target gene, but also allows identification of cells that have taken up the engineered gene. The embryonic stem cells are then injected into mouse embryos, where they have the potential to develop into all the different mouse cell types. The resulting animals are then bred, and those whose germ cells are derived from the ES cells pass the inactivated gene to their progeny.

Further, alternative methods are available to produce conditional knockouts of the tumor suppressor gene of interest, or tissue specific knockouts.

The bacteriophage P1 Cre-loxP recombination system is capable of mediating loxP site-specific recombination in both ES cells and transgenic mice. The site-specific recombinase Cre can also be used in a predefined cell lineage or at a certain stage of development. See, for example, Gu, H. et al., Science 265:103-106, 1994, in which a DNA polymerase .beta. gene segment was deleted from T cells; see also Tsien, J. Z. et al., Cell 87:1317-1326, 1996, in which Cre/loxP recombination was restricted to cells in the mouse forebrain.) The impact of the mutation on these cells can then be analyzed.

The Cre recombinase catalyzes recombination between 34 bp loxP recognition sequences (Sauer, B. and Henderson, N., Proc. Natl. Acad. Sci. USA 85:5166-5170, 1988). The loxP sequences can be inserted into the genome of embryonic stem cells by homologous recombination such that they flank one or more exons of a gene of interest (making a "floxed" gene). It is crucial that the insertions do not interfere with normal expression of the gene. Mice homozygous for the floxed gene are generated from these embryonic stem cells by conventional techniques and are crossed to a second mouse that harbors a Cre transgene under the control of a tissue type- or cell type-specific transcriptional promoter. In progeny that are homozygous for the floxed gene and that carry the Cre transgene, the floxed gene will be deleted by Cre/loxP recombination, but only in those cell types in which the Cre gene-associated promoter is active.

A myc transgene can be delivered selectively into B cells in culture by methods for transfection, or by infection with a retrovirus which expresses myc under the control of a retroviral promoter. See, Pear, W. S. et al., J. Exp. Med. 183:2283-2291, 1996, for an example of retroviral transduction used to induce clonal leukemias.

Proteins which act to have the effect of mimicking the phenotype caused by tumor suppressor mutations can also be used to achieve the same effect as knockouts in tumor suppressor genes.

A dominant negative mutant can be used to achieve loss of function of a tumor suppressor gene, instead of using a tumor suppressor knockout. See, for example the description of the mutant Val-135 p53 allele in Harvey, M. et al., Nat. Genet. 9:305-311, 1995. For example, a transgenic E.mu.-myc mouse which expresses a dominant negative p53 allele specifically in B cells can be made by a cross of an E.mu.-myc mouse to a mouse having a dominant negative p53 gene under the control of a B-cell specific promoter, such as the IgH enhancer E.mu.. A cell line expressing E.mu.-myc and a dominant negative p53 can be made by introducing a dominant negative p53 gene into an E.mu.-myc/p53+ lymphoma cell line (e.g., by a retroviral construct, transformation, etc.).

The R24C mutation in the gene encoding cyclin-dependent kinase 4 (CDK4) has been found to prevent binding of the CDK4 inhibitor (and tumor suppressor) p16 (Wolfel, T. et al., Science 269:1281-1284 (1995)). As an alternative to an E.mu.-myc/p16 (knockout) mouse, a transgenic mouse can be made in which CDK4-R24C is expressed, thereby producing the phenotype of an E.mu.-myc/p16-/- mouse.

Expression in E.mu.-myc mice of viral proteins that bind the Rb gene product and inactivate its function (e.g., human papilloma virus-16 E7 and adenovirus E1A) can be used to produce an E.mu.-myc/Rb-/- phenotype. See, regarding adenovirus E1A protein, Whyte, P. et al., Nature 334:124-129, 1988. For the human papilloma virus-16 E7 oncoprotein, see Dyson, N. et al., Science 243:934-937, 1989.

Testing to identify the p53, Rb and p16 mutant or wild type alleles, or for the identification of other alleles, can be done by PCR on isolated genomic DNA, using appropriate primers, or by Southern blots using appropriate hybridization probes, by a combination of these procedures, or by other methods. For instance, hybridization probes to distinguish the p16 alleles (by detection of the insertion of the NEO cassette) have been published in Serrano, M. et al., Cell 85:27-37 (1996).

It is also an object of the invention to provide cell lines that are derived from lymphomas arising in E.mu.-myc/mutant suppressor gene mice or in mice which by a different genotype mimic the phenotype of E.mu.-myc/mutant suppressor gene mice. For example, cell lines of the invention can be stably dividing cells that grow in cell culture conditions, arising from mouse strains of the invention. Preferred genotypes of cell lines are E.mu.-myc/p53-/- (e.g., cell line L1624), E.mu.-myc/Rb+/- (e.g. cell line L1790) and E.mu.-myc/p16-/- (e.g., cell line L1213). Cell lines can be made by using feeder layers of cells, as in the method described in Example 7, or by culturing the cells in a variety of media, including chemically defined media, without feeder layers.

An E.mu.-myc/tumor suppressor gene mutation transgenic mouse strain provides a useful model for studying apoptosis and cancer therapy, since: (i) tumor burden can be monitored externally by lymph node palpation and often by blood smears; (ii) lymphomas are detectable long before the animals die, so animals can be treated while otherwise healthy, (iii) large numbers of tumor cells can be isolated from mice undergoing therapy or treatment, (iv) therapy is performed in immunocompetent mice, and (v) lymphoma cells readily adapt to culture and/or can be transplanted into syngeneic mice.

A lymphoma arising in a mouse of a specific strain described herein can be tested for sensitivity to a treatment, by administering the treatment to the mouse (designated a "test" mouse), and monitoring the mouse for a decrease in signs of the lymphoma (remission). Full remission is a state of normalcy of the mouse in which no lymphoma can be detected by palpation, and white blood cell counts are indistinguishable (not statistically significantly different) from those of healthy mice. Sensitivity of a lymphoma to a treatment can be proportional to the length of time until relapse. An appropriate control mouse is one which is genetically identical or very similar, and which has been maintained under the same conditions as the test mouse, except that no treatment has been given.

The treatment to be tested can be one or more substances, for example, a known anti-cancer agent, such as adriamycin, cylophosphamide, prednisone, vincristine or a radioactive source. The substance can be administered preferably by intraperitoneal injection in a pharmaceutically acceptable vehicle, but also by other vehicles and routes, for example, orally, intranasally, by inhalation, intramuscular injection, hypodermic injection, intravenous injection or by surgical implantation, in topical creams, transdermal patches and the like, all optionally with pharmaceutically compatible carriers and solvents. The treatment can also be exposure to various kinds of energy or particles, such as gamma-irradiation, or can be a combination of approaches. In some cases, the treatment can also be administration of one or more substances or exposure to conditions, or a combination of both, wherein the effects of the treatment as anti-cancer therapy are unknown.

Another embodiment of the invention is a further method for testing a lymphoma arising from an E.mu.-myc/tumor suppressor gene mutation lymphoma for sensitivity to a treatment. Lymphoma cells are cultured in vitro, a treatment is administered to the cells (e.g., a drug is contacted with the cells), and the cells can be monitored for growth (e.g., by observing cell number, confluence in flasks, staining to distinguish viable from nonviable cells). A failure to increase in viable cell number, a slower rate of increase in cell number, or a decline in viable cell number, compared to cells which have been left untreated, or which have been mock-treated, is an indication of sensitivity to the treatment.

A further embodiment of the invention is a method employing secondary tumors arising from transplanted primary tumor cells. In this way, a statistically significant number of mice with the same lymphoma can be studied for their response to a regimen of therapy. One or more tumors arising in a E.mu.-myc/tumor suppressor gene mutation mouse can be harvested from an animal and transplanted into recipient mice by a suitable method, such as by tail vein injection. After a period of time, lymphomas arise in the recipient mice. A treatment is then administered to one population of recipient mice which have developed tumors (making a "treated" population). A second population of recipient mice can serve as controls and remain untreated or be sham-treated. Thereafter, the treated population of mice is monitored for remission (ordinarily, by palpation for tumors). Remissions among the treated population indicates sensitivity of the lymphomas of the E.mu.-myc/tumor suppressor gene mutation mice to the administered treatment.

A further embodiment of the invention is a test for the effect of a gene on sensitivity of a lymphoma to a treatment (e.g., anti-cancer drug). In one embodiment, a cell line can be produced from a lymphoma described herein. A gene to be tested for its effect on the sensitivity to a treatment of the lymphoma is introduced into cells of the cell line, such that the cells produce the gene product.

Introduction of the gene can be, for instance, by transformation, such as by electroporation, by calcium phosphate, DEAE-dextran, or by liposomes, using a vector which has been constructed to have an insertion of the gene. See Ausubel, F. M. et al, Current Protocols in Molecular Biology, chapter 9, containing supplements through Supplement 40, Fall, 1997, John Wiley & Sons, New York.

The introduction of a gene of interest can also be accomplished by viral infection, for example, by a retrovirus. Retroviral gene transfer has been used successfully to introduce genes into whole cell populations, thereby eliminating problems associated with clonal variation (McCurrach, M. E. et al., Proc. Natl. Acad. Sci. USA 94:2345-2349, 1997; Samuelson, A. V. and Lowe, S. W., Proc. Natl. Acad. Sci. USA:12094-12099, 1997; Serrano, M. et al., Cell 85:27037, 1997).

Cells so altered or transformed by the introduced gene, or unaltered cells, ["unaltered" including cells which have been transformed with a control vector, transfected with control DNA, or infected with a control virus (control constructs not carrying the gene of interest)], can be introduced into immunocompetent recipient mice ("test" mice receiving the gene and "control" mice not receiving the gene). Cells can be introduced by injection, for example, by injection into the tail vein of the mice. Lymphomas are allowed to form in both the test and control mice, and both test and control mice are monitored for the development of lymphomas. Both groups of mice are administered a treatment, preferably a drug given at a

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

CCAGCCGGCC ACAGTCG 17 dose with a known anti-tumor effect, and monitored for remission. A difference in the frequency of remissions in the test mice compared to that of the control mice (remissions are not anticipated in control mice) indicates an effect of the gene on the response of the lymphoma to the treatment.

This method can determine what the response of an animal would be to the same therapy, with and without the gene. Thus, genes that are important to drug sensitivity of a lymphoma can be identified.

Animal models provide a useful alternative to studies in humans and to human tumor cell lines grown as xenographs, since large numbers of genetically-identical individuals can be treated with identical regimens, and experimental strategies are not limited by the same ethical considerations applied to humans. Moreover, the ability to introduce oncogenic mutations into the mouse germline increases the power of mouse models.

The invention described herein exploits mouse models to generate tumors with specific genetic alterations, allowing the production of useful tools for future drug discovery programs applicable to human lymphomas. By understanding the regulation and execution of apoptosis in tumor cells it may be possible to selectively increase the chemosensitivity of tumor cells, or to develop novel therapies to activate apoptosis more directly. Cell lines that recapitulate the behavior of spontaneous E.mu.-myc lymphomas allow gene transfer studies in vitro and an evaluation of the impact of the gene on lymphomas produced in syngeneic recipients.

Claim 1 of 10 Claims

What is claimed is:

1. A transgenic mouse having a genome comprising a myc gene operably linked to an E.mu. IgH enhancer and further comprising one inactivated p16 allele, wherein said mouse exhibits accelerated development of lymphomas compared to E.mu.-myc mice.



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