WO2000010602A9 - Modeles animaux avec elimination de genes lats et leurs utilisations - Google Patents

Modeles animaux avec elimination de genes lats et leurs utilisations

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Publication number
WO2000010602A9
WO2000010602A9 PCT/US1999/019068 US9919068W WO0010602A9 WO 2000010602 A9 WO2000010602 A9 WO 2000010602A9 US 9919068 W US9919068 W US 9919068W WO 0010602 A9 WO0010602 A9 WO 0010602A9
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WO
WIPO (PCT)
Prior art keywords
lats
protein
cdc2
compound
complex
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PCT/US1999/019068
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English (en)
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WO2000010602A1 (fr
Inventor
Tian Xu
Wufan Tao
John Maie A R St
Xiaolan Fei
Royd K Fukumoto
Sheng Zhang
Gregory S Turenchalk
Rodney A Stewart
Original Assignee
Univ Yale
Tian Xu
Wufan Tao
John Maie A R St
Xiaolan Fei
Royd K Fukumoto
Sheng Zhang
Gregory S Turenchalk
Rodney A Stewart
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Publication date
Application filed by Univ Yale, Tian Xu, Wufan Tao, John Maie A R St, Xiaolan Fei, Royd K Fukumoto, Sheng Zhang, Gregory S Turenchalk, Rodney A Stewart filed Critical Univ Yale
Priority to AU57810/99A priority Critical patent/AU5781099A/en
Priority to CA002340456A priority patent/CA2340456A1/fr
Priority to EP99945126A priority patent/EP1105160A4/fr
Publication of WO2000010602A1 publication Critical patent/WO2000010602A1/fr
Publication of WO2000010602A9 publication Critical patent/WO2000010602A9/fr

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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the use of lats proteins, derivatives and fragments for the treatment of cancer, particularly for the treatment of cancer that is refractory to treatment by standard chemotherapy and radiation therapy protocols.
  • the present invention also relates to the use of lats proteins, derivatives and fragments for the treatment of diseases and disorders associated with an aberrantly high or aberrantly low level of cdc2 activity.
  • the present invention further provides complexes of lats and cdc2, and their production and uses.
  • the present invention also provides an animal model for cancer, particularly for skin cancer, soft tissue sarcomas, and ovarian tumors, and for pituitary disorders.
  • the animal model is preferably a mouse, in which a lats gene has been disrupted by homologous recombination, e.g., a lats knock-out mouse.
  • the present invention also provides methods of screening potential therapeutics for efficacy in the treatment and prevention of cancer and pituitary disorders using lats knock-out animals.
  • a neoplasm, or tumor is a neoplastic mass resulting from abnormal uncontrolled cell growth, which may cause swelling on the body surface, and which can be benign or malignant. Benign tumors generally remain localized. Malignant tumors are collectively termed cancers.
  • malignant generally means that the tumor can invade and destroy neighboring body structures and spread to distant sites to cause death (for review, see Robbins and Angell, 1976, Basic Pathology, 2d Ed.. W.B. Saunders Co., Philadelphia, pp. 68-122).
  • Treatment options, such as surgery, chemotherapy and radiation treatment, are either ineffective or present serious side effects.
  • the Cell Cycle and Tumor Suppressors Many cancers have been linked to perturbations in the regulation of the cell cycle, resulting in deregulation of cell growth. Briefly, the cell cycle occurs in four stages: Gl (for Gapl), the resting stage prior to DNA synthesis; S (for synthesis) phase, in which DNA synthesis occurs; G2 (for Gap2), the resting stage after DNA synthesis and prior to mitosis; and M phase, mitosis, in which cell division occurs. Progression of the cell cycle is driven by a group of cyclin-dependent kinases (CDKs) (Elledge, 1996, Science 274:1664-1672; Nasmyth, 1996, Science 274:1643-1645).
  • CDKs cyclin-dependent kinases
  • CDKs phosphorylation events and CDK inhibitors
  • CKIs CDK inhibitors
  • CDK2, CDK4 and CDK6 along with Cyclins D and E, regulate the progression from Gl into S phase
  • Cyclins A and B regulate the cell cycle progression from Gl into mitosis
  • Human tumor suppressors often act as negative regulators of the cell cycle, and several tumor suppressors are known to affect the activities of the CDK/cyclin complexes.
  • p53 activates the transcription of the p21 (p21 WAF1 c,pl ) CDK inhibitor in response to DNA damage signals, and p21 in turn binds and inactivates the CDK4 and CDK6 cyclin D complexes (Gartel et al., 1996, Proc. Soc. Exp. Biol. Med. 213:138-149).
  • Another CDK inhibitor, pi 6, is itself a potent tumor suppressor (Biggs and Kraft, 1995, J. Mol. Med. 73:509-514).
  • multiple members of the pl6 and p21 inhibitor families have been identified for other major CDKs, corresponding inhibitors that regulate the mitotic CDK, cdc2, have not previously been identified (Morgan, 1995, Nature 374:131- 134).
  • cancer therapy may involve surgery, chemotherapy and/or radiation treatment to eradicate neoplastic cells in a patient (see, for example, Stockdale, 1998, "Principles of Cancer Patient Management", in Scientific American: Medicine, vol. 3, Rubenstein and Federman, eds., Chapter 12, Section IV). All of these approaches pose significant drawbacks for the patient.
  • Surgery for example, may be contraindicated due to the health of the patient or may be unacceptable to the patient. Additionally, surgery may not completely remove the neoplastic tissue.
  • Radiation therapy is only effective when the neoplastic tissue exhibits a higher sensitivity to radiation than normal tissue, and radiation therapy can also often elicit serious side effects.
  • chemotherapeutic agents available for treatment of neoplastic disease.
  • a significant majority of cancer chemotherapeutics act by inhibiting DNA synthesis, either directly, or indirectly by inhibiting the biosynthesis of the deoxyribonucleotide triphosphate precursors, to prevent DNA replication and concomitant cell division (see, for example, Gilman et al., Goodman and Gilman's: The Pharmacological Basis of Therapeutics, Eighth Ed. (Pergamom Press, New York, 1990)).
  • agents which include alkylating agents, such as nitrosourea, anti- metabolites, such as methotrexate and hydroxyurea, and other agents, such as etoposides, campathecins, bleomycin, doxorubicin, daunorubicin, etc., although not necessarily cell cycle specific, kill cells during S phase because of their effect on DNA replication.
  • agents specifically colchicine and the vinca alkaloids, such as vinblastine and vincristine, interfere with microtubule assembly resulting in mitotic arrest.
  • Chemotherapy protocols generally involve administration of a combination of chemotherapeutic agents to increase the efficacy of treatment.
  • chemotherapeutic agents Despite the availability of a variety of chemotherapeutic agents, chemotherapy has many drawbacks (see, for example, Stockdale, 1998, "Principles Of Cancer Patient Management” in Scientific American Medicine, vol. 3, Rubenstein and Federman, eds., ch. 12, sect. 10). Almost all chemotherapeutic agents are toxic, and chemotherapy causes significant, and often dangerous, side effects, including severe nausea, bone marrow depression, immunosuppression, etc. Additionally, even with administration of combinations of chemotherapeutic agents, many tumor cells are resistant or develop resistance to the chemotherapeutic agents.
  • those cells resistant to the particular chemotherapeutic agents used in the treatment protocol often prove to be resistant to other drugs, even those agents that act by mechanisms different from the mechanisms of action of the drugs used in the specific treatment; this phenomenon is termed pleiotropic drug or multidrug resistance.
  • drug resistance many cancers prove refractory to standard chemotherapeutic treatment protocols.
  • alternative cancer treatments particularly for treatment of cancer that has proved refractory to standard cancer treatments, such as surgery, radiation therapy, and chemotherapy.
  • the pituitary regulates numerous biological functions through its secretion of different hormones.
  • glycoprotein hormones include luteinizing hormone (LH) and follicle stimulating hormone (FSH); LH and FSH regulate ovarian and testicular development as well as reproductive functions such as ovulation and spermatogenesis.
  • LH or FSH secretion has dramatic consequences for reproductive function, particularly for ovulation in the female.
  • the pituitary also releases somatomammotropic hormones, including growth hormone and prolactin.
  • Growth hormone promotes linear growth and is involved in the regulation of certain metabolic functions such as sugar and amino acid uptake and use of fat stores.
  • Prolactin stimulates and maintains lactation in post-parturition females. Although an increase or decrease in prolactin levels does not appear to have significant biological consequences beyond an effect on lactation, disruption of growth hormone secretion stunts growth and has other metabolic effects.
  • Other pituitary hormones include corticotropin (ACTH), thyroid stimulating hormone (TSH), and endorphins and related peptides. Although in some situations, hormone replacement therapy is available, there is a need for additional therapeutics to treat or prevent pituitary dysfunctions.
  • the large tumor suppressor or lats gene (also known as warts), a tumor suppressor gene, was previously isolated from Drosophila using a mosaic screen. Inactivation of lats in somatic cells causes dramatic overproliferation phenotypes (Xu et al., 1995, Development 121 :1053-1063; Justice et al., 1995, Genes & Devel. 9:534-546). Somatic cells that are mutant for lats undergo extensive proliferation and form large tumors in many tissues of mosaic flies (Xu et al., 1995, Development 121 :1053-1063). Tumors that result from inactivation of lats display many features of human neoplasms.
  • Lats mutant cells grow aggressively, and a single mutant cell can develop into a tumor that is 1/5 the size of the animal, and these fly tumors are highly irregular in shape and size and are often poorly differentiated (St. John and Xu, 1997, Am. J. Hum. Genet 61 :1006-1010).
  • Drosophila that are homozygous for the various lats alleles display a wide range of developmental defects including embryonic lethality, overproliferation of both neural and epidermal tissues, rough eyes, and sterility.
  • Molecular characterization of lats indicates that it contains a putative kinase domain (Xu et al., 1995, Development 121 : 1053-1063; Justice et al., 1995, Genes & Devel. 9:534-546).
  • the present invention relates to therapeutic and prophylactic methods and compositions for the treatment and prevention of cancers based on lats proteins, and therapeutically and or prophylactically effective analogs and fragments of lats protein.
  • the lats protein interacts with the cell cycle-dependent kinase cdc2, which is involved in the regulation of the G2 to M transition of the cell cycle, and thereby provides a means to regulate the G2 to M transition of the cell cycle and to treat cancers that have proven refractory to other cancer treatments, including chemotherapy and radiation therapy treatments.
  • the invention provides for treatment and prevention of cancer by administration of a therapeutic compound of the invention.
  • the therapeutic compounds of the invention useful for treatment of cancer refractory to a chemotherapy and/or radiation therapy protocol include: lats proteins, and therapeutically effective analogs and derivatives (including fragments) of lats, nucleic acids encoding lats proteins and therapeutically effective analogs and derivatives of lats, and lats agonists.
  • the invention further provides assays, both in vivo and in vitro, for testing the efficacy of the therapeutics of the invention for treatment of cancer, particularly cancer that has been shown to be refractory to chemotherapy and radiation therapy treatments.
  • the invention provides compositions and methods of production of complexes of lats and cdc2 proteins ("lats-cdc2 complexes"), including complexes of lats analogs or derivatives and cdc2 analogs and derivatives (including complexes of lats proteins with cdc2 analogs and derivatives and vice versa), where the analogs and derivatives have the ability to interact with the other member of the complex.
  • the lats-cdc2 complexes contain phosphorylated lats protein, specifically lats protein phosphorylated on a serine or threonine residue within 20 residues upstream of an Ala-Pro-Glu consensus in subdomain eight of a lats kinase domain, e.g., corresponding to serine 909 of human lats, as depicted in Figure 12 (SEQ ID NO:2).
  • the lats protein in the lats-cdc2 complex has a glutamate or aspartate residue substituted for a serine or threonine residue within 20 residues upstream of an Ala-Pro-Glu consensus in subdomain eight of a lats kinase domain, e.g., corresponding to serine 909 of human lats, as depicted in Figure 12 (SEQ ID NO:2).
  • the lats-cdc2 complex contains a portion of lats protein corresponding to amino acids 15-585 of human lats, as depicted in Figure 12 (SEQ ID NO:2).
  • the invention further provides methods of modulating the activity of cdc2 using lats proteins, as well as lats derivatives and fragments able to interact with cdc2 protein, lats- cdc2 complexes, and antibodies against lats-cdc2 complexes.
  • the invention provides methods for treating or preventing disorders involving an aberrant level of cdc2 in a subject.
  • Therapeutically effective amounts of compounds are administered to promote or inhibit LATS function, as required.
  • the invention provides recombinant non-human animals in which a lats gene has been inactivated, preferably recombinant mice in which a lats gene (preferably a gene having lats coding sequence of SEQ ID NO:3) has been inactivated, i.e., a lats knock-out mouse.
  • a lats gene preferably a gene having lats coding sequence of SEQ ID NO:3
  • the invention provides a lats knock-out mouse in which the inactivated lats gene had the coding sequence of SEQ ID NO:3, prior to disruption, and in a more preferred embodiment, the inactivated lats gene is deleted for the Lats C-terminal domain 1 (LCD1), the Lats C-terminal domain 2 (LCD2), the Lats C-terminal domain 3 (LCD3), and all or a portion of the kinase domain, and retains the Lats flanking domain (LFD), the Lats split domain 1 (LSD1), the Lats split domain 2 (LSD2), and the putative SH3-binding domain, in a most preferred embodiment the lats gene is disrupted by replacement of a non-lats sequence for the sequence encoding the amino acids corresponding to amino acids 756 to 1130 of human lats, as depicted in Figure 12 (SEQ ID NO:2).
  • the inactivated lats gene is deleted for all or a portion of the kinase domain (e.g., so as to inactivate kinase activity).
  • a lats "knock-out" animal is an animal in which at least one genomic copy of a lats gene has been inactivated by insertional mutagenesis, e.g., by homologous recombination, for example, as described and exemplified herein.
  • the invention further provides methods for screening potential therapeutics for activity in the treatment or prevention of cancer, preferably soft tissue sarcomas and ovarian tumors, using the lats knock-out animals of the invention.
  • the invention also provides methods for screening potential therapeutics for activity in the treatment or prevention of pituitary dysfunctions, using the lats knock-out animals of the invention.
  • the invention provides methods for screening potential therapeutics for activity in the treatment or prevention of skin cancer using a non-human lats knock-out animal, preferably a lats knock-out mouse, in which skin tumors have been induced with carcinogens.
  • underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its encoded protein product which is indicated by the name of the gene in the absence of any underscoring or italicizing.
  • "lats” shall mean the lats gene, whereas “lats” shall indicate the protein product of the lats gene.
  • FIGURES Figures 1 A-H Human lats can functionally replace the fly gene.
  • A Adult Drosophila in which lats homozygous mutant cells have been induced in the imaginal tissues of the lats heterozygous larvae, exhibit lats mosaic phenotype, and have mutant cells which have undergone extensive proliferation and formed tumors in various body parts of the mosaic adults.
  • B Adult Drosophila that express human lats (hs-h-lats) completely do not exhibit tumor development (compare to A).
  • FIG. 1 Scanning Election Micrograph view of a lats mosaic fly.
  • H A lats tumor on the wing (indicated by an arrow in panel G) is enlarged, showing that cells in the overproliferated mutant clone have differentiated into wing cells with hair structures.
  • Figures 2A-E Phosphorylation of lats oscillates with the cell cycle.
  • A The phosphorylation of lats protein in HeLa cells after exposure of the cells to certain conditions was assayed by immunoprecipitation and blotted with an anti-h-lats monoclonal antibody.
  • the proteins are converted into a fast-migrating form when incubated with Calf Intestinal Phosphatase ("CIP") (lanes 2-4).
  • CIP Calf Intestinal Phosphatase
  • ⁇ -gp ⁇ -glycerol phosphate
  • Lats proteins from 125 min ARN cells have both the slow-migrating and fast-migrating forms (lane 6) and OP-treatment converts all lats proteins into the fast-migrating form (lanes 7-9).
  • B Immunowestern blot shows that phosphorylation of the lats protein oscillates with the cell cycle.
  • Cell cycle stages GO, Gl, S, and G2 are indicated above each lane and cells in different mitotic stages (M) are indicated by min. (minutes ARN). The faint bands are degradation products of lats. The progression of the cell cycle was verified by DAPI staining.
  • C-E These panels show fluorescent micrographs of DAPI staining for cells at three time points (50' (C), 75' (D), and 100' (E) ARN). Arrows indicate cells at metaphase (50' ARN (panel C)), anaphase (75' ARN (panel D)), or telophase (100' ARN (panel E)), respectively.
  • FIGS 3A-E Lats directly complexes with cdc2 during mitosis and the lats/cdc2 complex is inactive for HI kinase activity.
  • Cdc2 is co-immunoprecipitated with lats from mitotic CHO cell lysates (M) but not from quiescent CHO cell lysates (GO).
  • Anti-h- lats polyclonal antibodies or anti-human cyclin B monoclonal antibodies were used for immunoprecipitation, and anti-human cdc2 monoclonal antibodies were used to visualize cdc2.
  • B Cdc2 co-immunoprecipitated with human lats proteins at early mitosis. The stages of the cell cycle are indicated above each lane as "min. ARN" or "GO”. Lats proteins were immunoprecipitated using anti-h-lats monoclonal antibodies and were separated on
  • H-lats proteins were precipitated with anti-h-lats monoclonal antibodies and probed with anti- human cdc2 monoclonal antibodies (upper panel, labeled "Cdc 2") and cdc2 proteins were precipitated with anti-human cdc2 monoclonal antibodies and probed with anti-h-lats monoclonal antibodies (lower panel, labeled "h-lats”).
  • Cdc 2 human cdc2 monoclonal antibodies
  • cdc2 proteins were precipitated with anti-human cdc2 monoclonal antibodies and probed with anti-h-lats monoclonal antibodies (lower panel, labeled "h-lats”).
  • Lats-associated cdc2 is inactive for HI kinase activity.
  • ARN HeLa cell lysates were divided for western quantification of cdc2 (upper panel, labeled "IP- Western") and for the histone HI kinase assay (lower panel, labeled "HI Kinase assay”), respectively.
  • IP- Western Western
  • HI Kinase assay Protein G-agarose beads incubated with equal amounts of cell lysates only were also used for the HI kinase assay (indicated by "-” in the legend).
  • Figures 4A-F Genetic interaction between lats, cdc2, and cyclin A in Drosophila.
  • A lats p8 llats PS homozygotes die at the pupal stage.
  • B Removal of one copy of the cdc2 gene rescues lats ps lethality (lats ps llats PS ; rlcdc2 B47 ).
  • C A typical rough, overproliferated eye dissected from a lats P8 llats PS dead pupa.
  • D An eye from the fly in panel (B), showing that the eye phenotype has been almost completely suppressed.
  • FIGS 5A-N Effect of inactivation and overexpression of lats on the cell cycle in Drosophila.
  • Drosophila third instar eye imaginal disc contains a homozygous lats l clone (arrowhead indicates the lack of Myc staining) that crosses the morphogenetic furrow (MF) (arrow).
  • Cyclin A staining (indicated by arrowhead) in the clone exhibits expression that spans the MF (arrow).
  • C Composite staining of the same disc shown in panels A and B showing Myc stains and propidium iodide staining which more clearly delineates the MF and the lats mutant clone (indicated by the arrow) that spans it.
  • a BamHI/EcoRV double digest generates a 3.5 kb fragment from the wild-type allele and a 5.8 kb fragment from the disrupted allele, both of which are recognized by the probe shown, which is not contained in the targeting vector.
  • the PGK-TK gene cassette and the PGK-neo fragment are denoted by open boxes labeled accordingly.
  • C Southern blot of genomic DNA isolated from individual embryonic stem cell clones.
  • genotypes of the clones are indicated above the lanes with the "+/+” indicating wild-type clones, "+/-” indicating clones heterozygous for the mutant allele, and "-/-” indicating clones homozygous for the mutant allele.
  • D Western blot using anti-h-lats polyclonal antibody on lysates from 13.5 dpc (days post coitus) mouse embryonic fibroblasts indicating the absence of lats protein in the knock-out mice.
  • the genotype of the clones is indicated above the lanes as in panel C.
  • Figures 7A and B Growth retardation of lats ' ' ' mice.
  • FIGS 8 A-D Ovarian phenotypes of lats' ⁇ mice. Histopathological sections of ovaries derived from lats + (panels A and C) and lats " (panels B and D) females. Overview images are in panels A and B while high magnification views are in panels C and D. The paraffin sections were stained with hematoxylin and eosin. An absence of corpora lutea (CL) is evident in the lats ' ' '' ovary. Ovarian stromal cell tumors (SC) which obliterate the normal structure of the ovary, eliminating follicles (FC) progressively, are readily apparent.
  • SC stromal cell tumors
  • FIGS 9A-F Absence of mammary gland development in lats ' ' ' Mice.
  • A,B Lats / " female with normal mammary gland and nipple development.
  • D,E Lats ' ' ' female displaying absence of mammary gland and nipple formation.
  • C,F Hematoxylin and eosin stained histopathological sections of mammary glands derived from lats +/+ (C) and lats ' ' ' (F) mice. The amount of breast epithelial tissue was markedly decreased in lats ' ' ' females, resulting in mammary fat pads, devoid of an epithelial component.
  • FIGS 10A-E Pituitary hyperplasia and dysfunction in lats ' ' ' mice.
  • a and B Histopathological sections of pituitaries derived from lats */ ⁇ (A) and lats ' ' ' (B) mice. The paraffin sections were stained with hematoxylin and eosin. The normal pituitary gland from a ⁇ ⁇ ⁇ mouse demonstrates the organized architecture of the gland. Hyperplastic changes are visible in lats ' ' ' pituitaries. Multiple atypical cells showing irregularly shaped nuclei, and variability in shape and size are readily apparent.
  • C-E The graphs indicate the amount of LH (C), PRL (D), and FSH (E) production in the mice. Hormone levels are plotted as ng/ml with the results from the lats + ' ⁇ mice represented by the right bar of each bar graph and the results from the lats ' ' ' mice represented by the left bar of each bar graph.
  • FIGS 1 1 A-C Soft tissue sarcoma development in lats ' ' mice.
  • A,B Typical soft tissue sarcomas in lats' ' mice (A, 6.5 months old; B, 4.5 months old).
  • C Histopathological section of the soft tissue sarcoma shown in panel B stained with hematoxylin and eosin revealing pleiomorphic, spindle-shaped cells characteristic of this tumor.
  • FIG. 1 Nucleotide and amino acid sequences of mouse lats2 (m-lats2) (SEQ ID NOS:5 and 6, respectively).
  • Figure 15 Nucleotide and amino acid sequences of Drosophila lats (SEQ ID NOS:7 and 8, respectively).
  • the present invention provides lats-cdc2 protein complexes, including complexes that contain lats analogs and fragments and/or cdc2 analogs and fragments, as well as methods of producing these complexes and nucleic acids encoding the two members of the complex.
  • the invention also provides antibodies that bind immunospecifically to a lats- cdc2 complex, but do not bind the individual binding partners immunospecifically.
  • the invention also provides methods for the modulation of cdc2 activity using lats proteins and lats analogs and derivatives that are able to interact with cdc2.
  • methods are provided for treating or preventing diseases and disorders associated with aberrant cdc2 activity by administration of a therapeutic compound of the invention.
  • the present invention further provides recombinant non-human animals, preferably mice, having at least one copy of (preferably both copies of, i.e., is homozygous for) an inactivated lats gene, i.e., lats knock-out animals.
  • these lats knock-out animals are generated by homologous recombination, i.e., have a gene disrupted by insertional mutagenesis induced by homologous recombination with a nucleic acid containing non-lats sequences flanked by lats genomic sequences.
  • the invention further provides methods of screening for compounds effective to treat or prevent cancer, preferably soft tissue sarcomas or ovarian tumors, more preferably skin cancer, using the recombinant non-human animals of the invention.
  • the invention also provides methods of screening for compounds effective to treat or prevent pituitary dysfunction using the recombinant non-human animals of the invention.
  • Therapeutics of the invention that can be used to treat or prevent diseases and disorders associated with an aberrant level of cdc activity include those therapeutics that promote lats function (e.g., lats proteins and lats derivatives and analogs that supply lats function, nucleic acids encoding lats, lats derivatives and analogs, and lats-cdc2 complexes), and those therapeutics that inhibit or antagonize lats function (e.g., lats derivatives and analogs that inhibit or antagonize lats function) and nucleic acids encoding these lats derivatives and analogs, anti-lats antibodies and anti-lats-cdc2 complex antibodies, lats antisense nucleic acids, and lats inhibitors and antagonists.
  • lats function e.g., lats proteins and lats derivatives and analogs that supply lats function, nucleic acids encoding lats, lats derivatives and analogs, and lats-
  • the present invention also provides therapeutic methods and compositions for the treatment and prevention of cancer based on lats proteins and therapeutically or prophylactically effective analogs and fragments of lats proteins.
  • the invention provides for treatment and prevention of cancer by administration of a therapeutic compound of the invention.
  • the therapeutic compounds of the invention that can be used to treat or prevent cancer include: lats proteins, including human lats proteins, therapeutically or prophylactically effective lats analogs and fragments, and nucleic acids encoding the lats proteins, analogs and fragments.
  • the invention provides therapeutic and prophylactic methods for the treatment or prevention of cancer that has been shown to be or may be refractory to chemotherapy or radiation therapy treatments or treatments based on tumor suppressor genes other than lats.
  • the invention provides for treatment or prevention of cancers refractory to chemotherapy or radiation therapy by administration of a therapeutic compound (termed herein "Therapeutic”).
  • the invention also provides for treatment or prevention of diseases or disorders that can be treated by modulation of cdc2 activity by administration of a Therapeutic of the invention.
  • Such "Therapeutics” include lats proteins and therapeutically or prophylactically effective analogs and fragments thereof; lats-cdc2 complexes; antibodies thereto; nucleic acids encoding the lats proteins, analogs, or fragments, and lats-cdc2 complexes; lats antisense nucleic acids, and lats agonists and antagonists .
  • the therapeutic is a lats protein or lats-cdc2 complex containing a lats protein that is phosphorylated, particularly a lats protein phosphorylated on a serine or threonine residue within 20 residues upstream of an Ala-Pro-Glu consensus in subdomain eight of a lats kinase domain, e.g., a serine corresponding to serine 909 of human lats, as depicted in Figure 12 (SEQ ID NO:2).
  • the therapeutic is a lats derivative or lats-cdc2 complex containing a lats derivative, in which derivative a serine or threonine residue within 20 residues upstream of an Ala-Pro-Glu consensus in subdomain eight of a lats kinase domain is substituted with a glutamate or aspartate residue, preferably the serine corresponding to serine 909 of human lats is replaced with a glutamate residue.
  • the therapeutic is a fragment of a lats protein or a lats-cdc2 complex containing a fragment of a lats protein comprising or consisting of the amino acid sequence of a lats protein corresponding to amino acids 15-585 of human lats, as depicted in Figure 12 (SEQ ID NO:2).
  • a human lats protein, derivative, or fragment, or nucleic acid, or an antibody to a human lats protein is therapeutically or prophylactically administered to a human patient.
  • Cancers including neoplasms, tumors, metastases, or any disorder characterized by uncontrolled cell growth, that have been shown to be refractory to a chemotherapy or radiation therapy can be treated or prevented by administration of a Therapeutic of the invention that promotes (i.e., increases or supplies) lats function.
  • Examples of such a Therapeutic include lats proteins, derivatives or fragments that are functionally active, particularly have a lats functional activity of inhibiting cell overproliferation (e.g., as demonstrated in in vitro assays or in an animal model), and nucleic acids encoding a lats protein or a functionally active derivative or analog thereof (e.g., for use in gene therapy).
  • Other Therapeutics that can be used, e.g., lats agonists can be identified using in vitro assays or animal models, examples of which are described in Examples section.
  • That a cancer is refractory to chemotherapy or radiation therapy means that at least some significant portion of the cancer cells are not killed or their cell division arrested by the particular chemotherapeutic agent or combination of chemotherapeutic agents or the level of radiation employed in a therapeutic protocol.
  • the determination of whether the cancer cells are refractory to the chemotherapy or the radiation therapy can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of treatment on cancer cells.
  • cancer that is refractory to radiation therapy, chemotherapy or combination chemotherapy, or combination of radiotherapy and chemotherapy is treated or prevented by administration of a Therapeutic of the invention.
  • cancer that is refractory to treatment with a chemotherapeutic agent that is cell cycle specific or said cancer is refractory to treatment with a chemotherapeutic agent that kills or arrests the cells in the S phase of the cell cycle, or said cancer is refractory to treatment with a chemotherapeutic agent that kills or arrests cells during the M phase of the cell cycle is treated using a Therapeutic of the invention.
  • the Therapeutic of the invention can be administered along with radiation therapy and/or one or a combination of chemotherapeutic agents, or as an alternative to other forms of therapy.
  • the chemotherapy or radiation therapy administered concurrently with or subsequent to the administration of the therapeutic of the invention can be administered by any method known in the art.
  • the chemotherapeutic agents are preferably administered in a series of sessions, any one or a combination of the chemotherapeutic agents listed above can be administered.
  • any radiation therapy protocol can be used depending upon the type of cancer to be treated.
  • x-ray radiation can be administered; in particular, high-energy megavoltage (radiation of greater that 1 MeV energy) can be used for deep tumors, and electron beam and orthovoltage x-ray radiation can be used for skin cancers.
  • Gamma ray emitting radioisotopes, such as radioactive isotopes of radium, cobalt and other elements may also be administered to expose tissues to radiation. Malignancies
  • Malignancies and related disorders that may become refractory to chemotherapy and/or radiation therapy and that can be treated or prevented by administration of a Therapeutic that promotes lats function include blood-related cancers and solid tumors (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).
  • diseases and disorders associated with aberrant levels of cdc2 activity can be treated or prevented by administration of a therapeutic of the invention able to modulate the activity of cdc2.
  • those diseases and disorders associated with an aberrantly high cdc2 activity are treated or prevented by administration of a Therapeutic that promotes lats activity.
  • those diseases and disorders associated with an aberrantly low cdc2 activity are treated or prevented by administration of a Therapeutic that inhibits lats activity, e.g., lats derivatives and analogs that inhibit or antagonize lats activity, anti-lats antibodies, lats antisense nucleic acids, lats inhibitors and antagonists, antibodies that specifically recognize a lats-cdc2 complex, etc.
  • a Therapeutic that inhibits lats activity e.g., lats derivatives and analogs that inhibit or antagonize lats activity, anti-lats antibodies, lats antisense nucleic acids, lats inhibitors and antagonists, antibodies that specifically recognize a lats-cdc2 complex, etc.
  • diseases and disorders that may be associated with an increased level of cdc2 activity include diseases and disorders associated with increased cell proliferation, such as malignancies.
  • diseases and disorders that may be associated with a decreased level of cdc2 activity include diseases and disorders associated with decreased cell proliferation.
  • the Therapeutics of the invention that reduce cdc2 activity can be administered to treat premalignant conditions and to prevent progression to a neoplastic or malignant state.
  • Such prophylactic or therapeutic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred.
  • the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or displayed in vitro by a cell sample from a patient can indicate the desirability of prophylactic/therapeutic administration of a Therapeutic that inhibits cdc2 activity.
  • Some characteristics of a transformed phenotype include morphology changes, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the
  • Therapeutics that can be used include anti-lats antibodies (and fragments and derivatives thereof containing the binding region thereof), lats derivatives or fragments that are dominant-negative kinases, lats antisense nucleic acids, and lats nucleic acids that are dysfunctional (e.g., due to a heterologous (non-lats sequence) insertion within the lats coding sequence) that are used to "knockout" endogenous lats function by homologous recombination (see, e.g., Capecchi, 1989, Science 244:1288-1292), as described herein.
  • Therapeutics that inhibit lats function can be identified by use of known convenient in vitro assays, e.g., based on their ability to inhibit binding of lats to another protein (e.g., cdc2), or inhibit any known lats function, as preferably assayed in vitro or in cell culture. Methods for screening for compounds that prevent or reduce lats binding to cdc2 are described herein. Preferably, suitable in vitro or in vivo assays are utilized to determine the effect of a specific Therapeutic (i.e., its ability to promote cdc2 activity or increase cdc2 levels) and whether its administration is indicated for treatment of the affected tissue.
  • a specific Therapeutic i.e., its ability to promote cdc2 activity or increase cdc2 levels
  • Diseases and disorders involving a deficiency in cell proliferation or in which cell proliferation is desired for treatment or prevention, and that can be treated or prevented by promoting cdc2 function include degenerative disorders, growth deficiencies, hypoproliferative disorders, physical trauma, lesions, and wounds; for example, to promote wound healing, or to promote regeneration in degenerated, lesioned or injured tissues, etc.
  • Gene therapy refers to therapy performed by the administration of a nucleic acid to a subject.
  • the nucleic acid produces its encoded protein that mediates a therapeutic effect by promoting lats function.
  • the Therapeutic comprises a lats nucleic acid that is part of an expression vector that expresses a lats protein or fragment or chimeric protein thereof in a suitable host.
  • a nucleic acid has a promoter operably linked to the lats coding region, said promoter being inducible or constitutive, homologous or heterologous, and, optionally, tissue-specific.
  • a nucleic acid molecule is used in which the lats coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the lats nucleic acid, as described (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
  • a nucleic acid or combination of nucleic acids containing both a lats and a cdc2 nucleic acid, preferably where each is operably linked to a promoter is delivered by gene therapy methods.
  • Delivery of the nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
  • a lats nucleic acid or both lats and cdc2 nucleic acids are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
  • stem or progenitor cells are used.
  • stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used, such as hematopoietic stem cells (HSC), stem cells of epithelial tissues such as the skin and the lining of the gut, embryonic heart muscle cells, liver stem cells (PCT Publication WO 94/08598, dated April 28, 1994), neural stem cells (Stemple and Anderson, 1992, Cell 71 :973-985), or epithelial stem cells (ESCs) (Rheinwald, 1980, Meth. Cell Bio. 21A:229; Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).
  • HSC hematopoietic stem cells
  • stem cells of epithelial tissues such as the skin and the lining of the gut
  • embryonic heart muscle cells embryonic heart muscle cells
  • liver stem cells PCT Publication WO 94/08598, dated April 28, 1994
  • neural stem cells Step and Anderson, 1992, Cell 71 :973-985
  • ESCs epithelial
  • Lats function may be inhibited by use of lats antisense nucleic acids.
  • the present invention provides the therapeutic or prophylactic use of nucleic acids of at least six nucleotides and are preferably oligonucleotides (ranging from 6 to about 200 oligonucleotides), that are antisense to a gene or cDNA encoding a lats protein, or portions thereof.
  • a lats "antisense" nucleic acid as used herein refers to a nucleic acid capable of hybridizing to a portion of a lats nucleic acid (preferably mRNA) by virtue of some sequence complementarity.
  • the antisense nucleic acid may be complementary to a coding and or noncoding region of a lats mRNA.
  • the oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 200 nucleotides.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotide can be modified at any position (examples of such modifications can be found in: Bailey, Ullmann's
  • antisense nucleic acids have utility as Therapeutics that inhibit lats function or activity, and can be used in the treatment or prevention of disorders characterized by an aberrantly low cdc2 level or activity.
  • lats antisense nucleic acids can be directly administered to a cell, or can be produced intracellularly by transcription of exogenous, introduced sequences. Alternatively, lats antisense nucleic acids are produced intracellularly by transcription from an exogenous sequence.
  • a vector can be introduced in vivo such that it is taken up by a cell, within which cell the vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention.
  • RNA antisense nucleic acid
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense
  • RNA Ribonucleic acid
  • vectors can be constructed by recombinant DNA technology methods standard in the art.
  • the antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a lats gene, preferably a human lats gene.
  • compositions of the invention comprising an effective amount of a lats antisense nucleic acid in a pharmaceutically acceptable carrier can be administered to a patient having a disease or disorder which is characterized by aberrantly low cdc2 activity.
  • the amount of lats antisense nucleic acid that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Where possible, it is desirable to determine the antisense cytotoxicity in vitro, and then in useful animal model systems prior to testing and use in humans.
  • compositions comp ⁇ sing lats antisense nucleic acids are administered via liposomes, microparticles, or microcapsules.
  • lats proteins and nucleic acids, and lats derivatives and fragments can be produced by any method known in the art.
  • the nucleic acid containing all or a portion of the nucleotide sequence encoding the protein can be inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted protein coding sequence.
  • the regulatory elements e.g., promoter
  • the regulatory elements are heterologous (i.e., not the native gene promoter).
  • Promoters which may be used include the SV40 early promoter (Bernoist and Chambon, 1981, Nature 290: 304-310), and the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al, 1980, Cell 22: 787-797), among others.
  • host- vector systems may be utilized to express the protein coding sequence. These include mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g. baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
  • virus e.g., vaccinia virus, adenovirus, etc.
  • insect cell systems infected with virus e.g. baculovirus
  • microorganisms such as yeast containing yeast vectors
  • bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA e.g., vaccinia virus, adenovirus, etc.
  • lats protein Once a lats protein, or derivative or fragment, has been recombinantly expressed, it may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • a lats protein may also be purified by any standard purification method from natural sources.
  • a lats protein, analog or derivative can be synthesized by standard chemical methods known in the art (e.g., see Hunkapiller et al., 1984, Nature 310:105-111).
  • the Therapeutics of the invention also include derivatives and fragments related to lats.
  • the derivative or fragment is functionally active, i.e., capable of exhibiting one or more functional activities associated with a full-length, wild- type lats protein, e.g., able to inhibit cell proliferation in in vitro and/or in vivo assays.
  • derivatives or fragments that inhibit lats activity e.g., promote cell proliferation, may also have a use in the methods of the invention. Derivatives or analogs of lats can be tested for the desired activity by procedures known in the art.
  • the Therapeutic is a lats protein that is phosphorylated, preferably that is phosphorylated on a serine or threonine residue within 20 amino acids upstream of an Ala-Pro-Glu consensus sequence in subdomain eight of a lats kinase domain, more preferably that is phosphorylated on a serine residue corresponding to serine 909 of human lats, as depicted in Figure 12 (SEQ ID NO:2).
  • the therapeutic is a lats derivative in which a serine or threonine residue within 20 residues upstream of an Ala-Pro-Glu consensus in subdomain eight of a lats kinase domain is substituted with a glutamate or aspartate residue, preferably, in which a serine residue corresponding to serine 909 of human lats is replaced with a glutamate residue.
  • the therapeutic is a fragment of a lats protein comprising or consisting of the amino acid sequence corresponding to amino acids 15 to 585 of human lats, as depicted in Figure 12 (SEQ ID NO:2).
  • lats derivatives can be made by altering lats sequences by substitutions, additions or deletions that provide for functionally equivalent molecules.
  • nucleotide coding sequences Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as a lats gene may be used in the practice of the present invention. These include nucleotide sequences comprising all or portions of lats genes which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • the lats derivatives of the invention include those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a lats protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • proteins consisting of or comprising a fragment of a lats protein consisting of at least 10 (continuous) amino acids of the lats protein is provided.
  • the fragment consists of at least 20 or 50 amino acids of the lats protein.
  • such fragments are not larger than 35, 100 or 200 amino acids.
  • the fragment of a lats protein is from the N-terminal portion of the protein, preferably including all or a portion of the amino acids corresponding to amino acids 15-585 of human lats.
  • Derivatives or fragments of lats include but are not limited to those molecules comprising regions that are substantially homologous to lats or fragments thereof (e.g., in various embodiments, at least 60% or 70% or 80%) or 90%> or 95%> identity over an amino acid sequence of identical size with no insertions or deletions considered, or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, e.g., the blastp program) or whose encoding nucleic acid is capable of hybridizing to the inverse complement (the inverse complement of a nucleic acid strand has the complementary sequence running in reverse orientation to the strand so that the inverse complement would hybridize without mismatches to the nucleic acid strand; thus, for example, where the coding strand is hybridizable to a nucleic acid with no mismatches between the coding strand and the hybridizable strand, then the inverse complement of the hybridizable strand is identical to the coding
  • lats derivatives and fragments of the invention can be produced by various methods known in the art.
  • the manipulations which result in their production can occur at the gene or protein level.
  • the cloned lats gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
  • the lats-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • Any technique for mutagenesis known in the art can be used, including chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson, C, et al., 1978, J. Biol. Chem 253:6551), use of TAB ® linkers (Pharmacia), etc.
  • lats sequence may also be made at the protein level. Included within the scope of the invention are lats protein fragments or other derivatives which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, etc. Any of numerous chemical modifications may be carried out by known techniques, including specific chemical cleavage by cyanogen bromide, trypsin, oxidation, reduction; etc.
  • analogs and fragments of lats can be chemically synthesized.
  • a peptide corresponding to a portion of a lats protein which comprises the desired domain, or which mediates the desired activity in vitro can be synthesized by use of a peptide synthesizer.
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the lats sequence.
  • the lats derivative is a chimeric, or fusion, protein comprising a lats protein or fragment thereof (preferably consisting of at least a domain or motif of the lats protein, or at least 15, preferably 20, amino acids of the lats protein) joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein.
  • a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising a lats-coding sequence joined in-frame to a coding sequence for a different protein).
  • Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art.
  • a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.
  • Chimeric genes comprising portions of lats fused to any heterologous protein-encoding sequences may be constructed.
  • a specific embodiment relates to a chimeric protein comprising a fragment of lats of at least six amino acids.
  • the lats derivative is a chimeric protein comprising a fragment of lats corresponding to amino acids 15-585 of human lats.
  • the lats derivative is a molecule comprising a region of homology with a lats protein.
  • a first protein region can be considered "homologous" to a second protein region when the amino acid sequence of the first region is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%o identical, when compared to any sequence in the second region of an equal number of amino acids as the number contained in the first region with no insertions or deletions considered, or when compared to an aligned sequence of the second region that has been aligned by a computer homology program known in the art.
  • a molecule can comprise one or more regions homologous to a lats domain or a portion thereof.
  • the methods of the invention use lats derivatives and fragments that comprise, or alternatively consist of, one or more domains of a lats protein, including but not limited to a lats C-terminal domain 3 (LCD3), lats C-terminal domain 2
  • LFD lats flanking domain
  • LFD lats split domain 1
  • LSD2 lats split domain 2
  • SH3-binding domain functional (e.g., binding) fragments of any of the foregoing, or any combination of the foregoing.
  • the LCD3 domain is the last three amino acids of the protein, which are Val-Tyr-Val in all four proteins.
  • the LCD2 domain is amino acid residues 1077-1086 and 1075-
  • Drosophila lats amino acid sequences depicted in Figures 12 and 14, respectively (SEQ ID NOS:2 and 8, respectively)); the LCDl domain is amino acid residues 1032-1043 and 1035-
  • the kinase domain is amino acid residues 703-1014 and 711-1018, respectively;
  • the LFD domain is amino acid residues 607-702 and 612-710 respectively; and
  • the putative SH3-binding domain is amino acids 247-268 and 196-217, respectively.
  • the LSD1 is amino acid residues 365-392 and the
  • LSD2 is amino acids 536-544. In human lats, the LSD1 and LSD2 domains are split into anterior and posterior portions such that the LSD1 is amino acid residues 328-334 and 498-
  • LSD2 is amino acid residues 28-31 and 555-559.
  • the Therapeutics of the invention include molecules comprising specific fragments of lats that are those fragments in the respective lats protein most homologous to specific fragments of a human or mouse lats protein.
  • a lats protein, derivative or fragment is provided that has a kinase domain and has a phosphorylated or dephosphorylated serine situated within 20 residues upstream of an Ala-Pro-Glu consensus in subdomain eight of its kinase domain, or in which the serine situated within 20 residues upstream of that consensus has been deleted or substituted by another amino acid.
  • the invention provides vanous phosphorylated and dephosphorylated forms of the lats protein, de ⁇ vative, or fragment that are active or inactive kinase forms. Both phosphorylation and dephosphorylation of lats at different residues could potentially activate or inactivate lats.
  • Phosphorylation can be carried out by any methods known in the art, e.g., by use of a kinase.
  • Dephosphorylation can be carried out by use of any methods known in the art, e.g., by use of a phosphatase.
  • Another specific embodiment relates to a derivative or fragment of a lats protein that is a dominant-active protein kinase.
  • a derivative or analog comprises a lats kinase domain that has been mutated so as to be dominantly active (exhibit constitutively active kinase activity).
  • acidic residues such as Glu and Asp sometimes mimic a phosphorylated residue, and changing the phosphorylatable Ser or Thr residue in subdomain eight into a Glu or Asp residue has been previously used to produce constitutively active kinases (Mansour et al., 1994, Science 265:966-970).
  • changing a serine or threonine residue situated within 20 residues upstream of an Ala-Pro-Glu consensus in subdomain eight of a lats kinase domain into another residue may be used to make a dominant-active lats protein kinase.
  • another residue e.g., Glu, Asp
  • changing Ser914 in Drosophila lats, or changing Ser909 in human lats, into a Glu residue could produce a dominant active lats kinase.
  • Another specific embodiment relates to a derivative or fragment of lats that is a dominant-negative protein kinase.
  • Protein kinases can be mutated into dominant negative forms. Expression of a dominant negative protein kinase can suppress the activity of the wild-type form of the same kinase.
  • Dominant negative forms of protein kinases are often obtained by expressing an inactive form of a kinase (Milarski and Saltiel, 1994, J. Biol. Chem. 269(33):21239-21243) or by expressing a noncatalytic domain of a kinase (Lu and Means, 1994, EMBO J. 12:2103-21 13; Yarden et al., 1992, EMBO J.
  • a lats dominant-negative kinase can be obtained by mutating the kinase domain so as to be inactive (e.g., by deletion and/or point mutation).
  • a lats derivative that is a dominant-negative kinase is a lats protein that lacks a kinase domain but comprises one or more of the other domains of the lats protein; e.g., a lats protein derivative truncated at about the beginning of the kinase domain (i.e., a lats fragment containing only sequences amino-terminal to the kinase domain).
  • a lats derivative that is a dominant-negative kinase is a lats protein in which one of the residues conserved among serine/threonine kinases (see Hanks et al, 1988, Science 241:42-52) is mutated (deleted or substituted by a different residue).
  • a molecule that comprises one or more domains (or functional portion thereof) of a lats protein but that also lacks one or more domains (or functional portion thereof) of a lats protein.
  • a protein may lack all or a portion of the kinase domain, but retain at least the SH3-binding domain of a lats protein.
  • a molecule is provided that comprises one or more domains (or functional portion thereof) of a lats protein, and that has one or more mutant (e.g., due to deletion or point mutation(s)) domains of a lats protein (e.g., such that the mutant domain has decreased function).
  • the kinase domain may be mutant so as to have reduced, absent, or increased kinase activity.
  • the invention provides lats-cdc2 complexes.
  • the lats- cdc2 complexes are complexes of human proteins.
  • fragment or derivative of a lats-cdc2 complex includes complexes where one or both members of the complex are fragments or derivatives of the wild-type lats or cdc2 protein. Such derivatives and fragments can be generated as described for lats derivatives and fragments above.
  • the lats-cdc2 complexes in which one or both members of the complex are a fragment or derivative of the wild type protein are functionally active lats-cdc2 complexes.
  • the native proteins, derivatives or analogs of lats and/or cdc2 are of animals, e.g. mouse, rat, pig, cow, dog, monkey, human, fly, frog, or of plants.
  • "Functionally active lats-cdc2 complex” as used herein refers to that material displaying one or more known functional attributes of a complex of full length lats with a full length cdc2, including but not exclusive to control of cell cycle progression, cell proliferation, etc.
  • the lats-cdc2 complex contains a lats protein that is phosphorylated, preferably that is phosphorylated on a serine or threonine residue within 20 amino acids upstream of an Ala-Pro-Glu consensus subdomain eight of a lats kinase domain, more preferably that is phosphorylated on a serine residue corresponding to serine 909 of human lats, as depicted in Figure 12 (SEQ ID NO:2).
  • the lats-cdc2 complex contains a lats derivative in which a serine or threonine residue within 20 residues upstream of an Ala-Pro-Glu consensus subdomain eight of a lats kinase domain is substituted with a glutamate or aspartate residue, preferably, in which a serine residue corresponding to serine 909 of human lats is replaced with a glutamate residue.
  • the therapeutic is a fragment of a lats protein comprising or consisting of the amino acid sequence corresponding to amino acids 15 to 585 of human lats, as depicted in Figure 12 (SEQ ID NO:2).
  • a specific embodiment relates to a lats-cdc2 complex of a fragment of lats and/or a fragment of cdc2 that can be bound by an anti-lats and/or anti-cdc2 antibody or antibody specific for a lats-cdc2 complex when such a fragment is included within a lats- cdc2 complex.
  • the lats-cdc2 complexes can be obtained by any method known in the art.
  • the cdc2 nucleotide and amino acid sequence is available from GenBank, accession no. Y00272 (see also, Lee and Nurse, 1987, Nature 327:31-35).
  • the lats-cdc2 complexes can be obtained, for example, by expressing an entire lats coding sequence and a cdc2 coding sequence in the same cell, either under the control of the same promoter or two separate promoters.
  • a derivative, fragment or homolog of lats and/or a derivative, fragment or homolog of cdc2 are recombinantly expressed.
  • the derivative, fragment or homolog of lats and or the cdc2 protein form a complex with a binding partner identified by a binding assay, such as co-immunoprecipitation with an anti-lats or anti-cdc2 antibody, or interaction in a yeast two-hybrid assay (Fields and Song, 1989, Nature 340:245-246; and Finley and Brent, in DNA Cloning 2, Rickwood and Hames, eds (Oxford University Press, Oxford, 1995)).
  • a binding assay such as co-immunoprecipitation with an anti-lats or anti-cdc2 antibody, or interaction in a yeast two-hybrid assay (Fields and Song, 1989, Nature 340:245-246; and Finley and Brent, in DNA Cloning 2, Rickwood and Hames, eds (Oxford University Press, Oxford, 1995)).
  • fusion or chimeric proteins contain the domains of a lats protein, or, in a specific embodiment, the amino acid sequence corresponding to amino acids 15 to 585 of human lats, and a cdc2 protein that directly form a lats-cdc2 complex and, optionally, a heterofunctional reagent, such as a peptide linker, linking the two domains, where such a heterofunctional reagent, such as a reagent or linker promotes the interaction of the lats and cdc2 binding domains.
  • a heterofunctional reagent such as a peptide linker
  • LATS proteins including functional derivatives and fragments thereof (e.g. a LATS protein encoded by a sequence of any one of SEQ ID NOs:2, 4, 6, or 8, or a subsequence thereof) may be used as an immunogen to generate monoclonal or polyclonal antibodies and antibody fragments or derivatives (e.g., chimeric, single chain, Fab fragments, etc.).
  • antibody fragments or derivatives e.g., chimeric, single chain, Fab fragments, etc.
  • antibodies to a particular domain of a lats protein may be desired.
  • fragments of a lats protein identified as hydrophilic are used as immunogens for antibody production using art-known methods.
  • Lats-cdc2 complexes may be markers of specific disease states involving disruption of physiological processes, such as cell cycle progression and cell proliferation, and pathological processes, such as hyperproliferative disorders, including tumorigenesis and tumor progression, and hypoproliferative disorders, and thus have diagnostic utility. Detecting levels of lats-cdc2 complexes, or individual lats and cdc2 proteins or the mRNA encoding lats and cdc2 may be used in diagnosis or prognosis, to follow the course of disease states, or to follow therapeutic response, etc.
  • Lats-cdc2 complexes, lats and cdc2 proteins, and derivatives, and sub-sequences thereof, lats and/or cdc2 nucleic acids (and sequences complementary thereto), and anti- lats-cdc2 complex antibodies and combinations of antibodies directed against lats and cdc2 have uses in diagnostics.
  • Such molecules can be used in assays, such as immunoassays, to detect, prognose, diagnose, or monitor various conditions, diseases, and disorders characterized by aberrant levels of lats-cdc2 complexes or monitor the treatment thereof.
  • such an immunoassay is carried out by a method comprising contacting a sample derived from a patient with an anti-lats-cdc2 complex antibody under conditions such that immunospecific binding can occur, and detecting or measuring the amount of any immunospecific binding by the antibody.
  • an anti-lats-cdc2 complex antibody under conditions such that immunospecific binding can occur, and detecting or measuring the amount of any immunospecific binding by the antibody.
  • binding of antibody, in tissue sections can be used to detect aberrant lats-cdc2 complex localization or aberrant (e.g., high, low or absent) levels of lats-cdc2 complex.
  • an antibody to a lats-cdc2 complex can be used to assay in a patient tissue or serum sample for the presence of a lats-cdc2 complex where an aberrant level of lats-cdc2 complex is an indication of a diseased condition.
  • aberrant levels is meant an increased or decreased level relative to that present, or a standard level representing that present, in an analogous sample from a portion of the body or from a subject not having the disorder.
  • the immunoassays which can be used include competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, etc.
  • Nucleic acids encoding lats and cdc2 proteins and related nucleotide sequences and sub-sequences, including complementary sequences, can also be used in hybridization assays.
  • the lats and cdc2 nucleotide sequences, or sub-sequences thereof comprising about at least 8 nucleotides, can be used as hybridization probes.
  • Hybridization assays can be used to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with aberrant levels of the mRNAs encoding the components of a lats-cdc2 complex.
  • such a hybridization assay is carried out by a method comprising contacting a sample containing nucleic acid with a nucleic acid probe capable of hybridizing to lats and cdc2 DNAs or RNAs, under conditions such that hybridization can occur, and detecting or measuring any resulting hybridization.
  • levels of lats-cdc2 complexes and lats and cdc2 proteins can be detected by immunoassay
  • levels of lats and cdc2 mRNA can be detected by hybridization assays (e.g., Northern blots, dot blots)
  • binding of lats to cdc2 can be done by binding assays commonly known in the art
  • translocations and point mutations in lats and/or cdc2 can be detected by Southern blotting, RFLP analysis, PCR using primers that preferably generate a fragment spanning at least most of the lats and/or cdc2 gene, sequencing of the lats and/or cdc2 genomic DNA or cDNA obtained from the patient, etc.
  • This embodiment includes cell sorting of prokaryotes such as but not restricted to, bacteria (Davey and Kell, 1996, Microbiol. Rev. 60:641-696), primary cultures and tissue specimens from eukaryotes, including mammalian species such as human (Steele et al., 1996, Clin. Obstet. Gynecol 39:801-813), and continuous cell cultures (Orfao and Ruiz-Arguelles, 1996, Clin. Biochem. 29:5-9).
  • prokaryotes such as but not restricted to, bacteria (Davey and Kell, 1996, Microbiol. Rev. 60:641-696)
  • primary cultures and tissue specimens from eukaryotes including mammalian species such as human (Steele
  • Kits for diagnostic use comprise in one or more containers an anti-lats-cdc2 complex antibody and, optionally, a labeled binding partner to the antibody.
  • the anti-lats-cdc2 complex antibody can be labeled (with a detectable marker, e.g., a chemiluminescent, enzymatic, fluorescent, or radioactive moiety).
  • a kit is also provided that comprises in one or more containers a nucleic acid probe or probes capable of hybridizing to lats and cdc2 mRNAs.
  • a kit can comprise in one or more containers a pair of primers (e.g., each in the size range of 6-30 nucleotides) that are capable of priming amplification [e.g., by polymerase chain reaction (see e.g., Innis et al., 1990, PCR Protocols, Academic Press, Inc., San Diego, CA), ligase chain reaction (see EP 320,308) use of ⁇ -replicase, cyclic probe reaction, or other methods known in the art], under appropriate reaction conditions of at least a portion of a lats nucleic acid and a cdc2 nucleic acid.
  • a kit can optionally further comprise in a container a predetermined amount of a purified lats-cdc2 complex, lats and cdc2 proteins or nucleic acids thereof, e.g., for use as a standard or control.
  • the Therapeutics of the invention are preferably tested in vitro, and then in vivo, for the desired therapeutic or prophylactic activity, prior to use in humans.
  • in vitro assays which can be used to determine whether administration of a specific Therapeutic is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a Therapeutic, and the effect of such Therapeutic upon the tissue sample is observed. A lower level of proliferation or survival of the contacted cells indicates that the Therapeutic is effective to treat the condition in the patient.
  • culturing cells from a patient instead of culturing cells from a patient,
  • Therapeutics may be screened using cells of a tumor or malignant cell line. Many assays standard in the art can be used to assess such survival and/or growth; for example, cell proliferation can be assayed by measuring H-thymidine incorporation, by direct cell count, by detecting changes in transcriptional activity of known genes such as proto-oncogenes (e.g., fos, myc) or cell cycle markers; cell viability can be assessed by trypan blue staining, differentiation can be assessed visually based on changes in morphology, etc.
  • a Therapeutic of the invention is screened for activity to modulate (e.g., promote, inhibit or antagonize) cdc2 levels and/or activity.
  • cdc2 protein and mRNA and cdc2 activity can be determined by any method well known in the art.
  • cdc2 protein can be quantitated by known immunodiagnostic methods such as western blotting immunoprecipitation using any antibody against cdc2 (for example, anti-cdc2 antibodies are commercially available from Santa Cruz Inc.)
  • Cdc2 mRNA can be quantitated by methods that are well known and routine in the art, for example by northern analysis, RNase protection, the polymerase chain reaction in connection with the reverse transcription, etc.
  • Cdc2 activity can also be assayed by any method known in the art, for example, by the histone-Hl kinase assay.
  • Compounds for use in therapy can be tested in suitable animal model systems prior to testing in humans, including but not limited to in rats, mice, chicken, cows, monkeys, rabbits, etc.
  • lats knock-out mice e.g., as described in the Examples, are used to test therapeutics of the invention for activity to treat or prevent cancers, or to modulate cdc2 activity.
  • the invention provides recombinant non-human animals in which one or more lats genes have been inactivated, e.g., "knock-out animals".
  • the recombinant non-human animal can be any animal, e.g., mouse, rats, rodents, hamster, sheep, pig, cow, Drosophila, C. elegans, insects, worms, primates, dogs, etc., and is preferably a mouse.
  • Such an animal can be generated by any method known in the art for disrupting a gene on the chromosome of an animal.
  • Lats knock-out animals do not include animals in which one or more lats genes have been inactivated by naturally occurring mutations.
  • a lats knock-out animal can be produced by promoting homologous recombination between a lats gene in its chromosome and an exogenous lats gene that has been rendered biologically inactive (preferably by insertion of a heterologous sequence, e.g., an antibiotic resistance gene).
  • Homologous recombination methods for disrupting genes in the mouse genome are described, for example, in Capecchi (1989, Science 244: 1288-1292) and Mansour et al. (1988, Nature 336:348-352).
  • a lats knock-out mouse may be produced by the method described in the Examples section.
  • a lats genomic clone is isolated from genomic DNA from the same species as the knock-out animal.
  • the lats genomic clone can be isolated by any method known in the art for isolation of genomic clones (e.g., by probing a genomic library with a probe derived from a lats sequence, such as those sequences provided in Figures 12- 15, i.e., SEQ ID NOS:l, 3, 5, or 7).
  • a probe derived from a lats sequence such as those sequences provided in Figures 12- 15, i.e., SEQ ID NOS:l, 3, 5, or 7.
  • the portion of the clone introduced into the vector that contains at least a portion of an exon of the lats gene i.e., contains a lats protein coding sequence.
  • a sequence not homologous to the lats sequence preferably a positive selectable marker, such as a gene encoding an antibiotic resistance gene, is then introduced into the lats gene exon.
  • the selectable marker is preferably operably linked to a promoter, more preferably a constitutive promoter.
  • the non- homologous sequence is introduced anywhere in the lats coding sequence that will disrupt lats activity, e.g., at a position where point mutations or other mutations have been demonstrated to inactivate lats protein function.
  • the non-homologous sequence can be inserted for the coding sequence for the portion of the lats protein containing all or a portion of the kinase domain (e.g., the nucleotide sequence coding for at least 50, 100, 150, 200 or 250 amino acids of the kinase domain), the Lats C-terminal domain 1, the Lats C-terminal domain 2, and the Lats C-terminal domain 3, or, more preferably, for the sequence coding for the amino acids corresponding to 756 to 1 130 of human lats (as depicted in Figure 12 (SEQ ID NO:2) and as indicated in the alignment of human and mouse lats in Figure 6A).
  • the coding sequence for the portion of the lats protein containing all or a portion of the kinase domain e.g., the nucleotide sequence coding for at least 50, 100, 150, 200 or 250 amino acids of the kinase domain
  • the Lats C-terminal domain 1 e
  • the positive selectable marker is preferably a neomycin resistance gene (neo gene) or a hygromycin resistance gene (hygro gene).
  • the promoter may be any promoter known in the art; by way of example the promoter may be the phosphoglycerate kinase (PKG) promoter (Adra et al.,1987, Gene 60:65-74), the PolII promoter (Soriano et al., 1991. Cell 64:693-701), or the MCI promoter, which is a synthetic promoter designed for expression in embryo-derived stem cells (Thomas & Capecchi, 1987, Cell 51 :503-512).
  • a selectable marker such as an antibiotic resistance gene
  • the targeting vector for example, the expression of the neo gene product confers resistance to G418, and expression of the hygro gene product confers resistance to hygromycin.
  • a negative selectable marker for a counterselection step for homologous, as opposed to non-homologous, recombination of the vector is inserted outside of the lats genomic clone insert, e.g., as shown in Figure 6B.
  • a negative selectable marker is the HSV thymidine kinase gene (HSV-tk), the expression of which makes cells sensitive to ganciclovir.
  • HSV-tk HSV thymidine kinase gene
  • the negative selectable marker is preferably under the control of a promoter such as the PGK promoter, the PolII promoter or the MCI promoter.
  • the portions of the vector that are homologous to the lats gene, as well as the non-homologous insert within the lats gene sequences, are incorporated into the lats gene in the chromosome, and the remainder of the vector is lost.
  • the negative selectable marker is outside the region of homology with the lats gene, cells in which homologous recombination has occurred (or their progeny), will not contain the negative selectable marker.
  • the negative selectable marker is the HSV-tk gene, the cells in which homologous recombination has occurred will not express thymidine kinase and will survive exposure to ganciclovir.
  • the targeting vector is linearized with a restriction enzyme for which there is a unique site in the targeting vector, and the linearized vector is introduced into embryo-derived stem (ES) cells (Gossler et al., 1986, Proc. Natl. Acad. Sci. USA 83:9065-9069) by any method known in the art, for example by electroporation. If the targeting vector includes a positive selectable marker and a negative, counterselectable marker, the ES cells in which homologous recombination has occurred can be selected by incubation in selective media.
  • ES embryo-derived stem
  • the selectable markers are the neo resistance gene and the HSV-tk gene
  • the cells are exposed to G418 (e.g., approximately 300 ⁇ g/ml) and ganciclovir (e.g., approximately 2 ⁇ M).
  • any technique known in the art for genotyping for example Southern blot analysis or the polymerase chain reaction, can be used to confirm that the disrupted lats sequences have homologously recombined into the lats gene in the genome of the ES cells. Because the restriction map of the lats genomic clone is known (see Figure 6b) and the sequence of the lats coding sequence is known (see Figure 13), the size of a particular restriction fragment or a PCR amplification product generated from DNA from both the disrupted and non-disrupted alleles can be determined.
  • the ES cells with the disrupted lats locus can then be introduced into mouse blastocysts by microinjection and then the blastocysts can be implanted into the uteri of pseudopregnant mice using routine techniques.
  • the mice that develop from the implanted blastocysts are chimeric for the disrupted allele.
  • the chimeric male mice can be crossed to female mice, and this cross can be designed such that germline transmission of the allele is linked to transmission of a certain coat color.
  • the germline transmission of the allele can be confirmed by Southern blotting or PCR analysis, as described above, of genomic DNA isolated from tail samples.
  • Clones comprising lats nucleotide sequences, particularly lats genomic clones, can be isolated by any method known in the art.
  • the nucleotide sequences encoding, and the corresponding amino acid sequences of, human lats, mouse lats, mouse lats2 and Drosophila lats are provided in Figures 12-15, respectively (SEQ ID NOS:l-8, respectively) and bacterial cells containing the plasmid pBS(KS)-h-lats, which contains the gene encoding human lats, were deposited on March 24, 1995 with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2201, and assigned Accession No. 69769.
  • Lats nucleic acids can be obtained by any method known in the art, e.g., from the deposited plasmid, by the polymerase chain reaction (PCR) using synthetic primers hybridizable to the 3' and 5' ends of a lats nucleotide sequence and/or by cloning from a cDNA or genomic library using an oligonucleotide probe specific for the gene sequence, such as a probe from the lats gene insert in plasmid pBS(KS)-h-lats.
  • PCR polymerase chain reaction
  • Genomic clones can be identified by probing a genomic DNA library under appropriate hybridization conditions, e.g., high stringency conditions, low stringency conditions or moderate stringency conditions, depending on the relatedness of the probe to the genomic DNA being probed. For example, if the lats probe and the genomic DNA are from the same species, then high stringency hybridization conditions may be used; however, if the lats probe and the genomic DNA are from different species, then low stringency hybridization conditions may be used. High, low and moderate stringency conditions are all well known in the art. Procedures for low stringency hybridization are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci.
  • Filters are incubated in hybridization mixture for 18-20 hours at 40°C, and then washed for 1.5 hours at 55°C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 hours at 60°C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68°C and reexposed to film.
  • Procedures for high stringency hybridizations are as follows: Prehybridization of filters containing DNA is carried out for 8 hours to overnight at 65°C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA. Filters are hybridized for 48 hours at 65°C in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X 10 6 cpm of 32 P-labeled probe.
  • Washing of filters is done at 37°C for 1 hour in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1 X SSC at 50°C for 45 minutes before autoradiography.
  • Moderate stringency conditions for hybridization are as follows: Filters containing DNA are pretreated for 6 hours at 55°C in a solution containing 6X SSC, 5X Denhardt's solution, 0.5%) SDS, and 100 ⁇ g/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution and 5-20 X 10 cmp 32 P-labeled probe is used. Filters are incubated in the hybridization mixture for 18-20 hours at 55°C, and then washed twice for 30 minutes at 60°C in a solution containing 1 X SSC and 0.1% SDS.
  • any eukaryotic cell potentially can serve as the nucleic acid source for the molecular cloning of the lats gene.
  • the nucleic acid sequences encoding lats can be isolated from vertebrate, mammalian, human, porcine, bovine, feline, avian, equine, canine, as well as additional primate sources, insects, etc.
  • the DNA may be obtained by standard procedures known in the art, preferably from cloned genomic DNA (e.g., a DNA "library”) from the desired cell (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II.).
  • the gene should be molecularly cloned into a suitable vector for propagation of the gene.
  • the genomic clone used to generate a recombinant, non- human animal by homologous recombination contains at least a portion of the lats coding sequence of SEQ ID NO:3; alternatively, the genomic clone contains at least a portion of the lats coding sequence of SEQ ID NO:5.
  • the invention provides methods for screening for compounds useful in the treatment or prevention of cancer or in the treatment and prevention of pituitary diseases and disorders by administration or application of the compound to be tested to a lats knock-out animal, preferably a lats knock-out mouse.
  • the invention provides a method for screening a potential therapeutic compound for activity in treating or preventing cancer.
  • the potential therapeutic compound is administered to a recombinant non-human animal having at least one inactivated lats gene (i.e., a lats knock-out animal, preferably a lats knock-out mouse), preferably two inactivated lats gene lats genes (i.e., is homozygous for the inactivated lats allele).
  • the size or progression of the cancer is then compared to that before the compound was added, or to a comparable recombinant animal without the administration of the compound, or to a normal, non-recombinant animal.
  • a decrease in the size or progression of the cancer in the recombinant non-human animal after the administration of the compound as compared to the same animal prior to the administration or to another recombinant non- human animal not so administered or the standard size or progression of the cancer indicates that the compound has activity in treating or preventing cancer
  • the screening method of the invention can be used to screen for potential therapeutic compounds for the treatment or prevention of any cancer, preferably a cancer or neoplastic disease that is caused by the lats knock-out mutation.
  • lats knock-out mice are susceptible to ovarian stromal tumors and soft tissue sarcomas that metastasize to vital organs.
  • the invention provides methods for screening compounds useful in treating or preventing ovarian tumors and soft tissue sarcomas.
  • Lats knock-out mutations in other animals or in other lats homologs may make the resulting knock-out animal susceptible to other types of neoplastic disease.
  • the invention also contemplates use of these other lats knock-out animals to screen compounds for efficacy in treating or preventing the types of neoplastic diseases found in these lats knock-out animals. Additionally, compounds effective to treat or prevent ovarian tumors and or soft tissue sarcomas in lats knock-out animals may also be effective to treat or prevent other types of cancers and neoplastic disease. Thus, lats knock-out animals may be used to screen for compounds that have activity to treat or prevent these other types of cancers and neoplastic disease.
  • the invention also provides methods of screening compounds for efficacy in treating or preventing skin cancer.
  • infra exposure to carcinogens induced, at a high frequency, skin tumors in the lats knock-out mice.
  • Many methods are known in the art for inducing skin carcinogenesis in animals (for review see DiGiovanni, 1992, Pharmac. Ther. 54:63-128).
  • mouse skin tumors can be elicited by application of a carcinogenic dose of tumor initiator, e.g., 600 to 800 nmole of a pure polycyclic aromatic hydrocarbon such as 9,10-dimethyl-l,2-benzanthracene (DMBA).
  • DMBA 9,10-dimethyl-l,2-benzanthracene
  • tumor initiators include, but are not limited to, arylamines, carbamates, haloalkylethers, haloaromatics, lactones, nitro-aromatics, nitrosamides and ureas.
  • mouse skin tumors can be induced by an initial application of a single sub-carcinogenic dose of a tumor initiator, e.g., DMBA, and then repeated doses or exposures to a tumor promoter, such as phorbolesters (e.g. , TPA), teleocidins, polyacetates, okadaic acid, calyculin A, palytoxin, and thapsigargin.
  • phorbolesters e.g. , TPA
  • UVB Ultraviolet B
  • such skin tumors are induced by a two-step process comprising a single treatment with DMBA, preferably 50 ⁇ l of a 0.5%> DMBA solution in acetone, to the dorsal surface of the mouse 1 to 5 days after birth followed by repeated exposure to UVB irradiation, e.g., exposures of approximately three times per week with an initial exposure of approximately 100 mJ/cm 2 per session, increasing the dosage by 10%o per treatment (unless erythema or scaling occurs) to a maximum of 700 mJ/cm2, with an average of about 27 treatment sessions per mouse (Serrano et al., 1996, Cell 85:27-37).
  • DMBA preferably 50 ⁇ l of a 0.5%> DMBA solution in acetone
  • the invention provides a method for screening a potential therapeutic compound for activity in treating or preventing skin cancer comprising administering the compound to a recombinant non-human animal in which one, preferably two, lats genes have been inactivated (i.e., a lats knock-out animal) and in which recombinant non-human animal tumors have been induced by exposure to at lease one carcinogen.
  • a recombinant non-human animal in which one, preferably two, lats genes have been inactivated (i.e., a lats knock-out animal) and in which recombinant non-human animal tumors have been induced by exposure to at lease one carcinogen.
  • the size or progression of the skin tumors are then compared before and after the administration of the compound.
  • the compound to be screened is administered by recombinantly expressing the compound in the recombinant non-human animal inactivated for the lats gene.
  • the administration of the compound to be tested can be carried out by any method known in the art, e.g., orally, intravenously, intramuscularly, intraperitoneally, subcutaneously, rectally, topically, etc.
  • the compound is preferably applied topically.
  • the tumors, sarcomas, and other cancers can be evaluated by any diagnostic or histopathological method for detecting and evaluating tumors and cancers, for example, by visual inspection of the tumors (particularly for skin tumors), manual palpitation of tumors, biopsy or surgical removal of the tumor tissue and subsequent inspection, and sacrifice and dissection of the recombinant non- human animal.
  • Morphological evaluation of tissue, either removed by biopsy or dissected from a sacrificed mouse may be performed by fixing the tissue by any method known in the art, for example, in 10%o neutral buffered formalin at 4°C, and subsequent dehydration, e.g., in ethanol.
  • the fixed and dehydrated tissue may be embedded in paraffin and then sectioned, for example into 4-5 mm sections by any method known in the art. Sections can be stained, for example, with a standard stain, such as hematoxylin and eosin, for microscopic inspection.
  • a standard stain such as hematoxylin and eosin
  • Another aspect of the invention provides methods for screening potential therapeutic compounds for efficacy in treating or preventing diseases or disorders associated with pituitary dysfunction.
  • Lats knock-out mice display a number of consequences of pituitary dysfunction, as described in the Examples section, infra.
  • the methods of the invention can be used to screen compounds for efficacy in treating or preventing such pituitary dysfunctions as pituitary hyperplasia, fertility defects, such as defective ovulation, lack of breast development, abnormal reproductive cycles in females, LH hypogonadotropic hypogonadism, reduced levels of pituitary hormones, specifically LH, GH and PRL, and reduced growth and metabolic abnormalities caused by reduced GH levels.
  • Therapeutics that are effective to treat one or more of these conditions associated with pituitary dysfunction may also be effective to treat or prevent other conditions, diseases or disorders associated with pituitary dysfunction.
  • potential therapeutic compounds to be screened for activity in treating or preventing diseases and disorders associated with pituitary dysfunction are administered to a recombinant non-human animal in which one or more chromosomal copies of the lats gene have been inactivated (i.e., a lats knock-out animal, preferably and lats knock-out mouse). Levels of an indicator of pituitary function or dysfunction are then compared in the recombinant non-human animal before and after the compound was administered.
  • the compound to be screened is administered by recombinantly expressing the compound in the recombinant non-human animal having an inactivated lats gene.
  • Indicators of pituitary function include fertility, ovulation, the female reproductive cycle (e.g., the estrus cycle), breast tissue development, growth or size of the animal, including weight, skeletal size, e.g., of the skull and/or longitudinal bones, and organ weight, and serum levels of LH, GH and PRL.
  • fertility may be evaluated by attempting to mate an animal and determining whether conception occurred, measuring sperm count in male animals or detecting ovulation in female animals.
  • the reproductive organ tissue may also be examined histopathologically (e.g.
  • morphological defects particularly in the testis, ovaries, and breast tissue.
  • Whether the animal goes through an estrus cycle may be determined by observation of the animal.
  • Hormone levels may be determined by any method known in the art, for example in serum samples by radio immunoassay using antibodies specific for the particular hormone. Lack of normal growth can be determined by measuring the animal e.g., the weight, size of the skull and/or longitudinal bones, or organ weight, during maturation.
  • Candidate therapeutics may come from any source of therapeutics known in the art.
  • these therapeutics can be proteins, nucleic acids (including anti-sense nucleic acids), antibodies, peptides, organic molecules, etc.
  • compounds may be screened first in in vitro assays to determine their potential as anti-cancer or anti-pituitary
  • chemical libraries may be screened for useful therapeutics.
  • Exemplary libraries are commercially available from several sources (ArQule, Tripos/PanLabs, ChemDesign, Pharmacopoeia). Many diversity libraries suitable for use are known in the art and can be used to provide compounds to be tested according to the present invention.
  • libraries can be constructed using standard methods.
  • Chemical (synthetic) libraries (Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski, 1994, Bio/Technology 12:709-710; Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251), recombinant expression libraries, or polysome-based libraries are exemplary types of libraries that can be used.
  • Other examples include combinatorial libraries (Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al.,
  • Non-naturally occurring amino acids can also be used.
  • Peptoids are polymers of non-natural amino acids that have naturally occurring side chains attached not to the alpha carbon but to the backbone amino nitrogen. Since peptoids are not easily degraded by human digestive enzymes, they are advantageously more easily adaptable to drug use. 35 Therapeutic/Prophylactic Administration and Compositions
  • the invention provides methods of treatment (and prophylaxis) by administration to a subject of an effective amount of a Therapeutic of the invention.
  • the Therapeutic is substantially purified.
  • the subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human. In a specific embodiment, a non-human mammal is the subject.
  • a Therapeutic of the invention e.g., encapsulation in liposomes (Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989)), microparticles, microcapsules, recombinant cells capable of expressing the Therapeutic, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a Therapeutic nucleic acid as part of a retroviral or other vector, etc.
  • Methods of introduction include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see
  • nucleic acid Therapeutic can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
  • compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the Therapeutic is administered.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W.
  • compositions will contain a therapeutically effective amount of the
  • Therapeutic preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the amount of the Therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight.
  • Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Lats nucleic acids, proteins, and derivatives may be used in screening assays to detect molecules that specifically bind to lats nucleic acids, proteins, or derivatives and thus have potential use as agonists or antagonists of lats, in particular, molecules that thus affect cell proliferation and/or cdc2 activity, or molecules that promote or inhibit formation of lats-cdc2 complexes.
  • such assays are performed to screen for molecules with potential utility as lead compounds for drug development, particularly as anti-cancer drugs.
  • the invention thus provides assays to detect molecules that specifically bind to lats nucleic acids, proteins, or derivatives or bind to or interfere with the formation of lats-cdc2 complexes.
  • recombinant cells expressing lats nucleic acids can be used to recombinantly produce lats proteins in these assays, to screen for molecules that bind to a lats protein
  • recombinant cells expressing lats and cdc2 nucleic acids can be used to recombinant produce both lats and cdc2 proteins in these assays, to screen for molecules that bind to or inhibit formation of a lats-cdc2 complex.
  • Molecules e.g., putative binding partners of lats
  • Molecules are contacted with the lats protein (or fragment thereof) under conditions conducive to binding, and then molecules that specifically bind to the lats protein or bind to or interfere with the formation of lats-cdc2 complexes are identified. Similar methods can be used to screen for molecules that bind to lats derivatives or nucleic acids. Methods that can be used to carry out the foregoing are commonly known in the art.
  • diversity libraries such as random or combinatorial peptide or nonpeptide libraries can be screened for molecules that specifically bind to lats.
  • libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phage display libraries), and in vitro translation-based libraries.
  • phage display libraries are described in Scott and Smith, 1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian, R.B., et al.,
  • In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058 dated April 18, 1991 ; and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91 :9022-9026.
  • a benzodiazepine library (see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91 :4708-4712) can be adapted for use.
  • Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can also be used.
  • Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA 91 :11138-1 1142).
  • Screening the libraries can be accomplished by any of a variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries:
  • screening can be carried out by contacting the library members with a lats protein (or nucleic acid or derivative) immobilized on a solid phase and harvesting those library members that bind to the protein (or nucleic acid or derivative).
  • lats protein or nucleic acid or derivative
  • harvesting those library members that bind to the protein (or nucleic acid or derivative).
  • the two-hybrid system for selecting interacting proteins in yeast can be used to identify molecules that specifically bind to a lats protein or derivative or that interfere with the formation of lats-cdc2 complexes.
  • the two hybrid system or co-immunoprecipitation of lats and cdc2 can be used as assays to screen for compounds that promote or inhibit formation of lats-cdc2 complexes.
  • the invention provides a method of screening for a molecule that modulates (i.e., inhibits, antagonizes or promotes) directly or indirectly, the formation of a complex of lats and cdc2 proteins comprising measuring the levels of said complex formed from lats and cdc2 proteins in the presence of said molecule (optionally, purified) under conditions conducive to formation of the complex; and comparing the levels of said complex with the levels of said complex that are formed in the absence of said molecule, lower or higher level of said complex in the presence of said molecule indicates that the molecule modulates formation of said complex.
  • lats is phosphorylated in a cell cycle-dependent manner and that it complexes with cdc2 in early mitosis.
  • Lats associated cdc2 has no mitotic cyclin partner and no kinase activity for histone HI.
  • lats mutant cells in Drosophila abnormally accumulate cyclin A.
  • h-lats cDNA was cloned into the vector pBacPAK8 and baculovirus were produced according to the protocols provided by Oontech.
  • IPLB-Sf21 cells were co-infected with equal amounts of h-lats and human cdc2- baculoviruses and were harvested 62 hours after infection for immunoprecipitation and immunoblot assay.
  • h-lats cDNA was cloned into the vector pCaSpeR-hs (Tummel and Pirrott, 1992, Drosophila Information Service 71 : 150). Multiple transformant lines were obtained and used in rescue experiments with lats ⁇ 32 , lats e26' ' , lats"', and lats x ⁇ alleles. Expression of hs-h-lats was induced as described in Xu et al. (1995, Development 121:1053-1063) — incubation at 37°C for one hour every day until eclosure.
  • cdc2c EI36E (a gift of Helena Richardson), cycA' wo " 4 , Df(2R)59A-B were used for cdc2c, cyclin A and cyclin B, respectively.
  • HeLa cells were synchronized at different cell cycle stages by va ⁇ ous treatments as described by Knehr et al.(1995, Exp. Cell. Res. 217:546-553). Briefly, cells were arrested at Gl by thymidine and hydroxyurea treatment; at S phase by thymidine double block
  • CHO cells were harvested at various time points after removal of nocodazole (herein "ARN") for further analysis.
  • CHO cells were grown in a-MEM medium plus 7% FBS and IPLB-SG1 cells were grown in sf-900 II SFM plus 10% FBS.
  • ARN nocodazole
  • Anti-human lats rat monoclonal and rabbit polyclonal antibodies were raised against a GST-N-h-Lats (GST fused to the N-terminal portion of lats, i.e., consisting of amino acids 15-585 of the human lats amino acid sequence as depicted in Figure 12 (SEQ ID NO:2) fusion protein.
  • Anti-human cdc2 (#sc 054), anti-human cyclin B (#sc 245), anti-human cyclin A (#sc 239) monoclonal antibodies were purchased from Santa Cruz Inc. Rabbit polyclonal an -Drosophila Cyclin A and B antibodies were gifts of David Glover.
  • Monoclonal mouse anti-BrdU antibodies (#347580) were purchased from Becton Dickinson and monoclonal mouse ant-c-myc antibodies (#OP 10) were purchased from Oncogene Sciences. Propidium iodide (Sigma) was used as a DNA marker. HeLa, CHO, or IPLB Sf21 cells were lysed in TG buffer (1% Triton, 10% glycerol)
  • HeLa cell lysates (50 minutes ARN) were precleaned by incubation in protein G- agarose. Immunoprecipitates were washed three times with TG buffer and twice with IX kinase buffer (50 mM Tris-HCl 7.5, 10 mM MgC12, 5 mM EGTA, 2 mM DTT) without DDT. The kinase assay was carried out on ice for 10 minutes in 35 ⁇ l of IX kinase buffer containing 15 ⁇ Ci of ⁇ - 32 P ATP, 1.6 ⁇ g of histone HI, and 1.5 ⁇ M ATP.
  • IX kinase buffer 50 mM Tris-HCl 7.5, 10 mM MgC12, 5 mM EGTA, 2 mM DTT
  • the kinase activities were measured by quantifying the intensities of histone-Hl phosphorylation using a Phosphorlmager (Molecular Dynamics).
  • the amounts of cdc2 in the immunoprecipitates were determined by anti-cdc2 immunoblotting and densitometer scanning (Molecular Dynamics).
  • the kinase assay experiments were repeated three times.
  • Lats is phosphorylated in a cell cycle-dependent manner
  • Lats immunoprecipitated from cells at different mitotic stages displayed varying amounts of the two forms (compare lanes 1 and 6 of Figure 2 A), suggesting that the phosphorylation state of lats may oscillate with the cell-cycle.
  • lats proteins were immunoprecipitated from extracts of HeLa cells at GO, Gl, S, and G2 phases, and different time points during mitosis (minutes after removal of nocodazole (ARN) block) (Knehr, et al., 1995, Exp. Cell Res. 217:546-553).
  • DAPI staining was used to verify the cell cycle progression.
  • Lats mutant cells in Drosophila mosaic for the lats mutation do differentiate, indicating that mutations in lats do not block cellular differentiation in general ( Figures 1G and H).
  • the lats mutant ove ⁇ roliferation phenotype and cell cycle-dependent phosphorylation of lats suggest that the protein could be directly involved in the regulation of the cell cycle.
  • Lats/cdc2 and cdc2/cyclin B complexes were immunoprecipitated separately from 50 minute ARN HeLa cell extracts using either anti-human lats or anti-cyclin B monoclonal antibodies and assayed for histone HI kinase activities.
  • the lats/cdc2 complex showed no detectable kinase activity for histone HI ( Figure 3D).
  • Densitometer readings indicated that the HI kinase activity of the lats/cdc2 complex does not differ from the background control and is at least 25 fold lower than the kinase activity of the cdc2/cyclin B complex. These results indicate that cdc2 molecules associated with lats are inactive or have dramatically reduced mitotic kinase activity.
  • the lack of HI kinase activity in the lats-associated cdc2 could be due to the inhibition of the kinase activity of the cdc2/cyclin complex by lats.
  • the lats/cdc2 complex may lack cyclin A and B which are the indispensable subunits for cdc2 kinase activity (Draetta et al., 1989, Cell 56:829-838; Solomon et al., 1990, Cell 63:1013- 1024).
  • cdc2 In Drosophila, cdc2 also complexes with cyclin A or B (Knobuz et al., 1994, Cell 77:107-120). We examined the potential genetic interactions between lats, cdc2, cyclin A and cyclin B in Drosophila. Animals heterozygous for the strong cdc2 allele, cdc2 B4? , or homozygous for the temperature sensitive cdc2' s mutation at permissive temperature are viable and mo ⁇ hologically normal (Clegg et al., 1993, Genome 36:676-685).
  • the lats PS mutation causes late pupal lethality in homozygous mutants ( Figure 4A), and reducing cdc2 activity in lats P8 homozygotes by introducing one copy of a cdc2 mutant allele (cdc2 B4? or cdc2'7+; lats P8 /lats P8 ) was sufficient to rescue the lats-associated lethality ( Figure 4B). Furthermore, the ove ⁇ roliferation phenotype of lats ps adult appendages were also suppressed. Rescued animals had near-wild type eyes in comparison to the ove ⁇ roliferated, large, rough eyes of the lats P8 mutants ( Figures 4C and D).
  • cyclin A and B are degraded when the cdc2/cyclin complexes are inactivated (Draetta et al., 1989, Cell 56:829-828; Murray et al, 1989, Nature 339:280-286; King et al., 1994, Cell 79:563- 571).
  • cyclin A and B are detected in cells anterior to the mo ⁇ hogenetic furrow (MF) as well as in a stripe of cells posterior to the MF which are undergoing the last round of cell division (the second mitotic wave) (Thomas et al., 1994, Cell 77:1003-1014).
  • anti-cyclin A or B antibody staining did not detect any obvious changes in levels of the two proteins ( Figures 5A-N; Whitfield et al., 1990, EMBO J.
  • the lats molecules are a novel family of conserved proteins
  • lats kinase domain contains all 1 1 subdomains previously found in other protein kinases (Hanks et al., 1988, Science 241 :42-45), suggesting that it is an active protein kinase.
  • lats alone and lats/cdc2 complex do not appear to have any autophosphorylation activity or phosphorylation activity for cdc2 and histone HI .
  • Yeast two-hybrid experiments showed that the N-terminal region of lats interacted with cdc2 much more strongly than did full-length lats (Figure 3E).
  • the ove ⁇ roliferation phenotype of lats behaves in a cell autonomous fashion: inactivating lats causes mutant cells to ove ⁇ roliferate (Xu et al., 1995, Development 121 :1053-1063). Furthermore, in mosaic discs containing lats mutant clones, there is an ove ⁇ roliferation of lats mutant cells as well as a reduction in the number of wild type cell. These observations are consistent with a regulatory mechanism where lats mutant cells are able to send signals inhibiting cell proliferation but are defective in receiving such signals.
  • cdc2/cyclin A functions at the Gl/S phase transition in addition to the G2/M phase transition.
  • Ectopic activation of cdc2/cyclin A by overexpressing cyclin A in Gl arrested cells can drive the Gl/S transition and induce S phase in cells lacking cyclin E (Dong et al., 1997, Genes & Devel. 11:94-105; Sprenger et al., 1997, Curr. Biol. 7:488- 499).
  • lats mutants from genetic alterations that affect multiple CDK cyclin complexes or that abnormally activate CDK/cyclin at a single cell-cycle stage or throughout the entire cell cycle, and provide an explanation for the extensive ove ⁇ roliferation phenotype of the Drosophila lats mutants.
  • the data provided herein indicate that cdc2/cyclin A activity is negatively regulated by the lats protein.
  • Yeast two-hybrid assays show that lats specifically interacts with cdc2 but not other CDKs.
  • Genetic data in Drosophila also show that lats interacts with cdc2 but not the fly CDK2 homolog, cdc2c.
  • cdc2 and the rest of the CDKs are negatively regulated by different families of proteins.
  • the activity of each CDK could be modulated by both types of negative regulators.
  • cdc2/cyclin A is inactivated during early mitosis by degradation of cyclin A, while degradation of cyclin B occurs later at the metaphase/anaphase transition (Minshull et al., 1990, EMBO J. 9:2865- 2875; Whitfield et al., 1990, EMBO J. 9:2563-2572).
  • the mechanism of such differential inactivation of cdc2/cyclin is unknown.
  • EXAMPLE 2 Mice Deficient for Lats Develop Soft Tissue Sarcomas, Ovarian Tumors and Pituitary Dysfunction Materials and Methods
  • Mouse lats genomic DNA was isolated by screening a 129 library (Stratagene) using a mouse lats cDNA as a probe. A Sail fragment from the cDNA was subcloned into a pBS vector. We cleaved this construct at the EcoRV site ( Figure 6B), and inserted a 1.8 kb fragment encoding PGK-neo. We subsequently digested with BamHI and Xhol and inserted a 3 kb PGK-TK gene cassette.
  • D3 embryonic stem (ES) cells were electroporated with the Sfil linearized vector, and selected in 0.3 mg/ml G418 and 2 ⁇ M ganciclovir media for inco ⁇ oration of the vector.
  • genomic DNA from the ES cells was digested with BamHI and EcoRV and analyzed by Southern blotting using the BamHI-EcoRI probe from the vector ( Figure 6C).
  • the double digest of the wild type allele generates a 3.5 kb fragment that hybridizes to the probe, while double digest of the disrupted allele generates a 5.8 kb fragment that hybridizes to the probe.
  • Lats heterozygous ES cells were microinjected into CS7BL/6 blastocysts which were transplanted into uteri of pseudopregnant ICR mice. Chimeric male progeny were crossed to CS7BL/6 females. Germline transmission of the disrupted allele was detected in agouti progeny by Southern blotting.
  • Proteins were extracted from whole-cell lysates of lats' ' mouse embryonic fibroblasts (MEFs) derived from 13-days post-coitum mouse embryos, separated using SDS-PAGE, transferred and probed with rabbit polyclonal anti-lats antibody, followed by enhanced chemiluminescence detection (Amersham). Histopathological examinations
  • tissues were fixed in 10% neutral buffered formalin at 4°C overnight, dehydrated with ethanol, embedded in paraffin, and sectioned into 4 to 5 mm sections. Paraffin sections were prepared by standard procedures and stained with hematoxylin and eosin.
  • mice were injected intraperitoneally with FSH administered in the form of 5 IU of pregnant mare serum gonadotropin (Sigma). 44-46 hours later, mice were injected intraperitoneally with LH in the form of 5 IU of human chorionic gonadotropin (Sigma).
  • mice were used 20 lats ' ' and 20 lats ⁇ ' age, sex, and estrus cycle matched females and males for these analyses.
  • Mouse serum levels of PRL, LH, GH, FSH, and TSH were determined in pooled serum samples by double antibody radioimmunoassays (RIAs). These sensitive, specific mouse pituitary hormone RIAs were developed by A. F. Parlow, and are distributed to the scientific research community via the National Hormone & Pituitary Program of NIDDK, NIH (see http://www.humc.edu/hormones).
  • UVB and DMBA treatments were performed as described by Serrano et al. (1996, Cell 85:27-37). Briefly, skin tumors were induced by first applying a single dose of 9,10- dimethyl-l,2-benzathralene (DMBA; 50 ⁇ l of an 0.5% solution in acetone) to the dorsal surface of the mouse 1 to 5 days after birth. This treatment was followed by exposure to ultraviolet B (UVB) irradiation approximately three times per week for, on average, 27 treatments, with an initial exposure of approximately 100 mJ/cm2, increasing the dosage by
  • UVB ultraviolet B
  • a 17.5 kilobase lats genomic clone obtained from a mouse 1295 V library was used to construct a targeting vector for homologous recombination by positive-negative selection (Mansour et al., 1988, Nature 336:348-352; Capecchi, 1989, Science 244:1288-1292) as shown in Figure 6B.
  • a PGK-neo cassette was inserted in inverse orientation into an exon of the lats clone resulting in the removal of amino acid sequence corresponding to amino acids 756-1130 of human lats ( Figure 6A).
  • We electroporated D3 embryonic stem cells (Gossler et al., 1986, Proc. Natl. Acad.
  • mice Male lats ' ' ' mice displayed decreased fertility although histopathological examination of the testis did not reveal obvious structural abnormalities. Lats ' ' ' females all displayed severe fertility defects, and approximately 60% of the females were completely sterile. Ovaries from all lats deficient females examined contained far fewer follicles than age and parity matched ovaries from lats " ' + females ( Figures 8A-D). The majority of follicles observed were primary and secondary follicles. Formation of the antrum was much less prominent than in normal mice. The follicles also contained fewer degenerative granulosa cells, which are common in atretic follicles in normal mice.
  • Estrus is another indicator of endocrine function. Vaginal smears taken from control ( + ⁇ ) mice showed that they cycled through proestrus, estrus, metestrus, and diestrus in 4 days as described previously for normal mice (Nelson et al., 1982, Biol. Reprod. 27:327- 339). In contrast, infertile lats' ' females did not cycle, and remained in continuous metestrus, an observation that further characterizes their infertility. The abnormal estrus cycle might reflect an underlying problem in signaling between the pituitary and the ovary.
  • the reduced serum GH level may contribute to the reduced size of lats' ' mice.
  • the diminished levels of serum LH could account for the lack of proper follicular maturation and differentiation, as well as the infertility observed in female lats ' ' ' animals, with greater atypia in the pituitary leading to the more severe phenotype.
  • the PRL and LH defects together, account for both the lack of mammary gland development and the co ⁇ us luteum insufficiency syndrome which these animals display.
  • serum levels of pituitary Follicle Stimulating Hormone (FSH) (Figure
  • Thyroid Stimulating Hormone Thyroid Stimulating Hormone
  • pituitary deficiencies of lats ' ' mice resembles those of other cell cycle regulator knock-out mice, such as the Rb ' , p53 ' , and p27 ' ' ' mice.
  • pituitary cells and other endocrine organs appear to be crucially dependent on cell cycle regulation for their proper development.
  • tumor suppressors may play such a key role in the pituitary because critical function in this tissue allows for a link between control of single cell proliferation and total organismal growth and survival.
  • stromal cell tumors are probably not resultant from pituitary dysfunction, as stromal cell tumors are most often local events (Clement, "Histology of the Ovary” in Histology For Pathologists, Second Ed., Steinberg, ed. (Lippincott-Raven, 1997) pp 934-935).
  • Some lats ' ' ' females were able to give birth to one litter, then became infertile as the stromal cell tumors expanded into the remaining functional ovary. To date, these stromal cell tumors have not yet displayed signs of malignancy.
  • lats- and p 16-knock-out mice are negative regulators of CDKs.
  • lats-knock-out mice resemble p 16-knock-out animals (Serrano et al., 1996, Cell 85:27-37) in that homozygotes develop tumors at an early age while heterozygotes do not.
  • different types of tumors are observed in these two mutants (e.g., ovarian tumors in lats' ' mice and lymphomas in pi 6 ' ' ' mice), both types of knock-out mice develop soft tissue sarcomas.

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Abstract

La présente invention concerne un animal humain non recombinant avec gène lats inactivé, en l'occurrence une souris. Comme les souris avec gène lats perturbé développent diverses tumeurs, risquent de développer des tumeurs de la peau en cas d'exposition à des agents cancérigènes et souffrent d'un dysfonctionnement de l'hypophyse, ces souris peuvent être utiles pour la recherche systématique de composés efficaces dans le traitement ou la prévention de cancers ou de troubles de l'hypophyse. On peut rechercher systématiquement des composés dont l'activité est utile dans le traitement ou la prévention de cancer de la peau chez des animaux non humains recombinants avec gène lats inactivé et chez lesquels des tumeurs de la peau ont été induites par exposition à des agents cancérigènes. L'invention porte également sur des méthodes de traitement dans le cas de cancers réfractaires à la chimiothérapie ou à la radiothérapie par action d'un agent thérapeutique faisant intervenir la fonction lats. Sont également décrites diverses méthodes de traitement de maladies ou de troubles associés à des niveaux anormaux d'activité cdc2 au moyen d'un agent thérapeutique qui soit favorise, soit inhibe, soit antagonise la fonction lats.
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