AU5781099A - Lats knock-out animal models and their uses - Google Patents

Description

WO 00/10602 PCTIUS99/19068 LATS KNOCK-OUT ANIMAL MODELS AND THEIR USES 5 This invention was made with Government support under Grant number NIH-NCI 1 R01 CA 69408 awarded by the National Institutes of Health. The Government has certain rights in the invention. RELATED APPLICATIONS 10 Priority is claimed to United States provisional application Serial Nos. 60/096,997 and 60/096,996, both filed on August 18, 1998, both of which are incorporated by reference herein in their entireties. FIELD OF THE INVENTION 15 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 20 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 25 provides methods of screening potential therapeutics for efficacy in the treatment and prevention of cancer and pituitary disorders using lats knock-out animals. BACKGROUND OF THE INVENTION Cancer 30 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. The term "malignant" generally means that the tumor can invade and destroy neighboring body structures and spread to distant sites to cause death (for review, 35 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 - 1- WO 00/10602 PCT/US99/19068 either ineffective or present serious side effects. Thus, there is a need for development of new drugs for the treatment of cancer. The Cell Cycle and Tumor Suppressors 5 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: GI (for Gap 1), 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 10 by a group of cyclin-dependent kinases (CDKs) (Elledge, 1996, Science 274:1664-1672; Nasmyth, 1996, Science 274:1643-1645). The kinase activities of CDKs require their positive subunits, the cyclins, and the activities of specific CDK/cyclin complexes are in turn positively and negatively regulated by phosphorylation events and CDK inhibitors (CKIs) (Hunter and Pines, 1995, Cell 80:225-236; Morgan, 1995, Nature 374:131-134). 15 While the specific CDKs, CDK2, CDK4 and CDK6, along with Cyclins D and E, regulate the progression from GI into S phase, cdc2, along with Cyclins A and B, regulate the cell cycle progression from GI into mitosis (Hunter and Pines, 1995, Cell 80:225-236). 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. 20 For example, p53 activates the transcription of the p21 (p2 1 WAFl/CIPI) 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, p16, is itself a potent tumor suppressor (Biggs and Kraft, 1995, J. Mol. Med. 73:509-514). Although multiple members of the p16 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 Currently, cancer therapy may involve surgery, chemotherapy and/or radiation 30 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. -2- WO 00/10602 PCT/US99/19068 With respect to chemotherapy, there are a variety of 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 5 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)). These 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. Other 10 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. Despite the availability of a variety of chemotherapeutic agents, chemotherapy has 15 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 20 resistance to the chemotherapeutic agents. In fact, 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. Thus, because of drug resistance, many cancers prove refractory to 25 standard chemotherapeutic treatment protocols. There is a significant need for alternative cancer treatments, particularly for treatment of cancer that has proved refractory to standard cancer treatments, such as surgery, radiation therapy, and chemotherapy. Pituitary Disorders The pituitary regulates numerous biological functions through its secretion of 30 different hormones. (For review see Frohman, "The Anterior Pituitary" in Cecil Textbook of Medicine, 18th Ed., Wyngaarden and Smith, eds. (W.B. Saunders Company, Philadelphia, 1988) pp. 1290-1305). In particular, the pituitary releases glycoprotein hormones, which 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. Disruption of 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 -3- WO 00/10602 PCT/US99/19068 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 5 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. 10 LATS 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 15 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 20 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). 25 Mouse and human homologs of the Drosophila lats have also been identified, and human lats was found to be down-regulated in a large number of human tumor cell lines. The nucleotide and amino acid sequences of human lats (h-lats), mouse lats (m-lats), mouse lats2 (m-lats2) and Drosophila lats are provided herein in Figures 12-15, respectively (SEQ ID NOS:1-8, respectively), and are described in PCT Publication WO 96/30402, published October 3, 1996, which is incorporated by reference herein in its entirety. 30 Citation of references hereinabove shall not be construed as an admission that such references are prior art to the present invention. SUMMARY OF THE INVENTION The present invention relates to therapeutic and prophylactic methods and 35 compositions for the treatment and prevention of cancers based on lats proteins, and therapeutically and/or prophylactically effective analogs and fragments of lats protein. This is due to the fact that although most tumor suppressor genes regulate the Gl/S phase of the -4- WO 00/10602 PCT/US99/19068 cell cycle, 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 5 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. 10 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. In another aspect, the invention provides compositions and methods of production of complexes of lats and cdc2 proteins ("lats-cdc2 complexes"), including complexes of lats 15 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 phosphorylated form of lats complexes with cdc2. Accordingly, in a preferred embodiment, the lats-cdc2 complexes contain phosphorylated lats protein, specifically lats protein phosphorylated on a shrine or threonine residue within 20 residues upstream of an 20 Ala-Pro-Glu consensus in subdomain eight of a lats kinase domain, e.g., corresponding to seine 909 of human lats, as depicted in Figure 12 (SEQ ID NO:2). Alternatively, the lats protein in the lats-cdc2 complex has a glutamate or aspartate residue substituted for a seine 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 seine 909 of human lats, as 25 depicted in Figure 12 (SEQ ID NO:2). In another embodiment, 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 30 cdc2 complexes, and antibodies against lats-cdc2 complexes. In particular, 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 35 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. -5 - WO 00/10602 PCT/US99/19068 In a preferred embodiment, 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 (LCD 1), the Lats C-terminal domain 2 (LCD2), the Lats C-terminal domain 3 5 (LCD3), and all or a portion of the kinase domain, and retains the Lats flanking domain (LFD), the Lats split domain 1 (LSD 1), 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). In other embodiments, the inactivated lats gene is deleted for all or a portion of the 10 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 15 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. In a preferred embodiment, 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 20 animal, preferably a lats knock-out mouse, in which skin tumors have been induced with carcinogens. Definitions As used herein, underscoring or italicizing the name of a gene shall indicate the 25 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. For example, "lats" shall mean the lats gene, whereas "lats" shall indicate the protein product of the lats gene. ARN = After Removal of Nocodazole CDK = Cyclin Dependent Kinase mlats = mouse lats 30 BRIEF DESCRIPTION OF THE FIGURES Figures IA-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 35 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). (C, D) High magnification views of the -6- WO 00/10602 PCT/US99/19068 flies in panel (B) show yellow bristles (white arrows), indicating lats cells (genetically marked by the yellow mutation) were produced and that they have developed into normal structures. (E, F) lats" 26 ' homozygous mutants display giant pupae and disc overproliferation phenotypes (left pupae in panels). Without heat-shock induction, the 5 leaky expression of the hs-h-lats transgene partially suppressed the lats" 2 6 ' phenotypes (middle pupae in panels). Daily induction of the hs-h-lats gene completely rescued the mutant phenotypes (right pupae in panels). (G) 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. 10 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. "CIP" indicates that the cells were incubated in calf intestinal phosphatase and "B-gp" indicates that the cells were incubated in B-glycerol phosphate. The "Time (min.)" indicates 15 time in minutes of incubation. "D-h-Lats" and "h-Lats" indicate the phosphorylated and dephosphorylated forms of h-lats, respectively. Lats proteins from mitotic HeLa cell lysates (50 min. ARN (After Removal of Nocodazole)) display a slow-migrating form on SDS PAGE (6%) (lane 1). The proteins are converted into a fast-migrating form when incubated with Calf Intestinal Phosphatase ("CIP") (lanes 2-4). When both CIP and a CIP inhibitor, B-glycerol phosphate ("B-gp"), are present, their mobility remains unchanged (lane 5). Lats 20 proteins from 125 min ARN cells have both the slow-migrating and fast-migrating forms (lane 6) and CIP-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, G1, 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 25 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. Figures 3A-E. Lats directly complexes with cdc2 during mitosis and the lats/cdc2 30 complex is inactive for HI kinase activity. (A) 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 8% SDS-PAGE. The western blot was sequentially probed with anti-human cdc2 -7- WO 00/10602 PCT/US99/19068 monoclonal antibodies (lower panel, labeled "Cdc2"), anti-h-lats monoclonal antibodies (upper panel, labeled "h-lats"), anti-human cyclin B and cyclin A monoclonal antibodies (middle panels, labeled "Cyclin B" and "Cyclin A", respectively). (C) Co immunoprecipitation of baculovirus-expressed human lats and cdc2 proteins. H-lats 5 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"). (D) Lats-associated cdc2 is inactive for HI kinase activity. Cdc2 proteins co-immunoprecipitated with either lats or cyclin B (indicated by "+" in the legend labeled "anti-Cyclin B" or "anti-h-lats") from 50 min. ARN 10 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. As a control, Protein G-agarose beads incubated with equal amounts of cell lysates only were also used for the H1 kinase assay (indicated by "-" in the legend). (E) Summarized are the results of yeast two-hybrid assays for interactions among full-length h 15 lats (h-lats), N-terminal region of h-lats (N-h-lats), C-terminal region of h-lats (C-h-lats), human cdc2, CDK2, and CDK4. +++, ++, +: indicate strong, intermediate, and weak interactions, respectively, while - and ND indicate no interaction and not determined, respectively. Figures 4A-F. Genetic interaction between lats, cdc2, and cyclin A in Drosophila. (A) lats/ 8 /latsP 8 homozygotes die at the pupal stage. (B) Removal of one copy of the cdc2 20 gene rescues lats' 8 lethality (lats" 8 /latsP 8 ; +/cdc2B 47 ). (C) A typical rough, overproliferated eye dissected from a lats' 8 /latsp" dead pupa. (D) An eye from the fly in panel (B), showing that the eye phenotype has been almost completely suppressed. (E) lats 2 6 I/latse 26 pupae (the pupa labeled 1) are much larger than wild-type (the pupa labeled 4). The giant-pupa phenotype is partially reduced by a cdc2 temperature sensitive mutant at room temperature (pupa labeled 2) (lats" 2 6 1 /latse 2 6 '; cdc2 EI-24 /cdc2 El-24 ) or by removal of one copy of the 25 cdc2 gene ([i[a labeled 3) (latse 2 l'/latse 26 -'; +/cdc2B 4 7 ). (F) The third instar larval wing discs dissected from animals of the same genotypes in (E) and labeled with the same number. The lats disc overproliferation phenotype (larva 1) is dramatically suppressed by mutations in the cdc2 gene (larvae 2 and 3). Removal of one copy of the cyclin A gene resulted in a phenotypic suppression of the lats mutants in a manner similar to removal of one cdc2 gene 30 as shown above. Figures 5A-N. Effect of inactivation and overexpression of lats on the cell cycle in Drosophila. (A) Drosophila third instar eye imaginal disc contains a homozygous lats"' clone (arrowhead indicates the lack of Myc staining) that crosses the morphogenetic furrow (MF) (arrow). (B) Cyclin A staining (indicated by arrowhead) in the clone exhibits 35 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. (D) -8- WO 00/10602 PCTIUS99/19068 Composite staining magnification of the same lats mutant clone shown in panels A-C. Cyclin A staining clearly spans the MF region (arrow). (E) Composite staining of the same region shown in panel D viewed with Myc and propidium iodide. (F) Composite staining magnification of the same clone shown in panel E viewed with Cyclin A staining and 5 propidium iodide. Cyclin A is degraded in lats mutant cells at late mitosis (as indicated by the arrowheads). (G) Drosophila third instar eye disc containing homozygous lats" clones (lack of Myc staining is indicated by arrowheads) that span the MF region. (H) The third instar eye disc shown in panel G was also stained for Cyclin B. The MF (indicated by the arrow) is well defined and free of cyclin B staining. (I and J) Scanning electron micrograph of a wild-type Drosophila adult eye (I) in comparison to an eye (J) of a GMR-d-lats 10 transgenic fly showing fewer and irregular ommatidia and missing bristles. (K) A section of a GMR-d-lats eye reveals that many pigment cells are missing, and that ommatidia occasionally lack a full complement of photoreceptor cells (arrow). (L) Propidium iodide staining of a GMR-d-lats third instar eye imaginal disc. A stripe of intensely stained nuclei which are tetraploid (indicated by the star) are seen in the region of the second mitotic 15 wave, which is immediately followed posteriorly by apoptotic cells with fragmented nuclei (indicated by the small arrows). (M) BrdU labeling (green) of a GMR-d-lats third instar eye disc reveals that S phase occurs in the second mitotic wave (indicated by the star) posterior to the MF (indicated by the arrow) just as it would in wild type. (N) A GMR-p21 eye disc is shown in which cell proliferation in the second mitotic wave is blocked before S phase and the stripe of BrdU labeling posterior to the MF (indicated by the arrow) is abolished. 20 Figures 6A-D. Targeting of the lats locus by homologous recombination. (A) Sequence alignment of human lats (h-lats) and mouse lats (m-lats, partial sequence). Arrow indicates the point at which the mouse lats gene was disrupted. (B) Targeting vector for positive-negative selection of homologous recombinants at the lats locus, with restriction map and the structure of the targeted lats locus. The vector is represented by the second line 25 from the top, while the wild-type and mutant (i.e., disrupted) lats alleles are indicated by the top and bottom lines, respectively. The BamHI sites are indicated by "B", the EcoRI sites are indicated by "R", and the EcoRV sites are indicated by "RV". Exons are represented by filled rectangles. 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. In the vector 30 and the mutant allele, 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. The 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) 35 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. -9- WO 00/10602 PCT/US99/19068 Figures 7A and B. Growth retardation of lats> mice. (A) Representative picture of a lats - mouse (agouti, the mouse on the right) with its wild-type littermate (black, the mouse on the left) (12-days-old). (B) Representative growth curve of lats*'', lats*', and lats- mice. Mice were weighed (grams (g)) at intervals and plotted against age in days. 5 Figures 8A-D. Ovarian phenotypes of lats'1 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 10 apparent. Figures 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) 15 mice. The amount of breast epithelial tissue was markedly decreased in lats- females, resulting in mammary fat pads, devoid of an epithelial component. Figures 1OA-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 lats'* mouse demonstrates the organized architecture of the gland. Hyperplastic 20 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. 25 . Figures 11 A-C. Soft tissue sarcoma development in lats' mice. (A,B) Typical soft tissue sarcomas in latsA 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. Figure 12. Nucleotide and amino acid sequences of human lats (h-lats) (SEQ ID NOS:1 and 2, respectively). 30 Figure 13. Nucleotide and amino acid sequences of mouse lats (m-lats) (SEQ ID NOS:3 and 4, respectively). Figure 14. 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 35 NOS:7 and 8, respectively). - 10 - WO 00/10602 PCT/US99/19068 DETAILED DESCRIPTION OF THE INVENTION 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 5 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. In particular, methods are provided for treating or preventing diseases and disorders associated with aberrant cdc2 activity by administration of a therapeutic compound of the invention. 10 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. Preferably, 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. 20 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 25 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. 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 30 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. 35 In a preferred embodiment, the invention provides therapeutic and prophylactic methods for the treatment or prevention of cancer that has been shown to be or may be - 11 - WO 00/10602 PCT/US99/19068 refractory to chemotherapy or radiation therapy treatments or treatments based on tumor suppressor genes other than lats. Also included in the invention are methods of screening lats proteins and lats derivatives and analogs for activity in treating cancer that has been shown to be or may be 5 refractory to chemotherapy or radiation therapy. Additionally, the invention provides methods of screening lats proteins, lats derivatives and fragments, anti-lats antibodies, lats antisense nucleic acids, lats antagonists and inhibitors, and lats-cdc2 complexes for activity in modulating the activity of cdc2. Therapeutic Uses 10 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 15 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 . In specific embodiments, 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 20 subdomain eight of a lats kinase domain, e.g., a seine corresponding to seine 909 of human lats, as depicted in Figure 12 (SEQ ID NO:2). In another embodiment, the therapeutic is a lats derivative or lats-cdc2 complex containing a lats derivative, in which derivative a seine 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 25 aspartate residue, preferably the shrine corresponding to seine 909 of human lats is replaced with a glutamate residue. In a further embodiment, 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). Generally, administration of products of a species origin or species reactivity (in the 30 case of antibodies) that is the same species as that of the subject is preferred. Thus, in a preferred embodiment, 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. 35 Treatment and Prevention of Cancers Refractory to Chemotherapy or Radiotherapy Cancers, including neoplasms, tumors, metastases, or any disorder characterized by uncontrolled cell growth, that have been shown to be refractory to a chemotherapy or - 12 - WO 00/10602 PCT/US99/19068 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 5 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 10 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. In various embodiments of the invention, 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. In a preferred embodiment, cancer that is refractory to treatment with a chemotherapeutic agent that is cell cycle specific, or said cancer is refractory to treatment with a 20 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. 25 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. With respect to radiation therapy, any radiation therapy protocol can be used depending upon the type of cancer to be treated. For example, but not by way of 30 limitation, 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. 35 - 13 - WO 00/10602 PCTIUS99/19068 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 5 a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia). Treatment and Prevention of Diseases by Modulation of Cdc2 Activity In a specific embodiment of the invention, diseases and disorders associated with aberrant levels of cdc2 activity, e.g., aberrantly high or aberrantly low levels of cdc2 protein 10 or activity, can be treated or prevented by administration of a therapeutic of the invention able to modulate the activity of cdc2. In particular, those diseases and disorders associated with an aberrantly high cdc2 activity are treated or prevented by administration of a Therapeutic that promotes lats activity. Alternatively, 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 15 antagonize lats activity, anti-lats antibodies, lats antisense nucleic acids, lats inhibitors and antagonists, antibodies that specifically recognize a lats-cdc2 complex, etc. Because cdc2 promotes cell division, diseases and disorders that may be associated with an increased level of cdc2 activity, as compared with the levels of cdc2 in a subject not afflicted with such a disease or disorder, include diseases and disorders associated with 20 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. Premalignant Conditions 25 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. Alternatively or in addition to the presence of abnormal cell growth characterized as 30 hyperplasia, metaplasia, or dysplasia, 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 250,000 dalton cell surface protein, etc. - 14 - WO 00/10602 PCT/US99/19068 Treatment and Prevention of Disorders in Which Cell Proliferation Is Desired Diseases and disorders involving a deficiency in cell proliferation (growth) or in which cell proliferation is otherwise desirable for treatment or prevention, are treated or prevented by administration of a Therapeutic that antagonizes (inhibits) lats function (in 5 particular, lats-mediated inhibition of cell proliferation and/or lats binding to cdc2). 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 10 recombination (see, e.g., Capecchi, 1989, Science 244:1288-1292), as described herein. Other 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 15 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. 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, 20 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 Gene therapy refers to therapy performed by the administration of a nucleic acid to a 25 subject. In this embodiment of the invention, the nucleic acid produces its encoded protein that mediates a therapeutic effect by promoting lats function. Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below. For general reviews of the methods of gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, 30 Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215. Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY. In a preferred aspect, 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 - 15 - WO 00/10602 PCT/US99/19068 suitable host. In particular, such 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. In another particular embodiment, a nucleic acid molecule is used in which the lats coding sequences and any other desired sequences are flanked by 5 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). In another embodiment, 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. 10 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. 15 Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92). In an embodiment in which recombinant cells are used in 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 20 administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any 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 25 (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). Antisense Therapy Lats function may be inhibited by use of lats antisense nucleic acids. The present 30 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 35 sequence complementarity. The antisense nucleic acid may be complementary to a coding and/or noncoding region of a lats mRNA. In specific aspects, the oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 200 nucleotides. - 16 - WO 00/10602 PCT/US99/19068 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 Encyclopedia of Industrial Chemistry (1998), 6th ed. Wiley and Sons). Such antisense 5 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. The 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 10 sequence. For example, 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. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. 15 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. However, absolute complementarity, although preferred, is not required. Pharmaceutical compositions of the invention, comprising an effective amount of a lats antisense nucleic acid in a pharmaceutically acceptable carrier can be administered to a 20 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. In a specific embodiment, pharmaceutical compositions comprising lats antisense nucleic acids are administered via liposomes, microparticles, or microcapsules. In various embodiments of the invention, it may be useful to use such compositions to achieve sustained release of the lats antisense nucleic acids. In a specific embodiment, it may be desirable to utilize liposomes targeted via antibodies to specific identifiable central nervous 30 system cell types (Leonetti et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87: 2448-2451; Renneisen et al., 1990, J. Biol. Chem. 265: 16337-16342). Lats Proteins, Derivatives, Fragments and Lats-cdc2 Complexes The lats proteins and nucleic acids, and lats derivatives and fragments can be produced by any method known in the art. For recombinant expression of lats proteins, and lats derivatives and fragments, the nucleic acid containing all or a portion of the nucleotide sequence encoding the protein can - 17 - WO 00/10602 PCT/US99/19068 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. In a preferred embodiment, the regulatory elements (e.g., promoter) are heterologous (i.e., not the native gene promoter). Promoters which may be used include the SV40 early promoter 5 (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. A variety of 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 10 such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. 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 15 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. Alternatively, 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. In particular embodiments, the derivative or fragment is functionally active, i.e., 20 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. Additionally, 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. 25 In specific embodiments of the invention, 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 seine residue corresponding to seine 909 of human lats, as depicted in Figure 12 (SEQ ID NO:2). In another specific embodiment, the therapeutic is a lats derivative in which a seine or threonine residue 30 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 seine residue corresponding to seine 909 of human lats is replaced with a glutamate residue. In another specific embodiment, the therapeutic is a fragment of a lats protein comprising or consisting of the amino acid sequence corresponding to amino acids 15 to 35 585 of human lats, as depicted in Figure 12 (SEQ ID NO:2). In particular, lats derivatives can be made by altering lats sequences by substitutions, additions or deletions that provide for functionally equivalent molecules. Due - 18- WO 00/10602 PCTIUS99/19068 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 5 equivalent amino acid residue within the sequence, thus producing a silent change. Likewise, 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. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity 10 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. For example, 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, 15 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. In a specific embodiment of the invention, 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. In other embodiments, the fragment consists of at least 20 or 50 amino 20 acids of the lats protein. In specific embodiments, such fragments are not larger than 35, 100 or 200 amino acids. In a specific embodiment, 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 25 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 30 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 strand) of a coding lats sequence, under high stringency, moderately stringency, or low stringency conditions, as discussed infra. The 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 - 19 - WO 00/10602 PCT/US99/19068 the gene or protein level. For example, 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). 5 Additionally, 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 10 (Pharmacia), etc. Manipulations of the 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 15 known techniques, including specific chemical cleavage by cyanogen bromide, trypsin, oxidation, reduction; etc. In addition, analogs and fragments of lats can be chemically synthesized. For example, 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. Furthermore, if desired, nonclassical amino acids or chemical amino 20 acid analogs can be introduced as a substitution or addition into the lats sequence. In a specific embodiment, 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 25 different protein. In one embodiment, such 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. Alternatively, 30 such 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. In another specific embodiment, the lats derivative is a chimeric protein comprising a fragment of lats corresponding to amino acids 15-585 of human lats. In another specific embodiment, the lats derivative is a molecule comprising a region of homology with a lats protein. By way of example, in various embodiments, a first - 20 - WO 00/10602 PCT/US99/19068 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% 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 5 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. For example, a molecule can comprise one or more regions homologous to a lats domain or a portion thereof. Derivatives of Lats Containing One or More Domains of the Protein In specific embodiments, the methods of the invention use lats derivatives and 10 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 (LCD2), lats C-terminal domain 1 (LCD 1), kinase domain, kinase subdomains, lats flanking domain (LFD) (amino-terminal to the kinase domain), lats split domain 1 (LSD1), lats split domain 2 (LSD2), SH3-binding domain, and opa repeat domain, functional (e.g., binding) 15 fragments of any of the foregoing, or any combination of the foregoing. In human lats (h-lats), m-lats, m-lats2, and Drosophila lats, the LCD3 domain is the last three amino acids of the protein, which are Val-Tyr-Val in all four proteins. For human lats and Drosophila lats, the LCD2 domain is amino acid residues 1077-1086 and 1075 1084, respectively (all amino acid residues provided in this paragraph are for the human and Drosophila lats amino acid sequences depicted in Figures 12 and 14, respectively (SEQ ID 20 NOS:2 and 8, respectively)); the LCD1 domain is amino acid residues 1032-1043 and 1035 1047, respectively; 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. For the lats split domains in Drosophila, the LSD1 is amino acid residues 365-392 and the 25 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 498 518 and LSD2 is amino acid residues 28-31 and 555-559. In particular, 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. 30 In a specific embodiment, a lats protein, derivative or fragment is provided that has a kinase domain and has a phosphorylated or dephosphorylated seine 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. In various specific embodiments, the invention 35 provides various phosphorylated and dephosphorylated forms of the lats protein, derivative, or fragment that are active or inactive kinase forms. Both phosphorylation and dephosphorylation of lats at different residues could potentially activate or inactivate lats. -21 - WO 00/10602 PCT/US99/19068 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 5 is a dominant-active protein kinase. Such a derivative or analog comprises a lats kinase domain that has been mutated so as to be dominantly active (exhibit constitutively active kinase activity). It is known that 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). Thus, changing a seine or threonine 10 residue situated within 20 residues upstream of an Ala-Pro-Glu consensus in subdomain eight of a lats kinase domain into another residue (e.g., Glu, Asp) may be used to make a dominant-active lats protein kinase. For example, changing Ser914 in Drosophila lats, or changing Ser909 in human lats, into a Glu residue could produce a dominant active lats kinase. 15 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 20 Means, 1994, EMBO J. 12:2103-2113; Yarden et al., 1992, EMBO J. 11:2159-2166). Thus, 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). For example, 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). As another example, 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). In another specific embodiment, a molecule is provided that comprises one or more 30 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. For example, such a protein may lack all or a portion of the kinase domain, but retain at least the SH3-binding domain of a lats protein. In another embodiment, 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 35 (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. - 22 - WO 00/10602 PCT/US99/19068 Lats-cdc2 Complexes The invention provides lats-cdc2 complexes. In a preferred embodiment, the lats cdc2 complexes are complexes of human proteins. As used herein, fragment or derivative of a lats-cdc2 complex includes complexes where one or both members of the complex are 5 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. Preferably, 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. In particular aspects, 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. 10 "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. In specific embodiments, the lats-cdc2 complex contains a lats protein that is phosphorylated, preferably that is phosphorylated on a seine or threonine residue within 20 15 amino acids upstream of an Ala-Pro-Glu consensus subdomain eight of a lats kinase domain, more preferably that is phosphorylated on a seine residue corresponding to seine 909 of human lats, as depicted in Figure 12 (SEQ ID NO:2). In another specific embodiment, the lats-cdc2 complex contains a lats derivative in which a seine 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 20 seine residue corresponding to seine 909 of human lats is replaced with a glutamate residue. In another specific embodiment, 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). Methods are presented for screening lats-cdc2 complexes, as well as derivatives and 25 fragments of the lats-cdc2 complexes for the ability to alter lats and/or cdc2 activity, e.g., to alter cell proliferation. For example, such derivatives or fragments which have the desired immunogenicity or antigenicity can be used in immunoassays, for immunization, for inhibition of lats-cdc2 complex activity, etc. Derivatives or fragments that retain, or alternatively lack or inhibit, a property of interest (e.g., participation in a lats-cdc2 complex) can be used as inducers, or inhibitors, respectively, of such a property and its physiological 30 correlates. 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 35 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 - 23 - WO 00/10602 PCT/US99/19068 same cell, either under the control of the same promoter or two separate promoters. In yet another embodiment, a derivative, fragment or homolog of lats and/or a derivative, fragment or homolog of cdc2 are recombinantly expressed. Preferably, the derivative, fragment or homolog of lats and/or the cdc2 protein form a complex with a binding partner 5 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)). In a specific embodiment, fusion or chimeric proteins are provided that contain the domains of a lats protein, or, in a specific embodiment, the amino acid sequence 10 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. These fusion proteins may be particularly useful where the stability of the interaction is desirable (due to the formation 15 of the complex as an intramolecular reaction), for example in production of antibodies specific to the lats-cdc2 complex. Generation of Antibodies to Lats Proteins and Lats-cdc2 Complexes and Derivatives 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 20 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.). For example, antibodies to a particular domain of a lats protein may be desired. In a specific embodiment, fragments of a lats protein identified as hydrophilic are used as immunogens for antibody production using art-known methods. Various known methods 25 for antibody production can be used including cell culture of hybridomas; production of monoclonal antibodies in germ-free animals (PCT/US90/02545); the use of human hybridomas (Cole et al., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:2026-2030; Cole et al., in Monoclonal Antibodies and Cancer Therapy (1985) Alan R. Liss, pp. 77-96), and production of humanized antibodies (Jones et al., Nature (1986) 321:522-525; US Pat. No. 5,530,101). 30 Diagnostic, Prognostic, and Screening Uses of Lats-cdc2 Complexes 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 35 tumor progression, and hypoproliferative disorders, and thus have diagnostic utility. - 24 - WO 00/10602 PCT/US99/19068 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 5 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. In particular, such an immunoassay is carried out by a method comprising 10 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. In a specific aspect, such 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. In a specific 15 embodiment, 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. By "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 20 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 25 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. In particular, such a hybridization assay is carried out by a method comprising contacting a sample containing nucleic acid with a nucleic acid probe capable of 30 hybridizing to lats and cdc2 DNAs or RNAs, under conditions such that hybridization can occur, and detecting or measuring any resulting hybridization. By way of example, 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 - 25 - WO 00/10602 PCT/US99/19068 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. Also embodied are methods to detect a lats-cdc2 complex in cell culture models that express a lats-cdc2 complex, or derivatives thereof, for the purpose of characterizing or 5 preparing the lats-cdc2 complex for harvest. 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). Kits for diagnostic use are also provided, that comprise in one or more containers an 10 anti-lats-cdc2 complex antibody and, optionally, a labeled binding partner to the antibody. Alternatively, 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. In a specific embodiment, a kit can comprise in one 15 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 B-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 20 of a purified lats-cdc2 complex, lats and cdc2 proteins or nucleic acids thereof, e.g., for use as a standard or control. Demonstration of Therapeutic Utility The Therapeutics of the invention are preferably tested in vitro, and then in vivo, for 25 the desired therapeutic or prophylactic activity, prior to use in humans. For example, 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 30 condition in the patient. Alternatively, 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 35 (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. - 26 - WO 00/10602 PCTIUS99/19068 In another embodiment of the invention, a Therapeutic of the invention is screened for activity to modulate (e.g., promote, inhibit or antagonize) cdc2 levels and/or activity. The levels of cdc2 protein and mRNA and cdc2 activity can be determined by any method well known in the art. For example, cdc2 protein can be quantitated by known 5 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-HI kinase assay. 10 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. In a preferred embodiment, 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 15 modulate cdc2 activity. Lats Knock-out Animals 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, 20 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. In a preferred aspect, a lats knock-out animal can be produced by promoting homologous recombination between a lats 25 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. 30 Briefly, all or a portion of 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:1, 3, 5, or 7). Once the genomic clone is isolated, all or a portion of 35 the clone is introduced into a recombinant vector. Preferably, 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, - 27 - WO 00/10602 PCT/US99/19068 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 5 lats activity, e.g., at a position where point mutations or other mutations have been demonstrated to inactivate lats protein function. For example, 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 10 preferably, for the sequence coding for the amino acids corresponding to 756 to 1130 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 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 15 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 PolIl 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). Use of a selectable marker, such as an antibiotic resistance gene, allows for the selection of cells that have incorporated the targeting vector (for example, the expression of the neo gene product 20 confers resistance to G418, and expression of the hygro gene product confers resistance to hygromycin). In a preferred embodiment, 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. For example, such a 25 negative selectable marker is the HSV thymidine kinase gene (HSV-tk), the expression of which makes cells sensitive to ganciclovir. The negative selectable marker is preferably under the control of a promoter such as the PGK promoter, the Poll promoter or the MC1 promoter. When homologous recombination occurs, the portions of the vector that are homologous to the lats gene, as well as the non-homologous insert within the lats gene 30 sequences, are incorporated into the lats gene in the chromosome, and the remainder of the vector is lost. Thus, since 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. For example, if the negative selectable marker is the HSV-tk gene, the cells in which homologous recombination has 35 occurred will not express thymidine kinase and will survive exposure to ganciclovir. This procedure permits the selection of cells in which homologous recombination has occurred, as compared to non-homologous recombination in which it is likely that the negative - 28 - WO 00/10602 PCT/US99/19068 selectable marker is also incorporated into the genome along with the lats sequences and the positive selectable marker. Thus, cells in which non-homologous recombination has occurred would most likely express thymidine kinase and be sensitive to ganciclovir. Once the targeting vector is prepared, it is linearized with a restriction enzyme for 5 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. For example, if the selectable markers are the neo resistance 10 gene and the HSV-tk gene, the cells are exposed to G418 (e.g., approximately 300 pg/ml) and ganciclovir (e.g., approximately 2 tM). 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 15 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. Thus, by assaying for a restriction fragment or PCR product, the size of which differs between the disrupted and non-disrupted lats gene, one can determine whether homologous recombination has occurred to disrupt the lats gene. 20 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 25 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. Isolating Lats Genes Clones comprising lats nucleotide sequences, particularly lats genomic clones, can 30 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:1-8, 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 35 Collection, 10801 University Boulevard, Manassas, Virginia 20110-2201, and assigned Accession No. 69769. Lats nucleic acids, either lats genomic clones or lats specific probes to identify lats genomic clones, can be obtained by any method known in the art, e.g., from - 29 - WO 00/10602 PCTIUS99/19068 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. Genomic clones can 5 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 10 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. USA 78:6789-6792): Filters containing DNA are pretreated for 6 hours at 40'C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 Ig/ml 15 denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 pg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 106 cpm 32 P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 hours at 40'C, and then washed for 1.5 hours at 55 0 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 20 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, 25 and 500 tg/ml denatured salmon sperm DNA. Filters are hybridized for 48 hours at 65'C in prehybridization mixture containing 100 tg/ml denatured salmon sperm DNA and 5-20 X 106 cpm of 32 P-labeled probe. Washing of filters is done at 37 0 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 30 DNA are pretreated for 6 hours at 55 0 C in a solution containing 6X SSC, 5X Denhardt's solution, 0.5% SDS, and 100 pg/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 0 C, and then washed twice for 30 minutes at 60'C in a solution containing 1 X SSC and 0.1% SDS. 35 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 - 30 - WO 00/10602 PCT/US99/19068 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; 5 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. In preferred embodiments, 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 10 the lats coding sequence of SEQ ID NO:5. Methods of Screening Therapeutics Using Lats Knock-out Mice 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 15 by administration or application of the compound to be tested to a lats knock-out animal, preferably a lats knock-out mouse. In a preferred embodiment, 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), 20 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 25 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. As described in the 30 Examples section, infra, lats knock-out mice are susceptible to ovarian stromal tumors and soft tissue sarcomas that metastasize to vital organs. Accordingly, in preferred embodiments, 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 35 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 -31 - WO 00/10602 PCT/US99/19068 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. 5 The invention also provides methods of screening compounds for efficacy in treating or preventing skin cancer. As demonstrated in Examples section, 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). Generally, mouse skin tumors can be elicited by application of a carcinogenic dose of tumor initiator, e.g., 600 to 800 nmole of a 10 pure polycyclic aromatic hydrocarbon such as 9 ,10-dimethyl-1,2-benzanthracene (DMBA). Other tumor initiators include, but are not limited to, arylamines, carbamates, haloalkylethers, haloaromatics, lactones, nitro-aromatics, nitrosamides and ureas. Alternatively, and preferably, 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 15 exposures to a tumor promoter, such as phorbolesters (e.g., TPA), teleocidins, polyacetates, okadaic acid, calyculin A, palytoxin, and thapsigargin. Ultraviolet B (UVB) radiation, skin abrasion and skin wounding are also strong tumor promoters. In a preferred embodiment, such skin tumors are induced by a two-step process comprising a single treatment with DMBA, preferably 50 ptl of a 0.5% DMBA solution in acetone, to the dorsal surface of the mouse 1 to 5 days after birth followed by repeated 20 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% 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). Accordingly, in a preferred embodiment, the invention provides a method for 25 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. The size or progression of the skin tumors are then compared before and after the administration of the compound. A reduction in the size or progression of the skin 30 tumors in the recombinant non-human animal administered the compound as compared to same animal prior to administration of the compound or to the animal not so administered or to the standard size or progression of the skin tumors, indicates that the compound has activity in treating or preventing skin cancer. In another embodiment, 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, - 32 - WO 00/10602 PCT/US99/19068 subcutaneously, rectally, topically, etc. For the screening of compounds for efficacy in treating or preventing skin cancer, the compound is preferably applied topically. After administration of the compound to be tested, the tumors, sarcomas, and other cancers can be evaluated by any diagnostic or histopathological method for detecting and 5 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% neutral buffered formalin at 4'C, and subsequent dehydration, 10 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. Another aspect of the invention provides methods for screening potential therapeutic 15 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 20 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. 25 In a preferred embodiment, 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 30 compound was administered. In one embodiment, 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 that may be assayed include fertility, ovulation, the female reproductive cycle (e.g., the estrus cycle), breast tissue development, growth or size 35 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. These indicators may be measured by any means known in the art for evaluating these indicators. For example, - 33 - WO 00/10602 PCT/US99/19068 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., by fixing, sectioning and staining the tissue for inspection) for morphological defects, 5 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. 10 Candidate Therapeutics Candidate therapeutics may come from any source of therapeutics known in the art. For example, these therapeutics can be proteins, nucleic acids (including anti-sense nucleic acids), antibodies, peptides, organic molecules, etc. In some instances, compounds may be 15 screened first in in vitro assays to determine their potential as anti-cancer or anti-pituitary dysfunction therapeutics. For example, 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. 20 Alternatively, 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 25 libraries (Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques 13:412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712), organic diversity (e.g., nonpeptide) libraries (Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712) may be used. Libraries of non-peptides, e.g., peptide derivatives (for example, that contain one or more 30 non-naturally occurring amino acids) can also be used. One example of these are peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371). 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. - 34 - WO 00/10602 PCT/US99/19068 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. In a preferred aspect, the Therapeutic is substantially purified. The subject is preferably an animal, including but not 5 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. Various delivery systems are known and can be used to administer 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 10 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, 15 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. In a specific embodiment where the Therapeutic is a nucleic acid encoding a protein Therapeutic, the nucleic acid can be administered in vivo to promote expression of its 20 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 U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), etc. Alternatively, a nucleic acid Therapeutic can be introduced intracellularly and incorporated within host cell DNA for expression, by 25 homologous recombination. The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "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 30 animals, and more particularly in humans. The term "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 35 pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the Therapeutic, preferably in purified form, together with a suitable amount of carrier so as to - 35 - WO 00/10602 PCT/US99/19068 provide the form for proper administration to the patient. The formulation should suit the mode of administration. In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration 5 to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, 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 10 condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. However, 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 15 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 20 the invention. Screening for Lats Agonists and Antagonists 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 25 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. In a preferred embodiment, 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 30 of lats-cdc2 complexes. For example, 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, and 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., 35 putative binding partners of lats) 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. - 36 - WO 00/10602 PCT/US99/19068 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. By way of example, diversity libraries, such as random or combinatorial peptide or nonpeptide libraries can be screened for molecules that specifically bind to lats. Many 5 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. Examples of chemically synthesized libraries are described in Fodor et al., 1991, Science 251:767-773; 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; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 10 90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques 13:412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381-5383. 15 Examples of 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., 1992, J. Mol. Biol. 227:711-718; Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/183 18 dated August 18, 1994. 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. 20 Natl. Acad. Sci. USA 91:9022-9026. By way of examples of nonpeptide libraries, 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 25 peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA 91:11138-11142). 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: Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 30 1992, Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell 76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Patent No. 5,096,815, U.S. Patent No. 5,223,409, and U.S. Patent No. 5,198,346, all to Ladner et al.; Rebar and Pabo, 1993, Science 263:671-673; and PCT Publication No. WO 94/18318. In a specific embodiment, 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 - 37 - WO 00/10602 PCT/US99/19068 harvesting those library members that bind to the protein (or nucleic acid or derivative). Examples of such screening methods, termed "panning" techniques are described by way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and in references cited hereinabove. In another embodiment, the two-hybrid system for selecting interacting proteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) 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. Additionally, the two hybrid system or co-immunoprecipitation of lats and cdc2 can be used as assays to 10 screen for compounds that promote or inhibit formation of lats-cdc2 complexes. In a preferred embodiment, 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, 15 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. 20 EXAMPLES EXAMPLE 1: Human Lats Modulates Cdc2/Cyclin A Activity Using mammalian cell culture assays, we have found that lats is phosphorylated in a cell cycle-dependent manner and that it complexes with cdc2 in early mitosis. Lats 25 associated cdc2 has no mitotic cyclin partner and no kinase activity for histone H 1. Furthermore, we have found that lats mutant cells in Drosophila abnormally accumulate cyclin A. These biochemical observations indicate that lats is a negative regulator of cdc2/cyclin A, a finding supported by in vivo genetic data demonstrating that lats specifically interacts with cdc2 and cyclin A in Drosophila. 30 Materials and Methods For yeast two-hybrid experiments, DNA encoding N-terminal h-lats (amino acid numbers 15 to 585 of the human lats protein sequence as depicted in Figure 12 (SEQ ID NO:2)), C-terminal h-lats (amino acid number 589 to 1130 of the human lats protein sequence as depicted in Figure 12 (SEQ ID NO:2)), and h-lats (amino acid numbers 15 to 35 1130 of the human lats protein sequence as depicted in Figure 12 (SEQ ID NO:2)) were cloned into pBTM1 16 (Bartel et al., Cellular Interactions in Development: A Practical Approach, ed. D. Hartley (Oxford University Press, Oxford, England (1993)). DNA - 38 - WO 00/10602 PCT/US99/19068 encoding human cdc2, CDK2, CDK4 and C-terminal h-lats were cloned in pACT (Durfee et al., 1993, Genes & Devel. 8:440-452). The constructs were transformed into yeast strain L40 (Vojtek et al., 1993, Cell 74:205-214). The transformants were tested for growth on SD his-ura-trp-leu medium (Biol01, California) and for -galactosidase activities (Vojtek et 5 al., 1993, Cell 74:205-214). For baculovirus experiments, full-length h-lats cDNA was cloned into the vector pBacPAK8 and baculovirus were produced according to the protocols provided by Clontech. IPLB-Sf2l 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. 10 Fly Genetics The full-length 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 latsem 3 2 , latse 26 -], lats"', and lats" alleles. 15 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. Since the induction of h-lats requires heat-shock treatments, X-ray irradiation was used to induce mitotic clones in y w P[hs-h-lats]/y w; P[FRT]82B lats'/P[y+)96E animals. The rest of the lats mutant mitotic clones were induced and labeled according to Xu and Rubin (1993, Development 117:1223-1237). The Drosophila lats and h-lats cDNAs were cloned into the 20 pGMR vector to generate multiple transformant lines (Hay et al., 1994, Development 120:2121-2129). Besides the mutations mentioned above, cdc2cE1 3 6 E (a gift of Helena Richardson), cycA""o"', Df(2R)59A-B were used for cdc2c, cyclin A and cyclin B, respectively. Tissue Culture 25 HeLa cells were synchronized at different cell cycle stages by various treatments as described by Knehr et al.(1995, Exp. Cell. Res. 217:546-553). Briefly, cells were arrested at GI by thymidine and hydroxyurea treatment; at S phase by thymidine double block (incubation in the presence of 2 mM thymidine for 24 hours, followed by recovery in the absence of thymidine for 12 hours, followed by another incubation in 2 mM thymidine for 30 14 hours) plus a 4 hour incubation in medium without thymidine; and at G2 by thymidine double block plus an 8 hour incubation in medium without thymidine. To arrest cells in M phase, cells were treated with 0.1 tg/ml nocodazole (Sigma) for 12 hours and mitotic cells were shaken off of the flask and washed twice with cold DMEM without serum. These mitotic cells were then resuspended in fresh warm medium without nocodazole and 35 incubated in suspension at 37C. 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-Sf21 cells were grown in sf-900 II SFM plus 10% FBS. - 39 - WO 00/10602 PCT/US99/19068 Antibodies and Immunochemistry 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) 5 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 anti-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. 10 HeLa, CHO, or IPLB Sf21 cells were lysed in TG buffer (1% Triton, 10% glycerol) (Sun et al., 1996, Genes & Devel. 10:395-406) plus freshly added proteinase inhibitors (1 mM PMSF, 10 pg/ml leupeptin, 1 ptg/ml pepstatin, and 2 pig/ml aprotinin). The lysates were centrifuged at 50 K, 4'C for 12 minutes. Supernatants were pre-cleaned by incubating with protein G-agarose. Immunoprecipitation and western blots were performed by the 15 procedures described by Sun et al. (1996, Genes & Devel. 10:395-406). Western blots were visualized by enhanced chemiluminescence (Amersham). Whenever necessary the blots were stripped following the procedure described by Edgar et al. (1994, Genes & Devel. 8:440-452). Calf Intestinal Phosphatase (CIP) treatments were carried out as described in Sun et al. (1996, Genes & Devel. 10:395-406). 20 HI Kinase Assay 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 MgCI2, 5 mM EGTA, 2 mM DTT) without DDT. The kinase assay was carried out on ice for 10 minutes in 35 pl of IX kinase buffer 25 containing 15 gCi of y-"P ATP, 1.6 pg of histone HI, and 1.5 ptM ATP. The kinase activities were measured by quantifying the intensities of histone-H1 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. 30 Results Human Lats Can Functionally Replace its Fly Counterpart Sequence conservation suggested that human lats could be a functional homolog of Drosophila lats. To test this, the human lats cDNA was introduced into the Drosophila genome under the control of the heat shock-inducible promoter (hs-h-lats) (Lis et al., 1983, 35 Cell 35:403-410) and expressed the transgene was expressed under the conditions previously established for rescue using the fly lats gene (Xu et al., 1995, Development 121:1053-1064; and PCT Publication WO 96/30402, published October 3, 1996). In - 40 - WO 00/10602 PCT/US99/19068 mosaic flies, clones of cells mutant for lats undergo extensive overproliferation and develop into large tumors in various tissues (Figure 1A and Xu et al., 1995, Development 121:1053 1064). Expression of human lats completely suppressed tumor formation in lats mosaic flies (Figures 1B-D). Instead, the lats mutant cells (genetically marked as yellow- cells) in 5 these human lats-expressing mosaic animals developed into normal adult structures (Figures 1 C and D). The ability of the human gene to support normal fly development was further examined. Expression of the human lats transgene in homozygous lats mutant Drosophila rescued all developmental defects including embryonic lethality found in homozygous lats mutants. Furthermore, the extent of phenotypic rescue correlated with the level of human lats expression. Complete phenotypic rescue required daily induction of human lats, and 10 leaky expression of lats controlled by the heat shock promoter at 25'C resulted in partial suppression of the lats mutant overproliferation phenotype (Figures 1E and F). These data demonstrate that human lats is an authentic homolog of the Drosophila lats tumor suppressor. 15 Lats is phosphorylated in a cell cycle-dependent manner To further explore the function of lats, the biochemical properties of the human lats protein were examined. Lats immunoprecipitated from HeLa cells had two major migrating forms (Figure 2A, lane 6). The slow-migrating form of lats was converted into the fast migrating form after the proteins were incubated with calf intestine alkaline phosphatase (CIP) (Figure 2A). Addition of a phosphatase specific inhibitor, 20 B-glycerophosphate, to the phosphatase reaction blocked this conversion (Figure 2A, lanes 5 and 10). These results indicate that the slow-migrating form is phosphorylated lats, while the fast-migrating form is dephosphorylated lats. Lats immunoprecipitated from cells at different mitotic stages displayed varying amounts of the two forms (compare lanes 1 and 6 of Figure 2A), suggesting that the 25 phosphorylation state of lats may oscillate with the cell-cycle. To verify this possibility, lats proteins were immunoprecipitated from extracts of HeLa cells at GO, G1, 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. All lats protein was phosphorylated at late prophase (0 minutes ARN; Figure 2B), and remained phosphorylated through metaphase (50 minutes 30 ARN; Figures 2B and C). Dephosphorylated lats could be detected when cells in the culture begin to enter anaphase (75 minutes ARN; Figures 2B and C), and by the start of telophase (100 minutes ARN) most of the lats protein was dephosphorylated (Figures 2B and C). In late mitosis, GI, S, G2 or GO phase, lats molecules were in the dephosphorylated form (Figure 2B). These observations strongly suggest that the lats protein undergoes two major 35 phosphorylation changes during the cell cycle. At the G2/M boundary or in early prophase, lats is phosphorylated, and lats becomes dephosphorylated at the metaphase/anaphase boundary or in early anaphase. - 41 - WO 00/10602 PCT/US99/19068 Lats complexes with cdc2 during mitosis 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 IG and H). The lats mutant overproliferation phenotype and cell cycle-dependent 5 phosphorylation of lats suggest that the protein could be directly involved in the regulation of the cell cycle. Immunoprecipitation experiments were carried out to examine whether lats proteins complex with known cell cycle regulators. Interestingly, cdc2 was found to co immunoprecipitate with lats in mitotic cells (Figures 3A and B). The co-precipitation of lats and cdc2 was confirmed in both murine and human cells using several polyclonal and 10 monoclonal anti-human lats antibodies (as described above in the Materials and Methods section, supra). Although similar amounts of lats were immunoprecipitated from HeLa cells during different stages of mitosis (Figure 3B, upper panel), the amount of co precipitated cdc2 varied (Figure 3B, lower panel). Co-precipitated cdc2 was most abundant at early mitosis (0 and 50 minutes ARN; Figure 3B; also see Figures 2B and C for cell cycle 15 progression). The amount of co-precipitated cdc2 then progressively decreased as the cell cycle progressed (Figure 3B--lanes 100', 150', and 200'). No cdc2 co-immunoprecipitation could be detected in quiescent cells in GO (Figure 3A and Figure 2B). The difference in the amount of co-precipitated cdc2 cannot be attributed to changes of cdc2 levels during the cell cycle, since it has previously been shown that the cdc2 protein is maintained at a nearly constant level in cycling cells (Dalton, 1992, EMBO J. 11:1797-1804; McGowan et al., 20 1990, Mol. Cell. Biol. 10:3847-3851). Indeed, equal amounts of cdc2 protein were precipitated from the 50 minutes and 200 minutes ARN extracts when anti-cdc2 antibodies were used. The interaction of lats and cdc2 proteins was studied by expressing human lats and cdc2 proteins in the baculovirus expression system. The baculovirus-expressed cdc2 and 25 lats proteins could be co-immunoprecipitated using either anti-human lats or anti-cdc2 antibodies (Figure 3C). This result suggests that the in vivo lats/cdc2 complex may result from direct binding of the two proteins. The interaction between lats and cdc2 was also examined using the yeast two-hybrid assay (Fields and Song, 1989, Nature 340:245-245; Finley and Brent, in DNA Cloning 2, Rickwood and Hames, eds (Oxford University Press (Oxford, 1995)). Consistent with the 30 co-immunoprecipitation results, full length lats and the N-terminal region of lats interacted with cdc2 in the assay (Figure 3E). Since the C-terminal kinase domain of lats did not interact with cdc2, these results indicate that lats associates with cdc2 through its N terminal domain. Furthermore, neither full length lats nor the N-terminal region of lats showed any interaction with two GI cell cycle kinases, CDK2 and CDK4 (Figure 3E), indicating that the association between lats and cdc2 is specific. - 42 - WO 00/10602 PCT/US99/19068 The lats/cdc2 complex is inactive for H1 kinase activity We examined whether the lats/cdc2 complex has any kinase activity on the substrate histone Hi. 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 5 monoclonal antibodies and assayed for histone HI kinase activities. In contrast to the cdc2/cyclin B complex, 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 10 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. Alternatively, 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 15 1024). Neither cyclin A nor cyclin B proteins could be detected in the lats/cdc2 immunocomplex when probed with anti-cyclin A and B antibodies (Figure 3B), indicating that lats modulates cdc2 activity in a way different from that of the known cyclin dependent kinase inhibitors (CDIs) (Sherr, 1996, Science 274:1672-1677; Harper, 1997, Cancer Surveys 29:91-107). 20 Lats genetically interacts with cdc2 and cyclin A during Drosophila development In Drosophila, cdc2 also complexes with cyclin A or B (Knoblich 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 ", or homozygous for the temperature sensitive cdc2" mutation at permissive temperature are 25 viable and morphologically normal (Clegg et al., 1993, Genome 36:676-685). The latsP 8 mutation causes late pupal lethality in homozygous mutants (Figure 4A), and reducing cdc2 activity in latsP 8 homozygotes by introducing one copy of a cdc2 mutant allele (cdc21 4 1 or cdc2t"/+; lats' 8 /lats' 8 ) was sufficient to rescue the lats-associated lethality (Figure 4B). Furthermore, the overproliferation phenotype of latsP 8 adult appendages were also 30 suppressed. Rescued animals had near-wild type eyes in comparison to the overproliferated, large, rough eyes of the latsP 8 mutants (Figures 4C and D). Reducing cdc2 activity also suppressed the giant larvae/pupae and disc overproliferation phenotypes of the latsE 2 6 - animals (Figures 4E and F). The Drosophila CDK2 homolog, cdc2c complexes with Cyclin E (Lehner and O'Farrell, 1990, Cell 61:535-547; Knoblich et al., 1994, Cell 35 77:107-120). Consistent with the result of the yeast two-hybrid assay for human lats and CDK2 (Figure 3C), inactivation of one copy of the cdc2 gene did not modify the phenotypes of the lats'8 mutant animals. We further examined the potential genetic - 43 - WO 00/10602 PCT/US99/19068 interaction between lats, cyclin A and cyclin B in Drosophila as described above. Interestingly, while cyclin B did not interact with lats, cyclin A behaved similarly to cdc2. Inactivation of one copy of the cyclin A gene resulted in almost identical phenotypic suppression of the latsp' and latsE 26 - mutants as did the cdc2 mutants (data not shown). 5 Thus, the specific genetic interactions between lats, cdc2, and cyclin A confirms the biochemical data indicating that lats regulates cell proliferation by negatively modulating cdc2/cyclin A activity. While cdc2 protein remains at a constant level during the cell cycle (Dalton, 1992, EMBO J. 11:1797-1804; McGowan et al., 1990, Mol. Cell. Biol. 10:3847-3851), cyclin A and B are degraded when the cdc2/cyclin complexes are inactivated (Draetta et al., 1989, 10 Cell 56:829-828; Murray et al., 1989, Nature 339:280-286; King et al., 1994, Cell 79:563 571). Accordingly, whether lats inactivation leads to accumulation of cyclin A and B was also determined. In the Drosophila eye imaginal disc, cyclin A and B are detected in cells anterior to the morphogenetic furrow (MF) as well as in a stripe of cells posterior to the MF 15 which are undergoing the last round of cell division (the second mitotic wave) (Thomas et al., 1994, Cell 77:1003-1014). In clones of lats cells located anterior to the MF, 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. 9:2563-2572), which might be due to the fact that cells in this region already accumulate high levels of both cyclin A and B. Cells in the MF are synchronized in G1 and so are the cells posterior to the MF which are 20 differentiating into neurons. Both populations of the G1 cells have no detectable cyclin A or B (Thomas et al., 1994, Cell 77:1003-1014; Figures 5B and H). However, in clones of lats* cells in the MF and in the region posterior to the MF, a high level of cyclin A was detected (Figures 5A-E). Interestingly, in lats' cells, cyclin A was still degraded in cells at late mitosis (Figure 5F), indicating that lats affects limited aspects of the cell cycle. Finally, 25 consistent with the genetic interaction results, lats- clones did not cause obvious changes in the staining pattern of cyclin B (Figures 5G and H). These results provide direct evidence indicating that inactivation of lats causes overproliferation by deregulating cdc2/cyclin A activity. The role of lats in cell cycle regulation was examined by overexpressing lats in the developing eye imaginal disc. When Drosophila and human lats cDNAs were expressed in 30 cells in and posterior to the MF under the direction of the GMR promoter (Hay et al., 1994, Development 120:2121-2129), they exhibited similar phenotypes. Adult eyes from these animals were smaller than wild type and had irregular architecture with missing bristles, a phenotype reminiscent to that of overexpressing p21 under the same promoter (GMR-p21; de Nooij and Hariharan, 1995, Science 270:983-985) (Figures 5I and J). Sections of GMR 35 lats adult retinas also revealed a phenotype identical to that observed in retinas from flies transformed with GMR-p21. While almost all of the ommatidia contained the full complement of photoreceptor cells, many pigment cells were missing (Figure 5K). This - 44 - WO 00/10602 PCT/US99/19068 phenotype suggested that, like overexpression of p21, overexpression of lats blocked the last round of cell division in the developing eye disc. Further examination of the GMR-lats eye discs revealed that in the region of second mitotic wave there was an accumulation of cells with intense propidium-iodide staining, indicating that the cells were tetraploid, which 5 was followed immediately posteriorly by apoptotic cells with fragmented nuclei (Figure 5L). In contrast to the overexpression of p21 which blocked entry into S phase, overexpressing lats arrested cells at G2/M or M phase. Consistent with this conclusion, BrdU labeling experiments revealed that the S phase of the cells in the second mitotic wave did occur in GMR-lats eye discs (Figures 5M and N). 10 Discussion The lats molecules are a novel family of conserved proteins Expression of human lats under the same conditions used for rescue by the fly gene completely suppressed tumor formation in lats mosaic flies and rescued all developmental defects in lats homozygous mutant Drosophila. These experiments provide definitive 15 evidence for functional conservation among the lats genes, indicating that human lats can perform all functions that are normally provided by the fly protein. A model for lats function Our biochemical and genetic data support the hypothesis that lats is a negative regulator of cdc2/cyclin A. Cdc2 co-immunoprecipitated with lats using either HeLa and 20 CHO cell extracts or baculovirus-expressed proteins. Cdc2 also interacted with lats in yeast two-hybrid assays. Moreover, lats-associated cdc2 has no cyclin A or B subunit and no histone HI kinase activity. In Drosophila, lats mutant cells abnormally accumulated cyclin A. Genetic data in Drosophila demonstrate that the overproliferation and lethality phenotypes of lats mutants can be suppressed by mutations in cdc2 and cyclin A genes. The 25 genetic interaction between lats, cdc2, and cyclin A is highly specific. While animals heterozygous for cdc2 or cyclin A did not display any defects, removal of one copy of either gene was sufficient to dominantly suppress the lats mutant phenotypes. Such a genetic interaction was not observed in hundreds of genes examined, including other positive cell cycle regulators such as cdc2c, cyclin B, and dE2F. The lats kinase domain contains all 11 subdomains previously found in other protein 30 kinases (Hanks et al., 1988, Science 241:42-45), suggesting that it is an active protein kinase. However, 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). This result indicates that the C 35 terminal kinase domain of lats has a negative effect on the binding between the lats N terminal region and cdc2. - 45 - WO 00/10602 PCT/US99/19068 The association of lats with cdc2 is directly correlated with its state of phosphorylation (compare Figures 2B and 3B). During early mitosis, lats is phosphorylated and associates with cdc2. At GO, lats is dephosphorylated and fails to associate with cdc2. Furthermore, the transition of the lats phosphorylation state during mitosis correlates with a 5 change in its ability to bind to cdc2. Phosphorylation is a common mechanism that regulates protein activities during the cell cycle (Hunter, 1995, Cell 80:225-236). Cell-cell communication mechanism regulating cell proliferation Many tumor suppressors probably evolved to play important regulatory roles during development. The study of the normal developmental functions of a tumor suppressor is 10 essential to our understanding of the mechanisms of tumorigenesis. A growing body of evidence suggests that proliferating cells in a developing Drosophila imaginal disc communicate to maintain a constant disc size, and that lats plays an important role in this process. Using mutations such as Minute and dE2F in Drosophila, it has been shown that, from a young mosaic disc containing cells of different genotypes, the number of progeny 15 cells from a given parental cell can vary dramatically in a mature disc, while the overall size of the mature disc is unaffected (Simpson, 1979, Devel. Biol. 69:182-193; Simpson and Morata, 1981, Devel. Biol. 85:299-308; Brook et al., 1996, EMBO J. 15:3676-3683). Imaginal discs can also undergo regeneration when a small region of a disc is surgically removed, a phenomenon similar to liver regeneration in mammals (French et al., 1976, Science 193:969-981; Meinhardt, 1994, Bioessays 16:627-632; Michalopoulos and 20 DeFrances, 1997, Science 276:60-66). Consistent with the notion that proliferation is regulated by local cell interaction, it has been shown that DNA replication and mitosis in growing discs occur in small, non-clonal clusters of cells throughout the disc (Adler and MacQueen, 1981, Exp. Cell Res. 133:452-456; Milan et al., 1996, Proc. Natl. Acad. Sci. USA 93:11687-11692; Milan et al., 1996, Proc. Natl. Acad. Sci. USA 93:640-645). 25 Furthermore, young discs transplanted into adult hosts grow until the disc reaches its normal size, indicating that such a size control mechanism is an intrinsic property of the cells in each disc (Bryant in The Genetics and Biology of Drosophila, Vol. 2c, Ashburner and Novitski, eds. (Academic Press, New York, 1978). Transplantation experiments in mice revealed a similar size control phenomenon with anlagen of some vertebrate organs (Leitina et al., 1971, Transplantation 11:499-502). 30 Lats mutations dramatically disrupt the size and shape of discs in Drosophila. Clones of lats mutant cells in mosaic discs overproliferate to form massive outgrowths that are sometimes larger than the mature discs themselves, and animals homozygous for many lats alleles also have dramatically overgrown discs (Figures 1A and F; Xu et al., 1995, Development 121:1053-1063). These lats phenotypes indicate that an inhibitory cell-cell 35 communication mechanism has been disrupted and suggest that the lats protein could be a component of this mechanism regulating cell proliferation. The overproliferation phenotype of lats behaves in a cell autonomous fashion: inactivating lats causes mutant cells - 46 - WO 00/10602 PCT/US99/19068 to overproliferate (Xu et al., 1995, Development 121:1053-1063). Furthermore, in mosaic discs containing lats mutant clones, there is an overproliferation 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 5 proliferation but are defective in receiving such signals. While the mammalian cdc2/cyclin A complex is involved in G2/M regulation (Hamaguchi et al., 1992, J. Cell Biol. 117:1041-1053; Hunter and Pines, 1994, Cell 79:573 582), Drosophila cdc2/cyclin A functions at the GUS phase transition in addition to the G2/M phase transition. Ectopic activation of cdc2/cyclin A by overexpressing cyclin A in GI arrested cells can drive the Gl/S transition and induce S phase in cells lacking cyclin E 10 (Dong et al., 1997, Genes & Devel. 11:94-105; Sprenger et al., 1997, Curr. Biol. 7:488 499). This GUS activity is greatly enhanced when both cyclin A and an activated form of cdc2 are overexpressed. In roughex (rux) mutants, cells accumulate cyclin A in early GI and progress into S phase precociously (Thomas et al., 1994, Cell 77:1003-1014; Thomas et al., 1997, Genes & Devel. 11:1289-1298). Loss of fizzy-related (fzr), a cdc2-related fly 15 gene, results in accumulation of mitotic cyclins in G1 cells and causes progression through an extra division cycle in the embryonic epidermis (Sigrist and Lehner, 1997, Cell 90:671 68 1). These observations have shown that in Drosophila extra cdc2/Cyclin A activity can cause overproliferation. Consistent with these observations, we find that Cyclin A is abnormally accumulated in lats mutant cells (Figures 5A-N) and lats phenotypes can be suppressed by cdc2 and cyclin A mutations. Several aspects of the lats phenotype are 20 unique. First, lats mutants deregulate cdc2/cyclin A activities which affects both the Gl/S and G2/M transitions. Second, while mutants such as rux, fizzy(fzy), andfzr accumulate multiple mitotic cyclins and thus affect activities of several cdc2/cyclin complexes (Thomas et al., 1994, Cell 77:1003-1014; Dawson et al., 1995, J. Cell Biol. 129:725-737; Sigrist and Lehner, 1997, Cell 90:671-68 1), lats mutants appear to only affect cdc2/cyclin A. Finally, 25 in lats mutant cells, cyclin A is degraded at late mitosis (Figure 5F), further indicating that many aspects of the cell cycle are normal in lats mutants. These properties distinguish 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 overproliferation phenotype of the Drosophila lats mutants. 30 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. Given that p16- and p21-like CDK inhibitors have not been found for cdc2, it is possible that cdc2 and the rest of the CDKs are negatively 35 regulated by different families of proteins. Alternatively, the activity of each CDK could be modulated by both types of negative regulators. In both flies and mammals, cdc2/cyclin A is inactivated during early mitosis by degradation of cyclin A, while degradation of cyclin B - 47 - WO 00/10602 PCTIUS99/19068 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. Our data indicate that lats specifically modulates cdc2/cyclin A activity but not cdc2/cyclin B activity: cyclin A but not cyclin B mutants 5 interact with lats genetically; lats mutant cells abnormally accumulate cyclin A but not cyclin B (Figures 5A-N). Finally, overexpression of cdc2 and cyclin A has been reported in multiple types of human tumors (Wang et al., 1990, Nature 343:555-557; Keyomarsi and Pardee, 1993, Proc. Natl. Acad. Sci. USA 90:1112-1116; Arany et al., 1994, Surg. Onc. 3:153-159). Negative regulators of CDK/Cyclins (e.g., p16) have been shown to function as tumor suppressors in 10 mammals (Serrano et al., 1996, Cell 85:27-37). The biochemical and genetic data for lats provided herein suggest that lats would behave as a tumor suppressor in mammals. EXAMPLE 2: Mice Deficient for Lats Develop Soft Tissue Sarcomas, Ovarian Tumors and Pituitary Dysfunction Materials and Methods 15 Generation of lats-'' mice Mouse lats genomic DNA was isolated by screening a 129 library (Stratagene) using a mouse lats cDNA as a probe. A SalI 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 XhoI and inserted 20 a 3 kb PGK-TK gene cassette. D3 embryonic stem (ES) cells were electroporated with the SfiI linearized vector, and selected in 0.3 mg/ml G418 and 2 ptM ganciclovir media for incorporation of the vector. The ES cell clones analyzed underwent homologous recombination at the lats locus (Figure 6B). For genotyping, genomic DNA from the ES cells was digested with BamHI and 25 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 30 disrupted allele was detected in agouti progeny by Southern blotting. Cell culture and protein analysis Proteins were extracted from whole-cell lysates of lats' mouse embryonic fibroblasts (MEFs) derived from 13-days post-coitum mouse embryos, separated using 35 SDS-PAGE, transferred and probed with rabbit polyclonal anti-lats antibody, followed by enhanced chemiluminescence detection (Amersham). -48- WO 00/10602 PCT/US99/19068 Histopathological examinations For morphological evaluation, 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 5 hematoxylin and eosin. Gonadotropin treatment 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). 10 Pituitary hormone measurements We 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). 15 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 tumorigenic treatments UVB and DMBA treatments were performed as described by Serrano et al. (1996, 20 Cell 85:27-37). Briefly, skin tumors were induced by first applying a single dose of 9,10 dimethyl-1,2-benzathralene (DMBA; 50 1 .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 25 10% per treatment to a maximum of 700 mJ/cm2. Results and Discussion Targeted Disruption of the mouse lats gene A 17.5 kilobase lats genomic clone obtained from a mouse 1295V library was used to construct a targeting vector for homologous recombination by positive-negative selection 30 (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. Sci USA 83:9065-9069) with the lats-neo construct; 35 single clones resistant to G418 and ganciclovir were screened by Southern blot hybridization using a mouse lats probe 5' to the portion of the lats gene contained in the targeting vector (Figure 6B). Restriction enzyme digestion of the wild-type lats locus with - 49 - WO 00/10602 PCT/US99/19068 BamHI and EcoRV generated a 3.5 kb fragment, while the correctly targeted, disrupted locus generated a 5.8 kb fragment (Figures 6B and C). A targeting frequency of approximately 1 in 100 was observed. Male chimeras transmitted the targeted lats allele through the germline, as demonstrated by Southern blot analysis of tail DNA. 5 Immunoprecipitation and western blotting of lats-/- mouse embryonic fibroblast lysates (derived from 13.5 days post-coitum embryos (dpc)) with polyclonal anti-human lats antibody confirmed the absence of lats protein in homozygous null embryos (Figure 6D). Growth and viability of lats-'- mice When assessed at 3 weeks of age, the number of lats mutant animals was drastically 10 lower than expected; 8% of pups genotyped from double heterozygote matings were lats, significantly lower than the expected frequency of 25%. Just before birth at embryonic day 18.5 (dpc), however, live homozygous embryos were found at the frequency predicted by Mendelian law (25%). The majority of homozygotes died within the first day of life, and their death was associated with internal hemorrhage into vital organs. The reason that some 15 lats- mice survived while most did not is uncertain, but the mixed genetic background (strains 129 and C57BL/6) of the mice could be contributory. The weight of homozygous embryos at birth was approximately 70% of that of wild-type embryos (lats", 1.5 ± 0.3 g; lats~'-, 1.3 ± 0.3 g; lats~-, 1.1 ± 0.1 g). All of these lats-'- mice were growth retarded. Most of the surviving lats homozygous mutant animals gained weight slowly, attaining only 60 70% of normal weight as adults (Figures 7A and B). A representative growth curve for lats 20 deficient mice is shown in Figure 7B. To determine if there was a correlation between weight and growth, skeletal growth and organ weight was examined. By radiography, we observed differences in the skull and longitudinal bones that corresponded to the decrease in the size of the mouse. The decreased body weight was not due to the animals being leaner than their wild-type siblings, because the weight of most organs tested had decreased in 25 proportion to the whole body weight. Exceptions were seen in a few particularly reduced organs, including the testis and the ovary. Pituitary dysfunction Male lats' mice displayed decreased fertility although histopathological examination of the testis did not reveal obvious structural abnormalities. Lats-' females all 30 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 35 granulosa cells, which are common in atretic follicles in normal mice. Moreover, the formation of a corpus luteum was not detected (Figures 8A-D). These histological findings suggest an impairment of ovulation in lats-'- ovaries, and potential endocrine dysfunction. - 50 - WO 00/10602 PCT/US99/19068 The amount of breast epithelial tissue was markedly decreased in lats' females, with some females displaying a complete lack of macroscopic nipple-formation (Figures 9D and E). Histologically, the mammary glands of lats' mice were frequently reduced to a "fat pad" devoid of a ductular component (Figure 9F). This too suggested an endocrine 5 component to the phenotype, as loss of lats may alter the levels of hormones that affect breast development. 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 10 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. To determine if lats-- ovaries could respond to appropriate gonadotropin stimulation, young adult mice (7 lats' females; 2 control females) were injected with a Follicle-Stimulating Hormone (FSH) analog (pregnant mare serum), and 46 hours later with a Luteinizing 15 Hormone (LH) preparation (human chorionic gonadotropin). This treatment allowed lats females to cycle into a prolonged period of estrus, confirming that the temporal synchronization of the levels of endogenous FSH and/or LH in lats-' females is deficient. In all lats knock-out mouse pituitaries examined (n=20), hyperplastic changes were readily apparent. There were multiple foci of atypical cells showing irregularly shaped nuclei with an increased content of chromatin and variability in size (Figures 10A and B). 20 This histological atypia was accompanied by a pathological dysfunction of the pituitary. For example, hormone measurements in sera from lats' females revealed: the levels of LH were 3-fold lower than controls (Figure 0C); the PRL levels were less than half of controls (Figure IOD); and Growth Hormone (GH) levels were reduced by 25%. These deficiencies may have resulted from an unbalanced increase in the number of certain types of cells in 25 this normally highly organized tissue. 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 corpus luteum insufficiency syndrome which these animals display. 30 Interestingly, serum levels of pituitary Follicle Stimulating Hormone (FSH) (Figure 1OE) and Thyroid Stimulating Hormone (TSH) are normal. The observation that lats' mice display a selective LH deficiency while sustaining normal FSH production supports a model of differential regulation of the two gonadotropins. This is in accordance with data from Lee and coworkers demonstrating that the transcription factor NGFI-A specifically 35 regulates LH-8 (Lee et al., 1996, Science 273:1219-1221). Lats' mice thereby provide a model for the human reproductive dysfunction of isolated LH hypogonadotropic hypogonadism. -51 - WO 00/10602 PCTIUS99/19068 It is interesting to note that the pituitary deficiencies of lats' mice resembles those of other cell cycle regulator knock-out mice, such as the Rb ', p53-, and p2 7 /- mice. In these examples, pituitary cells and other endocrine organs appear to be crucially dependent on cell cycle regulation for their proper development. Indeed, tumor suppressors may play 5 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. Tumor development in lats' mice Another cause for the infertility of lats-deficient females is that they developed ovarian stromal cell tumors by the age of 3 months. The body of the normal ovary consists 10 of spindle-shaped cells, reticular fibers and ground substance which together constitute the ovarian stroma, in which numerous follicles are embedded (Wheater et al., 1987, Functional Histology; Figure 8A). Stromal cell tumors in lats' mice obliterated the normal structure of the ovary, eliminating follicles progressively (Figures 8B and D). These stromal cell tumors are probably not resultant from pituitary dysfunction, as stromal cell tumors are 15 most often local events (Clement, "Histology of the Ovary" in Histology For Pathologists, Second Ed., Sternberg, 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. Stromal cell tumors were observed in all lats deficient mice examined (n=22), and extended throughout the entire ovary as determined by serial 20 sectioning. Additional evidence supporting the role of lats in mammalian tumorigenesis is that thus far, approximately 15% of all lats' females (n=28) between 4 to 10 months of age have developed large soft tissue sarcomas with metastases to vital organs (e.g. the lungs) (Figures I lA-C). Taking genetic backgrounds into consideration and using litter mates as controls, 25 approximately 57% of all latsA animals developed soft tissue sarcomas. Histology revealed that these sarcomas consist of pleiomorphic, spindle-shaped cells with mitotic figures (Figure 11 C), and they displayed immunohistochemical features of fibrosarcomas. No tumors were detected in control mice (n=80). It is possible that the spontaneous rate of tumor formation is even higher in lats-- mice as some tumors may be occult, and therefore not readily identifiable. Lats"- mice remained tumor free up to 8 months of age. This 30 phenotype is consistent with that observed in heterozygotes for the CDK inhibitors, p27 and p16 (Fero et al., 1996, Cell 85:733-744; Kiyokawa et al., 1996, Cell 85:721-732; Nakayama et al., 1996, Cell 85:707-720; and Serrano et al., 1996, Cell 85:27-37). The susceptibility of lats' mice to tumor induction by carcinogens was assessed using a standard two-stage protocol consisting of a single application of 9,10-dimethyl-1,2 35 benzanthracene (DMBA) followed by repeated exposure to ultraviolet B rays (Serrano et al., 1996, Cell 85:27-37). By 7 weeks of age, over 71% of lats- animals developed soft tissue sarcomas, whereas all of the control animals remained free of obvious tumors. The - 52 - WO 00/10602 PCT/US99/19068 frequency of tumor development in these lats-' animals is particularly impressive, given the fact that C57BL/6 mice are poorly susceptible to the development of skin tumors (as reviewed by DiGiovanni, 1991, Pharm. & Ther. 54:63-128). The spontaneous and induced tumors observed in lats' animals provide clear evidence of the role of lats in mammalian 5 tumorigenesis, and attest to the functional conservation of lats. Although both Drosophila and mice develop tumors when lats is inactivated, the correlation between genotype and phenotype differs between the two organisms. While every lats- cell overproliferates in mosaic flies (Xu et al. 1995, Development 121:1053 1063), only certain tissues in lats' mice develop tumors. This phenotypic difference could be due to the increased complexity in cell cycle control in mammals. For example, in 10 Drosophila, cdc2/Cyclin A functions at both the Gl/S and G2/M transitions in the cell cycle (Whitfield et al., 1990, EMBO J. 9:2563-2573; Knoblich and Lehner, 1993, EMBO J. 12:65-74; Knoblich et al., 1994, Cell 77:117-120; Sauer et al., 1995, Genes & Devel. 9:1237-1239; Sigrist and Lehner, 1997, Cell 90:671-681; Sprenger et al., 1997, Curr. Biol. 7:488-499; Thomas et al., 1997, Genes & Dev. 11:1289-1298). In mammals, however, a 15 different CDK, CDK2, complexes with cyclin A to regulate the Gl/S transition, while cdc2/cyclin A functions in the G2/M transition (Girard et al., 1991, Cell 67:1169-1179; Tsai et al., 1991, Nature 353:174-177; Hamaguchi et al., 1992, J. Cell. Biol. 117:1041-1053; Pagano et al., 1992, EMBO J. 11:961-967; Rosenblatt et al., 1992, Proc. Natl. Acad. Sci. USA 89:2824-2828; Hunter and Pines, 1994, Cell 79:573-582; Resnitzky et al., 1995, Mol. Cell Biol. 15:4347-4352). We have shown that lats negatively regulates cdc2/cyclin A, but 20 does not appear to interact with CDK2. Thus, while inactivation of lats in Drosophila affects regulation of both the Gl/S and G2/M transitions of the cell cycle, the removal of lats function in mammalian cells only affects control of the G2/M transition. This result is consistent with the fact that human cancers are often caused by mutations in multiple, non homologous cell cycle control pathways (Kinzler and Vogelstein, 1996, Cell 87:159-170; 25 Weinberg, 1996, Sci. Am. 275:62-70). The higher degree of redundancy in mammalian genomes could also contribute to the phenotypic difference between fly and mouse lats mutants. Although most human tumor suppressors that have been characterized function in the regulation of Gl/S (Brown, 1997, Adv. Genet. 36:45-135), there are indications that deregulation of G2/M and M controls may also contribute to tumorigenesis in mammals. 30 For example, p53 is involved in the regulation of both Gl/S and G2/M (and M) (Cross et al., 1995, Science 267:1353-1356; Hermeking et al., 1997, Mol. Cell 1:3-11). Inactivation of p53 in mice leads to impressive tumor development, while the disruption of p21, the p53 downstream effector for G1/S, has no tumorigenic effect (Deng et al., 1995, Cell 82:675 684). Recent data also suggests that Rb plays a role in G2/M in addition to its Gl/S 35 involvement (Niculescu et al., 1998, Mol. Cell Biol. 18:629-643). Furthermore, overexpression of cdc2 and mitotic cyclins has been reported in multiple types of human tumors (Arany et al., 1994, Surg, Oncol. 3:153-159; Keyomarsi and Pardee, 1993, Proc. - 53 - WO 00/10602 PCTIUS99/19068 Natl. Acad. Sci USA 90:1112-1116; Wang et al., 1990, Nature 343:555-557). Signaling through the RHAMM receptor affects cell proliferation by down-regulation of cdc2 and cyclin B transcripts and proteins and results in reversal of tumorigenesis (Mohapatra et al., 1996, J. Exper. Med. 183:1663-1668). The tumorigenic phenotype of lats- mice further 5 supports the notion that the regulation of G2/M or M also plays a role in mammalian tumor development. A comparison between lats- and p16-knock-out mice is interesting in several respects. Both lats and p16 are negative regulators of CDKs. In addition, lats-knock-out mice resemble p16-knock-out animals (Serrano et al., 1996, Cell 85:27-37) in that homozygotes develop tumors at an early age while heterozygotes do not. Although 10 different types of tumors are observed in these two mutants (e.g., ovarian tumors in lats mice and lymphomas in p16/ mice), both types of knock-out mice develop soft tissue sarcomas. The frequencies of spontaneous soft tissue sarcomas between these two knock out mutants cannot be directly compared due to protocol differences, however, induced tumors in these animals were obtained using the same induction protocol (Serrano et al., 15 1996, Cell 85:27-37). Interestingly, the frequency of induced tumor formation in lats knock-out mice is even higher than that observed for p16' mice. Over 71% of lats' animals developed soft tissue sarcomas by 7 weeks of age. Only 10% of the p]6' mice developed tumors by 7 weeks of age, and 60% of them displayed tumors between the ages of 6-12 weeks (Serrano et al., 1996, Cell 85:27-37). The present invention is not to be limited in scope by the specific embodiments 20 described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. 25 30 35 - 54 -

Claims (113)

1. A recombinant non-human animal in which a lats gene has been inactivated by a method comprising introducing a nucleic acid into the animal, or an ancestor thereof, 5 which nucleic acid comprises a non-lats sequence flanked by lats genomic sequences that promote homologous recombination, such that said non-lats sequence replaces the nucleotide sequence encoding the Lats C-terminal domain 1, the Lats C-terminal domain 2, the Lats C-terminal domain 3, and a portion of the kinase domain of the lats protein encoded by the lats gene. 10

2. The recombinant non-human animal of claim I in which the non-lats sequence replaces the nucleotide sequence encoding the amino acids that correspond to amino acids 756-1130 of human lats, as depicted in Figure 12 (SEQ ID NO:2).

3. The recombinant non-human animal of claim I which is a mouse. 15

4. The recombinant non-human animal of claim 3 in which the lats gene contains a lats coding sequence of SEQ ID NO:3.

5. The recombinant non-human animal of claim 1 in which both alleles of the lats gene have been inactivated. 20

6. A method for screening a potential therapeutic compound for activity in treating or preventing cancer comprising administering the compound to the recombinant non-human animal of claim 1; and comparing the size or progression of the cancer in the recombinant non-human animal to which the compound was administered with the size or 25 progression of the cancer in the same recombinant non-human animal prior to administration of the compound or in a recombinant non-human animal that was not so administered or to a standard size or progression of the cancer for such same or a recombinant non-human animal that was not so administered, wherein a decrease in the size or progression of the cancer in the recombinant non-human animal administered the compound as compared to the same animal prior to the administration or to the recombinant 30 non-human animal not so administered or to the standard size or progression of the cancer, indicates that the compound has activity in treating or preventing cancer.

7. The method of claim 6 in which the non-lats sequence replaces the nucleotide sequence encoding the amino acids that correspond to amino acids 756-1130 of 35 human lats, as depicted in Figure 12 (SEQ ID NO:2). - 55 - WO 00/10602 PCT/US99/19068

8. The method of claim 6 in which the recombinant non-human animal is a mouse.

9. The method of claim 8 in which the lats gene contains the lats coding 5 sequence of SEQ ID NO:3.

10. The method of claim 6 in which both alleles of the lats gene have been inactivated.

11. The method of claim 6 in which the compound is screened for activity in 10 treating or preventing soft tissue sarcomas.

12. The method of claim 6 in which the compound is screened for activity in treating or preventing ovarian tumors. 15

13. A method for screening a potential therapeutic compound for activity in treating or preventing cancer comprising recombinantly expressing the compound in the recombinant non-human animal of claim 1; and comparing the size or progression of the cancer in the recombinant non-human animal in which the compound was expressed with the size or progression of the cancer in the same recombinant non-human animal prior to expression of the compound or in a recombinant non-human animal in which the compound 20 was not so expressed or to a standard size or progression of the cancer for such same or a recombinant non-human animal in which the compound was not so expressed, wherein a decrease in the size or progression of the cancer in the recombinant non-human animal in which the compound was expressed as compared to the same animal prior to the expression of the compound or to the recombinant non-human animal in which said compound was not 25 so expressed or to the standard size or progression of the cancer, indicates that the compound has activity in treating or preventing cancer.

14. A method for screening a potential therapeutic compound for activity in treating or preventing skin cancer comprising administering the compound to a lats knock out animal having skin tumors induced by exposure to at least one carcinogen; and 30 comparing the size or progression of the skin tumors on the lats knock-out animal to which the compound was administered with the size or progression of skin cancers on the same lats knock-out animal prior to administration of the compound or on a lats knock-out animal in which skin tumors have also been induced by exposure to said at least one carcinogen but which has not been administered the compound or to a standard size or 35 progression of the skin tumors for such same or a lats knock-out animal that was not so administered, wherein a reduction in the size or progression of the skin tumors in the lats knock-out animal administered the compound as compared to the same animal prior to - 56 - WO 00/10602 PCT/US99/19068 administration of the compound or to the animal not so administered or to the standard size or progression of the skin tumors, indicates that the compound has activity in treating or preventing skin cancer. 5

15. The method of claim 14 in which the lats knock-out animal has at least one lats gene which was inactivated by promoting homologous recombination between lats genomic sequences and a nucleic acid having non-lats sequences flanked by genomic sequences.

16. The method of claim 15 in which the non-lats sequence replaces the 10 nucleotide sequence encoding the Lats C-terminal domain 1, the Lats C-terminal domain 2, the Lats C-terminal domain 3, and a portion of the kinase domain of the lats protein encoded by the lats gene.

17. The method of claim 15 in which the non-lats sequence replaces the 15 nucleotide sequence encoding the amino acids that correspond to amino acids 756-1130 of human lats, as depicted in Figure 12 (SEQ ID NO:2).

18. The method of claim 14 in which the lats knock-out animal is a mouse.

19. The method of claim 18 in which the lats gene contains the lats coding 20 sequence of SEQ ID NO:3.

20. The method of claim 14 in which both alleles of the lats gene have been inactivated. 25

21. The method of claim 14 in which the skin tumors were induced by 9,10 dimethyl-1,2-benzanthracene and repeated exposure to ultraviolet B radiation.

22. The method of claim 14 in which the potential therapeutic compound is administered topically. 30

23. A method for screening a potential therapeutic compound for activity in treating or preventing skin cancer comprising recombinantly expressing the compound in a lats knock-out animal having skin tumors induced by exposure to at least one carcinogen; and comparing the size or progression of the skin tumors on the lats knock-out animal in which the compound was expressed with the size or progression of skin cancers on the same 35 lats knock-out animal prior to expression of the compound or on a lats knock-out animal in which skin tumors have also been induced by exposure to said at least one carcinogen but in which the compound has not been expressed or to a standard size or progression of the skin - 57 - WO 00/10602 PCT/US99/19068 tumors for such same or a lats knock-out animal in which the compound was not so expressed, wherein a reduction in the size or progression of the skin tumors in the lats knock-out animal in which the compound was expressed as compared to the same animal prior to expression of the compound or to the animal in which the compound was not so 5 expressed or to the standard size or progression of the skin tumors, indicates that the compound has activity in treating or preventing skin cancer.

24. A method for screening a potential therapeutic compound for activity in treating or preventing a disease or disorder associated with pituitary dysfunction comprising administering the compound to a lats knock-out animal; and comparing the level of an 10 indicator of pituitary dysfunction in the lats knock-out animal to which the compound has been administered to the level of the indicator in the same lats knock-out animal prior to administration of the compound or to a lats knock-out animal that has not been administered the compound or to a standard level of the indicator for such same or a lats knock-out animal that was not so administered, wherein a change in the indicator toward the 15 level of the indicator in a wild type animal not afflicted with a pituitary dysfunction as compared to the same animal prior to administration of the compound or to the animal not so administered or to the standard level of the indicator, indicates that the compound is active to treat or prevent a disease or disorder associated with pituitary dysfunction.

25. The method of claim 24 in which the lats knock-out animal has at least one 20 lats gene which was inactivated by promoting homologous recombination between lats genomic sequences and a nucleic acid having non-lats sequences flanked by genomic sequences.

26. The method of claim 25 in which the non-lats sequence replaces the 25 nucleotide sequence encoding the Lats C-terminal domain 1, the Lats C-terminal domain 2, the Lats C-terminal domain 3, and a portion of the kinase domain of the lats protein encoded by the lats gene.

27. The method of claim 25 in which the non-lats sequence replaces the nucleotide sequence encoding the amino acids that correspond to amino acids 756-1130 of 30 human lats, as depicted in Figure 12 (SEQ ID NO:2).

28. The method of claim 24 in which the lats knock-out animal is a mouse.

29. The method of claim 28 in which the lats gene contains a lats coding 35 sequence of SEQ ID NO:3. - 58 - WO 00/10602 PCT/US99/19068

30. The method of claim 24 in which both alleles of the lats gene have been inactivated.

31. The method of claim 24 in which the indicator is fertility. 5

32. The method of claim 24 in which the indicator is ovulation.

33. The method of claim 24 in which the indicator is linear growth.

34. The method of claim 24 in which the indicator is serum levels of luteinizing 10 hormone, growth hormone or prolactin.

35. The method of claim 24 in which the disease or disorder is LH hypogonadotropic hypogonadism. 15

36. A method for screening a potential therapeutic compound for activity in treating or preventing a disease or disorder associated with pituitary dysfunction comprising recombinantly expressing the compound in a lats knock-out animal; and comparing the level of an indicator of pituitary dysfunction in the lats knock-out animal in which the compound has been expressed to the level of the indicator either in the same lats knock-out animal prior to expression of the compound or to a lats knock-out animal in which the 20 compound has not been expressed or to a standard level of the indicator for such same or a lats knock-out animal in which the compound was not so expressed, wherein a change in the indicator toward the level of the indicator in a wild type animal not afflicted with a pituitary dysfunction as compared to the same animal prior to expression of the compound or to the animal in which the compound was not so expressed or to the standard level of the indicator, 25 indicates that the compound is active to treat or prevent a disease or disorder associated with pituitary dysfunction.

37. The method of claim 6, 14 or 24 in which the compound is purified.

38. A method for treating a cancer that has been shown to be refractory to a 30 chemotherapy or radiation therapy in a subject in need of such treatment comprising administering to the subject a therapeutically effective amount of a molecule that promotes lats function.

39. The method of claim 38 in which the subject is a human.

40. The method of claim 38 in which the molecule is a lats protein. - 59 - WO 00/10602 PCT/US99/19068

41. The method of claim 38 in which the molecule is a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8. 5

42. The method of claim 38 in which the molecule is a protein having the amino acid sequence of SEQ ID NO:2.

43. The method of claim 38 in which the molecule is a lats analog or derivative that has activity to promote lats function. 10

44. The method of claim 38 in which the molecule is a protein encoded by a first nucleic acid that is hybridizable under conditions of low stringency to a second nucleic acid having a nucleotide sequence that is the reverse complement of a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, said protein having activity to inhibit cell overproliferation. 15

45. The method of claim 38 in which the molecule is a protein consisting of at least 20 contiguous amino acids of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, said protein having activity to inhibit cell overproliferation. 20

46. The method of claim 38 in which the molecule is a protein comprising a domain of a lats protein selected from the group consisting of a lats C-terminal domain 3 (LCD3), lats C-terminal domain 2 (LCD2), lats C-terminal domain 1 (LCD 1), kinase domain, lats flanking domain (LFD), lats split domain 1 (LSDI), lats split domain 2 (LSD2), and SH3-binding domain, said protein having activity to inhibit cell 25 overproliferation.

47. The method of claim 40 in which the lats protein is phosphorylated.

48. The method of claim 47 in which the lats protein is phosphorylated on a seine or threonine residue within 20 residues upstream of the amino acid sequence Ala-Pro 30 Glu in a subdomain eight of a kinase domain of said lats protein.

49. The method of claim 48 in which the lats protein is phosphorylated at a seine residue corresponding to shrine 909 of the human lats amino acid sequence, as depicted in Figure 12 (SEQ ID NO:2).

50. The method of claim 43 in which the lats analog or derivative has a threonine or seine residue within 20 residues upstream of the amino acid sequence Ala-Pro-Glu in a - 60 - WO 00/10602 PCT/US99/19068 subdomain eight of a kinase domain of said lats analog or derivative substituted with an aspartate or glutamate residue.

51. The method of claim 50 in which the lats analog or derivative has a 5 glutamate residue substituted for a seine residue at the residue corresponding to serine 909 of the human lats amino acid sequence, as depicted in Figure 12 (SEQ ID NO:2).

52. The method of claim 38 in which said molecule is a chimeric protein comprising a fragment of a lats protein, said fragment consisting of at least 20 contiguous amino acids of said lats protein, fused via a covalent bond to an amino acid sequence of a 10 second protein, said second protein not being a lats protein, said chimeric protein having activity to inhibit cell overproliferation.

53. The method of claim 38 in which said cancer has been shown to be refractory to radiation therapy. 15

54. The method of claim 38 in which said cancer has been shown to be refractory to chemotherapy.

55. The method of claim 54 in which said chemotherapy kills cancer cells during S phase of the cell cycle. 20

56. The method of claim 54 in which said chemotherapy kills cancer cells during mitosis.

57. The method of claim 38 which further comprises administering one or more 25 chemotherapeutic agents to the subject.

58. The method of claim 57 in which said one or more chemotherapeutic agents are administered concurrently with the administration of said molecule.

59. The method of claim 57 in which said one or more chemotherapeutic agents 30 are administered subsequent to the administration of said molecule.

60. The method of claim 38 in which said molecule is a nucleic acid comprising a nucleotide sequence encoding a lats protein. 35 61. The method of claim 60 in which said nucleotide sequence is SEQ ID NO:1.

- 61 - WO 00/10602 PCT/US99/19068

62. The method of claim 60 in which said nucleotide sequence encodes a protein having the amino acid sequence of SEQ ID NO:2.

63. The method of claim 38 in which said molecule is a first nucleic acid that 5 hybridizes under low stringency conditions to a second nucleic acid that is the reverse complement of a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7.

64. A method for treating a cancer that has been shown to be refractory to a chemotherapy or radiation therapy in a subject in need of such treatment comprising 10 administering to the subject a therapeutically effective amount of a cell that expresses a recombinant nucleic acid that promotes lats function.

65. The method of claim 64 in which said nucleic acid comprises the nucleotide sequence of SEQ ID NO:1. 15

66. The method of claim 64 in which said nucleic acid comprises a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:2.

67. A kit comprising in one or more containers a therapeutically effective amount of a molecule selected from the group consisting of a lats protein, a lats derivative, a 20 lats analog, a nucleic acid encoding a lats protein, a nucleic acid encoding a lats derivative, and a nucleic acid encoding a lats analog; and at least one chemotherapeutic agent.

68. A purified complex of a lats protein and a cdc2 protein. 25

69. The purified complex of claim 68 in which the proteins are human proteins.

70. The purified complex of claim 68 in which the lats protein is phosphorylated.

71. The purified complex of claim 70 in which the lats protein is phosphorylated on a serine or threonine residue within 20 residues upstream of the amino acid sequence 30 Ala-Pro-Glu in a subdomain eight of a kinase domain of said lats protein.

72. The purified complex of claim 71 in which the lats protein is phosphorylated at a seine residue corresponding to seine 909 of the human lats amino acid sequence, as depicted in Figure 12 (SEQ ID NO:2).

73. A purified complex selected from the group consisting of a complex of a derivative of a lats and a cdc2 protein, a complex of a lats protein and a derivative of a cdc2, - 62 - WO 00/10602 PCTIUS99/19068 and a complex of a derivative of a lats protein and a derivative of a cdc2 protein, in which the derivative of the lats protein is able to form a complex with a wild-type cdc2 protein and the derivative of the cdc2 is able to form a complex with a wild-type lats protein. 5

74. The purified complex of claim 73 in which the derivative of the lats protein and/or the cdc2 protein is fluorescently labeled.

75. The purified complex of claim 73 in which the lats derivative has a threonine or seine residue within 20 residues upstream of the amino acid sequence Ala-Pro-Glu in a subdomain eight of a kinase domain of said lats derivative substituted with an aspartate or 10 glutamate residue.

76. The purified complex of claim 75 in which the lats derivative has a glutamate residue substituted for a serine residue at the residue corresponding to serine 909 of the human lats amino acid sequence, as depicted in Figure 12 (SEQ ID NO:2). 15

77. The purified complex of claim 73 in which the lats derivative is a fragment of a lats protein consisting of the amino acid sequence corresponding to amino acids 15-585 of the amino acid sequence of human lats, as depicted in Figure 12 (SEQ ID NO:2).

78. A chimeric protein comprising a fragment of a lats protein consisting of at 20 least 6 amino acids fused via a covalent bond to a fragment of a cdc2 protein consisting of at least 6 amino acids.

79. The chimeric protein of claim 78 in which the fragment of the lats protein is a fragment capable of binding the cdc2 protein and in which the fragment of the cdc2 25 protein is a fragment capable of binding the lats protein.

80. The chimeric protein of claim 78 in which the fragment of the lats protein has an amino acid sequence corresponding to amino acids 15 to 585 of the amino acid sequence of human lats, as depicted in Figure 12 (SEQ ID NO:2). 30

81. The chimeric protein of claim 79 in which the fragment of the lats protein and the fragment of the cdc2 protein form a lats-cdc2 complex.

82. An antibody which immunospecifically binds the complex of claim 68 or a fragment or derivative of said antibody containing the binding domain thereof.

83. The antibody of claim 82 which does not immunospecifically bind a lats protein or a cdc2 protein that are not part of a lats-cdc2 complex. - 63 - WO 00/10602 PCT/US99/19068

84. An isolated nucleic acid or an isolated combination of nucleic acids comprising a nucleotide sequence encoding a lats protein and a nucleotide sequence encoding a cdc2 protein. 5

85. The isolated nucleic acid or isolated combination of nucleic acids of claim 84 which are nucleic acid vectors.

86. An isolated nucleic acid that comprises a nucleotide sequence encoding the chimeric protein of claim 78. 10

87. A cell containing the nucleic acid of claim 84, which nucleic acid is recombinant.

88. A cell containing the nucleic acid of claim 86, which nucleic acid is recombinant. 15

89. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the complex of claim 68; and a pharmaceutically acceptable carrier.

90. The pharmaceutical composition of claim 89 in which the proteins are human 20 proteins.

91. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the complex of claim 73; and a pharmaceutically acceptable carrier. 25

92. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the chimeric protein of claim 79; and a pharmaceutically acceptable carrier.

93. A pharmaceutical composition comprising a therapeutically or 30 prophylactically effective amount of the antibody of claim 83 or a fragment or derivative of said antibody containing the binding domain thereof; and a pharmaceutically acceptable carrier.

94. A pharmaceutical composition comprising a therapeutically or 35 prophylactically effective amount of the nucleic acids or combination of nucleic acids of claim 84; and a pharmaceutically acceptable carrier. - 64 - WO 00/10602 PCT/US99/19068

95. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the isolated nucleic acid of claim 86; and a pharmaceutically acceptable carrier. 5

96. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the recombinant cell of claim 87; and a pharmaceutically acceptable carrier.

97. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the recombinant cell of claim 88; and a 10 pharmaceutically acceptable carrier.

98. A method of producing a complex of a lats protein and a cdc2 protein comprising growing a recombinant cell containing the nucleic acid of claim 84 such that the encoded lats and cdc2 proteins are expressed and bind to each other, and recovering the 15 expressed complex of the lats protein and the cdc2 protein.

99. A method of diagnosing or screening for the presence of or a predisposition for developing a disease or disorder characterized by an aberrant level of a complex of a lats protein and a cdc2 protein in a subject comprising measuring the level of said complex, RNA encoding the lats and the cdc2 proteins, or functional activity of said complex, in a 20 sample derived from the subject, in which an increase or decrease in the level of said complex, said RNA encoding lats and cdc2, or functional activity of said complex in the sample, relative to the level of said complex, said RNA encoding lats and cdc2, or functional activity of said complex found in an analogous sample from a subject not having the disease or disorder or a predisposition for developing the disease or disorder, indicates 25 the presence of the disease or disorder or a predisposition for developing the disease or disorder.

100. A kit comprising in one or more containers a substance selected from the group consisting of a complex of a lats and a cdc2 protein, an antibody against said complex, nucleic acid probes capable of hybridizing to RNA of lats and RNA of cdc2, or 30 pairs of nucleic acid primers capable of priming amplification of at least a portion of a gene for lats and a gene for cdc2.

101. A method for modulating the activity of cdc2 comprising administering a molecule that promotes, inhibits, or antagonizes lats function.

102. A method for inhibiting the activity of cdc2 comprising administrating a molecule that promotes lats function. - 65 - WO 00/10602 PCT/US99/19068

103. A method for increasing the activity of cdc2 comprising administering a molecule that inhibits or antagonizes lats function.

104. A method for treating or preventing a disease or disorder associated with an 5 aberrantly high level of cdc2 in a subject in need of such treatment or prevention comprising administering to the subject a therapeutically effective amount of a molecule that promotes lats function.

105. The method of claim 104 in which said molecule is selected from the group consisting of a lats protein, a lats derivative or analog that promotes lats function, a nucleic 10 acid encoding a lats protein, and nucleic acid encoding a lats derivative or analog that promotes lats function, and a lats agonist.

106. A method for treating or preventing a disease or disorder associated with an aberrantly low level of cdc2 activity in a subject in which such treatment or prevention is 15 desired comprising administering to the subject a therapeutically effective amount of a molecule that inhibits or antagonizes lats function.

107. The method of claim 106 in which said molecule is selected from the group consisting of a lats analog or derivative that inhibits or antagonizes lats function, an anti-lats antibody, and a lats antisense nucleic acid. 20

108. A method for screening a molecule for efficacy in treating or preventing a cancer refractory to chemotherapy or radiation therapy, said method comprising contacting cancer cells that are refractory to treatment with chemotherapeutic agents or radiation with the molecule and comparing the proliferation or survival of the contacted cells with the 25 proliferation or survival of cells not so contacted, wherein a lower level of proliferation or survival of the contacted cells indicates that the molecule is effective to treat or prevent the cancer.

109. The method of claim 108 in which said cells are cultured in vitro from a tissue sample of a patient. 30

110. A method for screening a molecule for activity to modulate cdc2 levels or activity comprising contacting cells with the molecule, and comparing the level of cdc2 protein, mRNA or activity in cells contacted with the molecule to the amount of cdc2 protein, mRNA, or activity in cells not so contacted, wherein an increase or decrease in the 35 amount of cdc2 protein, mRNA, or activity in the contacted cells relative to the amount of cdc2 protein, mRNA, or activity in the cells not so contacted indicates that the molecule has activity to modulate cdc2 levels or activity. - 66 - WO 00/10602 PCT/US99/19068

111. A method for screening a molecule for activity to modulate, 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 under conditions conducive to formation of the complex; and comparing the levels of said 5 complex with the levels of said complex that are formed in the absence of said molecule, wherein a lower or higher level of said complex in the presence of said molecule indicates that the molecule modulates formation of said complex.

112. The method of claim 111 in which the molecule inhibits formation of the complex. 10

113. The method of claim 111 in which the molecule promotes formation of the complex. 15 20 25 30 35 - 67 -