Patent Publication Number: US-2006010505-A1

Title: High throughput cancer pharmaceutical screening using drosophila

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
      This application claims priority to U.S. Ser. No. 60/580,769 filed Jun. 18, 2004 and U.S. Ser. No. 60/580,897 filed Jun. 18, 2004; 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT  
      Not applicable.  
     REFERENCE TO A SEQUENCE LISTING  
      The Sequence Listing, which is a part of the present disclosure, includes a text file comprising nucleotide and/or amino acid sequences of the present invention on a floppy disk. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.  
     BACKGROUND OF THE INVENTION  
      1. Field of the Invention  
      The present invention relates in general to the field of drug assaying techniques, and in particular to a novel high throughput screening assay for screening libraries of candidate compounds for treating human diseases and conditions including cancer and cancer-related conditions.  
      2. Description of the Related Art  
      Recent scientific and technological advances have introduced new opportunities and challenges for drug discovery research. The increased availability of chemical libraries, including peptide and oligonucleotide libraries, and robotic systems enable virtually simultaneous synthesis and testing of hundreds or thousands of compounds. However, while screening of large numbers of candidate compounds is a critical early step in drug discovery and development, it can also be a bottleneck.  
      High throughput screening (HTS) assays and techniques of various types are typically used to screen chemical libraries consisting of large numbers of small molecules for their ability to suppress or enhance disease processes. Cell-free assays provide, for example, identification of putative drug targets implicated in a specific disease condition, such as a specific enzymatic reaction. Cell-based assays, for example, can provide insights into mechanisms underlying disease pathogenesis, and can also provide information on possible toxicity of candidate compounds. In either case, the goal of such screening is to identify the most likely candidates or “lead compounds” for use in further drug discovery and developments efforts, and not to identify a specific drug. The strength of a particular screening technique lies substantially in its ability to rapidly and efficiently screen large libraries of compounds while remaining cost effective.  
      Automated HTS assays and techniques and robotic systems for drug discovery have been described. The ability to perform a wide variety of biochemical and molecular biology tests using automated systems is widely known, including the ability to perform tests utilizing enzymatic activity, ELISA, receptor binding, macromolecular interactions, protein expression, and protein folding and assembly. Screens are typically carried out using multi-well microtiter plates. In drug discovery, a typical example of high throughput capacity is about one hundred to a few hundred plates per week depending on desired number of data points, the time required for all underlying biochemical reactions to occur, and the relative complexity of the scoring system used to determine whether a compound has an effect. A premium therefore exists on methods that simplify and speed detection of assay results.  
      A small molecular weight compound high throughput screening system using genetically modified  Drosophila melanogaster  has been described in U.S. Pub. No.: U.S. 2002/0026648 A1. Compounds of interest are microinjected into the open hemolymph of genetically manipulated  Drosophila  that have been modified with mutations within a selected signaling pathway of interest. However, microinjection of compounds of interest into numerous  Drosophila  is technically difficult, and is particularly so in a high throughput context where the ability to automate is especially important. In addition, delivery of candidate compounds by microinjection occurs more slowly and can miss orally absorbable drugs.  
      Accordingly, there remains a need for methods and related articles of manufacture that improve the ability to screen through chemical libraries consisting of large numbers of candidate compounds to rapidly and easily identify the most likely candidate compounds for further drug discovery and research efforts, and particularly with respect to potential therapeutics for treating human cancer and cancer-related conditions.  
     SUMMARY OF THE INVENTION  
      The present invention is based in part on the discovery that screenably distinct characteristics can be induced by targeted expression of oncogenes or tumor suppressors in wild-type  Drosophila . These induced characteristics reflect basic mechanisms underlying the development of cancer and cancer-related conditions in animals, and are therefore useful in high throughput screening of candidate compounds for cancer therapy. Candidate compounds which demonstrate the ability to modify expression of these characteristics according to the methods of the invention are thereby identified as suitable candidates for further testing as therapeutic alternatives for cancer treatment of animals including humans. The methods and related apparatus and kits are easily practiced, avoid the need for complex microinjection systems, identify orally absorbable drugs, and are readily adapted to automated high throughput systems.  
      Accordingly, in one embodiment there is provided a method a method for high throughput screening of compounds comprising inducing a screenably distinct characteristic in wild-type  Drosophila  using targeted expression of  Drosophila  genes to mimic a human cancer or cancer-related condition, feeding to the  Drosophila  larvae a compound that putatively modifies the screenably distinct characteristic, and screening the  Drosophila  to determine whether the compound modifies the screenably distinct characteristic. The screenably distinct characteristic is, for example, apoptosis, tissue degeneration or abnormal tissue growth. Inducing a screenably distinct characteristic in wild-type  Drosophila  using targeted expression of  Drosophila  genes involves, for example, using targeted expression of oncogenes or tumor suppressors or orthologs of oncogenes or tumor suppressors. More specifically, the targeted expression of oncogenes involves, for example, reducing or eliminating the dCsk gene (SEQ ID NO: 1) expression in the developing  Drosophila  eye using an RNA interference construct. Alternatively, targeted expression of a tumor suppressor involves, for example, targeting to the eye of the  Drosophila  an altered form of  Drosophila  dRet receptor (GenBank Accession No. CG1061; (SEQ ID NO: 2). The method can further include screening the  Drosophila  to determine whether the compound has a toxic effect on the  Drosophila.    
      In another embodiment there is provided a method of using  Drosophila  in a high throughput screening assay of compounds putatively modifying a screenably distinct characteristic in the  Drosophila , the method comprising inducing the screenably distinct characteristic in a plurality of  Drosophila  embryos by modifying expression of an oncogene or a tumor suppressor in the  Drosophila , plating at least one of the plurality of  Drosophila  embryos in each of multiple wells in a multi-well microtiter plate, administering a candidate compound to the at least one  Drosophila  embryo in each well and screening the  Drosophila  to determine whether a candidate compound modifies the induced screenably distinct characteristic. Modifying expression of an oncogene or a tumor suppressor in the  Drosophila  includes, for example, reducing or eliminating dCsk gene (SEQ ID NO: 1) expression in the developing  Drosophila  eye using an RNA interference construct, or targeting to the eye of the  Drosophila  an altered form of  Drosophila  dRet receptor comprising CG1061 (SEQ ID NO: 2). Modifying expression of an oncogene or a tumor suppressor in the  Drosophila  produces a  Drosophila  phenotype that, for example, mimics a human cancer or cancer-related condition. The screenably distinct characteristic is, for example, apoptosis, tissue degeneration or abnormal tissue growth. The method further optionally includes screening the  Drosophila  to determine whether the compound has a toxic effect on the  Drosophila.    
      In another embodiment there is provided apparatus for use in a high throughput screening assay method, the apparatus including a multi-well microtiter plate, an amount of a standard  Drosophila  growth medium placed into said multiple wells of said multi-well microtiter plate, an amount of a candidate compound added to said multiple wells, and at least one  Drosophila  in each of said multiple wells, said  Drosophila  with modified expression of an oncogene or tumor suppressor so that the  Drosophila  expresses a screenably distinct characteristic. The  Drosophila  with modified expression of an oncogene or a tumor suppressor comprises, for example, a  Drosophila  with reduced or eliminated expression of dCsk gene (SEQ ID NO: 1) in the developing eye, or a  Drosophila  with an altered form of  Drosophila  dRet receptor comprising CG1061 (SEQ ID NO: 2) targeted to the eye of the  Drosophila . The  Drosophila  expressing a screenably distinct characteristic expresses, for example, a characteristic that mimics cancer or a cancer-related condition, such as apoptosis, tissue degeneration or abnormal tissue growth. The tissue degeneration may be is, for example, neurodegeneration. The apparatus may optionally further include an inverted lid with an oxygen-permeable base for sealing each well of the microtiter plate.  
      In another embodiment there is provided a kit for use in a method for high throughput screening of compounds, the kit including the apparatus as described above, and further including instructions setting forth instructions for selecting an inducible screenably distinct characteristic in  Drosophila  wherein the inducible screenably distinct characteristic mimics a human disease or condition, instructions for plating at least one  Drosophila  embryo expressing the selected inducible screenably distinct characteristic in each of multiple wells in a multi-well microtiter plate, instructions for administering to the  Drosophila  embryos a compound that putatively modifies the screenably distinct characteristic, and instructions for screening the  Drosophila  to determine whether the compound modifies the screenably distinct characteristic. The kit optionally includes further instructions for determining whether the compound has a toxic effect on the  Drosophila.    
      These and other features, aspects and advantages of the present invention will become better understood with reference to the following description, examples and appended claims. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       FIG. 1  is an exemplary multiwell microtiter plate apparatus for high throughput screening of compounds using  Drosophila;    
       FIG. 2  is a sampling of photomicrographs of  Drosophila  omatidia showing wild type omatidia and overgrowth phenotype resulting from targeting MEN2A-analogous and MEN2B-analogous forms of dRet; and  
       FIG. 3  shows electron photomicrographs of omatidia illustrating modification of a screenably distinct phenotype by a candidate compound, in which the candidate compound strongly inhibits in dose-dependent fashion the severity of the rough eye phenotype of both dRet and dRet MEN2B . 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
      Abbreviations and Definitions  
      To facilitate understanding of the invention, a number of terms and abbreviations as used herein are defined below as follows:  
      “Altered form”: As used herein with respect to a gene, the term “altered form” refers to a gene which differs from a given gene sequence by one or more mutations such as a single point mutation, such that the activity of the gene is modified but not eliminated.  
      “ Drosophila ”: As used herein, “ Drosophila ” refers to an insect or insects belonging to the fruit fly species  Drosophila melanogaster , without regard to developmental stage thereof and including embryos (eggs), larvae, pupae and mature adult flies of the species.  
      “Mimic”: as used herein, the term “mimic” refers to the action of resembling or imitating a human disease or condition by producing characteristic symptoms of the disease, for example in the way that abnormal tissue growth is said to mimic cancer.  
      “Wild type”: As used herein, “wild type” refers to  Drosophila  having a genome that has not been genetically modified or manipulated in a laboratory, for example by recombinant techniques.  
      “To screen”: As used herein, “to screen” refers to the act of examining a group of organisms, such as  Drosophila , and using the expression of a selected characteristic as a criterion for separating the organisms into at least two groups.  
      “Screenably distinct”: As used herein, the term “screenably distinct” refers to a characteristic of a  Drosophila  individual or individuals, or to the  Drosophila  individual per se, that deviates from the of wild type individual  Drosophila  in such a way that visual inspection or other simple detection methods can be used to detect the presence of the characteristic, wherein the presence or absence of the characteristic is used as the criterion for screening the organisms into at least two groups. A screenably distinct characteristic may be a feature of a genotypic variant of wild-type  Drosophila  in the sense that the characteristic may result from a  Drosophila  gene or transcript that is orthologous to a human oncogene or tumor suppressor and is stably placed within the  Drosophila  and expressed in the  Drosophila.    
      “Variant”: As used herein, the term “variant” refers to a  Drosophila  individual that deviates from wild type individual  Drosophila  with respect to at least one characteristic.  
      “Oncogene”: As used herein, “oncogene” refers to a gene or transcript that is capable, when it has higher than normal activity, of inducing abnormal tissue growth due to effects on the biology of a cell, for example on the cell cycle or cell death process.  
      “Tumor suppressor”: As used herein, “tumor suppressor” refers to a gene or transcript that is capable, when it has lower than normal activity, of inducing abnormal tissue growth due to effects on the biology of a cell, for example on the cell cycle or cell death process.  
      “Activity”: As used herein, “activity” refers to the level of functioning in which a gene or transcript participates; for example, high activity of a gene or gene product refers to an increase in the gene&#39;s function relative to its normal level of functioning.  
      “Targeted expression”: As used herein, “targeted expression” refers to the manipulation of a gene or transcript through the use of a transgene to induce its expression in one or more tissues within the  Drosophila.    
      “Transgene”: As used herein, “transgene” refers to an artificially constructed stretch of DNA that, for example, can be placed into a  Drosophila  by stable integration in the  Drosophila &#39;s genome.  
      “Embryo(s)”: As used herein, “embryo” and “embryos” refer to the egg stage of  Drosophila melanogaster.    
      “Toxic”: As used herein, “toxic” and “toxicity” refer to a characteristic of a compound that through its chemical action kills, injures or impairs an organism.  
      “dCsk”: As used herein, “dCsk” refers to the gene or transcript having a sequence of GenBank accession no. CG17309 (SEQ ID NO: 1) in Flybase (http://flybase.bio.indiana.edu/.bin/fbidq.html?FBgn0037925) or the protein encoded by said locus.  
      “Csk”: As used herein, “Csk” refers to a gene or transcript or protein that is an ortholog of dCsk and is found in organisms other than  Drosophila.    
      “dRet”: As used herein, “dRet” refers to the gene or transcript having a sequence of GenBank accession no CG1061 (SEQ ID NO: 2) in Flybase (http://flybase.bio.indiana.edu/.bin/fbidq.html?FBgn0011829&amp;content=full-report) or the protein encoded by said locus.  
      “Ret”: As used herein, “Ret” refers to a gene or transcript or protein that is an ortholog of dRet and is found in organisms other than  Drosophila.    
      “To plate”: As used herein, “to plate” refers to the act of placing material, including growth medium, candidate compounds, and  Drosophila  embryos, into wells of a microtiter plate.  
      “Phenotype”: As used herein, “phenotype” refers to the outward manifestation of the action of a gene due to the gene&#39;s gain or reduction in activity, for example the aberrant development of the  Drosophila  eye due to reduction of dCsk activity.  
      The present invention provides methods and related are based in part on the discovery that targeted expression of oncogenes or tumor suppressors, or orthologs thereof, produces screenably distinct characteristics in  Drosophila  that then serve as a basis for discriminations within the context of a high throughput screening system. The present invention takes advantage of the novel combined use of a  Drosophila  line having a transgene-induced screenable characteristic, and a technique for high-throughput compound screening.  
      More specifically, expression of a transgene in  Drosophila  is modified, such that the functionality of dRet in  Drosophila  is increased, or alternatively, the dCsk functionality in  Drosophila  is reduced. The transgene expression is modified, for example, by engineering a single point mutation into a transgene, and establishing a stable transgenic line of individuals having the transgene. The transgene expression can also be modified using an RNAi construct, such as siRNA&#39;s as known in the art to produce targeted inhibition of gene expression. In either case, the modified gene expression that alters dRet or dCsk functionality in  Drosophila , leads to the formation of an abnormal retina in the  Drosophila . The abnormal retina is a screenably distinct characteristic in the  Drosophila , in that it is a characteristic of a  Drosophila  individual or individuals that deviates from wild type individual  Drosophila  so clearly that visual inspection or other simple detection methods can be used to detect the presence or absence of the abnormal retina. The presence or absence, and comparative level of abnormality when present, is then assessed and compared between  Drosophila  to which a candidate therapeutic compound has been administered, and  Drosophila  to which no compound or a control compound has been administered, and the comparison used to determine whether the candidate compound has any effect on the screenably distinct characteristic.  
      Accordingly, methods, related apparatus and kits for high throughput screening assays involve the preparation of microtiter plates each with multiple wells, wherein each well initially contains one or more  Drosophila  embryos with a transgene and an amount of a  Drosophila  growth medium. The embryos develop while feeding on the growth medium. The precise age of the embryos at the time they are plated matters less than the fact that they are all about the same age, to permit accurate evaluation of the possible effects of the candidate compound on larval and pupal development.  
      To prepare the microtiter plates, for example, 96-well microtiter plates are used, such as those commonly commercially available and typically used for various laboratory assay techniques, including other high throughput drug assay techniques. Into each well is pipetted 50-100 μl of standard  Drosophila  growth medium. A exemplary range of about 50 to about 100 μl is a balance between (i) providing sufficient food so as not to place undue feeding stress on the developing flies and (ii) providing sufficient air space for the third larval instars to find sufficient wall space to pupate and for minimal stress on the developing larvae and pupae. Any one of several standard  Drosophila  growth medium recipes as known in the art of breeding  Drosophila  for research can be used.  
      In an exemplary embodiment, a candidate compound, or cocktail of more than one compound, that has been selected for screening is prepared in EtOH or DMSO/aqueous solution. In an exemplary embodiment, EtOH is used. Although DMSO can be used, it can be toxic if it reaches final concentrations of more than 0.3% of the growth medium. The compound in solution is added and allowed to diffuse through the growth medium for an initial period of about 16 to about 24 hours. Alternatively, the compound in solution is mixed with the food by pipetting, by shaking, or by sonicating.  Drosophila  embryos of the desired genotype or containing the desired transgene are collected en masse and, after the initial period of diffusion of the candidate compound through the growth medium, sorted several to a well. In an exemplary embodiment, five to six embryos are sorted to each well. However, the number of embryos in each well can easily vary, provided that no more embryos than will flourish in the well are used. The number of embryos per well will also be influenced by the need to obtain a sufficient number of data points to make the test meaningful.  
      Once the  Drosophila  embryos are placed into each well on the growth medium, they hatch out and begin feeding after a second period of about 24 hours, bringing the final amount of diffusion time for the subject compound to about 40 to about 48 hours. A period of about 24 to about 48 hours is sufficient for full diffusion of most compounds. In some cases where adequate diffusion of the compound does not occur within a period of about 48 hours, the growth medium in the plate can be warmed and then sonicated to facilitate mixing of the candidate compound with the growth medium. Finally, each well is sealed by placing a second microwell plate in inverted orientation so that the opening at the top of each well is closely apposed; this second microwell will ideally have a membrane or covering at the base of each well that will permit (i) flow of sufficient oxygen to allow the developing  Drosophila  to thrive and (ii) the containment of the  Drosophila  within each compartment formed by the apposition of the two plates. An exemplary such covering is the Millipore Multiscreen-FC MAFCNOB10. In an exemplary embodiment, the two plates are further aligned and sealed by an intervening adaptor to yield the configuration as shown in  FIG. 1 . It is anticipated that other configurations and components can be utilized that will yield the same or suitably similar results.  
      Assaying Methods  
      In one embodiment of the methods, a method for high throughput screening of compounds includes inducing a screenably distinct characteristic in  Drosophila  by modifying expression of an oncogene or a tumor suppressor in the  Drosophila , feeding to embryos of such altered  Drosophila  a compound that putatively modifies the screenably distinct characteristic, and screening the  Drosophila  to determine whether the compound modifies the screenably distinct characteristic. In one exemplary embodiment, reducing the activity of dCsk in the developing  Drosophila  retina with an introduced transgene results in a screenably distinct retina. In another exemplary embodiment, expressing an activated form of dRet in the  Drosophila &#39;s retina with an introduced transgene results in a screenably distinct retina. It is anticipated that other approaches that alter the development of the eye can be utilized that yield a similar result.  
      The  Drosophila  retinae can be screened as described in the Examples, infra. For example, the screenably distinct characteristic of a  Drosophila  retina with a reduction in dCsk can be examined after (i) growing  Drosophilas  with said distinct characteristic in microwells containing standard  Drosophila  media plus a compound that putatively modifies the distinct characteristic, (ii) permitting said  Drosophila  to advance in their development in said microwells, and (iii) screening the ability of said compound to alter the perceived severity of the retina&#39;s distinct characteristic.  
      The severity of a  Drosophila  retina&#39;s distinct characteristic can be easily determined by a screening step involving examining the retina surface through a standard dissecting microscope plus a suitable light source. In one exemplary example, the severity of a  Drosophila  retina&#39;s distinct characteristic can be assessed by determining overall size of the retina, the total number of ommatidia, the proper alignment of the constituent ommatidia, whether two neighboring ommatidia are abnormally close together or fused, and whether the retina folds abnormally within its normal niche on the head.  
      Lethality of candidate compounds for  Drosophila  can be used to detect and quantify toxicity of candidate compounds. Well known standard statistical methods are used to help distinguish chance results from real toxic effects. Lethality is quantified, for example, by determining the number of  Drosophila  that fail to develop successfully to adulthood and applying suitable statistical analyses to determine statistical significance. Lethal dose evaluations can be used to quantify the extent of toxicity. For example, once a candidate compound demonstrates a mediating effect on a reduction of dCsk activity or an increase in dRet activity, the toxicity of the compound is evaluated by varying dosage levels across a broad range and quantifying the lethality of the compound at each dose to obtain an LD 50  value In an exemplary example, (i) a consistent and significant reduction in the number of adults within a microwell or (ii) the presence of dead or dying  Drosophila  within a microwell is taken as evidence that a compound is significantly toxic. In another exemplary example, the emergence of most  Drosophila  adults within a microwell indicates a lowered probability that an introduced compound is toxic.  
      Apparatus for Use in HTS Methods  
      In another aspect, the invention provides apparatus for use in high throughput screening methods as described herein. The apparatus includes a multi-well microtiter plate, an amount of a standard  Drosophila  growth medium placed into multiple wells of the multi-well microtiter plate, an amount of a candidate compound added to the multiple wells, and a plurality of screenably distinct  Drosophila  in the multiple wells, the screenably distinct  Drosophila  having developed from  Drosophila  embryos altered in a manner useful for studying a specific oncogene or tumor suppressor. The screenably distinct  Drosophila  include, for example,  Drosophila  with a reduced level of dCsk activity, or  Drosophila  with an increased level of dRet activity. In one embodiment,  Drosophila  with a screenably distinct characteristic is then placed into a multi-well microtiter plate with a suitable lid to (i) permit  Drosophila  survival and development and (ii) prevent escaping of developing  Drosophila.    
      Automated Screening  
      Preparation of the microtiter plates with the growth medium,  Drosophila  embryos and candidate compounds can be performed manually or using a robotic system or systems. For example, plating of the growth medium and of candidate compounds in solution on the microtiter plates can be readily adapted to known robotic systems that can be configured to repeatedly inject a predetermined volume of the growth medium and of the test solutions into each well of the microtiter plate. Similarly, the assay results can be determined manually, or can be adapted to automated or robotic analyzers.  
      Kits  
      Further, the present invention provides a kit for use in a method for high throughput screening of compounds. The kit includes instructions for the following: instructions for inducing a screenably distinct characteristic in  Drosophila  containing a mutation or transgene that creates a screenably distinct characteristic, instructions for feeding to the  Drosophila  embryos a compound that putatively modifies the screenably distinct characteristic, and instructions for screening the  Drosophila  to determine whether the compound modifies the screenably distinct characteristic. In one embodiment, the instructions set forth more specifically instructions for screening the  Drosophila  to determine whether the compound modifies alterations in the screenably distinct phenotype in the  Drosophila . In still another embodiment, the instructions set forth instructions for determining whether the compound has a toxic effect on the  Drosophila . In yet another embodiment, the kit further includes a multi-well microtiter plate, and an amount of a  Drosophila  growth medium for placement into multiple wells of the multi-well microtiter plate. The kit can still further include the lid for sealing each well of the multi-well microtiter plate.  
      Relationship to Multiple Endocrine Neoplasias  
      Multiple Endocrine Neoplasias (MENs) are dominant, inherited, familial cancer syndromes. They are characterized by a variety of tumors of the endocrine glands arising from neuroendocrine cells. Multiple Endocrine Neoplasia II (MEN II, or MEN2) is a hereditary disorder in which patients develop a type of thyroid cancer accompanied by recurring cancer of the adrenal glands. One type of this disease (MEN IIa, or MEN2a) is also associated with overgrowth (hyperplasia) of the parathyroid gland. MEN2 syndromes are defined by medullary thyroid carcinoma (MTC), a potentially aggressive tumor prone to widespread metastases that is generally refractory to radiation and chemotherapy. The cause of MEN2 is a mutation in a gene called Ret. The disorder affects all ages and both genders equally. A family history of MEN2 is the primary risk factor.  
      The Ret gene encodes a tyrosine kinase receptor for neurotrophic molecules. Gene rearrangements, including specific point mutations, activate the oncogenic potential of Ret in human thyroid papillary carcinomas. Different point mutations activate Ret in familial multiple endocrine neoplasia syndromes. Inactivating mutations of Ret are present in some Hirschsprung&#39;s disease patients. Increasingly detailed knowledge of the specific Ret mutations responsible for human tumors provides important tools for the clinical management of these diseases.  
      “C-ret” is a proto-oncogene (normal gene having the potential for change into an oncogene) of Ret, which encodes a 120 kD transmembrane receptor with a tendency to rearrange during transfection (Takahashi et al., 1985). C-Ret is expressed in a variety of tissues, primarily derivatives of the neural crest such as components of the autonomic and enteric nervous system and regions of the Wolffian duct and ureteric bud epithelium (Takahashi et al., 1998; Tsuzuki et al., 1995). Deletion of Ret activity in mice leads to renal dysgenesis and loss of enteric neurons (Schuchardt et al., 1994). This and a variety of related work has indicated that Ret plays a central role in the proliferation, differentiation, and migration of cells during renal organogenesis and enteric neurogenesis and likely a variety of other organs as well. In addition, the c-ret locus represents a ‘hotspot’ for oncogenic mutations.  
      Ligand-mediated activation of Ret leads to dimerization, auto-phosphorylation, and activation of the receptor. MEN2A mutations achieve ligand-independent activation by promoting dimerization; MEN2B mutations can bypass requirement for dimerization. The intracellular domain of Ret contains a tyrosine kinase catalytic domain that is necessary for its activity. Ligand-mediated activation of Ret leads to tyrosine phosphorylation and subsequent binding of a phospholipase C, and the Shc, SNT/FRS2, IRS1, Dok, and GRB2 adapters: in addition to ras, activated Ret can stimulate jnk, PI-3K/AKT, src, and p38 signaling (Alberti et al., 1998; Arighi et al., 1997; Besset et al., 2000; Borrello et al., 1994; Califano et al., 2000; Hayashi et al., 2000; Kurokawa et al., 2001; Melillo et al., 2001a; Melillo et al., 2001b; Ohiwa et al., 1997; Pelicci et al., 2002; Soler et al., 1999), and Enigma can bind and promote signaling in a phosphorylation-independent manner (Durick et al., 1998). The short form of Ret can bind both the PTB and SH2 domains of Shc, whereas the long form binds exclusively the PTB domain; the functional significance of this difference is not well understood. Interestingly, the hRet MEN2B  mutant forms, described below, bind exclusively to the PTB domain; again, the functional significance of its inability to bind the SH2 domain is also unclear (Ohiwa et al., 1997).  
      Five human syndromes are associated with mutations within the c-ret locus; in addition, somatic c-ret mutations are associated with sporadic medullary thyroid cancer. Hirschsprung&#39;s disease represents point mutations or breakpoints that reduce receptor activity, leading to intestinal aganglionosis and renal dysplasia. Activating point mutations can be classified into four groups: FMTC, Ret/PTC, MEN2A, and MEN2B. Familial Medullary Thyroid Carcinoma (FMTC) is characterized by one of several point mutations that lead to medullary thyroid carcinomas (MTCs). Mutations associated with FMTC appear to be weakly activating; most alter extracellular cysteines that provoke spontaneous activation, though some mutations target residues within the tyrosine kinase domain (Donis-Keller et al., 1993; Eng et al., 1996; Mulligan et al., 1994; Pasini et al., 1997; Pasini et al., 1996). Papillary thyroid carcinomas are commonly linked to rearrangements that create a chimeric receptor and spurious activation of a number of downstream targets (reviewed in Tallini, 2002).  
      Nearly all MEN2A patients contain a mutation that alters one of five cysteines (C609, C611, C618, C620, or C634) within the extracellular domain. The result is ligand-independent dimerization and strong activation of the receptor (Donis-Keller et al., 1993; Mulligan et al., 1994; Mulligan et al., 1993). This leads to a series of oncogenic events, particularly MTCs, pheochromocytomas (adrenal medulla tumors), and parathyroid adenomas.  
      A more severe form of MEN2 is typically the result of a methionine-to-threonine substitution at position 918 (M918T) within the tyrosine kinase catalytic domain of hRet (Carlson et al., 1994a; Hofstra et al., 1994); rarely (&lt;5%), other residues in Ret are targeted (Menko et al., 2002). The result is MEN2B, a debilitating disease also characterized by medullary thyroid carcinomas and pheochromocytomas; in addition, ganglioneuromas, mucosal neuromas, megacolon, a generalized neural hypertrophy, early defects in bone structure including marfinoid habitus, and possibly other developmental defects are commonly observed (reviewed in Takahashi, 1997). MEN2B mutations have also been associated with aganglionosis leading to congenital megacolon, more commonly associated with Hirschsprung&#39;s disease (Romeo et al., 1998). In both MEN2A and MEN2B, studies indicate the importance of prophylactic thyroidectomies: multifocal MTC and C cell hyperplasia were consistently found in youth as young as 6 years (Lallier et al., 1998).  
      Although MTC is a relatively uncommon form of thyroid cancer, the morbidity and mortality rates are significant. At present, there are no effective non-surgical therapies for the treatment of medullary thyroid carcinoma. Pre-symptomatic or prophylactic thyroidectomy in hRet disease allele carriers may be curative. However, most patients with MEN2B have metastatic disease involving nearby lymph nodes (levels II-V) at the time of diagnosis. Although there have been significant advances in the detection and surgical excision of metastatic disease in the neck region, surgery rarely provides a cure (Lips et al., 1994; Moley et al., 1998; Wells, 1994).  
      Early detection is central to the successful management of medullary thyroid carcinoma. Unfortunately, pre-symptomatic diagnosis and early surgical intervention is rarely possible for most MEN2B patients with MTC. There are two impediments to early identification and treatment. The first relates to the frequency with which new MEN2B mutations appear in the population. More than half of all patients with MEN2B have de novo disease (Carlson et al., 1994b); the lack of similarly affected family members leads to diagnosis at an age that is typically later than in multi-generational MEN2B kindreds. The second obstacle to early detection is a lack of specific symptoms in patients with MTC, an issue especially common to sporadic disease. Sporadic MTC usually presents as a palpable neck mass at a later age and at a higher stage than inherited forms of MTC (Wells, 1994).  
      Currently, surgery remains the only effective therapy for MTC; metastatic MTC is not responsive to radiation or chemotherapy. These surgeries are complex and tedious, and repeat procedures are common. A better understanding of the abnormal signaling that occurs in tumors with the hRet MEN2B  mutant receptor would help us identify better therapeutic targets. New agents to control and cure MTC are needed for the successful management of this group of patients. Inherited forms of MEN2B are not especially common; however, it is of note that the M918 mutation is likely the most frequent hRet defect seen in sporadic (somatic) MTC. As is the case with MEN2B patients, surgery is rarely curative.  
      The human M918T allele is the most malignant of the hRet mutations described to date. M918T accounts for more than 95% of MEN2B patients characterized, and 30%-80% of sporadic MTCs (Eng et al., 1996; Eng et al., 1994). Although mutations other than threonine at position 918 can lead to weak activation of the receptor, only threonine appears able to transform Ret into an oncogenic form (Cirafici et al., 1997). In addition, unlike hRet MEN2A  mutations, M918T-mediated receptor activation does not lead to or require dimerization of the receptor. When an analogous mutation was made in the Ron (M1254T) and Met (M1250T) receptor tyrosine kinases, the result was activation of the Ras signaling pathway and—similar to Ret MEN2B —apparent activation of another signal transduction pathway (Bardelli et al., 1998; Santoro et al., 1998); this is likely due to alteration of the ‘activation loop’, which regulates access to the kinase domain (Miller et al., 2001).  
      The cysteine mutations seen in Ret MEN2A  are likely to open the structure to spontaneous disulfide bonding and dimerization. Second-site mutagenesis studies indicated that Ret MEN2A  receptors require tyrosine 905 for signaling whereas Ret MEN2B  receptors require tyrosines 864 and 952, suggesting the potential for differences in the signaling targets of these two receptors (Takahashi et al., 1998). Ret MEN2B  receptors also fail to phosphorylate the tyrosine at position 1096, normally required for binding of the Grb2 adapter protein (Liu et al., 1996). Finally, Ret MEN2A  and Ret MEN2B  demonstrate different responses to GDNF ligand. In the presence of GDNFR□, Ret MEN2B  proved responsive to GDNF and phosphorylated the downstream target Shc, whereas Ret MEN2A  was poorly responsive (Bongarzone et al., 1998; Carlomagno et al., 1998).  
      The precise pathway(s) activated by the M918T mutation in Ret MEN2B  is unknown. Several possible pathways have been suggested, including Src-like and JNK signaling, Nck, Crk, and Paxillin (Bocciardi et al., 1997; Marshall et al., 1997; Songyang et al., 1995); however, the evidence for activation of any of these pathways in vivo has been lacking, and our own in vivo work failed to detect differences in intracellular signaling between the two MEN2 isoforms (see below).  
      Engineering MEN2A-analogous and MEN2B-analogous oncogenic forms of Ret into mice has yielded mixed results. Targeted expression of Ret MEN2A  isoforms directs MTC formation in mice, although the penetrance is variable; they also developed C-cell and follicle tumors (Acton et al., 2000; Michiels et al., 1997; Reynolds et al., 2001). Attempts to create an MEN2B model mouse has also been partially successful: introducing the M918T mutation into endogenous Ret led to C-cell hyperplasia, pheochromocytomas, and occasional ganglioneuromas, although the penetrance for many of the defects was low and other abnormalities seen in humans such as developmental defects were absent (Smith-Hicks et al., 2000). The normal development observed in homozygous M918T mice indicated that the MEN2B form of Ret still signals normally in addition to its transforming potential.  
     EXAMPLES  
      Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following specific examples are offered by way of illustration and not by way of limiting the remaining disclosure.  
     Example 1  
     dRet: Targeted Expression and Association with Cancer  
      To mimic the MEN2B mutation, a single methionine-to-threonine point mutation was engineered into a full-length dRet cDNA at codon 1007 (analogous to position 918 within human hRet subdomain VIII). To mimic the human MEN2A mutation, the cysteine at position 695 in dRet was altered to an arginine (C695R) that is in a position most analogous to hRet 634, one of the most commonly mutated sites in MEN2A patients. All mutated fragments were sequenced and returned to the original dRet clone to produce dRet MEN2A  and dRet MEN2B . dRet, dRet MEN2A  and dRet MEN2B  were then fused 3′ to a GMR promoter construct that directs expression exclusively and at high levels in the eye (Moses and Rubin, 1991); stable transgenic lines were then created by standard protocol to yield GMR-dRet, GMR-dRet MEN2A , and GMR-dRet MEN2B .  
      Targeting MEN2A-analogous and MEN2B-analogous forms of dRet by standard methods for expression within the developing  Drosophila  retina also led to an overgrowth phenotype that mimics aspects of the human MEN2A and MEN2B diseases.  FIG. 2  presents typical examples from each phenotype. A wild type eye is included for reference: note how the ommatidia are organized into smooth rows (top left).  
      Expression of one copy of the GMR-dRet construct gave either a normal phenotype or some led to a mildly roughened eye (Figure, top row, center panel). Two copies of the same transgene in all GMR-dRet insertions led to a strongly roughened eye ( FIG. 2 , top row, right panel). As indicated in the lower panels, the phenotypes of GMR-Ret MEN2A  and GMR-Ret MEN2B , designed to mimic MEN2A and MEN2B are more severe both as single copies (bottom row) and as multiple copies (not shown). They typically contain fused ommatidia with severe patterning defects. The variable number of ommatidia suggest alterations in cell proliferation and cell death, aspects commonly observed in human tissue with constituent tumors.  
     Example 2  
     Screening of ZD6474  
      A candidate therapeutic compound identified as ZD6474, obtained from AstraZeneca International, had previously been tested and found to reduce Ret activity in a tissue culture model (Carlomagno et al., 2002); this drug also shows some efficacy for VEGF-class receptors (Ciardiello et al., 2004; Ciardiello et al., 2003; Glade-Bender et al., 2003; Hennequin et al., 2002; Wedge et al., 2002).  FIG. 3  illustrates in part the results of screening compound ZD6474 according to the screening methods of the present invention. Screening demonstrated the ability of ZD6474 to strongly inhibit the severity of the rough eye phenotype of both dRet and dRet MEN2B , indicating that the overgrowth and phenotypic defects were ameliorated. The panels in  FIG. 3  demonstrate that the ZD6474 compound can rescue the dRet MEN2B  phenotype in a concentration-dependent fashion. Overall, toxicity was observed at concentrations at and above 2.5 mM, and at least partial rescue was observed with doses as low as 0.04 mM. Therefore, the estimated therapeutic index (ratio of concentrations that are toxic to  Drosophila  to concentrations that reduce the retinal phenotype is 2.5/0.04=31.  
      These data demonstrate that using the screening methods of the present invention, candidate therapeutic compounds can be screened for ability to reduce inhibit or prevent the effects of oncogenic forms of proteins. These results therefore support the application of the  Drosophila  screening method to the identification of candidate compounds, other drugs or genes that might ameloriate overgrowth and other defects in tissues that contain abnormal biochemical activity. It is recognized that this approach of screening altered  Drosophila  with compounds in the microwell-based approach described above can be utilized in other  Drosophila  models of animal disease, and particularly human disease.  
     Example 3  
       Drosophila  Ortholog of C-Terminal Src Kinase (Csk) Regulates Cell Growth and Proliferation Through Inhibition of the Src, JNK, and STAT Pathway  
      The Src family cytoplasmic tyrosine kinases play important roles in a wide variety of cellular processes including proliferation and differentiation. Their major regulation is by C-terminal Src kinase (Csk), which encodes a negative regulator of Src tyrosine kinase signaling. The  Drosophila  ortholog of Csk, dCsk, functions as a tumor suppressor: dCsk mutants demonstrated increased body size and over-proliferation of adult tissues. Src family kinases regulate multiple cellular processes including proliferation and oncogenesis. Csk encodes a critical negative regulator of Src family kinases. We demonstrate that the  Drosophila  Csk ortholog, dCsk, functions as a tumor suppressor: dCsk mutants display organ overgrowth and excess cellular proliferation. Results of genetic analysis revealed that the dCsk phenotype depends primarily on activation of the Src, Jun kinase, and STAT signal transduction pathways. Blockade of Stat92E function in dCsk mutants severely reduced Src dependent overgrowth and activated apoptosis of mutant tissue. These data confirm work in mammalian tissue culture that links Src transforming activity to STAT function and provides an in vivo model for the interplay of Csk and Src kinases.  
      Src Family Cytoplasmic Tyrosine Kinases (SFKs) and Disease  
      Normal development requires strict spatial and temporal control of cellular processes such as proliferation and differentiation in order for properly sized and functioning organisms to form. This control is achieved through a network of signal transduction pathways that coordinate developmental events between cells, tissues, and organs. Inappropriate activation of these signal transduction networks can cause diseases such as oncogenesis in which individual cells respond to aberrant internal cues to overproliferate and overgrow. Src family cytoplasmic tyrosine kinases (SFKs) play important roles within these networks to regulate both developmental events and disease states. Humans and mice have at least eight SFKs, including Src, Fyn, and Yes. Many of these kinases have been linked to developmental events such as morphogenesis and to diseases such as oncogenesis, but the exact roles of SFKS in these processes remain ambiguous.  
      SFKs are composed of a tyrosine kinase domain, an SH2 domain, an SH3 domain, and a regulatory C-terminal region. They can be activated by receptor tyrosine kinases (RTKs), cytokine and immune receptors, G-protein coupled receptors, and integrins. SFK activation can cause cell cycle entry, cytoskeletal rearrangements, and alterations in cell adhesion, while disruption of SFK function can inhibit cell migration. Mammalian tissue culture models have identified numerous downstream effectors of SFK functions; these include signaling molecules in the Ras/ERK, Jun kinase, Jak/STAT, PI-3 kinase, and Rac/Rho pathways. However, SFK activities have not been well explored in vivo, in part due to functional redundancy among SFKs. For example, src−/− mice show only subtle osteoclast defects, while src−/−; fyn−/−; yes−/− mouse embryos show early lethality and multiple developmental anomalies including neural tube defects and dramatically reduced size. Fibroblasts derived from src−/−; fyn−/−; yes−/− mice show reduced proliferation, suggesting that some of the phenotypes of compound knock-out embryos are caused by proliferative defects during development. However, the precise role of Src, Fyn, and Yes in cell cycle during development remains unknown.  
      SFKs are maintained in an inactive state through tyrosine phosphorylation of their C-terminal region by the negative regulator C-terminal Src kinase (Csk), which itself is closely related to SFKs. Deletion or mutation of the Csk target site leads to upregulation of SFK kinase activity. Mammals have two Csk family members, Csk and Chk. Mice deficient for Csk show hyperactivation of SFKs and a striking embryonic phenotype also characterized by early lethality, neural tube defects, and reduced size. Surprisingly, csk−/− fibroblasts do not show increased proliferation, which conflicts with data indicating that increased SFK activity leads to cell cycle entry. This may reflect functional compensation by Chk, which also negatively regulates SFKs. This redundancy between multiple SFKs and Csk kinases as well as the early lethality of Csk and compound SFK knockouts has impeded detailed evaluation of SFK function in developing mammalian tissues.  
      Abnormal constitutive activation of SFKs has been implicated in oncogenesis, but its precise role is also ambiguous. Numerous human tumors possess activated SFKs, but SFK mutations have been found in only a fraction of these tumors. Some human colon cancers harbor mutations that abolish the ability of the C-terminal domain to inhibit Src kinase activity. The transforming v-Src oncogene shows deletion of the Csk target site. Since SFKs can be abnormally activated through disregulation of the C-terminal region, reduced Csk family kinase activity could promote oncogenesis. Yet, the role of Csk and/or Chk in tumors is controversial or unclear. Large deletions within the region of chromosome 15 that harbors Csk have been observed in colon cancers, the tumor types that commonly show elevated SFK activity, but no specific loss-of-function Csk mutations have been found in tumors to date. Reduced Csk expression and function is correlated with Src activation in primary hepatocellular tumors, primary colorectal tumors, and colon carcinoma cell lines. However, others have reported elevated Csk in tumors with high SFK activity. In addition, Csk −/−  primary mouse fibroblasts do not show a transformed phenotype. Perhaps mutations in other loci, such as Chk, are required to reveal a tumor suppressor function for Csk. A detailed exploration of Csk&#39;s function in vivo is required to better understand its role in disease and development, but, again, such studies have been impeded by the early lethality of Csk −/−  mice.  
      The imaginal discs of  Drosophila  provide a powerful model system for the study of signal transduction. Imaginal discs share several properties with mammalian epithelial tissues: both are composed of epithelial cells that must maintain proportional growth, differentiation, and renewal in order to form functional tissues and organs. Cells within imaginal discs undergo proliferation and differentiation in response to molecular pathways that have been highly conserved across species and that function in oncogenesis. For example, studies of the eye imaginal disc have provided important evidence that the Ras and Jak/STAT signal transduction pathways are crucial for normal growth, proliferation, and differentiation. Recent genetic analyses of ‘tumor suppressor’ mutations have led to new insights about known human tumor suppressors and identification of new putative human tumor suppressors such as lats and Salvador.  
      The  Drosophila  genome contains two SFKs, Src42A and Src64B, that are functionally similar to their mammalian counterparts. Src42A and Src64B loss-of-function mutations disrupt cytoskeletal regulation within developing oocytes and embryos. Yet, the full repertoire of SFK functions remains to be elucidated in  Drosophila . Src42A and Src64B are regulated by a Csk-like activity in flies, but until now the gene responsible for that activity was unknown. In this report, we present the cloning and characterization of the  Drosophila  Csk ortholog, dCsk. Loss of dCsk function led primarily to overgrowth phenotypes in developing tissues such as the eye; genetic data indicated that excess proliferation was due to upregulation of SFKs. We provide evidence that this overgrowth required the JNK and STAT signal transduction pathways. Reducing STAT function prevented growth and normal differentiation of dCsk mutant tissue, instead provoking dCsk −/−  cells to undergo apoptosis. Our data provide in vivo evidence for a Src-dependent pro-apoptotic pathway triggered by reduced STAT function. They are consistent with results from Stewart et al (Stewart, 2003). Together, these results connect SFK signaling to the cell cycle and suggest an approach for restraining its proliferative potential.  
      Fly Stocks and Genetics  
      Flies were grown at 25° C. Fly stocks were obtained from the Bloomington Stock Center unless otherwise noted. S030003 and S017909 were from the Szeged Stock Center. Src64B P1  was a gift of M. Simon. Stat92E j6C8  was a gift of S. Hou. Src42A SuI  and Src42A 18-2  were gifts of X. Lu. To create EGUF clones, we established y w: ey-Gal4 UAS-FLP/+; FRT82B GMR-hid l(3)CL-R/FRT82B dCsk flies by standard crosses; w; FRT82B GMR-hid l(3)CL-R/FRT82B Ubi-GFPnlsS65T flies were utilized as controls for minor artifacts inherent in the EGUF system. dCsk j1D8/S030003  trans-heterozygotes showed an intermediate phenotype and were used to examine genetic interactions between dCsk and candidate genes.  
      Genomic and EST Analysis  
      Sequence flanking the j1D8 and S030003 P-element insertions was generated and mapped by the Berkeley  Drosophila  Genome Project (BDGP) and Szeged Stock Center, respectively. The following CG17309 ESTs were obtained from BDGP and fully sequenced: LD36541, LP09923, GH10267, LD22810, and LD33364. Sequences were assembled, compared, and analyzed with BLAST, MultAlin, PROSCAN, and Genestream.  
      Rescue and Reversion  
      To create the heat-shock inducible dCsk transgene hs-dCsk, the LD22810 cDNA was cloned into pPCaSpeR-hs, and stable insertions were created. dCsk j1D8 , dCsk S030003 , and dCsk S017909  were extensively out-crossed to remove observed background mutations. w; hs-dCsk/+; dCsk/dCsk and w; +/+; dCsk/dCsk embryos were collected for 3-4 days in vials. Larvae were heat shocked at 37° C. for 30 minutes every 10-16 hour to induce dCsk expression. For reversion, S017909 and S030003 were excised by standard crosses; over 10 independent excisions were scored for reversion of lethality.j1D8 failed to excise.  
      Larval and Pupal Body Size Measurements  
      Embryos were collected for 4 hours and larvae were grown at similar densities. For mass measurements, larvae were cleaned and weighed in groups of 15-20 on a Mettler AE50 balance. A minimum of 3 groups was measured for each genotype at each time point. Average body mass was calculated by determining the average of the sum of the average body mass per group. Values for each time point were normalized to the average mass of wild-type control larvae. For pupal measurements, pupae were photographed and relative length measurements were taken from printed enlargements. Values were normalized to wild-type pupae.  
      Clonal Analysis and Flow Cytometry  
      Flow cytometry was performed generally as described {Neufeld, 1998 #4340}. Dissociated imaginal discs cells were run on a Cytomation MoFlo Cytometer. Data was analyzed in Summit v3.1 (Cytomation). For analysis of loss-of-function clones, the genotypes were: y w hs-FLP/+: FRT82B Ubi-GFPnlsS65T/FRT82B dCsk j1D8  and y w hs-FLP/+; FRT82B Ubi-GFPnlsS65T/FRT82B dCsk S030003 . Clones were induced by heat shock at 48 and 72 hours AED and dissected at 120 hours. GFP positive and negative tissues were used to control for GFP detection. FACS experiments were repeated at least 3 times. We did not rely on direct scoring of clonal patches within the eye disc in part because we were not able to reliably distinguish the boundaries of the clones with the reagents available.  
      Histology, Immunohistochemistry, and SEM  
      In situ hybridization was performed as described (Tautz, 1989) using a probe to the 5′ end of both dCsk transcripts bounded by an Nco1 and Bsg1 site. Negative controls lacked probe. Digoxigenin was detected with an alkaline phosphatase conjugated antibody (Behringer Manheim).  
      For adult sections, heads were fixed in 1% glutaraldehyde/2% osmium tetroxide/PBS, dehydrated and washed, and incubated 4 hours in 1:1 propylene oxide Durcupan ACM resin, overnight in 100% resin, and finally at 65° C. to harden. Serial sections were stained with 0.5% methylene blue/0.1% toluidine blue. Digital photographs were taken on a Zeiss Axioplan.  
      For immunohistochemistry, tissue was fixed for 20 minutes in 4% paraformaldehyde with 1×PBS or 1×PEM and stains were performed in 1×PBS, 10% FBS, 0.3% Triton-X100. Antibodies to affinity purified anti-Stat92E was used at 1:500 {Chen, 2002 #4507}, anti-phospho-histone H3 (Upstate Biotechnology) at 1:200, and 22C10 and active-capase-7 (New England Biolabs) at 1:4 and 1:50, respectively. Secondary antibodies were conjugated to Alexa Red or Green (Molecular Probes). For dCsk mitotic clones, we used ey-FLP/+; FRT82B Ubi-GFPnlsS65T/FRT82 dCsk. Digital photographs were taken on a Zeiss Axioplan.  
      To estimate mitotic activity we examined printed enlargements of phospho-histone stains of EGUF discs. We controlled for tissue mass by counting phospho-histone positive nuclei within a quadrant of fixed size such that we recorded the number of positive nuclei within identically sized fields of tissue for each genotype. Nuclei were counted in 3 quadrants per disc and the average number of mitotic nuclei per quadrat was determined.  
      For SEM, adult flies were fixed in 95% ethanol, re-hydrated, treated with 1% osmium tetroxide, dried, and sputter coated. Ommatidia were counted on printed enlargements of SEM micrographs. For dCsk j1D8  EGUF clones, estimates of ommatidia were made using SEMs of the entire eye plus separate SEMs to visualize folds.  
      dCsk Encodes a Negative Regulator of Growth and Proliferation  
      In a screen for mutations that genetically modify an over-expressed, oncogenic form of the Ret receptor tyrosine kinase in  Drosophila  we identified three P transposable elements that enhanced the activated Ret phenotype. Fly lines j1D8, S030003, and S017909 contain P-element insertions within the CG17309 locus. We fully sequenced 5 of 50 known CG17309 ESTs and determined that CG17309 encodes two nearly identical predicted proteins that differ only at the N-terminus. The predicted proteins contain a tyrosine kinase domain and an SH2 domain that, together, show the highest homology with Csk family kinases. In fact, CG17309 proteins show a higher homology to Csk orthologs from other species such as mouse,  Xenopus , and Hydra than to any other  Drosophila  tyrosine kinase. They also contain a glutamine-rich region in place of the SH3 domain found in mammalian Csk proteins. Consistent with other members of the Csk family, CG17309 proteins lack an N-terminal myristoylation signal and lack a C-terminal negative regulatory tyrosine present in SFKs. Also, CG17309 proteins lack plextrin homology and Tec-homology domains, which distinguish them from the closely related Tec-Btk family tyrosine kinases. Previous analyses of the  Drosophila  genome have concluded that CG17309 encodes the sole  Drosophila  Csk ortholog. Based on these data and data presented below, we will refer to this locus as  Drosophila  Csk ortholog, or dCsk, and the three insertion lines as dCsk j1D8 , dCsk S030003 , and dCsk S017909 .  
      All three dCsk lines are lethal and displayed a stronger phenotype when in trans to a deficiency. dCsk j1D8  exhibited the earliest lethal phase, dying within 6-18 hours after pupation, a lethal phase which overlapped with that of dCsk j1D8  in trans to deficiency, illustrating that dCsk j1D8  is a strong hypomorphic mutation. Excision of the dCsk S030003  and dCsk S017909  insertions reverted their lethality and/or non-complementation with dCsk j1D8 . In situ hybridization indicated that dCsk mRNA is ubiquitously expressed within developing larval tissues. dCsk j1D8 , dCsk S030003 , and dCsk S017909  mutant tissues showed reduced dCsk expression by in situ hybridization. Finally, heat shock-induced expression of a dCsk cDNA rescued the lethality and mutant phenotypes in all three dCsk alleles. By itself, ectopic, ubiquitous expression had no detectable effect on the adult phenotype. These data demonstrate that all three P element insertions disrupt the dCsk locus.  
      During fly development, embryos hatch to progress through three larval stages followed by pupation and metamorphosis. dCsk mutants occasionally survived through later pupal development, allowing for characterization of dCsk larvae and pupae. The most striking phenotype of dCsk mutants was their increased body size relative to wild-type animals. Early third instar dCsk larvae weighed 30% more than age-matched wild-type larvae and eventually grew to weigh 84% more than wild-type larvae due to a prolonged larval stage in which they continued to feed and grow long after wild-type controls had pupated. dCsk pupae displayed a 21% increase in body length vs. controls. Wandering dCsk mutant larvae showed enlargement of tissues such as the brain, ventral ganglion, and salivary glands, and enlargement of the wing, leg, and eye imaginal discs.  
      Pharate adults are animals that attain a near adult morphology but die within the pupal case. The eyes and heads of the occasional dCsk j1D8/S030003  and dCsk S030003  mutants that survived as pharate adults were frequently enlarged and posterior ommatidia were sometimes misaligned. Histological sections indicated that individual mutant ommatidia were morphologically normal (data not shown) but contained more ommatidia than wild type controls. Rarely, the eyes were replaced with duplicated antennae. In addition, the wings and legs were severely malformed, the notum was sometimes ‘split’, and the head, legs, and notum often contained cuticle outgrowths.  
      To resolve the origin of the retinal defects, we utilized the EGUF system to generate ‘whole eye clones’ in which all adult eye tissue is homozygous for dCsk mutations in an otherwise heterozygous animal. This approach permitted us to isolate dCsk activity within the retina from, e.g., effects of the prolonged larval stage; flies with eyes homozygous for dCsk developed along a normal time course. dCsk EGUF clones were also enlarged in comparison to controls, with some dCsk j1D8  clones so enlarged that the eyes became malformed in order to pack onto a normally sized head. Occasionally, dCsk EGUF clones resulted in antennal duplication and cuticle overgrowth, phenotypes that recapitulated defects seen in dCsk pharate adults.  
      The enlarged dCsk EGUF eyes contained an increased number of ommatidia. The cells within these retinae were normal in morphology and size, though some ommatidia exhibited planar polarity inversions. Retinal cell proliferation occurs almost exclusively within the embryonic and larval eyes, and the observed extra cells most likely derive from excess proliferation during these stages. Importantly, previous studies show that blocking apoptosis does not affect eye size. Consistent with over-proliferation, late larval eye-antennal imaginal discs from dCsk EGUF clones were enlarged compared to age-matched controls and showed an increase in proliferating cells. These data indicate that dCsk acts to regulate organ size and cell proliferation within the developing eye field.  
      To further explore dCsk&#39;s cell proliferation defects and to determine whether it acts autonomously within individual cells, we utilized fluorescence-assisted cell sorting (FACS) analysis in whole eyes and Flp-FRT-generated clones. First, FACS analysis demonstrated that dissociated cells from whole dCsk mutant eye-antennal and wing discs consistently exhibited a decrease in the G0-G1 population and an increase in the G2-M population when compared to cells from age-matched control tissues; these results are consistent with a similar analysis in the wing. We found these differences in cell cycle profiles in mutant larvae over a range of ages, from 120 hr to 130 hr AED. Similar results were observed in dCsk EGUF larval eyes. To assess whether the defects observed in dCsk mutants are cell autonomous, we used the Flp-FRT system to generate mutant clones within the eye; to rigorously score the effects on individual cells, we then dissociated the cells and used FACS analysis to segregate the dCsk homozygous clonal cells from their wild-type and heterozygous neighbors. Again, dCsk mutant clones contained an increased G2-M population and a decreased G0-G1 population relative to surrounding control tissue, a cell cycle defect indicative of increased proliferation. Non-dCsk cells were unaffected. Forward scatter measurements confirmed that dCsk homozygous clonal cells and their neighbors were the same average cell size even in different phases of the cell cycle. Together, these data argue that dCsk controls tissue growth cell autonomously by negatively regulating cellular proliferation without affecting cell size, although we cannot rule out subtle non-autonomous effects.  
      dCsk Acts in Opposition to the Src and JNK Pathways  
      We utilized a dCsk j1D8/S030003  trans-heterozygote combination to test candidate loci for an in vivo role in dCsk function. Several candidate genes such as members of the Ras pathway failed to genetically interact with dCsk. The dCsk phenotype was suppressed by mutations in the  Drosophila  Src ortholog Src64B. Normally, 10-40% of developing dCsk flies survived to pharate stages and only 0-1% eclosed (emerged) from their pupal cases. Removing one copy of Src64B led to fully 61% surviving at least as pharate adults, and 26% of these eclosed from their pupal cases. The eclosed adults often displayed wing and leg defects, and typically died within 24-48 hours. Mutations in the Src ortholog Src42A weakly suppressed dCsk phenotypes: 56% of dCsk mutants either eclosed or lived to the pharate stages when one copy of Src42A was removed using the Src42A 18-2  allele.  
      The Btk29A locus encodes the sole Tec-Btk family kinases in the  Drosophila  genome, which function downstream of fly Src kinases such as Src64B. Mutations in Btk29A strongly suppressed dCsk: 70% of Btk29A/+; dCsk flies fully eclosed as nearly normal adults ( FIG. 4A, 4D ) and exhibited only mild wing defects. In addition, reduced Btk29A function also noticeably suppressed the increased body size and prolonged larval phase observed in dCsk mutants (data not shown). FACS analysis of dissociated wing and eye-antennal imaginal discs derived from Btk29A/+; dCsk larvae indicated that removal of a copy of Btk29A suppressed the increase in G2-M cells observed in dCsk mutants, demonstrating that Btk29A mediates the cell cycle defects observed in dCsk mutants.  
      The Jun N-terminal kinase (JNK) signaling pathway has also been identified as a mediator of Src signaling in both mammals and  Drosophila . Consistent with this data, removing one copy of the JNK ortholog basket (bsk) also suppressed the dCsk phenotype. 60% of bsk 1 /+; dCsk j1D8/S030003  flies formed viable adults that fully or partially eclosed. Similar to Src64B; dCsk survivors, these adults exhibited leg and wing defects and died shortly after eclosion. Larvae and pupae also showed suppression of the increased body size (data not shown). FACS analysis indicated that larval eye-antennal discs contained an increased G0-G1 and decreased G2-M population relative to control discs, demonstrating that mutations in bsk suppress the cell cycle defects caused by loss of dCsk.  
      dCsk Negatively Regulates Jak/Stat Signaling  
      Another pathway linked to Src signaling in mammalian tissue culture models is the Jak/Stat signal transduction pathway: Src can directly phosphorylate and activate STAT3 in vitro, and STAT3 function and activation are required for Src transforming activity in multiple tissue culture cell lines. In the  Drosophila  eye, the Jak/Stat pathway controls proliferation and planar polarity. The  Drosophila  Jak/Stat pathway is composed of the ligand Unpaired (Upd), the receptor Domeless, the single Jak ortholog Hopscotch (Hop), and the single STAT ortholog Stat92E. Recent work has demonstrated that over-expression of Upd leads to STAT pathway-dependent overproliferation and ommatidial polarity defects in the eye very similar to those seen in our dCsk EGUF clones. Removing one copy of Stat92E suppressed the Upd overexpression phenotype, indicating that the Upd phenotype was sensitive to alterations in Jak/Stat function. Conversely, removing one copy of dCsk enhanced eye overgrowth caused by Upd over-expression, demonstrating that dCsk negatively regulates the Jak/Stat pathway in this paradigm.  
      One indicator of  Drosophila  Jak/Stat activity is Stat92E protein levels: upd and hop mutant flies show decreased Stat92E protein expression and Upd over-expression in the eye leads to increased Stat92E protein. Cells fully mutant for dCsk exhibited a clear elevation in Stat92E protein levels relative to wild type or heterozygous eye tissue. This increase indicates that the Jak/Stat pathway is up-regulated in dCsk mutants and suggests that this up-regulation may provoke some of the cellular defects observed in dCsk eyes.  
      The dCsk Phenotype Requires Stat92E Function  
      To further explore the role of Stat92E in dCsk function, we utilized the EGUF system to create eyes fully mutant for both dCsk and Stat92E. Eyes mutant for Stat92E alone were mostly normal, showing a slight reduction in size, some misaligned ommatidia and, infrequently, missing antennal structures. Genotypically dCsk j1D8 ; Stat92E 06346  EGUF eyes—the two loci are linked on the same chromosomal arm—were consistently and often significantly smaller than either dCsk j1D8  or Stat92E 06346  EGUF eyes alone, demonstrating a block in the overgrowth phenotype. In addition, dCsk; Stat92E adult eyes were frequently fragmented, with scars and/or patches of eye tissue separated by patches of cuticle, suggesting that mutant tissue underwent localized programmed cell death during development. Doubly mutant flies also exhibited a loss of antennal structures and head cuticle malformations. The cuticle malformations were present on animals with small and scarred eyes suggesting that these malformations are secondary to retinal defects. All of these observations were confirmed in dCsk j1D8 ; Stat92E j6c8  flies, which demonstrated an even higher penetrance of eye tissue loss.  
      To determine if the defects we observed in dCsk; Stat92E EGUF clones were Src-dependent, we removed one copy of Btk29A in dCsk j1D8 ; Stat92E 06346  EGUF clones. If the reduced eye size of dCsk; Stat92E EGUF clones was due to Src hyperactivation, then reduced Btk29A function should ‘rescue’ the dCsk; Stat92E phenotype; if, however, the phenotype was the result of nonspecific synthetic lethality then it should not be sensitive to reduction of Btk29A function. Consistent with former possibility, reduced Btk29A suppressed and rescued the dCsk; Stat92E eye to a more normal phenotype. In particular, while 64% of all adult dCsk; Stat92E eyes were two-thirds or less of normal size, only 21% of all adult eyes from Btk29A k00206 /+; dCsk; Stat92E eyes were that small. Also, 77% of the Btk29A/+; dCsk; Stat92E eyes were normal or nearly normal in size, whereas only 32% of dCsk; Stat92E EGUF eyes were similarly normal. Indeed, most Btk29A/+; dCsk; Stat92E EGUF clones looked very similar to Stat92E EGUF clones, as both genotypes showed some misaligned ommatidia and, occasionally, missing antennal structures.  
      To determine the developmental origin of the dCsk; Stat92E EGUF phenotype, we examined eye-antennal imaginal discs. dCsk j1D8 ; Stat92E 06346  mutant larval eye-antennal discs frequently showed significantly reduced size relative to control, Stat92E, or dCsk EGUF clones, a reduction often also observed in developing antennal tissues. dCsk; Stat92E EGUF eyes showed reduced mitoses anterior to the morphogenetic furrow compared to control or dCsk j1D8  clones. In addition, doubly mutant eye tissue often exhibited patchy expression of neural markers and decreased proliferation relative to control or dCsk j1D8  tissue. Regions with reduced neural development harbored cells with abnormal and pyknotic nuclei as visualized with DAPI staining (data not shown), suggesting that cells within the eye were undergoing apoptosis. Consistent with this data, dCsk j1D8 ; Stat92E 06346  mutant larval eye tissue often exhibited increased programmed cell death and tissue loss within the developing eye field. This apoptosis primarily occurred in regions with reduced neural marker expression, indicating that defective neural differentiation may occur as a consequence of excessive apoptosis during development. Such extensive apoptosis is likely to account for much of the tissue loss and scarring observed in adult dCsk; Stat92E EGUF clones. In summary, reduced Stat92E activity inhibited SFK-mediated overgrowth in dCsk mutant tissue by reducing cell proliferation and promoting apoptotic cell death.  
      Discussion  
      Csk family kinases encode critical negative regulators of Src family kinases (SFKs). In this report we demonstrate that  Drosophila  dCsk is a vital negative regulator of growth and proliferation. Loss of dCsk activity leads to overgrowth of multiple tissues and this overgrowth requires the functions of Src-Btk, JNK, and STAT signal transduction pathways. A recent report has also linked dCsk to the Lats tumor suppressor (Stewart, 2003). Together, these results provide support for the long suspected role of human Csk kinases as tumor suppressors.  
      Partial reduction of Src64B, Src42A, or Btk29A activity suppressed the dCsk phenotype, providing functional data to support the view that the dCsk-mediated overgrowth phenotype results from inappropriate activation of the Src-Btk signal transduction pathways. Mutations in Btk29A more strongly suppressed dCsk than either Src42A or Src64B mutations, perhaps reflecting that (i) Src paralogs act redundantly to each other in  Drosophila  as in mammals and (ii) that Btk29A has previously been shown to act downstream of SFKs in flies and in mammals. Our results provide in vivo evidence that Tec-Btk family kinases are critical to SFK-mediated proliferation and suggest that partial reduction of Tec-Btk kinase activity could reduce proliferation in other cellular contexts in which overgrowth is driven by hyperactivated SFKs, such as in colon tumors.  
      Using a loss-of-function approach to identify effectors that mediate the dCsk overgrowth phenotypes, we failed to implicate some of these pathways in dCsk function. For example, SFKs upregulate the SOS-Ras-ERK pathway in multiple tissue culture studies and  Drosophila  overexpression models. However, although dRas1 signaling is active throughout retinal development, reduced dEGFR, drk (GRB2), Sos, and Jra (c-jun) gene dosage failed to affect the dCsk phenotype and dCsk failed to modify a hypermorphic allele of dEGFR. Levels of doubly-phosphorylated and activated ERK appeared unaltered in dCsk −/−  tissue (data not shown). These data argue that not every signal transduction pathway implicated in SFK tissue culture models necessarily functions as predicted within a developing epithelial tissue.  
      Our genetic studies emphasized the importance of two signaling pathways in dCsk and SFK function. Phenotypic and FACS analysis established that reduced JNK (bsk) function suppressed the phenotypes and cell cycle defects caused by loss of dCsk. These results support studies indicating that JNK functions downstream of the Src-Btk pathway in  Drosophila  and mammalian tissue culture cells. Components of the JNK pathway are required for Src-dependent cellular transformation, but the exact role of JNK in these cells is unknown. Importantly, our data shows that the JNK pathway mediates proliferative responses to Src signaling in vivo, but further work will be needed to precisely understand its mechanism.  
      dCsk proved a negative regulator of Jak/Stat signaling. For example, dCsk mutant tissues up-regulated Stat92E protein, a hallmark of Jak/Stat activation in  Drosophila . Stat92E, the sole  Drosophila  STAT ortholog, is most similar to mammalian STAT3. In mammalian cells, Src directly phosphorylates and activates STAT3, and STAT3 function and activation are required for Src transforming activity. Conversely, overexpression of Csk blocks STAT3 activation in v-Src transformed fibroblasts. However, the physiological significance of these interactions within developing epithelia has remained unclear.  
      dCsk; Stat92E double mutant clones demonstrated that loss of STAT function severely reduced Src-dependent overgrowth and promoted apoptosis of mutant tissue. dCsk −/− ; Stat92E −/−  EGUF adult eyes are strikingly similar to phenotypes caused by over-expression of Dacapo, the fly ortholog of the cdk inhibitor p21, and PTEN, a negative regulator of cell proliferation and growth. Importantly, removing Stat92E function in dCsk mutant tissue led to a synthetic small eye phenotype and did not simply rescue the dCsk −/−  proliferative phenotype. This outcome distinguishes Stat92E from mutations in Src64B, Btk29A, or bsk, which rescued dCsk-mediated defects toward a normal phenotype. The loss of tissue in dCsk −/− ; Stat92E −/−  clones indicates that Src signaling provokes apoptosis and blocks normal proliferation in the absence of Stat92E function. Consistent with this interpretation, reduced Btk29A function rescued the dCsk −/− ; Stat92E −/−  EGUF phenotype to a more normal phenotype, demonstrating that the reduced growth and apoptosis of the dCsk −/− ; Stat92E −/−  tissues is indeed Src-pathway-dependent.  
      Our data suggest the existence of a Src-dependent pro-apoptotic and anti-proliferative pathway that is normally suppressed by STAT. One possible component of this pathway is JNK given that JNK signaling is an important activator of apoptosis in both flies and mammals. Perhaps Src-dependent hyperactivation of Bsk (JNK) in dCsk −/− ; Stat92E −/−  tissue contributes to cell death in the absence of proliferative and/or survival signals provided by Stat92E. However, a number of other candidate pathways may also mediate this response. The further characterization and identification of these pathways may have important implications for interceding in Src-mediated oncogenesis.  
      Together, these observations indicate that, in tissue that contains hyperactive Src or reduced Csk, blocking STAT function is sufficient to decrease proliferation and trigger apoptosis in the absence of any further mutations or interventions. Reduced STAT3 function can promote apoptosis within breast and prostate cancer cells that show elevated SFK activity, but the molecular pathways driving apoptosis in these cells are unknown {Garcia, 2001 #4514; Mora, 2002 #4524}. These cells may require survival signals provided by STAT3 to counteract apoptosis due to chromosomal abnormalities or other defects. Alternatively, these cells may die because of pro-apoptotic signals provided by hyperactive SFKs in the absence of STAT3 function. Our data argue that the latter may be true, which suggests the intriguing possibility that therapeutic blockade of STAT function in tumors with activated Src may actively provoke Src-dependent apoptosis and growth arrest in tumor tissues.  
      Other Embodiments  
      When introducing elements of the present invention or the preferred embodiments thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.  
      As various changes could be made in the above constructions without departing from the scope of the invention, it is intended that all matter contained in the above description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description which do not depart from the spirit or scope of the present inventive discovery. Such modifications are also intended to fall within the scope of the appended claims.  
      References Cited  
      All publications, patents, patent applications and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.  
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