Patent Application
20090186815
The present invention relates to a method for identifying a compound capable of modulating the activity of the JAK/STAT pathway and to the use of different JAK/STAT pathway components as a target for the modulation of the activity of the JAK/STAT pathway. Moreover, the present invention is concerned with a method for modulating the activity of the JAK/STAT pathway. Furthermore, the present invention pertains to a pharmaceutical composition and to the use of different JAK/STAT pathway components and/or effector molecules thereof for the manufacture of such composition for the diagnosis, prevention or treatment of a JAK/STAT pathway associated disorder.
Signalling pathways mediating the transduction of information between cells are essential for development, cellular differentiation and homeostasis (Brivanlou, A. H. & Darnell, J. E., Jr., Science 295, 813-8. (2002)). Their dysregulation is also frequently associated with human malignancies. The JAK/STAT pathway represents one such signalling cascade whose evolutionarily conserved roles include cell proliferation and haematopoiesis (Hombria, J. C. & Brown, S., Curr Biol 12, R569-75 (2002)).
Developmental genetic screens in Drosophila have identified multiple JAK/STAT pathway components on the basis of their segmentation phenotype (Binari, R. & Perrimon, N., Genes Dev 8, 300-12. (1994); Harrison, D. A., McCoon, P. E., Binari, R., Gilman, M. & Perrimon, N., Genes Dev 12, 3252-63. (1998); Hou, X. S., Melnick, M. B. & Perrimon, N., Cell 84, 411-9 (1996)) and subsequent analysis of the pathway has characterised evolutionarily conserved roles during immune responses, haematopoiesis and cellular proliferation (Lagueux, M., Perrodou, E., Levashina, E. A., Capovilla, M. & Hoffmann, J. A., Proc Natl Acad Sci USA 97, 11427-32. (2000); Boutros, M., Agaisse, H. & Perrimon, N., Dev Cell 3, 711-22. (2002); Meister, M. & Lagueux, M., Cell Microbiol 5, 573-580 (2003); Mukherjee, T., Castelli-Gair Hombria, J. & Zeidler, M. P., Oncogene in press (2005)). The JAK/STAT signalling cascade in Drosophila is comprised of the extracellular ligand Unpaired (Upd) (Harrison, D. A., McCoon, P. E., Binari, R., Gilman, M. & Perrimon, N., Genes Dev 12, 3252-63. (1998)), a trans-membrane receptor with homology to the IL6 receptor family termed Domeless (Dome) (Brown, S., Hu, N. & Castelli-Gair Hombria, J., Curr Biol 11, 1700-5. (2001)), a single Janus tyrosine kinase (JAK) called Hopscotch (Hop) (Binari, R. & Perrimon, N., Genes Dev 8, 300-12. (1994)) and the STAT92E transcription factor (Hou, X. S., Melnick, M. B. & Perrimon, N., Cell 84, 411-9 (1996); Yan, R., Small, S., Desplan, C., Dearolf, C. R. & Darnell, J. E., Jr., Cell 84, 421-30 (1996)) (
In order to identify novel pathway components and circumvent limitations of classical genetic screens, the inventors of the present invention have undertaken a genome-wide RNA interference (RNAi) screen, a powerful technique for the identification of new components of diverse cellular pathways (Kamath, R. S. et al., Nature 421, 231-7 (2003); Kittler, R. et al., Nature 432, 1036-40 (2004); Berns, K. et al., Nature 428, 431-7 (2004); Paddison, P. J. et al., Nature 428, 427-31 (2004); Boutros, M. et al., Science 303, 832-5 (2004)). Using this screen, a systematic genome-wide survey for genes required for JAK/STAT pathway activity could be performed. Analysis of 20,026 RNAi-induced phenotypes in cultured Drosophila melanogaster haemocyte-like cells identified interacting genes encoding 4 known and 84 previously uncharacterised proteins. Subsequently, cell based epistasis experiments have been used to classify these based on their interaction with known components of the signalling cascade. In addition to multiple human disease gene homologues, the inventors of the present invention have identified the tyrosine phosphatase Ptp61F and the Drosophila homologue of BRWD3, a bromo-domain containing protein disrupted in leukaemia. Moreover, in vivo analysis demonstrates that disrupted dBRWD3 and overexpressed Ptp61F function as suppressors of leukaemia-like blood cell tumours. This screen represents a comprehensive identification of novel loci required for JAK/STAT signalling and provides molecular insights into an important pathway relevant for human diseases.
A first aspect of the present invention, therefore, relates to a method for identifying a compound capable of modulating the activity of the JAK/STAT pathway, comprising
(a) contacting a compound with at least one target molecule selected from
In accordance with the present invention, it is to be understood, that the term “modulating the activity of the JAK/STAT pathway”, when used herein, means activating or inhibiting the activity of the JAK/STAT signalling pathway. An activation or inhibition of the activity of the JAK/STAT signalling pathway may e.g. be mediated by an activation or inhibition of at least one component of the JAK/STAT pathway, either directly or indirectly.
According to the present invention, step (a) of the method for identifying a compound capable of modulating the activity of the JAK/STAT pathway comprises contacting a compound with at least one target molecule selected from the nucleic acid molecules of (i) and the polypeptide molecules of (ii).
The nucleic acid molecules of (i) used according to the method for identifying a compound capable of modulating the activity of the JAK/STAT pathway comprise in one embodiment of the present invention a nucleotide sequence of (i.1) as show in SEQ ID NOs. 88 to 265. Preferably, the nucleic acid molecules of (i) comprise a nucleic acid sequence of (i.1) as shown in SEQ ID NOs. 88 to 174. More preferably, the nucleic acid molecules of (i) comprise a nucleic acid sequence of (i.1) as shown in SEQ ID NOs. 91, 116, 124, 133, 136, 152, 154, 155 to 174.
It is to be understood that the Drosophila gene sequences of SEQ ID Nos. 175-265 encompasse respective splice variants.
Moreover, nucleic acid molecules of (i) used according to the method for identifying a compound capable of modulating the activity of the JAK/STAT pathway comprise in another embodiment of the present invention a nucleotide sequence of (i.2) which is complementary to a nucleotide sequence of (i.1). Preferably, the nucleic acid molecules of (i) comprise a nucleic acid sequence of (i.2) which is complementary to a nucleotide sequence of (i.1) as shown in SEQ ID NOs. 88 to 174. More preferably, the nucleic acid molecules of (i) comprise a nucleic acid sequence of (i.2) which is complementary to a nucleotide sequence of (i.1) as shown in SEQ ID NOs. 91, 116, 124, 133, 136, 152, 154, 155 to 174.
In a further embodiment of the present invention, the nucleic acid molecules of (i) used according to the method for identifying a compound capable of modulating the activity of the JAK/STAT pathway comprise a nucleotide sequence of (i.3) which has an identity of at least 65, preferably at least 70, more preferably at least 75 and most preferably at least 80% to a nucleotide sequence of (i.1) or (i.2). Within the context of the present application, the term “has an identity of at least 65, preferably at least 70, more preferably at least 75 and most preferably at least 80%”, as used herein, means that the sequence identity is at least 65, 66, 67, 6, 69, preferably at least 70, 71, 72, 73, 74, more preferably at least 75, 76, 77, 78, 79 and most preferably at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. Preferably, the nucleic acid molecules of (i) comprise a nucleotide sequence of (i.3) which has an identity of at least 65, preferably at least 70, more preferably at least 75 and most preferably at least 80% to a nucleotide sequence of (i.1) as shown in SEQ ID NOs. 88 to 174 or a nucleotide sequence of (i.2) which is complementary to a nucleotide sequence of (i.1) as shown in SEQ ID NOs. 88 to 174. More preferable, the nucleic acid molecules of (i) comprise a nucleotide sequence of (i.3) which has an identity of at least 65, preferably at least 70, more preferably at least 75 and most preferably at least 80% to a nucleotide sequence of (i.1) as shown in SEQ ID NOs. 91, 116, 124, 133, 136, 152, 154, 155 to 174 or a nucleotide sequence of (i.2) which is complementary to a nucleotide sequence of (i.1) as shown in SEQ ID NOs. 91, 116, 124, 133, 136, 152, 154, 155 to 174.
Finally, the nucleic acid molecules of (i) used according to the method for identifying a compound capable of modulating the activity of the JAK/STAT pathway comprise in a further embodiment of the present invention a nucleotide sequence of (i.4) which hybridizes under stringent conditions to a nucleotide sequence of (i.1), (i.2) or (i.3). The term “hybridizes under stringent conditions” according to the present application is used as described in Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, Laboratory Press (1989), 1.101-1.104. Consequently, hybridization under stringent conditions occurs when a positive hybridization signal is still detected after washing for 1 h with 1×SSC and 0.1% SDS at 55° C., preferably at 62° C. and most preferably at 68° C., in particular for 1 h in 0.2×SSC and 0.1% SDS at 55° C., preferably at 62° C. and most preferably at 68° C. It is preferred that the nucleic acid molecules of (i) comprise a nucleotide sequence of (i.4) which hybridizes under stringent conditions to a nucleotide sequence of (i.1) as shown in SEQ ID NOs. 88 to 174, a nucleotide sequence of (i.2) which is complementary to a nucleotide sequence of (i.1) as shown in SEQ ID NOs. 88 to 174 or a nucleotide sequence of (i.3) which has an identity of at least 65, preferably at least 70, more preferably at least 75 and most preferably at least 80% to a nucleotide sequence of (i.1) as shown in SEQ ID NOs. 88 to 174 or a nucleotide sequence of (i.2) which is complementary to a nucleotide sequence of (i.1) as shown in SEQ ID NOs. 88 to 174. More preferably, the nucleic acid molecules of (i) comprise a nucleotide sequence of (i.4) which hybridizes under stringent conditions to a nucleotide sequence of (i.1) as shown in SEQ ID NOs. 91, 116, 124, 133, 136, 152, 154, 155 to 174, a nucleotide sequence of (i.2) which is complementary to a nucleotide sequence of (i.1) as shown in SEQ ID NOs. 91, 116, 124, 133, 136, 152, 154, 155 to 174 or a nucleotide sequence of (i.3) which has an identity of at least 65, preferably at least 70, more preferably at least 75 and most preferably at least 80% to a nucleotide sequence of (i.1) as shown in SEQ ID NOs. 91, 116, 124, 133, 136, 152, 154, 155 to 174 or a nucleotide sequence of (i.2) which is complementary to a nucleotide sequence of (i.1) as shown in SEQ ID NOs. 91, 116, 124, 133, 136, 152, 154, 155 to 174.
The nucleic acid molecules of (i) used according to the method for identifying a compound capable of modulating the activity of the JAK/STAT pathway may be present in single-stranded or double-stranded form and may be selected from RNA, DNA or nucleic acid analog molecules, such as sugar- and backbone-modified ribonucleic acids or deoxyribonucleic acids. It should be noted, however, that other nucleic acid analogs, such as peptide nucleic acids (PNA) or locked nucleic acids (LNA), are also suitable.
Moreover, according to the present invention, the nucleic acid molecules of (i) used according to the present invention may be non-recombinant nucleic acid molecules, recombinant nucleic acid molecules generated by recombinant methods, e.g. by known amplification procedures such as PCR, or chemically synthesized nucleic acid molecules. The nucleic acid molecules of (i) may be present in isolated, i.e. purified, form or in non-isolated form, i.e. in a cellular environment.
In a preferred embodiment of the present invention, the nucleic acid molecules of (i) used according to the present invention are present in a vector, which may be any prokaryotic or eukaryotic vector, on which the nucleic acid sequence is present preferably under control of a suitable expression signal, e.g. promoter, operator, enhancer etc. Examples for prokaryotic vectors are chromosomal vectors, such as bacteriophages, and extrachromosomal vectors, such as plasmids, wherein circular plasmid vectors are preferred. Examples for eukaryotic vectors are yeast vectors or vectors suitable for higher cells, e.g. insect cells or mammalian cells, plasmids or viruses.
The polypeptide molecules of (ii) used according to the method for identifying a compound capable of modulating the activity of the JAK/STAT pathway are encoded by the nucleic acid molecules of (i) described above and or have a sequence as shown in SED ID Nos. 1-87. According to a preferred embodiment of the present invention, the polypeptide molecules of (ii) have an amino acid sequence as shown in SEQ ID NO. 4, 29, 37, 46, 49, 65, 67 to 87.
The compound used in step (a) of the method for identifying a compound capable of modulating the activity of the JAK/STAT pathway may be selected from compounds capable of directly and/or indirectly inhibiting or activating the transcription or translation of a nucleic acid molecule of (i). Preferably, the compounds capable of directly and/or indirectly inhibiting or activating the transcription or translation of a nucleic acid molecule of (i) comprise polypeptides such as proteins, enzymes, antibodies, polypeptide inhibitors, polypeptide activators, agonist, antagonists, mimetics, low molecular weight substances, antisense molecules, RNAi molecules and ribozymes. More preferably, the compounds capable of directly and/or indirectly inhibiting or activating the transcription or translation of a nucleic acid molecule of (i) are antisense molecules directed against a nucleic acid molecule of (i) or RNAi molecules. The antisense molecules and RNAi molecules may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesising oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, said molecules may be generated by in vitro and in vivo transcription of DNA sequences.
Moreover, the compound used in step (a) of the method for identifying a compound capable of modulating the activity of the JAK/STAT pathway may also be selected from compounds capable of directly and/or indirectly inhibiting or activating a polypeptide molecule of (ii). Preferably, the compounds capable of directly and/or indirectly inhibiting or activating a polypeptide molecule of (ii) comprise polypeptides such as proteins, enzymes, antibodies, polypeptide inhibitors, polypeptide activators, agonist, antagonists, mimetics, oligopeptides, low molecular weight substances and polypeptide cofactors. More preferably, the compounds capable of directly and/or indirectly inhibiting or activating a polypeptide molecule of (ii) are antibodies or fragments thereof directed against a polypeptide molecule of (ii). Within the context of the present invention, the term “antibody”, as used herein, encompasses polyclonal antibodies, monoclonal antibodies, e.g. chimeric antibodies, humanized antibodies, human antibodies or recombinant antibodies, e.g. single-chain antibodies. Further, the term “antibody fragment” encompasses common antibody fragments, e.g. proteolytic fragments such as Fab, F(ab)2, Fab′ or recombinant fragments such as scFv. The antibodies or fragments thereof may be obtained using hybridoma cell lines or recombinant DNA methods using techniques well known in the art. However, the antibodies or fragments thereof may also be isolated from phage antibody libraries using techniques described in the art.
According to the present invention, step (b) of the method for identifying a compound capable of modulating the activity of the JAK/STAT pathway comprises determining the degree of modulation of the at least one target molecule by the compound. Preferably, the degree of modulation of the at least one target molecule by the compound may be determined either by measuring the amount and/or expression rate of the nucleic acid molecules of (i) or by measuring the amount and/or activity of the polypeptide molecules of (ii). A variety of protocols including, for example, ELISA, RIA, and FACS, for measuring nucleic acid molecules and/or proteins are known in the art and provide a basis for measuring the amount and/or expression rate of a nucleic acid molecule or the amount and/or activity of a polypeptide molecule. Particularly, the capability of a substance to modulate the activity of the JAK/STAT pathway is determined as described in the Example.
According to the present invention, the method for identifying a compound capable of modulating the activity of the JAK/STAT pathway may be a molecular based assay or a cellular assay. Therefore, the at least one target molecule may be provided either in vivo in a cellular system, preferably a cellular system overexpressing the at least one target molecule, or in vitro in cell fractions containing the at least one target molecule or with the at least one target molecule in a substantially isolated and purified form. Methods for providing the at least one target molecule are well known in the art and may be used in performing the present invention. According to the present invention, it is preferred that the method for identifying a compound capable of modulating the activity of the JAK/STAT pathway is performed in a high-throughput format.
A second aspect of the present invention pertains to the use of at least one molecule selected from
(i) nucleic acid molecules, comprising
Within the context of the present invention, the nucleic acid molecules of (i), comprising (i.1) a nucleotide sequence as shown in SEQ ID NOs. 88 to 265, (i.2) a nucleotide sequence which is complementary to a nucleotide sequence of (i.1), (i.3) a nucleotide sequence which has an identity of at least 65% to a nucleotide sequence of (i.1) or (i.2), and/or (i.4) a nucleotide sequence which hybridizes under stringent conditions to a nucleotide sequence of (i.1), (i.2) or (i.3), and the polypeptide molecules of (ii) encoded by the nucleic acid molecules of (i) used as afore-mentioned are as described above.
A third aspect of the present invention relates to a method for modulating the activity of the JAK/STAT pathway comprising contacting a cell with at least one molecule selected from
(i) nucleic acid molecules, comprising
The method for modulating the activity of the JAK/STAT pathway may suitably be performed as molecular based assay or cellular assay. Preferably, the cell used in the method for modulating the activity of the JAK/STAT pathway is a cell showing the JAK/STAT pathway, e.g. an animal cell.
According to the present invention, the nucleic acid molecules of (i), comprising (i.1) a nucleotide sequence as shown in SEQ ID NOs. 88 to 265, (i.2) a nucleotide sequence which is complementary to a nucleotide sequence of (i.1), (i.3) a nucleotide sequence which has an identity of at least 65% to a nucleotide sequence of (i.1) or (i.2), and/or (i.4) a nucleotide sequence which hybridizes under stringent conditions to a nucleotide sequence of (i.1), (i.2) or (i.3), and the polypeptide molecules of (ii) encoded by the nucleic acid molecules of (i) used according to the method for modulating the activity of the JAK/STAT pathway are as described above.
Moreover, the effector molecules of (i) and/or (ii) used according to the method for modulating the activity of the JAK/STAT pathway are selected from polypeptides such as proteins, enzymes, antibodies, polypeptide inhibitors, polypeptide activators, agonist, antagonists, mimetics, oligopeptides, cofactors, low molecular weight substances, antisense molecules, RNAi molecules and ribozymes. Preferably, the effector molecules of (i) and/or (ii) are compounds identified by the method for identifying compounds of modulating the activity of the JAK/STAT pathway described above. More preferably, the effector molecules of (i) and/or (ii) are antibodies or fragments thereof directed against a polypeptide molecule of (ii), antisense molecules directed against a nucleic acid molecule of (i) and/or RNAi molecules.
Further, the present invention is concerned in a fourth aspect with a pharmaceutical composition comprising as an active agent at least one molecule selected from
(i) nucleic acid molecules, comprising
According to the present invention, the nucleic acid molecules of (i), comprising (i.1) a nucleotide sequence as shown in SEQ ID NOs. 88 to 265, (i.2) a nucleotide sequence which is complementary to a nucleotide sequence of (i.1), (i.3) a nucleotide sequence which has an identity of at least 65% to a nucleotide sequence of (i.1) or (i.2), and/or (i.4) a nucleotide sequence which hybridizes under stringent conditions to a nucleotide sequence of (i.1), (i.2) or (i.3), the polypeptide molecules of (ii) encoded by the nucleic acid molecules of (i) and the effector molecules (of (iii)) of (i) and/or (ii) comprised in the pharmaceutical composition of the invention are as described above.
In addition to the at least one active ingredient, the pharmaceutical composition of the invention may contain suitable pharmaceutically acceptable carriers, diluents and/or adjuvants, which facilitate processing of the active ingredient into preparations, which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
The pharmaceutical composition of the present invention is particularly suitable for the diagnosis, prevention or treatment of a JAK/STAT pathway associated disorder. Preferably, the JAK/STAT pathway associated disorder is selected from the group consisting of papillary thyroid carcinoma, Refsum disease, blood-brain barrier glucose transport defect, X-linked nonsyndromic mental retardation, long QT syndrome 4, subcortical laminar heterotopia, leukemia, steroid-resistant nephrotic syndrome, invasive pituitary tumor, sporadic Sotos syndrome, autosomal dominant iron overload, hereditary pancreatitis, stomatocytosis I, atypical Rett syndrome, phosphoglycerate dehydrogenase deficiency, Wolman disease, neurophysiologic defect in schizophrenia, autosomal recessive SCID (T-negative/B-positive type), atelostogenesis (type I), Larson syndrome, spondylocarpotarsal synostosis syndrome, frontometaphyseal dysplasia, diabetes mellitus (type II), susceptibility to insulin resistance, Griscelli Syndrome, limb-girdle muscular dystrophy (type 2A), growth hormone insensitivity with immunodeficiency and breast cancer.
In one embodiment of the present invention the pharmaceutical composition is used for the prevention or treatment of a JAK/STAT pathway associated disorder. Pharmaceutical compositions suitable for the prevention or treatment of a JAK/STAT pathway associated disorder include compositions wherein the at least one active ingredient is contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art. For any compounds, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
The actual amount of the pharmaceutical composition administered, will of course, be dependent on the subject being treated, on the subject's weight, the severity of the JAK/STAT pathway associated disorder, the manner of administration and the judgement of the prescribing physician. For the pharmaceutical composition of the invention, a daily dosage of 1 to 200 mg of the at least one active ingredient per kg and day, particularly 10 to 100 mg of the at least one active ingredient per kg and day, is suitable. Suitable routes of administration may, for example, include oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administrations. Preferably, the subject being treated is an animal, in particular a human being.
In another embodiment of the present invention the pharmaceutical composition is used for the diagnosis of a JAK/STAT pathway associated disorder, e.g. a disorder characterized by or associated with the over- or underexpression of a nucleic acid molecule of (i) or a polypeptide molecule of (ii). Diagnostic assays include methods which utilize the pharmaceutical composition and a label to detect the nucleic acid molecule of (i) or polypeptide molecule of (ii) in human body fluids or extracts of cells or tissues.
Finally, a further aspect of the present invention relates to the use of at least one molecule selected from
(i) nucleic acid molecules, comprising
According to the present invention, the nucleic acid molecules of (i), comprising (i.1) a nucleotide sequence as shown in SEQ ID NOs. 88 to 265, (i.2) a nucleotide sequence which is complementary to a nucleotide sequence of (i.1), (i.3) a nucleotide sequence which has an identity of at least 65% to a nucleotide sequence of (i.1) or (i.2), and/or (i.4) a nucleotide sequence which hybridizes under stringent conditions to a nucleotide sequence of (i.1), (i.2) or (i.3), the polypeptide molecules of (ii) encoded by the nucleic acid molecules of (i) and the effector molecules (of (iii)) of (i) and/or (ii) used according to the present invention for the manufacture of a pharmaceutical composition for the diagnosis, prevention or treatment of a JAK/STAT pathway associated disorder are as described above.
Moreover, according to the present invention, the pharmaceutical composition and the JAK/STAT pathway associated disorder are as described above.
Methods for the manufacture of a pharmaceutical composition, comprising the step of admixing at least one molecule selected from nucleic acid molecules of (i), comprising (i.1) a nucleotide sequence as shown in SEQ ID NOs. 88 to 265, (i.2) a nucleotide sequence which is complementary to a nucleotide sequence of (i.1), (i.3) a nucleotide sequence which has an identity of at least 65% to a nucleotide sequence of (i.1) or (i.2), and/or (i.4) a nucleotide sequence which hybridizes under stringent conditions to a nucleotide sequence of (i.1), (i.2) or (i.3), polypeptide molecules of (ii) encoded by the nucleic acid molecules of (i) and effector molecules (of (iii)) of (i) and/or (ii) with a pharmaceutically acceptable excipient, vehicle or carrier and optionally other ingredients are well known to those skilled in the art and may be used in performing the present invention.
Further, the present invention shall be explained by the following Tables, Figures and Example.
Table 1 shows the RNAi JAK/STAT phenotypes.
Table 2 shows the functional groups classified by InterPro prediction and GO.
Table 3 shows the genetic interactions with hopTuml.
Table 4 shows sequence and cytological information.
Table 5 shows human homologues of Drosophila genes with JAK/STAT phenotypes.
Table 6 shows human disease homologues of Drosophila genes with JAK/STAT phenotypes.
Supplementary Table 7 shows the expected and observed phenotype frequency.
Table 7 shows preferred human JAK/STAT homologues ranked according to their involvement in a human disease.
Table 8 shows evolutionary and functional conservation of JAK/STAT pathway components.
Signalling pathways mediating the transduction of information between cells are essential for development, cellular differentiation and homeostasis (Brivanlou, A. H. & Darnell, J. E., Jr., Science 295, 813-8. (2002)). Their dysregulation is also frequently associated with human malignancies. The JAK/STAT pathway represents one such signalling cascade whose evolutionarily conserved roles include cell proliferation and haematopoiesis (Hombria, J. C. & Brown, S., Curr Biol 12, R569-75 (2002)). Here, the inventors of the present invention describe a systematic genome-wide survey for genes required for JAK/STAT pathway activity. Analysis of 20,026 RNAi-induced phenotypes in cultured Drosophila melanogaster haemocyte-like cells identified interacting genes encoding 4 known and 84 previously uncharacterised proteins. Subsequently, cell based epistasis experiments have been used to classify these based on their interaction with known components of the signalling cascade. In addition to multiple human disease gene homologues, the inventors of the present invention have identified the tyrosine phosphatase Ptp61F and the Drosophila homologue of BRWD3, a bromo-domain containing protein disrupted in leukaemia (Kalla, C. et al., Genes Chromosomes Cancer 42, 128-43 (2005)). Moreover, in vivo analysis demonstrates that disrupted dBRWD3 and overexpressed Ptp61F function as suppressors of leukaemia-like blood cell tumours. This screen represents a comprehensive identification of novel loci required for JAK/STAT signalling and provides molecular insights into an important pathway relevant for human diseases.
The JAK/STAT firefly luciferase reporter 6x2xDrafLuc was constructed by multimerisation of a molecularly characterised STAT92E binding site present in the promoter of the endogenous target genes Draf (Kwon, E. J. et al., J Biol Chem 275, 19824-19830 (2000)) while the 4xsocsLuc reporter is based on a single region containing four potential STAT92E binding sites present within the first intron of socs36E (Karsten, P., Hader, S. & Zeidler, M. P., Mech Dev 117, 343-6 (2002)). A Renilla luciferase reporter gene under the control of the constitutively active Actin5C promoter was co-transfected and used to monitor cell number.
Strictly speaking, the JAK/STAT reporter 6x2xDrafLuc was constructed by multimerisation of STAT92E binding sites. Specifically, a 165 bp blunted BamHI/Xbal fragment from the original p2xDrafSTAT(wt) (Kwon, E. J. et al., J Biol Chem 275, 19824-19830 (2000)) (a kind gift of M. Yamaguchi and M.A. Yoo) was inserted into the Smal cut p2xDrafSTAT(wt). The same fragment was amplified by PCR with NotI sites on both ends and inserted into compatible sites to yield the 3x2xDrafLuc reporter containing six STAT92E binding sites. These fragments were amplified again and the resulting 540 bp fragment was inserted into the Sacl cut 3x2xDrafLuc vector to generate the 6x2xDrafLuc reporter with an enhancer of approximately 1000 bp containing a total of 12 STAT92E binding sites. A second independent JAK/STAT pathway reporter, 4xsocsLuc, was generated by amplifying a 745 bp product from genomic DNA using the primers 5′-GTTAGGTACCGGGTCGCAGTATCGTTGGCG-3′ and 5′-CGMGGATCC CTGTCACTTCTCAGAAATCGGTC-3′. This was then cut with EcoRI/BamHI to give a 285 bp fragment, subcloned into pBS(KS+) (Stratagene) and re-excised with Asp718/BamHI. This 340 bp fragment, containing four predicted STAT92E binding sites (Karsten, P., Hader, S. & Zeidler, M., Mech Dev 117, 343. (2002)), was cloned into Asp718/BgIII sites of pGL3 vector (Promega).
The pAct-RL vector expressing Renilla luciferase from a constitutive reporter was generated by cloning a 974 bp fragment coding for Renilla luciferase from pRLSV40 (Invitrogen) into the BamHI/Xbal cut pPAc5c-PL vector (a kind gift from Dan Curtis). To generate the pAct-UpdGFP vector, a cDNA coding for Upd (Harrison, D. A., Binari, R., Nahreini, T. S., Gilman, M. & Perrimon, N., Embo J 14, 2857-65 (1995)) was fused in frame to EGFP via a BamHI site and inserted into the BamHI/Xbal cut pPAc5c-PL vector. A vector expressing the dominant gain-of-function allele HopTumL was cloned by inserting the open reading frame obtained from pUAS-hopTumL (Harrison, D. A., Binari, R., Nahreini, T. S., Gilman, M. & Perrimon, N., Embo J 14, 2857-65 (1995)) into the NotI/Xbal cut pAc5.1A vector (Invitrogen). A pAc5.1-Sid-1 expression construct which was used to facilitate uptake of dsRNA was a gift of Craig Hunter (Feinberg, E. H. & Hunter, C. P., Science 301, 1545-7 (2003)).
To generate Ptp61F. expression constructs, cDNAs encoding Ptp61Fc (LP01280) and Ptp61Fa (RE01370) were obtained from the DrosophilaGenomics Resource Center (University of Indiana). cDNA clones were analysed by restriction analysis and end sequencing to confirm their integrity before subcloning into pAc5.1A and pUAST (Brand, A. H. & Perrimon, N., Development 118, 401-15 (1993)). For Ptp61Fc, the coding region of LP01280 was excised as an EcoRI/XhoI (partial digest) fragment of 1.8 kb and cloned into pUAST. Subsequently, the insert was re-excised with EcoRI/Xbal and cloned into pAc5.1A (Invitrogen). For Ptp61Fa, the coding region of RE01370 was cut out with EcoRI/Asp718(filled) and cloned into pAc5.1A cut EcoRI/Xbal(filled). The generate a pUAST construct, an EcoRI/Asp718 fragment was used.
To clone p[w+,UAS-dPIAS-GFP], the EST clone LD09022 was used as a template in conjunction with the oligos 5′-CATCGGATCCTGCAAAAAGGGG TCCAACGTACC GGAT-3′ and 5′-GGGGTACCAAAAATGGTGCATATGCTT CGA-3′ to amplify a region coding for 522 amino acids. The resulting product was sequenced, cut with Asp718/BamHI and subcloned into pBS-EGFP-B to generate an in frame C-terminal EGFP fusion protein. This gene was then subcloned as an Asp718/Xbal fragment into pUAST (Brand, A. H. & Perrimon, N., Development 118, 401-15 (1993)).
Multiple independent transgenic Drosophila stocks of each transformation vector construct were generated by microinjection of embryos using standard techniques (Spradling, A. C. & Rubin, G. M., Science 218, 341-347 (1982)).
A genome-wide RNAi library based on PCR templates with an average length of 408 bp flanked by T7-promotor binding sites was generated by in vitro transcription (Boutros, M. et al., Science 303, 832-5 (2004)). Therefore, PCR fragments containing T7 promoter sequences on each end (Hild, M. et al., Genome Biol 5, R3 (2003)) were used as templates to generate 20,026 dsRNAs by in vitro transcription (Boutros, M. et al., Science 303, 832-5 (2004)). After DNAse I treatment, dsRNAs were purified by ethanol precipitation and individually quality controlled by gel electrophoresis. RNAs were diluted to a working stock concentration and aliquoted in ready-to-screen 384-well tissue culture plates (Greiner). Computational mapping predict that the 20,026 RNA fragments target >91% of all predicted genes in the Drosophila genome (Annotation 4.0) (Misra, S. et al., Genome Biol 3, RESEARCH0083-3 (2002)). Protocols and supplemental material can be found at http://www.dkfz-heidelberg.de/signaling/jak-pathway/. Complete primer and amplicon sequence information for double-stranded RNAs including calculation of predicted efficiency and off-target effects for the RNAi library is publicly accessible at http://rnai.dkfz.de.
For screening experiments, Drosophila Kc167 cells were maintained in Schneider's medium (Invitrogen) supplemented with 10% foetal bovine serum (PAA) and 100 μg/ml penicillin-streptomycin (Invitrogen). Cells were grown at 25° C. at subconfluent densities. The RNAi screening experiments were performed in white, polystyrene 384-well tissue culture plates (Greiner 781 073). A total of fiftyseven 384-well screening plates were loaded with an average of 75 nM (500 ng) dsRNA in 5 μl of 1 mM Tris pH 7. Kc167 cells were transfected in batch in 6-well plates with 0.25 μg of the 6x2xDrafLuc JAK/STAT signalling reporter, 0.6 μg of pAct-UpdGFP expression vector, 0.25 μg pAc5.1-Sid-1 (to facilitate RNA uptake (Feinberg, E. H. & Hunter, C. P., Science 301, 1545-7 (2003))) and 0.025 μg of pAct-RL vector as a co-reporter. The total plasmid amount was normalised to 2 μg with a pAc5.1 plasmid (Invitrogen) and 5×106 cells were transfected with Effectene (Qiagen). After 7 hours incubation at 25° C., batch transfected cells were resuspended in serum-free medium. Subsequently 15,000 cells in 20 μl were dispensed per dsRNA containing well using an automated liquid dispenser (MultiDrop, Thermo Labsystems). Cells were incubated for 45 min and 30 μl of serum-containing medium was added to each well. Cells were grown for 5 days to allow for protein depletion. Pathway activity was measured for using a luminescence assay for firefly and Renilla luciferase on a Mithras LB940 plate reader (Berthold Technologies). Luminescence of the Renilla luciferase was measured using a 490 nm filter set. Screens were performed in duplicate. Each plate contained dsRNA targeting stat92E, dome, hop and socs36E in A1, A2, B1, B2 which were used as positive controls (see also
To identify candidate genes that significantly increase or decrease JAK/STAT signalling pathway activity, the raw luciferase results were normalised by median centering of each 384-well plate (separately by channel). Z-scores were calculated as the number standard deviation that a particular well differed from the median of the 384-well plate. To minimise false negatives, the inventors of the present invention applied a set of low-stringency criteria to generate a list of candidate genes to be used in specific retests. First, the inventors filtered dsRNA treatments with z-scores >2 for negative regulators or <−2 for positive regulators, respectively. Treatments that showed a high variability between duplicates were excluded. Further, RNAi experiments that showed z-scores of >2 or <−2 in the control channel were not selected for retesting. The inventors also filtered against previously identified cell viability modifiers that show a phenotype in cultured Drosophila cells (Boutros, M. et al., Science 303, 832-5 (2004)). The inventors also excluded genes that showed phenotypes in other screens. These filtering steps led to a final list of approximately 107 candidates that were selected for retesting. New dsRNA was re-synthesized as described above and repeat assays were performed in quadruplicate. 89 of the candidates were confirmed using a second JAK/STAT reporter assay (4xsocsLuc) employed to exclude reporter-specific artefacts. Data analysis and representation were performed using R and Bioconductor (Gentleman, R. C. et al., Genome Biol 5, R80 (2004)).
The predicted genes targeted by 91 dsRNAs were classified according to InterPro (Mulder, N. J. et al., Nucleic Acids Res 33 Database Issue, D201-5 (2005)) and GO (Harris, M. A. et al., Nucleic Acids Res 32, D258-61 (2004); Drysdale, R. A. et al., Nucleic Acids Res 33 Database Issue, D390-5 (2005)) and manual inspection was used to order genes into functional groups. Predicted proteins without InterPro domain or GO annotation were classified as “Unknown” although these sequences might encode structurally conserved proteins. To determine whether Drosophila proteins have homologues in other species, the inventors used BLASTP searches against the protein predictions from H. sapiens (NCBI build 35) with a cut-off of E<10−10. Databases were obtained from Ensembl (http://www.ensembl.org) (Clamp, M. et al., Nucleic Acids Res 31, 38-42 (2003)) and Flybase (hftp://www.flybase.org) (Drysdale, R. A. et al., Nucleic Acids Res 33 Database Issue, D390-5 (2005)). Reciprocal best BLASTP analysis was used to identify the human homologue of CG31132. CG31132 and human BRWD3 are classified as orthologous pairs by InParanoid (http://inparanoid.cgb.ki.se/).
To undertake epistasis experiments, cells were transfected with vectors to stimulate pathway activity (see below) for 7 hours and 30,000 cells in 50 μl of serum-free medium were seeded into wells of clear bottom 96-well plates (Greiner), which contained 1.5 μg of the dsRNAs to be tested (listed in
Each dsRNA was tested for its ability to suppress pathway activity under three conditions: (1) in Upd-expressing cells (screening conditions), (2) in cells treated with Upd-conditioned medium (Upd-CM), and (3) in cells expressing the activated form of JAK, HopTuml (Harrison, D. A., Binari, R., Nahreini, T. S., Gilman, M. & Perrimon, N., Embo J 14, 2857-65 (1995); Luo, H., Hanratty, W. P. & Dearolf, C. R., Embo J 14, 1412-20 (1995)). Specifically, for Upd overexpression 5×106 Kcl67 cells were transfected with 600 ng pAct-UpdGFP, 500 ng 6x2xDrafLuc reporter, 250 ng pAc5.1-Sid-1, 25 ng pAct-RL and pAc5.1 to a total of 2 μg DNA. For HopTumL overexpression, 5×106 Kc167 cells were transfected with 200 ng pAct-hopTumL, 500 ng 6x2xDrafLuc reporter, 250 ng pAc5.1-Sid-1, 25 ng pAct-RL and pAc5.1 to a total of 2 μg DNA. To analyse processes upstream of Upd, two batches of cells were transfected separately to generate ‘responder’ and ‘Upd-producer’ cells. The ‘responder’ cells were batch transfected with 500 ng 6x2xDrafLuc reporter, 250 ng pAc5.1-Sid-1, 25 ng pAct-RL and pAc5.1 to a total of 2 μg plasmid DNA and subsequently seeded into 96-well plates containing the respective dsRNAs as described above. The ‘Upd-producing’ cells were transfected with 2 μg pAct-UpdGFP and cultured in 10 cm dishes (Falcon). Three days after transfection, cells were treated with 50 μg/ml Heparin (Sigma). After 24 hours, the supernatant was harvested, cleared by centrifugation and passed through a 0.2 μm filter (Millipore). 50 μl of this Upd-conditioned medium were then used to stimulate pathway activity in the ‘responder’ cells for 24 hours. Control medium from untransfected Heparin treated cells did not elicit pathway activity (not shown).
Experiments were performed in eight replicates and repeated at least twice. Reporter activity in the firefly luciferase channel was divided by the Renilla luciferase channel to normalise for cell number. Z-scores were calculated as the multiples of the standard deviation that a specific RNAi treatment differed from cells treated with lacZ dsRNA as negative controls. Z-scores were subsequently transformed into a false-colour representations as depicted in
RNA controls as shown in
For epistasis analysis of the putative negative regulator ptp61F, cells were batch transfected with reporter and Upd inducer as described above. Subsequently, these cells were treated with 1.5 μg of dsRNA targeting the ptp61F transcript and 1.5 μg of dsRNA against lacZ, dome, hop or stat92E. In parallel, cells from the same transfection batch were treated with lacZ, dome, hop or stat92E dsRNAs alone. After normalisation, the values of experiments with control dsRNA alone were set to one. To examine the JAK/STAT phenotype of ptp61F in cells, 5×106 Kc167 cells were transfected with 0.6 μg pAct-UpdGFP, 0.5 μg 6x2xDrafLuc reporter, 0.25 μg pAc5.1-Sid-1, 0.025 μg pAct-RL and pAc5.1 to a total of 2 μg DNA. To assess the effects of the different Ptp61F splice forms, cells were transfected as described before with additional 0.5 μg of pAct-Ptp61Fa, pAct-Ptp61Fc or vector control, respectively. JAK/STAT pathway activation was expressed in relation to control cells.
A P-element insertion termed I(3)05842 (Spradling, A. C. et al., Genetics 153, 135-77 (1999)) was identified in the fourth intron of dBRWD3/CG31132 as part of a Flybase search (Drysdale, R. A. et al., Nucleic Acids Res 33 Database Issue, D390-5 (2005)). A I(3)05842 stock was obtained from the Bloomington stock centre (University of Indiana). The P-element insertion I(3)05842 is homozygous lethal and fails to complement the Df(3R)crb874 and Df(3R)crb87-5 deficiencies. Twenty three independent stocks in which the ry+ marker present in the P[ry+,PZ] insertion had been lost following a cross to a transposase source were established. Of these, seven were viable revertants (30%) and include two stocks with the wing vein phenotype (
For genetic interaction assays, females of the stock y,w,hopTuml/FM7; P [w+,cg-Gal4.A]2 (Harrison, D. A., Binari, R., Nahreini, T. S., Gilman, M. & Perrimon, N., Embo J 14, 2857-65 (1995)) were crossed to wild type controls (OreR and w1118) and mutations in stat92E and I(3)05842. The haemocyte specific Gal4 driver line P[w+,cg-Gal4.A]2 allowed specific UAS insertions to be tested for their potential influence on tumour formation. Transgenic animals expressing UAS-EGFP or UAS-β-galactosidase were used as negative controls while UAS-dPIAS-EGFP served as a positive control (Betz, A., Lampen, N., Martinek, S., Young, M. W. & Darnell, J. E., Jr., Proc Natl Acad Sci USA 98, 9563-8 (2001)) (see Table 3).
Crosses were incubated at 25° C. and adult females heterozygous for the hopTuml chromosome were scored within 24 hours of eclosion for the presence of tumours classified as small (one or two small melanotic spots as shown in
Genetic interaction with P[w+,GMR-updΔ3′]′19 was undertaken as described in Genetics 165, 1149-66 ((2003), Bach, E. A., Vincent, S., Zeidler, M. P. & Perrimon, N.) using OreR and STAT92E06346 as negative and positive controls, respectively. Suppression of P[w+,GMR-updΔ3′]′19 induced eye overgrowth by dBRWD305842 was observed in multiple independent experiments in a majority of individuals of the appropriate genotype. Drosophila heads were photographed using a Zeiss STEMI 2000-C binocular microscope and Axiocam camera.
Developmental genetic screens in Drosophila have identified multiple JAK/STAT pathway components on the basis of their segmentation phenotype (Binari, R. & Perrimon, N., Genes Dev 8, 300-12. (1994); Harrison, D. A., McCoon, P. E., Binari, R., Gilman, M. & Perrimon, N., Genes Dev 12, 3252-63. (1998); Hou, X. S., Melnick, M. B. & Perrimon, N., Cell 84, 411-9 (1996)) and subsequent analysis of the pathway has characterised evolutionarily conserved roles during immune responses, haematopoiesis and cellular proliferation (Lagueux, M., Perrodou, E., Levashina, E. A., Capovilla, M. & Hoffmann, J. A., Proc Natl Acad Sci USA 97, 11427-32. (2000); Boutros, M., Agaisse, H. & Perrimon, N., Dev Cell 3, 711-22. (2002); Meister, M. & Lagueux, M., Cell Microbiol 5, 573-580 (2003); Mukherjee, T., Castelli-Gair Hombria, J. & Zeidler, M. P., Oncogene in press (2005)). The JAK/STAT signalling cascade in Drosophila is comprised of the extracellular ligand Unpaired (Upd) (Harrison, D. A., McCoon, P. E., Binari, R., Gilman, M. & Perrimon, N., Genes Dev 12, 3252-63. (1998)), a trans-membrane receptor with homology to the IL6 receptor family termed Domeless (Dome) (Brown, S., Hu, N. & Castelli-Gair Hombria, J., Curr Biol 11, 1700-5. (2001)), a single Janus tyrosine kinase (JAK) called Hopscotch (Hop) (Binari, R. & Perrimon, N., Genes Dev 8, 300-12. (1994)) and the STAT92E transcription factor (Hou, X. S., Melnick, M. B. & Perrimon, N., Cell 84, 411-9 (1996); Yan, R., Small, S., Desplan, C., Dearolf, C. R. & Darnell, J. E., Jr., Cell 84, 421-30 (1996)) (
In order to identify novel pathway components and circumvent limitations of classical genetic screens, the inventors of the present invention have undertaken a genome-wide RNA interference (RNAi) screen, a powerful technique for the identification of new components of diverse cellular pathways (Kamath, R. S. et al., Nature 421, 231-7 (2003); Kittler, R. et al., Nature 432, 1036-40 (2004); Berns, K. et al., Nature 428, 431-7 (2004); Paddison, P. J. et al., Nature 428, 427-31 (2004); Boutros, M. et al., Science 303, 832-5 (2004)). To this end, the inventors devised a quantitative assay for JAK/STAT signalling activity in cultured Drosophila cells by multimerising a STAT92E-binding site from the Draf promotor (Kwon, E. J. et al., J Biol Chem 275, 19824-19830 (2000)) to generate the 6x2xDrafLuc firefly luciferase reporter. Given the role for JAK/STAT signalling in haematopoiesis (Meister, M. & Lagueux, M., Cell Microbiol 5, 573-580 (2003)), the inventors used Drosophila hemocyte-like Kc167 cells due to their endogenous ability to respond to pathway activation (
The inventors then set out to systematically identify genes required for JAK/STAT signalling by generating a library of 20,026 dsRNAs targeting 91% of the predicted transcripts in the Drosophila genome. Using this library the inventors performed duplicate genome-wide screens as outlined in
Based on InterPro and GO annotations, pathway modifiers were classified according to their predicted functions. Signalling factors, enzymes mediating post-translational protein modifications and transcription factors cumulatively represent 47% of the genes identified (
A genetic technique to characterise signalling molecules is the determination of their epistatic relationship with respect to defined pathway components. The inventors therefore performed cell-based epistatic assays to determine the pathway response to Upd expression, Upd conditioned medium or expression of the constitutively active JAK allele hopTuml (Harrison, D. A., McCoon, P. E., Binari, R., Gilman, M. & Perrimon, N., Genes Dev 12, 3252-63. (1998); Sefton, L., Timmer, J. R., Zhang, Y., Beranger, F. & Cline, T. W., Nature 405, 970-3 (2000)) while simultaneously targeting a subset of positive regulators. In this way, dsRNA-inactivated genes required upstream in the pathway can be characterised on the basis of their rescue by pathway activation further downstream (
In order to confirm the function of candidate genes in vivo, the inventors tested examples of both positive and negative regulators of the JAK/STAT signalling pathway. One positive regulator mentioned above is CG31132 which encodes a 2232 amino acid WD40 and bromo-domain containing protein homologous to human BRDW3 (
A previously uncharacterised mutagenic P-element inserted in the fourth intron of CG31132 (henceforth termed dBRDW305842) has been deposited in public stock collections as part of the Drosophila genome project and remobilisation of this transposon indicates that the insertion is responsible for late embryonic lethality. The inventors therefore tested for genetic interactions between dBRDW3 and JAK/STAT signalling by crossing the dBRDW305842 allele to GMR-updΔ3′ (Bach, E. A., Vincent, S., Zeidler, M. P. & Perrimon, N., Genetics 165, 1149-66 (2003)). The GMR-upd□3′ transgene ectopically misexpress Upd during eye development resulting in cellular overproliferation and an enlarged adult eye (
One phenotypic consequence of constitutive JAK/STAT activation caused by the gain-of-function JAK allele hopTuml is the overproliferation of haemocytes and the frequent formation of melanotic tumours, a phenotype previously described as a Drosophila model for leukaemia (Luo, H., Hanratty, W. P. & Dearolf, C. R., Embo J 14, 1412-20 (1995); Harrison, D. A., Binari, R., Nahreini, T. S., Gilman, M. & Perrimon, N., Embo J 14, 2857-65 (1995)). The inventors found that the removal of one copy of dBRWD3 is sufficient to reduce the size and the frequency of hopTuml induced melanotic tumours (
As a second example the inventors analysed the ptp61F gene which encodes a protein tyrosine phosphatase. dsRNA knocking down all mRNA splice forms transcribed from this locus leads to an increase in JAK/STAT signalling activity. The inventors performed epistasis analysis in which the inventors removed known pathway components and tested for the effect of simultaneously targeting ptp61F. Double RNAi against ptp61F together with lacZ, dome or hop results in pathway stimulation (
Aberrant JAK/STAT signalling has been implicated in multiple human malignancies and its components have been proposed as molecular targets for the development of therapeutic compounds (O'Shea, J. J., Pesu, M., Borie, D.C. & Changelian, P. S., Nat Rev Drug Discov 3, 555-64 (2004)). The genome-wide screen presented here identified known and previously unknown genes and the inventors have characterised their likely level of interaction with defined pathway components using cell-based epistasis analysis. Of the 89 JAK/STAT modifiers identified, many have human homologues that remain to be characterised. The inventors have here performed an analysis of two examples in vivo and demonstrate their roles in regulating the pathway during development and tumour genesis in Drosophila. One of these is a homologue of human BRWD3, a gene recently identified at the break-point of a translocation isolated from multiple B-CLL patients (Kalla, C. et al., Genes Chromosomes Cancer 42, 128-43 (2005)). Given our functional analysis of dBRWD3 and the known roles for JAK/STAT signalling during normal haematopoiesis, it is possible that a breakdown in BRWD3 mediated STAT regulation may represent a key molecular mechanism leading to the development of B-CLL. Thus, comprehensive reverse genetic surveys for signalling pathway components using Drosophilaas a model organism represent a potentially powerful approach with which insights relevant to human disease can be obtained.
Novel components regulating the JAK/STAT pathway in Drosophilamelanogaster have been previously been identified using a robust STAT92E responsive reporter assay in combination with genome-wide RNAi (Müller, P., Kuttenkeuler, D., Gesellchen, V. Zeidler, M.P. and Boutros M. (2005) “Identification of JAK/STAT signalling components by genome-wide RNAi” Nature 436 871-875). Having identified the essential components in Drosophila, a second crucial step is the identification of human functional orthologs. Given that many of the potential human orthologs have been implicated in human disease, these proteins, and the mRNAs that encode them, may represent targets for therapeutic interventions by small molecules or RNAi based approaches. Using a HeLa cell model we have assayed the activity of endogenous STAT1 and STAT3 following treatment with siRNA targeting potential pathway modulating genes. Assays of hSTAT phosphorylation state and the expression levels of their targets, have identified 27 human genes, which function as modulators of human JAK/STAT signal transduction. These have been ranked on the basis of potential significance and are listed in Table 7 together with the human diseases they have previously been associated with.
Compared to Drosophila, the JAK/STAT pathway in mammalians is much more complex in that multiple paralogs exist for the pathway ligand, receptor, JAK and STAT. As an initial approach towards identifying regulators of human JAK/STAT signaling, we have analyzed phenotypes caused by siRNA-mediated knockdown of candidate pathway modifiers in human cells. Human genes for this analysis were selected based on their homology to Drosophila JAK/STAT pathway regulators previously identified (Müller et al. 2005). Homology prediction by a variety of methods yielded 73 candidates homologous to 56 Drosophila genes. Pools of 4 siRNAs per candidate (Dharmacon SMARTpools) were used to ensure the efficiency and specificity of knockdown. As an easily tractable model, we have used human cancer-derived HeLa cells which express multiple STATs and which respond to stimulation by a variety of cytokine ligands (Ehret G.B., Reichenbach P., Schindler U., Horvath C.M., Fritz S., Nabholz M., Bucher P. (2001) “DNA binding specificity of different STAT proteins. Comparison of in vitro specificity with natural target sites” J Biol Chem 276 6675-6688).
Two approaches have been used to determine the activity of STAT1 and STAT3 in the HeLa cell system tested. Firstly, the levels of tyrosine-701-phosphorylated STAT1 (pSTAT1) and tyrosine-705-phosphorylated STAT3 (pSTAT3) were determined in HeLa cell lysates that had been stimulated with human Interferon gamma (INFγ) or Oncostatin M (OSM) for 15 min, respectively. These cells had previously been treated with siRNA targeting either controls or the putative pathway interactors for 72hs. After determination of the overall level of STAT1/3, the western blots were stripped and re-probed with pSTAT1 and pSTAT3 antibodies and with antibodies to determine β-ACTIN levels as a normalization control. The relative levels of pSTAT1/3 versus STAT1/3 were assessed with regard to the overall level of β-ACTIN detected and a call made representing either an increase in PSTAT levels (+), a decrease in PSTAT (−) or no change (
As a second independent approach to determine the level of STAT1 and STAT3 activity, the expression levels of the previously characterized pathway target genes GBPI (a STAT1 target) and SOCS3 (a STAT3 target) were determined 6 hrs after stimulation of HeLa cells with INFy and OSM, respectively. As before, cells had previously been treated with siRNA targeting either controls or putative pathway regulators for 72 hrs. Target gene levels were determined using branched DNA technology (QuantiGene, Panomics) and normalized to the level of β-actin mRNA. Results from duplicate assays are expressed as fold changes in target gene expression levels relative to cells treated with control siRNA. Statistically significant changes in response (p<0.05) are shown in black (decrease in expression level) or white (increase in expression) (FIG. 7 column 2 & 3, Table 8 column 5 & 6). In this table the scores relating to hSTAT1 and hSTAT3 target genes are expressed such that 1 is the expression level induced by pathway ligands following treatment with a control siRNA. Numbers below 1 therefore indicate a reduction in expression while scored above 1 represent an increase. Scores for Drosophila STAT92E are expressed as z-scores—a measure of statistical significance in which significant suppression of activity is represented by numbers <−2.0 while significant enhancement is represented by values >+2.0. Statistically significant changes are indicated by the change in colour of the boxes shown in
Analysis of these two independent data sets, in conjunction with the scores originally obtained for the Drosophila orthologs (
†InterPro and GO results classified into one of functionally related groups. See Table 1 for complete list of genes, specific IPR domains and GO assigned within each group.
(*)‘wild type’ results used to calculate z-scores
(1)Hanratty, W. P. & Dearolf, C. R. The Drosophila Tumorous-lethal hematopoietic oncogene is a dominant mutation in the hopscotch locus. Mol Gen Genet 238, 33-7 (1993).
(2)Silver, D. L. & Montell, D. J. Paracrine signaling through the JAK/STAT pathway activates invasive behavior of ovarian epithelial cells in Drosophila. Cell 107, 831-841 (2001).
(3)Hou, X. S., Melnick, M. B. & Perrimon, N. Marelle acts downstream of the Drosophila HOP/JAK kinase and encodes a protein similar to the mammalian STATs. Cell 84, 411-9 (1996).
(4)Betz, A., Lampen, N, Martinek, S., Young, M. W. & Darnell, J. E. Jr. A Drosophila PIAS homologue negatively regulates stat92E. Proc Natl Acad Sci USA 98, 9563-8 (2001).
CAG CAG TAA AGC ACT TTC AA
CCG ATT CCG GCA TGG C
Drosophila
Drosophila
Drosophila
D. melanogaster
sapiens
Drosophila
melanogaster
Homo sapiens
| Number | Date | Country | Kind |
|---|---|---|---|
| 05 012 934.5 | Jun 2005 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2006/005744 | 6/14/2006 | WO | 00 | 12/16/2008 |
