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  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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  • C12N2310/00—Structure or type of the nucleic acid
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• • C—CHEMISTRY; METALLURGY
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/50—Physical structure
  • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

• HEC1 antagonists in the treatment of proliferative disorders and cancer
• the present invention relates to the use of (an) anti-HEC1 compound(s), (an) HEC1 -complex antagonist(s) and/or (an) HEC1 -complex inhibitor(s) for the preparation of a pharmaceutical composition for the prevention, amelioration and/or prevention of a hyperproliferative disorder/disease. Furthermore, the invention provides for a pharmaceutical composition comprising at least one anti-HEC1 compound, at least one H EC 1 -complex antagonist and/or at least one HEClcomplex inhibitor. Additionally, the invention relates to a method for identifying an anti-Hed compound, an HEC1 -complex antagonist or an HEC1 -complex inhibitor.
• Prometaphase is characterized by a further condensation of chromosomes and the developing microtubules are captured by the kinetochores that are spezialized proteinaceous structures associated with centromere DNA on mitotic chromosomes. Subsequently, the chromosomes begin to congress to an equatorial plane and during metaphase they align on the metaphase plate. After the chromosomes have undergone proper bipolar attachment by the spindle apparatus, the cohesion of the sister chromatids in the centromere region is severed and they are pulled apart due to motor activity and a shortening of the kinetochor-microtubules towards the poles of a cell, i.e. anaphase A.
• CIN aneuploid
• hNuf2R a 54 kDa protein (human Nuf2p-related) (Wigge (2001), loc. cit.) and the other is MPP1, a 225 kDa protein (Fritzler (2000), J. Invest. Med. 48, 28-39) which were both identified by database searches (Janke (2001), loc. cit.; Wigge (2001), loc. cit).
• hNuf2R is also known as Nuf2, a conserved protein implicated in connecting the centromere to the spindle during chromosome segregation (Nabetani (2001), Chromosoma 110, 322-334).
• hNuf2R localizes to centromeres (Wigge (2001), loc. cit.). From these data it was concluded that these proteins and also so far non-identified homologues of the budding yeast proteins are associated with the kinetochore and may have a similar function as assumed for the yeast proteins.
• HEC1 was proposed to localize to the nuclei of interphase cells and a portion of the protein to centromeres/kinetochores during M- phase (Chen (1999), loc. cit.). The authors speculate that HEC1 is involved in spindle attachment to chromosomes during prophase as well as indirectly in subsequent chromosome movement, i.e. in chromosome segregation. In particular, the study of Chen (1999), loc. cit. showed severe mitotic anomalies following microinjection of anti-HEC1 antibodies and it was concluded that HEC1 inactivation causes defects in chromosome segregation but does not arrest cells in mitosis.
• the technical problem underlying the present invention was to provide means and methods for pharmaceutical intervention of disorders related to the pathological proliferation of eukaryotic cells.
• the solution to said technical problem is achieved by providing embodiments characterized in the claims.
• the present invention relates to the use of (an) anti-HEC1 compound(s), (an) HEC1 -complex antagonist(s) and/or HEC1 -complex inhibitor(s) for the preparation of a pharmaceutical composition for the prevention, amelioration and/or treatment of a hyperproliferative disease/disorder.
• anti-HEC1 compound(s) relates to compound(s), (a) molecule(s) or (a) substance(s) that at least inactivate(s), intervene(s), interfere(s) with, inhibit(s), disturbe(s), lead(s) to dissociation or destroy(s) the HEC1 -complex, thereby inactivating its function.
• said inactivation, intervention, interference, inhibition is a transient event and the HEC1 function in a subject to be treated is restored after a treatment regime with the anti-HEC1 compounds, HEC1 antagonists or inhibitors of the HEC1-complex as defined herein.
• HEC1 and/or the HEC1 -complex is permanently inactivated, intervened, interfered or inhibited in cells effected by an hyperproliferative disease/disorder or cancer.
• said anti- HECl compound, HEC1-compIex antagonist and/or HEC1-complex inhibitor may, inter alia, comprise an antibody or derivative or fragment thereof or an aptamer.
• said anti-HEC1 compound, HEC1 -complex antagonist or HEClcomplex inhibitor may also comprise an inhibiting nucleic acid, such as an antisense oligonucleotide, antisense DNA, antisense RNA, iRNA, ribozyme or siRNA.
• an inhibiting nucleic acid such as an antisense oligonucleotide, antisense DNA, antisense RNA, iRNA, ribozyme or siRNA.
• small peptides or peptide-like molecules are envisaged as HEC1/HEC1- complex inhibitor(s) or antagonist(s). Such small peptides or peptide-like molecules bind to and occupy the active site of a protein thereby making the catalytic site inaccessible to substrate such that normal biological activity is prevented.
• HEC1/HEC1 -complex inhibitor or antagonist any biological or chemical composition(s) or substance(s) may be envisaged as HEC1/HEC1 -complex inhibitor or antagonist.
• said specific HEC1/HEC1-complex inhibitor(s) or antagonist(s) may be employed in accordance with this invention as HEC1/HEC1 -complex inhibitors or antagonist which may be tested for their specific inhibitory/antagonistic function by methods known in the art and by methods described herein.
• Such methods comprise interaction assays, like immunoprecipitation assays, ELISAs, RIAs as well as specific inhibition assays, like the assays provided in the appended Examples.
• the present invention shows that elimination/inactivation of the human kinetochore protein HEC1 interferes with correct chromosome segregation and - in cells with an intact spindle checkpoint - triggers a mitotic arrest.
• Chen (1999), loc. cit. proposes that HEC1 inactivation does not arrest cells in mitosis but allows them to proceed aberrantly.
• Chen (1999), loc. cit. proposes that HEC1 inactivation does not arrest cells in mitosis but allows them to proceed aberrantly.
• HEC1 and/or HEC1 -complex causes hyperproliferative or tumorous cells to undergo catastrophic mitotic exit, i.e. to die.
• normal cells in the human or animal subject to be treated with an anti-HEC1 compound, a HEC1 antagonist and/or a HEC1-complex inhibitor comprise an active spindle checkpoint and merely pause in mitosis, until HEC1 function is restored. Accordingly, it is in particular preferred to administer the above mentioned anti-HEC compound, HEC1 antagonist and/or HEC1 -complex inhibitor transiently.
• the present invention provides for the medical use of transient or permanent inactivation of HEC1 and/or the HEC1 -complex in such cells.
• the use of said inactivators/inhibitors causes such cells to undergo catastrophic mitotic exit, i.e. to die, while cells with an intact spindle checkpoint in the body should activate the checkpoint and merely pause in mitosis until HEC1 function is restored.
• the present invention identifies HEC1 and/or HEC1 -complex partners as an attractive target for the selective elimination of spindle checkpoint deficient cells, for example cells showing aneuploidy, a common event in cancer. Moreover, several lines of evidence indicate that aneuploidy might contribute to malignant transformation.
• the HEC1-compIex as defined herein comprises HEC1 and hNuf2R which is also known as Nuf2R or Nuf2 (Wigge (2001), loc. cit.; Nabetani (2001), loc. cit.).
• the experimental data provided in the appended Examples for the inhibition of HEC1 may also be generated by methods known in the art for Nuf2/hNuf2R.
• the person skilled in the art can, for Example, carry out RNAi experiments or the like to completely knock-out/silence or transiently silence hNuf2R expression as shown in the appended Examples. Based on the phenotype that is observed when carrying out such experiments, the person skilled in the art can demonstrate that hNuf2R (Nuf2) is a component of the HEC1 -complex.
• hNuf2R is a component of the HEC1-complex.
• Said Nuf2p is a component of the Ndc80/Nuf2p/Spc24/Spc25 complex of S. cerevisiae. Accordingly, hNuf2R is expected to be a component of the HEC1 -complex.
• MPP1 (Fritzler (2000), loc. cit.; Kamimoto (2001), J. Biol. Chem. 276, 37520-37528) was found in data base searches. Since Nuf2p is a component of the Ndc80/Spc24/Spc25 complex in S. cerevisiae, it can be speculated whether MPP1 is a component of the HEC1 -complex. The person skilled in the art can determine whether MPP1 is a component of the HEC1 -complex by the approach as described hereinabove for HEC1 or hNuf2R and as described in the Examples hereinbelow. Thus, in another preferred embodiment the HEC1 -complex comprises HEC1, hNuf2R and MPP1. However, it is particularly preferred that said HEC1-complex comprises HEC1 and hNuf2R.
• HEC1 -complex means a complex, preferably a protein complex, comprising at least HEC1 and/or hNuf2R (Nuf2).
• said complex may also comprise MPP1 as well as further, so far not identified polypeptides.
• spindle checkpoint proteins like Mad1/Mad2 are also capable of interacting with HEC1. Accordingly, corresponding interaction partners of HEC1 and/or HEC1 -complex partners may be deduced and found by methods known in the art, like, e.g. two hybrid screening and a corresponding verification by biochemical/ immunobiochemical methods.
• said HEClcomplex comprises merely (a) functional parts(s) or fragment(s) of HEC1 or hNuf2R.
• MPP1 may also be a component of said HEClcomplex.
• said HEC1 -complex comprises HEC1 and hNuf2R.
• said HEC1-complex localizes to the kinetochores that are proteinaceous structures associated with centromere DNA of mitotic chromosomes. In yeasts further components involved in HEC1-homologous complexes are known, but so far only a few of them are identified, e.g.
• homologues of said yeast polypeptides may be part of an HEC1 -complex as defined herein.
• the anti-HEC1 compound, HEC1 -complex antagonist and/or HEC1-complex inhibitor is an inhibiting nucleic acid molecule.
• potential antagonistic or inhibiting compounds comprise nucleic acid molecules that are capable of reducing HEC1-complex activity in a cell by way of interfering the gene expression of HEC1 and/or gene expression of interaction partner(s) of HEC1 comprised in an HEC1-complex.
• Such antagonists/inhibitors are, inter alia, antisense oligonucleotides, antisense DNA, antisense RNA, iRNA, ribozymes or siRNA. Said inhibiting nucleic acids will be described herein below in more detail.
• An inhibiting/antagonistic nucleic acid molecule is preferably complementary to any HEC1 sequence, for example 5'-untranslated regulatory region, the open reading frame or 3'-untranslated region. Mutatis mutandis, these nucleic acid molecules may also target the corresponding region(s) of genes encoding HEC1 -complex proteins as defined herein.
• Said inhibiting nucleic acid molecules are preferably used for repression of expression of a gene comprising such sequences, for example due to an antisense or triple helix effect or for the construction of appropriate ribozymes (see e.g., EP-B1 0 291 533, EP-A1 0 321 201 , EP-B1 0 360 257) which specifically cleave the (pre)-mRNA of a gene comprising a sequence of the HEC1 gene or genes encoding members of the HEC1 -complex. Selection of appropriate target sites and corresponding ribozymes can be done as described for example in Steinecke, Ribozymes, Methods in Cell Biology 50, Galbraith et al. eds Academic Press, Inc. (1995), 449-460.
• Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, whereby the inhibitory effect is based on specific binding of a nucleic acid molecule to DNA or RNA.
• the 5' coding portion of a nucleic acid molecule encoding a HEC1 protein, hNuf2R protein or MPP1 protein or any other protein of the HEC1-complex to be inhibited can be used to design an antisense oligonucleotide, e.g., of at least 10 nucleotides in length.
• the antisense DNA or RNA oligonucleotide hybridises to the mRNA in vivo and blocks translation of said mRNA and/or leads to destabilization of the mRNA molecule (Okano, J. Neurochem. 56 (1991), 560; Oligodeoxynucleotides as antisense inhibitors of gene expression, CRC Press, Boca Raton, FL, USA (193 ⁇ ).
• a DNA oligonucleotide can be designed to be complementary to a region of the gene encoding a HEC1-protein, a hNuf2R-protein or a MPP1-protein or any other protein of the HEC1 -complex to be inhibited according to the principles laid down in the prior art (see for example Lee, Nucl. Acids Res. 6 (1979), 3073; Cooney, Science 241 (19 ⁇ ), 456; and Dervan, Science 251 (1991), 1360).
• Such a triple helix forming oligonucleotide can then be used to prevent transcription of the specific gene.
• the oligonucleotides described above can also be delivered to target cells via a gene delivery vector as described above in order to express such molecules in vivo to inhibit gene expression of the respective protein.
• antisense molecules are oligonucleotides specifically hybridising to a polynucleotide encoding a polypeptide having HEC1, hNuf2R, MPP1 activity or any activity of proteins of the HEC1 -complex.
• Such oligonucleotides have a length of preferably at least 10, in particular at least 15, and particularly preferably of at least 50 nucleotides. They are characterized in that they specifically hybridise to said polynucleotide, that is to say that they do not or only to a very minor extent hybridise to other nucleic acid sequences.
• RNAi refers to the introduction of homologous double stranded RNA (dsRNA) to specifically target a gene's product, resulting in null or hypomorphic phenotypes.
• dsRNA homologous double stranded RNA
• Introduction of dsRNA into a cell results in the loss of the targeted endogenous HEC1-mRNA and/or mRNAs encoding components of the HEC1-complex as defined herein.
• dsRNA is also remarkably potent (i.e., only a few dsRNA molecules per cell are required to produce effective interference), the dsRNA must be either replicated and/or work catalytically.
• RNAi constructs a sense portion comprising the coding region of the gene to be inactivated (or a part thereof, with or without non-translated region) is followed by a corresponding antisense sequence portion. Between both portions, an intron not necessarily originating from the same gene may be inserted. After transcription, RNAi constructs form typical hairpin structures.
• the RNAi technique may be carried out as described by Smith (Nature 407 (2000), 319-320) or Marx (Science 238 (2000), 1370-1372).
• RNA molecules with ribozyme activity which specifically cleave transcripts of a gene encoding HEC1, hNuf2R, MPP1 or any other protein of the HEClcomplex can be used.
• Said ribozymes may also target DNA molecules encoding the corresponding RNAs.
• Ribozymes are catalytically active RNA molecules capable of cleaving RNA molecules and specific target sequences. By means of recombinant DNA techniques it is possible to alter the specificity of ribozymes.
• the first group is made up of ribozymes which belong to the group I intron ribozyme type.
• the second group consists of ribozymes which as a characteristic structural feature exhibit the so-called "hammerhead” motif.
• the specific recognition of the target RNA molecule may be modified by altering the sequences flanking this motif. By base pairing with sequences in the target molecule these sequences determine the position at which the catalytic reaction and therefore the cleavage of the target molecule takes place. Since the sequence requirements for an efficient cleavage are low, it is in principle possible to develop specific ribozymes for practically each desired RNA molecule.
• a DNA sequence encoding a catalytic domain of a ribozyme is bilaterally linked with DNA sequences which are homologous to sequences encoding the target protein.
• Sequences encoding a catalytic domain and DNA sequence flanking the catalytic domain are preferably derived from the polynucleotides encoding HEC1, hNuf2R, MPP1 or any other protein of the HEC1-complex.
• the expression of ribozymes in order to decrease the activity in certain proteins is also known to the person skilled in the art and is, for example, described in EP-B1 0 321 201 or EP-B1 0 360 257.
• the inhibiting nucleic acid molecule is siRNA as dislosed in Elbashir ((2001), Nature 411 , 494-498)) and as illustrated in the appended Examples.
• shRNAs short hairpin RNAs
• shRNAs short hairpin RNAs
• the shRNA approach for gene silencing is well known in the art and may comprise the use of st (small temporal) RNAs; see, inter alia, Paddison (2002) Genes Dev. 16, 948-95 ⁇ .
• approaches for gene silencing are known in the art and comprise "RNA"-approaches like RNAi or siRNA. Successful use of such approaches has been shown in Paddison (2002), loc. cit, Elbashir (2002) Methods 26, 199-213; Novina (2002) Nat. Med.
• siRNA is targeted to deplete HEC1.
• siRNA is targeted to deplete hNuf2R (Nuf2).
• targeted means that (an) siRNA duplex(es) is/are specifically targeted to a coding sequence of HEC1 or Nuf2R (Nuf2) or any other component of the HEC1-complex, for example, MPP1, to cause gene silencing by RNA interference (RNAi) since said siRNA duplex(es) is/are homologous in sequence to a gene desired to be silenced, for example, the HEC1 gene, hNuf2R (Nuf2) gene or any other gene encoding a component of the HEClcomplex, for example, MPP1.
• RNA interference RNA interference
• “Homologous in sequence” in the context of the present invention means that said siRNA duplex(es) is/are homologous in the sequence to a gene, for example the HEC1 gene, the hNuf2R (Nuf2) gene or any other gene encoding a component of the HEC1-complex, desired to be silenced by the mechanism/pathway of RNA interference (RNAi). It is envisaged that the degree of homology between the siRNA duplex(es) and the sequence of the gene desired to be silenced is sufficient that said siRNA duplex(es) is/are capable to cause gene silencing of said desired gene initiated by double-stranded RNA (dsRNA), for example, (an) siRNA duplex(es).
• dsRNA double-stranded RNA
• the person skilled in the art is readily in a position to determine whether the degree of homology is sufficient to deplete HEC1, hNuf2R (Nuf2) or any other component of the HEC1-complex.
• the appended Examples provide methods and guidance how to determine whether HEC, hNuf2R (Nuf2) or any other component of the HEC1 -complex is depleted.
• the Figures hereinbelow show, inter alia, the phenotype of HEC1 or hNuf2R (Nuf2) depletion.
• to deplete when using the term "to deplete” in the context of the present invention, it means that due to a process of sequence-specific, post-transcriptional gene silencing (PTGS) expression of a desired gene, for example, HEC1 gene expression or hNuf2R (Nuf2) gene expression or expression of any other gene encoding a component of the HEC1-complex, for example, MPP1 , is suppressed.
• PTGS sequence-specific, post-transcriptional gene silencing
• the RNA encoding for example, HEC1 , hNuf2R (Nuf2) or any other component of the HEC1 -complex may be partially or completely degraded by the mechanism/pathway of RNAi and, thus, may not be translated or only translated in insufficient amounts which causes a phenotype almost resembling or resembling that of a knock-out of the respective gene. Consequently, for example, no or at least to less HEC1 protein, hNuf2R (Nuf2) protein or any other protein that is a component of the HEC1 -complex will be produced.
• the appended Examples and the Figures hereinbelow provide guidance to the person skilled in the art whether HEC1 , hNuf2R (nuf2) or any other component of the HEC1-complex is depleted and/or functionally inhibited.
• the siRNA which is targeted to deplete HEC1 comprises (an) siRNA duplex(es) formed by the nucleic acid sequence shown in SEQ ID NOs: 1 and 2.
• the siRNA which is targeted to deplete hNuf2R (Nuf2) comprises (an) siRNA duplex(es) formed by the nucleic acid sequence shown in SEQ ID NOs: 3 and 4.
• siRNA treatment is performed and how siRNA duplex(es) is/are formed or how (an)other siRNA duplex(es), except that/those which is/are particularly preferred is/are used to deplete HEC1 , hNuf2R (Nuf2) or any other component of the HEC1 -complex.
• inhibiting molecules acting as HEC1 -complex antagonists/inhibitors or as anti-HEC1 compounds may be introduced via gene therapy approaches (e.g. the introduction of heavy and/or light chains or at least the variable- regions " thereof or the introduction of scFvs by use of corresponding vector systems).
• Inhibiting RNAs as defined herein and to be employed as anti-HEC1 compounds may also be introduced by vector systems and/or "gene therapy" approaches yet further introduction systems for the herein defined anti-HEC1 compounds and HEC1 -complex antagonists/inhibitors are envisaged.
• liposomes may also be employed in this context.
• Liposomes as transfection systems have been described in the art and have not only been employed for the introduction of genes and proteins/peptides but also for the transfection with oligonucleotides, like RNA; see, inter alia, Paul (2002), loc. cit.
• 20- to 50-nucleotide RNAs preferably 15, 18, 20, 21 , 25, 30, 35, 40, 45 and 50- nucleotide RNAs are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
• siRNAs and the like are obtained from commercial RNA oligo synthesis suppliers, which sell RNA-synthesis products of different quality and costs.
• RNAs are not too difficult to synthesize and are readily provided in a quality suitable for RNAi.
• specific gene silcencing may also be obtained by longer RNA, for example long dsRNA which may comprise even 500 nt; see, inter alia, Paddison (2002), PNAS 99, 1443-1448.
• the preferred targeted region is selected from a given nucleic acid sequence beginning, inter alia, 50 to 100 nt downstream of the start codon.
• One approach to therapy of human cancer cells is to introduce vectors expressing constructs providing for anti-HEC1 compounds (like e.g.
• a method for inhibiting proliferation of cells characterized by potential for continuous increase in cell number e.g., neoplastic cells, which comprises obtaining a DNA expression vector containing a cDNA sequence having the sequence of human HEC1 , hNuf2R or MPP1 or other proteins comprised in HEC1 -complexes mRNA which is operably linked to a promoter such that it will be expressed in antisense orientation, and transforming the neoplastic cells with the DNA vector.
• the expression vector material is generally produced by culture of recombinant or transfected cells and formulated in a pharmacologically acceptable solution or suspension, which is usually a physiologically-compatible aqueous solution, or in coated tablets, tablets, capsules, suppositories, inhalation aerosols, or ampules, as described in the art, for example in U.S. Pat. No. 4,446,128.
• the vector-containing composition is administered to a mammal in an amount sufficient to transfect a substantial portion of the target cells of the mammal.
• Administration may be any suitable route, including oral, rectal, intranasal or by intravesicular (e.g. bladder) instillation or injection where injection may be, for example, transdermal, subcutaneous, intramuscular or intravenous.
• the expression vector is administered to the mammal so that the tumor cells of the mammal are preferentially transfected. Determination of the amount to be administered will involve consideration of infectivity of the vector, transfection efficiency in vitro, immune response of the patient, etc.
• a typical initial dose for administration would be 10-1000 micrograms when administered intravenously, intramuscularly, subcutaneously, intravesicularly, or in inhalation aerosol, 100 to 1000 micrograms by mouth, or 10.sup.5 to 10.sup.10 plaque forming units of a recombinant vector, although this amount may be adjusted by a clinician doing the administration as commonly occurs in the administration of other pharmacological agents.
• a single administration may usually be sufficient to produce a therapeutic effect, but multiple administrations may be necessary to assure continued response over a substantial period of time. Further description of suitable methods of formulation and administration according to this invention may be found in U.S. Pat. Nos. 4,592,002 and 4,920,209.
• the anti-HEC1 compound, HEC1 -complex antagonist and/or HEC1 -complex inhibitor may also be an antibody or a derivative or a fragment thereof or an aptamer.
• Antibodies useful as antagonists can be monoclonal or polyclonal and can be prepared according to methods well known in the art.
• the term “antibody” also comprises derivatives or fragments thereof or aptamers of an antibody which still retain the binding specificity.
• the HEC1 -polypeptide, the hNuf2R, the MPP1 or any further so far not identified polypeptide of the HEC1 -complex, their fragments or other derivatives thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto.
• chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of a Fab expression library Various procedures known in the art may be used for the production of such antibodies and fragments.
• Antibodies directed against a polypeptide as described above can be obtained, e.g., by direct injection of the protein into an animal or by administering the polypeptide to an animal, preferably a non-human animal. The antibody so obtained will then bind the protein itself. In this manner, even a sequence encoding only a fragment of the protein can be used to generate antibodies binding the whole native polypeptide.
• any technique which provides antibodies produced by continuous cell line cultures can be used.
• the antibodies/antibody constructs as well as antibody fragments or derivatives to be employed in accordance with this invention or capable to be expressed in a cell may, inter alia, be achieved by direct injection of the corresponding proteineous molecules or by injection of nucleic acid molecules encoding the same.
• gene therapy approaches are envisaged.
• the term "antibody molecule” relates to full immunoglobulin molecules as well as to parts of such immunoglobulin molecules.
• the term relates, as discussed above, to modified and/or altered antibody molecules, like chimeric and humanized antibodies.
• the term also relates to monoclonal or polyclonal antibodies as well as to recombinantly or synthetically generated/synthesized antibodies.
• the present invention relates to an aptamer specifically recognizing an epitope of nucleic acid molecule/polynucleotide coding for HEC1 or a member of the HEC1 -complex as defined herein above or coding for a fragment of HEC1 or another member of the HEC1-complex or an aptamer specifically directed to the RNA.
• said cancer is a leukemia or lymphoma
• said leukemia or lymphoma may be selected from the group consisting of T-cell leukemia, T-cell lymphoma, B-cell leukemia and B-cell lymphoma, such as, inter alia, Burkitt's lymphoma, Hodgkin's lymphoma and non- Hodgkin's lymphoma.
• T-cell leukemia T-cell lymphoma
• B-cell leukemia and B-cell lymphoma such as, inter alia, Burkitt's lymphoma, Hodgkin's lymphoma and non- Hodgkin's lymphoma.
• transforming protein Tax of the human T cell leukaemia virus type 1 HTLV-1
• Mad1 transforming protein Tax of the human T cell leukaemia virus type 1
• Mad1 a mitotic checkpoint control protein
• cells from adult T-cell leukemias show phenotypes, i.e.
• the cancer cells and/or aneuploid cancer cells are spindle assembly checkpoint deficient.
• Said spindle pole checkpoint deficiency may be due to modifications and/or mutations in a gene/gene product selected from the group consisting of Mad1, Mad2, Mad1/2-compIex, Bub1 , BubR1, Bub3, Mps1, CENP-E, CENP-F, ZW10, Rod, HsCdc20, HsCdhl, dynein/dynactin- complex, CLIP170, EB1 , APC, Polo-like Kinase 1 , Aurora-A, Aurora-B, INCENP and/or survivin.
• a gene/gene product selected from the group consisting of Mad1, Mad2, Mad1/2-compIex, Bub1 , BubR1, Bub3, Mps1, CENP-E, CENP-F, ZW10, Rod, HsCdc20, HsCdhl, dynein/dynactin- complex, CLIP170, EB1 , APC, Polo-like Kinase
• hCdhl is described in Taguchi ((2002), FEBS. Lett. 519, 59-65), CLIP170, EB1 , APC and dynein/dynactin are described, inter alia, in Perez (1999), Cell 96, 517-527; Nakamura (2001), Curr. Biol. 11, 1062-1067; Wassmann (2001), Curr. Opin. Genet. Dev. 11 , 83-90; Howell (2001), J. Cell. Biol. 155, 1159-1172.
• aneuploidy is a common event in cancer, and several lines of evidence indicate that it could contribute to malignant transformation. Should the spindle assembly checkpoint be the surveillance mechanism responsible to ensure that chromosomes have been properly aligned before progression to anaphase, it is reasonable to speculate that aneuploidy correlates frequently with inactivation of the checkpoint machinery. Accordingly, Cahill (1998), loc. cit. reported the identification of dominantly acting Bub1 mutations in some colorectal tumor cell lines.
• compositions of the invention are administered to cells.
• administered herein is meant administration of a therapeutically effective dose of the agents of the invention, e.g. anti-Hed compounds and/or HEC1 -complex antagonists/inhibitors, to a cell either in cell culture or in a patient.
• therapeutically effective dose herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques.
• a "patient” or “subject” for the purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human.
• the pharmaceutical compositions are in a water soluble form, such as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
• the term "candidate compound” in accordance with the present invention may comprise any biologically active substance that has an effect on inactivating, intervening, inhibiting, interfering with, disturbing, destroying the HEC1 -complex.
• Preferred compounds are nucleic acids, preferably coding for a peptide, polypeptide, antisense RNA, iRNA, siRNA or a ribozyme or nucleic acids that act independently of their transcription respective their translation as for example an antisense RNA or ribozyme; natural or synthetic peptides, preferably with a relative molecular mass of about 1.000, especially of about 500, peptide analogs polypeptides or compositions of polypeptides, proteins, protein complexes, fusion proteins, preferably antibodies, especially murine, human or humanized antibodies, single chain antibodies, Fab fragments or any other antigen binding portion or derivative of an antibody, including modifications of such molecules as for example glycosylation, acetylation, phosphorylation, famesylation, hydroxy
• Screening methods for identifying compounds that influence the activity of given proteins are well known in the art and can be taken from literature. Preferably, such a screening is carried out in a high-throughput fashion with a degree of automation as high as possible.
• candidate compounds may for instance be provided from libraries of chemical or biological substances that are routinely taken for such approaches and are known in the art.
• the effect of exposing a spindle assembly checkpoint deficient cell to a candidate compound may be determined whether said exposition causes a catastrophic mitotic exit, as shown in the appended examples.
• reagents may be included in the screening assays disclosed herein. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also, reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.
• FIG. 1B Figure 1B summarizes all results in schematic form. Domains of Mad1 required for Heel binding (residues 203 to 378) and Mad2 binding (residues 475 to 557) are indicated. CC, NLS, LZ and RLK refer to the ends of the coiled-coil domain, a putative nuclear localization signal, a leucine zipper and a putative Bub 1 -binding region, respectively.
• C HeLa cells triple labeled with anti-Hed (green) and anti-CENP-B (red) antibodies and DAPI (4,6 Diamidino-2-phenylindole; blue). Merge shows the overlay of the three colors. Top row, cell in prometaphase. Bottom row, cell in anaphase. Cells at similar stages in mitosis labeled with anti-Mad 1 antibodies are shown for comparison; inset: DNA staining. Bar represents 10 ⁇ m.
• CENP-E siRNA produces spindle checkpoint arrest with Heel and Mad1/Mad2 on kinetochores.
• CENP-E-depIeted mitotic HeLa cells were double-stained with anti- CENP-B and either anti-Mad 1 or anti-Mad2 antibodies.
• DNA staining shows the typical arrest phenotype of CENP-E-depleted cells, with most chromosomes aligned at the metaphase plate and a few trailing, misaligned chromosomes (arrows). Note that the kinetochores of unaligned chromosomes are positive for both Mad1 and Mad2. Bar represents 10 ⁇ m.
• Nuf2/hNuf2R siRNA causes spindle checkpoint-mediated prometaphase arrest.
• HeLa cells were treated for 48 hours with a control siRNA duplex (GL2; Elbashir et al., (2001), Nature. 2001 411 , 494-498) (A) or a duplex targeting Nuf2/hNuf2R (B and C).
• Kinetochores were stained with anti- Nuf2/hNuf2R and anti-CENP-B antibodies, and DNA was stained with DAPI, as indicated. Bar represents 10 ⁇ m.
• the coiled-coil protein Heel is a putative mammalian homolog of budding yeast Ndc ⁇ Op (Wigge, J. Cell Biol. 141 (1998), 967-977).
• the exact functions of Heel and Ndc ⁇ Op are unknown, but both Ndc ⁇ Op and Heel were recently localized to kinetochores.
• Yeast Ndc ⁇ Op forms a complex with other proteins, termed Nuf2p, Spc24p and Spc25p, and mutational inactivation of this complex causes severe defects in chromosome segregation.
• microinjection of anti-Hed antibodies into mammalian cells disrupts mitotic progression.
• siRNA small interfering RNA
• rabbit antibodies were raised against hMadl (affinity-purified; 1 ⁇ g/ ⁇ l), hBubl (1:500), and hHed (1 :500).
• Immunofluorescence microscopy was performed using a Zeiss Axioplan-ll microscope and 40x or 63x oil immersion objectives. Photographs were taken using a Micromax (Princeton Instruments) CCD camera and Metaview (Universal Imaging Corp.) software. Whole HeLa cell extracts and Western blots were prepared as described (Stucke (2002), loc. cit.).
• Primary antibodies were: affinity purified rabbit anti-hMadl (1 ⁇ g/ ⁇ l), rabbit anti-hMad2 (BAbCO, 1:500), anti-mHed (1:500; 23), anti-hHed (1:500) or mouse monoclonal anti- ⁇ -tubulin (Sigma Aldrich, 1:2000). Horseradish peroxidase conjugated goat anti-rabbit or goat anti-mouse IgG (Amersham, 1 :3000) were used as secondary antibodies, prior to detection by ECL (Amersham).
• siRNA duplexes for depleting, for example, HEC1, Nuf2 or MPP1 or any other component of the HEClcomplex.
• siRNA treatment see, for example, Elbashir, loc. cit.
• siRNA duplex(es) may, inter alia, be used to deplete HEC1:
• the nucleic acid sequence of the upper strand shown in SEQ ID NO: 1 base pairs with the nucleic acid sequence of the lower strand shown in SEQ ID NO: 2.
• the first G at the 5'-end of the upper strand base pairs with the first C at the 3'-end of the lower strand or the U at the second position calculated from the 5'-end of the upper strand base pairs with the A at the second position calculated from the 3'-end of the lower strand etc. to build siRNA duplexes.
• the above siRNA duplex which is generated by complementary base pairing of the nucleic acid sequences shown in SEQ ID NOs: 1 and 2 is used for depleting HEC1.
• any other suitable siRNA duplex(es) is/are used for depleting HEC1.
• the person skilled in the art is readily in a position to deduce corresponding sequences by techniques known in the art (for example, Elbashir (2001), loc. cit).
• techniques described herein may be employed to test for the usefulness of said siRNAs (as well as other HEC1 antagonists) in vitro for their capacity of, inter alia, cell cycle arrest(s). Upon simultaneous depletion of both proteins (Fig.
• the above siRNA duplex is generated by complementary base pairing of the nucleic acid sequence of the upper strand shown in SEQ ID NO: 3 with the nucleic acid sequence of the lower strand shown in SEQ ID NO: 4.
• the first G at the 5'-end of the upper strand base pairs with the first C at the 3'-end of the lower strand etc. to build siRNA duplexes.
• the above siRNA duplex which is generated by complementary base pairing of the nucleic acid sequences shown in SEQ ID NOs: 3 and 4 is used for depleting Nuf2/hNuf2R.
• any other suitable siRNA duplex(es) is/are used for depleting Nuf2/hNuf2R.
• the nucleic acid sequences shown in SEQ ID NOs: 1 to 4 are RNA, with the exception that the "dT" nucleotide residues at the 3'-end of the respective SEQ ID NO. are DNA.
• Said siRNA duplexes may, for example, be synthesized by methods well known in the art and may or may not be subsequently mixed under conditions known to the person skilled in the art to form siRNA duplex(es).
• CENP-E could no longer be detected at kinetochores, but Hed was still present (Fig. 4A).
• Fig. 4A In the absence of CENP-E, about 20-25 % of cells displayed a mitotic arrest, and this arrest could also be overridden by concurrent elimination of either Mad2 or BubRI , confirming that a checkpoint response is triggered upon interference with CENP-E in mammalian cells (Yao (2000), loc. cit; Chan, J. Cell Biol. 146 (1999), 941-954).
• kinetochore-MT interactions and checkpoint signaling in vertebrate cells may involve two distinct pathways, one centered on Hed interacting with Mad1/Mad2, the other on CENP-E interacting with CENP-F and BubRI , both converging onto APC/C (Chan (1999), loc. cit; McEwen, Mol. Biol. Cell 12 (2001), 2776-2789). With Ndc ⁇ Op the former pathway has a clear counterpart in budding yeast, but the latter appears to have arisen later in evolution, as yeast lacks an obvious homolog of CENP-E.
• Hed -depleted cells display persistent spindle checkpoint activity, although they lack significant amounts of Mad1 or Mad2 at kinetochores.
• checkpoint-deficient cells Any interference with Hed function in checkpoint-deficient cells, be it through siRNA or any other specific agent, would be expected to result in a catastrophic exit from mitosis, thereby causing the demise of most progeny. In contrast, normal, checkpoint-proficient cells would be expected to reversibly arrest in response to Hed inhibition. From this perspective, Hed constitutes an attractive target for therapeutic intervention in cancer and other hyperproliferative diseases.

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