EP1519736A2 - Use of hec1 antagonists in the treatment of proliferative disorders and cancer - Google Patents

Abstract

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 HEC1-complex antagonist and/or at least one HEC1complex inhibitor. Additionally, the invention relates to a method for identifying an anti-Hec1 compound, an HEC1-complex antagonist or an HEC1-complex inhibitor.

Description

Use of 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.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated by reference.

Since more than 100 years it has fascinated scientists to understand mitosis, i.e. how cells divide and how they faithfully transmit their genetic information from one generation to the next (Paweletz (2001), Nature Rev. Mol. Cell. Biol. 2, 72-75). More than 20 years research on the cell cycle has contributed to define the molecular basis of mitosis. A typical cell cycle comprises interphase, i.e. G1 , S and G2 phase and mitosis, i.e. M phase. In particular, in a typical somatic cell cycle, cells proceed from G1 phase due to the action of cyclin-dependent kinases (CDKs) which become activated after association with cyclins into S phase in which the DNA of the chromosomes is to be properly replicated resulting in identical sister chromatids. Afterwards, again CDK/cyclin complexes drive cells from a seemingly quiescent post-replicative stage (G2) into a highly dynamic reorganisation of chromatin topology and cytoskeletal architecture during M-phase which comprises mitosis and cytokinesis. The main purpose of mitosis is to segregate sister chromatids into two nascent cells, whereby it has to be ensured that each cell inherits one complete set of chromosomes. Mitosis is usually divided into five distinct stages: prophase, prometaphase, metaphase, anaphase and telophase. During prophase, interphase chromatin condenses into well-defined chromosomes and duplicated centrosomes migrate apart, thereby defining the poles of the future spindle apparatus that is responsible for the mechanic segregation of the sister chromatids. Moreover, the centrosomes begin nucleating highly dynamic microtubules to build the bipolar spindle and the nuclear envelope breaks down. 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. The poles themselves separate further due to motor activity and prolongation of the polar microtubules which coincides with stretching of the cell, i.e. anaphase B. Finally, telophase in which the spindle apparatus disintegrates, the nuclear envelope reforms around daughter chromosomes and chromatin decondensation takes place is followed by cytokinesis.

The major task of mitosis is to ensure the genetic stability of a cell, which depends on the correct segregation of chromosomes during cell division (Nicklas (1997), Science 275, 632-637; Nasmyth (2000), Science 288, 1379-1385) since errors in this process can lead to an unequal distribution of chromosomes and as a result from this cancer may occur. Thus, eukaryotic cells have evolved a control mechanism which monitors the accuracy of the correct segregation of chromosomes. This surveillance mechanism is known as the "spindle assembly checkpoint", "spindle checkpoint", "mitotic spindle checkpoint", "kinetochore checkpoint" or as "mitotic checkpoint" (Millbrand (2002), Trends Cell. Biol. 12, 205- 209; Shah (2000), Cell 103, 997-1000; Hoyt (2001), Journal of Cell Biol. 154, 909- 911) and prevents anaphase until all kinetochores have been captured by one or more spindle microtubules. This checkpoint is able to detect a single unaligned chromosome, causing a cell to arrest in prometaphase until proper bipolar attachment is achieved, i.e. the so called "wait anaphase" signal. Several key components of this checkpoint were originally identified by genetic analyses in the budding yeast Saccharomyces cerevisiae (Mad 1-3, Bub1, Bub3, and Mps1) and functional homologues of these yeast proteins are known to exist in other, multicellular organisms (Hoyt (1991), Cell 66, 507-517; Li (1991), Cell 66, 519-531; Kitagawa (2001), Nat. Rev. Mol. Cell Biol. 2, 678-687; Rieder (1998), Trends Cell Biol. 8, 310-318). In humans, these include the protein kinases Bub1, BubR1 and Mps1, the Bub1/R1-partner Bub3 (Stucke (2002), EMBO J. 21, 1723-1732; Taylor (1998) J. Cell Biol. 142, 1-11) and Mad1 , Mad2 or the Mad1-Mad2 complex (Luo (2002), Mol. Cell 9, 59-71 ; Sironi (2001), EMBO J. 20, 6371-6382). So far, it could be shown that all these checkpoint components are localized to kinetochores, particularly during early stages of mitosis (Milbrand (2002), loc. cit.). The prevailing model of spindle checkpoint function holds that the absence of an appropriate kinetochore-microtubule interaction generates a signal that inhibits the activity of an ubiquitin ligase known as anaphase promoting complex/cyclosome (APC/C). In turn, APC/C controls ubiquitination and proteolytic degradation of securin (Pdslp in S. cerevisiae), an inhibitor of sister chromatid separation (Nasmyth (2000), loc. cit.). Both Mad2 (Fang (1998), Genes Dev. 12, 1871-1883; Kallio (1998), J. Cell Biol. 141 , 1393-1406) and multiprotein complexes comprising Mad2, BubR1 and Bub3 (Sudakin (2001), J. Cell Biol. 154, 925-936; Tang (2001), Developmental Cell 1, 227-237) have been implicated in the inhibition of APC/C. However, the exact contributions of these inhibitory complexes remain to be clarified, but they all seem to target specifically a form of APC/C that is activated by the WD repeat protein HsCdc20 (Hwang (1998), Science 279, 1041 ff).

Upon proper attachment of the last kinetochore, the APC/C-inhibitory signal is extinguished, and anaphase ensues.

One model for the spindle assembly checkpoint proposes that the checkpoint proteins are recruited to unattached kinetochores in distinct complexes. From deletion studies of the centromere-associated kinesin-related protein CENP-E it is speculated that CENP-E directly bridges between spindle microtubules and the checkpoint kinase BubR1 thereby localizing it together with Mps1 and Bub1 to kinetochores. This suggests a role for CENP-E together with dynein in linking the attachment of spindle microtubules to kinetochores and the mitotic checkpoint signalling cascade (Yao (2000), Nat. Cell Biol. 2, 484-491; Abrieu (2000), Cell 102, 817-826; Sharp-Baker (2001), J. Cell Biol. 153, 1239-1250; Chan (1999), J. Cell Biol. 146, 941-954). This interaction is thought to regulate the recruitment of Mad1- Mad2 complexes and translate structural information, i.e. the presence or absence of appropriate microtubule-kinetochore substrates into a chemical signal, like phosphorylation (Nigg (2001), Nat. Rev. Mol. Cell Biol. 2, 21-32). Mad2 might then be released from kinetochores in a modified state due to the binding of Bub1 and Bub3 to Mad1 (Millbrand (2002), loc. cit.) and becomes, together with Bub3, BubR1 and HsCdc20, a potent inhibitor of soluble APC/C-inhibitory complexes that already exist in interphase cells prior to the assembly of mitotic kinetochores (Sudakin (2001), loc. cit.). Additionally, it was demonstrated that the zeste-white 10 protein (Zw10) and its binding partner, rough deal (Rod) are required for activation of the spindle checkpoint in human cells. These proteins have been previously shown to recruit dynein and dynactin to the kinetochore and, upon microtubule attachment, to "stream" along kinetochore microtubules to the spindle poles. Moreover, it was shown that Mps1 phosphorylates Mad1 in vitro (Hardwick (1996), Science 273, 953- 956). On attachment of the last kinetochore, the production of modified Mad2 would cease and activation of APC/C ensues. Up to now, it remains to be elucidated how a cell cycle-inhibitory signal is generated at unattached kinetochores and how this signal is extinguished upon attachment of the last kinetochore. Defects in the checkpoint can lead to chromosomal instability (CIN) which leads to an abnormal chromosome number (aneuploidy) and, thus, cancer. It was found that such changes of chromosome numbers are nearly ubiquitous in all major human tumor types, e.g. colorectal cancer, brain cancer, breast tumor, prostate tumor, oropharynx tumor, lung tumor (Lengauer (1998), Nature 396, 643-649; Jallepalli (2001), Nat. Rev. Cancer 1 , 109-117). The molecular basis of CIN tumors is currently elucidated. So far the assumption exists that certain genes, when altered, can lead to CIN. These genes include those involved in chromosome condensation, sister-chromatid cohesion, kinetochore structure and function and centrosome/microtubule formation and dynamics, as well as spindle checkpoint genes (Lengauer (1998), loc. cit.). Based on experimental data Tighe (2001), infra, concluded that mutations in genes not directly involved in the spindle checkpoint but involved in microtubule dynamics and/or spindle positioning may be the cause of CIN in colon cancer cells (Tighe (2001), EMBO Rep. 21 , 609-614). It was shown that CIN colon cancer lines undergo transient mitotic arrest upon spindle damage which argues for a robust spindle checkpoint. From these data the authors concluded that it is unlikely that mutations in spindle checkpoint genes underlie the aneuploidies associated with human tumors.

However, involvement of spindle checkpoint genes in the above mentioned group of genes involved in CIN is prompted by the fact that some aneuploid (CIN) lines responded aberrantly to spindle-disrupting agents such as nocodazole and colcemid, i.e CIN lines appeared to exit mitosis prematurely and begin again with DNA synthesis and that alterations in the expression or sequence of human mitotic- checkpoint genes have been detected in human cancers (Cahill (1998), Nature 392, 300-303; Li (1996), Science , 246-248).

Yet, the difference between the study of Tighe (2001), loc. cit. and Cahill (1998), loc. cit. may be explained in that defects in the spindle checkpoint might be partial. This means that tumor cells display a spectrum comprising the range of an almost intact spindle checkpoint to an almost complete defect spindle checkpoint. This assumption is prompted by the fact that it is postulated that mutations in many different "checkpoint genes" can contribute to the development of tumors which could be the reason that so far no "master-gene" in a large number of tumors has been identified (Jallepalli (2001), loc. cit.).

The spindle checkpoint becomes even more complex in view of the existence of another complex that presumably controls kinetochore/microtubule attachment. Recently, in budding yeast NdcδOp, Nuf2p, Spc24p and Spc25p were found to interact as a complex at the kinetochore and may thereby function in the attachment of kinetochores to microtubules and in the checkpoint control (Janke (2001), EMBO J. 20, 777-791; Wigge (2001), J. Cell Biol. 152, 349-360).

It is known that NdcδOp, Nuf2p and Spc24p have homologues in other organisms, e.g. in the fission yeast Schizosaccharomyces pombe, namely SpNdc80, SpNuf2 and SpSpc24 (Wigge (2001), loc. cit.). The human homologue of NdcδOp is HEC1 (highly expressed in cancer) which shares 30% identity on amino acid level. HEC1 is a coiled-coil protein that contains 642 amino acids and a long series of leucine heptad repeats at its C-terminus (Chen (1999), Mol Cell Biol. 8. 5417-5428.). Two homologous proteins for Nuf2p in humans are described. One is 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). Additionally, it was shown that 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.

Mutational inactivation of the NdcδOp, Nuf2p, Spc24p and Spc25p complex in yeast causes severe defects in chromosome segregation. Yet, inactivation of NDC80 in S. cerevisiae does not trigger a checkpoint arrest, demonstrating that there are some important differences in the chromosome-microtubule interactions between yeast and mammals.

It was recently reported that microinjection of anti-HEC1 antibodies into mammalian cells disrupts mitotic progression. 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. This would imply that the checkpoint was partially or completely bypassed in anti-HEC1 antibody-injected cells. The authors further suggest that HEC1 may play roles other than those directly related to spindle attachment at the centromere. Furthermore it was concluded that this implies a checkpoint control problem in H EC 1 -inactivated cells.

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.

Accordingly, 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.

In accordance with this invention, the term "anti-HEC1 compound(s)", "HEClcomplex antagonist(s)" and "HEC1-complex inhibitor(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. Preferably, 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. Yet, it is also envisaged that HEC1 and/or the HEC1 -complex is permanently inactivated, intervened, interfered or inhibited in cells effected by an hyperproliferative disease/disorder or cancer. As will be explained in detail herein below, 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. Additionally, 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. Moreover, 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. Moreover, any biological or chemical composition(s) or substance(s) may be envisaged as HEC1/HEC1 -complex inhibitor or antagonist. Additionally, 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.

Surprisingly, and in contrast to the study by Chen (1999), loc. cit. 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. Furthermore, Chen (1999), loc. cit. speculates that "there is a problem with checkpoint control in cells in which HEC1 has been inactivated." However, as demonstrated herein and in particular in the appended Examples, an inactivation of HEC1 or the HEC1-complex leads to a persistent activation of the spindle checkpoint. However, if HEC1 is eliminated/inactivated in a cell with the inability to mount a spindle checkpoint, a catastrophic mitotic exit follows. Such a catastrophic exit from mitosis will ultimately lead to cell death. In light of the fact that tumor cells appear to be deficient in spindle checkpoint, it is envisaged, in accordance with this invention, that an inactivation of HEC1 and/or HEC1 -complex causes hyperproliferative or tumorous cells to undergo catastrophic mitotic exit, i.e. to die. Without being bound by theory, 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.

As illustrated in the appended Examples, here it was found that when the spindle checkpoint is deliberately inactivated (e.g. inactivation of a critical checkpoint component in connection with elimination of HEC1) a catastrophic mitotic exit results. Accordingly, here it was shown that the fate of cells with impaired HEC1 function depends on the spindle checkpoint status of said cell. In view of the fact that certain tumor cells appear to be already deficient in the spindle checkpoint, 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.

Therefore, 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.

Preferably, 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. In particular, 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. Namely, as shown in the Figures hereinbelow it is confirmed that the phenotype observed when knocking out/silencing hNuf2R is identical or at least similar to the phenotype observed when knocking out/silencing HEC1 expression. The corresponding phenotype is described in the appended Examples and shown in the Figures hereinbelow. The assumption that hNuf2R is a component of the HEC1-complex is also based on the fact that hNuf2R is homologous to Nuf2p from S. cerevisiae. 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.

Further, based on its homology to the S. cerevisiae protein Nuf2p, 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.

In accordance with the present invention, the term "HEC1 -complex" means a complex, preferably a protein complex, comprising at least HEC1 and/or hNuf2R (Nuf2). However, as mentioned hereinabove said complex may also comprise MPP1 as well as further, so far not identified polypeptides. As illustrated in the appended Examples, also 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. It is also envisaged that said HEClcomplex comprises merely (a) functional parts(s) or fragment(s) of HEC1 or hNuf2R. As mentioned hereinabove, MPP1 may also be a component of said HEClcomplex. However, it is particularly preferred that said HEC1 -complex comprises HEC1 and hNuf2R.. Preferably, 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. Spc24p or Spc25p in budding yeast or SpSpc24 in fission yeast. Thus, homologues of said yeast polypeptides may be part of an HEC1 -complex as defined herein. In a preferred embodiment the anti-HEC1 compound, HEC1 -complex antagonist and/or HEC1-complex inhibitor is an inhibiting nucleic acid molecule. Accordingly, 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.

The person skilled in the art can easily deduce corresponding nucleic acid sequences to be employed in this context, since the sequences for, inter alia, HEC1, hNuf2R or MPP1 are well known, see Chen (1999), loc. cit.; Wigge (2001), loc. cit. and Fritzler (2000), loc. cit.

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. For example, 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 (193δ). For applying a triple-helix approach, 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 (19δδ), 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.

Examples for 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.

Another suitable approach is the use of nucleic acid molecules mediating an RNA interference (RNAi) effect. RNAi refers to the introduction of homologous double stranded RNA (dsRNA) to specifically target a gene's product, resulting in null or hypomorphic phenotypes. 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. Because RNAi 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. Thereby, the formation of double-stranded RNA leads to an inhibition of gene expression in a sequence-specific fashion. More specifically, in 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. In accordance with the method of the present invention, the RNAi technique may be carried out as described by Smith (Nature 407 (2000), 319-320) or Marx (Science 238 (2000), 1370-1372).

Likewise, 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. There are various classes of ribozymes. For practical applications aiming at the specific cleavage of the transcript of a certain gene, use is preferably made of representatives of two different groups 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. In order to produce DNA molecules encoding a ribozyme which specifically cleaves transcripts of a gene encoding HEC1 , hNuf2R, MPP1 or any other protein of the HEC1-complex, for example 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. In a preferred embodiment, the inhibiting nucleic acid molecule is siRNA as dislosed in Elbashir ((2001), Nature 411 , 494-498)) and as illustrated in the appended Examples.

It is also envisaged in accordance with this invention that for example short hairpin RNAs (shRNAs) are employed in accordance with this invention as anti-HEC1 compounds, HEC1-complex antagonists or HEC1 -complex inhibitors. 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, 948-95δ. As mentioned above, 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. June 3, 2002; Donze (2002) Nucl. Acids Res. 30, e46; Paul (2002) Nat. Biotech 20, 505-508; Lee (2002) Nat. Biotech. 20, 500- 505; Miyagashi (2002) Nat. Biotech. 20, 497-500; Yu (2002) PNAS 99, 6047-6052 or Brummelkamp (2002), Science 296, 550-553. These approaches may be vector- based, e.g. the pSUPER vector, or RNA pollll vectors may be employed as illustrated, inter alia, in Yu (2002), loc. cit.; Miyagishi (2002), loc. cit. or Brummelkamp (2002), loc. cit.

In a more preferred embodiment of the present invention said siRNA is targeted to deplete HEC1.

In a further more preferred embodiment of the present invention said siRNA is targeted to deplete hNuf2R (Nuf2).

In accordance with the present invention the term "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. "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). 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. Namely, the appended Examples provide methods and guidance how to determine whether HEC, hNuf2R (Nuf2) or any other component of the HEC1 -complex is depleted. Additionally, the Figures hereinbelow show, inter alia, the phenotype of HEC1 or hNuf2R (Nuf2) depletion.

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. Accordingly, 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.

In a particularly preferred embodiment 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. In another particularly preferred embodiment 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.

As described in more detail in the appended Examples hereinbelow, the person skilled in the art knows, inter alia, how 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.

As mentioned herein, 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. For example, 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. Most conveniently, siRNAs and the like are obtained from commercial RNA oligo synthesis suppliers, which sell RNA-synthesis products of different quality and costs. In general, 20 to 50-nucleotide RNAs are not too difficult to synthesize and are readily provided in a quality suitable for RNAi. However, 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. vι_ and VH regions of anti- HECl antibodies) antisense sequences or inhibiting RNAs as defined herein to block expression of HEC1 and/or components of the HEC1-complex. In one embodiment of this invention, a method is provided 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. Preferably, 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.

As discussed herein above, 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. In^" particular, also included are 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. For the preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples for such techniques include the hybridoma technique (Kohler and Milstein (1975), Nature 256, 495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96). Techniques describing the production of single chain antibodies (e.g., U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptides as described above. Furthermore, transgenic mice may be used to express humanized antibodies directed against said immunogenic polypeptides. It is in particular preferred that 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. This may, inter alia, be achieved by direct injection of the corresponding proteineous molecules or by injection of nucleic acid molecules encoding the same. Furthermore, gene therapy approaches are envisaged. Accordingly, in context of the present invention, the term "antibody molecule" relates to full immunoglobulin molecules as well as to parts of such immunoglobulin molecules. Furthermore, 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 term also relates to intact antibodies as well as to antibody fragments thereof, like, separated light and heavy chains, Fab, Fab/c, Fv, Fab', F(ab')₂. The term "antibody molecule" also comprises bifunctional antibodies and antibody constructs, like single chain Fvs (scFv) or antibody-fusion proteins-. It is also envisaged in context of this invention that the term "antibody" comprises antibody constructs which may be expressed in cells, e.g. antibody constructs which may be transfected and/or transduced via, inter alia, viruses or vectors. It is in particular envisaged that such antibody constructs specifically recognize HEC1 and/or a member of the HEC1-complex and that said expressed antibody constructs inhibit and/or antagonize the function of HEC1 and/or the HEC1 -complex. It is, furthermore, envisaged that said antibody construct is employed in gene therapy approaches.

In another embodiment, 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.

In accordance with the present, invention, the term "aptamer" means nucleic acid molecules that can bind to target molecules. Aptamers commonly comprise RNA, single stranded DNA, modified RNA or modified DNA molecules. The preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sides (Gold (1995), Ann. Rev. Biochem 64 , 763-797). In accordance with the present invention, the hyperproliferative disease/disorder to be ameliorated and/or treated with the anti-HEC1 compound and/or HEC1 antagonist or inhibitor of the HEC1 -complex may be cancer and/or a tumorous disease.

In a preferred embodiment the cancer and/or tumorous disease is characterized as leading to or as comprising aneuploid cancer cells. In accordance with the present application, the term "aneuploid" means alterations in chromosome numbers involving losses or gains of whole chromosomes or structural rearrangement(s) of chromosomes.

Preferably, the cancer to be treated may, inter alia, be colorectal cancer, breast cancer, lung cancer, non-small cell lung cancer, small cell lung cancers, prostate cancer, cervical cancer, bladder cancer, mammary/breast cancer, brain cancer, oropharynx cancer, nasopharyngeal cancer or head and neck squamous cell carcinoma (HNSCC). It is also envisaged that said cancer is a leukemia or lymphoma, wherein 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. For example, evidence exists that transforming protein Tax of the human T cell leukaemia virus type 1 (HTLV-1) interacts with Mad1, a mitotic checkpoint control protein (lha (2000), AIDS Res. Hum. Retroviuses 16, 1633-1633; Jin (1993), Cell 93, 31-91). Moreover, cells from adult T-cell leukemias show phenotypes, i.e. cells with more nuclei which are throughout comparable to cells having a defect in the spindle checkpoint.

Further tumorous diseases or cancers to be treated with the anti-HEC1 compounds described herein are described herein below. Yet, the present invention is not limited to the treatment of the distinct cancers disclosed herein.

In a preferred embodiment, the cancer cells and/or aneuploid cancer cells are spindle assembly checkpoint deficient.

The term "checkpoint" means a point where the cell division can be halted until conditions are suitable for the cell to proceed to the next stage. In particular, the term "spindle assembly checkpoint" comprises a control mechanism which monitors the accuracy of the correct segregation of chromosomes and acts to prevent anaphase until all kinetochores have been captured one or more spindle microtubules. Thereby, this checkpoint is able to detect a single unaligned chromosome, causing a cell to arrest in prometaphase until proper bipolar attachment is achieved, i.e. the so called "wait anaphase" signal. This surveillance mechanism is also named "spindle checkpoint", "mitotic spindle checkpoint", "kinetochore checkpoint" or "mitotic checkpoint" (Millbrand (2002), loc. cit; Shah (2000), loc. cit.; Hoyt (2001), loc. cit.) 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. It is of note that, in accordance with this invention, also cancer cells are to be treated which comprise a deficiency in spindle assembly checkpoint which is due to (a) modification(s), (a) mutation(s), or (a) variant(s) of the spindle checkpoint genes/expressed proteins described herein and to be elucidated.

Known spindle checkpoint proteins comprise Mad1 , Mad2, Mad1/Mad2-complex, Bub1 , BubR1 , Bub3, Mps1 and are described in Millberg (2002), loc. cit. and Nigg (2001), loc. cit. Furthermore, CENP-E and HsCdc20 are described in Shah (2000), loc. cit., CENP-F is described in Chan ((1993), J Cell Biol. 143, 49-63, Polo-like Kinase 1, Aurora-A, Aurora-B , INCENP and survivin in Nigg (2001), loc. cit. and Jallepalli (2001), loc. cit. ZW10, Rod in Scaerou ((2001), J. Cell Sci. 114, 3103- 3114), Chan ((2000), Nat. Cell. Biol. 2, 944-947) and Basto ((2000), Nat. Cell. Biol. 2, 939-943). 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. As indicated above, 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. Further investigation has shown that about 50% of the cancer cell lines checked have a defective spindle checkpoint when tested using drugs that disrupt the spindle machinery (Takahashi (1999), Oncogene 18, 4295 ff.; Scolnick (2000), Nature 406, 430-435; Matsuura (2000), Am. J. of Human Gen. 67, 483-436; Wang (2000), Carcinogenesis 21 , 2293-2297; Weitzel (2000), Cell & Tissue Res. 300, 57-65). Additionally, it has been shown that mutations in the spindle checkpoint genes, e.g. Bub1 , Mad1 , Mad2, BubR1 are present resulting in colorectal cancer, digestive tract cancer, head and neck squamous cell carcinoma, lung cancer, non-small lung cancer, small lung cancer, breast cancer, nasopharyngeal cancer, adenocarcinoma, bladder cancer, T-cell leukemia or B-cell leukemia, see, inter alia, Cahill (1998), loc. cit.; Imai (1999), Jpn. J. Cancer 90, 837-340; Yamaguchi (1999), Cancer Lett. 139, 183-187; Takahashi (1999), Oncogene 18, 4295 ff.; Nomoto (1999), Oncogene 18, 71 δ0 ff.; Sato (2000), Jpn. J. Cancer Res. 91, 504-509; Ohshima (2000), Cancer Lett. 15δ, 141-150; Gemma (2000), Genes, Chromosomes & Cancer 29, 213-213; Jaffrey (2000), Cancer Res. 60, 4349-4352; Percy (2000), Genes, Chromosomes & Cancer 29, 356-362; Myrie (2000), Cancer Lett. 152, 193-199; Wang (2000), Carcinogenesis 21 , 2293-2297; Weitzel (2000), Cell & Tissue Res. 300, 57-65; Mimori (2001), Oncology Reports 8, 39-42; Haruki (2001), Cancer Lett. 162, 201- 205; Oelsen (2001), Carcinogenesis 22, 813-315.

The present invention also relates to a pharmaceutical composition comprising at least one anti-HEC1 compound, at least one HEC1-complex antagonist and/or at least one HEC1 -complex inhibitor as characterized and defined herein above. As discussed herein, anti-HEC1 compounds/antagonists/inhibitors and/or inhibitors/antagonists of the HEC1 -complex are in particular useful for the treatment of hyperproliferative disorders and/or cancer.

The pharmaceutical composition of the present invention may, in addition to the compound described above, e.g. at least one HEC1/HEC1-complex antagonist or inhibitor, comprise a pharmaceutically acceptable carrier and/or diluent. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 μg (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. Dosages of nucleic acid molecules, inter alia administered in form of a gene therapy approach, will vary, but a preferred dosage for intravenous administration of DNA is from approximately 10⁶ to 10¹² copies of the DNA molecule. The compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

In a preferred embodiment of this present invention, the HEC1 -complex antagonist/inhibitor or the anti-HEC1 compound described herein is administered to a subject, preferably a human subject, in need of such a treatment in form of a pharmaceutical composition.

Furthermore, the pharmaceutical composition of the invention may comprise further agents, depending on the intended use of the pharmaceutical composition. It is also envisaged that the anti-HEC1 -compound or antagonist/inhibitor of HEC1 /HEClcomplex is employed in co-therapy approaches, i.e. in co-administration with other medicaments or drugs, for example anti-cancer drugs.

In this context, it is also of note, that the anti-HEC1 compound, the HEC1 -complex antagonist(s) or inhibitor(s) may also be administered by gene therapy approaches. For gene therapy applications, nucleic acid molecules encoding said anti- HEC1/HEC1-complex compounds may be cloned into a gene delivering system, such as a virus or further vectors. Suitable vectors, methods or gene-delivering systems for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 30δ-δ13; Isner, Lancet 343 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1036; Onodua, Blood 91 (1998), 30-36; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-2251 ; Verma, Nature 339 (1997), 239-242; Anderson, Nature 392 (Supp. 1993), 25-30; Wang, Gene Therapy 4 (1997), 393-400; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957; US 5,580,859; US 5,589,466; US 4,394,448 or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and references cited therein.

The invention also provides for a method for preventing, ameliorating and/or treating a hyperproliferative disorder/disease, like cancer, comprising the step of administering to a subject in need of such a prevention, amelioration and/or treatment an anti-HEC1 compound, HEC1-complex antagonist and/or HEC1- complex inhibitor as characterized and described above. It is also envisaged that compounds identified by the methods disclosed herein below are employed in the method for preventing, ameliorating and/or treating the disorders ans diseases disclosed herein.

The compositions and methods provided herein are particularly deemed useful for the treatment of cancer including solid tumors such as skin, breast, brain, cervical carcinomas, testicular carcinomas, etc. More particularly, cancers that may be treated by the compositions and methods of the invention include, but are not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinorna, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli- Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma], fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma. Thus, the term "cancerous cell" as provided herein, includes a cell afflicted by any one of the above identified conditions.

Accordingly, the compositions of the invention are administered to cells. By "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. By "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. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art. By "cells" herein is meant almost any cell in which mitosis or meiosis can be altered. 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 compounds described herein as well as the candidate agents as disclosed herein below having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a patient, as described herein. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways as discussed below. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt %. The agents may be administered alone or in combination with other treatments, i.e., radiation, or other chemotherapeutic agents.

In a preferred embodiment, 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 pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents. The pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol. Additives are well known in the art, and are used in a variety of formulations.

The administration of the candidate agents of the present invention can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In some instances, for example, in the treatment of wounds and inflammation, the candidate agents may be directly applied as a solution or spray.

Furthermore, the invention relates to a method for identifying an anti-HEC1 compound, an HEC1-complex antagonist or an HEC1-complex inhibitor comprising the steps of

(a) exposing a spindle (assembly) checkpoint deficient cell to a candidate compound; and

(b) determining whether the exposition to said candidate compound causes a catastrophic mitotic exit.

In accordance with the present invention, the term "exposing" means that spindle assembly checkpoint deficient cells as described herein (also as illustrated in the appended examples) are contacted with a candidate compound as defined below. In particular, a candidate compound may be added to the culture medium in which the cells are growing or is microinjected into said cells. Furthermore, said candidate compound may be exposed to said cell by transfection or transduction approaches.

It is, inter alia, feasible to employ in a screening method described herein above a cell which comprises a mutation/modification in a checkpoint compound as described herein above or described in Cahill (1998), loc. cit., Takahashi (1999), loc. cit., Scolnick (2000), loc. cit., Matsuma (2000), loc. cit., Wang (2000), loc. cit. or Weitzel (2000), loc. cit. Furthermore, the appended Examples illustrate how checkpoint-deficient cells may be obtained, inter alia by siRNA approaches. Further approaches to obtain cells/cell lines which are deficient in a certain gene product are known in the art. These comprise, inter alia, antisense-technology, or the use of RNAi, siRNAs or shRNAs as described herein above or as illustrated in Paddison (2002), loc. cit.; Paddison (2002) PNAS 99, 1443-1448; Donze (2002) Nuc. Acids Res. 30, e46, Brummelkamp (2002) Science 296, 550-553. A spindle assembly checkpoint deficient cell may be produced, inter alia, by the methods provided in the appended Example. In addition, it is envisaged to construct corresponding "indicator-cell lines" comprising a dominant-negative mutation in (a) checkpoint- proteins as defined herein above. Such dominant-negative checkpoint mutants are known in the art, see, e.g. Tsukasaki (2002), J. Clin. Immunol. 22, 57-63. Accordingly, the method provided herein above is not limited to exposing the candidate compound to a spindle assembly checkpoint deficient cell, but the assay system may also be employed by use of further spindle checkpoint deficient cells as defined herein above.

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, hydroxylation, methylation or esterification, hormones, organic or inorganic molecules or compositions, preferably small molecules with a relative molecular mass of about 1.000, especially of about 500.

Yet, candidate agents/candidate compounds encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Candidate agents/compounds comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. In a preferred embodiment, the candidate agents are organic chemical moieties, a wide variety of which are available in the literature.

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. As defined herein above, 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.

Compounds tested positive for being capable to cause a catastrophic mitotic exit are prime candidates for the direct use as a medicament or as lead compounds for the development of a medicament. Naturally, the toxicity of the compound identified and other well-known factors crucial for the applicability of the compound as a medicament will have to be tested. Methods for developing a suitable active ingredient of a pharmaceutical composition on the basis of the compound identified as a lead compound are described elsewhere in this specification.

The invention also relates to a method for identifying a checkpoint antagonist comprising the steps of:

(a) exposing a cell arrested in prometaphase to a candidate compound; and

(b) determining whether the exposition to said candidate compound causes a catastrophic mitotic exit.

Preferably, the prometaphase arrest is due to inhibition of the HEC1-complex by an inhibiting nucleic acid, an antibody or a derivate or a fragment thereof or an aptamer or any compound described herein or identified by the aforementioned method. For example, cells arrested in prometaphase by HEC1 inhibitors or antagonists, like anti-HEC1 siRNA, may be employed in screening methods for pharmacological or genetic checkpoint antagonists.

Accordingly, the cell/cell population to be employed in this screening method may be arrested in its checkpoint due to HEC1 inhibition. The corresponding read-out, after application of the candidate compound, would be whether said candidate compound leads to a "mitotic exit" of said arrested cell/cell population.

The embodiments for the candidate compound for identifying a checkpoint antagonist by the method provided herein are already illustrated in further methods disclosed herein and are to be applied, mutatis mutandis.

The identification/screening methods described herein may be conventionally employed in form of "high throughput screenings", whereby the term "high throughput screening" refers to (an) assay(s) which provide(s) for multiple candidate agents or samples to be screened simultaneously. The candidate agents- may be isolated agents to be tested, but may also constitute mixtures of candidate agents. The candidate agent(s) may also comprise extracts, like cellular or plant extracts. The candidate agents may also be tested in conjunction with a carrier. In the assays and test provided herein the modulation of mitotic events, in particular in catastrophic mitotic exit (as demonstrated in the appended examples) may be tested. It is preferred that said methods/assays comprise in vitro assays, wherein also (and furthermore) alterations in cell cycle distribution, cell viability, morphology, and/or distribution of chromosomes or mitotic spindles be assayed.

A variety of other 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.

Additionally, the invention provides for a method for the production of a pharmaceutical composition comprising the steps of the method of the aforementioned methods and a further step, wherein the candidate compound identified as an anti-HEC1 compound, an HEC1 -complex antagonist or HEClcomplex inhibitor is mixed with a pharmaceutically acceptable carrier.

The Figures show:

Figure 1 Interaction between Heel and Mad1 at kinetochores.

(A) Mad1 and Heel interact in the yeast two-hybrid system. Different fragments of hMadl were introduced into a pDBD (DNA-binding domain) vector and tested for interaction with either pACT (activation domain)- Hec1 or pACT-Mad2, both on non-selective (-LW; minus leucine, tryptophan) and selective (-LWAH; minus leucine, tryptophan, adenine, histidine) plates. Figure 1A illustrates growth of selected DBD-Mad1 constructs tested against ACT-Hec1.

(B) 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 μm.

Figure 2 Requirements for kinetochore localization of spindle checkpoint proteins.

(A) Heel is required for kinetochore localization of Mad1/Mad2 but not Wee-versa. HeLa cells were treated for 42 hours with siRNA duplexes specific for Heel (left panels) or Mad1 (right panels). Prometaphase cells were co-stained with anti-Hed and anti-CENP-B antibodies or with anti- Mad1 or anti-Mad2 antibodies, as indicated. Bar represents 10 μm.

(B) Western blot demonstrating depletion of Hed and Mad1. Total HeLa cell extracts treated with appropriate siRNA duplexes were probed with the antibodies indicated. Anti-α-tubulin antibodies were used to demonstrate equal loading. (C) Table summarizing results of siRNA experiments. Following depletion by siRNA of Heel, Mad1 or CENP-E, the kinetochore association was tested for the proteins listed, (x): expected elimination of siRNA target, (+): persistent kinetochore association, (-): loss of kinetochore association, (n.d.): no data.

Figure 3 Depletion of Heel causes spindle checkpoint-mediated prometaphase arrest.

(A) HeLa cells were treated for 42 hours with siRNA duplexes as indicated and stained with DAPI (blue) and α-tubulin antibodies (red). Top row: merged colors. Bottom row: enlarged views, illustrating DNA condensation states. Bars represent 20 μm.

(B) Western blot demonstrating depletion of Heel and Mad2. Total HeLa cell extracts treated with appropriate siRNA duplexes were probed with the antibodies indicated. Anti-α-tubulin antibodies were used to demonstrate equal loading.

(C) Mitotic indices of HeLa cells treated for 24 or 42 hours with siRNA duplexes targeted at the proteins indicated. Histograms show average results and standard deviations from several independent experiments, counting at least 100 cells in at least three different fields in each experiment.

Figure 4 CENP-E siRNA produces spindle checkpoint arrest with Heel and Mad1/Mad2 on kinetochores.

(A) Mitotic HeLa cells treated with or without siRNA duplex specific for CENP-E were double-stained with anti-CENP-E (top row) and anti-Hed antibodies (bottom row).

(B) 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 μm.

Figure 5: 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 μm.

The examples illustrate the invention.

Example 1: Two-hybrid screen for Mad1 -interacting proteins

In a yeast two-hybrid screen for novel human Mad 1 -interacting proteins that might localize to the kinetochore, not only the expected Mad1 and Mad2, two known interactors of Mad1 were isolated, but also a cDNA coding for full-length human Heel (highly expressed in cancer) (Chen (1999), loc. cit.) (Fig. 1A). A hMadl cDNA was obtained from Dr. K.T.Jeang (Jeang, Cell 93 (1998), 81-91). It was recloned into pUNHO (SM009), the entry vector for the Univector system (Liu, Curr. Biol. 8 (1998), 1300-1309) and completely sequenced. Its sequence matched accession number AF123318 (see Campbell, J. Cell Sci. 114 (2001), 953-963). Deletion mutants and constructs for yeast two-hybrid screens (James, Genetics 144 (1996), 1425-1436) were prepared following standard procedures. A human lymphocyte cDNA library (Durfee, Genes Dev. 7 (1993), 555-569) was screened. Of 1 ,4x10⁵ transformants, 92 colonies grew on selective plates. Following analysis by restriction digests and partial sequencing, several interactors of hMadl were identified. These included fragments of Mad1, CENP-F, and Smc-3, as well as full-length Mad2 and Heel . As discussed in the introduction, supra, the coiled-coil protein Heel is a putative mammalian homolog of budding yeast NdcδOp (Wigge, J. Cell Biol. 141 (1998), 967-977). The exact functions of Heel and NdcδOp are unknown, but both NdcδOp and Heel were recently localized to kinetochores. Yeast NdcδOp forms a complex with other proteins, termed Nuf2p, Spc24p and Spc25p, and mutational inactivation of this complex causes severe defects in chromosome segregation. Similarly, microinjection of anti-Hed antibodies into mammalian cells disrupts mitotic progression. This prompted the speculation that the NdcδOp complex, and by implication a similar HEC1-complex in vertebrates, may contribute to mediate kinetochore-MT interactions. As shown by double immunofluorescence microscopy with antibodies against Heel and CENP-B, a marker for centromeric heterochromatin, Heel was present on kinetochores throughout mitosis (Fig. 1 B). In contrast, Mad1 was released upon alignment of chromosomes at the metaphase plate (Fig. 1 B, see also Campbell (2001), loc. cit). To examine the possibility that Heel might transiently recruit a Mad1/Mad2 complex to kinetochores, both the Hed and the Mad2 binding sites were mapped on Mad1 , using the yeast two-hybrid assay. As shown in Fig. 1A, the regions of Mad1 required for binding Hed and Mad2 were clearly distinct, indicating that Mad1 could indeed bind simultaneously to both proteins.

To directly explore the functional significance of the Hec1-Mad1 interaction in HeLa S3 cells, gene silencing by small interfering RNA (siRNA) as described by Elbashir (2001), loc. cit. was used. Efficient silencing of the Hed gene was demonstrated by both immunofluorescence microscopy (Fig. 2A) and immunoblotting (Fig. 2B). The siRNA sequence targeting Hed was from position 1517-1539 relative to the start codon (accession number NM_006101), Mad1 (AF123313) 1δ43-1365. Duplexes targeting CENP-E and lamin A have been described previously (Harborth, J. Cell Sci. 114 (2001), 4557-4565). Following siRNA treatment of HeLa S3cells, as described in Elbashir (2001), loc. cit, cells were analyzed by immunofluorescence microscopy (Stucke, EMBO J. 21 (2002), 1723-1732), using the following antibodies: rabbit anti-XMad2 (1 :1000; from T.U.Mayer), anti-mHed (1 :500; 23), anti-CENP-F (Abeam, product ab5; 1:1000), and mouse monoclonal anti-CENP-B (1:1000; from W. Earnshaw), anti-CENP-E (1:500; from D. Brown), and anti-α-tubulin (Sigma Aldrich, 1 :2000). Additionally, rabbit antibodies were raised against hMadl (affinity-purified; 1 μg/μl), 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 μg/μl), rabbit anti-hMad2 (BAbCO, 1:500), anti-mHed (1:500; 23), anti-hHed (1:500) or mouse monoclonal anti-α-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).

Importantly, for all siRNA experiments reported herein, Western blotting demonstrated the selective depletion of the targeted protein, whereas the levels of other checkpoint components remained unchanged (Figure 2B). Furthermore, siRNA phenotypes displayed very little variation from cell to cell, indicating that 33 protein depletion was rather uniformly efficient. In the absence of Hed , neither Mad1 nor Mad2 could be detected on mitotic kinetochores (Fig. 2A), although anti- Mad 1 and anti-Mad2 antibodies produced strong kinetochore staining in control cells (Fig. 1B, and data not shown). Furthermore, the localization of CENP-B was unaffected by the absence of Hed (Fig. 2A). It was further addressed whether the kinetochore-association of Hed was dependent on the presence of Mad Depletion of Mad1 by siRNA abolished the kinetochore-association of Mad2, as expected, but it did not interfere with Hed localization (Fig. 2A). Thus, the recruitment of the Mad1/Mad2 complex to kinetochores depends on Hed but not vice-versa. To determine whether the depletion of Hed would interfere with the kinetochore-association of other proteins, Hed -depleted cells were double-stained with antibodies against Mpsl, Bub1 and CENP-B or CENP-E and CENP-F. In cells lacking Hed, the kinetochore-association of Mpsl was completely -abolished and that of Bub1 was reduced by about 50 %, while CENP-B, -E and -F still localized to kinetochores efficiently (Fig. 2C). This demonstrates that the elimination of Hed interferes with the kinetochore-association of a specific subset of spindle checkpoint proteins, rather than causing a total disruption of the kinetochore.

Example 2: Hec1-and hNuf2R (Nuf2)-siRNAtreatment of cultured cells results in activation of the spindle check point

The described yeast two-hybrid studies did not indicate whether the interaction between Hed and Mad1 was direct or indirect. However, it is likely that additional components might be involved in mediating the interaction between these proteins in vivo. In support of this possibility, a recent study has implicated the Mpsl kinase in the kinetochore-association of the Mad1/Mad2 complex in a Xenopus egg extract (Abrieu (2001), Cell 106, 33-93). The siRNA-mediated depletion of human Mpsl also abolished the kinetochore-association of Mad1 , whereas it did not influence the localization of CENP-B or Hed . These data show that Hed is required for the kinetochore- association of Mpsl, and that both Hed and Mpsl are required for recruiting the Mad1/Mad2 complex. Strikingly, cultures subjected to Hed siRNA treatment displayed a high percentage of mitotic cells with condensed chromosomes, suggesting a prometaphase arrest (Figs. 3Ab,e and C). These chromosomes were attached to the spindle apparatus, as kinetochore MTs selectively resisted short calcium treatment (Kapoor, J. Cell Biol. 150 (2000), 975-93δ) and kinetochores stained positively for the MT plus end- binding protein EB1. However, virtually none of the cells displayed chromosomes aligned in a metaphase plate, pointing to an underlying defect in chromosome congression. To examine whether this phenotype was dependent on a functional spindle checkpoint, cells were subjected to siRNA for both Hed and Mad2. The person skilled in the art knows how to design appropriate siRNA duplexes for depleting, for example, HEC1, Nuf2 or MPP1 or any other component of the HEClcomplex. Moreover, the person skilled in the art knows how to perform siRNA treatment; see, for example, Elbashir, loc. cit. For carrying out HEC1 -siRNA treatment the following siRNA duplex(es) may, inter alia, be used to deplete HEC1:

GUU CAAAAG CUG GAU GAU C dT dT (SEQ ID NO: 1) dT dTCAAGUU UUC GAC CUA CUAG (SEQ ID NO: 2).

Within the siRNA duplex 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. For example, 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.

Particularly preferred, 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. However, it is also envisaged that 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). Furthermore, 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. 3B), the accumulation of mitotic cells was abolished (Figs. 3Ac,f and C). Instead, cells passed through mitosis (Fig. 3C), giving rise to frequent and severe nuclear aberrations, including multinucleation and nuclear fragmentation (Fig. 3Ac,f). A very similar reduction in the mitotic index was seen after simultaneously depleting Hed and BubRI , another component of the spindle checkpoint, but not upon co-depletion of Hed and lamin A (Fig. 3C). These results show that depletion of Hed results in persistent activation of the spindle checkpoint. Conversely, if Hed is eliminated from a cell with the inability to mount a spindle checkpoint, a catastrophic mitotic exit ensues. It is of great interest that similar results may be obtained when siRNA against the H EC 1 -interaction partner (human) Nuf2 is employed. Such results can be obtained by carrying out similar experiments as shown herein for HEC1. It is expected that a high percentage of cells appears to be arrested in prometaphase since hNuf2R is expected to be a component of the HEC1 -complex. As is shown in Figure 5B,C, Nuf2/hNuf2R siRNA causes spindle checkpoint-mediated prometaphase arrest. For carrying out Nuf2/hNuf2R-siRNA treatment the following siRNA duplex(es) may, inter alia, be used to deplete Nuf2/hNuf2R:

GCA UGC CGU GAA ACG UAU A dT dT (SEQ ID NO: 3) dT dT CGU ACG GCA CUU UGC AUA U (SEQ ID NO: 4)

As already described hereinabove, 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. For example, 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.

Particularly preferred, 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. However, as already described hereinabove, mutatis mutandis, it is also envisaged that 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).

The person skilled in the art knows that similar experiments and results can be obtained for any other component which is comprised by the HEC1 -complex, e.g., MPP1.

Both the defect in chromosome congression and the activation of the spindle checkpoint in Hed -depleted cells could be explained if Hed was required to mediate MT-kinetochore interactions. Such a role is strongly supported by data obtained on NdcδOp, the S. cerevisiae homologue of Hed (He, Cell 106 (2001), 195-206). In vertebrate cells, however, a key role in kinetochore-MT interactions has previously been attributed to the kinesin-related motor CENP-E (Yao, Nat. Cell Biol. 2 (2000), 4δ4-491). Thus, the consequences of eliminating CENP-E by siRNA was examined. Following 42 hours of treatment with a CENP-E specific duplex, CENP-E could no longer be detected at kinetochores, but Hed was still present (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). Although the checkpoint arrest phenotypes observed in response to depletion of Hed and CENP-E were superficially similar, it has to be emphasized that they differed in two important aspects: first, chromosome congression to a metaphase plate occurred to a greater extent in cells lacking CENP-E than in cells lacking Hed (compare Figs. 4B and 3Ae), consistent with earlier studies showing formation of robust metaphase plates in cells microinjected with anti-CENP-E antibodies. Second, the localization of the Mad1/Mad2 complex was in agreement with the prevailing paradigm for checkpoint signaling in CENP-E depleted cells but not in Hed -depleted cells: in cells lacking CENP-E, chromosomes already aligned at the metaphase plate showed neither Mad1 nor Mad2 on kinetochores, but the kinetochores of unattached, misaligned chromosomes stained strongly positive for both proteins (Fig. 4B, arrows). In striking contrast, Hed depleted cells displayed a checkpoint arrest although neither Mad1 nor Mad2 could be detected at kinetochores (see above). This surprising finding upsets the notion that high steady- state levels of kinetochore-associated Mad1/Mad2 complexes constitute a reliable marker for the activation of the spindle checkpoint (see also Waters, (1993) J. Cell Biol. 141, 1181-1191; Skoufias, (2001) PNAS 98, 4492-4497). Considering the short half-life of the Mad1/Mad2 complex at the kinetochore, as discussed in Howell (2000), J. Cell Biol. 150, 1233-1250 it is conceivable that undetectably low levels of kinetochore-associated Mad1/Mad2 complexes are sufficient for checkpoint signaling. Alternatively, it is possible that a factor whose association with kinetochores does not depend on Hed is able to communicate with diffusible Mad2 complexes to signal checkpoint activation. In cells depleted of Hed, this activity could be CENP-E/BubR1.

The data presented here clearly show that depletion of Hed in human cells activates the spindle assembly checkpoint. In apparent contrast, injection of anti- Hed antibodies into T24 bladder carcinoma cells was reported to cause aberrant mitotic progression and extensive cell death, but no checkpoint arrest (Chen, (1999), loc. cit).

In S. cerevisiae, mutations in the apparent Hed homologue, Ndc80, also caused severe chromosome segregation defects but did not significantly arrest the cell cycle, indicating that the spindle checkpoint had not been triggered (Wigge, J. Cell Biol. 141 (1998), 967-977; He, Cell 106 (2001), 195-206). This difference may reflect the evolution of an increasing complexity in both kinetochore-MT interactions and their links to the checkpoint machinery. Whereas kinetochores in budding yeast only bind a single MT, those in vertebrate cells bind multiple MTs (Rieder, Trends Cell Biol. 8 (1998), 310-318). Furthermore, as discussed above, 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 NdcδOp 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. As a consequence, depletion of either Hed or CENP-E from vertebrate cells would still leave checkpoint signaling intact, whereas any mutation abolishing the NdcδOp- based pathway in yeast would also destroy the spindle checkpoint (Wigge (199δ), loc. cit; Wigge, J. Cell Biol. 152 (2001), 349-360; He (2001), loc. cit; Janke, EMBO J. 20 (2001), 777-791 ; Gardner, Genetics 157 (2001), 1493-1502). In conclusion, the data presented here show that the human kinetochore protein Hed is required for recruiting the Mad1/Mad2 complex to kinetochores. Most interestingly, Hed -depleted cells display persistent spindle checkpoint activity, although they lack significant amounts of Mad1 or Mad2 at kinetochores. This observation contrasts with models emphasizing the importance of high steady-state levels of kinetochore-associated Mad1/Mad2 complexes in checkpoint activation. Instead, it suggests that some protein that does not depend on Hed for kinetochore localization is able to communicate with diffusible Mad2 complexes (see also Sudakin, J. Cell Biol. 154 (2001), 925-936). Finally, it is interesting to consider the implications of our findings for those tumor cells that are thought to be defective in the spindle checkpoint. 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.

Claims

Claims

1. 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.

2. The use of claim 1 , wherein the HEC1-complex comprises HEC1 , MPP1 , hNuf2R (Nuf2).

3. The use of claim 1 or 2, wherein said anti-HEC1 compound, HEC1 -complex antagonist and/or HEC1 -complex inhibitor is an inhibiting nucleic acid molecule.

4. The use of claim 3, wherein said inhibiting nucleic acid molecule is selected from the group consisting of antisense oligonucleotide, antisense DNA antisense RNA, iRNA, ribozyme, shRNA and siRNA.

5. The use of claim 4, wherein said siRNA is targeted to deplete HEC1.

6. The use of claim 4, wherein said siRNA is targeted to deplete hNuf2R (Nuf2).

7. The use of claim 4 or 5, wherein said siRNA comprises (an) siRNA duplex(es) formed by the nucleic acid sequences shown in SEQ ID NOs: 1 and 2.

3. The use of claim 4 or 6, wherein said siRNA comprises (an) siRNA duplex(es) formed by the nucleic acid sequences shown in SEQ ID NOs: 3 and 4.

9. The use of claim 1 or 2, wherein said anti-HEC1 compound, HEC1 -complex antagonist and/or HEC1 -complex inhibitor is an antibody or a derivative or a fragment thereof or an aptamer.

10. The use of any one of claims 1 to 9, wherein said hyperproliferative disease/disorder is cancer and/or a tumorous disease.

11. The use of claim 10, wherein said cancer and/or tumorous disease is characterized as leading to or as comprising aneuploid cancer cells.

12. The use of claim 11, wherein said cancer is colorectal cancer, stomach cancer, breast cancer, lung cancer, non-small cell lung cancer, small cell lung cancers, small or large bowel cancer, prostate cancer, cervical cancer, tester cancer, ovarial cancer, bladder cancer, liver cancer, bone cancer, mammary/breast cancer, brain cancer, oropharynx cancer, skin cancer, nasopharyngeal cancer, head and neck squamous cell carcinoma (HNSCC), sarcoma, pancreas cancer, kidney cancer, cancer of the nervous system, neurofibroma.

13. The use of claim 10, wherein said cancer is a leukemia or lymphoma.

14. The use of claim 13, wherein said leukemia or lymphoma is selected from the group consisting of T-cell leukemia, T-cell lymphoma, B-cell leukemia and B- cell lymphoma such as Burkitt's lymphoma, Hodgkin's lymphoma and non- Hodgkin's lymphoma.

15. The use of any one of claim 11 to 14, wherein said aneuploid cancer cells are spindle assembly checkpoint deficient.

16. The use of claim 15, wherein said spindle pole checkpoint deficiency is due to modifications and/or mutations in a gene/gene product selected from the group consisting of Mad1, Mad2, Mad1/2-complex, Bub1, BubRI, Bub3, Mpsl, CENP-E, CENP-F, ZW10, Rod, HsCdc20, HsCdhl, dynein/dynactin- complex, CLIP170, EB1 , APC, Polo-like Kinase 1 , Aurora-B, INCENP and/or survivin.

17. A pharmaceutical composition comprising at least one anti-HEC1 compound, at least one HEC1 -complex antagonist and/or at least one HEC1 -complex inhibitor as characterized in any one of claims 3 to 9.

18. A method for preventing, ameliorating and/or treating a hyperproliferative disorder/disease comprising the step of administering to a subject in need of such a prevention, amelioration and/or treatment an anti-HEC1 compound, HEC1 -complex antagonist and/or HEC1 -complex inhibitor as characterized in any one of claims 3 to 9.

19. A method for identifying an anti-HEC1 compound, an HEC1 -complex antagonist or an HEC1-complex inhibitor comprising the steps of

(a) exposing a spindle (assembly) checkpoint deficient cell to a candidate compound; and

(b) determining whether the exposition to said candidate compound causes a catastrophic mitotic exit.

20. A method for identifying a checkpoint antagonist comprising the steps of:

(a) exposing a cell arrested in prometaphase to a candidate compound; and

(b) determining whether the exposition to said candidate compound causes a catastrophic mitotic exit.

21. A method for the production of a pharmaceutical composition comprising the steps of the method of claim 19 or 20 and a further step, wherein the candidate compound identified as an anti-HEC1 compound, an HEClcomplex antagonist or HEC1 -complex inhibitor is mixed with a pharmaceutically acceptable carrier.