WO2008023841A1

WO2008023841A1 - Breast cancer-associated gene, melk, and its interactions with bcl-g

Info

Publication number: WO2008023841A1
Application number: PCT/JP2007/066826
Authority: WO
Inventors: Yusuke Nakamura; Toyomasa Katagiri; Shuichi Nakatsuru
Original assignee: Oncotherapy Science, Inc.
Priority date: 2006-08-25
Filing date: 2007-08-23
Publication date: 2008-02-28

Abstract

The present invention relates to the discovery that MELK, a human maternal embryonic leucine zipper kinase, is involved in cell growth of breast cancers through interaction with Bcl-G, a pro-apoptotic member of Bcl-2 family. In particular, the binding and phosphorylation of Bcl-G by MELK is described herein. As MELK has been shown to be overexpressed in breast, bladder and lung cancer, it appears that it may be a promising molecular target for the treatment and prevention of various types of cancer. Accordingly, objective screening methods for identifying therapeutic agents useful in the treatment of cancer, e.g., breast cancer, bladder cancer, and lung cancer, that use the interaction of MELK and Bcl-G as an index are described herein. The present invention also provides therapeutic agents or methods for treating cancer using the polypeptides. The polypeptides of the present invention are composed of an amino acid sequence which comprises polypeptide which comprises SEQ ID NO: 39. The polypeptides of the present invention can be introduced into cancer cells by modifying the polypeptides with transfection agents such as poly-arginine.

Description

DESCRIPTION

BREAST CANCER-ASSOCIATED GENE, MELK, AND ITS INTERACTIONS WITH BCL-G

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional Application No.

60/840, 120, filed August 25, 2006, the entire disclosure of which is hereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of cancer treatment and prevention, particularly, to methods and kits for identifying agents useful in the treatment and prevention of cancer, more particularly breast, bladder and lung cancer, as well as methods and compositions for treating and preventing same. More particularly, the present method relates to the discovery that MELK, a cancer specific gene up-regulated in breast, bladder, and lung cancer, interacts with and phosphorylates BcI-G, a pro-apoptotic member of the Bcl-2 family of proteins.

BACKGROUND OF THE INVENTION

Breast cancer is one of leading causes of cancer death in women worldwide. According to estimations in 2002, approximately 1,151,298 patients were newly diagnosed to have breast cancer, and 410,712 patients died of the disease (Veronesi U. et al., Lancet. 365: 1727-41 2005). Recent improvements in detection of breast cancer, through mammographic screening, for example, have contributed to a decrease in breast cancer- associated mortality, primarily due to diagnosis at an early stage. Mastectomy is the first concurrent option for the treatment of the localized breast cancer. However, despite surgical removal of the primary tumors, relapse at local or distant sites occurs in a subset of patients, most likely as a result of undetectable micrometastasis at the time of diagnosis (Bast RC et al. J Clin Oncol, 19: 1865-78, 2001.).

Cancer therapy directed at specific, frequently occurring molecular alterations in signaling pathways of cancer cells has been validated through the clinical development and regulatory approval of agents such as Tamoxifen, aromatase inhibitors and Trastuzumab (Herceptin) for treatment of advanced breast cancer (Navolanic PM et al. Int J Oncol 27: 1341-4 2005.). While Tamoxifen was developed to interfere with the estrogen receptor, aromatase inhibitors function by inhibiting estrogen production altogether. Conversely, as the first approved monoclonal antibody for blocking the human epidermal growth factor 2 (HER- 2, ErbB-2) signaling (Fendly BM et al. Cancer Res., 50: 1550-1558, 1990 ), Herceptin utilizes a different mechanism of action. Nevertheless, in order to achieve great responses and overall survival, a subject must express estrogen and HER-2 receptors. Accordingly, the effectiveness of these therapies is substantially limited. Moreover, concerns have recently been raised as to their side effects. For example, long term tamoxifen treatment may be associated wtih endometrial cancer. In addition, the deleterious effects of bone fracture in postmenopausal women has been noted in aromatase prescribed patients (Fendly BM et al. Cancer Res., 50: 1550-1558, 1990.). Thus, given the known limitations and negative side effects as well as the emergence of drug resistance, there is a clear need for novel targets for molecularly-orientated drugs on the basis of characterized mechanism of action.

BRIEF SUMMARY OF THE INVENTION To that end, the precise expression profiles of 81 breast tumors using a combination of laser-microbeam microdissection and a cDNA microarray consisting of 23,040 genes (Nishidate T et al. Int J Oncol 2004;25:797-819) were examined. Through those expression profiles of breast cancers and 29 normal human tissues (Saito-Hisaminato A, et al. DNA Res 9:35-45. 2002 6), the present inventors focused a gene termed maternal embryonic leucine zipper kinase (MELK) (GenBank Accession NO. NM 014791) (SEQ ID NO: 1) that was significantly overexpressed in the great majority of breast cancer cases examined. The present inventors further identified MELK (SEQ ID NO: 2) as a cancer-specific protein kinase, the down-regulation of which leads to growth suppression of breast cancer cells.

In the course of their research, the present inventors discovered that MELK physically interacted and phosphorylated BcI-G, a pro-apoptotic member of the Bcl-2 family (Lin ML et al. Breast Cancer Res. 2007;9(l):R17). Intriguingly, immune complex kinase assays showed that BcI-G was an ideal in vitro substrate for MELK kinase. Furthermore, the introduction of wild-type MELK was shown to rescue apoptosis induced by BcI-G, whereas kinase-dead of MELK could not. These findings are consistent with the conclusion that the inhibition of BcI-G by overexpression of MELK is likely to be involved in breast carcinogenesis through anti-apoptotic manners. Accordingly, MELK is a promising molecular target for treatment of breast cancer. Thus, the present invention provides novel methods for identifying therapeutic agents that prevent the onset and/or slow or arrest the progression of cancer, e.g., breast, bladder and/or lung cancer, by interfering with MELK/Bcl-G interaction or by inhibiting MELK-mediated phosphorylation of BcI-G. Accordingly, it is an objective of the present invention to provide methods of screening for agents useful in the treatment and/or prevention of cancer, particularly breast, bladder, and lung cancer. In one embodiment, the method includes the steps of:

(a) contacting a polypeptide having a Bcl-G-binding domain of a MELK polypeptide with a polypeptide having a MELK-binding domain of a BcI-G polypeptide in the presence of a test;

(b) detecting binding between the polypeptides; and

(c) selecting the test agent that inhibits binding between the polypeptides.

In one embodiment, the MELK binding domain of BcI-G comprises residues 12 - 294 of the amino acid sequence for BcI-G (as set forth in SEQ ID NO: 8). In one embodiment, the MELK binding domain of BcI-G comprises residues 12 - 72 of the amino acid sequence for BcI-G. In one embodiment, the MELK binding domain of BcI-G comprises residues 12 - 60 of the amino acid sequence for BcI-G. In one embodiment, the MELK binding domain of BcI-G comprises residues 12 - 48 of the amino acid sequence for BcI-G. In one embodiment, the MELK binding domain of BcI-G comprises residues 12 - 34 of the amino acid sequence for BcI-G. In one embodiment, the MELK binding domain of BcI-G comprises residues 21 - 36 of the amino acid sequence for BcI-G. In one embodiment, the MELK binding domain of BcI-G comprises residues 24-34 of the amino acid sequence for BcI-G.

The present invention also provides kits for screening for an agent useful in treating or preventing cancer. In one embodiment, such a kit includes: (a) a polypeptide having a Bcl-G-binding domain of a MELK polypeptide; (b) a polypeptide having a MELK-binding domain of a BcI-G polypeptide; and (c) means to detect the interaction between the polypeptides. In a preferred embodiment, the polypeptide having the Bcl-G-binding domain is a MELK polypeptide and the polypeptide having the MELK-binding domain is a BcI-G polypeptide.

In an alternative embodiment, the kit for screening for an agent useful in treating or preventing cancer, for example, breast, bladder and non-small cell lung cancer, may include (a) a polypeptide having a phosphorylation site of a BcI-G polypeptide; and (b) means to detect the phosphorylation of the polypeptide. In a preferred embodiment, the polypeptide having the phosphorylation site of a BcI-G is a BcI-G polypeptide. The kit may optionally further include a MELK polypeptide.

It is a further object of the present invention to provide a method of screening for an agent that induces apoptosis of cancer cells. In one embodiment, such a method may include the steps of:

(a) contacting a polypeptide selected from the group consisting of:

(i) a polypeptide having the amino acid sequence of SEQ ID NO: 2 (full- length MELK);

(ii) a polypeptide having the amino acid sequence of SEQ ID NO: 2 wherein one or more amino acids are added, substituted, deleted, or inserted, and that has a biological activity equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; (iii) a polypeptide having an amino acid sequence that has at least about

80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the full-length of amino acid sequence of SEQ ID NO: 2, wherein the polypeptide has a biological activity equivalent to a polypeptide consisting of the amino acid sequence of SEQ ED NO: 2; (iv) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 (full-length MELK), wherein the polypeptide has a biological activity equivalent to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; and (v) a polypeptide encoded by a polynucleotide that shares at least about

80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the full-length of the nucleotide sequence of SEQ ID NO: 1 (full-length MELK), wherein the polypeptide has a biological activity equivalent to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 with a substrate phosphorylated by the polypeptide and an agent under conditions that allow phosphorylation of the substrate;

(b) detecting the phosphorylation level of the substrate; (c) comparing the phosphorylation level of the substrate with the phosphorylation level of the substrate detected in the absence of the agent; and

(d) selecting the agent that reduces the phosphorylation level of the polypeptide as an agent that induces apoptosis of cancer cells.

In one embodiment, the substrate is BcI-G or a fragment thereof that includes, at a minimum, its phosphorylation site. The phosphorylation site is preferably included in a MELK-binding region of BcI-G. In one embodiment, the fragment includes residues 12 - 294 or 12 - 215 of the amino acid sequence for BcI-G set forth in SEQ ID NO: 8.

The present invention further provides a method of screening for an agent for preventing or treating cancer, an example of which includes the steps of:

(a) contacting a polypeptide selected from the group consisting of:

(ii) a polypeptide having the amino acid sequence of SEQ ID NO: 2 wherein one or more amino acids are added, substituted, deleted, or inserted, and that has a biological activity equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; (iii) a polypeptide having an amino acid sequence that has at least about 80% 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the full-length of the amino acid sequence of SEQ ID NO: 2, wherein the polypeptide has a biological activity equivalent to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; (iv) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 (full-length MELK), wherein the polypeptide has a biological activity equivalent to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; and (v) a polypeptide encoded by a polynucleotide that shares at least about

80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the full-length of the nucleotide sequence of SEQ ID NO: 1 (full-length

MELK), wherein the polypeptide has a biological activity equivalent to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 with a substrate phosphorylated by the polypeptide and an agent under conditions that allow phosphorylation of the substrate;

(b) detecting the phosphorylation level of the substrate;

(c) comparing the phosphorylation level of the substrate with the phosphorylation level of the substrate detected in the absence of the agent; and

It is a further object of the present invention to provide a composition for treating and/or preventing cancer, such as breast, bladder or lung cancer. In one embodiment, such a composition includes a pharmaceutically effective amount of a test agent selected by one of the screening methods set forth above in combination with a pharmaceutically acceptable carrier. In an alternate embodiment, the composition includes a pharmaceutically effective amount of an agent that inhibits the phosphorylation of a peptide consisting of the amino acid sequence of SEQ ED NO: 8 (full-length BcI-G) as an active ingredient in combination with a pharmaceutically acceptable carrier. In a particularly preferred embodiment, the agent inhibits the phosphorylation of a peptide consisting of the amino acid sequence of SEQ ID NO: 8 by MELK.

The present invention further provides a method for treating and/or preventing cancer, such as breast, bladder or lung cancer, that includes the step of inhibiting the phosphorylation of a peptide consisting of the amino acid sequence of SEQ ID NO: 8 by MELK, for example, by administrating an agent that inhibits the phosphorylation of the peptide by MELK.

In addition, the present invention provides for the use of an agent that inhibits the phosphorylation of a peptide consisting of the amino acid sequence of SEQ ID NO: 8 (full- length BcI-G) by MELK, for manufacturing a composition for treating and/or preventing cancer, for example, breast, bladder and non-small cell lung cancer.

The present invention also relates to methods for treatment and/or prevention of cancer comprising the step of administering an inhibitory polypeptide that contains FKILAYYTRHH (SEQ ID NO: 39), for example an inhibitory polypeptide having at least a fragment of the amino acid sequence TIEFKILA YYTRHHVF (SEQ ID NO: 37); or a polynucleotide encoding the same. Furthermore, the present invention relates to the use of polypeptides of the invention; or the use of nucleotides encoding the same, in manufacturing pharmaceutical formulations for the treatment and/or prevention of cancer.

It is to be understood that both the foregoing summary of the invention and the following detailed description are of preferred embodiments, and not restrictive of the invention or other alternate embodiments of the invention. Accordingly, other features, advantages and objects of the invention will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 (A-F) depicts the expression and distribution of MELK in human tissues and breast cancer cell lines. Figure IA depicts the expression of MELK in breast cancer specimens by semi-quantitative RT-PCR. N, MG, LUN, HEA, LIV and KID indicate microdissected normal breast ductal cells, mammary gland, lung, heart, liver and kidney, respectively. Figure IB depicts the results of northern blot analysis carried out using a hybridization probe near the 3' end of untranslated region of mRNA. P. B. L. indicate peripheral blood leukocytes. Figure 1C depicts the results of northern blot analysis of breast cancer cell-lines, particularly the revelation of an approximately 2.5 kb size variant uniquely expressed in cancer cell lines. Each lane was normalized by loading equal amount of poly A RNA. cDNA library screening using T47D breast cancer cell lines identified five transcripts using C-terminal fragments of variant 1 as a probe {see Materials and Methods). Figure ID depicts the results of cDNA library screening using T47D breast cancer cell-lines. Five variant transcripts were identified {see Materials and methods). White boxes indicate coding region, and black boxes indicate non-coding region. Figure IE depicts the results of northern blot analysis using a probe that located the common region (exon 12-14) among the Vl, V2 and V3, particularly the verification of cancer specific variants. As shown in Figure IF, each variant isolated from cDNA library screening was subjected to in vitro translation assay.

Vector expressed luciferase protein served as a positive control. The bracketed figure below the title of each construct represented the predicted molecular weight.

Figure 2 (A-B) depicts the knockdown effect of MELK by small-interfering RNA (siRNA) on cell viability and proliferation. Four psiHl promoter-based siRNA constructs were introduced into T47D {A) and MCF-7 {B) cell-lines. SC indicates scramble as a control for siRNA experiment. Gene silencing was evaluated by semi-quantitative RT-PCR at 7-day s after transfection. β2-microtubulin (β2MG) served as a control for normalization. MTT assays were performed to evaluate cell viability at 10 days and graphed after standardization by Mock to 1.0. Colony formation assays were carried out three weeks after selection (see the Materials and Methods). Two siRNA constructs (si-#3 and -#4) showed significantly knockdown effects against internal MELK expression and inhibited cell growth in both of T47D (A) and MCF-7 (B). Values represent the average from triplicate experiments. Error bars indicate standard deviation.

Figure 3 (A-B) depicts the generation of active MELK recombinant protein (WT and D 150A). In Figure 3A, an aliquot (10 μl) of wild-type (WT) and kinase-dead (D 150A) recombinant protein of MELK were electrophoresed on 10 % SDS-PAGE, and visualized by Coomassie blue staining. In Figure 3B, the kinase activity of recombinant proteins was examined by in vitro kinase assay using histone Hl (34 kDa) as a general substrate. No substrate shows no addition of substrates in this reaction mixture.

Figure 4 (A-E) depicts the interaction of MELK with BcI-G. Long isoform in vivo. Figure 4A depicts the expression of BcI-G protein in eight breast cancer cell-lines as well as human mammary epithelial cells (HMEC). β-actin was served as a control for normalization. In Figure 4B, extracts from HeLa-cells transfected with pCAGGS-MELK-HA, pCAGGS- Flag-Bcl-G, or a combination, were harvested 36 hours after transfection. The cell lysates were immunoprecipitated with anti-Flag M2 antibody. Precipitated proteins were separated by SDS-PAGE and western blotting analysis was performed with anti-HA antibody. Figure 4C depicts the co-localization of MELK and BcI-G in mammalian cells. Exogenous MELK and BcI-G were co-transfected into COS7 cells at 48 hours before immunocytochemical staining experiments. Nucleus was stained with DAPI (blue); HA-tagged MELK was labeled with Alexa-488 (green); Flag-tagged BcI-G was labeled with Alexa-594 (red); Co-localization of both proteins manifested as yellow. Figure 4D is a schematic representation of BcI-G N- terminal and C-terminal deletion mutants. C-4 construct is deleted of BH3 domain. Prefix N and C indicate N-terminal and C-terminal truncation, respectively. Figure 4E depicts the results of immunoprecipitation assays used to determine the regions of BcI-G that bind with wild-type MELK (WT-MELK). The HA-tagged WT-MELK and a set of Flag-tagged BcI-G were pulled down by immunoprecipitation with Flag M2 antibody and immunoblotted with Flag-rabbit or HA-rat antibodies, respectively. The expression of HA-tagged WT-MELK in total cell lysates was confirmed by Western blotting analysis. As a control, mock immunoprecipitation was performed from cells co-transfected with pCAGGSn3FC (Mock) and HA-tagged WT-MELK through all steps.

The results shown in Figure 5 (A-G) demonstrate that MELK phosphorylates BcI- G in vitro. In Figure SA, the expression of exogenous Flag-tagged BcI-G was immunoprecipitated from HeLa cells with Flag-M2 antibody and confirmed by immunoblot analysis using anti-HA antibody. In Figure SB, immunoprecipitates were subjected to immune complex kinase assay with wild-type (WT) or kinase-dead (D 150A) MELK recombinant proteins. The single arrowhead indicates phosphorylated BcI-G, and the double arrowhead reveals autophosphorylated MELK protein. W and D indicate wild-type-MELK and kinase-dead MELK, respectively. Figure SC depicts the phosphorylation of bacterial recombinant BcI-G protein by recombinant MELK protein. Figure SD depicts the expression of a set of N-terminal truncated constructs of BcI-G by immunoblotting analysis. Each truncated construct was transfected into HeLa cells. After immunoprecipitation with mouse Flag-antibody, immunoblotting with rabbit Flag-antibody was performed. Figure SE depicts the in vitro phosphorylation of N-terminal truncated constructs of BcI-G (N-I, N-3, N-4 and N-5; see Figure 4D by MELK kinase. Figure SF depicts the expression of C-terminal truncated constructs of BcI-G by immunoblot after immunoprecipitation. Figure SG depicts the in vitro phosphorylation of BcI-G and its C-terminal truncated constructs by MELK. W and D represents wild-type and kinase-dead of MELK, respectively, and a 75 kDa band indicates autophosphorylation of MELK protein. The arrowheads indicate each C-terminal construct.

The results depicted in Figure 6 (A-C) demonstrate that MELK is involved in the apoptosis cascade through BcI-G in mammalian cells. MELK and BcI-G expression vectors were co-transfected into COS7 cells for 24 hours. Figure 6A depicts the results of western blot analysis examining the expression of MELK and a set of truncated constructs of BcI-G proteins. Figure 6B depicts the results of FACS analysis of cells collected after transfection with MELK (WT and D 150A) and BcI-G expression vectors, respectively. Proportions of apoptotic cells are indicated as percentages of sub-Gl populations. Figure 6C depicts the results of FACS analysis of cells collected after transfection with N-terminal truncated (N-I, N-3 and N-4) and C-terminal truncated constructs (C-I, C-2 and C-4) of BcI-G, respectively. Proportions of apoptotic cells are indicated as percentages of sub-Gl populations. The results shown in Figure 7 demonstrate the growth curve of T47D breast cancer cells treated with the 6.25 μM BcI-G peptide (RRRRRRRRRRRGGGTIEFKILAYYTRHHVF: 21-36), SCl (RRRRRRRRRRRGGGEHITAFTRKLIHVFYY), SC2 (RRRRRRRRRRR GGGIYTVARFTHIHFKLYE) or nothing.

DETAILED DESCRIPTION OF THE INVENTION I. Overview:

Medical applications of microarray technologies include (i) discovery of genes that contribute to carcinogenesis, (ii) discovery of novel molecular target(s) for anti-cancer agents and useful diagnostic biomarker(s) and (iii) identification of genes involved in conferring chemosensitivity. Clinical applications have begun to emerge from such experiments; for example, novel drugs that target molecules associated with development of certain types of cancers have proven to be very effective. Although various cytotoxic agents and hormone- targeting drug such as Herceptin or tamoxifen in breast cancer have been developed, effects of these therapies to the advanced- stage patients are very limited (Fendly BM et al. Cancer Res. 50: 1550-8, 1990.). Hence, it is urgent to develop new anti-cancer drugs that will be specific to malignant cells to avoid or minimize adverse effects.

Through the genome-wide expression profiles of breast cancer (Nishidate T et al. Int J Oncol 25:797-819 2004.) and normal human tissues (Knebel A, et al. EMBO J 2001;20:4360-9.), the present inventors identified maternal embryonic leucine zipper kinase, MELK, one of whose transcripts was specifically up-regulated in a great majority of clinical breast cancer patients and not expressed in 29 normal human tissues examined except testis and thymus.

MELK was previously identified as a new member of the snfl/AMPK serine- threonine kinase family that involved in mammalian embryonic development (Heyer BS et al. Dev Dyn 215:344-51 1999; Blot J et al. Dev Biol 241 :327-38 2002.). It has been reported that MELK interacts with several substrates for various biological functions. It played a role in the hematopoiesis (Saito R et al. MoI Cell Biol 25:6682-93 2005,; Gil M et al. Immunol Lett 64:79-83. 1998.) and was thought to participate in cell cycle regulation as interacting with nuclear inhibitor of Ser/Thr phosphatase- 1 (NIPPl) to end the pre-mRNA splicing just before mitosis through the phosphorylation of threonine 478 residue (Vulsteke V et al. J Biol Chem 279:8642-7 2004 ). It phosphorylated cdc25B, a phosphatase that is required for entering mitosis phase (Davezac N βt al. Oncogene 21 :7630-41 2002 ). Interestingly, its expression was also detected in the self-renewal of multipotent neural progenitors as well as proliferation of undifferentiated cells (Nakano I et al. J Cell Biol 170:413-27 2005 ). Despite the discoveries of interacting proteins, the significance of phosphorylation of these substrates still remained unclear.

The present invention involves characterization of MELK gene as a candidate of molecular-targets for breast cancer therapy. To that end, the present inventors first demonstrated, by means of the siRNA expression vector system that knocked down the expression of endogenous MELK, that suppression of MELK expression resulted in growth suppression of breast cancer cell-lines (Figure 2). Intriguingly, the established NIH3T3- derivative cells that stably expressed wild-type-MELK (WT-MELK) showed that overexpression of WT-MELK had no significant enhancement of cell growth as well as overexpression of exogenous D 150A kinase-dead mutant (data not shown). These findings are consistent with the conclusion that the lack of MELK gene product has a critical effect on the survival of breast cancer cells although overexpression of this gene alone does not have growth enhancing activity. Particularly, NIH3T3 cells appears to have no specific substrates for MELK kinase activity. It has been previously demonstrated that MELK is also commonly up-regulated in bladder cancers, osteosarcoma and non-small cell lung cancers as well as breast cancers through cDNA microarray data (See, for example, WO2004/31413,

PCT/JP2006/302684, and PCT/JP2005/014369, the contents of which are incorporated by reference herein in their entirety). Together, these findings are consistent with the conclusion that MELK has an oncogenic role in not only breast cancers but also bladder cancers and lung cancers, particularly non-small cell lung cancer.

To assess biological function of MELK in cancer cells, the present inventors screened substrates for MELK kinase by means of pull-down with wild-type and "kinase- dead" -MELK recombinant proteins and proteomics analyses. Herein, "kinase-dead" refers to inactivation of kinase activity. Especially, "kinase-dead mutant MELK" is D 150A mutant of MELK; asparate changed to alanine at the 150 residue within the ATP binding site of kinase domin (11-263). As a result, a long isoform of BcI-G, a pro-apoptotic member of Bcl-2 family, was identified as a potential substrate for MELK kinase, and demonstrated by a co- immunoprecipitation assay that the N-terminal end of BcI-G (24-34 residues) bound to MELK (Figure 4, 5, 7). Immunocytochemical staining revealed that both proteins were diffusely co- localized in the cytoplasm of COS7 cells, supporting that MELK physically bound to BcI-G in vivo. Furthermore, in vitro immunocomplex kinase assays showed that BcI-G protein was specifically phosphorylated by WT-MELK, but not phosphorylated by D 150A kinase-dead- MELK. In addition, in vitro kinase assays also revealed that bacterial recombinant MELK protein phosphorylated recombinant BcI-G protein. Together, MELK can directly phosphorylate BcI-G in vitro.

In confirmation with the report by Guo B et al. (J Biol Chem 276:2780-5 2001.), the present inventors also demonstrated by FACS analysis that overexpression of full-length BcI-G protein reproducibly induced increases apoptosis (Figure 6). On the other hand, the overexpression of MELK-WT inhibited apoptosis induced by BcI-G protein, but MELK- D 150A did not inhibit it (Figure 6B), indicating anti-apoptotic function of MELK. Furthermore, as shown in Figure 6C, MELK was discovered to clearly suppress apoptosis through phosphorylation of the N-terminal region of BcI-G. These findings are consistent with the conclusion that MELK can promote cell growth through inhibition of the pro- apoptotic function of BcI-G.

In a previous report, the BcI-G gene was described as encoding two isoforms, a long isoform and a short isoform, that were produced through alternative mRNA splicing, consisting of 327 and 252 amino acids, respectively (Guo B et al. J Biol Chem 276:2780-5 2001 ). Both isoforms of BcI-G revealed the presence of BH3 domain, similar to several other pro-apoptotic Bcl2 family proteins (Guo B et al. J Biol Chem 276:2780-5 2001; Reed JC. Oncogene 17:3225-36 1998, Xu Q et al. MoI Cell 1:337-46 1998 ). However, only the short isoform was reported to be co-immunoprecipitated with BCI-X_L via BH3 domain (Guo B et al. J Biol Chem 276:2780-5 2001 ). This isoform is capable of triggering apoptosis via sequestering of survival protein BCI-X_L and subsequent activation of BAK protein (Guo B et al. J Biol Chem 276:2780-5 2001, Willis SN et al. Genes Dev 19: 1294-305 2005.). Interestingly, as shown in Figure 6, overexpression of BH3 domain-deleted peptide showed no induction of apoptosis, in spite of this peptide possess a binding region of BcI-G with MELK, indicating BH3 domain is essential for apoptosis through BcI-G function. Furthermore, Guo et al. reported that BcI-G long isoform contains an extra BH2 domain at the C-terminus end (308-315 residues; see Figure 4D), which was proposed to suppress its apoptotic capability by interfering binding to BcI-XL (Guo B, et al. J Biol Chem 276:2780-5 2001 ). However, the present inventors observed significant differences in sub-Gl cell population between FL-BcI-G and C-terminal truncated constructs (C-4 and C-5) in this studies (Figure 6C). The results herein are consistent with the conclusion that the activity of the pro-apoptotic BcI-G long isoform is regulated by interaction with other molecules or post- translational modification of BcI-G through the MELK kinase. For instance, the Bad protein, another member of the Bcl-2 family, is inactivated by phosphorylation though several kinases including PKA, Akt and Rafl, which thereby prevent the dimerization of Bcl-2 and BCI-X_L (Reed JC. Oncogene 17:3225-36 1998 ). The modification and regulation of these Bcl-2 families, thus, may set up a model for future analysis to unravel the anti-apoptotic mechanism of MELK. Taken together, these findings are consistent with the conclusion that MELK can promote cell growth by inhibiting the pro-apoptotic function of BcI-G.

As further demonstrated herein, both proteins were detected in breast cancer clinical samples and breast cancer cells through our expression profiles of cDNA microarray (data not shown), and by western blot analysis using anti-Bcl-G short (data not shown) and long isoform specific antibody (Figure 4A). Since the N-terminal region of both isoforms of BcI-G is identical according to their amino-acids sequence, it was discovered that MELK also interacted with and phosphorylated the short isoform of BcI-G in vitro (data not shown). The instant findings are consistent with the conclusion that MELK may also suppress pro- apoptotic function of the short isoform of BcI-G through their interaction or phosphorylation of it. The ready phosphorylation of BcI-G by MELK in vitro provides elucidation to the activity of many cell-killing BH3 containing proteins as being regulated by phosphorylation (Puthalakath H et al. Cell Death Differ 9:505-12 2002 ). For example, phosphorylation of BAD at serine 112 or serine 136 disassociated Bad from BcI-XL, allowed its sequestration in the cytoplasm by binding to the 14-3-3 chaperon protein and protected cells from apoptosis (Zha J et al. Cell 87:619-28 1996, Muslin AJ et al. Cell 84:889-97 1996, Yaffe MB et al. Cell 91 :961-71 1997 ).

In conclusion, the findings of the present invention are consistent with the conclusion that MELK is overexpressed in both breast cancer specimens and cancer cell-lines, and its kinase activity plays a significant role in breast cancer cell growth. Recent anti-cancer drug development has focused on targeting important molecules involved in the oncogenic pathways, for example, imatinib mesylate and trastuzumab. Herein, it was discovered that the down-regulation of MELK by treatment with siRNA significantly suppressed the cell growth of breast cancer, indicating its crucial role in proliferation and tumorigenesis of breast cancer. In particular, the present inventors demonstrated a new biological function for MELK in breast carcinogenesis by impairing inhibition of apoptosis though interaction and phosphorylation of BcI-G, pro-apoptotic member of Bcl-2 family. Thus, the data provided herein contribute to a better understanding of carcinogenesis, and demonstrate that MELK is a promising molecular target for cancer treatment.

II. Definitions:

The words "a", "an" and "the" as used herein mean "at least one" unless otherwise specifically indicated.

In the context of the present invention, "MELK" refers to the Maternal Embryonic

Leucine zipper Kinase, identified elsewhere as "A2282". MELK is a member of the Snfl/AMPK related kinase family that has been implicated in stem cell renewal, cell cycle progression and pre-m-RNA splicing. MELK is also a marker for self-renewing multipotent neural progenitors, and may function in embryonic and postnatal forebrain development. Recent studies on human tumor samples and cell lines are consistent with the conclusion that MELK expression is frequently elevated in cancer relative to normal tissues. MELK may provide a growth advantage for neoplastic cells, and may be a potential target for anti-cancer therapies. Nucleotide and amino acid sequences for human MELK are set forth in SEQ ID NOs: 1 and 2, respectively.

Herein, "BcI-G" refers to a recently identified pro-apoptotic member of the Bcl-2 family. Nucleotide and amino acid sequences for human BcI-G are set forth in SEQ ED NOs: 7 and 8, respectively. The human BCL-G gene (SEQ ID NO: 7) consists of 6 exons, resides on chromosome 12pl2, and encodes two proteins through alternative mRNA splicing, BcI- G(L) (long) and BcI-G(S) (short) consisting of 327 and 252 amino acids in length, respectively. BcI-G(L) and BcI-G(S) have identical sequences for the first 226 amino acids but diverge thereafter. BcI-G(L) mRNA is expressed widely in adult human tissues, whereas BcI-G(S) mRNA is primarily limited to testis. Overexpression of BcI-G(L) or BcI-G(S) in cells has been demonstrated to induce apoptosis, although BcI-G(S) appears to be more potent than BcI-G(L).

In the context of the present invention, the phrase "inhibition of binding" of two proteins refers at least to a reduction in the binding between the proteins. Thus, in some cases, the percentage of binding pairs in a sample will be decreased compared to an appropriate (e.g., not treated with test compound or from a non-cancer sample, or from a cancer sample) control. The reduction in the amount of proteins bound may be, e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 25%, 10%, 5%, 1% or less (e.g., 0%), than the pairs bound in a control sample.

The terms "isolated" and "biologically pure" refer to material that is substantially or essentially free from components which normally accompany it as found in its native state. However, the term "isolated" is not intended to refer to the components present in an electrophoretic gel or other separation medium. An isolated component is free from such separation media and in a form ready for use in another application or already in use in the new application/milieu.

The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, such as an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that similarly functions to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, γ- carboxyglutamate, and O-phosphoserine). The phrase "amino acid analog" refers to compounds that have the same basic chemical structure (an α carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium). The phrase "amino acid mimetic" refers to chemical compounds that have different structures but similar functions to general amino acids.

Amino acids may be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

The terms "polynucleotides", "nucleotides", "nucleic acids", and "nucleic acid molecules" are used interchangeably unless otherwise specifically indicated and are similarly to the amino acids referred to by their commonly accepted single-letter codes. Similar to the amino acids, they encompass both naturally-occurring and non-naturally occurring nucleic acid polymers.

As noted above, the nucleotide and amino acid sequences for human MELK are shown in SEQ ID NOs: 1 and 2, respectively, and are available through GenBank under Accession No. NM 014791. Similarly, the nucleotide and amino acid sequences for human BcI-G are shown in SEQ ID NOs: 7 and 8, respectively.

The present invention contemplates the use not only of wild-type proteins but also of functional equivalents thereof. In the context of the present invention, the term "functional equivalent" means that the subject polypeptide retains the relevant biological activity of a reference polypeptide. For example, a functional equivalent of BcI-G would have the ability to bind to MELK and/or promote apoptosis. Similarly, a functional equivalent of MELK would have the ability to interact with and/or phosphorylate a substrate, such as BcI-G. Assays for determining such activities are well known in the art.

It is generally known that modifications of one or more amino acids in a protein do not influence the function of the protein (Mark DF et al, Proc Natl Acad Sci USA 1984, 81 : 5662-6; Zoller MJ & Smith M, Nucleic Acids Res 1982, 10: 6487-500; Wang A et al, Science 1984, 224: 1431-3; Dalbadie-McFarland G et al, Proc Natl Acad Sci USA 1982, 79: 6409-13). Thus, one of skill in the art will recognize that individual additions, deletions, insertions, or substitutions to an amino acid sequence which alters a single amino acid or a small percentage of amino acids constitute "conservative modifications" that yield a protein that is similar in structure and function to the base protein of interest. Those modifications that result in the retention of the properties of particular amino acid side chain are particularly preferred. Examples of properties of amino acid side chains include: hydrophobic amino acids (alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, valine), hydrophilic amino acids (arginine, aspartic acid, aspargine, cysteine, glutamic acid, glutamine, glycine, histitidine, lysine, serine, threonine), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (glycine, alanine, valine, leucine, isoleucine, praline); a hydroxyl group containing side-chain (serine, threonine, tyrosine); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (aspartic acid, aspargine, glutamic acid, glutamine); a base containing side-chain (arginine, lysine, histidine); and an aromatic containing side-chain (histidine, phenylalanine, tyrosine, tryptophan). Furthermore, conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following eight groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E);

3) Aspargine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins 1984).

Such conservatively modified polypeptides are included in the present invention. However, the present invention also contemplates non-conservative modifications so long as they retain the relevant biological activity of the base protein (i.e., give rise to a "functional equivalent" of the base protein). The number of amino acids to be mutated in a modified protein may vary but preferably constitutes no more than 5% of the total residues. Accordingly, the number of amino acids to be mutated is generally 20-30 amino acids or less, preferably 10 amino acids or less, more preferably 5-6 amino acids or less, even more preferably 2-3 amino acids or less.

An example of a protein modified by addition of one or more amino acids residues is a fusion protein. Fusion proteins are fusions of the base protein of interest (e.g., MELK or BcI-G) and other peptides or proteins, which also can be used in the present invention. Fusion proteins can be made by techniques well known to a person skilled in the art, such as by linking the DNA encoding the base protein with a DNA encoding other peptides or proteins, so that the frames match, inserting the fusion DNA into an expression vector and expressing it in a host. There is no restriction as to the peptides or proteins fused to the base protein so long as the resulting fusion protein retains the requisite biological activity of the base protein.

Known peptides that can be used as peptides to be fused include, for example, FLAG (Hopp TP et al, Biotechnology 1988 6: 1204-10), 6xF£is containing six His (histidine) residues, lOxHis, Influenza agglutinin (HA), human c-myc fragment, VSP-GP fragment, pi 8HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, Protein C fragment, and the like. Examples of proteins that may be fused to a protein of the invention include GST (glutathione-S-transferase), Influenza agglutinin (HA), immunoglobulin constant region, β-galactosidase, MBP (maltose-binding protein), and such.

Fusion proteins can be prepared by fusing commercially available DNA, encoding the fusion peptides or proteins discussed above, with the DNA encoding the MELK or BcI-G protein and expressing the fused DNA prepared.

Additional examples of modified proteins include polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.

An alternative method known in the art to isolate functionally equivalent proteins utilizes, for example, the hybridization technique (Sambrook J et ah, Molecular Cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. Press, 1989). For example, one skilled in the art can readily isolate a DNA having high homology with a whole or part of the human MELK DNA sequence {e.g., SEQ ID NO: 1) encoding the human MELK protein, and isolate functional equivalent proteins to the human MELK protein from the isolated DNA. Thus, the proteins used for the present invention include those that are encoded by DNA that hybridize under stringent conditions with a whole or part of the DNA sequence encoding the human MELK protein and are functional equivalent to the human MELK protein. These proteins include mammal homologues corresponding to the protein derived from human or mouse (for example, a protein encoded by a monkey, rat, rabbit and bovine gene). In isolating a cDNA highly homologous to the DNA encoding the human MELK protein from animals, it is particularly preferable to use tissues from testis, or breast cancer.

The condition of hybridization for isolating a DNA encoding a protein functional equivalent to protein of interest can be routinely selected by a person skilled in the art. The phrase "stringent (hybridization) conditions" refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not detectably to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).

Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength pH. The T_m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times of background, preferably 10 times of background hybridization.

For example, hybridization may be performed by conducting prehybridization at 68⁰C for 30 min or longer using "Rapid-hyb buffer" (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68⁰C for 1 h or longer. The following washing step can be conducted, for example, in a low stringent condition. A low stringent condition is, for example, 42⁰C, 2x SSC, 0.1% SDS, or preferably 5O⁰C, 2x SSC, 0.1% SDS. More preferably, high stringent condition is used. A high stringent condition is, for example, washing 3 times in 2x SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in Ix SSC, 0.1% SDS at 37⁰C for 20 min, and washing twice in Ix SSC, 0.1% SDS at 5O⁰C for 20 min. However, several factors such as temperature and salt concentration can influence the stringency of hybridization and one skilled in the art can suitably select the factors to achieve the requisite stringency.

In place of hybridization, a gene amplification method, for example, the polymerase chain reaction (PCR) method, can be utilized to isolate a DNA encoding a protein that is functionally equivalent to the human MELK or BcI-G protein, using a primer synthesized based on the sequence information of the DNA (e.g., SEQ ID NOs. 1 to 7) encoding the protein of interest (e.g., SEQ ID NOs: 2 or 8).

Functionally equivalent proteins isolated through the above hybridization techniques or gene amplification techniques, normally have a high homology (also referred to as sequence identity) to the amino acid sequence of the base protein of interest. "High homology" (also referred to as "high identity") typically refers to the degree of identity between two optimally aligned sequences (either polypeptide or polynucleotide sequences). Typically, high homology or identity refers to homology of 40% or higher, for example, 60% or 80% or higher, for example, 85%, 90%, 95%, 98%, 99%, or higher. The degree of homology or identity between two polypeptide or polynucleotide sequences can be determined by following the algorithm in "Wilbur, WJ & Lipman DJ, Proc Natl Acad Sci USA 1983, 80: 726-30".

A protein useful in the context of the present invention may have variations in amino acid sequence, molecular weight, isoelectric point, the presence or absence of sugar chains, or form, depending on the cell or host used to produce it or the purification method utilized. Nevertheless, so long as it has the requisite biological activity of the base protein of interest (e.g., the phosphorylating activity of MELK; the pro-apoptotic activity of BcI-G) it is useful in the present invention.

The present invention also encompasses the use of partial peptides. A partial peptide has an amino acid sequence specific to the protein interest and consists of less than about 400 amino acids, usually less than about 200 and often less than about 100 amino acids, and at least about 7 amino acids, preferably about 8 amino acids or more, and more preferably about 9 amino acids or more. A partial peptide useful in the context of the screening methods of the present invention suitably contains at least the phosphorylation site of the protein of interest. Thus, an example of a partial peptide of MELK useful in the context of the screening assays of the present invention is a polypeptide that includes at least the BcI-G binding domain of MELK. Likewise, an exemplary partial peptide of BcI-G useful in the context of the screening assays of the present invention is a polypeptide that includes at least the MELK binding domain of BcI-G. Such partial peptides are fall under the heading of "functional equivalents".

Accordingly, in the context of the present invention, the phrase "MELK gene" encompasses polynucleotides that encode the MELK protein or any of the functional equivalents of the MELK protein. Likewise, the phrase "BcI-G gene" encompasses polynucleotides that encode the BcI-G protein or any of the functional equivalents of the BcI- G protein

In the context of the present invention, a "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (e.g., a polypeptide of the invention), which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

The terms "identical" or percent "identity", in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides or amino acids in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides or amino acids in length, or over the full-length of the sequence.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) Adv. Appl Math. 2:482-489, by the homology alignment algorithm of Needleman and Wunsch (1970) J. MoI. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat 7. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection {see, e.g., Ausubel et al, Current Protocols in Molecular Biology (1995 supplement)).

Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (199O) J. MoI Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix {see Henikoff and Henikoff (1989) Proc. Natl Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences {see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-7). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

In the context of the present invention, agents to be identified through the present screening methods may be any compound or composition including several compounds that disrupt the interaction between MELK and BcI-G discussed in detail herein. Furthermore, the test agent exposed to a cell or protein according to the screening methods of the present invention may be a single compound or a combination of compounds. When a combination of compounds is used in the methods, the compounds may be contacted sequentially or simultaneously.

Any test agent, for example, cell extracts, cell culture supernatant, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micromolecular compounds (including nucleic acid constructs, such as antisense RNA, siRNA, ribozymes, etc.) and natural compounds can be used in the screening methods of the present invention. The test agent of the present invention can be also obtained using any of the numerous approaches in combinatorial library methods known in the art, including (1) biological libraries, (2) spatially addressable parallel solid phase or solution phase libraries, (3) synthetic library methods requiring deconvolution, (4) the "one-bead one-compound" library method and (5) synthetic library methods using affinity chromatography selection. The biological library methods using affinity chromatography selection is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des 1997, 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al, Proc Natl Acad Sci

USA 1993, 90: 6909-13; Erb et al, Proc Natl Acad Sci USA 1994, 91 : 11422-6; Zuckermann et al, J Med Chem 37: 2678-85, 1994; Cho et al, Science 1993, 261 : 1303-5; Carell et al, Angew Chem Int Ed Engl 1994, 33: 2059; Carell et al, Angew Chem Int Ed Engl 1994, 33: 2061; Gallop et al, J Med Chem 1994, 37: 1233-51). Libraries of compounds may be presented in solution (see Houghten, Bio/Techniques 1992, 13: 412-21) or on beads (Lam, Nature 1991, 354: 82-4), chips (Fodor, Nature 1993, 364: 555-6), bacteria (US Pat. No. 5,223,409), spores (US Pat. No. 5,571,698; 5,403,484, and 5,223,409), plasmids (Cull et al, Proc Natl Acad Sci USA 1992, 89: 1865-9) or phage (Scott and Smith, Science 1990, 249: 386-90; Devlin, Science 1990, 249: 404-6; Cwirla et al, Proc Natl Acad Sci USA 1990, 87: 6378-82; Felici, J MoI Biol 1991, 222: 301-10; US Pat. Application 2002103360).

A compound in which a part of the structure of the compound screened by any of the present screening methods is converted by addition, deletion and/or replacement, is included in the agents obtained by the screening methods of the present invention. Such compounds preferably have a molecular weight of less than 1,500 daltons, and in some cases less than 1,000, 800, 600, 500, or 400 daltons.

Furthermore, when the screened test agent is a protein, for obtaining a DNA encoding the protein, either the whole amino acid sequence of the protein may be determined to deduce the nucleic acid sequence coding for the protein, or partial amino acid sequence of the obtained protein may be analyzed to prepare an oligo DNA as a probe based on the sequence, and screen cDNA libraries with the probe to obtain a DNA encoding the protein. The obtained DNA finds use in preparing the test agent which is a candidate for treating or preventing cancer.

Test agents useful in the screening described herein can be antisense oligonucleotides that inhibit the expression of the MELK or BcI-G proteins. The term "antisense oligonucleotides" as used herein means, not only those in which the nucleotides corresponding to those constituting a specified region of a DNA or mRNA are entirely complementary, but also those having a mismatch of one or more nucleotides, as long as the DNA or mRNA and the antisense oligonucleotide can specifically hybridize with the nucleotide sequence of interest {e.g., SEQ ID NO: 1).

Such polynucleotides are contained as those having, in the "at least 15 continuous nucleotide sequence region", a homology of at least 70% or higher, preferably at 80% or higher, more preferably 90% or higher, even more preferably 95% or higher. The algorithm stated herein can be used to determine the homology. Such polynucleotides are useful as probes for the isolation or detection of DNA encoding the polypeptide of the invention as stated in a later example or as a primer used for amplifications.

Test agents useful in the screening described herein can also be antibodies or non- antibody binding proteins that specifically bind to the MELK or BcI-G proteins or functional equivalents thereof. For example, antibodies {e.g., monoclonal antibodies) can be tested for their ability to block phosphorylation of the MELK protein or binding of the protein with its BcI-G substrate.

The term "antibody" as used herein encompasses naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof, (e.g., Fab', F(ab')₂, Fab, Fv and rlgG). See also, Pierce Catalog and Handbook, 1994- 1995 (Pierce Chemical Co., Rockford, IL). See also, e.g., Kuby, J., Immunology, 3^rd Ed., W H. Freeman & Co., New York (1998). Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al, Science 246: 1275-81 (1989), which is incorporated herein by reference. These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14:243-6 (1993); Ward et al., Nature 341:544-6 (1989); Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor

Laboratory publications, New York, 1988; Hilyard et al, Protein Engineering: A practical approach (IRL Press 1992); Borrebaeck, Antibody Engineering, 2d ed. (Oxford University Press 1995); each of which is incorporated herein by reference).

The term "antibody" includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). The term also refers to recombinant single chain Fv fragments (scFv). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described in, e.g., Kostelny et al. (1992) J Immunol 148: 1547, Pack and Pluckthun (1992) Biochemistry 31: 1579, Holliger e/ α/. (1993) Proc Natl Acad Sci U S A. 90:6444, Gruber et al. (1994) J Immunol :5368, Zhu et al. (1997) Protein Sci 6:781, Hu et al. (1997) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.

Typically, an antibody has a heavy and light chain. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains"). Light and heavy chain variable regions contain four "framework" regions interrupted by three hyper-variable regions, also called "complementarity-determining regions" or "CDRs". The extent of the framework regions and CDRs have been defined. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three dimensional spaces.

The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDRl, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a V_H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a V_L CDRl is the CDRl from the variable domain of the light chain of the antibody in which it is found.

References to "V_H" refer to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv , or Fab. References to "V_L" refer to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.

The phrase "single chain Fv" or "scFv" refers to an antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain. Typically, a linker peptide is inserted between the two chains to allow for proper folding and creation of an active binding site.

A "chimeric antibody" is an immunoglobulin molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

A "humanized antibody" is an immunoglobulin molecule that contains minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al, Nature 321 : 522- 5 (1986); Riechmann et al, Nature 332:323-7 (1988); and Presta, Curr. Op. Struct. Biol

2:593-6 (1992)). Humanization can be essentially performed following the method of Winter and co-workers (Jones et al, Nature 321 :522-5 (1986); Riechmann et al, Nature 332:323-7 (1988); Verhoeyen et al, Science 239:1534-6 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (US Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.

The terms "epitope", "antigenic" and "determinant" refer to a site on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, X-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).

The terms "non-antibody binding protein" or "non-antibody ligand" or "antigen binding protein" interchangeably refer to antibody mimics that use non-immunoglobulin protein scaffolds, including adnectins, avimers, single chain polypeptide binding molecules, and antibody-like binding peptidomimetics, as discussed in more detail below. Other compounds have been developed that target and bind to targets in a manner similar to antibodies. Certain of these "antibody mimics" use non-immunoglobulin protein scaffolds as alternative protein frameworks for the variable regions of antibodies.

For example, Ladner et al. (U.S. Patent No. 5,260,203) describe single polypeptide chain binding molecules with binding specificity similar to that of the aggregated, but molecularly separate, light and heavy chain variable region of antibodies. The single-chain binding molecule contains the antigen binding sites of both the heavy and light variable regions of an antibody connected by a peptide linker and will fold into a structure similar to that of the two peptide antibody. The single-chain binding molecule displays several advantages over conventional antibodies, including, smaller size, greater stability and are more easily modified.

Ku et al. (Proc. Natl. Acad. ScL U.S.A. 92(14):6552-6556 (1995)) discloses an alternative to antibodies based on cytochrome b562. Ku et al. (1995) generated a library in which two of the loops of cytochrome b562 were randomized and selected for binding against bovine serum albumin. The individual mutants were found to bind selectively with BSA similarly with anti-BSA antibodies.

Lipovsek et al. (U.S. Patent Nos. 6,818,418 and 7,1 15,396) discloses an antibody mimic featuring a fibronectin or fibronectin-like protein scaffold and at least one variable loop. Known as Adnectins, these fibronectin-based antibody mimics exhibit many of the same characteristics of natural or engineered antibodies, including high affinity and specificity for any targeted ligand. Any technique for evolving new or improved binding proteins can be used with these antibody mimics.

The structure of these fibronectin-based antibody mimics is similar to the structure of the variable region of the IgG heavy chain. Therefore, these mimics display antigen binding properties similar in nature and affinity to those of native antibodies. Further, these fibronectin-based antibody mimics exhibit certain benefits over antibodies and antibody fragments. For example, these antibody mimics do not rely on disulfide bonds for native fold stability, and are, therefore, stable under conditions which would normally break down antibodies. In addition, since the structure of these fibronectin-based antibody mimics is similar to that of the IgG heavy chain, the process for loop randomization and shuffling can be employed in vitro that is similar to the process of affinity maturation of antibodies in vivo. Beste et al. (Proc. Natl. Acad. Sci. U.S.A. 96(5): 1898-1903 (1999)) discloses an antibody mimic based on a lipocalin scaffold (Anticalin®). Lipocalins are composed of a β- barrel with four hypervariable loops at the terminus of the protein. Beste (1999), subjected the loops to random mutagenesis and selected for binding with, for example, fluorescein. Three variants exhibited specific binding with fluorescein, with one variant showing binding similar to that of an anti-fluorescein antibody. Further analysis revealed that all of the randomized positions are variable, indicating that Anticalin® would be suitable to be used as an alternative to antibodies.

Anticalins® are small, single chain peptides, typically between 160 and 180 residues, which provides several advantages over antibodies, including decreased cost of production, increased stability in storage and decreased immunological reaction.

Hamilton et al. (U.S. Patent No. 5,770,380) discloses a synthetic antibody mimic using the rigid, non-peptide organic scaffold of calixarene, attached with multiple variable peptide loops used as binding sites. The peptide loops all project from the same side geometrically from the calixarene, with respect to each other. Because of this geometric confirmation, all of the loops are available for binding, increasing the binding affinity to a ligand. However, in comparison to other antibody mimics, the calixarene-based antibody mimic does not consist exclusively of a peptide, and therefore it is less vulnerable to attack by protease enzymes. Neither does the scaffold consist purely of a peptide, DNA or RNA, meaning this antibody mimic is relatively stable in extreme environmental conditions and has a long life span. Further, since the calixarene-based antibody mimic is relatively small, it is less likely to produce an immunogenic response.

Murali et al. {Cell. MoI. Biol. 49(2):209-216 (2003)) discusses a methodology for reducing antibodies into smaller peptidomimetics, they term "antibody like binding peptidomemetics" (ABiP) which can also be useful as an alternative to antibodies.

Silverman et al. (Nat. Biotechnol. (2005), 23: 1556-1561) discloses fusion proteins that are single-chain polypeptides comprising multiple domains termed "avimers." Developed from human extracellular receptor domains by in vitro exon shuffling and phage display the avimers are a class of binding proteins somewhat similar to antibodies in their affinities and specificities for various target molecules. The resulting multidomain proteins can comprise multiple independent binding domains that can exhibit improved affinity (in some cases sub- nanomolar) and specificity compared with single-epitope binding proteins. Additional details concerning methods of construction and use of avimers are disclosed, for example, in U.S. Patent App. Pub. Nos. 20040175756, 20050048512, 20050053973, 20050089932 and 20050221384.

In addition to non-immunoglobulin protein frameworks, antibody properties have also been mimicked in compounds comprising RNA molecules and unnatural oligomers (e.g., protease inhibitors, benzodiazepines, purine derivatives and beta-turn mimics) all of which are suitable for use with the present invention.

An agent isolated by any of the methods of the invention can be administered as a pharmaceutical or can be used for the manufacture of pharmaceutical (therapeutic or prophylactic) compositions for humans and other mammals, such as mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees for treating or preventing breast cancer. Preferred cancers to be treated or prevented by the agents screened through the present methods include breast, bladder and lung cancer.

The isolated agents can be directly administered or can be formulated into dosage form using known pharmaceutical preparation methods, for example, in combination with a "pharmaceutically acceptable carrier". Herein, the phrase "pharmaceutically acceptable carrier" refers to an inert substance used as a diluent or vehicle for a drug. For example, pharmaceutical formulations may include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub- cutaneous and intravenous) administration, or for administration by inhalation or insufflation. For example, according to the need, the agents can be taken orally, as sugar-coated tablets, capsules, elixirs and microcapsules; or non-orally, in the form of injections of sterile solutions or suspensions with water or any other pharmaceutically acceptable liquid. For example, the agents can be mixed with pharmaceutically acceptable carriers or media, specifically, sterilized water, physiological saline, plant-oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, and such, in a unit dose form required for generally accepted drug implementation. The amount of active ingredients in these preparations makes a suitable dosage within the indicated range acquirable.

Examples of additives that can be mixed to tablets and capsules are, binders such as gelatin, corn starch, tragacanth gum and arabic gum; excipients such as crystalline cellulose; swelling agents such as corn starch, gelatin and alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose or saccharin; and flavoring agents such as peppermint, Gaultheria adenothrix oil and cherry. When the unit-dose form is a capsule, a liquid carrier, such as an oil, can also be further included in the above ingredients. Sterile composites for injections can be formulated following normal drug implementations using vehicles such as distilled water used for injections.

Physiological saline, glucose, and other isotonic liquids including adjuvants, such as D-sorbitol, D-mannnose, D-mannitol, and sodium chloride, can be used as aqueous solutions for injections. These can be used in conjunction with suitable solubilizers, such as alcohol, specifically ethanol, polyalcohols such as propylene glycol and polyethylene glycol, non-ionic surfactants, such as Polysorbate 80 (TM) and HCO-50.

Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may be used in conjunction with benzyl benzoate or benzyl alcohol as a solubilizer and may be formulated with a buffer, such as phosphate buffer and sodium acetate buffer; a pain-killer, such as procaine hydrochloride; a stabilizer, such as benzyl alcohol and phenol; and an anti-oxidant. The prepared injection may be filled into a suitable ampoule.

Pharmaceutical formulations suitable for oral administration may conveniently be presented as discrete units, such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; or as a solution, a suspension or as an emulsion. The active ingredient may also be presented as a bolus electuary or paste, and be in a pure form, i.e., without a carrier. Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrant or wetting agents. A tablet may be made by compression or molding, optionally with one or more formulational ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be coated according to methods well known in the art. Oral fluid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives. The tablets may optionally be formulated so as to provide slow or controlled release of the active ingredient therein.

Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline, water-for-injection, immediately prior to use. Alternatively, the formulations may be presented for continuous infusion.

Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter or polyethylene glycol. Formulations for topical administration in the mouth, for example buccally or sublingually, include lozenges, comprising the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a base such as gelatin and glycerin or sucrose and acacia. For intra-nasal administration the compounds obtained by the invention may be used as a liquid spray or dispersible powder or in the form of drops. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs.

For administration by inhalation the compounds are conveniently delivered from an insufflator, nebulizer, pressurized packs or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichiorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, the compounds may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form, in for example, capsules, cartridges, gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflators.

When desired, the above described formulations, adapted to give sustained release of the active ingredient, may be employed. The pharmaceutical compositions may also contain other active ingredients such as antimicrobial agents, immunosuppressants or preservatives.

Preferred unit dosage formulations are those containing an effective dose, as recited below, or an appropriate fraction of the active ingredient. Herein, a "pharmaceutically effective amount" of an agent is defined a quantity that is sufficient to treat and/or ameliorate disease in an individual. An example of a pharmaceutically effective amount may an amount needed to decrease the interaction between MELK and BcI-G when administered to an animal. The decrease in interaction may be, e.g., at least a 5%, 10%, 20%, 30%, 40%, 50%, 75%, 80%, 90%, 95%, 99%, or 100% change in binding.

Methods well known to one skilled in the art may be used to administer an agent screened by the present methods to patients, for example, as intraarterial, intravenous, or percutaneous injections and also as intranasal, intramuscular or oral administrations. The dosage and method of administration vary according to the body- weight and age of a patient and the administration method; however, one skilled in the art can routinely select a suitable method of administration. If said agent is encodable by a DNA, the DNA can be inserted into a vector for gene therapy and the vector administered to a patient to perform the therapy. The dosage and method of administration vary according to the body- weight, age, and symptoms of the patient but one skilled in the art can suitably select them.

Generally, an efficacious or effective amount of one or more inhibitors of MELK/Bcl-G binding and/or BcI-G phosphorylation is determined by first administering a low dose or small amount of a MELK/Bcl-G inhibitor and then incrementally increasing the administered dose or dosages, and/or adding a second MELK/Bcl-G inhibitor as needed, until a desired effect of inhibiting or preventing a cancer mediated by MELK/Bcl-G binding and/or BcI-G phosphorylation is observed in the treated subject, with minimal or no toxic side effects. Applicable methods for determining an appropriate dose and dosing schedule for administration of a pharmaceutical composition of the present invention is described, for example, in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., Brunton, et al., Eds., McGraw-Hill (2006), and in Remington: The Science and Practice of Pharmacy, 21st Ed., University of the Sciences in Philadelphia (USEP), Lippincott Williams & Wilkins (2005), both of which are hereby incorporated herein by reference.

Although the dose of an agents screened by the present methods depends on the symptoms and such, the compositions may be administered at a dose of from about 0.1 to about 250 mg/kg per day. The dose range for adult humans is generally from about 5 mg to about 17.5 g/day, preferably about 5 mg to about 10 g/day, and most preferably about 100 mg to about 3 g/day. Tablets or other unit dosage forms of presentation provided in discrete units may conveniently contain an amount which is effective at such dosage or as a multiple of the same, for instance, units containing about 5 mg to about 500 mg, usually from about 100 mg to about 500 mg.

When administering parenterally, in the form of an injection to a normal adult (weight 60 kg), although there are some differences according to the patient, target organ, symptoms and method of administration, it is convenient to intravenously inject a dose of about 0.01 mg to about 30 mg per day, preferably about 0.1 to about 20 mg per day and more preferably about 0.1 to about 10 mg per day. Also, in the case of other animals, an amount converted to 60 kgs of body-weight can be administered.

The agents are preferably administered orally or by injection (intravenous or subcutaneous), and the precise amount administered to a subject will be determined under the responsibility of the attendant physician, considering a number of factors, including the age and sex of the subject, the precise disorder being treated, and its severity. Also the route of administration may vary depending upon the condition and its severity.

As noted above, the agents identified by the screening methods of the present methods find utility in the treatment and prevention of cancer, for example, breast, bladder or lung cancer, in a subject. Accordingly, administration can be prophylactic or therapeutic to a subject at risk of (or susceptible to) developing cancer. When a treatment of interest is applied prophylactically, the term "efficacious" means that the treatment retards or prevents a tumor from forming or retards, prevents, or alleviates a symptom of clinical cancer. Similarly, a therapy is deemed therapeutically "efficacious" if it leads to a reduction in the expression of a pathologically up-regulated gene, an increase in the expression of a pathologically down- regulated gene or a decrease in size, prevalence, or metastatic potential of cancer, e.g., breast, bladder or lung cancer, in a subject. Assessment of tumors can be made using standard clinical protocols.

The terms "label" and "detectable label" are used herein to refer to any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Such labels include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DYNABEADS™), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting, the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label.

The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, e.g., recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. By the term "recombinant nucleic acid" herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases and endonucleases, in a form not normally found in nature. In this manner, operable linkage of different sequences is achieved. Thus an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention. Similarly, a "recombinant protein" is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as depicted above.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

III. Identifying Agents that Inhibit the Binding Between MELK and BcI-G:

As discussed above, agents that interfere with the interaction between MELK and BcI-G, for example by inhibiting the binding there between, are expected to induce apoptosis and, therefore, find utility as anti-cancer agents. Accordingly, one aspect of the invention involves the identification of agents that inhibit the binding between MELK and BcI-G or functional equivalents thereof.

Methods for determining MELK/Bcl-G binding include any methods for determining interactions of two proteins. Such assays include, but are not limited to, traditional approaches, for example, cross-linking, co-immunoprecipitation, and co- purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored using a yeast-based genetic system described by Fields and co- workers (Fields and Song, Nature 340:245-6 (1989); Chien et al, Proc. Natl. Acad. ScL USA 88, 9578-82 (1991)) and as disclosed by Chevray and Nathans (Proc. Natl. Acad. Sci. USA 89:5789-93 (1992)). Many transcriptional activators, such as yeast GALA, consist of two physically discrete modular domains, one acting as the DNA-binding domain, while the other one functions as the transcription activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GALl-lacZ reporter gene under control of a GALA-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.

While the present application refers to "MELK" and "BcI-G" it is understood that where the interaction of the two is analyzed or manipulated, the binding portions (i.e., binding domains) of one or both of the proteins in place of the full-length copies of the proteins can be used. Fragments of MELK that bind to BcI-G may be readily identified using standard deletion analysis and/or mutagenesis of MELK to identify fragments that bind to BcI-G. Similar analysis may be used to identify BcI-G -binding fragments of MELK. A test agent will be deemed to "bind" MELK, BcI-G, or a functional equivalent thereof if it has a K₃ of at least about 10⁵ mol^"1, 10⁶ mol^"1 or greater, 10⁷ mol^"1 or greater, 10⁸ mol ¹ or greater, or 10⁹ mol^"1 or greater under physiological conditions.

In one embodiment, a BcI-G polypeptide comprising residues 12-72, 12-60, 12-48, 12-34, 21-36 or 24-33 of BcI-G (as set forth in SEQ ID NO:8) is used.

As noted above, any test agents, including, e.g., proteins (including antibodies and non-antibody binding proteins), muteins, polynucleotides, nucleic acid aptamers, and peptide and nonpeptide small organic molecules, may serve as test agents of the present invention. Test agents may be isolated from natural sources, prepared synthetically or recombinantly, or any combination of the same.

For example, peptides may be produced synthetically using solid phase techniques as described in "Solid Phase Peptide Synthesis" by G. Barany and R. B. Merrifield in Peptides, Vol. 2, edited by E. Gross and J. Meienhoffer, Academic Press, New York, N. Y., pp. 100-18 (1980). Similarly, nucleic acids can also be synthesized using the solid phase techniques, as described in Beaucage, S. L., & Iyer, R P. (1992) Tetrahedron, 48, 2223-311; and Matthes et al., EMB0 J., 3:801-5 (1984).

Where inhibitory peptides are identified, modifications of peptides of the present invention with various amino acid mimetics or unnatural amino acids are particularly useful in increasing the stability of the peptide in vivo. Stability can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, e.g., Verhoef et ah, Eur. J. Drug Metab Pharmacokin. 11 :291-302 (1986). Other useful peptide modifications known in the art include glycosylation and acetylation.

Both recombinant and chemical synthesis techniques may be used to produce test compounds of the present invention. For example, a nucleic acid test compound may be produced by insertion into an appropriate vector, which may be expanded when transfected into a competent cell. Alternatively, nucleic acids may be amplified using PCR techniques or expression in suitable hosts (cf. Sambrook et al, Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory, New York, USA).

Peptides and proteins may also be expressed using recombinant techniques well known in the art, e.g., by transforming suitable host cells with recombinant DNA constructs as described in Morrison J. Bact, 132:349-51 (1977); and Clark-Curtiss & Curtiss, Methods in Enzymology, 101 :347-62 (1983).

The polypeptides comprising the selected amino acid sequence of the present invention, can be of any length, so long as the polypeptide inhibits cancer cell proliferation. Specifically, the length of the amino acid sequence may range from 8 to 70 residues, for example, from 8 to 50, preferably from 8 to 30, more specifically from 8 to 20, further more specifically from 8 to 16 residues. For example, the amino acid sequence RRRRRRRRRRR GGGTIEFKILAYYTRHHVF(SEQ ID NO: 38) is preferable as the above-described selected amino acid sequence. Therefore, a polypeptide comprising or consisting of the amino acid sequence TIEFKILAYYTRHHVF(SEQ ID NO: 37) is a preferred example of the polypeptides in the present invention. The polypeptides of the present invention, which are characterized by containing the amino acid sequence FKILAYYTRHH(SEQ ED NO: 39). The polypeptides of the present invention may contain two or more "selected amino acid sequences". The two or more "selected amino acid sequences" may be the same or different amino acid sequences. Furthermore, the "selected amino acid sequences" can be linked directly. Alternatively, they may be disposed with any intervening sequences among them. Furthermore, the present invention relates to polypeptides homologous (i.e., share sequence identity) to the FKILAYYTRHH(SEQ ED NO: 39) polypeptide specifically disclosed here. In the present invention, polypeptides homologous to the FKILAYYTRHH(SEQ ID NO: 39) polypeptide are those which contain any mutations selected from addition, deletion, substitution and insertion of one or several amino acid residues and are functionally equivalent to the FKILAYYTRHH(SEQ ID NO: 39) polypeptide. The phrase "functionally equivalent to the FKILAYYTRHH(SEQ ID NO: 39) polypeptide" refers to having the function to inhibit the binding of MELK to BcI-G. The FKILAYYTRHH(SEQ ID NO: 39) sequence is preferably conserved in the amino acid sequences constituting polypeptides functionally equivalent to FKILAYYTRHH(SEQ ID NO: 39) polypeptide. Therefore, polypeptides functionally equivalent to the FKILAYYTRHH(SEQ ID NO: 39) peptide in the present invention preferably have amino acid mutations in sites other than the FKILAYYTRHH(SEQ ID NO: 39) sequence. Amino acid sequences of polypeptides functionally equivalent to the FKILAYYTRHH(SEQ ID NO: 39) peptide in the present invention conserve the FKILAYYTRHH(SEQ ID NO: 39) sequence, and have 60% or higher, usually 70% or higher, preferably 80% or higher, more preferably 90% or higher, or 95% or higher, and further more preferably 98% or higher homology to a "selected amino acid sequence". Amino acid sequence homology can be determined using algorithms well known in the art, for example, BLAST or ALIGN set to their default settings.

Alternatively, the number of amino acids that may be mutated is not particularly restricted, so long as the FKILAYYTRHH(SEQ ID NO: 39) peptide activity is maintained.

Generally, up to about 50 amino acids may be mutated, preferably up to about 30 amino acids, more preferably up to about 10 amino acids, and even more preferably up to about 3 amino acids. Likewise, the site of mutation is not particularly restricted, so long as the mutation does not result in the disruption of the FKILAYYTRHH(SEQ ID NO: 39) peptide activity. In a preferred embodiment, the activity of the RRHRRRRRRRR

GGGTIEFKILAYYTRHHVF(SEQ ID NO: 37) peptide comprises growth inhibition effect in a MELK expressing cell, i.e. breast cancer cell, prostatic cancer cell, breast, bladder and lung cancer cell. Growth inhibition effect of cancer cells may encompass the apotosis. Apoptosis means cell death caused by the cell itself and is sometimes referred to as programmed cell death. Aggregation of nuclear chromosome, fragmentation of nucleus, or condensation of cytoplasm is observed in a cell undergoing apoptosis. Methods for detecting apoptosis are well known. For instance, apoptosis may be confirmed by TUNEL staining (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling; Gavrieli et al., (1992) J. Cell Biol. 119: 493-501, Mori et al., (1994) Anat. & Embryol. 190: 21-28). Alternatively, DNA ladder assays, Annexin V staining, caspase assay, electron microscopy, or observation of conformational alterations on nucleus or cell membrane may be used for detecting apoptosis. Any commercially available kits may be used for detecting these behaviors in cells which are induced by apoptosis. For example, such apoptosis detection kits may be commercially available from the following providers:

LabChem Inc., Promega, BD Biosciences Pharmingen,

Calbiochem,

Takara Bio Company (CLONTECH Inc.), CHEMICON International, Inc, Medical & Biological Laboratories Co., Ltd. etc. The polypeptides of the present invention can be chemically synthesized from any position based on selected amino acid sequences. Methods used in the ordinary peptide chemistry can be used for the method of synthesizing polypeptides. Specifically, the methods include those described in the following documents and Japanese Patent publications:

Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol. 2, Academic Press Inc., New York, 1976;

Peputido gousei (Peptide Synthesis), Maruzen (Inc.), 1975;

Peputido gousei no kiso to jikken (Fundamental and Experimental Peptide Synthesis), Maruzen (Inc.), 1985;

Iyakuhin no kaihatsu (Development of Pharmaceuticals), Sequel, Vol. 14: Peputido gousei (Peptide Synthesis), Hirokawa Shoten, 1991;

International Patent Publication WO99/67288.

The polypeptides of the present invention can be also synthesized by known genetic engineering techniques. An example of genetic engineering techniques is as follows. Specifically, DNA encoding a desired peptide is introduced into an appropriate host cell to prepare a transformed cell. The polypeptides of the present invention can be obtained by recovering polypeptides produced by this transformed cell. Alternatively, a desired polypeptide can be synthesized with an in vitro translation system, in which necessary elements for protein synthesis are reconstituted in vitro.

When genetic engineering techniques are used, the polypeptide of the present invention can be expressed as a fused protein with a peptide having a different amino acid sequence. A vector expressing a desired fusion protein can be obtained by linking a polynucleotide encoding the polypeptide of the present invention to a polynucleotide encoding a different peptide so that they are in the same reading frame, and then introducing the resulting nucleotide into an expression vector. The fusion protein is expressed by transforming an appropriate host with the resulting vector. Different peptides to be used in forming fusion proteins include the following peptides:

FLAG (Hopp et al., (1988) BioTechnology 6, 1204-10),

6xHis consisting of six His (histidine) residues, lOxHis,

Influenza hemagglutinin (HA),

Human c-myc fragment, VSV-GP fragment, pi 8 HIV fragment,

T7-tag,

HSV-tag,

E-tag, SV40T antigen fragment, lck tag, α-tubulin fragment,

B-tag,

Protein C fragment, GST (glutathione-S-transferase),

HA (Influenza hemagglutinin),

Immunoglobulin constant region, β-galactosidase, and

MBP (maltose-binding protein). The polypeptide of the present invention can be obtained by treating the fusion protein thus produced with an appropriate protease, and then recovering the desired polypeptide. To purify the polypeptide, the fusion protein is captured in advance with affinity chromatography that binds with the fusion protein, and then the captured fusion protein can be treated with a protease. With the protease treatment, the desired polypeptide is separated from affinity chromatography, and the desired polypeptide with high purity is recovered.

The polypeptides of the present invention include modified polypeptides. In the present invention, the term "modified" refers, for example, to binding with other substances. Accordingly, in the present invention, the polypeptides of the present invention may further comprise other substances such as cell-membrane permeable substance. The other substances include organic compounds such as peptides, lipids, saccharides, and various naturally- occurring or synthetic polymers. The polypeptides of the present invention may have any modifications so long as the polypeptides retain the desired activity of inhibiting the binding of MELK to BcI-G. In some embodiments, the inhibitory polypeptides can directly compete with BcI-G binding to MELK. Modifications can also confer additive functions on the polypeptides of the invention. Examples of the additive functions include targetability, deliverability, and stabilization. Preferred examples of modifications in the present invention include, for example, the introduction of a cell-membrane permeable substance. Usually, the intracellular structure is cut off from the outside by the cell membrane. Therefore, it is difficult to efficiently introduce an extracellular substance into cells. Cell membrane permeability can be conferred on the polypeptides of the present invention by modifying the polypeptides with a cell- membrane permeable substance. As a result, by contacting the polypeptide of the present invention with a cell, the polypeptide can be delivered into the cell to act thereon.

The "cell-membrane permeable substance" refers to a substance capable of penetrating the mammalian cell membrane to enter the cytoplasm. For example, a certain liposome fuses with the cell membrane to release the content into the cell. Meanwhile, a certain type of polypeptide penetrates the cytoplasmic membrane of mammalian cell to enter the inside of the cell. For polypeptides having such a cell-entering activity, cytoplasmic membranes and such in the present invention are preferable as the substance. Specifically, the present invention includes polypeptides having the following general formula.

[R]-[D]; wherein,

[R] represents a cell-membrane permeable substance; [D] represents a fragment sequence containing FKILAYYTRF£H(SEQ ID NO: 39). In the above-described general formula, [R] and [D] can be linked directly or indirectly through a linker. Peptides, compounds having multiple functional groups, or such can be used as a linker. Specifically, amino acid sequences containing -GGG- can be used as a linker. Alternatively, a cell- membrane permeable substance and a polypeptide containing a selected sequence can be bound to the surface of a minute particle. [R] can be linked to any positions of [D].

Specifically, [R] can be linked to the N terminal or C terminal of [D], or to a side chain of amino acids constituting [D]. Furthermore, more than one [R] molecule can be linked to one molecule of [D]. The [R] molecules can be introduced to different positions on the [D] molecule. Alternatively, [D] can be modified with a number of [R]s linked together. For example, there have been reported a variety of naturally-occurring or artificially synthesized polypeptides having cell-membrane permeability (Joliot A. & Prochiantz A., Nat Cell Biol. 2004; 6: 189-96). All of these known cell-membrane permeable substances can be used for modifying polypeptides in the present invention. In the present invention, for example, any substance selected from the following group can be used as the above-described cell-permeable substance: poly-arginine; Matsushita et al., (2003) J. Neurosci.; 21, 6000-7.

[Tat / RKKRRQRRR] (SEQ ID NO: 42) Frankel et al., (1988) Cell 55,1189- 93.

Green & Loewenstein (1988) Cell 55, 1179-88. [Penetratin / RQIKIWFQNRRMKWKK] (SEQ ID NO: 43)

Derossi et al., (1994) J. Biol. Chem. 269, 10444-50.

[Buforin II / TRS SRAGLQFP VGRVHRLLRK] (SEQ ID NO: 44)

Park et al., (2000) Proc. Natl Acad. Sci. USA 97, 8245-50.

[Transportan / GWTLNSAGYLLGKINLKALAALAKKIL] (SEQ ID NO: 45)

Pooga et al., (1998) FASEB J. 12, 67-77.

[MAP (model amphipathic peptide) / KLALKLALKALKAALKLA] (SEQ ID NO: 46)

Oehlke et al., (1998) Biochim. Biophys. Acta. 1414, 127-39. [K-FGF / AAVALLPAVLLALLAP] (SEQ ID NO: 47)

Lin et al., (1995) J. Biol. Chem. 270, 14255-8.

[Ku70 / VPMLK] (SEQ ID NO: 48 Sawada et al., (2003) Nature Cell Biol. 5, 352-7.

[Ku70 / PMLKE] (SEQ ID NO: 49)

Sawada et al., (2003) Nature Cell Biol. 5, 352-7.

[Prion / MANLGYWLLALFVTMWTDVGLCKKRPKP] (SEQ ID NO: 50) Lundberg et al., (2002) Biochem. Biophys. Res. Commun. 299, 85-90.

[pVEC / LLIILRRRIRKQAHAHSK] (SEQ ID NO: 55)

Elmquist et al., (2001) Exp. Cell Res. 269, 237-44.

[Pep-1 / KETWWETWWTEWSQPKKKRKV] (SEQ ID NO: 52)

Morris et al., (2001) Nature Biotechnol. 19, 1173-6. [SynBl / RGGRLSYSRRRFSTSTGR] (SEQ ID NO: 53)

Rousselle et al., (2000) MoI. Pharmacol. 57, 679-86.

[Pep-7 / SDLWEMMMVSLACQY] (SEQ ID NO: 54)

Gao et al., (2002) Bioorg. Med. Chem. 10, 4057-65.

[HN-I / TSPLNIHNGQKL] (SEQ ID NO: 56) Hong & dayman (2000) Cancer Res. 60, 6551-6.

In the present invention, the poly-arginine, which is listed above as an example of cell- membrane permeable substances, is constituted by any number of arginine residues. Specifically, for example, it is constituted by consecutive 5-20 arginine residues. The preferable number of arginine residues is 11 (SEQ ID NO: 56). Herein, a test agent is deemed to "decrease" or "inhibit" the interaction of MELK and BcI-G when the level of detectable complex of MELK/Bcl-G decreases by, for example, 10%, 25%, or 50%, or at least 1-fold, at least 2-fold, at least 5-fold, or at least 10-fold or more compared to the level of detected MELK/Bcl-G complexes not contacted with the test agent. For example, Student's t-test, the Mann- Whitney U-test, or ANOVA may be used for statistical analysis.

IV. Identifying Agents that Reduce the Phosphorylation Level of a MELK Substrate:

As discussed above, agents that interfere with the interaction between MELK and BcI-G, for example by suppressing or inhibiting the phosphorylation activity of MELK or reducing the phosphorylation level of its BcI-G substrate, are expected to induce apoptosis and, therefore, find utility as anti-cancer agents. Accordingly, one aspect of the invention involves the identification of such useful anti-cancer agents using the phosphorylation level of a MELK substrate, such as BcI-G as index.

The phosphorylation level of a MELK substrate may be detected according to any method known in the art. For example, immunological techniques that utilize an antibody recognizing the phosphorylated MELK substrate can be used for the detection. ELISA or immunoblotting with antibodies recognizing phosphorylated BcI-G polypeptide can be used for the present invention. Phosphorylation of a MELK substrate can also be detected using a radioactive phosphorus isotope, for example ³²P or ³³P, in standard in vitro phosphorylation assays and then detecting phosphorylated MELK substrate resolved by electrophoresis on an autoradiograph.

If the detected amount of phosphorylated MELK substrate in a cell contacted with a test agent is decreased to the amount detected in a cell not contacted with the test agent, the test agent is deemed to inhibit a MELK phosphorylation and thus have apoptosis inducing ability. Herein, a phosphorylation level can be deemed to be "decreased" when it decreases by, for example, 10%, 25%, or 50% from, or at least 0.1 -fold, at least 0.2-fold, at least 1-fold, at least 2-fold, at least 5-fold, or at least 10-fold or more compared to that detected for cells not contacted with the test agent. For example, Student's Mest, the Mann- Whitney U-test, or ANOVA may be used for statistical analysis.

Alternatively, the phosphorylation level of the MELK substrate thereof may be detected by detecting the cell cycle of the cell. Specifically, the cell cycle of a cell can be determined by using conventional methods known in the art including FACS and so on. When detecting the cell cycle of a cell for determining the phosphorylation level of the polypeptide, after the contact of the cell with a test agent, it is preferred to incubate the cell for a sufficient time, for example, for 12 h or more, till normal cells path through the G2/M phase. According to this procedure, a test agent can be determined to have the ability to induce apoptosis of breast cancer cells, when the cell cycle is detected to be trapped at the G2/M phase.

Alternatively, the phosphorylation level of the MELK substrate thereof may be detected by detecting the amount of apoptotic cells. Specifically, the apoptotic cells can be determined by using conventional methods known in the art including FACS and so on. When detecting apoptotic cells for determining the phosphorylation level of the polypeptide, after the contact of the cell with a test agent, it is preferred to incubate the cell for a sufficient time. According to this procedure, a test agent can be determined to have the ability to induce apoptosis of breast cancer cells, when the sub-Gl cell population is detected.

The MELK polypeptide and MELK substrate used in the screening methods of the present invention can be prepared as recombinant proteins or natural proteins, by methods well known to those skilled in the art. The polypeptides may be obtained adopting any known genetic engineering methods for producing polypeptides (e.g., Morrison J., J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101 : 347-62). For example, a recombinant protein can be prepared by inserting a DNA , which encodes a protein of the present invention (for example, the DNA comprising the nucleotide sequence of SEQ ID NO: 1), into an appropriate expression vector, introducing the vector into an appropriate host cell, obtaining the extract, and purifying the protein by subjecting the extract to chromatography, for example, ion exchange chromatography, reverse phase chromatography, gel filtration, or affinity chromatography utilizing a column to which antibodies against the protein of the present invention is fixed, or by combining more than one of aforementioned columns.

Also when the protein useful in the context of the present invention is expressed within host cells (for example, animal cells and E. colϊ) as a fusion protein with glutathione-S- transferase protein or as a recombinant protein supplemented with multiple histidines, the expressed recombinant protein can be purified using a glutathione column or nickel column.

After purifying the fusion protein, regions other than the objective protein can be excluded, for example, by cutting with thrombin or factor-Xa as required.

A natural protein can be isolated by methods known to a person skilled in the art, for example, by contacting the affinity column, in which antibodies binding to the protein of interest are bound, with the extract of tissues or cells expressing the protein of the present invention. The antibodies can be polyclonal antibodies, monoclonal antibodies, or any modified antibodies so long as it binds to the relevant protein. The proteins of the present invention may also be produced in vitro adopting an in vitro translation system.

As noted above, a partial peptide of MELK may also be used for the invention so long as it retains the phosphorylating activity of the full length protein. Such partial peptides can be produced by genetic engineering, by known methods of peptide synthesis, or by digesting the natural MELK / protein with an appropriate peptidase. For peptide synthesis, for example, solid phase synthesis or liquid phase synthesis may be used. Conventional peptide synthesis methods that can be adopted for the synthesis include:

Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol. 2, Academic Press, New York, 1976; - Peptide Synthesis (in Japanese), Maruzen Co., 1975;

Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985;

Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991;

WO99/67288; and - Barany G. & Merrifield R B , Peptides Vol. 2, "Solid Phase Peptide Synthesis",

Academic Press, New York, 1980, 100-118.

The polypeptide or fragments thereof may be further linked to other substances so long as the polypeptide and fragments retains its original ability to phosphorylate a substrate. Usable substances include: peptides, lipids, sugar and sugar chains, acetyl groups, natural and synthetic polymers, etc. These kinds of modifications may be performed to confer additional functions or to stabilize the polypeptide and fragments.

The MELK polypeptide or functional equivalent thereof to be contacted with a test agent and substrate can be, for example, a purified polypeptide, a soluble protein, or a fusion protein fused with other polypeptides.

An MELK substrate is any compound capable of accepting a phosphorus group such as a protein, a nucleic acid (RNA or DNA) or a small molecule. In the context of the present invention, the preferred MELK substrate is BcI-G or a fragment thereof containing the phosphorylation site. In some embodiments, the fragment of BcI-G {e.g., amino acids 12-295 or 12-215 of SEQ ID NO: 8) is used as a MELK substrate. Accordingly, BcI-G or a fragment thereof containing amino acids 12-295 orl2-215 of SEQ ED NO: 8 can be useful as the substrate.

Similarly to MELK polypeptide, BcI-G substrate can be prepared as a recombinant protein or natural protein. Furthermore, similarly to the MELK polypeptide, BcI-G may be prepared as a fusion protein so long as the resulting fusion protein can be phosphorylated by the MELK polypeptide.

In the present invention, a substance enhancing phosphorylation of the MELK polypeptide or the substrate can be added to the reaction mixture of screening. When phosphorylation of the substrate is enhanced by the addition of the substance, phosphorylation level of a substrate can be determined with higher sensitivity.

The contact of the MELK polypeptide or functional equivalent thereof, its substrate, and a test agent may be conducted in vivo or in vitro. The screening in vitro can be carried out in buffer, for example, but are not limited to, phosphate buffer and Tris buffer, so long as the buffer does not inhibit the phosphorylation of the substrate by the MELK polypeptide or functional equivalent thereof.

In the present invention, the phosphorylation level of a substrate can be determined by methods known in the art. For example, the MELK polypeptide or a functional equivalent thereof a substrate can be incubated with a labeled phosphorus donor, under suitable assay conditions. For example, ³²P-γ-ATP can be used as the phosphorus donor. Transfer of the radiolabel to the substrate can be detected, for example, by SDS-PAGE electrophoresis and autoradiography. Alternatively, following the reaction the substrate can be separated from the reaction mixture by filtration, and the amount of radiolabel retained on the filter may be quantitated by scintillation counting. Other suitable labels that can be attached to phosphorus donors, including enzymatic, chromogenic and fluorescent labels, and methods of detecting transfer of these labels to the substrates are known in the art.

Alternatively, the phosphorylation level of a substrate can be determined using reagents that selectively recognize phosphorylated substrates. For example, after the contact and incubation of the substrate with the MELK polypeptide under the presence of a test agent and the condition allowing phosphorylation of the substrate, phosphorylated substrate can be detected by an immunological method. Any immunological techniques, including ELISA and immunoblotting, using antibodies that distinguish phosphorylated and non-phosphorylated substrate can be used for the detection. For example, when BcI-G is used as the substrate, antibody against phosphorylated BcI-G may be used to detect phosphorylation.

Instead of using antibodies, phosphorylated substrates can be detected using reagents that selectively bind with high affinity to the phosphorylated substrates. Such reagents may be labeled for detection or may be detected using antibodies against the reagents.

Various low-throughput and high-throughput enzyme assay formats are known in the art and can be readily adapted for detection or measuring of the phosphorylation level of the substrate of the MELK polypeptide. For high-throughput assays, the substrate can conveniently be immobilized on a solid support. Following the reaction, the phosphorylated substrate can be detected on the solid support by the methods described above. Alternatively, the contact step can be performed in solution, after which the substrate can be immobilized on a solid support, and the phosphorylated substrate detected. To facilitate such assays, the solid support can be coated with streptavidin and the substrate labeled with biotin, or the solid support can be coated with antibodies against the substrate. The skilled person can determine suitable assay formats depending on the desired throughput capacity of the screen. The assays of the invention are also suitable for automated procedures which facilitate high-throughput screening.

Examples of supports that may be used for binding substrates include insoluble polysaccharides, such as agarose, cellulose, and dextran; and synthetic resins, such as polyacrylamide, polystyrene, and silicon; preferably commercially available beads, slides, chips and plates (e.g., multi-well plates, biosensor chip, etc.) prepared from the above materials may be used. When using beads, they may be filled into a column.

The binding of a substrate to a support may be conducted according to routine methods, such as chemical bonding, and physical adsorption. Alternatively, the substrate may be bound to a support via antibodies specifically recognizing the substrate. Moreover, binding of a substrate to a support can be also conducted by means of avidin and biotin binding.

According to one aspect of the present invention, the components necessary for the present screening methods may be provided as a kit for screening agents that induces apoptosis of breast cancer cells or agents for treating or preventing breast cancer. The kit may contain, for example, a cell expressing MELK polypeptide or a function equivalent thereof, or a MELK polypeptide and its substrate or functional equivalents thereof. Further, the kit may include control reagents (positive and/or negative), detectable labels, cell culture medium, containers required for the screening, instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the method, and so on. The components and reagents may be packaged in separate containers. V. Suitable Test Agents & Test Agent Libraries: Antibodies:

As noted above, in some aspects of the present invention, test agents are anti-MELK or anti-Bcl-G antibodies In some embodiments, the antibodies are chimeric, including but not limited to, humanized antibodies. In some cases, antibody embodiments of the present invention will bind either MELK or BcI-G at the interface where one of these proteins associates with the other. In preferred embodiments, these antibodies bind MELK or BcI-G with a K_a of at least about 10⁵ mol^"1, 10⁶ mol ¹ or greater, 10⁷ mol^"1 or greater, 10⁸ moi^"1 or greater, or 10⁹ mol^"1 or greater under physiological conditions. Such antibodies can be purchased from a commercial source, for example, Chemicon, Inc. (Temecula, CA), or can be raised using as an immunogen, such as a substantially purified MELK or BcI-G protein, e.g., a human protein, or an antigenic fragment thereof. Methods of preparing both monoclonal and polyclonal antibodies from provided immunogens are well-known in the art. For purification techniques and methods for identifying antibodies to specific immunogens, see e.g., PCT/US02/07144 (WO/03/077838) incorporated by reference herein in its entirety Methods for purifying antibodies using, for example, antibody affinity matrices to form an affinity column are also well known in the art and available commercially (AntibodyShop, Copenhagen, Denmark). Identification of antibodies capable of disrupting the MELK/Bcl-G association is performed using the same test assays detailed below for test agents in general.

Converting enzymes:

Converting enzymes may act as test agents of the present invention. In the context of the present invention, converting enzymes are molecular catalysts that perform covalent post-translational modifications to either MELK or BcI-G, or both Converting enzymes of the present invention will covalently modify one or more amino acid residues of MELK and/or BcI-G in a manner that causes either an allosteric alteration in the structure of the modified protein, or alters the MELK/Bcl-G molecular binding site chemistry or structure of the modified protein in a manner that interferes with binding between MELK and BcI-G. Herein, interference with binding between the two molecules refers to a decrease in the K₃ of binding by at least 25%, 30%, 40%, 50%, 60%, 70% or more relative to the Ka of binding between the proteins measured at 3O⁰C and an ionic strength of 0.1 in the absence of detergents. Exemplary converting enzymes of the invention include kinases, phosphatases, amidases, acetylases, glycosidase and the like. Constructions of test agent libraries:

Although the construction of test agent libraries is well known in the art, the present section provides additional guidance in identifying test agents and construction libraries of such agents for screening of effective inhibitors of MELK/Bcl-G interaction.

Molecular modeling

Construction of test agent libraries is facilitated by knowledge of the molecular structure of compounds known to have the properties sought, and/or the molecular structure of the target molecules to be inhibited, i.e., MELK and BcI-G. One approach to preliminary screening of test compounds suitable for further evaluation is computer modeling of the interaction between the test compound and its target. In the present invention, modeling the interaction between MELK and/or BcI-G provides insight into both the details of the interaction itself, and provides strategies for disrupting the interaction, including designing molecular inhibitors of the interaction.

Computer modeling technology allows visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule. The three-dimensional construct typically depends on data from X-ray crystallographic analysis or NMR imaging of the selected molecule. The molecular dynamics require force field data. The computer graphics systems enable prediction of how a new compound will link to the target molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity. Prediction of what the molecule-compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.

An example of the molecular modeling system described generally above consists of the CHARMm and QUANTA programs, Polygen Corporation, Waltham, Mass. CHARMm performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive with specific proteins, such as; Ripka, New Scientist 54-7 (Jun. 16, 1988); McKinlay and Rossmann, Annu. Rev. Pharmacol. Toxiciol. 29, 111-22 (1989); Perry and Davies, Prog Clin Biol Res. 291:189- 93(1989); Lewis and Dean, Proc. R. Soc. Lond. 236, 125-40 and 141-62 (1989); and, with respect to a model receptor for nucleic acid components, Askew, et al, J. Am. Chem. Soc. 111, 1082-90 (1989).

Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc., Pasadena, Calif, Allelix, Inc, Mississauga, Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario. See, e.g., DesJarlais et al. (1988) J. Med. Chem. 31 :722; Meng et al. (1992) J. Computer Chem. 13:505; Meng et al. (1993) Proteins 17:266; Shoichet et al. (1993) Science 259: 1445.

Once a putative inhibitor of the MELK/Bcl-G interaction has been identified, combinatorial chemistry techniques can be employed to construct any number of variants based on the chemical structure of the identified putative inhibitor, as detailed below. The resulting library of putative inhibitors, or "test compounds" may be screened using the methods of the present invention to identify test compounds of the library that disrupt the MELK/Bcl-G association.

Combinatorial chemical synthesis

Combinatorial libraries of test compounds may be produced as part of a rational drug design program involving knowledge of core structures existing in known inhibitors of the MELK/Bcl-G interaction. This approach allows the library to be maintained at a reasonable size, facilitating high throughput screening. Alternatively simple, particularly short polymeric molecular libraries may be constructed by simply synthesizing all permutations of the molecular family making up the library. An example of this latter approach would be a library wherein all peptide members are, for example, six amino acids in length. Such a peptide library could include every 6 amino acid sequence permutation. This type of library is termed a linear combinatorial chemical library.

Preparation of combinatorial chemical libraries is well known to those of skill in the art, and may be generated by either chemical or biological synthesis. Combinatorial chemical libraries include, but are not limited to, peptide libraries {see, e.g., US Patent 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-93 (1991) and Houghten et al., Nature 354:84-6 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptides (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (DeWitt et al, Proc. Natl. Acad. ScL USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al, J. Amer. Chem. Soc.

114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al, J. Amer. Chem. Soc. 114:9217-8 (1992)), analogous organic syntheses of small compound libraries (Chen et al, J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al, Science 261 : 1303 (1993)), and/or peptidyl phosphonates (Campbell et al, J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083), antibody libraries (see, e.g., Vaughan et al, Nature Biotechnology, 14(3):309-14 (1996) and PCT/US96/ 10287), carbohydrate libraries (see, e.g., Liang et al, Science, 274: 1520-2 (1996) and U.S. Patent 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Gordon, Curr Opin Biotechnol. 1995 Dec l;6(6):624-31; isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, and the like).

Phage display

Another approach uses recombinant bacteriophage to produce libraries. Using the "phage method" (Scott and Smith, Science 249:386-90, 1990; Cwirla, et al, Proc. Natl. Acad. ScL, 87:6378-82, 1990; Devlin et al, Science, 249:404-6, 1990), very large libraries can be constructed (e.g., 10⁶ -10⁸ chemical entities). A second approach uses primarily chemical methods, of which the Gey sen method (Geysen et al, Molecular Immunology 23:709-15, 1986; Geysen et al. J. Immunologic Method 102:259-74, 1987; and the method of Fodor et al (Science 251 :767-73, 1991) are examples. Furka et al (14th International Congress of

Biochemistry, Volume #5, Abstract FR: 013, 1988; Furka, Int. J. Peptide Protein Res. 37:487- 93, 1991), (US Pat. No. 4,631,211) and. (US Pat. No. 5,010, 175) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.

Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced ChemTech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N J. , Tripos, Inc., St. Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

VI. Screening Methods:

Screening methods of the present invention provide efficient and rapid identification of test agents that have a high probability of interfering with the interaction between MELK and BcI-G (i.e., the binding between MELK and BcI-G and/or the phosphorylation level of BcI-G) Generally, any method that determines the ability of a test agent to inhibit the binding between MELK and BcI-G or reduce the phosphorylation level of BcI-G is suitable for use with the present invention. For example, competitive and non- competitive inhibition assays in an ELISA format may be utilized. Control experiments should be performed to determine maximal binding capacity of system (e.g., contacting bound MELK with BcI-G and determining the amount of BcI-G that binds to MELK in the examples below).

Competitive assay format Competitive assays may be used for screening test agents of the present invention.

By way of example, a competitive ELISA format may include MELK (or BcI-G) bound to a solid support. Other detection assay formats are suitable. The bound MELK (or BcI-G) would be incubated with BcI-G (or MELK) and a test agent. After sufficient time to allow the test agent and/or BcI-G (or MELK) to bind MELK (or BcI-G), the substrate would be washed to remove unbound material. The amount of BcI-G (or MELK) bound to MELK (or BcI-G) is then determined. This may be accomplished in any of a variety of ways known in the art, for example, by using a BcI-G (or MELK) species tagged with a detectable label, or by contacting the washed substrate with a labeled anti-Bcl-G (or MELK) antibody. The amount of BcI-G (or MELK) bound to MELK (or BcI-G) will be inversely proportional to the ability of the test compound to interfere with the Bcl-G/MELK association. In some embodiments, one or both of the MELK and BcI-G polypeptides are labeled. Protein, including but not limited to, antibody, labeling is described in Harlow & Lane, Antibodies, A Laboratory Manual (1988). In some competitive assay formats, e.g. , detection using mass spectrometry, neither the MELK nor BcI-G polypeptides need to be labeled.

In a variation, MELK (or BcI-G) is labeled with an affinity tag. Labeled MELK (or

BcI-G) is then incubated with a test compound and BcI-G (or MELK), then immunoprecipitated. The immunoprecipitate is then subjected to Western blotting using an anti-Bcl-G (or MELK) antibody. As with the previous competitive assay format, the amount of BcI-G (or MELK) found associated with MELK (or BcI-G) is inversely proportional to the ability of the test compound to interfere with the MELK/Bcl-G association.

Non-competitive assay format

Non-competitive binding assays also find utility as an initial screen for test agent libraries constructed in a format that is not readily amenable to screening using competitive assays, such as those described herein. An example of such a library is a phage display library {See, e.g., Barrett, et al. (1992) Anal. Biochem 204,357-64).

Phage libraries find utility in being able to produce quickly working quantities of large numbers of different recombinant peptides. Phage libraries do not lend themselves to competitive assays of the invention, but can be efficiently screened in a non-competitive format to determine which recombinant peptide test compounds bind MELK or BcI-G. Test compounds identified as binding can then be produced and screened using a competitive assay format. Production and screening of phage and cell display libraries is well-known in the art and discussed in, for example, Ladner et al., WO 88/06630; Fuchs et al. (1991) Biotechnology 9: 1369-72; Goward et al. (1993) Trends Biochem Sci. 18: 136-40; Charbit et al. (1986) EMBO J 5, 3029-37. Cull et al. (1992) PNAS USA 89: 1865-9; Cwirla, et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6378-82.

An exemplary non-competitive assay would follow an analogous procedure to the one described for the competitive assay, without the addition of one of the components (MELK or BcI-G). However, as non-competitive formats determine test agent binding to MELK or BcI-G, the ability of test compound to both MELK and BcI-G needs to be determined for each candidate. Thus, by way of example, binding of the test agent to immobilized MELK may be determined by washing away unbound test compound; eluting bound test compound from the support, followed by analysis of the eluate; e.g., by mass spectroscopy, protein determination (Bradford or Lowry assay, or Abs. at 280nm determination ). Alternatively, the elution step may be eliminated and binding of test compound determined by monitoring changes in the spectroscopic properties of the organic layer at the support surface. Methods for monitoring spectroscopic properties of surfaces include, but are not limited to, absorbance, reflectance, transmittance, birefringence, refractive index, diffraction, surface plasmon resonance, ellipsometry, resonant mirror techniques, grating coupled waveguide techniques and multipolar resonance spectroscopy, all of which are known to those of skill in the art. A labeled test compound may also be used in the assay to eliminate need for an elution step. In this instance, the amount of label associated with the support after washing away unbound material is directly proportional to test compound binding.

A number of well-known robotic systems have been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett Packard, Palo Alto, Calif), which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available {see, e.g., ComGenex, Princeton, N. J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

Screening converting enzymes

Test agents that are converting enzymes may be assayed in a noncompetitive format, using co-factors and auxiliary substrates specific for the converting enzyme being assayed. Such co-factors and auxiliary substrates are known to one of skill in the art, given the type of converting enzyme to be investigated.

One exemplary screening procedure for converting enzymes involves first contacting MELK and/or BcI-G with the converting enzyme in the presence of co-factors and auxiliary substrates necessary to perform covalent modification of the protein characteristic of the converting enzyme, preferably under physiologic conditions. The modified protein(s) is then tested for its ability to bind to its binding partner {i.e., binding of MELK to BcI-G). Binding of the modified protein to its binding partner is then compared to binding of unmodified control pairs to determine if the requisite change in K₃ noted above has been achieved. To facilitate the detection of proteins in performing the assay, one or more proteins may be labeled with a detectable label as described above, using techniques well known to those of skill in the art.

VII. Therapeutic Compositions for Treating and/or Preventing Cancer:

Accordingly, the present invention includes therapeutic compositions and methods useful in preventing or treating cancer, particularly a breast, bladder, and lung cancer, as well as other cancers characterized by cells displaying elevated expression levels of MELK. These compositions and methods include at least one test agent of the present invention identified as disruptive to the MELK/Bcl-G interaction in an amount effective to resume the induction of apoptosis characteristic of native, non-phosphorylated BcI-G. More specifically, a therapeutically effective amount means an amount effective to prevent the development of or to alleviate existing symptoms of the subject being treated.

Individuals to be treated with methods of the present invention may be any individual afflicted with cancer, including, e.g., cancer characterized by aberrantly elevated (i.e., abnormally high) expression of marker protein MELK. Such an individual can be, for example, a vertebrate such as a mammal, including a human, non-human primate, dog, cat, horse, cow, goat; sheep, pig, rodent, or any other animal, particularly a commercially important (e.g., agricultural) animal, a domesticated animal or a laboratory animal. For purposes of the present invention, elevated expression of marker proteins refers to a mean cellular marker protein concentration for one or both marker proteins that is at least 10%, preferably 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or more above normal mean cellular concentration of the marker protein(s).

As noted above, determination of an effective dose range for the therapeutic compositions of the present invention is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. The therapeutically effective dose for an active test agent can be estimated initially from cell culture assays and/or animal models. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 (the dose where 50% of the cells show the desired effects) as determined in cell culture. Toxicity and therapeutic efficacy of test compounds also can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (i.e., the ratio between LD50 and ED50). Compounds which exhibit high therapeutic indices may be used. The data obtained from these cell culture assays and animal studies may be used in formulating a dosage range for use in humans. The dosage of such compounds may lie within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, e.g., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 1 lth Ed., Brunton, et al., Eds., McGraw-Hill (2006). Dosage amount and interval may be adjusted individually to provide plasma levels of the active test compound sufficient to maintain the desired effects.

Also as noted above, therapeutic compositions of the present invention to be administered to a mammal (e.g., a human) may contain a pharmaceutically-acceptable excipient, or carrier. Suitable excipients and their formulations are described in Remington: The Science and Practice of Pharmacy, 21st Ed., University of the Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins (2005), edited by Oslo et al. For aqueous preparations an appropriate amount of a pharmaceutically-acceptable salt is typically used in the formulation to render the formulation isotonic. Examples of the pharmaceutically- acceptable isotonic excipients include liquids such as saline, Ringer's solution, Hanks's solution and dextrose solution. Isotonic excipients are particularly important for injectable formulations.

For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

Excipients may be used to maintain the correct pH of the formulation. For optimal shelf life, the pH of solutions containing test compounds preferably ranges from about 5 to about 8, and more preferably from about 7 to about 7.5. The formulation may also comprise a lyophilized powder or other optional excipients suitable to the present invention including sustained release preparations such as semi-permeable matrices of solid hydrophobic polymers, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain excipients may be more preferable depending upon, for instance, the route of administration, the concentration of test compound being administered, or whether the treatment uses a medicament that includes a protein, a nucleic acid encoding the test compound, or a cell capable of secreting a test compound as the active ingredient

The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen.

For oral administration, carriers enable the active test agents of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by formulating a test compound with a solid dispersable excipient, optionally grinding a resulting mixture and processing the mixture of granules after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Many of the active test agents of the present invention may be optionally provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc, depending upon the application. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.

In addition to acceptable excipients, formulations of the present invention may include therapeutic agents other than identified test agents. For example, formulations may include anti-inflammatory agents, pain killers, chemotherapeutics, mucolytics {e.g. n-acetyl- cysteine) and the like. In addition to including other therapeutic agents in the medicament itself, the medicaments of the present invention may also be administered sequentially or concurrently with the one or more other pharmacologic agents. The amounts of medicament and pharmacologic agent depend, for example, on what type of pharmacologic agent(s) is are used, the disease being treated, and the scheduling and routes of administration.

The therapeutic compositions comprising FKILAYYTRHH(SEQ ID NO: 39) are also provided. Because the polypeptides of the present invention inhibit proliferation of cancer cells, the present invention provides therapeutic and/or preventive agents for cancer which comprise as an active ingredient a polypeptide which comprises FKILAYYTRHH(SEQ ID NO: 39); or a polynucleotide encoding the same. Alternatively, the present invention relates to methods for treating and/or preventing cancer comprising the step of administering a polypeptide of the present invention. Furthermore, the present invention relates to the use of the polypeptides of the present invention in manufacturing pharmaceutical compositions for treating and/or preventing cancer. Cancers which can be treated or prevented by the present invention are not limited, so long as expression of MELK is up-regulated in the cancer cells. For example, the polypeptides of the present invention are useful for treating breast cancer, lung cancer or bladder cancer. Among them, breast cancer is particularly preferable as a target for treatment or prevention in the present invention.

Alternatively, the inhibitory polypeptides of the present invention can be used to inhibit proliferation of cancer cells. Therefore, the present invention provides agents for inhibition of cell proliferation, which comprise as an active ingredient a polypeptide which comprises FKILAYYTRHH(SEQ ID NO: 39); or a polynucleotide encoding the same. The proliferation inhibiting agents of the present invention may be used for treating cell proliferative diseases such as cancer. Cancers which can be treated or prevented by the present invention are not limited, so long as expression of MELK is up-regulated in the cancer cells. For example, the polypeptides of the present invention are useful in treating breast cancer, bladder cancer or lung cancer.. Among them, breast cancer is particularly preferable as a target for treatment or prevention in the present invention. Alternatively, the present invention relates to methods for inhibition of cell proliferation which comprise the step of administering the polypeptides of the present invention. Furthermore, the present invention relates to the use of polypeptides of the present invention in manufacturing pharmaceutical compositions for inhibition of cell proliferation. The inhibitory polypeptides of the present invention inhibit cell proliferlation in

MELK-expressing cells such as breast cancer. In the meantime, MELK expression has not been observed in most of normal organs. In some normal organs, the expression level of MELK is relatively low as compared with cancer tissues. Accordingly, the polypeptides of the present invention may induce apoptosis specifically in cancer cells.

When the polypeptides of the present invention are administered, as a prepared pharmaceutical, to human and other mammals such as mouse, rat, guinea pig, rabbit, cat, dog, sheep, pig, cattle, monkey, baboon and chimpanzee for treating cancer or inducing apoptosis in cells, isolated compounds can be administered directly, or formulated into an appropriate dosage form using known methods for preparing pharmaceuticals. For example, if necessary, the pharmaceuticals can be orally administered as a sugar-coated tablet, capsule, elixir, and microcapsule, or alternatively parenterally administered in the injection form that is a sterilized solution or suspension with water or any other pharmaceutically acceptable liquid. For example, the compounds can be mixed with pharmacologically acceptable carriers or media, specifically sterilized water, physiological saline, plant oil, emulsifier, suspending agent, surfactant, stabilizer, corrigent, excipient, vehicle, preservative, and binder, in a unit dosage form necessary for producing a generally accepted pharmaceutical. Depending on the amount of active ingredient in these formulations, a suitable dose within the specified range can be determined.

Examples of additives that can be mixed in tablets and capsules are binders such as gelatin, corn starch, tragacanth gum, and gum arabic; media such as crystalline cellulose; swelling agents such as corn starch, gelatin, and alginic acid; lubricants such as magnesium stearate; sweetening agents such as sucrose, lactose or saccharine; and corrigents such as peppermint, wintergreen oil and cherry. When the unit dosage from is capsule, liquid carriers such as oil can be further included in the above-described ingredients. Sterilized mixture for injection can be formulated using media such as distilled water for injection according to the realization of usual pharmaceuticals. Physiological saline, glucose, and other isotonic solutions containing adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used as an aqueous solution for injection. They can be used in combination with a suitable solubilizer, for example, alcohol, specifically ethanol and polyalcohols such as propylene glycol and polyethylene glycol, non-ionic surfactants such as Polysorbate 80TM and HCO-50. Sesame oil or soybean oil can be used as an oleaginous liquid, and also used in combination with benzyl benzoate or benzyl alcohol as a solubilizer. Furthermore, they can be further formulated with buffers such as phosphate buffer and sodium acetate buffer; analgesics such as procaine hydrochloride; stabilizers such as benzyl alcohol and phenol; and antioxidants. Injections thus prepared can be loaded into appropriate ampoules.

Methods well-known to those skilled in the art can be used for administering pharmaceutical compounds of the present invention to patients, for example, by intraarterial, intravenous, or subcutaneous injection, and similarly, by intranasal, transtracheal, intramuscular, or oral administration. Doses and administration methods are varied depending on the body weight and age of patients as well as administration methods. However, those skilled in the art can routinely select them. DNA encoding a polypeptide of the present invention can be inserted into a vector for the gene therapy, and the vector can be administered for treatment. Although doses and administration methods are varied depending on the body weight, age, and symptoms of patients, those skilled in the art can appropriately select them. For example, a dose of the compound which bind to the polypeptides of the present invention so as to regulate their activity is, when orally administered to a normal adult (body weight 60 kg), about 0.1 mg to about 100 mg/day, preferably about 1.0 mg to about 50 mg/day, more preferably about 1.0 mg to about 20 mg/day, although it is slightly varied depending on symptoms.

When the compound is parenterally administered to a normal adult (body weight 60 kg) in the injection form, it is convenient to intravenously inject a dose of about 0.01 mg to about 30 mg/day, preferably about 0.1 mg to about 20 mg/day, more preferably about 0.1 mg to about 10 mg/day, although it is slightly varied depending on patients, target organs, symptoms, and administration methods. Similarly, the compound can be administered to other animals in an amount converted from the dose for the body weight of 60 kg.

Following administration of a medicament of the invention, the mammal's physiological condition can be monitored in various ways well known to the skilled practitioner.

VIII. Gene Therapy:

Protein and peptide test compounds identified as disruptors of the MELK/Bcl-G association may be therapeutically delivered using gene therapy to patients suffering from cancer, e.g., breast, bladder, and lung cancer. Exemplary test agents amenable to gene therapy techniques include, but are not limited to, converting enzymes as well as peptides that directly alter the MELK/Bcl-G association by steric or allosteric interference. In some aspects, gene therapy embodiments include a nucleic acid sequence encoding a suitable identified test compound of the invention. In some embodiments, the nucleic acid sequence includes those regulatory elements necessary for expression of the test compound in a target cell. The nucleic acid may be equipped to stably insert into the genome of the target cell (see e.g., Thomas KR and Capecchi MR (1987) Cell 51:503 for a description of homologous recombination cassettes vectors).

Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

For general reviews of the methods of gene therapy, see Gold spiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 33:573-96; Mulligan, 1993, Science 260:926-32; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191-217; 1993, TIBTECH 11(5): 155-215). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds ), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.

IX. Screening Kits: The present invention also provides an article of manufacture or kit containing materials for screening for an agent useful in treating or preventing cancer, particularly breast, bladder, or lung cancer. Such an article of manufacture may comprise one or more labeled containers of materials described herein along with instructions for use. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic.

In one embodiment, the screening kit comprises: (a) a first polypeptide comprising a Bcl-G-binding domain of a MELK polypeptide; (b) a second polypeptide comprising a MELK-binding domain of a BcI-G polypeptide, and (c) means (e.g., a reagent) to detect the interaction between the first and second polypeptides.

In some embodiments, the first polypeptide, i.e., the polypeptide comprising the

Bcl-G-binding domain, comprises a MELK polypeptide. Similarly, in other embodiments, the second polypeptide, i.e., the polypeptide comprising the MELK-binding domain, comprises a BcI-G polypeptide.

In some embodiments, the polypeptide comprising a Bcl-G-binding domain is expressed in a living cell.

The present invention further provides articles of manufacture and kits containing materials useful for treating the pathological conditions described herein are provided. Such an article of manufacture may comprise a container of a medicament as described herein with a label. As noted above, suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In the context of the present invention, the container holds a composition having an active agent which is effective for treating a cell proliferative disease, for example, breast, bladder or lung cancer. The active agent in the composition may be an identified test compound (e.g., antibody, small molecule, etc.) capable of disrupting the MELK/Bcl-G association in vivo. The label on the container may indicate that the composition is used for treating one or more conditions characterized by abnormal cell proliferation. The label may also indicate directions for administration and monitoring techniques, such as those described herein.

In addition to the container described above, a kit of the present invention may optionally comprise a second container housing a pharmaceutically-acceptable diluent. It may further include other materials desirable from a commercial end-user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising an agent of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

Hereinafter, the present invention is described in more detail by reference to the Examples. However, the following materials, methods and examples only illustrate aspects of the invention and in no way are intended to limit the scope of the present invention. As such, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

EXAMPLES

MA TERIALS AND METHODS: (A) Breast Cancer Cell-Lines And Clinical Samples:

Human-breast cancer cell lines HBLlOO, HCC 1937, MCF-7, MDA-MB-435S, SKBR3, T47D and YMBl, and COS7 cells were purchased from American Type Culture Collection (ATCC, Rockville, MD), and were cultured under their respective depositors' recommendation. HBC4, HBC5 and MDA-MB-231 cells lines were kind gifts from Dr. Yamori of Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research. All cells were cultured in appropriate media; i.e. RPMI- 1640 (Sigma- Aldrich, St. Louis, MO) for HBC4, HBC5, SKBR3, T47D, YMBl, and HCC1937 (with 2mM L-glutamine); Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) for HBLlOO, COS7; EMEM (Sigma-Aldrich) with O. lmM essential amino acid (Roche, Basel, Switzerland), ImM sodium pyruvate (Roche), O.Olmg/ml Insulin (Sigma- Aldrich) for MCF-7; L- 15 (Roche) for MDA-MB-231 and MDA-MB-435S. Each medium was supplemented with 10% fetal bovine serum (Cansera) and 1% antibiotic/antimycotic solution (Sigma-Aldrich). MDA-MB-231 and MDA-MB-435S cells were maintained at 37⁰C an atmosphere of humidified air without CO₂. Other cell-lines were maintained at 37⁰C an atmosphere of humidified air with 5% CO₂. Tissue samples from surgically-resected breast cancers, and their corresponding clinical information were obtained after obtaining written informed consent.

(B) Northern-Blot Analysis:

Total RNAs were extracted from all breast cancer cell lines using RNeasy kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. After treatment with DNase I (Nippon Gene, Osaka, Japan), mRNA was isolated with mRNA purification kit (GE Healthcare, Buckinghamshire, United Kingdom) following the manufacturer's instructions. A 1-μg aliquot of each mRNA, along with polyA(+) RNAs isolated from normal mammary gland, lung, heart, liver, kidney, brain (Takara Clontech, Kyoto, Japan), were separated on 1% denaturing agarose gels and transferred to nylon membranes (Breast cancer-Northern blots). Breast cancer and Human multiple-tissue Northern blots (Takara Clontech) were hybridized with [γ³²P]-dCTP-labeled PCR products of MELK prepared by RT-PCR (see below). Pre- hybridization, hybridization and washing were performed according to the supplier's recommendations. The blots were autoradiographed with intensifying screens at -80⁰C for 14 days. Common probe for MELK and for the specific sequence among variant 1, 2 and 3 of MELK were prepared by PCR using the following primer sets; 5 ' -TTATC ACTGTGCTC ACC AGGAG-3 ' (SEQ ID NO : 9) and

5'-CAGTAACATAATGACAGATGGGC-S ' (SEQ ID NO: 10), and were radioactively labeled using megaprime DNA labeling system (GE Healthcare).

(C) cDNA Library Screening:

A cDNA library was constructed using and superscript™ plasmid system with gateway™ technology for cDNA synthesis and cloning kit (Invitrogen, Carlsbad, CA) and poly(A)+ RNA obtained from breast cancer cell line T47D, and screened 3x 10⁶ independent clones of this library with cDNA probes corresponding to nucleotide 1251-2094 (843 bp) of Vl variant of MELK.

(D) In Vitro Translation Assay: The five variants (V 1 , V2, V3 , V4 and V5) of MELK which were cloned into pSPORT-1 expression vector during a cDNA library screening described as above were used as templates for transcription/translation experiments in vitro. The plasmids (lμg) were transcribed and translated using TNT Coupled Reticulocyte Lysate Systems (Promega, Madison, WI) in the presence of ε-labeled biotinylated lysine-tRNA according to the manufacturer's instructions. Proteins were electrophoresed on 5-20 % gradient SDS- polyacrylamide gels. After electroblotting, the biotinylated proteins are visualized by binding streptavidin-horseradish peroxidase, follows by chemiluminescent detection (GE Healthcare).

(E) Plasmid constructions:

For MELK expression vectors, the corresponding coding sequences of all constructs were cloned and amplified by PCR using KOD-Plus DNA polymerase (Toyobo, Osaka,

Japan) and ligated into the EocR I and Xho I sites of pCAGGSnHC expression vector in frame with C-terminal HA-tag unless stated elsewhere.

The primer sets used for cloning were listed as followings: Wildtype MELK-Vl forward: 5'-CGGAATTCACTATGAAAGATTATGATGAAC-S' (SEQ ID NO: 3), and reverse 5'-AAACTCGAGTACCTTGCAGCTAGATAGGAT-S ' (SEQ ID NO: 4). Kinase- dead mutant MELK (D 150A) was generated by QuickChange Site-Directed Mutagenesis Kit (Stratagene) using following set of primers; D 150A forward:

5'-CATAAATTAAAGCTGATTGCCTTTGGTCTCTGTGCAAAACC-S ' (SEQ ID NO: 5), reverse: 5'-GGTTTTGCACAGAGACCAAAGGCAATCAGCTTTAATTTATG-S' (SEQ ID NO: 6).

For constructing of BcI-G (long isoform) (GenBank Accession No. AAG59793) (SEQ ID NO: 8 encoded by SEQ ID NO: 7), the corresponding ORFs were generated by RT- PCR using human testis mRNA as a template and the primers as shown in Table. 2. The ORFs were then ligated into Notl and Xho\ sites of pCAGGSn3FH vector in frame with N- terminal Flag-tag. All of constructs were confirmed by DNA sequencing (AB 13700, PE Applied Biosystems, Foster, CA).

Table 1. MELK-siRNA sequences

Table 2. The sequences of primers for constructs of BcI-G expresion vectors

The underline sequences indicate restriction enzyme site, Xhol (CTCGAG)and Notl (GCGGCCGC).

For constructs of C-I, C-2, C-3 and C-4, WT-forward primer is commonly used, and for constructs of N-I, N-2 N-3, N-4, N-5, N-6, N-7 and N-8, WT-reverse primer is commonly used.

(F) Construction OfMELK Specific-siRNA Expression Vector Using Psihlbx: A vector-based RNAi (RNA interference) system was established using psiHlBX3.0 siRNA expression vector according to the previous report (Shimokawa T et al. Cancer Res 63 :6116-20 2003 ). siRNA expression vectors against MELK (psiH IBX-MELK) were prepared by cloning of double-stranded oligonucleotides into the Bbsl site of the psiHlBX3.0 vector (Table. 1). Control plasmids, psiHlBX-SC (scrambled control) was prepared by cloning the following double-stranded oligonucleotidesinto the Bbsl site in the psiHlBX3.0 vector:

5'-TCCCGCGCGCTTTGTAGGATTCGTTCAAGAGACGAATCCTACAAAGCGCGC-S ' (SEQ ID NO: 27) and

5'-AAAAGCGCGCTTTGTAGGATTCGTCTCTTGAACGAATCCTACAAAGCGCGC-B ' (SEQ ID NO: 28)

All constructs were confirmed by DNA sequencing.

(G) Gene-Silencing Effect OfMELK By siRNA:

Human breast cancer cells lines, T47D and MCF-7 was plated onto 15-cm dishes (4xlO⁶ cells/dish) and transfected with 16μg of psiH IBX-MELK si-#3 (SEQ ID NO: 11), si- #4 (SEQ ID NO: 14) and psiHlBX-SC (scrambled control) as negative control using FuGENEό reagent (Roche) according to the supplier's recommendations. At 24 hours after transfection, cells are re-seeded again for colony formation assay (3x10⁶ cells/ 10 cm dish), semi-quantitative RT-PCR (IxIO⁶ cells / 10 cm dish) and MTT assay (5xlO⁵ cells / well). The MELK siRNA-transfected T47D or MCF7 cells were selected with medium containing 0.7 mg/ml or 0.6mg/ml of neomycin (Geneticin, Invitrogen), respectively. Total RNA was extracted from cells and an aliquot of 0.5μg was reverse-transcribed into cDNA (superscript II; Invitrogen) 4 days after neomycin selection, and then the knockdown effect of siRNAs was confirmed by a semi-quantitative RT-PCR using specific primer sets for MELK and β2MG\

5'-TTAGCTGTGCTCGCGCTACT-S' (SEQ ID NO: 29) and 5'-TCACATGGTTCACACGGCAG-S ' (SEQ ID NO: 30) for β '2MG as an internal control, and 5'-TTATCACTGTGCTCACCAGGAG-S ' (SEQ ID NO: 9) and

5'-CAGTAACATAATGACAGATGGGC-S ' (SEQ ID NO: 10) ϊoτMELK.

Transfectants expressing siRNA were grown for 3 weeks in selective media containing neomycin, then fixed with 4% paraformaldehyde for 15 min before staining with Giemsa's solution (Merck, Whitehouse Station, NJ) to assess colony number. To quantify cell viability, MTT assays were performed at 7 days after selection with Cell Counting Kit-8 (Wako, Osaka, Japan) according to manufacture's protocols. Absorbance at 570 nm wavelength was measured with a Microplate Reader 550 (Bio-Rad). For colony formation assay, cells were allowed to grow for 28 days in selective media containing neomycin. These experiments were performed in triplicate. (H) Generation And Purification Of His-Tagged MELK Recombinant Protein:

The entire coding sequence of WT-MELK and D150A-MELK were subcloned into the pET2 Ia vector (Merck, Novagen, Darmstadt, Germany), respectively. The recombinant WT- and D150A-MELK expression vectors were expressed in BL21 codon-plus NIL competent cells (Stratagene), respectively. Inductions were carried out at 0.5 mM IPTG, and then cells were further cultured with the TBG-M9 buffer at 27⁰C for 4 hours. Purification was performed using Ni-NTA superflow (Qiagen) under the native condition according to the supplier's instructions.

(I) In Vitro Kinase Assay: 0.5 μM His-tagged recombinant MELK proteins (WT-MELK and D 150A-MELK) were incubated for 30 minutes at 30 ⁰C with 5 μg recombinant histone Hl protein (UPSTATE, Lake Placid, NY) in 0.05 mL of kinase buffer containing 30 mM Tris-HCL pH 7.5, 0.1 mM EGTA, 10 mM DTT, 4OmM NaF, 40 mM sodium β-glycerophosphate (Sigma- Aldrich), 50 μM cold ATP, lOCi of [γ-³²P] ATP (GE Healthcare) and 10 mM MgCL₂. The reaction was terminated by SDS-sample buffer and boiled for 3 minutes prior to 10 % SDS-PAGE electrophoresis. The gel was then dried, and autoradiographed with intensifying screens at room temperature overnight.

(J) Immunocomplex Kinase Assay:

HeLa cells were transfected with 16 μg of pC AGGS-MELK-HA expression vector plasmid. Transfected cells were lysed with lysis buffer (50 mM Tris-HCL (pH 7.5), 150 mM NaCL, 1% NP-40, 40 mM NaF, 40 mM β-glycerophosphate, protease inhibitor cocktail set III (Calbiochem), and then immunoprecipitated with anti-HA rat antibody (Roche). The protein bound Rec-protein G sepharose 4 B beads (ZYMED, S. San Francisco California USA) were washed twice with ImI of lysis buffer and once with ImI of kinase buffer (50 mM Tris pH 7.5, 1OmM MgCL, 25 mM NaCl and 1 mM DTT) as kinase enzyme source.

To prepare the kinase substrate, HeLa cells were transfected with BcI-G (long isoform) and then were immunoprecipitated with Flag-conjugated agarose M2 gel (Sigma) at 4 °C for one hour as the potential substrate. Immunoprecipitates were washed five times with lysis buffer containing 50 mM Tris-HCL (pH 7.5), 150 mM NaCL, 1% NP-40 and protease inhibitor cocktail (Calbiochem). Aliquot of immunoprecipitates were subjected to immunoblotting using anti-Flag antibody rabbit origin (Sigma) to confirm the success of immunoprecipitation. In vitro kinase assay was carried out as described above except that general substrate histone Hl was replaced by 20 μl of immunoprecipiates.

(K) Protein Pull Down A ssay:

HBC4 cells were lysed with lysis buffer (40 mM Tris-HCl (pH 7.5), 1% Trition X- 100, 2.5 mM EDTA, 15 mM DTT and protease inhibitor) as previously described (Knebel A et al. EMBO J 20 4360-9 2001.) with minor modification. An aliquot (7.5 μg) of the lysate was mixed for 5 minutes at 30 ⁰C with 480 μg (0.5 μM) of MELK recombinant protein immobilized on lOOμl of Ni-NTA agarose beads (QIAGEN), 2 μM cold ATP and 12.5 ml of kinase buffer. The beads were washed with lysis buffer before mixing with SDS-sample buffer. Eluted proteins were analyzed by SDS-PAGE, and subjected to silver staining and mass spectrometry.

(L) Co-Immunoprecipitation Assay:

HEK 293 cells in a 15-cm dish were transiently co- transfected for 48 h with pCAGGSn3FC-Bcl-G (16 μg) and pCAGGS-MELK-HA (16 μg) as experimental design using FuGENEό transfection reagent (Roche Applied Science). Cells were lysed with a lysis buffer as described in above section. The exact was pre-cleaned with normal mouse IgG (1.2 μg) and 30 μl of rec-Protein G Sepharose 4B (Zymed, S. San Francisco California USA) at 4 °C for 30 minutes. The lysate was incubated with 30 μl anti-flag agarose M2 gel (Sigma) at 4 ⁰C for 12 hours. After washing three times with lysis buffer, proteins on beads were solubilized by SDS-sample buffer.

(M) Immunocytochemical Staining:

COS7 cells (lxlO⁴)were co-transfected with pCAGGS-Flag-full-length BcI-G and pCAGGS-WT-MELK-HA. Forty-eight hours after transfection, cells were fixed with 4% paraformaldehyde solution at 4⁰C for 30 min, and rendered permeable with PBS (-) containing 0.1% Triton-X for 2.5 min at room temperature. Cells were then washed with PBS (-) before covering with blocking solution (3% BSA) for 1 hour at room temperature. Exogenous MELK expression was detected with anti-HA rat antibody 3F10 (Roche, Applied Science), and labeled with Alexa-488 (green). By contrast, exogenous BcI-G expression was detected with anti-Flag monoclonal M2 antibody (Sigma) and subsequently labeled with Alexa-594 (red). Nucleus was stained with DAPI (blue). Expression of proteins was observed by confocal microscope (Leica DMRXA2). (N) Apoptosis And Flow Cytometry Analysis:

COS7 cells (IxIO⁶) grow on 10 cm dish was co-transfected with 10 μg of pCAGGS-WT-MELK-HA and a set of pCAGGS-Flag-Bcl-G expression vectors (N-I, N-3, N- 4, N-5, C-I, C-2 and C-4) using Fugeneό (Roche Applied Science) according to supplier's protocol. For FACS analysis, cells were harvested with trypsin and fixed with 70% ethanol at room temperature for 30 min. After centrifuge for removal of 70% ethanol, cells were treated with 0.5 ml of PBS (-) containing 2μg/ml of RNase I for 30 min followed by staining in 1 ml of PBS (-) containing 20 mg/ml of propidium iodide (PI) for 30 min. The percentages of each fraction of cell cycle phases were determined by at least 10,000 cells through flow cytometer (FACSCalibur; Becton Dickinson, San Diego, CA).

(O) Peptide inhibition assay:

T47D breast cancer cells were seeded in a 96-well plated (Corning Costar) at the concentration of 2500 cells/100 ul /well. The following day, cells were incubated with the medium (RPMI) containing 6.25 μ M of BcI-G peptide (RRRRRRRRRRRGGGTIEFKILAYYTRHHVF) and two scramble controls (SCl : RRRRRRRRRRRGGGEHITAFTRKLIHVFYY;

SC2: RRRRRRRRRRRGGGIYTVARFTHIHFKLYE) for 8 days. The medium containing peptide was changed every 2days. The treatment was carried out in triplicate for each peptide. The effect of peptide on cell growth was measured by using cell counting kit 8 according to manufacture's instruction.

RESULTS:

(A) Up-Regidation OfMELK In Breast Cancer:

The genome-wide expression profiles of 81 clinical breast cancers by means of cDNA microarray analysis representing 23,040 genes in combination with laser microbean microdissection system was previously reported (Nishidate T et al. Int J Oncol 25:797-819 2004 ). Among genes highly over-expressed in breast cancer, the MELK gene was found to be transactivated in the majority of the breast cancers examined. The subsequent semiquantitative RT-PCR analysis confirmed elevated expression of MELK in clinical breast cancer specimens (Figure IA).

To further examine the expression pattern of this gene in breast cancer cell lines and normal tissues, northern blot analyses were performed using mRNAs from multiple human tissues and breast cancer cell lines. Using a cDNA fragment (554 bp) corresponding to the 3' UTR portion of the MELK gene as a probe, two bands of approximately 1.3 and 2.5 kb were unexpectedly detected, indicating the existence of alternating splicing variants. The expression of approximately 1.3 kb transcripts were observed in skeletal muscle, while approximately 2.5 kb transcripts was detected in testis and thymus, small intestine, stomach (Figure IB). Interestingly, 2.5 kb transcripts were significantly over-expressed in breast cancer cells, but not expressed in vital organs with breast cancer cell lines-northern blot analysis using a same probe (Figure 1C).

To characterize these variants specifically transcribed in breast cancer cells, a cDNA library constructed from poly(A)+ RNA obtained from breast cancer cell line T47D was screened. Subsequent cDNA sequencing analysis identified five different transcriptional variants (Figure ID). Among them, three cDNAs, designated as Vl, V2 and V3, are 2501bp, 2368bp, and 225 lbp, respectively. The other two transcripts, V4 and V5, are respectively, 1212bp and 520bp, but expression of these variants are very low because only one independent clone derived from each variant, respectively, was isolated in cDNA library screening. Moreover, Melk2 transcript that has been reported was not included among those transcripts (Heyer BS et al. Dev Dyn 215:344-51 1999, Gray D et al. Cancer Res 65:9751-61 2005.). To confirm results of northern blot analysis, breast cancer cell lines-northern blot analysis was performed using the common sequence (within exonl3) among Vl, V2 and V3 as a probe. As expected, these transcripts were specifically over-expressed in all of breast cancer cell lines, but not detectably expressed in normal tissues (Figure IE).

To examine whether all five transcripts were translated into proteins, an in vitro translation assay was performed. Three transcripts Vl, V2 and V3 were translated into proteins of 75, 71 and 66 kDa respectively but proteins corresponding to the two remaining shorter transcripts of V4 and V5 variants were not detected (Figure IF). The subsequent semi-quantitative RT-PCR analysis confirmed that Vl transcript was dominantly expressed in breast cancer cells rather than those of V2 and V3 transcripts (data not shown).

Sequence analysis indicated that the V2 variant lacked a part of exon 3 and the V3 variant also lacked of exons 3 and 4 due to alternative splicing. The exon skipping of these two variants, V2 and V3 caused a premature translational termination. An alternative initiation codon was found downstream of the stop codon in the same reading frame and must have been used for translation of these two variants. Since the V2 and V3 proteins lose a part of the kinase domain, it is difficult to speculate their kinase activities. They represent proteins with altered signaling characteristics, or they act as dominant negative splice variants. In this study, only kinase activity possessing Vl transcript is focused in the hope to identify the kinase substrates.

(B) Growth-Inhibitory Effects Of Small-Interfering RNA (siRNΛ) Designed To Reduce Expression OfMELK:

To assess the role of MELK in breast cancer cell growth, the expression of endogenous MELK was knocked down in breast cancer lines, T47D and MCF7 (Figure 2 A and 2B), which showed overexpression of MELK, by means of the mammalian vector-based RNA interference technique (see Materials and Methods). Expression levels of MELK were examined by semi-quantitative RT-PCR experiments and found that MELK-siKNA si-#3 and si-#4 suppressed expression, compared with scramble-siRNA (SC) as a control (Figure 2). Colony-formation and MTT assays revealed that both of MELK-siKNA (si-#3 and si-#4) significantly suppressed cell growth of T47D and MCF7 cells, concordant with the results of the knock-down effect of this gene. These results are consistent with the conclusion that MELK has a significant role in the growth of the breast cancer cells.

(C) Bacterial MELK Recombinant Proteins Possess Kinase A ctivity:

To characterize kinase activity of MELK protein, active forms of wildtype (WT) and kinase-dead mutant MELK were generated. The latter has replacement of an asparate to alanine at 150 residues within DFG triplet in the kinase subdomain VII, which is required for the ATP binding as described previously (Davezac N et al. Oncogene 21 :7630-41 2002,

Vulsteke V et al. J Biol Chem 279:8642-7 2004, Beullens M et al. J Biol Chem 280:40003- 11 2005. 11-3). As shown in Figure 3A, Coomassie Blue staining of SDS-PAGE revealed an additional retarded band above the predicted 75kDa band of MELK in WT but not in D 150A kinase-dead (D 150A) recombinant protein. It was confirmed that an additional retarded band of MELK in WT was disappeared after treatment of λ-phosphatase (data not shown), supporting that this bacterial WT-MELK recombinant protein phosphorylated by itself as described previously (Davezac N et al. Oncogene 21 : 7630-41 2002. Blot J et al. Dev Biol 241 :327-38 2002. Lizcano JM et al. EMBO J 23:833-43 2004.).

To further confirm whether these recombinant proteins are active, their phosphorylating ability was tested by an in vitro kinase assay using histone Hl that has used as a general substrate for measurement of MELK kinase activity (Davezac N et al. Oncogene 21 :7630-41 2002, Beullens M et al. J Biol Chem 280:40003-11 2005 ). The results showed that bacterial WT but not D 150A recombinant proteins possessed kinase activity and phosphorylated histone Hl protein in vitro (Figure 3B). Additionally a 75kDa band was observed in the reaction mixture without histone Hl substrate indicating bacterial WT can autophosphorylate itself as descried previously (Davezac N et al. Oncogene 21 :7630-41 2002, Beullens M et al. J Biol Chem 280:40003-11 2005 ).

(D) Identification OfBcI-G As A MELK-Interacting Protein:

Although substrates that bind to MELK have been reported {e.g., cdc25B (Davezac N et al. Oncogene 21:7630-41 2002); NIPPl (Vulsteke V et al. J Biol Chem 279:8642-7 2004); and ZPR9 (Seong HA et al. Biochem J 361:597-604 2002)), the significance of their phosphorylation and their biological functions in cancer cells remains unclear. Therefore, to investigate the biological functions of MELK protein in breast cancer cells, the present inventors searched for novel substrates for MELK by means of an in vitro protein pull-down assay that used recombinant WT-MELK and kinase-dead MELK (D 150A) proteins as described above section. Silver staining of SDS-PAGE gels that contained the pull-downed cell lysates identified a 27-kDa protein only in proteins immunoprecipitated with WT proteins but not with D 150A MELK protein (data not shown).

MALDI-TOF mass spectrometry defined this 27-kDa protein to be a partial portion of long isoform of BcI-G that belongs to the Bcl-2 protein family (Guo B et al. J Biol Chem 276:2780-5 2001.). The expression of BcI-G at mRNA and protein levels in almost all of breast cancer cell-lines was confirmed by western blot and semi-quantitative RT-PCR analyses (Figure 4A). Subsequently, to validate the interaction between MELK and BcI-G, constructs designed to express following proteins were generated : Flag-tagged BcI-G and HA-tagged MELK. These constructs were co-transfected into HeLa cells, and then the proteins were immunoprecipitated with anti-Flag antibody. Immunoblotting of the precipitates using anti-HA antibodies indicated that Flag-Bcl-G was co-precipitated with HA- MELK (Figure 4B).

To provide additional proof that MELK co-localize with BcI-G in COS7 cells, immunocytochemical staining experiments were performed. WT-MELK and BcI-G were transiently co-transfected into COS7 cells, and both exogenous proteins were observed to be co-localized diffusely in the cytoplasm (Figure 4C). These findings indicate that MELK can physically interact with BcI-G in vivo. To further determine which regions of BcI-G can interact with MELK protein, co- immunoprecipitation analyses were performed using a series truncated forms of Flag-tagged BcI-G (N-I to N-8 and C-I to C-4) and HA-tagged WT-MELK that were co-transfected into HeLa cells (Figure 4D). As shown in Figure 4E, immunoblotting analysis with anti-Flag or anti-HA antibodies showed that N-I (12-327) and N-6 (24-327) constructs bound to MELK as well as full-length BcI-G, but N-2 (35-327), N-7 (49-327), N-8 (61-327) and N-3 (72-324) did not. Additionally, C-I, C-2 and C-4 constructs including N-terminal regions also bound to MELK. These findings are consistent with the conclusion that the N-terminal portion of BcI-G (24-34 residues) bound to MELK.

(E) MELK Phosphorylates BcI-G:

Next, to examine whether MELK phosphorylated BcI-G as a substrate, an immune complex kinase assay was performed using MELK recombinant protein (WT-MELK and D150A-MELK) and BcI-G expression vector. Firstly, the amount of immunoprecipitated protein of exogenously expressed BcI-G was confirmed by western blot analysis (Figure 5A). It was demonstrated that only WT-MELK but not D 15 OA-MELK phosphorylated 4OkD of BcI-G protein (Figure 5B). In addition, an approximately 75kDa-MELK protein was detected as autophosphorylated by itself (Figure 5B). Furthermore, it was confirmed that recombinant WT-MELK protein also phosphorylated recombinant GST-fused BcI-G protein but D 150A did not phosphorylate it (Figure 5C). These findings are consistent with the conclusion that BcI-G is an ideal substrate for MELK kinase.

To further determine which regions of BcI-G protein are necessary for phosphorylation by MELK, an immune complex kinase assay was performed using a WT- MELK and a various set of truncated forms of BcI-G constructs as shown in Figure 4D. Firstly, the amount of immunoprecipitated protein of N-terminal truncated constructs of BcI- G (N-I, N-3, N-4 and N-5) was confirmed by western blot analysis (Figure 5D). The results showed that phosphorylated BcI-G proteins were detected when overexpressed with the full- length BcI-G (FL) or N-I constructs, whereas were completely abolished when overexpressed with N-3, N-4 and N-5 constructs, respectively (Figure 5E). These results indicate that MELK constructs that do not bind to BcI-G will not phosphorylateBCL-G. Subsequently, C- terminal regions of BcI-G were examined to determine if they were phosphorylated by MELK, particularly C-terminal truncated constructs (C-I, C-2 and C-4). It was further confirmed that the amount of immunoprecipitated protein of C-terminal truncated BcI-G constructs by western blot analysis (Figure 5F). Eventually, Figure 5G showed that all of C-terminal constructs (C-I, C-2 and C-4) were phosphorylated by MELK. Taken together, these findings indicate that N-terminal MELK-binding region of BcI-G (24-34 residues) is necessary for phosphorylation of BcI-G protein by MELK.

(F) MELK Involved In Apoptotic Pathway Through BcI-G:

Since MELK physically interacted with and phosphorylated BcI-G, it was hypothesized that MELK is involved in the apoptosis cascade through BcI-G pathway. To verify this hypothesis, HA-tagged MELK (WT or D 150A) and a Flag-tagged BcI-G constructs were transiently co-transfected into COS7 cells, respectively, and then FACS analysis was performed to measure the percentage of sub-Gl cell population {see Material and Methods).

Firstly, the expression of MELK and BcI-G proteins was confirmed by western blot analysis (Figure 6A). As shown in Figure 6B, the overexpression of full-length (FL) BcI-G protein reproducibly induced increases of sub-Gl cell population (20.32%; lower left panel) as compared with Mock-transfection (5.78%; upper left panel), indicating BcI-G tend to induce apoptosis as described previously (Reed JC. Oncogene 17:3225-36 1998 ). In contrast, interestingly the co-overexpression with MELK-WT and FL-BcI-G proteins reduced sub-Gl cell population (16.48%; lower middle panel), while MELK-D 150A increased it (22.69%; lower right panel). These findings are consistent with the conclusion that kinase activity of MELK involves regulation of the pro-apoptotic function of BcI-G.

Furthermore, to confirm which regions of BcI-G required for regulation of apoptosis in more detail, the sub-Gl cell population was examined by FACS analysis when overexpressed with a set of truncated BcI-G constructs (Figure 4D), respectively. As shown in Figure 6C, FACS analysis showed that the N-3 and N-4 constructs which can not bind to WT-MELK induced clearly increases of sub-Gl cell population (21.9% and 22.4%; middle panels), as compared with sub-Gl cell population by introduction of FL-BcI-G protein and N- 1 which can bind to WT-MELK (18.2; upper panel and 19.7%; middle panel) (Figure 6C). Furthermore, each sub-Gl cell population was not changed when expressed with C-I or C-2, as compared with FL-BcI-G construct, supporting that C-terminal region of BcI-G (295-327 residues) is not necessary for apoptosis.

However, interestingly, C-4 which has no BH3 domain showed no increase of subGl population (7.8%), as compared with Mock-induced subGl population (6.5%) in spite of binding region of BcI-G with MELK, representing BH3 domain is necessary for apoptosis through BcI-G function. Thus, MELK suppress increases of sub-Gl cell population through interaction with N-terminal region of BcI-G Taken together, these data are consistent with the conclusion that MELK regulates apoptosis through phosphorylation of N-terminal region (1-215 residues) of BcI-G and then promote cancer cell growth of breast cancer though block of pro-apoptotic function of BcI-G.

(G) Inhibibtion of the interaction between BcI-GL and MELK by cell-permeable peptide:

To investigate the biological significance of an interaction between BcI-GL and MELK in breast carcinogenesis, the present inventers attempted to inhibit the interaction by the use of the dominant-negative peptide. Since the binding region of BcI-G was determined as described above, the present inventers designed the 16-amino acid including this binding region (amino acids 21 to 36) that was conjugated with arginine (R)-repeat to facilitate cell permeability. The inventers investigated whether this peptide could inhibit cancer cell growth or not, by treatment of breast cancer cells with this peptide. This peptide treatment suppressed the growth of cancer cells in time-dependent manner. The finding suggests that this dominant-negative peptide is attractive candidates for disrupting proten-protein interactions.

INDUSTRIAL APPLICABILITY:

The data reported herein add to a comprehensive understanding of breast cancer, facilitate development of novel diagnostic strategies, and provide clues for identification of molecular targets for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of breast tumorigenesis, and provides indicators for developing novel strategies for diagnosis, treatment, and ultimately prevention of breast cancer.

In particular, the gene-expression analysis of breast cancer samples described herein, obtained through a combination of laser-capture dissection and genome-wide cDNA microarray, has identified MELK as a specific molecular target for cancer prevention and therapy. As demonstrated herein, MELK interacts with and specifically phosphorylates BcI-G, thereby inhibiting its pro-apoptotic function of BcI-G. Thus, agents that inhibit the binding of MELK and BcI-G and prevent their respective activities may find therapeutic utility as anticancer agents, particularly anti-cancer agents for the treatment of breast, bladder and lung cancers. For example, agents that block the expression of MELK, or prevent its activity, particularly with respect to its phosphorylation of pro-apoptotic protein BcI-G, find therapeutic utility as anti-cancer agents, particularly anti-cancer agents for the treatment of breast, lung and bladder cancers. Examples of such agents include antisense oligonucleotides, small interfering RNAs, and ribozymes against the MELK gene, and antibodies that recognize MELK.

All publications, databases, Genbank sequences, patents, and patent applications cited herein are hereby incorporated by reference.

While the invention has been described in detail and with reference to specific embodiments thereof, it is to be understood that the foregoing description is exemplary and explanatory in nature and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, one skilled in the art will readily recognize that various changes and modifications can be made therein without departing from the spirit and scope of the invention. Thus, the invention is intended to be defined not by the above description, but by the following claims and their equivalents.

Claims

1. A method of screening for an agent useful in treating or preventing cancer, said method comprising the steps of:

(a)contacting a polypeptide comprising a Bcl-G-binding domain of a MELK polypeptide with a polypeptide comprising a MELK-binding domain of a BcI-G polypeptide in the presence of a test agent;

(b)detecting binding between the polypeptides; and (c) selecting the test agent that inhibits binding between the polypeptides.

2. The method of claim 1, wherein the polypeptide comprising the Bcl-G-binding domain comprises a MELK polypeptide.

3. The method of claim 1, wherein the polypeptide comprising the MELK-binding domain comprises a BcI-G polypeptide.

4. The method of claim 1, wherein the cancer is selected from the group consisting of breast, bladder and non-small cell lung cancer.

5. A kit for screening for an agent useful in treating or preventing cancer, wherein the kit comprises:

(a)a polypeptide comprising a Bcl-G-binding domain of a MELK polypeptide; (b)a polypeptide comprising a MELK-binding domain of a BcI-G polypeptide; and (c)means to detect the interaction between the polypeptides.

6. The kit of claim 5, wherein the polypeptide comprising the Bcl-G-binding domain comprises a MELK polypeptide.

7. The kit of claim 5, wherein the polypeptide comprising the MELK-binding domain comprises a BcI-G polypeptide.

8. The kit of claim 5, wherein the cancer is selected from the group consisting of breast, bladder and non-small cell lung cancer.

9. A method of screening for an agent that induces apoptosis of cancer cells comprising the steps of: a) contacting a polypeptide selected from the group consisting of: i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (full-length MELK); ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 wherein one or more amino acids are added, substituted, deleted, or inserted, and that has a biological activity equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; iii) a polypeptide comprising an amino acid sequence that has at least about 80% homology to the amino acid sequence of SEQ ID NO: 2 wherein the polypeptide has a biological activity equivalent to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; and iv) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID

NO: 1, wherein the polypeptide has a biological activity equivalent to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; with a substrate phosphorylated by the polypeptide and a test agent under a condition that allows phosphorylation of the substrate; b) detecting the phosphorylation level of the substrate; c) comparing the phosphorylation level of the substrate with the phosphorylation level of the substrate detected in the absence of the test agent; and d) selecting the test agent that reduces the phosphorylation level of the substrate as an agent that induces apoptosis of cancer cells.

10. The method of claim 9, wherein the cancer is selected from the group consisting of breast, bladder and non-small cell lung cancer.

11. The method of claim 9, wherein the substrate is BcI-G or a fragment thereof that comprises at least its phosphorylation site.

12. The method of claim 11, wherein the phosphorylation site is included in MELK-binding region of BcI-G.

13. The method of claim 11, wherein the fragment comprises 12 - 294 residues of BcI-G.

14. The method of claim 13, wherein the fragment comprises 12 - 215 residues of BcI-G.

15. A method of screening for an agent for preventing or treating cancer comprising the steps of: a) contacting a polypeptide selected from the group consisting of: i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 wherein one or more amino acids are added, substituted, deleted, or inserted, and that has a biological activity equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; iii) a polypeptide comprising the amino acid sequence that has at least about 80% homology to SEQ ID NO: 2 wherein the polypeptide has a biological activity equivalent to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; and iv) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID

NO: 1, wherein the polypeptide has a biological activity equivalent to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; with a substrate phosphorylated by the polypeptide and a test agent under a condition that allows phosphorylation of the substrate; b) detecting the phosphorylation level of the substrate; c) comparing the phosphorylation level of the substrate with the phosphorylation level of the substrate detected in the absence of the test agent; and d) selecting the test agent that reduces the phosphorylation level of the substrate as an agent for treating or preventing cancer.

16. A composition for treating or preventing a cancer, wherein the composition comprises a pharmaceutically effective amount of an agent agent selected by the method of claim 1, 9 or 15, and a pharmaceutically acceptable carrier.

17. A kit for screening for an agent useful in treating or preventing cancer, wherein the kit comprises: (a) a polypeptide comprising a phosphorylation site of a BcI-G polypeptide; and

(b) means to detect the phosphorylation of the polypeptide.

18. The kit of claim 17, wherein the polypeptide comprising the phosphorylation site of a BcI-G comprises a BcI-G polypeptide.

19. The kit of claim 17, wherein the kit further comprises a MELK polypeptide.

20. The kit of claim 17, wherein the cancer is selected from the group consisting of breast, bladder and non-small cell lung cancer.

21. A composition for treating and/or preventing cancer, said composition comprising a pharmaceutically effective amount of an agent which inhibits the phosphorylation of a peptide consisting of the amino acid sequence of SEQ ID NO: 8 as an active ingredient, and a pharmaceutically acceptable carrier.

22. The composition of claim 21, wherein the agent inhibits the phosphorylation of a peptide consisting of the amino acid sequence of SEQ ID NO: 8 by MELK.

23. The composition of claim 21, wherein the cancer is selected from the group consisting of breast, bladder and non-small cell lung cancer.

24. A method for treating and/or preventing cancer, said method comprising the step of inhibiting the phosphorylation of a peptide consisting of the amino acid sequence of SEQ

ID NO: 8 by MELK.

25. The method of claim 24, wherein the step comprises an administrating an agent that inhibits the phosphorylation of the peptide by MELK.

26. Use of an agent that inhibits the phosphorylation of a peptide consisting of the amino acid sequence of SEQ ID NO: 8 by MELK, for manufacturing a composition for treating and/or preventing cancer.

27. Use of claim 26, wherein the cancer is selected from the group consisting of breast, bladder and non-small cell lung cancer.

28. A polypeptide comprising SEQ ID NO: 39; or an amino acid sequence of a polypeptide functionally equivalent to the polypeptide, wherein the polypeptide inhibits the biological function of a peptide consisting of SEQ ID NO: 2.

29. The polypeptide of claim 28, wherein the biological function is anti-apoptotic activity.

30. The polypeptide of claim 28, wherein the polypeptide consists of 8 to 50 residues.

31. The polypeptide of claim 28, wherein the polypeptide comprises the amino acid sequence SEQ ID NO: 37.

32. The polypeptide of claim 28, wherein the polypeptide consists of the amino acid sequence SEQ ID NO: 37.

33. The polypeptide of claim 28, wherein the polypeptide is modified with a cell-membrane permeable substance.

34. The polypeptide of claim 33, which has the following general formula:

[R]-[D]; wherein [R] represents the cell-membrane permeable substance; and [D] represents the amino acid sequence of a fragment sequence which comprises SEQ ID NO: 39; or the amino acid sequence of a polypeptide functionally equivalent to the polypeptide comprising said fragment sequence, wherein [R] and [D] are linked directly or indirectly through a linker, wherein the polypeptide inhibits the biological function of a peptide consisting of SEQ ID NO: 2.

35. The polypeptide of claim 34, wherein the linker has the amino acid sequence of "GGG".

36. The polypeptide of claim 34, wherein the cell-membrane permeable substance is any one selected from the group consisting of: poly-arginine;

Tat / RKKRRQRRR/SEQ ID NO: 42;

Penetratin / RQKIWFQNRRMKWKK/SEQ ID NO: 43; Buforin II / TRS SRAGLQFP VGRVHRLLRK/SEQ ID NO: 44;

Transportan / GWTLNSAGYLLGKINLKALAALAKKIL/SEQ ID NO: 45;

MAP (model amphipathic peptide) / KLALKLALKALKAALKLA/SEQ ID NO: 46;

K-FGF / AAV ALLP AVLLALLAP/SEQ ID NO: 47;

Ku70 / VPMLK/SEQ ID NO: 48 Ku70 / PMLKE/SEQ ID NO: 49;

Prion / MANLGYWLLALFVTMWTDVGLCKKRPKP/SEQ ID NO: 50; pVEC / LLIILRRRIRKQ AHAHSK/SEQ ID NO: 51;

Pep-1 / KETWWETWWTEWSQPKKKRKV/SEQ ID NO: 52;

SynBl / RGGRLSYSRRRFSTSTGR/SEQ ID NO: 53; Pep-7 / SDLWEMMMVSLACQY/SEQ ID NO: 54; and

HN-I / TSPLNIHNGQKL/SEQ ID NO: 55.

37. The polypeptide of claim 36, wherein the poly-arginine is Arg 11 (SEQ ID NO: 56).

38. The polypeptide of claim 37, wherein the polypeptide comprises the amino acid sequence SEQ ID NO: 38.

39. An agent for either or both of treating and preventing cancer comprising as an active ingredient a polypeptide which comprises SEQ ID NO: 39; a polypeptide functionally equivalent to the polypeptide; or polynucleotide encoding those polypeptides, wherein the polypeptide inhibits the biological function of a peptide consisting of SEQ ID NO: 2.

40. The agent of claim 39, wherein the biological function is anti-apoptotic activity.

41. The agent of claim 39, wherein the polypeptide consists of 8 to 50 residues.

42. The agent of claim 39, wherein the polypeptide comprises the amino acid sequence SEQ ID NO: 37.

43. The agent of claim 39, wherein the polypeptide consists of the amino acid sequence SEQ ID NO: 37.

44. The agent of claim 39, wherein the polypeptide is modified with a cell-membrane permeable substance.

45. The agent of claim 44, wherein the polypeptide has the following general formula:

[R]-[D]; wherein [R] represents the cell-membrane permeable substance; and [D] represents the amino acid sequence of the fragment sequence which comprises SEQ ID NO: 39; or a polypeptide functionally equivalent to the polypeptide, wherein [R] and [D] are linked directly or indirectly through a linker, wherein the polypeptide inhibits the biological function of a peptide consisting of SEQ ED NO: 2.

46. The agent of claim 45, wherein the linker has the amino acid sequence of "GGG".

47. The agent of claim 45, wherein the cell-membrane permeable substance is any one selected from the group consisting of: poly-arginine;

Tat / RKKRRQRRR/SEQ ID NO: 42;

Penetratin / RQIKIWFQNRRMKWKK/SEQ ID NO: 43;

Buforin II / TRS SRAGLQFP VGRVHRLLRK/SEQ ID NO: 44; Transportan / GWTLNSAGYLLGKINLKALAALAKKIL/SEQ ID NO: 45;

MAP (model amphipathic peptide) / KLALKLALKALKAALKLA/SEQ ID NO: 46;

K-FGF / AAVALLPAVLLALLAP/SEQ ID NO: 47;

Ku70 / VPMLK/SEQ ID NO: 48

Ku70 / PMLKE/SEQ ID NO: 49; Prion / MANLGYWLLALFVTMWTDVGLCKKRPKP/SEQ ID NO: 50; pVEC / LLIILRRRIRKQ AHAHSK/SEQ ID NO: 51; Pep-1 / KETWWETWWTEWSQPKKKRKV/SEQ ID NO: 52; SynBl / RGGRLSYSRRRFSTSTGR/SEQ ID NO: 53; Pep-7 / SDLWEMMMVSLACQY/SEQ ID NO: 54; and HN-I / TSPLNIHNGQKL/SEQ ID NO: 55.

48. The agent of claim 47, wherein the poly-arginine is Arg 11 (SEQ ID NO: 56).

49. The agent of claim 48, wherein the polypeptide comprises the amino acid sequence SEQ ID NO: 38.

50. The agent of claim 39, wherein the cancer is any one selected from the group consisting of breast cancer, lung cancer and bladder cancer.

51. A method for either or both of treating and preventing cancer comprising the step of administering a polypeptide comprising SEQ ID NO: 39; a polypeptide functionally equivalent to the polypeptide; or polynucleotide encoding these polypeptides, wherein the polypeptide inhibits the biological function of a peptide consisting of SEQ ID NO: 2.

52. Use of a polypeptide comprising SEQ ID NO: 39; a polypeptide functionally equivalent to the polypeptide; or polynucleotide encoding those polypeptides in manufacturing a pharmaceutical composition for either or both of treating and preventing cancer, wherein the polypeptide inhibits the biological function of a peptide consisting of SEQ ID NO: 2.

53. A pharmaceutical composition comprising a polypeptide comprising SEQ ID NO: 39; or a polypeptide functionally equivalent to the polypeptide; and a pharmaceutically acceptable carrier, wherein the polypeptide inhibits the biological function of a peptide consisting of SEQ ID NO: 2.