CA2706075A1 - Cancer diagnostic and therapeutic methods that target plk4/sak - Google Patents

Abstract

Methods of assessing expression of PLK4 in a cancer patient for use in determining if the patient is a candidate for PLK4antagomst therapy, predicting cancer metastasis and if the patient is a candidate for aggressive cancer therapy. Also provided is the use of PLK4 antagonists in the treatment of cancer and metastasis.
Preferred cancers for treatment are breast cancer (basal sub-type and luminal B sub-type) and soft tissue cancers.

Description

CANCER DIAGNOSTIC AND THERAPEUTIC METHODS THAT TARGET

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.
61/003,825, filed on November 20, 2007.
The entire teachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

It has long been appreciated that cancers are diverse in their cellular origin and vary widely in responsiveness to treatments. However, cancers of a particular tissue can also be diverse in cellular origin and have varying responsiveness to treatments. For example, breast cancer has been diagnosed by clinicians for many decades, however only relatively recent studies have demonstrated that breast cancers have phenotypic diversity among different breast cancer sub-types as demonstrated by DNA microarray profiling (see, e.g., Kapp et al., BMC Genomics (2006) 7:23 1, and references cited therein). Breast cancer has at least five sub-types:
basal, ERRB2-overexpressing, luminal A, luminal B, and normal breast-like (Sorlie et al., PNAS (2003) 100: 8418-8423; and Kapp et al., BMC Genomics (2006) 7:231).
The prognosis and chemotherapy sensitivity of the different molecular subgroups are different. The basal subtype breast cancer does not typically express the estrogen receptor (ER) nor overexpress HER2, and has been associated with poor a clinical outcome (Carey et al., JAMA. (2006) 295:2492-2502). Compared with luminal B tumors, luminal A tumors express higher levels of ER and GATA3 and show more favorable patient outcomes, whereas luminal B tumors more often express human epidermal growth factor receptor-I (HER I), HER2, and/or cyclin (Carey et al., JAMA. (2006) 295:2492-2502, and references cited therein). It has been reported that patients with luminal A type tumors lived considerably longer before they developed metastatic disease, whereas basal and ERBB2-overexpressing patients have a much shorter disease-free time (Sorlie et al., PNAS (2003) 100:
8418-8423). The reasons for these differing prognoses among breast cancer sub-types is not understood. Indeed, although many cancers, such as breast cancer, have multiple sub-types, the diagnosis, prognosis, and treatment of the different sub-types of cancers are poorly understood or even appreciated. Thus, a need exists for methods of diagnosis, prognosis and treatment of specific sub-types of cancer.
The polo-like kinase (PLK) family of serine/threonine kinases comprises at least four known members: PLKI, PLK2 (also known as Snk), PLK3 (also known as Fnk or Prk) and PLK4 (also known as Sak). PLKI is the best characterized member of the family, whereas PLK2, PLK3 and PLK4 are less well characterized. Of the four members, PLK 1, PLK2, and PLK3 all share a characteristic, highly conserved N-terminal catalytic domain and tandem C-terminal polo-box (PB) sequences that have a conserved function as phosphopeptide binding domains which serve to localize the kinases and regulate their activity (Johnson et al., Biochemistry (2007) 46(33): 9551-9563, and references cited therein). PLKI is a mitotic kinase and has been the target of therapeutic treatment of cancer (Johnson et al., Biochemistry (2007) 46(33): 9551-9563, and references cited therein).
PLK4 is the least understood and most divergent PLK member of the PLK
family. The N-terminal catalytic domain of PLK4 has a different substrate specificity from that of PLK1-3. PLK4 also has a divergent C-terminus comprising only a single polo-box sequence, not tandem PB sequences like in PLK1-3, that appears to act as a homodimerization domain rather than a localization domain (Lowery et al., (2005) Oncogene 24: 248-259).
PLK4 is involved in the control of mitotic entry and exit. It is a regulator of centrosome duplication (Habedanck et al. Nature Cell Biology 7: 1140-1146, 2005).
PLK4 transcripts increase from S through M phase, and the protein is ubiquitylated and destroyed by the anaphase promoting complex (APC) (Hudson et al. Curr.
Biol.
11: 441-446, 2001; Fode et al. Mol. Cell. Biol. 16: 4665-4672, 1996). PLK4 is required for late mitotic progression (Fode et al. PNAS. 91: 6388-6392, 1994;
Hudson et al. Curr. Biol. 11: 441-446, 2001), cell survival and postgastrulation embryonic development (Hudson et al. Curr. Biol. 11: 441-446, 2001). PLK4 knockout mice are embryonic lethal (E7.5), with a marked increase in mitotic and apoptotic cells (Hudson et al. Curr. Biol. 11: 441-446, 2001). PLK4 is transcriptionally repressed by p53 (Li et al. Neoplasia 7: 312-323, 2005).
This repression is likely mediated through the recruitment of histone deacetylase (HDAC) repressors and repression appears to contribute to p53-induced apoptosis (Li et al.
Neoplasia 7: 312-323, 2005). PLK4 has been reported to be overexpressed in colorectal tumors with expression reported as low in adjacent normal intestinal mucosa (Macmillian et al. Ann. Surg. Oncol. 8: 729-740, 2001). In addition, mRNA has been reported to be overexpressed in some tumor cell lines (Hitoshi, et al., U.S. Patent Application No. US 2003/0027756). But, PLK4 overexpression has not been demonstrated in cancers other than colorectal tumors. In particular, overexpression has not been demonstrated in association with specific sub-types of cancers.
Thus, the role of PLK4 in other cancers and other proliferative diseases is poorly understood. A need, therefore, exists to understand the role of PLK4 in other cancers and other proliferative diseases, and for methods of diagnosis, prognosis and treatment of such conditions.

SUMMARY OF THE INVENTION

The invention provides diagnostic and therapeutic methods that target PLK4.
Such methods are particularly useful for diagnosis and therapeutic treatments of patients with breast cancer, and in particular, basal sub-type breast cancer or luminal B sub-type breast cancer. The methods are also particularly useful for diagnosis and therapeutic treatments of metastatic disease and angiogenesis disorders.
Furthermore, the methods are also particularly useful for diagnosis and therapeutic treatments of soft tissue cancers. The methods are also useful as prognostic indicators, and in particular, as a predictor of disease relapse.
One aspect of the invention is a method for identifying a cancer patient candidate for anti-cancer therapy using a PLK4 antagonist, wherein the cancer patient is not a colorectal cancer patient. The method comprises providing a suitable sample from the patient (e.g., a tumor sample, a cancer tissue sample, a soft tissue cancer sample) and assessing expression of PLK4 in the sample. Expression of PLK4 in the sample indicates that the cancer patient is a candidate for anti-cancer therapy using a PLK4 antagonist. Alternatively, increased expression of PLK4 in the sample relative to a suitable control indicates that the cancer patient is a candidate for anti-cancer therapy using a PLK4 antagonist. In one embodiment, the cancer patient is a patient who has been diagnosed with breast cancer. In another embodiment, the breast cancer patient is a patient diagnosed with basal sub-type breast cancer or luminal B sub-type breast cancer. In one embodiment, the cancer patient is a patient who has been diagnosed with a soft tissue cancer. In another embodiment, the cancer patient is a patient who has been diagnosed with a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleomorphic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
Another aspect of the invention is a method for identifying a breast cancer patient candidate for anti-cancer therapy using a PLK4 antagonist. The method comprises providing a suitable sample from the patient (e.g., a tumor sample, a breast cancer tissue sample) and assessing expression of PLK4 in the sample.
Expression of PLK4 in the sample indicates that the breast cancer patient is a candidate for anti-cancer therapy using a PLK4 antagonist. Alternatively, increased expression of PLK4 in the sample relative to a suitable control indicates that the breast cancer patient is a candidate for anti-cancer therapy using a PLK4 antagonist.
In one aspect, the breast cancer patient is a patient who has been diagnosed with basal sub-type breast cancer or luminal B sub-type breast cancer.
A further aspect of the invention is a method for selecting a cancer patient, such as a breast cancer patient, a soft tissue cancer patient, for PLK4 therapy. The method comprises providing a suitable sample from the patient (e.g., a tumor sample, a breast cancer tissue sample, a soft tissue cancer sample) and assessing expression of PLK4 in the sample. Expression of PLK4 in the sample indicates selecting the patient for anti-cancer therapy using a PLK4 antagonist.
Alternatively, increased expression of PLK4 in the sample relative to a suitable control indicates selecting the patient for anti-cancer therapy using a PLK4 antagonist. In one aspect, the cancer patient is a patient who has been diagnosed with basal sub-type breast cancer or luminal B sub-type breast cancer. In another aspect, the cancer patient is a patient who has been diagnosed with a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleomorphic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
Another aspect of the invention is a method for treating a basal sub-type breast cancer, luminal B sub-type breast cancer or a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleomorphic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma in a patient. The method comprises administering to the patient a therapeutically effective amount of a PLK4 antagonist. In another aspect, the method comprises administering to the patient a pharmaceutical composition comprising a PLK4 antagonist in an amount that is sufficient to treat the basal sub-type breast cancer, luminal B sub-type breast cancer, or a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleomorphic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma, in a patient. In a further aspect, the method further comprises administering another therapeutic agent, such as a PLK1 antagonist. The PLK4 antagonist and the other therapeutic agent, e.g., PLKI antagonist, can be administered simultaneously or sequentially.
A further aspect of the invention is a method for predicting cancer metastasis in a cancer patient. The method comprises providing a suitable sample from the patient (e.g., a tumor sample, a cancer tissue sample) and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control is indicative of an increased likelihood of developing cancer metastasis in the patient. In one aspect, the cancer patient has breast cancer. In a further aspect, the breast cancer patient has basal sub-type breast cancer or luminal B sub-type breast cancer.
In another aspect, the invention is a method for screening a cancer patient, such as a breast cancer patient, a soft tissue cancer sarcoma patient, as an aid for selecting aggressive cancer therapy for the patient. The method comprises obtaining a suitable sample from the patient (e.g., a tumor sample, a breast cancer tissue sample, a soft tissue cancer sample) and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control indicates the need or clinical desirability for aggressive cancer therapy. In one aspect, the cancer patient has basal sub-type breast cancer or luminal B
sub-type breast cancer. In another aspect, the cancer patient has a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
Another aspect of the invention is a method for inhibiting angiogenesis in a patient. The method comprises administering to the patient a therapeutically effective amount of a PLK4 antagonist. In another aspect, the method comprises administering to the patient a pharmaceutical composition comprising a PLK4 antagonist in an amount that is sufficient to inhibit angiogenesis. In a further aspect, the method further comprises administering another therapeutic agent, such as a PLK1 antagonist. The PLK4 antagonist and the other therapeutic agent, e.g., antagonist, can be administered simultaneously or sequentially.
A further aspect of the invention is a method for treating a cancer metastasis disease (such as a metastatic tumor) in a patient. The method comprises administering to the patient a therapeutically effective amount of a PLK4 antagonist.
In another aspect, the method comprises administering to the patient a pharmaceutical composition comprising a PLK4 antagonist in an amount that is sufficient to treat a cancer metastasis disease in a patient. In a further aspect, the method further comprises administering another therapeutic agent, e.g., a PLK1 antagonist. The PLK4 antagonist and the other therapeutic agent, e.g., PLK1 antagonist, can be administered simultaneously or sequentially.
In another aspect, the invention is a method for identifying a patient that is likely to be responsive to PLK4 antagonist therapy. The method comprises providing a suitable sample (e.g., a tissue sample, a tumor sample) from the patient and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control indicates that the patient is likely to be responsive to PLK4 antagonist therapy. In one aspect, the tissue sample is a breast tissue sample and the patient has a breast cancer. In another aspect, the patient has a basal sub-type breast cancer or a luminal B sub-type breast cancer. In a further aspect of the invention, the patient has a soft tissue cancer. In one embodiment, the soft tissue cancer patient has a sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma. In another aspect of the invention, the patient has a disorder associated with angiogenesis. In a still further aspect of the invention, the patient has a cancer metastasis disease, such as a metastatic tumor.
In another aspect, the invention is a method for predicting whether a patient will be responsive to PLK4 antagonist therapy. The method comprises providing a suitable sample (e.g., a tissue sample, a tumor sample) from the patient and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control indicates that the patient will be responsive to PLK4 antagonist therapy. In one aspect, the tissue sample is a breast tissue sample and the patient has a breast cancer. In another aspect, the patient has a basal sub-type breast cancer or a luminal B sub-type breast cancer. In a further aspect of the invention, the patient has a soft tissue cancer. In one embodiment, the soft tissue cancer is a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma. In another aspect of the invention, the patient has a disorder associated with angiogenesis. In a still further aspect of the invention, the patient has a cancer metastasis disease.
In a further aspect, the invention is a method for treating a cancer patient.
The method comprises administering to the patient a therapeutically effective amount of a small molecule PLK4 antagonist, such as an antagonist selected from the group consisting of:
jNH2 [1 ]
N
S

H [2]
NHZ aN
N
C\ / O
N I
S
H H

[4]
H
CH, bN--Br F

[5]

/ IOI
N \ 0 N
S N
H H
NHZ / \
!N\ CH3 [6]
N--N

H3C -i--- CH3 HN

[7]

N

/ \

N
/N

S
H

N O
H

F NN
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[9]
HN

N
H

NH
/
rill [14]
N
N

H

N N
I

N--NH

NH
H
N [15]
y~IN N S

` J
H3C v H
N
O

\ / I [16]
N N

NC
H
N
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N N [17].

~NH

and a physiologically acceptable salt of any of the foregoing. In one embodiment, the cancer patient is diagnosed with breast cancer. In another embodiment, the cancer patient is diagnosed with or has a basal sub-type breast cancer. In another embodiment, the breast cancer patient is diagnosed with or has a luminal B sub-type breast cancer. In one embodiment, the cancer patient is diagnosed with a soft tissue cancer. In another embodiment, the cancer patient is diagnosed with a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color.
Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
FIG. 1 is the nucleotide sequence of human PLK4 (SEQ ID NO: 1).

FIG. 2 is the amino acid sequence of human PLK4 (SEQ ID NO: 2).
FIG. 3 is a schematic diagram of PLKI and PLK4. The consensus sequence for serine-threonine kinase activity of each PLK is shown below the corresponding schematic kinase domain (SEQ ID NOS: 3 and 4) (where 4 denotes a large hydrophobic residue and is a charged residue dependent on the context of the surrounding sequence). PB is an abbreviation for "Polo-Box" domain.
FIG. 4 is a table summarizing PLKI, PLK2, PLK3 and PLK4 expression levels in breast cancer tissue samples as compared with normal breast, correlation of PLK expression with breast cancer patient survival and growth inhibition induced by depletion of PLK expression using small interference RNA.
FIG. 5 are graphs showing the transcript levels of PLK4 expression in various normal tissues and various sarcoma tumor samples. As can be seen, PLK4 expression is increased in soft tissue sarcoma tumor cells. The data analysis indicates that the PLK4 mRNA expression is elevated in majority of the tumors tissue samples in comparison to those in normal tissues. The PLK4 expression analysis was done using the Gene Expression Omnibus database that contains datasets for levels of human gene expression in human cancers at the National Centre for Biotechnology information.
FIGS. 6A and 6B are graphs illustrating PLK4 overexpression in breast and lung cancer cell lines. In FIG. 6A, the mRNA transcript levels of PLK4 in breast cancer cell lines were measured by quantitative RT-PCR and compared to those in normal human mammary epithelial cells (HMECs) from three separate donors. In FIG. 6B, the mRNA transcript levels of PLK4 in lung cancer cell lines were measured by quantitative RT-PCR and compared to those in normal human bronchial epithelial cells (NHBEs) from three separate donors. The primers used are: 5'-CCACAGACAACAATGCCAAC-3' (forward) (SEQ ID NO: 5) and 5'-GGTCTGCAAATGGAAAAGGA-3' (reverse) (SEQ ID NO: 6).
FIG. 7 is a graph illustrating the efficiency of PLK4 knockdown with different PLK4 siRNAs. As can be seen from the graph and indicated by the red arrows, PLK4 knockdown efficiency of greater than 70% was achieved with multiple siRNAs. The sequences for the siRNAs are: siRNA#1 5'-AAGCCATGTACAAAGCAGGAA-3' (SEQ ID NO: 7); siRNA#2 5'-ACTCCTTTCAGACATATAAG-3' (SEQ ID NO: 8); siRNA#3 5'-AACTATCTTGGAGCTTTATAA-3' (SEQ ID NO: 9); and siRNA#4 5'-CTGGTAGTACTAGTTCACCTA-3' (SEQ ID NO: 10).
FIG. 8 is a table of results illustrating that siRNA depletion of PLK4 reduces cell viability in breast cancer cell lines. The sulforhodamine B (SRB) assay is used for cell viability determination. Cells in 96-well plates were transfected with the indicated siRNAs (40 nM) using Lipofectamine 2000 Transfection Reagent (Invitrogen Corporation) and cultured at 37 C. After 5 days, the cells were washed with PBS, fixed with 10% ice-cold trichloroacetic acid at 4 C and dried at room temperature. Proteins were stained with SRB in I% acetic acid at room temperature, washed with I% acetic acid and dried at 37 C. To dissolve the SRB bound proteins, 10 mM Tris base was added to each well and incubated at room temperature with mechanical agitation. SRB bound to protein was measured by absorbance using a SpectraPlus microplate reader (Molecular Devices Corporation).
FIG. 9 is a series of cell cycle profiles in FACS analysis graphs. As shown, PLK4 knockdown using siRNA, specifically siRNA#3 and a pooled siRNA of siRNA#1-4, increases G2/M and/or sub-GI cell cycle populations in breast cancer cells. Depletion of PLK1 also causes a similar cellular phenotype. Statistical shifts in cell cycle population at the different stages of the cell cycle with different treatments, as compared to the siRNA control (siControl). Increase in G2/M
population indicates cells arrested at G2/M phase of the cell cycle. Increase in sub G1 population indicates cell death. Cells in 6-well plates were transfected with the indicated siRNAs (40 nM) using Lipofectamine 2000 Transfection Reagent (Invitrogen Corporation) and cultured at 37 C. After 3-4 days, the cells were trypsinized, washed in PBS and centrifuged. Cell pellets were resuspended in propidium iodide in Hepes buffer, mixed and incubated at room temperature in the dark. Samples were read in a FACSCalibur flow cytometer (Becton, Dickinson and Company) and data analyzed using FloJo software (Tree Star, Inc.).
FIG. 10 is a series of cell cycle profiles in FACS analysis graphs illustrating that PLK4 knockdown does not affect the cell cycle profile of normal breast cells. A
similar observation was made for the cells depleted of PLKI expression.

FIG. 11 are graphs of two exemplary experiments illustrating siRNA
depletion of PLK4 and PLK 1 induces apoptosis in breast cancer cell lines but not in normal cells.
FIGS. 12A and 12B illustrate that siRNA depletion of PLK4 inhibits colony formation in soft agar. FIG. 12A graphs colony size with the indicated siRNA
treatments. FIG. 12B graphs colony number with the indicated siRNA treatments.
FIG. 13 illustrates the chemical structures of PLK inhibitors (compounds 1-21). Adapted from Johnson et al., Biochemistry (2007) 46(33): 9551-9563.
FIG. 14 is a Table demonstrating various Ki values for the PLK inhibitors (compounds 1-21) illustrated in FIG. 13 against PLK1-4. Adapted from Johnson et al., Biochemistry (2007) 46(33): 9551-9563.
FIG. 15 is a graph illustrating that high PLK4 expression is associated with reduced patient survival. The data analysis was done using the gene expression data from the references: Hu et al., BMC Genomics (2006) 7:96; van de Vijver et al., N.
Engl. J. Med. (2002) 347(25): 1999-2009; and Miller et al., PNAS (2005) 102(38):
13550-13555.
FIG. 16 is a graph illustrating that high PLK4 expression is associated with an increased incidence of metastasis.
FIGS. 17A and 17B are two data sets illustrating that high PLK4 expression may be associated with an increased risk of relapse.
FIG. 18 is a table detailing PLK4 overexpression in basal-like and luminal B
subtypes of breast cancer is significantly associated with poor prognosis.
FIGS. 19A and 19B illustrate that PLK4 knockdown suppresses breast tumor growth. FIG. 19A demonstrates the PLK4 protein levels in infected MDA-MB-468 cells at the start of the study (day 1). pSIREN-shPLK4 infected MDA-MB-468 cells have an approximately 60% knockdown of PLK4 expression as compared to control.
FIG. 19B is a graph charting tumor volume and weight over time (days) of nude mice (n=5) injected subcutaneously into the left and right hind limb with 5x pSIREN-shLUC (control) and pSIREN-shPLK4 infected MDA-MB-468, respectively.

DETAILED DESCRIPTION OF THE INVENTION

Definitions As used herein, a "therapy" is the administration of one or more therapeutic agents to a subject. A subject is any individual (e.g., a mammal, such as a primate (e.g., human), cow, sheep, goat, horse, dog, cat, rabbit, guinea pig, rat, mouse or other bovine, ovine, equine, canine feline, rodent or murine species) in need of therapy. Therapy can be the administration of a therapeutic agent in a single dose, in multiple doses, simultaneously with other agents, or sequentially with other agents.
In addition, the one or more therapeutic agents can be administered to a subject at a particular dose (e.g., level, amount, mass) and on a particular schedule or at particular intervals (e.g., in increments of minutes, days, weeks, months, etc.).
A "therapeutic agent" is an agent which is used in a medical treatment, such as a therapy, e.g., to treat or cure a disease or condition, and/or to treat or alleviate a symptom, and/or to prevent or mitigate a disease or condition.
A "therapeutically effective amount" is an amount sufficient to achieve the desired therapeutic effect (such as a curing effect, treatment, or a prophylactic effect) under the conditions of administration.
As used herein, "directly inhibit tumor cell growth" refers to specifically inhibiting tumor cell growth (e.g., reducing tumor cell proliferation, promoting tumor cell death), but not necessarily inhibiting conditions that promote or permit tumor cell growth (e.g., not necessarily inhibiting growth of new blood vessels (angiogenesis) to or within a tumor mass).
An "anti-tumor effective amount" is an amount of an agent that is sufficient to directly inhibit tumor cell growth (e.g., inhibit tumor cell proliferation), inhibit tumor survival and/or promote tumor cell death.
An "anti-metastasis effective amount" is an amount of an agent that is sufficient to directly inhibit cancer metastasis (e.g., inhibit metastatic cancer cell proliferation, inhibit spread of metastatic cancer cells, inhibit formation of metastatic cancer cells), inhibit metastatic cancer cell survival and/or promote metastatic cancer cell death.
An "anti-angiogenic effective amount" is an amount sufficient to inhibit angiogenesis.

An agent administered as a "primary therapy" is an agent that is the principal therapeutic agent in a therapy.
An adjunct therapy is another (e.g., secondary) therapy used together with the primary therapy, wherein the combination provides the desired treatment.
Adjunct therapy is also known in the art as "adjunctive therapy".
An adjuvant therapy is a therapy given after the primary therapy to increase the chances of a cure. Adjuvant therapy may include chemotherapy, radiation therapy, hormone therapy, biological therapy and the like.
As used herein, "aggressive therapy" is the administration of a therapeutic agent or agents at higher doses, more frequent doses, or a combination thereof, than is normally administered in a typical therapeutic regime. Aggressive therapy can also be the administration of a combination of therapeutic agents that are not typically administered in the same therapeutic regime. Aggressive therapy is often at or near the limit of tolerance for a subject receiving such therapy. For example, aggressive chemotherapy is sufficiently toxic that the subject's bone marrow is likely to fail (e.g., the bone marrow will no longer be able to produce hematopoeitic cells after aggressive therapy). To get around this anticipated side effect of aggressive therapy, the subject may receive an autologous bone marrow transplant, or receive a tissue-type matched bone marrow transplant.
As used herein, "PLK4 expression" means the expression (e.g., presence, absence, level, amount, etc.) of PLK4 nucleic acid sequence (e.g., mRNA), protein, and/or the activity of the protein (e.g., kinase activity, ability to bind substrate, ability to localize in the cell, etc.) in a sample. PLK4 expression can also mean the localization of the protein in a tissue sample (e.g., cellular localization).
An "antagonist" is an agent that binds the target molecule (e.g., PLK4, PLK1), and inhibits one or more activities of the target molecule; or an agent that inhibits (e.g., reduces, prevents) the expression of the target molecule gene and/or protein. Thus, a "PLK4 antagonist" is an agent (e.g., small molecule, protein, peptide, polypeptide, peptidemimetic, non-peptidic molecule, antibody, siRNA
molecule, antisense oligonucleotide, chemical compound, or a combination thereof) which specifically and preferably selectively binds PLK4, and inhibits one or more activities of PLK4; or an agent that inhibits (e.g., reduces, prevents) the expression of PLK4 gene and/or protein. Preferably, a PLK4 antagonist selectively binds or inhibits expression of PLK4 and, therefore, does not substantially bind other PLK
family members (e.g., PLK1, PLK2, PLK3, or a combination thereof) under physiological or therapeutic conditions. Furthermore, a "PLK1 antagonist" is an agent that specifically and preferably selectively binds PLK1, and inhibits one or more activities of PLK1; or an agent that inhibits (e.g., reduces, prevents) the expression of PLK1 gene and/or protein. Preferably, a PLK1 antagonist selectively binds or inhibits expression of PLKI and, therefore, does not substantially bind other PLK family members (e.g., PLK2, PLK3, PLK4, or a combination thereof) under physiological or therapeutic conditions.
The term "peptide", refers to a compound consisting of from about 2 to about 100 amino acid residues wherein the amino group of one amino acid is linked to the carboxyl group of another amino acid by a peptide bond. Such peptides are typically less than about 100 amino acid residues in length and preferably are about 10, about 20, about 30, about 40 or about 50 residues.
The term "peptidomimetic" or "peptide mimetic", refers to molecules which are not polypeptides, but which mimic aspects of their structures.
The term "antibody" is intended to encompass all types of polyclonal and monoclonal antibodies (e.g., human, chimeric, humanized, primatized, veneered, single chain, domain antibodies (dAbs)) and antigen-binding fragments of antibodies (e.g., Fv, Fc, Fd, Fab, Fab', F(ab'), dAb) (see e.g., Harlow et al., Antibodies A
Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
A "soft tissue cancer" is an art-recognized term that encompasses tumors derived from any soft tissue of the body. Such soft tissue connects, supports, or surrounds various structures and organs of the body, including, but not limited to, smooth muscle, skeletal muscle, tendons, fibrous tissues, fatty tissue, blood and lymph vessels, perivascular tissue, nerves, mesenchymal cells and synovial tissues.
Thus, soft tissue cancers can be of fat tissue, muscle tissue, nerve tissue, joint tissue, blood vessels, lymph vessels, and fibrous tissues. Soft tissue cancers can be benign or malignant. Generally, malignant soft tissue cancers are referred to as sarcomas, or soft tissue sarcomas. There are many types of soft tissue tumors, including lipoma, lipoblastoma, hibernoma, liposarcoma, leiomyoma, leiomyosarcoma, rhabdomyoma, rhabdomyosarcoma, neurofibroma, schwannoma (neurilemoma), neuroma, malignant schwannoma, neurofibrosarcoma, neurogenic sarcoma, nodular tenosynovitis, synovial sarcoma, hemangioma, glomus tumor, hemangiopericytoma, hemangioendothelioma, angiosarcoma, Kaposi sarcoma, lymphangioma, fibroma, elastofibroma, superficial fibromatosis, fibrous histiocytoma, fibrosarcoma, fibromatosis, dermatofibrosarcoma protuberans (DFSP), malignant fibrous histiocytoma (MFH), myxoma, granular cell tumor, malignant mesenchymomas, alveolar soft-part sarcoma, epithelioid sarcoma, clear cell sarcoma, and desmoplastic small cell tumor. In a particular embodiment, the soft tissue cancer is a sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleomorphic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
As described herein, it has been discovered that particular cancers have an increased expression of PLK4, and that increased PLK4 expression correlates with and is indicative of a surprisingly poor prognosis or clinical outcome for the patients. Thus, identifying patients that have an increased PLK4 expression is particularly useful for diagnosis, prognosis and determining a therapy for selected patients.
Methods for Identifying Patients for PLK4 Therapy In one aspect, the invention is a method for identifying a cancer patient candidate for anti-cancer therapy using a PLK4 antagonist, wherein the cancer patient is not a colorectal cancer patient. The method comprises providing a suitable sample from the patient (e.g., a tumor sample, a cancer tissue sample) and determining the PLK4 expression in the sample. Increased PLK4 expression in the sample relative to a suitable control indicates that the cancer patient is a candidate for anti-cancer therapy using a PLK4 antagonist. In one embodiment is a method for identifying a breast cancer patient candidate for anti-cancer therapy using a antagonist. The method comprises providing a suitable sample from the patient (e.g., a tumor sample, a breast cancer sample) and determining the PLK4 expression in the sample. Increased PLK4 expression in the sample relative to a suitable control indicates that the breast cancer patient is a candidate for anti-cancer therapy using a PLK4 antagonist. In another embodiment is a method for identifying a soft tissue cancer patient candidate for anti-cancer therapy using a PLK4 antagonist. The method comprises providing a suitable sample from the patient (e.g., a tumor sample, a soft tissue cancer sample) and determining the PLK4 expression in the sample. Increased PLK4 expression in the sample relative to a suitable control indicates that the soft tissue cancer patient is a candidate for anti-cancer therapy using a PLK4 antagonist.
A suitable sample can be obtained for example by cell or tissue biopsy. A
sample can also be obtained from other tissues, bodily fluids and products, e.g., from a blood sample, spinal tap, feces, tissue smear, tissue scrape, and the like.
Thus, the sample can be a biopsy specimen (e.g, tumor, polyp, mass (solid, cellular)), aspirate, smear, fecal sample and/or blood sample. The sample can be from a tissue that has a tumor (e.g., cancerous growth) and/or tumor cells, or is suspected of having a tumor and/or tumor cells. For example, a tumor biopsy can be obtained in an open biopsy, a procedure in which an entire (excisional biopsy) or partial (incisional biopsy) mass is removed from a target area. Alternatively, a tumor sample can be obtained through a percutaneous biopsy, a procedure performed with a needle-like instrument through a small incision or puncture (with or without the aid of a imaging device) to obtain individual cells or clusters of cells (e.g., a fine needle aspiration (FNA)) or a core or fragment of tissues (core biopsy). The biopsy samples can be examined cytologically (e.g., smear), histologically (e.g., frozen or paraffin section) or using any other suitable method (e.g., molecular diagnostic methods). A tumor sample can also be obtained by in vitro harvest of cultured human cells derived from an individual's tissue. Tumor samples can, if desired, be stored before analysis by suitable storage means that preserve a sample's protein and/or nucleic acid in an analyzable condition, such as quick freezing, or a controlled freezing regime.
If desired, freezing can be performed in the presence of a cryoprotectant, for example, dimethyl sulfoxide (DMSO), glycerol, or propanediol-sucrose. Tumor samples can be pooled, as appropriate, before or after storage for purposes of analysis.
Determining or assessing PLK4 expression can be performed using any suitable method, such methods are routine in the art. For example, screening a BAC
array data set, quantitative polymerase chain reaction (QPCR), including quantitative real-time PCR, in situ hybridization, western blot analysis, immunohistochemical staining, kinase assays, and the like. Other such methods to detect a PLK4 protein or peptide can include immunological and immunochemical methods like flow cytometry (e.g., FACS analysis), enzyme-linked immunosorbent assays (ELISA), including chemiluminescence assays, radioimmunoassay, and immunohistology, or other suitable methods such as mass spectroscopy. For example, antibodies to PLK4 can be used to determine the presence and/or expression level of PLK4 in a sample directly or indirectly using, for instance, immunohistology. For instance, paraffin sections can be taken from a biopsy, fixed to a slide and combined with one or more antibodies by suitable methods.
Methods to detect a PLK4 gene or expression thereof (e.g., DNA, mRNA) include PLK4 nucleic acid amplification and/or visualization. To detect a PLK4 gene or expression thereof, nucleic acid can be isolated from an individual by suitable methods which are routine in the art (see, e.g., Sambrook et al.
Molecular Cloning, 1989). Isolated nucleic acid can then be amplified (by e.g., polymerase chain reaction (PCR) (e.g., direct PCR, quantitative real time PCR, reverse transcriptase PCR), ligase chain reaction, self sustained sequence replication, transcriptional amplification system, Q-Beta Replicase, or the like) and visualized (by e.g., labeling of the nucleic acid during amplification, exposure to intercalating compounds/dyes, probes). PLK4 gene or expression thereof can also be detected using a nucleic acid probe, for example, a labeled nucleic acid probe (e.g., fluorescence in situ hybridization (FISH)) directly in a paraffin section of a tissue sample taken from, e.g., a tumor biopsy, or using other suitable methods. PLK4 gene or expression thereof can also be assessed by Southern blot or in solution (e.g., dyes, probes). Further, a gene chip, microarray, probe (e.g., quantum dots) or other such device (e.g., sensor, nanonsensor/detector) can be used to detect expression and/or differential expression of a PLK4 gene.
Increased PLK4 expression in a sample as compared with a suitable control indicates that the patient is a candidate for PLK4 antagonist therapy. PLK4 expression in a tissue sample, such as a cancer tissue sample, can be compared with a suitable control. Suitable controls are well recognized in the art and include, for example, normal cells or a non-neoplastic tissue sample such as one isolated from the donor of the cancer tissue sample, non-cancerous cells, non-metastatic cancer cells, non-malignant (benign) cells or the like, or any other suitable known or determined standard. In addition, the control can be a known or pre-determined typical, normal or normalized range or level of expression of a PLK4 protein or gene (e.g., an expression standard). Thus, the methods of the invention do not require that expression of the gene and/or protein be separately assessed in a suitable control each time a suitable sample from a patient is assessed for PLK4 expression.
Instead, whether PLK4 expression in a sample is increased or not can be determined using prior knowledge or comparison with a known or pre-determined typical, normal or normalized range or level of expression of a PLK4 protein or gene, such as a standard. The standard can be a lack or very low expression of PLK4 expression. It is noted that non-dividing cells (including, e.g., differentiated cells) have very low /nondetectable levels of PLK4 expression, and thus may be used as a suitable standard or referenced as the known standard. Thus, PLK4 expression can be compared to its expression in a known or a determined standard or it can be determined whether PLK4 expression exceeds a threshold, typical, normal, or normalized level. In one embodiment, a suitable control is a normal breast tissue sample obtained from the same donor of the breast cancer tissue sample.
In one embodiment, provided is a method for selecting a breast cancer patient for PLK4 therapy, wherein the patient is or has been diagnosed with a basal sub-type breast cancer. In another embodiment, the patient is or has been diagnosed with a luminal B sub-type breast cancer.
In another embodiment, provided is a method for selecting a soft tissue cancer patient for PLK4 therapy, wherein the patient is or has been diagnosed with a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
In another aspect, the invention is a method for screening a breast cancer patient as an aid for selecting aggressive cancer therapy of said patient (i.e., a method for determining if aggressive cancer therapy is indicated for a patient). The method comprises providing a suitable sample from the patient, e.g., a tumor sample, a breast cancer tissue sample, and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control indicates the need for aggressive cancer therapy. In one embodiment, the patient has basal sub-type breast cancer. In another embodiment, the patient has luminal B sub-type breast cancer.
In another aspect, the invention is a method for screening a soft tissue cancer patient as an aid for selecting aggressive cancer therapy of said patient (i.e., a method for determining if aggressive cancer therapy is indicated for a patient). The method comprises providing a suitable sample from the patient, e.g., a tumor sample, a soft tissue cancer tissue sample, and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control indicates the need for aggressive cancer therapy. In one embodiment, the patient has a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
In another aspect of the invention, provided is a method for identifying a patient that will be responsive to PLK4 antagonist therapy. The method comprises providing a suitable sample, e.g., a tissue sample, a tumor sample, from the patient and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control indicates that the patient will be responsive to PLK4 antagonist therapy. In one embodiment, the tissue sample is a breast tissue sample and the patient has breast cancer. For example, the patient that has breast cancer can have a basal sub-type breast cancer or a luminal B sub-type breast cancer. In another embodiment, the patient has a soft tissue cancer.
For example, the patient has a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma. In another embodiment, the patient has a cancer metastasis disease. In yet another embodiment, the patient has a disorder associated with angiogenesis.

Methods for Prognosis One aspect of the invention is a prognostic method for predicting (or indicating) a clinical outcome, e.g., relapse, cancer metastasis, survival, of a patient.
The method comprises providing a suitable sample from the patient, e.g., a tumor sample, a cancer tissue sample, and determining PLK4 expression in the sample.
Increased PLK4 expression in the sample as compared with a suitable control is indicative of the presence or increased likelihood of developing cancer metastasis in the patient. In one embodiment, the method predicts cancer metastasis in a patient diagnosed with a basal sub-type breast cancer. In another embodiment, the method predicts cancer metastasis in a patient diagnosed with luminal B sub-type breast cancer. In one embodiment, the method predicts cancer metastasis in a patient diagnosed with a soft tissue cancer. In another embodiment, the method predicts cancer metastasis in a patient diagnosed with a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
The prediction of cancer metastasis can be either an aid to diagnosis, prognosis, and/or assisting in the selecting a patient for a particular therapy.
A further aspect of the invention is a method for predicting (or indicating) reduced survival in a patient. The method comprises providing a suitable sample from the patient, e.g., a tumor sample, a cancer tissue sample, and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control predicts the reduced survival of the patient.
In one embodiment, the method predicts reduced survival in a patient diagnosed with a basal sub-type breast cancer. In another embodiment, the method predicts reduced survival in a patient diagnosed with luminal B sub-type breast cancer. In one embodiment, the method predicts reduced survival in a patient diagnosed with a soft tissue cancer. In a particular embodiment, the method predicts reduced survival in a patient diagnosed with a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.

Another aspect of the invention is a method for predicting (or indicating) an increased risk of relapse in a patient. The method comprises providing a suitable sample from the patient, e.g., a tumor sample, a cancer tissue sample, and determining PLK4 expression in the sample. Increased PLK4 expression in the sample as compared with a suitable control predicts an increased risk of relapse of the patient. In one embodiment, the method predicts an increased risk of relapse in a patient diagnosed with a basal sub-type breast cancer. In another embodiment, the method predicts an increased risk of relapse in a patient diagnosed with luminal B
sub-type breast cancer. In one embodiment, the method predicts an increased risk of relapse in a patient diagnosed with a soft tissue cancer. In a particular embodiment, the method predicts an increased risk of relapse in a patient diagnosed with a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
Antagonists As described above, a "PLK4 antagonist" is an agent (e.g., small molecule, protein, peptide, polypeptide, peptidemimetic, non-peptidic molecule, antibody, siRNA molecule, antisense oligonucleotide, chemical compound, or a combination thereof) which specifically and preferably selectively binds PLK4, and inhibits one or more activities of PLK4; or an agent that inhibits (e.g., reduces, prevents) the expression of PLK4 gene and/or protein. A PLK4 antagonist can, for example, inhibit binding of a ligand or substrate (e.g., ATP) to PLK4. A PLK4 antagonist can inhibit the activity of a PLK4 in response to ligand or substrate binding. A

antagonist that inhibits the expression and/or activity of PLK4 can be, for example, a natural or synthetic nucleic acid or nucleic acid analog, antisense molecule, small interfering RNA (siRNA), short hairpin RNA (shRNA), protein, peptide, peptidomimetic, antibody, chemical compound or the like. Preferably, a PLK4 antagonist selectively binds or inhibits expression of PLK4 and, therefore, does not substantially bind other PLK family members (e.g., PLK1, PLK2, and/or PLK3) under physiological or therapeutic conditions.

A composition comprising a PLK4 can be used in a such a screen or binding assay to detect and/or identify agents that can bind to a PLK4. Compositions suitable for use include, for example, cells which naturally express a PLK4.
Agents which bind PLK4 can be further evaluated for PLK4 antagonist activity.
An agent that binds a PLK4 can be identified in a competitive binding assay, for example, in which the ability of a test agent to inhibit the binding of a reference agent (e.g., a ligand or substrate) is assessed. The reference agent can be labeled with a suitable label (e.g., radioisotope, epitope label, affinity label (e.g., biotin and avidin or streptavidin), spin label, enzyme, fluorescent group, chemiluminescent group, dye, metal (e.g., gold, silver), magnetic bead) and the amount of labeled reference agent required to saturate the PLK4 in the assay can be determined.
The specificity of the formation of the complex between the PLK4 and the test agent can be determined using a suitable control (e.g., unlabeled agent, label alone).
The capacity of a test agent to inhibit formation of a complex between the reference agent and a PLK4 can be determined as the concentration of test agent required for 50% inhibition (IC50 value) of specific binding of labeled reference agent. Specific binding is preferably defined as the total binding (e.g., total label in complex) minus the non-specific binding. Non-specific binding is preferably defined as the amount of label still detected in complexes formed in the presence of excess unlabeled reference agent.
An agent which binds a PLK4 can be further studied to assess the ability of that agent to antagonize (reduce, prevent, inhibit) one or more functions of the PLK4. Functional characteristics of a PLK4 include binding activities (e.g., ligand or substrate binding), kinase activity (e.g., phosphorylation of a substrate) and/or an ability to stimulate a cellular response (e.g., mitosis). Such assays are standard in the art (see, e.g., Johnson et al., Biochemistry (2007) 46(33): 9551-9563 for a description of kinase assays for assessing activity of PLK 1, PLK 2, PLK 3 or PLK
4). For example, the agent can be incubated with PLK4 (purified, recombinant, or the like), in the presence of a suitable substrate (such as a peptide substrate, which can be labeled with a suitable label, e.g., an epitope label, an affinity label, such as biotin, avidin, streptavidin, and the like, magnetic bead, etc.) under conditions suitable for kinase activity. Suitable conditions include the presence of a kinase reaction buffer, with ATP. The ATP can be suitably labeled, e.g., with a radioisotope, epitope label, affinity label (e.g., biotin, avidin, streptavadin), spin label, enzyme, fluorescent group, chemiluminescent group, dye, metal (e.g., gold, silver), magnetic bead, or the like. Following incubation of the agent with PLK4, substrate and ATP, phosphorylation of the substrate can be assessed, e.g., by determining the amount of label from the ATP has transferred to the substrate.
Alternatively, a phosphorylated substrate can be detected using a phospho-specific antibody. The substrate peptide: TPSDSLIYDDGLS (SEQ ID NO: 15) can be used to selectively assay PLK4 activity (see Johnson et al., Biochemistry (2007) 46(33):
9551-9563) A PLK4 antagonist can be, for example, a small molecule, which can be found in nature (e.g., identified, isolated, purified) and/or artificially produced (e.g., synthesized). Small molecules can be tested for PLK4 binding specificity in a screen for example, a high-throughput screen of chemical compounds and/or libraries (e.g., chemical, peptide, nucleic acid libraries). Compounds or small molecules can be identified from numerous available libraries of chemical compounds from, for example, the Chemical Repository of the National Cancer Institute, the Molecular Libraries Small Molecules Repository (PubChem) and other libraries that are commercially available. Examples of small molecule antagonists of PLK, including PLK4, are described in Johnson et al., Biochemistry (2007) 46(33): 9551-9563 (see also FIGS. 17A, 17B and 18). Notably, some small molecule inhibitors exhibit PKL4 selectivity (see, e.g., compounds 1, 2, 4-9 and 14-17 disclosed herein and in Johnson et al., Biochemistry (2007) 46(33): 9551-9563;
see also FIGS. 17A, 17B and 18). Such libraries or collections of molecules can also be prepared using well-known chemical methods, such as well-known methods of combinatorial chemistry. The libraries can be screed to identify compounds that bind and inhibit PLK4. Identified compounds can serve as lead compounds for further diversification using well-known methods of medicinal chemistry. For example, a collection of compounds that are structural variants of the lead can be prepared and screened for PLK4 binding and/or inhibiting activity. This can result in the development of an structure activity relationship that links the structure of the compounds to biological activity. Compounds that have suitable binding and inhibitory activity can be further developed for in vivo use.
In one embodiment, small molecule PLK4 antagonists have the general structure [I], or pharmaceutically acceptable salts or solvates thereof:
H
H

N I [Il N \

H

wherein RI is a substituted or unsubstituted aryl or heteroaryl, or a group of the formula CH=CH-R3 or CH=N-R3 where R3 is a substituted or unsubstituted alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and R2 is a substituted or unsubstituted aryl or heteroaryl, or X-Y, where X is substituted or unsubstituted aryl, heteroaryl, NH-(alkyl), NH-(cycloalkyl), NH-(heterocycloalkyl), NH
(aryl), NH(heteroaryl), NH-(alkoxyl), or NH-(dialkylamide), and Y is 0, S, C=CH2, C=O, S=O, SO2, alkylidene, NH, or N-(CI - C8 alkyl) (see also U.S. Patent Nos.
6,531,491, 6,534,524).
In a further embodiment, small molecule PLK4 antagonists have the general structure [II], or pharmaceutically acceptable salts or solvates thereof:
H
O N

H
S R3 [II]
N\ \ I I /

wherein: R1 is CH=CH-R4, or CH=N-R4, and RI is substituted with 0 to 4 R5 groups; R2 is (Cl to C12) alkyl, (C3 to C12) cycloalkyl, (5 to 12-membered) heterocycloalkyl, (C6 to C12) aryl, (5 to 12-membered) heteroaryl, (Cl to C12) alkoxy, (C6 to C12) aryloxy, (C3 to C12) cycloalkoxy, NH-(C1 to C8 alkyl), NH-to C12 aryl), NH-(5 to 12-membered heteroaryl), N=CH-C1 to C12 alkyl), NH(C=O)R5, or NH2, and R2 is substituted with 0 to 4 R5 groups; each R3 is independently hydrogen, halogen, or (Cl to C8) alkyl, and the (Cl to C8) alkyl is substituted with 0 to 4 R5 groups; R4 is (Cl to C12) alkyl, (C3 to C12) cycloalkyl, (5 to 12-membered) heterocycloalkyl, (C6 to C12) aryl, (5 to 12-membered) heteroaryl, and R4 is substituted with 0 to 4 R5 groups; and each R5 is independently halogen, (C1 to C8) alkyl, -OH, NO2, -CN, -000H, -0-C1 to C8 alkyl), (C6 to C12) aryl, aryl (CI to C8) alkyl, -CO2CH3, -CONH2, -OCH2CONH2, NH2, -SO2NH2, halo (CI to C12) alkyl, or -0-halo (C1 to C12) alkyl. (see also US
Publication Patent Application No. 2006/0094881 Al).
Thus, small molecule PLK4 antagonists of the present invention used for treating a disease (e.g., a cancer, such as breast cancer, a cancer metastasis disease, such as a metastatic cancer, angiogenesis, such as pathological angiogenesis) include the following small molecule compounds or pharmaceutically acceptable salts or solvates thereof.

/
C [1]
N

H NH N \ I [2]
z \
N
Y
N I

H H
N

NY
0 I o [4]
H
N

Br / F

O [5]

N \ J~ N
S N N
H H

NH2 / \
\N I
[6]
N_N

HN

N
[7]

N

/ \
N
[8]

S N ~N
H

H

H~ \/ N V

HN [9]

N
H

NH
/ [14]
NI/
N / ~\
N
H / N I \

N
I

N --NH

--(";,~NH

H
\ N
N [15]
y~N N S

N` J
H3C v H
N
O

[16]
N N

NC and H
N
O

N N 1 / [17].
HN%%%"" 0 CH3 In one embodiment, the small molecule PLK4 antagonist is used to treat a cancer patient. In another embodiment, the cancer patient is a breast cancer patient.
In a further embodiment, the breast cancer patient has or is diagnosed with a basal sub-type breast cancer. In another embodiment, the breast cancer patient has or is diagnosed with a luminal B sub-type breast cancer. In one embodiment, the cancer patient is a soft tissue cancer patient. In a further embodiment, the soft tissue cancer patient has or is diagnosed with a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma. In a particular embodiment, the small molecule PLK4 antagonist used to treat a disease (e.g., a cancer, a cancer metastasis disease, pathological angiogenesis) is compound 8.
A PLK4 antagonist can be a peptide (e.g., synthetic, recombinant, fusion or derivatized) which specifically binds to and inhibits (reduces, prevents, decreases) the activity of the PLK4. The peptide can be linear, branched or cyclic, e.g., a peptide having a heteroatorn ring structure that includes several amide bonds.
Peptides, including cyclic peptides, that are selective for binding to a particular domain (e.g., unique domain) of a PLK4 can be produced. A peptide can be, for example, derived or removed from a native protein by enzymatic or chemical cleavage, or can be synthesized by suitable methods, for example, solid phase peptide synthesis (e.g., Merrifield-type synthesis) (see, e.g., Bodanszky et al.
"Peptide Synthesis," John Wiley & Sons, Second Edition, 1976). Peptides that are PLK4 antagonists can also be produced, for example, using recombinant DNA

methodologies or other suitable methods (see, e.g., Sambrook J. and Russell D.W., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001). PLK4 antagonists can also be fusion peptides fused, for example to a carrier protein (e.g., myc, his, glutathione sulthydryl transferase) and/or tagged (e.g., radiolabeled, fluorescently labeled).
A peptide can comprise any suitable L-and/or D-amino acid, for example, common a-amino acids (e.g., alanine, glycine, valine), non-a-amino acids (e.g., 13-alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statine), and unusual amino acids (e.g., citrulline, homocitruline, homoserine, norleucine, norvaline, ornithine). The amino, carboxyl and/or other functional groups on a peptide can be free (e.g., unmodified) or protected with a suitable protecting group.
Suitable protecting groups for amino and carboxyl groups, and methods for adding or removing protecting groups are known in the art and are disclosed in, for example, Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, 1991. The functional groups of a peptide can also be derivatized (e.g., alkylated) using art known methods.
Peptides can be synthesized and assembled into libraries comprising a few to many discrete molecular species. Such libraries can be prepared using methods of combinatorial chemistry, and can be screened using any suitable method to determine if the library comprises peptides with a desired biological activity. Such peptide antagonists can then be isolated using suitable methods. The peptide can comprise modifications (e.g., amino acid linkers, acylation, acetylation, amidation, methylation, terminal modifiers (e.g., cyclizing modifications)), if desired.
The peptide can also contain chemical modifications (e.g., N-methyl-a-amino group substitution). In addition, the peptide antagonist can be an analog of a known and/or naturally-occurring peptide, for example, a peptide analog having conservative amino acid residue substitution(s). These modifications can improve various properties of the peptide (e.g., solubility, binding), including its PLK4 antagonist activity.
Peptidomimetic antagonists can be prepared by conventional chemical methods (see e.g., Damewood J.R. "Peptide Mimetic Design with the Aid of Computational Chemistry" in Reviews in Computational Biology, 2007, Vol. 9, pp.1-80, John Wiley and Sons, Inc., New York, 1996; Kazmierski W.K., "Methods of Molecular Medicine: Peptidomimetic Protocols," Humana Press, New Jersey, 1999). Peptidomimetics can be prepared that are PLK4 antagonists. For example, polysaccharides can be prepared that have the same functional groups as peptides.
Peptidomimetics can be designed, for example, by establishing the three dimensional structure of a peptide agent in the environment in which it is bound or will bind to a target molecule. The peptidomimetic comprises at least two components, the binding moiety or moieties and the backbone or supporting structure.
The binding moieties are the chemical atoms or groups which will react or form a complex (e.g., through hydrophobic or ionic interactions) with a target molecule, for example, with the amino acid(s) at or near the ligand binding site. For example, the binding moieties in a peptidomimetic can be the same as those in a peptide or protein antagonist. The binding moieties can be an atom or chemical group which reacts with the PLK4 in the same or similar manner as the binding moiety in the peptide antagonist. Examples of binding moieties suitable for use in designing a peptidomimetic for a basic amino acid in a peptide include nitrogen containing groups, such as amines, ammoniums, guanidines and amides or phosphoniums. Examples of binding moieties suitable for use in designing a peptidomirnetic for an acidic amino acid include, for example, carboxyl, lower alkyl carboxylic acid ester, sulfonic acid, a lower alkyl sulfonic acid ester or a phosphorous acid or ester thereof.
The supporting structure is the chemical entity that, when bound to the binding moiety or moieties, provides the three dimensional configuration of the peptidomimetic. The supporting structure can be organic or inorganic. Examples of organic supporting structures include polysaccharides, polymers or oligomers of organic synthetic polymers (such as, polyvinyl alcohol or polylactide). It is preferred that the supporting structure possess substantially the same size and dimensions as the peptide backbone or supporting structure. This can be determined by calculating or measuring the size of the atoms and bonds of the peptide and peptidomimetic. In one embodiment, the nitrogen of the peptide bond can be substituted with oxygen or sulfur, for example, forming a polyester backbone.
In another embodiment, the carbonyl can be substituted with a sulfonyl group or sulfinyl group, thereby forming a polyamide (e.g., a polysulfonamide). Reverse amides of the peptide can be made (e.g., substituting one or more-CONH-groups for a-NHCO-group). In yet another embodiment, the peptide backbone can be substituted with a polysilane backbone.
These compounds can be manufactured by known methods. For example, a polyester peptidomimetic can be prepared by substituting a hydroxyl group for the corresponding a-amino group on amino acids, thereby preparing a hydroxyacid and sequentially esterifying the hydroxyacids, optionally blocking the basic and acidic side chains to minimize side reactions. Determining an appropriate chemical synthesis route can generally be readily identified upon determining the chemical structure.
Peptidomimetics can be synthesized and assembled into libraries comprising a few to many discrete molecular species. Such libraries can be prepared using well-known methods of combinatorial chemistry, and can be screened to determine if the library comprises one or more peptidomimetics which have the desired activity.
Such peptidomimetic antagonists can then be isolated by suitable methods.
PLK4 antagonists are also agents that inhibit (reduce, decrease, prevent) the expression of PLK4. Agents (molecules, compounds, nucleic acids, oligonucleotides) which inhibit PLK4 gene expression (e.g., transcription, mRNA
processing, translation) are effective PLK4 antagonists. For example, small interfering ribonucleic acids (siRNAs) and, similarly, short hairpin ribonucleic acids (shRNAs) which are processed into short siRNA-like molecules in a cell, can prevent the expression (translation) of the PLK4 protein. siRNA molecules can be polynucleotides that are generally about 20 to about 25 nucleotides long and are designed to bind specific RNA sequence (e.g., PLK4 mRNA). siRNAs silence gene expression in a sequence-specific manner, binding to a target RNA (e.g., an RNA
having the complementary sequence) and causing the RNA to be degraded by endoribonucleases. siRNA molecules able to inhibit the expression of the PLK4 gene product can be produced by suitable methods. There are several algorithms that can be used to design siRNA molecules that bind the sequence of a gene of interest (see e.g., Mateeva O. et al. Nucleic Acids Res. 35(8):Epub, 2007;
Huesken D. et al., Nat. Biotechnol. 23:995-1001; Jagla B. et al., RNA 11:864-872, 2005;
Shabalinea S.A. BMC Bioinformatics 7:65, 2005; Vert J.P. et al. BMC
Bioinformatics 7:520, 2006). Expression vectors that can stably express siRNA
or shRNA are available. (See e.g., Brummelkamp, T.R., Science 296: 550-553, 2002, Lee, NS, et al., Nature Biotechnol. 20:500-505, 2002; Miyagishi, M., and Taira, K.
Nature Biotechnol. 20:497-500, 2002; Paddison, P.J., et al., Genes & Dev.
16:948-958, 2002; Paul, C.P., et al., Nature Biotechnol. 20:505-508; 2002; Sui, G., et al., Proc. Natl. Acad. Sci. USA 99(6):5515-5520, 2002; Yu, J-Y, et al., Proc. Natl.
Acad.
Sci. USA 99(9):6047-6052, 2002; Elbashir, SM, et al., Nature 411:494-498, 2001.).
Stable expression of siRNA/shRNA molecules is advantageous in the treatment of cancer as it enables long-term expression of the molecules, potentially reducing and/or eliminating the need for repeated treatments.
Antisense oligonucleotides (e.g., DNA, riboprobes) can also be used as PLK4 antagonists to inhibit PLK4 expression. Antisense oligonucleotides are generally short (-13 to -25 nucleotides) single-stranded nucleic acids which specifically hybridize to a target nucleic acid sequence (e.g., mRNA) and induce the degradation of the target nucleic acid (e.g., degradation of the RNA through RNase H-dependent mechanisms) or sterically hinder the progression of splicing or translational machinery. (See e.g., Dias N. and Stein C.A., Mol. Can. Ther.
1:347-355, 2002). There are a number of different types of antisense oligonucleotides that can be used as PLK4 antagonists including methylphosphonate oligonucleotides, phosphorothioate oligonucleotides, oligonucleotides having a hydrogen at the 2'-position of ribose replaced by an O-alkyl group (e.g., a methyl), polyamide nucleic acid (PNA), phosphorodiamidate morpholino oligomers (deoxyribose moiety is replaced by a morpholine ring), PN (N3'--),P5' replacement of the oxygen at the 3' position on ribose by an amine group) and chimeric oligonucleotides (e.g., 2'-O-Methyl/phosphorothioate). Antisense oligonucleotides can be designed to be specific for PLK4 using predictive algorithms. (See e.g., Ding, Y., and Lawrence, C.
E., Nucleic Acids Res., 29:1034-1046, 2001; Sczakiel, G., Front. Biosci., 5:1) D201, 2000; Scherr, M., et al., Nucleic Acids Res., 28:2455-2461, 2000;
Patzel, V., et al. Nucleic Acids Res., 27:4328-4334,1999; Chiang, M.Y., et al., J. Biol.
Chem., 266:18162-18171,1991; Stull, R. A., et al., Nucleic Acids Res., 20:3501-3508, 1992;
Ding, Y., and Lawrence, C. E., Comput. Chem., 23:387-400,1999; Lloyd, B. H., et al., Nucleic Acids Res., 29:3664-3673, 2001; Mir, K. U., and Southern, E. M., Nat.
Biotechnol., 17:788-792,1999; Sohail, M., et al., Nucleic Acids Res., 29:2041 -2051, 2001; Altman, R. K., et al., J. Comb. Chem., 1:493-508, 1999). The antisense oligonucleotides can be produced by suitable methods; for example, nucleic acid (e.g., DNA, RNA, PNA) synthesis using an automated nucleic acid synthesizer (from, e.g., Applied Biosystems) (see also Martin, P., Hely. Chim. Acta 78:486-504, 1995). Antisense oligonucleotides can also be stably expressed in a cell containing an appropriate expression vector.
Antisense oligonucleotides can be taken up by target cells (e.g., tumor cells) via the process of adsorptive endocytosis. Thus, in the treatment of a subject (e.g., mammalian), antisense PLK4 can be delivered to target cells (e.g., tumor cells) by, for example, injection or infusion. For instance, purified oligonucleotides or siRNA/shRNA, can be administered alone or in a formulation with a suitable drug delivery vehicle (e.g., liposomes, cationic polymers, (e.g., poly-L-lysine' PAMAM
dendrimers, polyalkylcyanoacrylate nanoparticles and polyethyleneimine) or coupled to a suitable carrier peptide (e.g., homeotic transcription factor, the Antennapedia peptide, Tat protein of HIV-1, E5 CA peptide).
The PLK4 antagonist can be an antibody or antigen-binding fragment thereof which selectively binds a PLK4 protein. In a particular embodiment, the PLK4-specific antibody is a human antibody or humanized antibody. PLK4-specific antibodies can also be directly or indirectly linked to a cytotoxic agent.
Antibodies or antibody fragments which selectively bind to and inhibit the activity of a PLK4 can be produced, constructed, engineered and/or isolated by conventional methods or other suitable techniques. For example, antigen-specific antibodies can be raised against an appropriate immunogen, such as a recombinant mammalian (e.g., human) PLK4, or portion thereof (including synthetic molecules, e.g., synthetic peptides). A variety of methods have been described (see e.g., Kohler et al., Nature, 256: 495 497 (1975) and Eur. J. Immunol. 6: 511 519 (1976);
Milstein et al., Nature 266: 550 552 (1977); Koprowski et al., U.S. Patent No.
4,172,124;
Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY); Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer'94), Ausubel, F.M. et al., Eds., (John Wiley & Sons: New York, NY), Chapter 11, (1991)). Antibodies can also be raised by immunizing a suitable host (e.g., mouse) with cells that express PLK4 (e.g., cancer cells/cell lines) or cells engineered to express PLK4 (e.g., transfected cells) (see e.g., Chuntharapai et al., J. Immunol., 152:1783-1789 (1994);
Chuntharapai et al. U.S. Patent No. 5,440, 021). For the production of monoclonal antibodies, a hybridoma can be produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as SP2/0 or P3X63Ag8.653) with antibody producing cells. The antibody producing cells can be obtained from the peripheral blood, or preferably, the spleen or lymph nodes, of humans or other suitable animals immunized with the antigen of interest. The fused cells (hybridomas) can be isolated using selective culture conditions, and cloned by limited dilution. Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
Antibody fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab')2 fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab')2 fragments. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons has been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CHI domain and hinge region of the heavy chain. Single chain antibodies, and human, chimeric, humanized or primatized (CDR grafted), or veneered antibodies, as well as chimeric, CDR grafted or veneered single chain antibodies, comprising portions derived from different species, and the like are also encompassed by the present invention and the term "antibody". The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein.
See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent No.
0,125,023 B1; Boss et al., U.S. Patent No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, et al., WO 86/01533; Neuberger, et al., European Patent No. 0,194,276 B 1; Winter, U.S. Patent No. 5,225,539; Winter, European Patent No. 0,239,400 B 1; Queen et al., European Patent No. 0 451 216 B 1; and Padlan, et al., EP 0 519 596 Al. See also, Newman, R. et al., BioTechnology, 10:
1455 1460 (1992), regarding primatized antibody, and Ladner et al., U.S.
Patent No.
4,946,778 and Bird, R.E. et al., Science, 242: 423 426 (1988) regarding single chain antibodies.
Humanized antibodies can be produced using synthetic or recombinant DNA
technology using standard methods or other suitable techniques. Nucleic acid (e.g., cDNA) sequences coding for humanized variable regions can also be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see e.g., Kamman, M., et al., Nucl. Acids Res., 17: 5404 (1989));
Sato, K., et al., Cancer Research, 53: 851 856 (1993); Daugherty, B.L. et al., Nucleic Acids Res., 19(9): 2471 2476 (1991); and Lewis and Crowe, Gene, 101: 297 302 (1991)).
Using these or other suitable methods, variants can also be readily produced.
In one embodiment, cloned variable regions (e.g., dAbs) can be mutated, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al., U.S. 5,514,548; Hoogenboom et al., WO
93/06213, published April 1, 1993).
Other suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, for example, methods which select a recombinant antibody or antibody-binding fragment (e.g., dAbs) from a library (e.g., a phage display library), or which rely upon immunization of transgenic animals (e.g., mice).
Transgenic animals capable of producing a repertoire of human antibodies are well-known in the art (e.g., Xenomouse(k (Abgenix, Fremont, CA)) and can be produced using suitable methods (see e.g., Jakobovits et al., Proc. Natl. Acad. Sci.
USA, 90:
2551 2555 (1993); Jakobovits et al., Nature, 362: 255 258 (1993); Lonberg et al., U.S. Patent No. 5,545,806; Surani et al., U.S. Patent No. 5,545,807; Lonberg et al., WO 97/13852).
Similarly, as described above for PLK4 antagonists, a "PLKI antagonist" is an agent (e.g., small molecule, protein, peptide, polypeptide, peptidemimetic, non-peptidic molecule, antibody, siRNA molecule, antisense oligonucleotide, chemical compound, or a combination thereof) which specifically and preferably selectively binds PLKI, and inhibits one or more activities of PLK1; or an agent that inhibits (e.g., reduces, prevents) the expression of PLKI gene and/or protein. A PLKI
antagonist can, for example, inhibit binding of a ligand or substrate (e.g., ATP) to PLKI. A PLKI antagonist can inhibit the activity of a PLKI in response to ligand or substrate binding. A PLKI antagonist that inhibits the expression and/or activity of PLK 1 can be, for example, a natural or synthetic nucleic acid or nucleic acid analog, antisense molecule, small interfering RNA (siRNA), short hairpin RNA
(shRNA), protein, peptide, peptidomimetic, antibody, chemical compound or the like. Preferably, a PLK1 antagonist selectively binds or inhibits expression of PLK1 and, therefore, does not substantially bind other PLK family members (e.g., PLK2, PLK3, and/or PLK4) under physiological or therapeutic conditions.
A composition comprising a PLKI can be used in a such a screen or binding assay to detect and/or identify agents that can bind to a PLKI. Compositions suitable for use include, for example, cells which naturally express a PLKI .
Agents which bind PLKI can be further evaluated for PLKI antagonist activity.
An agent that binds a PLKI can be identified in a competitive binding assay, for example, in which the ability of a test agent to inhibit the binding of a reference agent (e.g., a ligand or substrate) is assessed. The reference agent can be labeled with a suitable label (e.g., radioisotope, epitope label, affinity label (e.g., biotin and avidin or streptavadin), spin label, enzyme, fluorescent group, chemiluminescent group, dye, metal (e.g., gold, silver), magnetic bead) and the amount of labeled reference agent required to saturate the PLKI in the assay can be determined.
The specificity of the formation of the complex between the PLK 1 and the test agent can be determined using a suitable control (e.g., unlabeled agent, label alone).
The capacity of a test agent to inhibit formation of a complex between the reference agent and a PLK 1 can be determined as the concentration of test agent required for 50% inhibition (IC50 value) of specific binding of labeled reference agent. Specific binding is preferably defined as the total binding (e.g., total label in complex) minus the non-specific binding. Non-specific binding is preferably defined as the amount of label still detected in complexes formed in the presence of excess unlabeled reference agent.
An agent which binds a PLKI can be further studied to assess the ability of that agent to antagonize (reduce, prevent, inhibit) one or more functions of the PLKI . Functional characteristics of a PLK4 include binding activities (e.g., ligand or substrate binding), kinase activity (e.g., phosphorylation of a substrate) and/or an ability to stimulate a cellular response (e.g., mitosis). Such assays are standard in the art (see, e.g., Johnson et al., Biochemistry (2007) 46(33): 9551-9563 for a description of kinase assays for assessing activity of PLKI, PLK 2, PLK 3 or PLK
4). For example, the agent can be incubated with PLK1 (purified, recombinant, or the like), in the presence of a suitable substrate (such as a peptide substrate, which can be labeled with a suitable label, e.g., an epitope label, an affinity label, such as biotin, avidin, streptavidin, and the like, magnetic bead, etc.) under conditions suitable for kinase activity. Suitable conditions include the presence of a kinase reaction buffer, with ATP. The ATP can be suitably labeled, e.g., with a radioisotope, epitope label, affinity label (e.g., biotin, avidin, streptavadin), spin label, enzyme, fluorescent group, chemiluminescent group, dye, metal (e.g., gold, silver), magnetic bead, or the like. Following incubation of the agent with PLKI, substrate and ATP, phosphorylation of the substrate can be assessed, e.g., by determining the amount of label from the ATP has transferred to the substrate.
Alternatively, a phosphorylated substrate can be detected using a phospho-specific antibody. The substrate peptide: LGEDQAEEISDDLLEDSLSDEDE (SEQ ID NO:
11) can be used to selectively assay PLK4 activity (see Johnson et al., Biochemistry (2007) 46(33): 9551-9563).
As also described above, a PLKI antagonist can be, for example, a small molecule, which can be found in nature (e.g., identified, isolated, purified) and/or artificially produced (e.g., synthesized). Small molecules can be tested for binding specificity in a screen for example, a high-throughput screen of chemical compounds and/or libraries (e.g., chemical, peptide, nucleic acid libraries).
Examples of small molecule antagonists of PLK, including PLK 1, are described in Johnson et al., Biochemistry (2007) 46(33): 9551-9563 (see also FIGS. 17A, 17B
and 18). Notably, some small molecule inhibitors are more selective at inhibiting PKL1-3 than PLK4 (see, e.g., compounds 20 and 21 disclosed herein and in Johnson et al., Biochemistry (2007) 46(33): 9551-9563; see also FIGS. 17A, 17B and 18).
Methods for Therapy One aspect of the invention relates to a method for inhibiting the growth of a tumor (e.g., by directly inhibiting tumor growth) that expresses a PLK4 comprising administering to a patient with the tumor a therapeutically effective amount (e.g., an anti-tumor effective amount) of a PLK4 antagonist. In one embodiment, a therapeutically effective amount is an amount that is sufficient to inhibit (e.g., reduce, prevent or retard) tumor cell growth (e.g., as measured by tumor cell proliferation, tumor size or mass, tumor differentiation or de-differentiation) and/or tumor progression (e.g., increased malignancy, tumor cell invasion, and/or cancer metastasis) for a particular cancer.
In one aspect, the PLK4 antagonist directly inhibits the growth of the tumor by inducing apoptosis of the tumor cells or by inhibiting proliferation of the tumor cells. The PLK4 antagonist can inhibit PLK4 gene expression (e.g., using siRNA, antisense oligonucleotides) or PLK4 protein activity (e.g., using an antibody, peptide, peptide mimetic) of PLK4, thereby directly inhibiting the growth of the cells of the tumor.
Another aspect of the invention is a method for treating a basal sub-type breast cancer, luminal B sub-type breast cancer, or a soft tissue cancer sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma, in a patient. The method comprises administering to the patient a therapeutically effective amount of a PLK4 antagonist. In one embodiment, the administered antagonist inhibits tumor growth directly by inducing the death (e.g., apoptosis) of the cells of the tumor or by inhibiting the growth (e.g., proliferation) of the cells of the tumor. In one embodiment, the method further comprises administering another therapeutic agent, such as a PLK1 antagonist. As described above, the PLK4 antagonist and the PLK1 antagonist can be administered simultaneously or sequentially.

In another aspect, the invention is a method for treating a patient with cancer metastasis. The method comprises administering to the patient a therapeutically effective amount of a PLK4 antagonist. In one embodiment, the administered antagonist inhibits cancer metastasis cell growth directly by inducing the death (e.g., apoptosis) of the cells of the cancer metastasis or by inhibiting the growth (e.g., proliferation) of the cells of the cancer metastasis. In one embodiment, the method further comprises administering another therapeutic agent, such as a PLKI
antagonist. For example, the PLK4 antagonist and the other therapeutic agent, such as a PLK1 antagonist, can be administered simultaneously or sequentially.
Another aspect, the invention is a method for inhibiting angiogenesis in a patient. In one embodiment, the method comprises administering to the patient a therapeutically effective amount of a PLK4 antagonist. In one embodiment, the method comprises administering to the patient an anti-angiogenic effective amount of a PLK4 antagonist. In another embodiment, the method further comprises administering another therapeutic agent, such as a PLK1 antagonist. The PLK4 antagonist and the other therapeutic agent, such as a PLK1 antagonist, can be administered simultaneously or sequentially, as described above.
Angiogenesis is associated with some cancers, more specifically it is associated with many solid tumors, but pathological angiogenesis can be unrelated to a cancer. For example, pathological angiogenesis contributes to diabetic retinopathy, rheumatoid arthritis and macular degeneration. Anti-angiogenic therapies can block angiogenic growth factor signals (e.g., endostatin, tumstatin, angiostatin) or the response of endothelial cells to those signals (e.g., avastatin).
Anti-angiogenic therapies may indirectly affect (inhibit, reduce) tumor growth by blocking the formation of new blood vessels that supply tumors with nutrients needed to sustain tumor growth and enable tumors to metastasize. Starving the tumor of nutrients and blood supply in this manner can eventually cause the cells of the tumor to die by necrosis and/or apoptosis. Previous work has indicated that the clinical outcomes (inhibition of endothelial cell-mediated tumor angiogenesis and tumor growth) of cancer therapies that involve the blocking of angiogenic factors (e.g., VEGF, bFGF, TGF-a, IL-8, PDGF) or their signaling have been more efficacious when lower dosage levels are administered more frequently, providing a continuous blood level of the antiangiogenic agent. (See Browder et al. Can.
Res.
60:1878-1886, 2000; Folkman J., Sem. Can. Biol. 13:159-167, 2003). This type of dosing can be referred to as an "anti-angiogenic" or "metronomic" schedule.
This anti-angiogenic dosing schedule is in contrast to the high dose, cyclic treatment (maximum tolerated dosing) regimen frequently used for therapies that directly inhibit tumor growth. An anti-angiogenic treatment regimen has been used with a targeted inhibitor of angiogenesis (thrombospondin I and platelet growth factor-4 (TNP-470)) and the chemotherapeutic agent cyclophophamide. Every 6 days, TNP-470 was administered at a dose lower than the maximum tolerated dose and cyclophophamide was administered at a dose of 170 mg/kg. This treatment regimen resulted in complete regression of the tumors. In fact, anti-angiogenic treatments are most effective when administered in concert with other anti-cancer therapeutic agents, for example, those agents that directly inhibit tumor growth (e.g., chemotherapeutic agents). Anti-angiogenic therapy can be used alone in the treatment of an angiogenesis disorder, or used in conjunction with anti-tumor therapy.
Accordingly, an anti-angiogenic effective amount of a PLK4 antagonist, for example, a small molecule, can be from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about 1 mg/kg, every 1 to 7 days over a period of about 4 to about 6 months. In addition, an anti-angiogenic effective amount of a PLK4 antagonist, for example, an antibody, can be from about 0.01 mg/kg to about 300 mg/kg body weight per treatment and preferably from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10 mg/kg every 1 to 7 days over a period of about 4 to about 6 months.
The anti-angiogenic effective amount of a PLK4 antagonist can be administered alone, as an adjuvant therapy to a primary cancer therapy (surgery, radiation), with anti-angiogenic therapies (e.g., avastatin, endostatin, tumstatin, angiostatin) or as a primary therapy with other adjuvant therapies (e.g., chemotherapeutic, hormone).
The effectiveness of a therapy (e.g., the reduction or elimination of a tumor, the prevention or inhibition of tumor growth, the treatment or prevention of an angiogenesis disorder, and/or the prevention or treatment of cancer metastasis) can be determined by any suitable method (e.g., in situ immunohistochemistry, imaging (MRI, NMR), 3H-thymidine incorporation).
The methods described herein comprise administering a PLK4 antagonist.
The PLK4 antagonist may be administered to the individual in need thereof as a primary therapy (e.g., as the principal therapeutic agent in a therapy or treatment regimen); as an adjunct therapy (e.g., as a therapeutic agent used together with another therapeutic agent in a therapy or treatment regime, wherein the combination of therapeutic agents provides the desired treatment; "adjunct therapy" is also referred to as "adjunctive therapy"); in combination with an adjunct therapy;
as an adjuvant therapy (e.g., as a therapeutic agent that is given to the subject in need thereof after the principal therapeutic agent in a therapy or treatment regimen has been given); or in combination with an adjuvant therapy (e.g., chemotherapy (e.g., dacarbazine (DTIC), Cis-platinum, cimetidine, tamoxifen, cyclophophamide), radiation therapy, hormone therapy (e.g., anti-estrogen therapy, androgen deprivation therapy (ADT), luteinizing hormone-releasing hormone (LH-RH) agonists, aromatase inhibitors (AIs, such as anastrozole, exemestane, letrozole), estrogen receptor modulators (e.g., tamoxifen, raloxifene, toremifene)), or biological therapy). Numerous other therapies can also be administered during a cancer treatment regime to mitigate the effects of the disease and/or side effects of the cancer treatment including therapies to manage pain (narcotics, acupuncture), gastric discomfort (antacids), dizziness (anti-veritgo medications), nausea (anti-nausea medications), infection (e.g., medications to increase red/white blood cell counts) and the like, all of which are readily appreciated by the person skilled in the art.
Thus, a PLK4 antagonist can be administered as an adjuvant therapy (e.g., with another primary cancer therapy or treatment). As an adjuvant therapy, the PLK4 antagonist can be administered before, after or concurrently with a primary therapy like radiation and/or the surgical removal of a tumor(s). In some embodiments, the method comprises administering a therapeutically effective amount of a PLK4 antagonist and one or more other therapies (e.g., adjuvant therapies, other targeted therapies). An adjuvant therapy (e.g., a chemotherapeutic agent) and/or the one or more other targeted therapies (e.g., a PLKI
antagonist) and the PLK4 antagonist can be co-administered simultaneously (e.g., concurrently) as either separate formulations or as a joint formulation. Alternatively, the therapies can be administered sequentially, as separate compositions, within an appropriate time frame (e.g., a cancer treatment session/interval such as 1.5 to 5 hours) as determined by the skilled clinician (e.g., a time sufficient to allow an overlap of the pharmaceutical effects of the therapies). The adjuvant therapy and/or one or more other targeted therapies (e.g., a PLK1 antagonist) and the PLK4 antagonist can be administered in a single dose or multiple doses in an order and on a schedule suitable to achieve a desired therapeutic effect (e.g., inhibition of tumor growth, inhibition of angiogenesis, and/or inhibition of cancer metastasis).
One or more agents that are an PLK4 antagonist can be administered in single or multiple doses. Suitable dosing and regimens of administration can be determined by a clinician and are dependent on the agent(s) chosen, pharmaceutical formulation and route of administration, various patient factors and other considerations. With respect to the administration of a PLK4 antagonist with one or more other therapies or treatments (adjuvant, targeted, cancer treatment-associated, and the like) the PLK4 antagonist is typically administered as a single dose (by e.g., injection, infusion, orally), followed by repeated doses at particular intervals (e.g., one or more hours) if desired or indicated.
The amount of the PLK4 antagonist to be administered (e.g., a therapeutically effective amount, an anti-tumor effective amount, an anti-angiogenesis effective amount, an anti-metastasis effective amount) can be determined by a clinician using the guidance provided herein and other methods known in the art and is dependent on several factors including, for example, the particular agent chosen, the subject's age, sensitivity, tolerance to drugs and overall well-being. For example, suitable dosages for a small molecule can be from about 0.00 1 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about I mg/kg body weight per treatment. Suitable dosages for antibodies can be from about 0.01 mg/kg to about 300 mg/kg body weight per treatment and preferably from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about mg/kg to about 10 mg/kg body weight per treatment. Where the PLK4 antagonist is a polypeptide (linear, cyclic, mimetic), the preferred dosage will result in a plasma concentration of the peptide from about 0.1 g/mL to about 200 g/mL.
Determining the dosage for a particular agent, patient and cancer is well within the abilities of one of skill in the art. Preferably, the dosage does not cause or produces minimal adverse side effects (e.g., immunogenic response, nausea, dizziness, gastric upset, hyperviscosity syndromes, congestive heart failure, stroke, pulmonary edema).
Methods for Administration In one aspect of the invention, an "anti-tumor effective amount" of a PLK4 antagonist is administered to a patient in need thereof. For example, agents which directly inhibit tumor growth (e.g., chemotherapeutic agents) are conventionally administered at a particular dosing schedule and level to achieve the most effective therapy (e.g., to best kill tumor cells). Generally, about the maximum tolerated dose is administered during a relatively short treatment period (e.g., one to several days), which is followed by an off-therapy period. In a particular example, the chemotherapeutic cyclophosphamide is administered at a maximum tolerated dose of 150 mg/kg every other day for three doses, with a second cycle given 21 days after the first cycle. (Browder et al. Can Res 60:1878-1886, 2000).
An anti-tumor effective amount of PLK4 which directly inhibits the expression or activity of PLK4 in a tumor cell (e.g., neutralizing antibodies, inhibitory nucleic acids (e.g., siRNA, antisense nucleotides)) can be administered, for example, in a first cycle in which the maximum tolerated dose of the antagonist is administered in one interval/dose, or in several closely spaced intervals (minutes, hours, days) with another/second cycle administered after a suitable off-therapy period (e.g., one or more weeks). Suitable dosing schedules and amounts for a PLK4 antagonist can be readily determined by a clinician of ordinary skill.
Decreased toxicity of a particular PLK4 antagonist as compared to chemotherapeutic agents can allow for the time between administration cycles to be shorter.
When used as an adjuvant therapy (to, e.g., surgery, radiation therapy, other primary therapies), an anti-tumor effective amount of a PLK4 antagonist is preferably administered on a dosing schedule that is similar to that of the other cancer therapy (e.g., chemotherapeutics), or on a dosing schedule determined by the skilled clinician to be more/most effective at inhibiting (reducing, preventing) tumor growth.
A treatment regimen for an anti-tumor effective amount of a small molecule PLK4 antagonist can be from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about 1 mg/kg, every 1 to 7 days over a period of about 4 to about 6 months.
A treatment regimen for an anti-tumor effective amount of an antibody PLK4 antagonist can be from about 0.01 mg/kg to about 300 mg/kg body weight per treatment and preferably from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10 mg/kg body weight per treatment, every 1 to 7 days over a period of about 4 to about 6 months.
A treatment regimen for an anti-angiogenesis effective amount of a small molecule PLK4 antagonist can be from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about 1 mg/kg, every 1 to 7 days over a period of about 4 to about 6 months.
A treatment regimen for an anti-angiogenesis effective amount of an antibody PLK4 antagonist can be from about 0.01 mg/kg to about 300 mg/kg body weight per treatment and preferably from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10 mg/kg body weight per treatment, every 1 to 7 days over a period of about 4 to about months.
A variety of routes of administration can be used including, for example, oral, dietary, topical, transdermal, rectal, parenteral (e.g., intravenous, intraaterial, intramuscular, subcutaneous injection, intradermal injection), intravenous infusion and inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops) routes of administration, depending on the agent and the particular cancer to be treated. Administration can be local or systemic as indicated. The preferred mode of administration can vary depending on the particular agent chosen. In one embodiment, intravenous infusion is preferred (e.g., to administer neutralizing PLK4 antibodies).

The agent (such as a PLK4 antagonist) can be administered to a mammalian subject as part of a pharmaceutical or physiological composition. For example, the agent can be administered as part of a pharmaceutical composition for inhibition of PLK4 expression (e.g., inhibition of PLK4 gene expression and/or inhibition of PLK4 activity) and a pharmaceutically acceptable carrier. Formulations or compositions comprising a PLK4 antagonist or compositions comprising a PLK4 antagonist and one or more other therapeutic agents (e.g., a PLKI antagonist) will vary according to the route of administration selected (e.g., solution, emulsion or capsule). Suitable pharmaceutical carriers can contain inert ingredients which do not interact with the PLK4 antagonist. Standard pharmaceutical formulation techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's lactate and the like.
Formulations can also include small amounts of substances that enhance the effectiveness of the active ingredient (e.g., emulsifying, solubilizing, pH
buffering, wetting agents). Methods of encapsulation compositions (such as in a coating of hard gelatin or cyclodextran) are known in the art. For inhalation, the agent can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer or nebulizer or pressurized aerosol dispenser).
For example, a nucleic acid-based PLK4 antagonist (e.g., siRNA, shRNA, antisense oligonucleotide, natural or synthetic nucleic acids, nucleic acid analogs, aptamers) can be introduced into a mammalian subject in a number of ways. For instance, chemically synthesized or in vitro transcribed nucleic acids can be transfected into cells in cell culture by any suitable method (e.g., viral infection).
The nucleic acids may also be expressed endogenously from expression vectors or PCR products in host cells or packaged into synthetic or engineered compositions (e.g., liposomes, polymers, nanoparticles) that can then be introduced directly into the bloodstream of a mammalian subject (by, e.g., injection, infusion). Anti-nucleic acids or nucleic acid expression vectors (e.g., retroviral, adenoviral, adeno-associated and herpes simplex viral vectors, engineered vectors, non-viral-mediated vectors) can also be introduced into a mammalian subject directly using established gene therapy strategies and protocols (see e.g., Tochilin V.P. Annu. Rev.
Biomed.
Eng. 8:343-375, 2006; Recombinant DNA and Gene Transfer, Office of Biotechnology Activities, National Institutes of Health Guidelines).
Similarly, where the agent is a protein or polypeptide, the agent can be administered via in vivo expression of recombinant protein. In vivo expression can be accomplished by somatic cell expression according to suitable methods (see, e.g., U.S. Patent No. 5,399,346). Further, a nucleic acid encoding the polypeptide can also be incorporated into retroviral, adenoviral or other suitable vectors (often a replication deficient infectious vector) for delivery, or can be introduced into a transfected or transformed host cell capable of expressing the polypeptide for delivery. In the latter embodiment, the cells can be implanted (alone or in a barrier device), injected or otherwise introduced in an amount effective to express the polypeptide in a therapeutically effective amount.

EXEMPLIFICATION

EXAMPLE 1: PLK4 Identification and Association With Breast and Lung Cancer Gene expression profiling and overexpression of PLK4 analysis (e.g., FIG
5): RNA was isolated from cells using Trizol Reagent (Invitrogen Corporation) as described by the manufacturer. Total RNA concentrations were assayed on a ND-1000 Spectrophotometer (Nanodrop Technologies). The amount of PLK4 mRNA
was measured by analyzing equivalent amounts of total RNA and normalized using (3-Actin as a reference gene.
Amplification primers for PLK4 are:
5'-CCACAGACAACAATGCCAAC-3' (forward) (SEQ ID NO: 5) and 5'-GGTCTGCAAATGGAAAAGGA-3' (reverse) (SEQ ID NO: 6).
Amplification primers for (3-Actin are:
5'-GGCACTCTTCCAGCCTTCCTT-3' (forward) (SEQ ID NO: 12) and 5'-TCTCCTTCTGCATCCTGTCG-3' (reverse) (SEQ ID NO: 13).
SYBR Green quantitation was performed on an Eppendorf Mastercycler ep realplex (Eppendorf Canada Ltd.) using OneStep RT-PCR Kit (Qiagen) following the manufacturer's protocol. Standard curves were used to ensure that all PCR
results fell within the linear range of the assay.

EXAMPLE 2: Identification of PLK4 Association with Soft Tissue Cancers (e.g.
FIG. 5).
Gene expression profiles of 39 human sarcoma samples and 15 control normal tissue samples were assessed using Affymetrix HG U133A oligonucleotide arrays by SS Yoon et al. (Detwiller et al., Cancer Res. (2005) 65(13) 5881-9;
Yoon et al., J. Surg. Res. (2006) 135(2):282-90). Fluorescence was converted to signal intensity by means of Affymetrix Microarray Suite v4.0 software. In addition to signal intensity, the Affymetrix software performed an absolute analysis that indicated if the transcript was present (P), absent (A), marginal (M), or no call (NC).
The absolute analysis was based on comparing intensities of perfect match probes to mismatch probes. If the intensity of the mismatch probes was higher than that of the perfect match probes, the transcript was called "absent". The data are available online through NCBI Gene Expression Omnibus repository as dataset GDS 1209.
PLK4 transcript was represented on the Affymetrix array by 3 probe sets:
204887_s_at, 204886_at, 211088_s_at. The latter probe set had "absent" calls in nearly all samples, therefore the analysis was limited to the first two probe sets.
Since all of the normal samples had "absent" calls on the first probe set and all but one on the second, it was concluded that PLK4 expression was not detectable in normal tissues and therefore no formal statistical tests comparing cancer tissues to normal ones were necessary or meaningful. FIG. 5 illustrates that the box plots of PLK4 mRNA expression levels represented by signal intensity values in normal tissues and sarcoma tissues. It is shown that PLK4 is over-expressed in sarcomas compared to its levels in the normal tissues tested.

EXAMPLE 3: Validation of PLK4 as an Anti-Tumor Target (e.g., FIGS 7-12) siRNAs and reagents: The sequences for the PLK4 siRNAs are:
siRNA#1 5'-AAGCCATGTACAAAGCAGGAA-3' (SEQ ID NO: 7);
siRNA#2 5'-ACTCCTTTCAGACATATAAG-3' (SEQ ID NO: 8);
siRNA#3 5'-AACTATCTTGGAGCTTTATAA-3' (SEQ ID NO: 9); and siRNA#4 5'-CTGGTAGTACTAGTTCACCTA-3' (SEQ ID NO: 10).
Transfection of siRNAs into cell lines: Cells were seeded in antibiotic-free growth medium one day before transfection so that their confluency was 30-40%
at the day of transfection. The indicated siRNAs (40 nM) and Lipofectamine 2000 Transfection Reagent (Invitrogen Corporation) were diluted separately in reduced-serum medium OptiMEM (GIBCO Life Sciences) and incubated for 5 minutes at room temperature. The two solutions were mixed and incubated for 20 minutes at room temperature. siRNA-Lipofectamine 2000 complexes were then added to the cells, and the plate was mixed by gentle rocking. Transfected cells were incubated at 37 C and 5% CO2 for varying amounts of time.
Cell viability (e.g., FIG 8): Cells in 96-well plates were transfected with the indicated siRNAs (40 nM) using Lipofectamine 2000 Transfection Reagent (Invitrogen Corporation) and cultured at 37 C. After 5 days, the cells were washed with PBS, fixed with 10% ice-cold trichloroacetic acid at 4 C and dried at room temperature. Proteins were stained with SRB in 1% acetic acid at room temperature, washed with 1% acetic acid and dried at 37 C. To dissolve the SRB bound proteins, 10 mM Tris base was added to each well and incubated at room temperature with mechanical agitation. SRB bound to protein was measured by absorbance using a SpectraPlus microplate reader (Molecular Devices Corporation).
Cell cycle analysis assays (e.g., FIGS. 9 and 10): Cells in 6-well plates were transfected with the indicated siRNAs (40 nM) using Lipofectamine 2000 Transfection Reagent (Invitrogen Corporation) and cultured at 37 C. After 3-4 days, the cells were trypsinized, washed in PBS and centrifuged. Cell pellets were resuspended in propidium iodide in Hepes buffer, mixed and incubated at room temperature in the dark. Samples were read in a FACSCalibur flow cytometer (Becton, Dickinson and Company) and data analyzed using FloJo software (Tree Star, Inc.).
Apoptosis assay (e.g., FIG 11): Apoptosis was measured using Cell Death Detection ELISAP''us Kit (Roche) according to the manufacturer's protocol.
Cells in 96-well plates were transfected with the indicated siRNAs (40 nM) using Lipofectamine 2000 Transfection Reagent (Invitrogen Corporation) and cultured at 37 C. After varying amounts of time, the cells were washed in PBS, lysed and added to streptavidin-coated microplates containing anti-histone-biotin and anti-DNA-POD antibodies. Nucleosomes were photometrically detected by measuring POD activity with 2,2'-Azino-di[3-ethyl-benz-thiazolin-sulfonat] as a substrate using a SpectraPlus microplate reader (Molecular Devices Corporation).
Cell growth inhibition (e.g., FIG 12): Cells were transfected with the indicated siRNAs (40 nM) using Lipofectamine 2000 Transfection Reagent (Invitrogen Corporation), mixed with culture medium containing 0.7% agar in 6-well plates and cultured at 37 C. After 3 weeks, the top layer of the culture was stained with 0.2% p-iodonitrotetrazolium violet and colonies were counted using a Sorcerer Colony Counter (Optomax).

EXAMPLE 4: PLK4 Overexpression is Associated with Reduced Patient Survival, Increased Incidence of Metastasis, Increased Risk of Relapse, and Poor Prognosis Patient data sets (e.g., FIGS. 15-18): All gene expression analyses were done in 3 individual datasets and in a combined dataset. The datasets were combined after normalizing expression values, e.g. logratios, of each gene by mean and standard deviation in each dataset. Regression models in the combined dataset contained a dataset term in addition to the logratio term. To verify that results did not depend on normality assumption, another combined dataset was assembled using logratio ranks, computed separately in each dataset and scaled to vary from 0 to 1.
The same analyses were performed and general agreement with the results from the normalized logratio dataset was verified.
Survival analysis was performed using Cox regression of survival times vs.
gene expression logratios, followed by the estimation of false discovery rates by Benjamini and Hochberg's method. Perou dataset provided data on recurrence and survival; NEJM295 on recurrence, metastasis, and survival; PNAS on survival only;
the combined dataset could be analyzed for survival, i.e. risk of death, only.
High expression of PLK4 was associated with higher risks of recurrence, metastasis, and death in all datasets where such data were available. These associations reached formal statistical significance with p values < 0.05 and false discovery rates < 0.2 in NEJM295 and the combined dataset. Kaplan-Meier curves were generated in individual datasets for illustration purposes. In order to generate the curves, logratios were rank-ordered and split into three equal groups. The curves for the highest and the lowest tertiles were displayed on the plots.
Tumor gene expression profiles were classified into subtypes defined by Hu et al. The classification of the Perou dataset was provided by its authors;

and PNAS datasets were classified into Luminal A, Luminal B, Basal-like and HER2+/ER- subtypes using the intrinsic gene set defined by Hu et al. Different subtypes were compared to each other and, where available, to normal controls.
Peron dataset was the only one that contained a normal control group. All cancers as a group and individual subtypes were compared to the control group using t tests. Significantly higher than normal expression of PLK4, with p values <
0.05 and false discovery rates < 0. 1, was detected in all cancers as a group and in Basal-like, Luminal B, and HER2+/ER- subtypes. The over-expression was the highest and most significant in the basal-like subtype, where it was, on average, 2-fold higher than normal.
An additional analysis was performed on the Perou dataset to determine if a fraction of cancers expressed a gene significantly outside of the normal range. This analysis was designed to detect over-expression in a subset of a potentially heterogeneous population of cancers. The normal range was defined as the mean +/-3 standard deviations of the normal control group. The fractions of tumor samples that fell above and below this range were recorded as percentages of the total number of tumors, in all cancers as a group and in individual subtypes. PLK4 was found to be over-expressed in 26.4% of all tumors and in 48.5% of basal-like tumors.

Different tumor subtypes were compared to each other using one way ANOVA and pair-wise t tests in all 3 datasets. There were statistically significant differences in PLK4 expression between different tumor subtypes in all 3 datasets, with estimated false discovery rates in 10'5-10"17 range for the ANOVA p values.
Expression was highest in basal-like and HER2+/ER- tumors and lowest in normal controls in Perou and in luminal A in the other two datasets. Therefore, higher PLK4 expression was associated with more aggressive subtypes of breast cancer.
EXAMPLE 5: PLK4 knockdown suppresses breast tumor growth: xenograft models (e.g., FIGS. 19A and 19B) Materials and methods: A double-stranded oligonucleotide encoding a human PLK4 gene-specific shRNA (sense insert sequence 5'-GTTCTATCTTGGAGCTTTAT-3'; SEQ ID NO: 14) was ligated into the RNAi-Ready-pSIREN-RetroQ-ZsGreen retroviral vector (Clontech). Amphotropic-Phoenix packaging cells (ATCC, Manassas, VA) were transiently transfected with either control RNAi-Ready-pSIREN-RetroQ-ZsGreen-shLUC (Clontech) or RNAi-Ready-pSIREN-RetroQ-ZsGreen-shPLK4 using FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN). Culture supernatants were collected 2 days after transfection and filtered through 0.45- m pore-size filters. MDA-MB-468 breast cancer cells (ATCC, Manassas, VA) were infected with retroviruses by culturing the cells for 24 hours in 1:1 Phoenix conditioned media (Dulbecco's Modified Eagle's Media, 10% FCS, supplemented with 8 g/ml Polybrene; Sigma-Aldrich). This transfection process was repeated three times to increase the transfection efficiency. One day after the final infection, the RNAi-Ready-pSIREN-RetroQ-ZsGreen-shLUC and RNAi-Ready-pSIREN-RetroQ-ZsGreen-shPLK4 infected cancer cells were trypsinized, counted and injected subcutaneously into the left and right hindlimb, respectively, of nude mice at concentrations of 5 x 106 cells (5 mice per group). The infected cells were also analyzed for PLK4 expression using Western blotting with an anti-PLK4 antibody. The tumors were measured and viable tumor area was calculated twice weekly for approximately 10 weeks.
Results: Infection of MDA-MB-468 cells with the PLK4 shRNA-encoding construct led to the reduction of PLK4 protein expression in these cells by approximately 60% compared to that of the control cells (FIG. 19A).
Furthermore, reduction of PLK4 protein levels resulted in a significant suppression of tumor growth in mice (FIG. 19B). At day 69 post cancer cell implantation, the average tumor volume of the PLK4 knockdown tumors was reduced by 60% in comparison to that of control (FIG. 19B). Taken together, these results suggest that inhibition of PLK4 activity in cancer cells inhibits tumor growth in tumors that express PLK4.

The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims (35)

1. A method for identifying a cancer patient candidate for anti-cancer therapy using a PLK4 antagonist, wherein the cancer patient is not a colorectal cancer patient, comprising:
(a) providing a suitable sample from said patient; and (b) assessing expression of PLK4 in said sample;
wherein increased expression of PLK4 in said sample relative to a suitable control indicates that the breast cancer patient is a candidate for anti-cancer therapy using a PLK4 antagonist.

2. The method of Claim 1, wherein the cancer patient is diagnosed with a basal sub-type breast cancer, a luminal B sub-type breast cancer, or a soft tissue cancer.

3. The method of Claim 2, wherein the soft tissue cancer is a sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.

4. A method for treating a basal sub-type breast cancer, luminal B sub-type breast cancer, or a soft tissue cancer in a patient, comprising administering to the patient a therapeutically effective amount of a PLK4 antagonist.

5. The method of Claim 4, wherein the method further comprises administering a PLK1 antagonist.

6. The method of Claim 5, wherein the PLK4 antagonist and the PLK1 antagonist are administered simultaneously or sequentially.

7. A method for predicting cancer metastasis in a cancer patient, comprising:
(a) providing a suitable sample from said patient; and (b) determining PLK4 expression in said sample;
wherein increased PLK4 expression in said sample as compared with a suitable control is indicative of an increased likelihood cancer metastasis in said patient.

8. The method of Claim 7, wherein the cancer patient is a breast cancer patient or a soft tissue cancer patient.

9. The method of Claim 8, wherein the breast cancer patient has basal sub-type breast cancer.

10. The method of Claim 8, wherein the breast cancer patient has luminal B sub-type breast cancer.

11. The method of Claim 8, wherein the a soft tissue cancer patient has a sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.

12. A method for screening a breast cancer patient as an aid for selecting aggressive cancer therapy of said patient, comprising:
(a) providing a suitable sample from said patient; and (b) determining PLK4 expression in said sample;
wherein increased PLK4 expression in said sample as compared with a suitable control indicates the patient is a candidate for aggressive cancer therapy of said patient.

13. The method of Claim 12, wherein said breast cancer patient has basal sub-type breast cancer.

14. The method of Claim 12, wherein the breast cancer patient has luminal B
sub-type breast cancer.

15. A method for screening a soft tissue cancer patient as an aid for selecting aggressive cancer therapy of said patient, comprising:
(a) providing a suitable sample from said patient; and (b) determining PLK4 expression in said sample;
wherein increased PLK4 expression in said sample as compared with a suitable control indicates the patient is a candidate for aggressive cancer therapy of said patient.

16. The method of Claim 15, wherein said soft tissue cancer patient has a sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.

17 A method for inhibiting angiogenesis in a patient, comprising administering to the patient a therapeutically effective amount of a PLK4 antagonist.

18. The method of Claim 17, wherein the method further comprises administering a PLK1 antagonist.

19. The method of Claim 18, wherein the PLK4 antagonist and the PLK1 antagonist are administered simultaneously or sequentially.

20. A method for treating a cancer metastasis disease in a patient, comprising administering to the patient a therapeutically effective amount of a PLK4 antagonist.

21. The method of Claim 20, wherein the method further comprises administering a PLK1 antagonist.

22. The method of Claim 21 wherein the PLK4 antagonist and the PLK1 antagonist are administered simultaneously or sequentially.

23. A method for identifying a patient that is likely to be responsive to PLK4 antagonist therapy, comprising:
(a) providing a suitable sample from said patient; and (b) determining PLK4 expression in said sample;
wherein increased PLK4 expression in said sample as compared with a suitable control indicates that the patient is likely to be responsive to PLK4 antagonist therapy.

24. The method of Claim 23, wherein the tissue sample is a breast tissue sample and the patient has breast cancer.

25. The method of Claim 24, wherein the patient has a basal sub-type breast cancer or luminal B sub-type breast cancer.

26. The method of Claim 23, wherein the patient has a soft tissue cancer.

27. The method of Claim 26, wherein the patient has a soft tissue cancer which is a sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.

28. The method of Claim 23, wherein the patient has a disorder associated with angiogenesis.

29. The method of Claim 23, wherein the patient has a cancer metastasis disease.

30. A method for treating a cancer patient, comprising administering to the patient a therapeutically effective amount of a small molecule PLK4 antagonist.

31. The method of Claim 30, wherein the small molecule PLK4 antagonist is selected from the group consisting of:

and a physiologically acceptable salt of any of the foregoing.

32. The method of Claim 30, wherein the cancer patient is diagnosed with breast cancer.

33. The method of Claim 32, wherein the breast cancer patient is diagnosed with a basal sub-type breast cancer or a luminal B sub-type breast cancer.

34. The method of Claim 30, wherein the cancer patient is diagnosed with a soft tissue cancer.

35. The method of Claim 34, wherein the soft tissue cancer is a sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleimoprhic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.